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Title:
HYBRIDIZED POLY(AMIDOAMINE)-AMINO ACID DENDRIMERS
Document Type and Number:
WIPO Patent Application WO/2021/046307
Kind Code:
A1
Abstract:
Disclosed herein are hybrid poly(amidoamine)-amino acid dendrimers, their methods of manufacture, and uses thereof. In accordance with the purposes of the disclosed materials and methods, as embodied and broadly described herein, the disclosed subject matter, in one aspect, relates to dendrimer compounds, methods for their manufacture, and uses thereof. More specifically, the subject matter disclosed herein relates to hybridized poly(amidoamine)-amino acid dendrimer compounds of Formula I as described herein, their methods of manufacture, and uses thereof.

Inventors:
SMITH RYAN (US)
MENEGATTI STEFANO (US)
GORMAN CHRISTOPHER B (US)
Application Number:
PCT/US2020/049338
Publication Date:
March 11, 2021
Filing Date:
September 04, 2020
Export Citation:
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Assignee:
UNIV NORTH CAROLINA STATE (US)
International Classes:
A61K9/50
Foreign References:
US5795582A1998-08-18
US20090047272A12009-02-19
US20160015823A12016-01-21
Attorney, Agent or Firm:
ANDREANSKY, Eric S. et al. (US)
Download PDF:
Claims:
CLAIMS

We claim:

1. A dendrimer defined by Formula I: or a salt thereof; wherein: (Core) is a is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11;

* indicates covalent attachment to a carbon moiety of (BUI); b is 4;

BU are building units;

BUx are building units of generation x, wherein the total number of building units within generation x of the dendrimer of Formula I is equal to b(2x-1); wherein BU within each generation x is selected from BUA or BUB: wherein BU in at least one generation x is selected from BUB;

# indicates covalent attachment to an amino moiety of Core and an amino moiety of BU;

+ indicates covalent attachment to a carbonyl moiety of BU or a covalent attachment to z;

A is selected at each occurrence in a generation x from a natural or non-natural amino acid moiety that is optionally protected with one or more protecting groups, wherein the carbonyl group of the amino acid moiety is covalently attached to the amino group @ of BUB and the amino group of the amino acid moiety is covalently attached to the carbonyl group $ of BUb; Z is selected at each occurrence from a monovalent capping moiety; or two Z groups attached to the same amino moiety may be brought together with the nitrogen they are attached to form an optionally substituted heterocycle ring.

2. The dendrimer of claim 1, wherein A is selected at each occurrence in a generation x from a natural amino acid moiety.

3. The dendrimer of any one of claims 1 or 2, wherein A is selected from: or a protected derivative thereof.

4. The dendrimer of any one of claims 1-3, wherein A is selected at each occurrence in a generation x from a non-natural amino acid moiety.

5. The dendrimer of any one of claims 1-4, wherein A is be selected from allylglycine, propargylglycine, azidolysine, or derivatives thereof.

6. The dendrimer of any one of claims 1-5, wherein A is selected from or a derivative thereof.

7. The dendrimer of any one of claims 1-6, wherein A is selected from or a derivative thereof.

8. The dendrimer of any one of claims 1-7, wherein Z is monovalent moiety substituted with one or more primary amine, secondary amine, amide, carbamate, or urea groups, or combinations thereof.

9. The dendrimer of any one of claims 1-8, wherein Z is selected from:

C is selected from: and and

R is independently selected from C1-C12 alkyl.

10. The dendrimer of any one of claims 1-8, wherein Z is selected from 11 The dendrimer of any one of claims 1-8, wherein Z is selected from

12. The dendrimer of any one of claims 1-8, wherein Z is selected from: and

C is selected from:

13. The dendrimer of any one of claims 1-8, wherein two Z groups attached to an amino acid moiety are brought together with the nitrogen to which they are attached to form , or a derivative thereof.

14. The dendrimer of claim 9, wherein Z is

15. The dendrimer of any one of claims 1-14 of Formula la: or a salt thereof.

16. The dendrimer of claim 15, wherein BUI is selected from:

17. The dendrimer of claim 15, wherein BUI is selected from:

18. The dendrimer of any one of claims 15-17, wherein Z is selected from:

19. The dendrimer of any one of claims 1-14 of Formula lb: or a salt thereof. 20. The dendrimer of claim 19, wherein BUI is selected from: and BU2 is BUA. 21. The dendrimer of claim 19, wherein BUI is selected from: and BU2 is BUA.

22 The dendrimer of claim 19, wherein BU2 is selected from: and BUI is BUA.

23. The dendrimer of claim 19, wherein BU2 is selected from: and BUI is BUA.

24. The dendrimer of any one of claims 19-23, wherein Z is selected from:

25. The dendrimer of claim 1, wherein the dendrimer is selected from:

and

or a salt thereof.

26. A conjugate comprising: a dendrimer of any one of claims 1-25; and a peptide covalently bound to the dendrimer.

27. A conjugate comprising: a dendrimer of any one of claims 1-25; and one or more therapeutic agents covalently bound to the dendrimer.

28. A drug delivery system comprising: a plurality of microgel particles, each microgel particle having a surface; and a plurality of dendrimers of any one of claims 1-25 adhered to the surface of the microgel particles.

Description:
HYBRIDIZED POLY (AMIDOAMINE)-AMINO ACID DENDRIMERS

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of priority to U.S. Provisional Application No. 62/895,647, filed September 4, 2019, the disclosure of which is incorporated herein by reference in its entirety.

TECHNICAL FIELD

The subject matter disclosed herein relates generally to dendrimer compounds and their methods of manufacture. More specifically, the subject matter disclosed herein related to hybridized poly(amidoamine)-amino acid dendrimer compounds and their manufacture and uses thereof.

BACKGROUND

Since the pioneering work of Fritz Vogtle and Donald Tomalia on dendritic macromolecules, now popularized under the name “Dendrimers”, the body of literature in this field has grown at a staggering rate, nearing 60,000 publications in 2020. PAMAM dendrimers (the structure that populates -25% of all dendrimer publications) are globular macromolecules that are densely decorated with terminal functional groups, a non-functional core with internal cavities, and tunable nano-metric size determined by the number of generations synthesized. Despite their scientific relevance, dendrimers have not yet received adequate translation attention and have made little inroad in pharmaceutical manufacturing and clinical setups. The list of commercial dendrimer products is indeed brief, with only two in vivo products as of 2015, namely OcuSeal ® , a liquid ocular bandage, and VivaGel ® , a vaginal microbicide gel. As a research platform, PAMAM dendrimers are prevalent. Considering that the aggregate number of “PAMAM” publications since 1990 is -16,000 articles, and assuming that each PAMAM publication requires 2 - 5 grams of material then roughly 32 - 80 kg of PAMAM dendrimer have been purchased for published academic research. Since each gram of PAMAM dendrimer costs -$500 then $16 - 40 million USD revenue has been generated strictly from PAMAM sales that translated to academic publications. This does not include non-published research and industrial research that has not materialized, and therefore the actual revenue is likely much greater.

There are a number of limitations associated with PAMAM dendrimers that have prevented further clinical adoption. Particularly sought after is an update in dendrimer architecture, especially focused on moving beyond terminal-only functionality. This disclosure addresses these as well as other needs.

SUMMARY

In accordance with the purposes of the disclosed materials and methods, as embodied and broadly described herein, the disclosed subject matter, in one aspect, relates to dendrimer compounds, methods for their manufacture, and uses thereof. More specifically, the subject matter disclosed herein relates to hybridized poly(amidoamine)-amino acid dendrimer compounds of Formula I as described herein, their methods of manufacture, and uses thereof.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the chemical structure of dendrimers of Formula I of the present disclosure.

FIG. 2 shows the chemical structure of G.3-Glu 8 -OH dendrimer as described herein.

FIG. 3 shows the chemical structure of G.3-Lys 8 -OH dendrimer as described herein.

FIG. 4 shows the chemical structure of G.2-Glu 4 -OH dendrimer as described herein.

FIG. 5 shows the chemical structure of G.2-Lys 4 -OH dendrimer as described herein.

FIG. 6 shows (a) Fgl-G3-Lys-OH radial distribution function; (b) Fgl-G3-Glu-OH radial distribution function; (c) Fgl-G3-Lys-OH average water solvation; and (d) Fgl-G3-Glu-OH average water solvation.

FIG. 7 shows (a) a photograph of microfluidic device; (b) a microscopic view of device geometry; (c) endothelial cells attached to device surfaces; (d) evaluate of nanoparticle binding/uptake by endothelial cells; and (e) correlation between nanoparticle binding in the devices and in the lungs.

FIG. 8 shows (a) a schematic of the device and inlet and outlet flow constituents; (b) microscopy view of the microfluidic surface containing co-cultured TNBC MDA-MB-231 breast cells and MCF 10A breast epithelial cells; evaluation of DDC performance by (c) binding/uptake of DDCs by fluorescent microscopy; and (d) toxicity against both cell lines using GFP -labeled cells.

FIG. 9 shows the values of R h of G.2-Lys 4 -OH, G.2-Glu 4 -OH, G.3-Lys 8 -OH, and G.3- G1U 8 -OH in 100 mM citrate buffer at pH 5, 100 mM phosphate buffer at pH 7, and 100 mM tris- HC1 buffer at pH 9, as measured by DLS analysis.

FIG. 10 shows the profiles of viscosity versus shear rate obtained with solutions of G.3- Lys 8 -OH and G.3-Glu 8 -OH Dendripeps, at 100 mg/ml in Milli-Q water. FIGs. 11A and 11B are snapshots of MD simulations of (11 A) G.3-Lys 8 -OH Dendripep with only primary amines protonated and (11B) G.3-Glu 8 -OH Dendripep with tertiary amines protonated and carboxyl groups deprotonated.

FIG. 12 is a representative schematic depicting a drug delivery system comprising a microgel particle and dendrimers of the present disclosure adhered to the surface of the microgel particle. The dendrimer coating is particularly sensitive to shear stress and is dissociated from the microgel surface upon exposure to mild shear stress.

FIG. 13 is a representative schematic depicting a drug delivery system comprising a microgel particle and dendrimers of the present disclosure adhered to the surface of the microgel particle. The dendrimer coating is particularly sensitive to shear stress and is dissociated from the microgel surface upon exposure to mild shear stress. This creates a dendrimer coating of selective thickness that can be controlled by the shear stress applied to the system.

FIGs. 14A-14C are confocal microscopy images of A) Dendripeps, B) Dendripeps, and C) Fluoresbrite ® 641 Carboxylate Microsphere 0.50 mm.

FIG. 15 is a series of confocal microscopy images showing Dendripeps adhered to the surface of Fluoresbrite ® 641 Carboxylate Microsphere 0.50 mm. The left-most images are taken using a red fluorescent channel to exclusively image the microparticles that fluoresce red. The middle images are taken using a green fluorescent channel to exclusively image the dendrimers that fluoresce green. The right most image is taken using both the red and green fluorescent channels to image both the microparticles and the dendrimers.

FIG. 16 is a series of confocal microscopy images showing PAMAM G2-NH 2 dendrimers adhered to Fluoresbrite ® 641 Carboxylate Microspheres 0.50 mm. The left most images are taken using a red fluorescent channel to exclusively image the microparticles that fluoresce red. The middle images are taken using a green fluorescent channel to exclusively image the dendrimers that fluoresce green. The right-most image is taken using both the red and green fluorescent channels to image both the microparticles and dendrimers.

FIG. 17 is a series of confocal microscopy images showing a Dendripep coating onto the surface of Fluoresbrite ® 641 Carboxylate Microspheres 0.50 mm being removed by the application of shear force. The left-most images are taken using a red fluorescent channel to exclusively image the microparticles that fluoresce red. The middle images are taken using a green fluorescent channel to exclusively image the dendrimers that fluoresce green. The right- most image is taken using both the red and green fluorescent channels to image both the microparticles and dendrimers. FIG. 18 is a plot that shows the release of a drug (doxorubicin) as a function of time as a free drug and also encapsulated by a Dendripep. When doxorubicin is encapsulated by a Dendripep, the rate of release is decreased when compared to free doxorubicin.

FIG. 19 is a representative schematic depicting a drug delivery system comprising a microgel particle, a therapeutic encapsulated within the microgel particle, and dendrimers of the present disclosure adhered to the surface of the microgel particle. The dendrimers are release upon exposure of the system to a strong shear stress, subsequently allowing release of the encapsulated therapeutic.

FIG. 20 is a representative schematic drawing showing drug-conjugated dendrimers as described herein adhered to the surface of a cell. Upon exposure of the cell to a strong shear stress, the drug-conjugated dendrimers are release from the cell surface.

FIG. 21 is a representative schematic drawing showing a drug covalently conjugated to the internal functional groups of Dendripeps. Upon exposure to an external stimuli (such as decreased pH, enzymatic cleavage, etc.) the drug is released from within the Dendripep.

FIG. 22 is a representative schematic drawing showing drug-conjugated Dendripeps as described herein adhered to the surface of a cell. When the cell reaches vasculature in vivo that has atherosclerosis, the decreased diameter of the vasculature increase the shear force experience by the blood cell, subsequently removing the Dendripep-drug coating from the surface of the cell.

DETAILED DESCRIPTION

The materials, compounds, compositions, and methods described herein may be understood more readily by reference to the following detailed description of specific aspects of the disclosed subject matter, the Figures, and the Examples included therein.

Before the present materials, compounds, compositions, and methods are disclosed and described, it is to be understood that the aspects described below are not limited to specific synthetic methods or specific reagents, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular aspects only and is not intended to be limiting.

Also, throughout this specification, various publications are referenced. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art to which the disclosed matter pertains. The references disclosed are also individually and specifically incorporated by reference herein for the material contained in them that is discussed in the sentence in which the reference is relied upon.

General Definitions

In this specification and in the claims that follow, reference will be made to a number of terms, which shall be defined to have the following meanings:

Throughout the specification and claims the word “comprise” and other forms of the word, such as “comprising” and “comprises,” means including but not limited to, and is not intended to exclude, for example, other additives, components, integers, or steps.

As used in the description and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise.

“Optional” or “optionally” means that the subsequently described event or circumstance can or cannot occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.

Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the disclosure are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. Any numerical value, however, inherently contain certain errors necessarily resulting from the standard deviation found in their respective testing measurements. Furthermore, when numerical ranges of varying scope are set forth herein, it is contemplated that any combination of these values inclusive of the recited values may be used. Further, ranges can be expressed herein as from “about” one particular value, and/or to “about” another particular value. When such a range is expressed, another aspect includes from the one particular value and/or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent “about,” it will be understood that the particular value forms another aspect. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint. Unless stated otherwise, the term “about” means within 5% (e.g, within 2% or 1%) of the particular value modified by the term “about.”

It is understood that throughout this specification the identifiers “first” and “second” are used solely to aid in distinguishing the various components and steps of the disclosed subject matter. The identifiers “first” and “second” are not intended to imply any particular order, amount, preference, or importance to the components or steps modified by these terms.

Chemical Definitions As used herein, the term “composition” is intended to encompass specified ingredients in the specified amounts, as well as any product which results, directly or indirectly, from combination of the specified ingredients in the specified amounts.

As used herein, the term “substituted” is contemplated to include all permissible substituents of organic compounds. In a broad aspect, the permissible substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, and aromatic and non-aromatic substituents of organic compounds. Illustrative substituents include, for example, those described below. The permissible substituents can be one or more and the same or different for appropriate organic compounds. For purposes of this disclosure, the heteroatoms, such as nitrogen, can have hydrogen substituents and/or any permissible substituents of organic compounds described herein which satisfy the valency of the heteroatoms. This disclosure is not intended to be limited in any manner by the permissible substituents of organic compounds. Also, the terms “substitution” or “substituted with” include the implicit proviso that such substitution is in accordance with permitted valence of the substituted atom and the substituent, and that the substitution results in a stable compound, e.g ., a compound that does not spontaneously undergo transformation such as by rearrangement, cyclization, elimination, etc.

The symbols A n is used herein as merely a generic substituent in the definitions below.

The term “amino acid” as used herein is a a-amino acid, a bΐ -amino acid or a p2-amino acid, a g-amino acid, or a d-amino acid, or the corresponding N-substituted amino acids. The amino acid as used herein can be a natural (i.e., protein-building) amino acid or a non-natural amino acid. Amino acid moieties as used herein can have a structure including, but not limited to:

The term “alkyl” as used herein is a branched or unbranched saturated hydrocarbon group of 1 to 18 carbon atoms, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, t- butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, dodecyl, tetradecyl, hexadecyl, eicosyl, tetracosyl, and the like.

The term “alkoxy” as used herein is an alkyl group bound through a single, terminal ether linkage; that is, an “alkoxy” group can be defined as — OA 1 where A 1 is alkyl as defined above. Throughout this specification “C(O)” is a short hand notation for C=O.

The terms “amine” or “amino” as used herein are represented by the formula NA 1 A 2 A 3 , where A 1 , A 2 , and A 3 can be, independently, hydrogen or an alkyl group described above.

The term “carboxylic acid” as used herein is represented by the formula — C(O)OH. A “carboxylate” as used herein is represented by the formula — C(O)O-.

The term “ester” as used herein is represented by the formula — OC(O)A 1 or — C(O)OA 1 , where A 1 can be an alkyl group described above.

The term “amide” as used herein is represented by the formula -C(O)N-A 1 A 2 , where A 1 or A 2 can be independently a hydrogen or an alkyl group described above.

The term “hydroxyl” as used herein is represented by the formula — OH.

The term “thiol” as used herein is represented by the formula — SH.

It is to be understood that the compounds provided herein may contain chiral centers. Such chiral centers may be of either the ( R- ) or (S-) configuration. The compounds provided herein may either be enantiomerically pure or be diastereomeric or enantiomeric mixtures. It is to be understood that the chiral centers of the compounds provided herein may undergo epimerization in vivo. As such, one of skill in the art will recognize that administration of a compound in its ( R- ) form is equivalent, for compounds that undergo epimerization in vivo , to administration of the compound in its (S-) form.

As used herein, substantially pure means sufficiently homogeneous to appear free of readily detectable impurities as determined by standard methods of analysis, such as nuclear magnetic resonance (NMR), high performance liquid chromatography (HPLC) and mass spectrometry (MS), gas-chromatography mass spectrometry (GC-MS), and similar, used by those of skill in the art to assess such purity, or sufficiently pure such that further purification would not detectably alter the physical and chemical properties, such as enzymatic and biological activities, of the substance. Both traditional and modern methods for purification of the compounds to produce substantially chemically pure compounds are known to those of skill in the art. A substantially chemically pure compound may, however, be a mixture of stereoisomers.

Unless stated to the contrary, a formula with chemical bonds shown only as solid lines and not as wedges or dashed lines contemplates each possible isomer, e.g ., each enantiomer, diastereomer, and meso compound, and a mixture of isomers, such as a racemic or scalemic mixture. Reference will now be made in details to specific aspects of the disclosed materials, compounds, compositions, articles, and methods, examples of which are illustrated in the accompanying Examples and Figures.

Compounds of Formula I

In one aspect, a dendrimer is provided of Formula I: or a salt thereof; wherein: (Core) is a is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11;

* indicates covalent attachment to a carbon moiety of (BUI); b is 4;

BU are building units;

BUx are building units of generation x, wherein the total number of building units within generation x of the dendrimer of Formula I is equal to b(2 x-1 ); wherein BU within each generation x is selected from BU A or BU B : wherein BU in at least one generation x is selected from BU B ;

# indicates covalent attachment to an amino moiety of Core and an amino moiety of BU; + indicates covalent attachment to a carbonyl moiety of BU or a covalent attachment to z;

A is selected at each occurrence in a generation x from a natural or non-natural amino acid moiety that is optionally protected with one or more protecting groups, wherein the carbonyl group of the amino acid moiety is covalently attached to the amino group @ of BU B and the amino group of the amino acid moiety is covalently attached to the carbonyl group $ of BU B ;

Z is selected at each occurrence from a monovalent capping moiety; or two Z groups attached to the same amino moiety may be brought together with the nitrogen they are attached to form an optionally substituted heterocycle ring.

In some embodiments, A is selected at each occurrence in a generation x from a natural amino acid moiety. In some embodiments, A is selected from: and or a protected derivative thereof.

In another embodiment, A is selected at each occurrence in a generation x from a non- natural amino acid moiety. The term “non-natural amino acid” refers to an organic compound that is a congener of a natural amino acid in that is has a structure similar to a natural amino acid so that it mimics the structure and reactivity of a natural amino acid. The non-natural amino acid moiety can be derived from a modified amino acid, and/or an amino acid analog, that is not one of the 20 common naturally occurring amino acids of the rare natural amino acids selenocysteine or pyrrolysine. Non-natural amino acids can also be the D-isomer of the natural amino acids. Examples of suitable amino acids include, but are not limited to, alanine, allosoleucine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, lysine, methionine, napthylalanine, phenylalanine, proline, pyroglutamic acid, serine, threonine, tryptophan, tyrosine, valine, or derivatives thereof. In some embodiments, A may be selected at each occurrence in a generation X from alanine, allosoleucine, arginine, asparagine, aspartic acid, cysteine, cyclohexylalanine, 2,3-diaminopropionic acid, 4-fluorophenylalanine, glutamine acid, glutamine, glycine, histidine, homoproline (i.e. pipecolic acid), isoleucine, leucine, lysine, methionine, napthylalanine, norleucine, phenylalanine, phenylglycine, 4- (phosphonodifluoromethyl)phenylalanine, proline, sarcosine, selenocysteine, serine, threonine, tyrosine, tryptophan, valine, or derivatives thereof. In some embodiments, A may be selected from ADDA, thialysine, 2-aminoisobutyric acid, aminolevulinic acid, azetidine-2-carboxylic acid, canaline, canavanine, carboxy glutamic acid, chloralanine, citrulline, dehydroalanine, diaminopimelic acid, dihydroxyphenylglycine, enduracididine, homocysteine, homoserine, 4- hydroxyphenylglycine, hydroxyproline, hypusine, lanthionine, beta-leucine, norleucine, norvaline, ornithine, penicillamine, pyroglutamic acid, quisqualic acid, sarcosine, theanine, tricholomic acid, and derivatives thereof. In some embodiments, A may be selected from allylglycine, propargylglycine, azidolysine, or derivatives thereof.

In some embodiments, A is selected from or a derivative thereof.

In some embodiments, A is selected from or a derivative thereof.

In some embodiments, Z may be selected at each occurrence from a monovalent capping moiety as would be deemed suitable for the intended purpose. In some embodiments, Z is monovalent moiety substituted with one or more primary amine, secondary amine, amide, carbamate, or urea groups, or combinations thereof.

In some embodiments, Z is selected from: wherein C is selected from: and and

R is independently selected from C 1 -C 12 alkyl.

In some embodiments, Z is selected from

In some embodiments, Z is selected from In some embodiments, C is selected from:

In some embodiments, two Z groups attached to an amino acid moiety are brought together with the nitrogen to which they are attached to form or a derivative thereof.

In some embodiments, In some embodiments, the dendrimer of Formula I is selected from a dendrimer of Formula la: or a salt thereof. In some embodiments of Formula la, BUI is selected from:

In some embodiments of Formula la, BUI is selected from: In some embodiments of Formula la, Z is selected from:

In some embodiments, the dendrimer of Formula I is selected from a dendrimer of

Formula Ib: or a salt thereof.

In some embodiments of Formula Ib, BUI is selected from: and BU2 is BU A

In some embodiments of Formula Ib, BUI is selected from: and BU2 is BU A

In some embodiments of Formula lb, BU2 is selected from: and BUI is BU A

In some embodiments of Formula lb, BU2 is selected from: and BUI is BU A

In some embodiments of Formula lb, Z is selected from:

Non-limiting examples of dendrimers of the present invention include, but are not limited to:

and

or a salt thereof. Dendrimer Conjugates

In some embodiments, a dendrimer of Formula I as described herein may be conjugated with a peptide. Representative examples of peptides which may be conjugated with the dendrimers described herein include, but are not limited to, bivalirudin, buserelin, corticotropin, cosyntropin, enfuvirtide, eptifibatide, exenatide, glatiramer, gramicidin D, lepirudin, leuprolide, liraglutide, lucinactant, nesuritide, oxytocin, pramlintide, salmon calcitonin, secretin, sermorelin, teduglutide, thymalfasin, or combinations thereof. In some embodiments, a dendrimer of Formula I as described herein may be conjugated with a therapeutic agent. As used herein, “therapeutic agent” can refer to any substance, compound, molecule, and the like, which can be biologically active or otherwise can induce a pharmacologic, immunogenic, biologic and/or physiologic effect on a subject to which it is administered to by local and/or systemic action. A therapeutic agent can be a primary active agent, or in other words, the component(s) of a composition to which the whole or part of the effect of the composition is attributed. A therapeutic agent can be a secondary therapeutic agent, or in other words, the component(s) of a composition to which an additional part and/or other effect of the composition is attributed. The term therefore encompasses those compounds or chemicals traditionally regarded as drugs, vaccines, and biopharmaceuticals including molecules such as proteins, peptides, hormones, nucleic acids, gene constructs and the like. Examples of therapeutic agents are described in well-known literature references such as the Merck Index (14th edition), the Physicians' Desk Reference (64th edition), and The Pharmacological Basis of Therapeutics (12th edition), and they include, without limitation, medicaments; vitamins; mineral supplements; substances used for the treatment, prevention, diagnosis, cure or mitigation of a disease or illness; substances that affect the structure or function of the body, or pro-drugs, which become biologically active or more active after they have been placed in a physiological environment. For example, the term “therapeutic agent” includes compounds or compositions for use in all of the major therapeutic areas including, but not limited to, adjuvants; anti-infectives such as antibiotics and antiviral agents; analgesics and analgesic combinations, anorexics, anti- inflammatory agents, anti-epileptics, local and general anesthetics, hypnotics, sedatives, antipsychotic agents, neuroleptic agents, antidepressants, anxiolytics, antagonists, neuron blocking agents, anticholinergic and cholinomimetic agents, antimuscarinic and muscarinic agents, antiadrenergics, antiarrhythmics, antihypertensive agents, hormones, and nutrients, antiarthritics, antiasthmatic agents, anticonvulsants, antihistamines, antinauseants, antineoplastics, antipruritics, antipyretics; antispasmodics, cardiovascular preparations (including calcium channel blockers, beta-blockers, beta-agonists and antiarrythmics), antihypertensives, diuretics, vasodilators; central nervous system stimulants; cough and cold preparations; decongestants; diagnostics; hormones; bone growth stimulants and bone resorption inhibitors; immunosuppressives; muscle relaxants; psychostimulants; sedatives; tranquilizers; proteins, peptides, and fragments thereof (whether naturally occurring, chemically synthesized or recombinantly produced); and nucleic acid molecules (polymeric forms of two or more nucleotides, either ribonucleotides (RNA) or deoxyribonucleotides (DNA) including both double- and single-stranded molecules, gene constructs, expression vectors, antisense molecules and the like), small molecules (e.g., doxorubicin) and other biologically active macromolecules such as, for example, proteins and enzymes. The agent may be a biologically active agent used in medical, including veterinary, applications and in agriculture, such as with plants, as well as other areas. The term therapeutic agent also includes without limitation, medicaments; vitamins; mineral supplements; substances used for the treatment, prevention, diagnosis, cure or mitigation of disease or illness; or substances which affect the structure or function of the body; or pro- drugs, which become biologically active or more active after they have been placed in a predetermined physiological environment.

DNA synthesis and thereby cell division. Purine analogs can also be incorporated into the DNA molecule itself during DNA synthesis, which can interfere with cell division. According to certain aspects, for example, the purine analog may be selected from the group consisting of acyclovir, allopurinol, 2-aminoadenosine, arabinosyl adenine (ara-A), azacitidine, azathiprine, 8- aza-adenosine, 8-fluoro-adenosine, 8 -m ethoxy-adenosine, 8-oxo-adenosine, cladribine, deoxycoformycin, fludarabine, gancylovir, 8-aza-guanosine, 8-fluoro-guanosine, 8- methoxy- guanosine, 8-oxo-guanosine, guanosine diphosphate, guanosine diphosphate-beta- L-2- aminofucose, guanosine diphosphate-D-arabinose, guanosine diphosphate-2- fluorofucose, guanosine diphosphate fucose, mercaptopurine (6-MP), pentostatin, thiamiprine, thioguanine (6- TG), and salts, analogs, and derivatives thereof.

In yet another particular aspect, for example, the antimetabolite agent is a pyrimidine analog. Similar to the purine analogs discussed above, pyrimidine-based antimetabolite agents block the synthesis of pyrimidine-containing nucleotides (cytosine and thymine in DNA; cytosine and uracil in RNA). By acting as “decoys,” the pyrimidine-based compounds can prevent the production of nucleotides, and/or can be incorporated into a growing DNA chain and lead to its termination. According to certain aspects, for example, the pyrimidine analog may be selected from the group consisting of ancitabine, azacitidine, 6-azauridine, bromouracil (e.g., 5- bromouracil), capecitabine, carmofur, chlorouracil (e.g. 5-chlorouracil), cytarabine (cytosine arabinoside), cytosine, dideoxyuridine, 3'-azido-3'-deoxythymidine, 3'- dideoxycytidin-2'-ene, 3'-deoxy-3'-deoxythymidin-2'-ene, dihydrouracil, doxifluridine, enocitabine, floxuridine, 5- fluorocytosine, 2-fluorodeoxycytidine, 3-fluoro-3'-deoxythymidine, fluorouracil (e.g., 5- fluorouracil (also known as 5-FU), gemcitabine, 5-methylcytosine, 5- propynylcytosine, 5- propynylthymine, 5-propynyluracil, thymine, uracil, uridine, and salts, analogs, and derivatives thereof. In one aspect, the pyrimidine analog is other than 5- fluorouracil. In another aspect, the pyrimidine analog is gemcitabine or a salt thereof.

In certain aspects, the antimetabolite agent is selected from the group consisting of 5- fluorouracil, capecitabine, 6-mercaptopurine, methotrexate, gemcitabine, cytarabine, fludarabine, pemetrexed, and salts, analogs, derivatives, and combinations thereof. In other aspects, the antimetabolite agent is selected from the group consisting of capecitabine, 6- mercaptopurine, methotrexate, gemcitabine, cytarabine, fludarabine, pemetrexed, and salts, analogs, derivatives, and combinations thereof. In one particular aspect, the antimetabolite agent is other than 5- fluorouracil. In a particularly preferred aspect, the antimetabolite agent is gemcitabine or a salt or thereof (e.g., gemcitabine HC1 (Gemzar®)).

Other antimetabolite anti-cancer agents may be selected from, but are not limited to, the group consisting of acanthifolic acid, aminothiadiazole, brequinar sodium, Ciba-Geigy CGP- 30694, cyclopentyl cytosine, cytarabine phosphate stearate, cytarabine conjugates, Lilly DATHF, Merrel Dow DDFC, dezaguanine, dideoxycytidine, dideoxyguanosine, didox, Yoshitomi DMDC, Wellcome EHNA, Merck & Co. EX-015, fazarabine, fludarabine phosphate, N-(2'-furanidyl)-5-fluorouracil, Daiichi Seiyaku FO-152, 5-FU-fibrinogen, isopropyl pyrrolizine, Lilly LY-188011; Lilly LY-264618, methobenzaprim, Wellcome MZPES, norspermidine, NCI NSC-127716, NCI NSC-264880, NCI NSC-39661, NCI NSC-612567, Warner-Lambert PALA, pentostatin, piritrexim, plicamycin, Asahi Chemical PL-AC, Takeda TAC-788, tiazofurin, Erbamont TIF, tyrosine kinase inhibitors, Taiho UFT and uricytin, among others.

In one aspect, the antimitotic agent is a microtubule inhibitor or a microtubule stabilizer. In general, microtubule stabilizers, such as taxanes and epothilones, bind to the interior surface of the beta-microtubule chain and enhance microtubule assembly by promoting the nucleation and elongation phases of the polymerization reaction and by reducing the critical tubulin subunit concentration required for microtubules to assemble. Unlike mictrotubule inhibitors, such as the vinca alkaloids, which prevent microtubule assembly, the microtubule stabilizers, such as taxanes, decrease the lag time and dramatically shift the dynamic equilibrium between tubulin dimers and microtubule polymers towards polymerization. In one aspect, therefore, the microtubule stabilizer is a taxane or an epothilone. In another aspect, the microtubule inhibitor is a vinca alkaloid.

In some embodiments, the therapeutic agent may comprise a taxane or derivative or analog thereof. The taxane may be a naturally derived compound or a related form, or may be a chemically synthesized compound or a derivative thereof, with antineoplastic properties. The taxanes are a family of terpenes, including, but not limited to paclitaxel (Taxol®) and docetaxel (Taxotere®), which are derived primarily from the Pacific yew tree, Taxus brevifolia, and which have activity against certain tumors, particularly breast and ovarian tumors. In one aspect, the taxane is docetaxel or paclitaxel. Paclitaxel is a preferred taxane and is considered an antimitotic agent that promotes the assembly of microtubules from tubulin dimers and stabilizes microtubules by preventing depolymerization. This stability results in the inhibition of the normal dynamic reorganization of the microtubule network that is essential for vital interphase and mitotic cellular functions.

Also included are a variety of known taxane derivatives, including both hydrophilic derivatives, and hydrophobic derivatives. Taxane derivatives include, but are not limited to, galactose and mannose derivatives described in International Patent Application No. WO 99/18113; piperazino and other derivatives described in WO 99/14209; taxane derivatives described in WO 99/09021, WO 98/22451, and U.S. Pat. No. 5,869,680; 6-thio derivatives described in WO 98/28288; sulfenamide derivatives described in U.S. Pat. No. 5,821,263; deoxygenated paclitaxel compounds such as those described in U.S. Pat. No. 5,440,056; and taxol derivatives described in U.S. Pat. No. 5,415,869. As noted above, it further includes prodrugs of paclitaxel including, but not limited to, those described in WO 98/58927; WO 98/13059; and U.S. Pat. No. 5,824,701. The taxane may also be a taxane conjugate such as, for example, paclitaxel-PEG, paclitaxel -dextran, paclitaxel-xylose, docetaxel -PEG, docetaxel- dextran, docetaxel-xylose, and the like. Other derivatives are mentioned in “Synthesis and Anticancer Activity of Taxol Derivatives,” D. G. I. Kingston et ah, Studies in Organic Chemistry, vol. 26, entitled “New Trends in Natural Products Chemistry” (1986), Atta-ur- Rabman, P. W. le Quesne, Eds. (Elsevier, Amsterdam 1986), among other references. Each of these references is hereby incorporated by reference herein in its entirety.

Various taxanes may be readily prepared utilizing techniques known to those skilled in the art (see also WO 94/07882, WO 94/07881, WO 94/07880, WO 94/07876, WO 93/23555, WO 93/10076; U.S. Pat. Nos. 5,294,637; 5,283,253; 5,279,949; 5,274,137; 5,202,448; 5,200,534; 5,229,529; and EP 590,267) (each of which is hereby incorporated by reference herein in its entirety), or obtained from a variety of commercial sources, including for example, Sigma-Aldrich Co., St. Louis, Mo.

Alternatively, the antimitotic agent can be a microtubule inhibitor; in one preferred aspect, the microtubule inhibitor is a vinca alkaloid. In general, the vinca alkaloids are mitotic spindle poisons. The vinca alkaloid agents act during mitosis when chromosomes are split and begin to migrate along the tubules of the mitosis spindle towards one of its poles, prior to cell separation. Under the action of these spindle poisons, the spindle becomes disorganized by the dispersion of chromosomes during mitosis, affecting cellular reproduction. According to certain aspects, for example, the vinca alkaloid is selected from the group consisting of vinblastine, vincristine, vindesine, vinorelbine, and salts, analogs, and derivatives thereof.

The antimitotic agent can also be an epothilone. In general, members of the epothilone class of compounds stabilize microtubule function according to mechanisms similar to those of the taxanes. Epothilones can also cause cell cycle arrest at the G2-M transition phase, leading to cytotoxicity and eventually apoptosis. Suitable epithiolones include epothilone A, epothilone B, epothilone C, epothilone D, epothilone E, and epothilone F, and salts, analogs, and derivatives thereof. One particular epothilone analog is an epothilone B analog, ixabepilone (Ixempra™).

In certain aspects, the antimitotic anti-cancer agent is selected from the group consisting of taxanes, epothilones, vinca alkaloids, and salts and combinations thereof. Thus, for example, in one aspect the antimitotic agent is a taxane. More preferably in this aspect the antimitotic agent is paclitaxel or docetaxel, still more preferably paclitaxel. In another aspect, the antimitotic agent is an epothilone (e.g., an epothilone B analog). In another aspect, the antimitotic agent is a vinca alkaloid.

Examples of cancer drugs that may be used in the present disclosure include, but are not limited to: thalidomide; platinum coordination complexes such as cisplatin (cis-DDP), oxaliplatin and carboplatin; anthracenediones such as mitoxantrone; substituted ureas such as hydroxyurea; methylhydrazine derivatives such as procarbazine (N- methylhydrazine, MIH); adrenocortical suppressants such as mitotane (o,p'-DDD) and aminoglutethimide; RXR agonists such as bexarotene; and tyrosine kinase inhibitors such as sunitimib and imatinib. Examples of additional cancer drugs include alkylating agents, antimetabolites, natural products, hormones and antagonists, and miscellaneous agents. Alternate names are indicated in parentheses. Examples of alkylating agents include nitrogen mustards such as mechlorethamine, cyclophosphainide, ifosfamide, melphalan sarcolysin) and chlorambucil; ethylenimines and methylmelamines such as hexamethylmelamine and thiotepa; alkyl sulfonates such as busulfan; nitrosoureas such as carmustine (BCNU), semustine (methyl-CCNU), lomustine (CCNU) and streptozocin (streptozotocin); DNA synthesis antagonists such as estramustine phosphate; and triazines such as dacarbazine (DTIC, dimethyl-triazenoimidazolecarboxamide) and temozolomide. Examples of antimetabolites include folic acid analogs such as methotrexate (amethopterin); pyrimidine analogs such as fluorouracin (5-fluorouracil, 5-FU, SFU), floxuridine (fluorodeoxyuridine, FUdR), cytarabine (cytosine arabinoside) and gemcitabine; purine analogs such as mercaptopurine (6-mercaptopurine, 6-MP), thioguanine (6-thioguanine, TG) and pentostatin (2'-deoxycoformycin, deoxycoformycin), cladribine and fludarabine; and topoisomerase inhibitors such as amsacrine. Examples of natural products include vinca alkaloids such as vinblastine (VLB) and vincristine; taxanes such as paclitaxel, protein bound paclitaxel (Abraxane) and docetaxel (Taxotere); epipodophyllotoxins such as etoposide and teniposide; camptothecins such as topotecan and irinotecan; antibiotics such as dactinomycin (actinomycin D), daunorubicin (daunomycin, rubidomycin), doxorubicin, bleomycin, mitomycin (mitomycin C), idarubicin, epirubicin; enzymes such as L-asparaginase; and biological response modifiers such as interferon alpha and interlelukin 2. Examples of hormones and antagonists include luteinising releasing hormone agonists such as buserelin; adrenocorticosteroids such as prednisone and related preparations; progestins such as hydroxyprogesterone caproate, medroxyprogesterone acetate and megestrol acetate; estrogens such as diethylstilbestrol and ethinyl estradiol and related preparations; estrogen antagonists such as tamoxifen and anastrozole; androgens such as testosterone propionate and fluoxymesterone and related preparations; androgen antagonists such as flutamide and bicalutamide; and gonadotropin- releasing hormone analogs such as leuprolide. Alternate names and trade-names of these and additional examples of cancer drugs, and their methods of use including dosing and administration regimens, will be known to a person versed in the art.

In some aspects, the anti-cancer agent may comprise a chemotherapeutic agent. Suitable chemotherapeutic agents include, but are not limited to, alkylating agents, antibiotic agents, antimetabolic agents, hormonal agents, plant-derived agents and their synthetic derivatives, anti- angiogenic agents, differentiation inducing agents, cell growth arrest inducing agents, apoptosis inducing agents, cytotoxic agents, agents affecting cell bioenergetics i.e., affecting cellular ATP levels and molecules/activities regulating these levels, biologic agents, e.g., monoclonal antibodies, kinase inhibitors and inhibitors of growth factors and their receptors, gene therapy agents, cell therapy, e.g., stem cells, or any combination thereof.

According to these aspects, the chemotherapeutic agent is selected from the group consisting of cyclophosphamide, chlorambucil, melphalan, mechlorethamine, ifosfamide, busulfan, lomustine, streptozocin, temozolomide, dacarbazine, cisplatin, carboplatin, oxaliplatin, procarbazine, uramustine, methotrexate, pemetrexed, fludarabine, cytarabine, fluorouracil, floxuridine, gemcitabine, capecitabine, vinblastine, vincristine, vinorelbine, etoposide, paclitaxel, docetaxel, doxorubicin, daunorubicin, epirubicin, idarubicin, mitoxantrone, bleomycin, mitomycin, hydroxyurea, topotecan, irinotecan, amsacrine, teniposide, erlotinib hydrochloride and combinations thereof. Each possibility represents a separate aspect of the invention.

According to certain aspects, the therapeutic agent may comprise a biologic drug, particularly an antibody. According to some aspects, the antibody is selected from the group consisting of cetuximab, anti-CD24 antibody, panitumumab and bevacizumab.

Therapeutic agents as used in the present disclosure may comprise peptides, proteins such as hormones, enzymes, antibodies, monoclonal antibodies, antibody fragments, monoclonal antibody fragments, and the like, nucleic acids such as aptamers, siRNA, DNA, RNA, antisense nucleic acids or the like, antisense nucleic acid analogs or the like, low-molecular weight compounds, or high-molecular-weight compounds, receptor agonists, receptor antagonists, partial receptor agonists, and partial receptor antagonists.

Additional representative therapeutic agents may include, but are not limited to, peptide drugs, protein drugs, desensitizing materials, antigens, factors, growth factors, anti -infective agents such as antibiotics, antimicrobial agents, antiviral, antibacterial, antiparasitic, antifungal substances and combination thereof, antiallergenics, steroids, androgenic steroids, decongestants, hypnotics, steroidal anti-inflammatory agents, anti-cholinergics, sympathomimetics, sedatives, miotics, psychic energizers, tranquilizers, vaccines, estrogens, progestational agents, humoral agents, prostaglandins, analgesics, antispasmodics, antimalarials, antihistamines, cardioactive agents, nonsteroidal anti-inflammatory agents, antiparkinsonian agents, anti -Alzheimer's agents, antihypertensive agents, beta-adrenergic blocking agents, alpha-adrenergic blocking agents, nutritional agents, and the benzophenanthridine alkaloids. The therapeutic agent can further be a substance capable of acting as a stimulant, a sedative, a hypnotic, an analgesic, an anticonvulsant, and the like.

Additional therapeutic agents may comprise CNS-active drugs, neuro-active drugs, inflammatory and anti-inflammatory drugs, renal and cardiovascular drugs, gastrointestinal drugs, anti-neoplastics, immunomodulators, immunosuppressants, hematopoietic agents, growth factors, anticoagulant, thrombolytic, antiplatelet agents, hormones, hormone-active agents, hormone antagonists, vitamins, ophthalmic agents, anabolic agents, antacids, anti-asthmatic agents, anti-cholesterolemic and anti-lipid agents, anti-convulsants, anti-diarrheals, anti-emetics, anti-manic agents, antimetabolite agents, anti-nauseants, anti-obesity agents, anti-pyretic and analgesic agents, anti-spasmodic agents, anti -thrombotic agents, anti-tussive agents, anti- uricemic agents, anti-anginal agents, antihistamines, appetite suppressants, biologicals, cerebral dilators, coronary dilators, bronchiodilators, cytotoxic agents, decongestants, diuretics, diagnostic agents, erythropoietic agents, expectorants, gastrointestinal sedatives, hyperglycemic agents, hypnotics, hypoglycemic agents, laxatives, mineral supplements, mucolytic agents, neuromuscular drugs, peripheral vasodilators, psychotropics, stimulants, thyroid and anti-thyroid agents, tissue growth agents, uterine relaxants, vitamins, antigenic materials, and so on. Other classes of therapeutic agents include those cited in Goodman & Gilman's The Pharmacological Basis of Therapeutics (McGraw Hill) as well as therapeutic agents included in the Merck Index and The Physicians’ Desk Reference (Thompson Healthcare).

Other therapeutic agents include androgen inhibitors, polysaccharides, growth factors (e.g., a vascular endothelial growth factor- VEGF), hormones, anti -angiogenesis factors, dextromethorphan, dextromethorphan hydrobromide, noscapine, carbetapentane citrate, chlophedianol hydrochloride, chlorpheniramine maleate, phenindamine tartrate, pyrilamine maleate, doxylamine succinate, phenyltoloxamine citrate, phenylephrine hydrochloride, phenylpropanolamine hydrochloride, pseudoephedrine hydrochloride, ephedrine, codeine phosphate, codeine sulfate morphine, mineral supplements, cholestryramine, N- acetylprocainamide, acetaminophen, aspirin, ibuprofen, phenyl propanolamine hydrochloride, caffeine, guaifenesin, aluminum hydroxide, magnesium hydroxide, peptides, polypeptides, proteins, amino acids, hormones, interferons, cytokines, and vaccines.

Further examples of therapeutic agents include, but are not limited to, peptide drugs, protein drugs, desensitizing materials, antigens, anti-infective agents such as antibiotics, antimicrobial agents, antiviral, antibacterial, antiparasitic, antifungal substances and combination thereof, antiallergenics, androgenic steroids, decongestants, hypnotics, steroidal anti- inflammatory agents, anti-cholinergics, sympathomimetics, sedatives, miotics, psychic energizers, tranquilizers, vaccines, estrogens, progestational agents, humoral agents, prostaglandins, analgesics, antispasmodics, antimalarials, antihistamines, antiproliferatives, anti- VEGF agents, cardioactive agents, nonsteroidal anti-inflammatory agents, antiparkinsonian agents, antihypertensive agents, b-adrenergic blocking agents, nutritional agents, and the benzophenanthridine alkaloids. The agent can further be a substance capable of acting as a stimulant, sedative, hypnotic, analgesic, anticonvulsant, and the like.

Further representative therapeutic agents include but are not limited to analgesics such as acetaminophen, acetylsalicylic acid, and the like; anesthetics such as lidocaine, xylocaine, and the like; anorexics such as dexadrine, phendimetrazine tartrate, and the like; antiarthritics such as methylprednisolone, ibuprofen, and the like; antiasthmatics such as terbutaline sulfate, theophylline, ephedrine, and the like; antibiotics such as sulfisoxazole, penicillin G, ampicillin, cephalosporins, amikacin, gentamicin, tetracyclines, chloramphenicol, erythromycin, clindamycin, isoniazid, rifampin, and the like; antifungals such as amphotericin B, nystatin, ketoconazole, and the like; antivirals such as acyclovir, amantadine, and the like; anticancer agents such as cyclophosphamide, methotrexate, etretinate, paclitaxel, taxol, and the like; anticoagulants such as heparin, warfarin, and the like; anticonvulsants such as phenyloin sodium, diazepam, and the like; antidepressants such as isocarboxazid, amoxapine, and the like; antihistamines such as diphenhydramine HCl, chlorpheniramine maleate, and the like; hormones such as insulin, progestins, estrogens, corticoids, glucocorticoids, androgens, and the like; tranquilizers such as thorazine, diazepam, chlorpromazine HC1, reserpine, chlordiazepoxide HCl, and the like; antispasmodics such as belladonna alkaloids, dicyclomine hydrochloride, and the like; vitamins and minerals such as essential amino acids, calcium, iron, potassium, zinc, vitamin B12, and the like; cardiovascular agents such as prazosin HC1, nitroglycerin, propranolol HC1, hydralazine HC1, pancrelipase, succinic acid dehydrogenase, and the like; peptides and proteins such as LHRH, somatostatin, calcitonin, growth hormone, glucagon-like peptides, growth releasing factor, angiotensin, FSH, EGF, bone morphogenic protein (BMP), erythopoeitin (EPO), interferon, interleukin, collagen, fibrinogen, insulin, Factor VIII, Factor IX, Enbrel®, Rituxam®, Herceptin®, alpha-glucosidase, Cerazyme/Ceredose®, vasopressin, ACTH, human serum albumin, gamma globulin, structural proteins, blood product proteins, complex proteins, enzymes, antibodies, monoclonal antibodies, and the like; prostaglandins; nucleic acids; carbohydrates; fats; narcotics such as morphine, codeine, and the like, psychotherapeutics; anti- malarials, L-dopa, diuretics such as furosemide, spironolactone, and the like; antiulcer drugs such as rantidine HC1, cimetidine HC1, and the like.

The therapeutic agent can also be an immunomodulator, including, for example, cytokines, interleukins, interferon, colony stimulating factor, tumor necrosis factor, and the like; immunosuppressants such as rapamycin, tacrolimus, and the like; allergens such as cat dander, birch pollen, house dust mite, grass pollen, and the like; antigens of bacterial organisms such as Streptococcus pneumoniae, Haemophilus influenzae, Staphylococcus aureus, Streptococcus pyrogenes, Corynebacterium diphteriae, Listeria monocytogenes, Bacillus anthracis, Clostridium tetani, Clostridium botulinum, Clostridium perfringens. Neisseria meningitides, Neisseria gonorrhoeae, Streptococcus mutans. Pseudomonas aeruginosa, Salmonella typhi, Haemophilus parainfluenzae, Bordetella pertussis, Francisella tularensis, Yersinia pestis, Vibrio cholerae, Legionella pneumophila, Mycobacterium tuberculosis, Mycobacterium leprae, Treponema pallidum, Leptspirosis interrogans, Borrelia burgddorferi, Campylobacter jejuni, and the like; antigens of such viruses as smallpox, influenza A and B, respiratory synctial, parainfluenza, measles, HIV, SARS, varicella-zoster, herpes simplex 1 and 2, cytomeglavirus, Epstein-Barr, rotavirus, rhinovirus, adenovirus, papillomavirus, poliovirus, mumps, rabies, rubella, coxsackieviruses, equine encephalitis, Japanese encephalitis, yellow fever, Rift Valley fever, lymphocytic choriomeningitis, hepatitis B, and the like; antigens of such fungal, protozoan, and parasitic organisms such as Cryptococcus neoformans, Histoplasma capsulatum, Candida albicans, Candida tropicalis, Nocardia asteroids, Rickettsia ricketsii, Rickettsia typhi, Mycoplasma pneumoniae, Chlamydia psittaci, Chlamydia trachomatis, Plasmodium falciparum, Trypanasoma brucei, Entamoeba histolytica, Toxoplasma gondii, Trichomonas vaginalis, Schistosoma mansoni, and the like. These antigens may be in the form of whole killed organisms, peptides, proteins, glycoproteins, carbohydrates, or combinations thereof.

In a further specific aspect, the therapeutic agent can comprise an antibiotic. The antibiotic can be, for example, one or more of Amikacin, Gentamicin, Kanamycin, Neomycin, Netilmicin, Streptomycin, Tobramycin, Paromomycin, Ansamycins, Geldanamycin, Herbimycin, Carbacephem, Loracarbef, Carbapenems, Ertapenem, Doripenem,

Imipenem/Cilastatin, Meropenem, Cephalosporins (First generation), Cefadroxil, Cefazolin, Cefalotin or Cefalothin, Cefalexin, Cephalosporins (Second generation), Cefaclor, Cefamandole, Cefoxitin, Cefprozil, Cefuroxime, Cephalosporins (Third generation), Cefixime, Cefdinir, Cefditoren, Cefoperazone, Cefotaxime, Cefpodoxime, Ceftazidime, Ceftibuten, Ceftizoxime, Ceftriaxone, Cephalosporins (Fourth generation), Cefepime, Cephalosporins (Fifth generation), Ceftobiprole, Glycopeptides, Teicoplanin, Vancomycin, Macrolides, Azithromycin,

Clarithromycin, Dirithromycin, Erythromycin, Roxithromycin, Troleandomycin, Telithromycin, Spectinomycin, Monobactams, Aztreonam, Penicillins, Amoxicillin, Ampicillin, Azlocillin, Carbenicillin, Cloxacillin, Dicloxacillin, Flucloxacillin, Mezlocillin, Meticillin, Nafcillin, Oxacillin, Penicillin, Piperacillin, Ticarcillin, Polypeptides, Bacitracin, Colistin, Polymyxin B, Quinolones, Ciprofloxacin, Enoxacin, Gatifloxacin, Levofloxacin, Lomefloxacin, Moxifloxacin, Norfloxacin, Ofloxacin, Trovafloxacin, Sulfonamides, Mafenide, Prontosil (archaic), Sulfacetamide, Sulfamethizole, Sulfanilimide (archaic), Sulfasalazine, Sulfisoxazole, Trimethoprim, Trimethoprim-Sulfamethoxazole (Co-trimoxazole) (TMP-SMX), Tetracyclines, including Demeclocy cline, Doxy cy cline, Minocycline, Oxytetracy cline, Tetracycline, and others; Arsphenamine, Chloramphenicol, Clindamycin, Lincomycin, Ethambutol, Fosfomycin, Fusidic acid, Furazolidone, Isoniazid, Linezolid, Metronidazole, Mupirocin, Nitrofurantoin, Platensimycin, Pyrazinamide, Quinupristin/Dalfopristin, Rifampicin (Rifampin in U.S.), Timidazole, or a combination thereof. In one aspect, the therapeutic agent can be a combination of Rifampicin (Rifampin in U.S.) and Minocycline.

Growth factors useful as therapeutic agents include, but are not limited to, transforming growth factor-a (“TGF-a”), transforming growth factors (“TGF-b”), platelet-derived growth factors (“PDGF”), fibroblast growth factors (“FGF”), including FGF acidic isoforms 1 and 2, FGF basic form 2 and FGF 4, 8, 9 and 10, nerve growth factors (“NGF”) including NGF 2.5s, NGF 7.0s and beta NGF and neurotrophins, brain derived neurotrophic factor, cartilage derived factor, bone growth factors (BGF), basic fibroblast growth factor, insulin-like growth factor (IGF), vascular endothelial growth factor (VEGF), granulocyte colony stimulating factor (G- CSF), insulin like growth factor (IGF) I and II, hepatocyte growth factor, glial neurotrophic growth factor (GDNF), stem cell factor (SCF), keratinocyte growth factor (KGF), transforming growth factors (TGF), including TGFs alpha, beta, betal, beta2, beta3, skeletal growth factor, bone matrix derived growth factors, and bone derived growth factors and mixtures thereof.

Cytokines useful as therapeutic agents include, but are not limited to, cardiotrophin, stromal cell derived factor, macrophage derived chemokine (MDC), melanoma growth stimulatory activity (MGS A), macrophage inflammatory proteins 1 alpha (MIP-1alpha), 2, 3 alpha, 3 beta, 4 and 5, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, TNF-a, and TNF-b. Immunoglobulins useful in the present disclosure include, but are not limited to, IgG, IgA, IgM, IgD, IgE, and mixtures thereof. Some preferred growth factors include VEGF (vascular endothelial growth factor), NGFs (nerve growth factors), PDGF-AA, PDGF- BB, PDGF-AB, FGFb, FGF a, and BGF.

Other molecules useful as therapeutic agents include but are not limited to growth hormones, leptin, leukemia inhibitory factor (LIF), tumor necrosis factor alpha and beta, endostatin, thrombospondin, osteogenic protein- 1, bone morphogenetic proteins 2 and 7, osteonectin, somatomedin-like peptide, osteocalcin, , interferon alpha, interferon alpha A, interferon beta, interferon gamma, interferon 1 alpha, and interleukins 2, 3, 4, 5 6, 7, 8, 9, 10, 11, 12,13, 15, 16, 17 and 18.

Drug Delivery Systems

In another aspect, a drug delivery system is provided comprising: a plurality of microgel particles, each microgel particle having a surface; a plurality of dendrimers of Formula I as described herein adhered to the surface of each microgel particle; and one or more therapeutic agents as described herein encapsulated within each microgel particle. The term “microgel particle” comprises a hydrogel particle having a size within the range of 1 nm to 100 mm and which comprises a cross-linked polymer formed by the polymerization of a plurality of cross-linked co-monomers.

The microgel particle may be considered as one macromolecule (i.e., the cross-linked polymer) comprising a molar mass of between about 10 6 and 10 10 daltons (for example, between 10 6 and 10 9 daltons). However, the individual co-monomers that were used during the preparation of the microgel particle may comprise a molar mass of between about 5 daltons and 5,000 daltons, for example between about 10 daltons and 1,000 daltons, between 50 daltons and 500 daltons, between 75 daltons and 400 daltons, or between 100 daltons and 300 daltons.

Representative examples of co-monomers which may be used in the microgel particles of the present disclosure include, but are not limited to, ethyl acrylate, methacrylic acid, 1,4- butanediol diacrylate, methylmethacrylate, ethyleneglycol dimethacrylate, N- isopropyl acrylamide, vinylcaprolactone, hydroxyethyl methacrylate, vinyl alcohol, ethylene glycol methacrylate, poly(ethylene glycol) methacrylate.

In another aspect, a drug delivery system is provided comprising: a cell having a surface; and a dendrimer-drug conjugate as described herein adhered to the cell surface.

The following examples are set forth below to illustrate the methods and results according to the disclosed subject matter. These examples are not intended to be inclusive of all aspects of the subject matter disclosed herein, but rather to illustrate representative methods, compositions, and results. These examples are not intended to exclude equivalents and variations of the present invention, which are apparent to one skilled in the art.

EXAMPLES

Example 1. Expanding Dendrimer Functionality by Hybridizing Poly(amidoamine) (PAMAM) Scaffolds with Peptide Segments

Since their inception in 1985, PAMAM dendrimers have been employed in a myriad of applications, ranging from drug and gene delivery to diagnostics and biosensing. [1_4] Yet, the design of their chemically inert backbone has remained unaltered for three decades. [5] In fact, the internal structure of nearly every class of dendrimers presented to date does not exhibit any functional groups other than the branch termini, exception made for a few examples. [6_8] Developing a modular method to furnish internally functionalized dendrimers offers a tremendous opportunity to advance dendrimer chemistry and unlock their full potential in the biomedical field. Recently, Svenson has stated that if the dendrimer field is to progress, more attention is required to develop internally functionalized scaffolds. [9] Therefore a synthetic procedure was developed for constructing PAMAM-like dendrimers displaying ancillary functional groups on their dendritic backbone. Owing to their inherent molecular flexibility and biocompatibility, PAMAMs represent an ideal “host” backbone to develop next-generation dendrimers. In particular, the “decoration” of the hyperbranched backbone with ionizable functional groups holds great potential to enable fine- tuning of physicochemical and biophysical properties ( e.g ., hydrodynamic radius, z-potential, colloidal stability, cell and tissue permeability, and transport and release of therapeutic payloads). For example, drug delivery research using PAMAM dendrimers presents an opportunity for improvement. Traditionally, drugs are either covalently bound to the terminal groups of the dendrimer’ s branches or non- covalently entrapped within the internal cavities of the PAMAM architecture. [10-19] Both approaches present disadvantages. Covalently conjugating therapeutics to the corona of the dendrimer significantly alters the z-potential, solubility characteristics, and generally changes the way the dendrimer interacts with its surrounding in vivo. [9] Additionally, because the topological peripheral groups of the dendrimer are often found at its geometric periphery, the drug is poorly encapsulated within the dendrimer, and non- specific cytotoxicity based on partial interaction of the drug with healthy cells is of concern. Absorption in a biocompatible and hydrophilic dendrimer seemingly solves all of these problems. When using this approach, however, undesired drug leakage is inevitable. [10-12] Since the therapeutic is only trapped by non-covalent interactions, its release in the systemic circulation and off-target effects are difficult to prevent. Thus, the precise release of a therapeutic, a highly desirable trait in therapeutic nanocarriers, to the target is not feasible using noncovalent encapsulation with the current PAMAM dendrimer architecture. Both of these shortcomings could be addressed through the use of a biocompatible internally functionalized dendrimer where the therapeutic is covalently tethered to the internal cavities of the structure.

Herein, a modular synthetic strategy is described to construct internally functionalized dendrimers by hybridizing the poly(amidoamine) scaffold of PAMAM with amino acids at prescribed positions and numbers (DendriPeps). The choice of amino acids as “guest” monomers is motivated by the wide chemical diversity of natural and non-natural amino acids available for use in Fmoc/tBu protected form. Furthermore, the use of functional diamines as building blocks was ruled out in the interest of synthetic yield; while in fact the formation of amide bonds between methyl ester and amine groups is feasible when using low molecular weight diamines - such as diaminoethane in traditional PAMAM chemistry - it is not viable with high molecular weight or structurally complex diamine building blocks due to challenges associated with purification and poor reaction kinetics. On the other hand, the conjugation of amino acids as pentafluorophenol (Pfp) activated esters onto an amine-terminated PAMAM host core is simple and efficient.

To demonstrate this strategy, the use of lysine and glutamic acid to respectively append primary amine or carboxyl groups on their PAMAM backbone is reported in this example. Briefly, Fmoc-Lys(Boc)-OPfp or Fmoc-Glu(OtBu)-OPfp are conjugated to the terminal primary amine groups of a G1 PAMAM dendrimer in DMSO for 72 hours. The amino acid capped dendrimers Fgl-Gl-Lys-P and Fgl-Gl-Glu-P were purified by trituration in a 1 % saline solution and diethyl ether, followed by centrifugation; it was noted that the products exhibited limited solubility in organic solvents, except for DMSO. An intermediate characterization by 1 H, 13 C, and 19 F NMR was performed; the absence of signals associated to pentafluorophenol in the 19 F NMR ensures the complete removal of unreacted amino acid. The Fmoc protection was removed with 20 % piperidine in DMF, and Fgl-Gl-Lys and Fgl-Gl-Glu were purified by trituration with diethyl ether followed by centrifugation. Both products, now exhibiting a free amine, were soluble in organic solvents, and were characterized via 1 H and 13 C NMR and electrospray ionization liquid chromatography mass spectroscopy (ESI-LC/MS). Fmoc-b-Ala- OPfp was subsequently installed following the same procedure. This alanine “spacer” is critical to reduce the steric congestion at the cr-amine position that would otherwise prevent the bis addition of methyl acrylate in the subsequent step. Following conjugation, the Fgl-G2-Lys-Ala- P and Fgl-G2-Glu-Ala-P were purified by trituration in a 1 % saline solution and diethyl ether, followed by centrifugation; once again, both products were found soluble only in DMSO, and were characterized by 1 H, 13 C, and 19 F NMR. The Fmoc protection was removed with 20 % piperidine in DMF, and Fgl-G2-Lys-Ala and Fgl-G2-Glu-Ala were purified by trituration with diethyl ether followed by centrifugation, and characterized with 1 H and 13 C NMR and ESI- LC/MS. The amine-terminated DendriPeps can now be expanded towards higher generations by traditional divergent synthesis. Accordingly, a Michael addition with methyl acrylate was performed, followed by rotary evaporation of excess methyl acrylate under vacuum, and characterization via 1 H and 13 C NMR and ESI-LC/MS. Final termination was accomplished with a large excess of ethanolamine, resulting in hydroxyl -terminated Fgl-G3-Lys-OH-P and Fgl- G3-Glu-OH-P. The products were repeatedly triturated into 1:1 dioxaneTHF, and characterized with 1 H and 13 C NMR and ESI-LC/MS. The Fgl-G3-Lys-OH-P and Fgl-G3-Glu-OH-P DendriPeps were purified by reverse phase C18 chromatography on a preparative high- performance liquid chromatography (HPLC), up to 70 - 92 % purity. Notably, the residual impurities were identified by ESI-LC/MS to be minor deviations from the target DendriPep (e.g. lacking an ethanolamine terminal); being almost identical to the final product, these truncated variants could not be removed by preparative HPLC. The chromatographic fractions with high purity (> 70-92 %) were pooled and added with ammonium hydroxide to neutralize the acidic pH of the mobile phase (0.1 % formic acid). The sample was dried under high vacuum, and the removal of Boc or OtBu protecting groups was performed using trifluoroacetic acid (TFA) in DCM followed by rotary and high vacuum evaporation of DCM and TFA. The resulting hygroscopic solid was dissolved in deionized water and dialyzed against deionized water for 4 days. The resulting Fgl-G3-Lys-OH and Fgl-G3-Glu-OH DendriPeps were validated by ESI- LC/MS analysis (purity by total ion count 95 %).

The synthetic procedure described above is amenable to the adoption of core PAMAM dendrimers of different generation, as well as the construction of a different number of PAMAM layers onto the dipeptide segment, although product purity is expected to decrease. The advantage to using core PAMAMs with different generations is that it provides control over the number and position of the amino acid residues as well as the final hydrodynamic radius of the product. Molecular dynamics simulations (MDS) indicate that the functional groups, either amine or carboxylic acid, are internalized in the dendrimer structure and are sterically shielded from the topological surface. To estimate the position and solvation of the primary e-amine groups and g-carboxylic acid groups in DendriPeps, 2-ns atomistic molecular dynamics simulations (MDS) of in Fgl-G3-Lys-OH and Fgl-G3-Glu-OH in explicit solvent conditions were performed. FIG. 6 compares the radial distribution functions of the nitrogen atoms in the 4 tertiary amines in the G1 layer, the oxygen atoms in the 16 terminal hydroxyl groups, the nitrogen atoms in the 8 primary e-amine groups in Fgl-G3-Lys-OH (panel A), and the oxygen atoms in the 8 g-carboxylic groups in Fgl-G3-Glu-OH (panel B). The comparison with the distribution of the terminal hydroxyl groups at the topological periphery of the DendriPeps indicates that the Lys e-amine groups and the Glu g-carboxylic groups consistently reside closer to center of the molecule and are therefore expected to be “encapsulated” within each DendriPep. Furthermore, FIG. 6 shows the average number of water molecules solvating the 8 e- amine groups of Fgl-G3-Lys-OH (panel C) and the 8 g-carboxylic acid groups of Fgl-G3-Glu- OH (panel C), compared to those solvating the hydroxyl groups located on the topological peripheral units. Fgl-G3-Lys-OH features equal solvation of primary amine groups and terminal hydroxyl groups, whereas Fgl-G3-Glu-OH shows a somewhat restricted solvation of the carboxylic acid groups. Nevertheless, in both cases, the appended groups are accessible to small molecules (water), and it is therefore reasonable to expect that they can be subjected to further reaction with other small molecules.

In summary, four distinct examples of DendriPeps, namely Fgl-G3-Lys-OH, Fgl-G3- Glu-OH, Fg0-G2-Lys-OH, and Fg0- G2-Glu-OH were synthesized in high purity with varying number, position, and type of functional groups displayed along their internal backbone. It is anticipated that DendriPeps will have far-reaching interdisciplinary implications in the fields of soft materials, biomedical engineering, and catalysis due to their unprecedented structure and modular synthetic strategy.

References Cited in Example 1

[1] S. Gurdag, J. Khandare, S. Stapels, L. Matherly, R. Kannan, Activity of Dendrimer- Methotrexate Conjugates on Methotrexate Sensitive and Resistant Cell Lines. Bioconjugate Chem. 2006, 17, 275-283.

[2] J. Li, H. Liang, J. Liu, Z. Wang, Poly (Amidoamine) (PAMAM) Dendrimer Mediated Delivery of Drug and PDNA/SiRNA for Cancer Therapy. Int. J. Pharm. 2018, 546 , 215-225.

[3] F. Abedi-Gaballu, G. Dehghan, M. Ghaffari, R. Yekta, S. Abbaspour-Ravasjani, B. Baradaran, J. Ezzati Nazhad Dolatabadi, R. Hamblin, PAMAM Dendrimers as Efficient Drug and Gene Delivery Nanosystems for Cancer Therapy. Appl. Mater. Today. 2018, 72, 177-190.

[4] S. Chandra, M. Mayer, J. Baeumner, PAMAM Dendrimers: A Multifunctional Nanomaterial for ECL Biosensors. Talanta, 2017, 768, 126-129.

[5] D. Tomalia, H. Baker, J. Dewald, M. Hall, G. Kallos, S. Martin, J. Roeck, J. Ryder, P. Smith, A New Class of Polymers: Starburst-Dendritic Macromoleculese. Polym. J. 1985, 77, 117-132.

[6] T. Kang, J. Amir, Khan, A.; K. Ohshimizu, J. Hunt, K. Sivanandan, M. Montanez, M. Malkoch, M. Ueda, C. Hawker, Facile Access to Internally Functionalized Dendrimers through Efficient and Orthogonal Click Reactions. Chem. Commun. 2010, 46, 1556-1558.

[7] P. Antoni, Y. Hed, A. Nordberg, D. Nystrom, H. Von Holst, A. Hult, M. Malkoch, Bifunctional Dendrimers: From Robust Synthesis and Accelerated One-Pot Postfunctionalization Strategy to Potential Applications. Angew. Chemie - Int. Ed. 2009, 48, 2126-2130.

[8] X. Deng, F. Du, Z. Li, Combination of Orthogonal ABB and ABC Multicomponent Reactions toward Efficient Divergent Synthesis of Dendrimers with Structural Diversity. ACS Macro Lett. 2014, 3,667-670.

[9] S. Svenson, The Dendrimer Paradox - High Medical Expectations but Poor Clinical Translation. Chem. Soc. Rev. 2015, 44, 4131-4144. [10] C. Kojima, K. Kono, K. Maruyama, T. Takagishi, Synthesis of Polyamidoamine Dendrimers Having Poly(Ethylene Glycol) Grafts and Their Ability To Encapsulate Anticancer Drugs. Bioconjug. Chem. 2000, 11, 910-917.

[11] M. Liu, K. Kono, J. Frechet, Water-Soluble Dendritic Unimolecular Micelles:: Their Potential as Drug Delivery Agents. J. Control. Release 2000, 65, 121-131.

[12] B. Noriega-Luna, L. Godinez, F. Rodriguez, A. Rodriguez, G. Zaldivar-Lelo de Larrea,

C. Sosa-Ferreyra, R. Mercado-Curiel, J. Manriquez, E. Bustos, Applications of Dendrimers in Drug Delivery Agents, Diagnosis, Therapy, and Detection. J. Nanomater. 2014, 2014, 1-19.

[13] R. Navath, Y. Kurtoglu, B. Wang, S. Kannan, R. Romero, R. Kannan, Dendrimer-Drug Conjugates for Tailored Intracellular Drug Release Based on Glutathione Levels. Bioconjug. Chem. 2008, 19, 2446-2455.

[14] Y. Kurtoglu, R. Navath, B. Wang, S. Kannan, R. Romero, R. Kannan, Poly(Amidoamine) Dendrimer-Drug Conjugates with Disulfide Linkages for Intracellular Drug Delivery. Int. J. Pharm. 2010, 384, 189-194.

[15] I. Majoros, A. Myc, T. Thomas, C. Mehta, J. Baker, PAMAM Dendrimer-Based Multifunctional Conjugate for Cancer Therapy: Synthesis, Characterization, and Functionality. Biomacromolecules, 2006, 7, 572-579.

[16] Y. Kurtoglu, M. Mishra, S. Kannan, R. Kannan, Pharmaceutical Nanotechnology Drug Release Characteristics of PAMAM Dendrimer-Drug Conjugates with Different Linkers. Int. J. Pharm. 2010, 384, 189-194.

[17] M. Najlah, Synthesis of Dendrimers and Drug-Dendrimer Conjugates for Drug Delivery.

Curr. Opin. Drug Discov. Devel. 2007, 10, 1367-6733.

[18] S. Kuruvilla, G. Tiruchinapally, A. Crouch, M. Elsayed, J. Greve, Dendrimer- Doxorubicin Conjugates Exhibit Improved Anticancer Activity and Reduce Doxorubicin- Induced Cardiotoxicity in a Murine Hepatocellular Carcinoma Model. Plos One, 2017, 1-24.

[19] B. Srinageshwar, S. Peruzzaro, M. Andrews, K. Johnson, A. Hietpas, B. Clark, C. McGuire, E. Petersen, J. Kippe, A. Stewart, O. Lossia, A. Al-Gharaibeh, A. Antcliff, R. Culver,

D. Swanson, G. Dunbar, A. Sharma, J. Rossignol, PAMAM Dendrimers Cross the Blood-Brain Barrier When Administered through the Carotid Artery in C57BL/6J Mice. Int. J. Mol. Sci. 2017, 18, 628-644.

Example 2. Synthesis of Compounds of Formula I Materials Methyl acrylate was purchased from Acros Organics. PAMAM dendrimers GO and G1 were purchased from Sigma-Aldrich. FMOC-b-Ala-OPfp was purchased from BA Chem. FMOC-Glu- OtBu-OPfp was purchased from A ChemTec Inc. FMOC-LYS(BOC)-OPfp was purchased from BA Chem. Ammonium hydroxide was purchased from VWR. Ethanolamine was purchased from Sigma Aldrich.

Experimental

Preparative HPLC Method:

Instrument: Agilent 1100 series UV Detector: HP 1100 series

Column: XBridge Prep C18 5um OBD, 19x100mm Column, PN 1806002978, SN 28213635511203

Solution A: 1 % ACN in water, 0.1 % Formic acid Solution B: 20 % ACN in Methanol, 0.1 % Formic acid Sample concentration: 50 mg/mL Injection Volume: 300 uL injection Method:

Linear gradient

Reset fraction collector before first run Fraction collection threshold of 200 mAU Fraction collector store for 215 nm

LCMS Method:

Analysis was carried out on a high-resolution mass spectrometer - the Thermo Fisher Scientific Exactive Plus MS , a benchtop full-scan Orbitrap™ mass spectrometer - using Heated Electrospray Ionization (HESI). Samples were diluted in water and analyzed via LC injection into the mass spectrometer at a flow rate of 500 mL/min. The mobile phase was acetonitrile with 0.1 % formic acid and water with 0.1 % formic acid. The column was a Thermo Accucore-150- C4, 100 x 2.1 mm, 2.6 m particle size. The mass spectrometer was operated in positive ion mode.

Gradient

Molecular Dynamics Simulations:

Atomistic molecular dynamics simulations (MDS) were performed as follows. The molecules were constructed using a modified version of the dendrimer building toolkit (DBT). 1 To construct the lys- and glu-based residues, appropriate models were constructed with endcaps as described previously. 1 These were subjected to geometry optimization first using the AMI

Hamiltonian in MOPAC 2016 2 and then further using Gaussian 09. 3 If using a version of

AMBER that has retired the top2mol2 program, it can be found at https://github.eom/choderalab/ambermini/blob/master/antecham ber/top2mol2.c. The process for two stage RESP fitting, removal of endcaps and saving a new residue in a library was followed as described previously. 1 Once the models were complete, they were solvated with water (box radius of 10.0 Å) using the TIP3PBOX model found in the ff99SB force field in AMBER. The

GAFF force field was used for the non-water atoms. These were then subjected to 1,000 steps of steepest descent minimization, followed by 4,000 steps of conjugate gradient minimization. The models were then equilibrated via 5,000 steps of MDS (time step of 2 fs), heating from a temperature of 100 K to 300 K, constant volume, with no pressure control, and employing the SHAKE algorithm. The models were then further equilibrated for a further 5,000 steps at constant pressure and constant temperature. Ensuring that the temperature was around 300 K and the density was around 1.0 g/mL, a further set of 50,000 steps of constant pressure, constant temperature equilibration was performed. Then, a 2 ns constant pressure, constant temperature production run was performed. These data were used in the analyses reported here.

Synthetic Methods:

Synthesis of FG1-G1-LYS-P

Under a nitrogen atmosphere, 2.111 g (3.3 mmol) of FMOC-Lys(Boc)-OPfp was dissolved in 8 mL of DMSO and stirred at room temperature in a round bottom flask. G1 PAMAM dendrimer, 0.238 g (0.17 mmol), was dissolved in 8 mL of DMSO and added dropwise to the stirring amino acid solution over 2 hours. The reaction was stirred for 72 hours at room temperature. The crude reaction mixture was triturated into 300 mL of 1% sodium chloride DI water, and this solution was centrifuged at 4000 rpm for 30 minutes. The supernatant was decanted leaving a yellow pellet, and 50 mL of fresh DI water was added to each centrifuge tube. The pellet and water were centrifuged once more at 3000 rpm for 2 minutes, and the water was decanted leaving a white pellet. The sample was placed on a high-vacuum for 24 h, and subsequently suspended in 20 mL of DCM. The crude suspension was triturated into cold ether, and the ether solution was centrifuged at 3000 rpm for 3 minutes. The supernatant was decanted leaving a waxy pellet, 20 mL of ether was added to each centrifuge tube, the sample was centrifuged at 3000 rpm for 3 minutes, and the supernatant was decanted leaving a waxy pellet. The pellet was suspended in DCM, transferred to a round bottom flask, and the DCM was removed via rotary evaporation and high vacuum overnight. The final product was confirmed with 1 H and 19 F NMR at a 73 % recovery to yield 0.626 g (0.124 mmol) of product.

1 H-NMR (400 MHz; DMSO-d 6 ): d 7.95 (br, 16H), 7.87-7.85 (d, 16H), 7.70 (t, 16H), 7.39 (m, 24H), 7.30 (t, 16H), 6.74 (t, 8H), 5.76 (s, DCM), 4.28 (m, 8H), 4.19 (br, 16H), 3.92-3.86 (q, 8H), 3.51 (s, MeOH), 3.35 (br, water), 3.08 (br, 40H), 2.88 (br, 24H), 2.67 (br, 16H), 2.50 (s, DMSO- de), 2.41 (br, 12H), 2.19 (br, 16H), 1.65-1.45 (br, 16H), 1.37 (br, 8H), 1.34 (s, 72H), 1.23 (br, 16H).

19 F-NMR (400MHZ; DMSO-d 6 ): no peaks found.

Synthesis of FG1-G1-LYS (FMOC Deprotection of FG1-G1-LYS-P):

Under a nitrogen atmosphere, 0.806 g (0.16 mmol) FG1-G1-LYS-P was dissolved in a 10 mL stirred 20 % piperidine/DMF solution at room temperature. This solution was allowed to stir for 20 minutes. To remove piperidine, DMF, and the fluorenyl group, the crude reaction mixture was triturated into cold ether, and the ether solution was centrifuged at 3000 rpm for 3 minutes. After the supernatant was removed, 20 mL of ether was added to each centrifuge tube, the sample was centrifuged at 3000 rpm for 3 minutes, and the supernatant was decanted. The remaining oily pellet was dissolved in methanol and transferred to a round bottom flask. The solvent was removed using a rotary evaporator and high vacuum, and the final product was confirmed with 1 H, 13 C NMR and LCMS at an 82 % to yield 0.429 g (0.132 mmol) of product. 1H-NMR (400 MHz; CDCl 3 ): d 8.00 (br, 16H), 7.26 (s, CDCl 3 ), 5.10 (br, 8H), 3.46 (s, MeOH), 3.32 (br, 34H), 3.21 (br, 8H), 3.08 (br, 16H), 2.95 (s, DMF), 2.87 (s, DMF), 2.71 (br, 24H), 2.52 (br, 12H), 2.42-2.18 (br, 48H), 1.75 (br, 8H), 1.48 (br, 24H), 1.41 (s, 72H).

13 C-NMR (00 MHz; CDCl 3 ): d 207.1, 175.6, 174.4, 173.3, 172.7 (DMF), 169.0, 156.2, 78.8, 77.16 (CDCl 3 ), 63.8, 55.0, 52.4, 50.3, 50.0, 40.2, 39.2, 37.5, 34.4, 34.1, 33.4, 30.9, 30.0, 29.7,

29.5, 28.4, 25.9, 22.8, 19.0. HRMS (ESI-TOF) Calc. for [C 150 H 288 N 42 O 36 ] [M+4H] 4+ = 813.5499, found = 813.5510 D, M (ppm) = 1.3520.

Synthesis of FG1-G2-LYS-ALA-P

Under a nitrogen atmosphere, 0.700 g (1.5 mmol) of Fmoc-Ala-OPfp was dissolved in 5 mL of DMSO and stirred at room temperature in a round bottom flask. FG1-G1-LYS, 0.239 g (0.073 mmol), was dissolved in 10 mL of DMSO and added dropwise to the stirring amino acid solution over 2 hours. The reaction was stirred for 96 hours at room temperature. The crude reaction mixture was triturated into 300 mL of 1 % sodium chloride DI water, and this solution was centrifuged at 4000 rpm for 30 minutes. The supernatant was decanted leaving a yellow pellet, and 50 mL of fresh DI water was added to each centrifuge tube. The pellet and water were centrifuged once more at 3000 rpm for 2 minutes, and the water was decanted leaving a white pellet. The sample was placed on a high vacuum for 24 h and was subsequently suspended in 20 mL of DCM. The crude suspension was triturated into cold ether, and the ether solution was centrifuged at 3000 rpm for 3 minutes. The supernatant was decanted leaving a waxy pellet, 20 mL of ether was added to each centrifuge tube, the sample was centrifuged at 3000 rpm for 3 minutes, and the supernatant was decanted. The pellet was suspended in DCM, transferred to a round bottom flask, and the DCM was removed via rotary evaporation and high vacuum overnight. The final product was confirmed with 1 H and 19 F NMR at an 86 % recovery (0.354 g, 0.063 mmol). 1 H-NMR (400 MHz; DMSO-d 6 : d 8.04-8.02 (d, 8H), 7.94 (br, 8H), 7.87 (t, 30H), 7.67 (t, 28H), 7.43-7.38 (q, 28H), 7.34-7.27 (br, 28H), 5.75 (s, DCM), 4.26 (t, 28H), 4.19 (q, 20H), 3.34 (s, water), 3.20 (t, 32H), 3.09 (br, 32H), 2.74 (br, 16H) 2.50 (s, DMSO-d 6 ), 2.42-2.31 (m, 28H), 2.25-2.18 (br, 28H), 1.86 (m, 8H), 1.71 (m, 8H), 1.35 (s, 72H). 1 9 F-NMR (400MHZ; DMSO-d 6 ): no peaks found.

Synthesis of FG1-G2-LYS-ALA (FMOC Deprotection of FG1-G2-LYS-ALA-P):

Under a nitrogen atmosphere, 0.614 g (0.110 mmol) FG1-G2-LYS-ALA-P was dissolved in a 10 mL 20 % piperidine/DMF solution stirring at room temperature. This solution was allowed to stir for 20 minutes. To remove piperidine, DMF, and the fluorenyl group, the crude reaction mixture was triturated into cold ether, and the ether solution was centrifuged at 3000 rpm for 3 minutes. After the supernatant was removed, 20 mL of ether was added to each centrifuge tube, the sample was centrifuged at 3000 rpm for 3 minutes, and the supernatant was decanted. The pellet was dissolved in methanol and transferred to a round bottom flask. The solvent was removed using a rotary evaporator and high vacuum, and the final product was confirmed with 1H, 13 C NMR and LCMS at a 92 % recovery (0.385 g, 0.101 mmol).

1 H-NMR (400 MHz; CDCl 3 ): d 8.06 (br, 16H), 7.26 (s, CDCl 3 ), 5.25 (br, 4H), 4.35 (br, 4H), 3.43 (s, MeOH), 3.29 (br, 28H), 3.05 (br, 18H), 2.71 (br, 22H), 2.50 (br, 12H), 2.40-2.30 (br, 22H), 1.85 (br, 8H), 1.63 (br, 8H), 1.41 (s, 72H).

13 C-NMR (100 MHz; CDCl 3 ): d, 173.6, 173.2, 172.8, 162.7, 156.4, 79.0, 77.4, 77.16 (CDCl 3 ), 58.1, 53.8, 50.3, 46.6, 40.2, 38.9, 38.0, 37.6, 36.6, 34.1, 31.7, 31.5, 29.5, 28.5, 28.0, 26.0, 24.4, 23.0. HRMS (ESI-TOF) Calc. for [C 174 H 328 N 50 O 44 ] [M+3H] 3+ = 1275.17279, found = 1275.17671, DM (ppm) = 3.071.

Synthesis of FG1-G2.5-LYS:

Freshly distilled methyl acrylate 0.278 g (3.22 mmol) was dissolved in 1 ml of methanol and cooled with an ice bath to 0 °C. Under nitrogen atmosphere, FG1-G2-LYS-ALA (0.385 g, 0.1 mmol) dissolved in 5 mL methanol was added dropwise over 10 minutes. The ice bath was kept for another 3 hours, and afterwards, the reaction was stirred for 48 hours at room temperature. The solvent and excess methyl acrylate was removed on a rotary evaporator and high vacuum. This gave the half-generation FG1-G2.5-LYS in the form of slightly yellowish oil in yields in a 95 % recovery. The final product was confirmed with NMR. 1H-NMR (400 MHz; CDCl 3 ) d: 7.85 (br, 24H), 7.26 (s, CDCl 3 ), 5.22 (br, 4H), 4.33 (br, 8H), 3.69 (s, 12H), 3.66 (s, 30H), 3.31 (br, 34H), 3.22 (br, 16H), 3.07 (br, 24H), 2.90 (t, 12H), 2.80 (br, 60H), 2.55 (t, 48H), 2.35 (br, 36H), 1.79 (br, 8H), 1.66 (br, 8H), 1.46 (br, 28H), 1.41 (s, 72H). 13 C-NMR (100 MHz; CDCl 3 ) d: 175.2, 173.4, 173.1, 172.6, 162.6, 156.2, 79.0, 77.16 (CDCl 3 ), 62.7, 54.3, 52.5, 51.9, 51.7, 50.3, 49.3, 43.8, 40.3, 39.5, 29.2, 37.6, 36.6, 34.4, 34.05, 32.3, 32.8, 32.1, 29.9, 28.5, 26.0, 24.3, 23.2. Synthesis of FG1-G3-LYS-OH-P

FG1-G2.5-LYS 0.240 g, 0.046 mmol was dissolved in 15 mL of methanol. Freshly distilled ethanolamine 1.407 g, 23 mmol was dissolved in 5 mL methanol and cooled to 0 °C, using an ice bath. The FG1-G2.5-LYS solution was added to this solution dropwise over 1 hour under nitrogen atmosphere. The ice bath was kept for another 3 hours, and afterwards, the reaction was stirred for 6 days at room temperature. Workup was done by removing methanol using a rotary evaporator. After that, the product/ethanolamine solution was triturated into cold dioxane. The product settled and stuck to the bottom of the beaker, and the dioxane/ethanolamine solution was decanted. The beaker was rinsed/decanted twice more with dioxane. The product was then dissolved in methanol followed by solvent removal using a rotary evaporator and high vacuum. This gave the hygroscopic hydroxyl -terminated product at 99 % recovery (0.264 g, 0.046 mmol) and 56 % purity as confirmed by LCMS TIC chromatogram. The final product was confirmed with 1 H, 13 C NMR and LCMS. 1 H-NMR (400 MHz; MeOD): d 4.89 (s, H 2 0), 4.25 (br, 8H), 3.67-3.59 (m, 80H), 3.31 (s, MeOD), 3.03 (br, 48H), 2.83-2.79 (m, 108H), 2.59 (br, 16H), 2.52 (br, 16H), 2.44-2.38 (m, 92H), 1.78 (br, 8H), 1.66 (br, 8H), 1.48 (br, 28H), 1.43 (s, 72H), 1.34 (br, 70H), 1.16 (br, 8H). 1 3 C-NMR (100 MHz; CDCl 3 ): d 178.5, 175.4, 175.1, 174.9, 174.7, 158.5, 79.8, 62.4, 61.62, 61.59, 51.51, 51.6, 51.0, 50.6, 49.0 (MeOD), 48.4, 46.8, 46.3, 45.7, 43.9, 43.0, 41.2, 40.3, 39.9,

38.7, 38.4, 36.7, 36.1, 35.5, 34.6, 30.7, 28.9, 24.4.

HRMS (ESI-TOF) Calc. for [C 254 H 472 N 66 O 76 ] [M+7H] 7+ = 810.07282, found = 810.07745, DM (ppm) = 5.716.

Synthesis of FG1-G3-LYS-OH (BOC deprotection of FG1-G3-LYS-OH-P)

FG1-G3-LYS-OH-P 0.075 g, 0.013 mmol) was purified using preparative liquid chromatography. The fractions were analyzed for purity with LCMS, and the purest fractions were combined and neutralized to pH 7 with ammonium hydroxide. The combined fractions were placed under high vacuum for 24 hours to evaporate the mobile phase. To deprotect the BOC groups, the remaining solid was dissolved in 50 % TFA/DCM and stirred for 3 hours at room temperature. The TFA and DCM were evaporated with the rotary evaporator and placed on the high vacuum overnight. The remaining solid was dissolved in 5 mL of deionized water and was dialyzed with 500-1000 MWCO dialysis tubing for 4 days against deionized water. The solution inside the dialysis tubing was removed and was evaporated under high vacuum for 24 hours. The product was confirmed with LCMS. The product was recovered (18 mg, 0.004 mmol) at 97 % purity by LCMS TIC chromatogram.

HRMS (ESI-TOF) Calc. for [C 214 H N 66 O 60 ] [M+7H] 7+ = 696.15571, found = 696.15991, DM (ppm) = 6.0331. Synthesis of FG0-G0-LYS-P

Under a nitrogen atmosphere, 2.850 g (4.5 mmol) of FMOC-Lys(Boc)-OPfp was dissolved in 8 mL of DMSO and stirred at room temperature in a round bottom flask. GO PAMAM dendrimer, 0.232 g (0.45 mmol), was dissolved in 8 mL of DMSO and added dropwise to the stirring amino acid solution over 2 hours. The reaction was stirred for 72 hours at room temperature. The crude reaction mixture was triturated into 300 mL of 1 % sodium chloride DI water, and this solution was centrifuged at 4000 rpm for 30 minutes. The supernatant was decanted leaving a yellow pellet, and 50 mL of fresh DI water was added to each centrifuge tube. The pellet and water were centrifuged once more at 3000 rpm for 2 minutes, and the water was decanted leaving a white pellet. The sample was placed on a high-vacuum for 24 h, and subsequently suspended in 20 mL of DCM. The crude suspension was triturated into cold ether, and the ether solution was centrifuged at 3000 rpm for 3 minutes. The supernatant was decanted leaving a waxy pellet, 20 mL of ether was added to each centrifuge tube, the sample was centrifuged at 3000 rpm for 3 minutes, and the supernatant was decanted leaving a waxy pellet. The pellet was suspended in DCM, transferred to a round bottom flask, and the DCM was removed via rotary evaporation and high vacuum overnight. The final product was confirmed with 1 H and 19 F NMR at a 98 % recovery (1.026 g).

1 H-NMR (400 MHz; DMSO-d 6 ): d 7.98-7.90 (br, 8H), 7.89-7.84 (d, 8H), 7.71 (t, 8H), 7.44-7.36 (t, 12H), 7.34-7.27 (t, 8H), 6.74 (t, 4H), 5.75 (s, DCM), 4.30-4.17 (m, 12H), 3.92-3.84 (q, 4H), 3.43-3.24 (b, H 2 0/ether), 3.15-3.01 (b, 16H), 2.94-2.81 (b, 12H), 2.74-2.61 (b, 8H), 2.50 (s, DMSO-d 6 ), 2.26-2.13 (b, 8H), 1.64-1.45 (b, 8H), 1.36 (s, 16H), 1.35 (s, 36H), 1.09 (t, ether). 1 9 F-NMR (400MHZ; DMSO-d 6 ): no peaks found.

FMOC Deprotection of FG0-G0-LYS: Under a nitrogen atmosphere, 0.763 g (0.329 mmol) of FG0-G0-LYS was dissolved in a 10 mL stirred 20 % piperidine/DMF solution at room temperature. This solution was allowed to stir for 20 minutes. To remove piperidine, DMF, and the fluorenyl group, the crude reaction mixture was triturated into cold ether, and the ether solution was centrifuged at 3000 rpm for 3 minutes. After the supernatant was removed, 20 mL of ether was added to each centrifuge tube, the sample was centrifuged at 3000 rpm for 3 minutes, and the supernatant was decanted. The remaining oily pellet was dissolved in methanol and transferred to a round bottom flask. The solvent was removed using a rotary evaporator and high vacuum, and the final product was confirmed with 1 H, 13 C NMR and LCMS at a 79 % recovery (0.372 g, 0.26 mmol) and 77 % purity by LCMS chromatogram.

1 H-NMR (400 MHz; CDCl 3 ): d 7.91-7.77 (br, 8H), 7.26 (s, CDCl 3 ), 5.02-4.84 (br, 4H), 3.47 (s, MeOH), 3.39-3.26 (br, 8H + IMP), 3.14-3.02 (q, 8H), 2.72-2.63 (t, 8H), 2.46 (s, 4H), 2.37-2.27 (t, 8H), 1.96-1.82 (br, 12H), 1.82-1.70 (m, 4H), 1.56-1.44 (br, 16H), 1.43-1.40 (s, 36H), 1.40- 1.32 (br, 8H). 13 C-NMR (100 MHz; CDCl 3 ): d 176.3, 173.4, 162.6 (DMF), 156.25, 79.0, 77.5 (CDCl 3 ), 77.16 (CDCl 3 ), 76.84 (CDCl 3 ), 55.3, 51.2, 50.3 (MeOH), 46.9, 40.3 (IMP), 39.4, 36.5 (DMF), 34.9, 34.1, 31.5 (DMF), 29.9, 28.5, 26.4, 24.7, 23.1.

HRMS (ESI-TOF) Calc. for [C 66 H 128 N 18 O 16 ] [M-2H] 2+ = 715.49506, found = 715.49166, DM (ppm) = -4.753.

Synthesis of FG0-G1-LYS-ALA-P

Under a nitrogen atmosphere, 0.995 g (0.002 mol) of Fmoc-Ala-OPfp was dissolved in 5 mL of DMSO and stirred at room temperature in a round bottom flask. FG0-G0-LYS, 0.298 g (0.21 mmol), was dissolved in 10 mL of DMSO and added dropwise to the stirring amino acid solution over 2 hours. The reaction was stirred for 96 hours at room temperature. The crude reaction mixture was triturated into 300 mL of 1 % sodium chloride DI water, and this solution was centrifuged at 4000 rpm for 30 minutes. The supernatant was decanted leaving a yellow pellet, and 50 mL of fresh DI water was added to each centrifuge tube. The pellet and water were centrifuged once more at 3000 rpm for 2 minutes, and the water was decanted leaving a white pellet. The sample was lyophilized for 24 h, and subsequently suspended in 20 mL of DCM. The crude suspension was triturated into cold ether, and the ether solution was centrifuged at 3000 rpm for 3 minutes. The supernatant was decanted leaving a waxy pellet. The pellet was suspended in DCM, transferred to a round bottom flask, and the DCM was removed via rotary evaporation and high vacuum overnight. The final product was confirmed with 1 H and 19 F NMR at a 97 % recovery (0.532 g, 0.204 mmol).

1 H-NMR (400 MHz; DMSO-d 6 ): d 7.98 (br, 8H), 7.90-7.86 (d, 8H), 7.67 (t, 8H), 7.40-7.32 (t,

12H), 7.34-7.27 (t, 8H), 6.73 (t, 4H), 5.75 (s, DCM), 4.26-4.22 (d, 8H), 4.20 (t, 4H), 4.13 (d,

4H), 3.35 (br, H 2 0), 3.20 (br, 12H), 3.08 (br, 16H), 2.86 (br, 8H), 2.74 (br, 8H), 2.50 (s, DMSO- de), 2.33 (br, 8H), 2.26 (br, 8H), 1.59 (br, 4H), 1.46 (br, 4H), 1.34 (s, 36H), 1.19 (br, 16H). 1 9 F-NMR (400MHZ; DMSO-d 6 ): no peaks found. FMOC Deprotection of FG0-G1-LYS-ALA:

Under a nitrogen atmosphere, FG0-G1-LYS-ALA (0.637 g, 0.245 mmol) was dissolved in a 10 mL stirred 20 % piperidine/DMF solution at room temperature. This solution was allowed to stir for 20 minutes. To remove piperidine, DMF, and the fluorenyl group, the crude reaction mixture was triturated into cold ether, and the ether solution was centrifuged at 3000 rpm for 3 minutes. After the supernatant was removed, 20 mL of ether was added to each centrifuge tube, the sample was centrifuged at 3000 rpm for 3 minutes, and the supernatant was decanted. The remaining oily pellet was dissolved in methanol and transferred to a round bottom flask. The solvent was removed using a rotary evaporator and high vacuum, and the final product was confirmed with 1 H NMR and LCMS at a 92 % recovery (0.385 g, 0.225 mmol) and 80 % purity. 1 H-NMR (400 MHz; CDCl 3 ): d 8.02 (br, 8H), 7.26 (s, CDCl 3 ), 4.41 (br, 4H), 3.49 (s, MeOH), 3.33 (br, 16H, 3.06 (br, 12H), 2.70 (br, 8H), 2.38 (br, 16H), 1.85 (br, 8H), 1.68 (br, 8H), 1.49 (br, 8H), 1.43 (s, 36H). HRMS (ESI-TOF) Calc. for [C 78 H 148 N 22 O 20 ] [M+2H] 2+ = 856.5620, found = 856.56464, DM (ppm) = 3.08209.

Synthesis of FG0-G1.5-LYS:

Freshly distilled methyl acrylate 0.195 g, 0.002 mol was dissolved in 1 ml of methanol and cooled with an ice bath to 0 °C. Under nitrogen atmosphere, FG0-G1-LYS-ALA (0.352 g, 0.206 mmol) dissolved in 5 mL methanol was added dropwise over 1 hour. The ice bath was kept for another 3 hours, and afterwards, the reaction was stirred for 48 hours at room temperature. The solvent and excess methyl acrylate was removed on a rotary evaporator and high vacuum. This gave the half-generation FG0-G1.5-LYS in the form of slightly yellowish oil at 88 % recovery (0.435 g, 0.181 mmol). The final product was confirmed with 1 H, 13 C NMR and LCMS at 50 % purity. 1 H-NMR (400 MHz; CDCl 3 ): d 7.84 (br, 8H), 7.26 (s, CDCl 3 ), 5.13 (br, 4H), 4.35 (q, 4H), 3.69 (s, 6H), 3.66 (s, 18H), 3.30 (br, 16H), 3.08 (br, 12H), 2.90 (t, 4H), 2.80 (t, 24H), 2.55 (t, 6H), 2.50 (t, 18H), 2.41 (br, 12H), 1.80 (br, 4H), 1.63 (br, 4H), 1.48 (br, 8H), 1.41 (s, 36H), 1.33 (br, 8H).

13 C-NMR (100 MHz; CDCl 3 ): d 173.0, 172.9, 172.2, 156.3, 79.0, 77.4, 77.17 (CDCl 3 ), 53.5, 52.1, 51.9, 50.1, 49.7, 48.7, 48.6, 40.4, 39.0, 33.7, 32.1, 31.9, 31.4, 29.7, 28.6, 23.1.

HRMS (ESI-TOF) Calc. for [C 110 H 196 N 22 C 36 ] [M-4H] 4+ = 601.36255, found = 601.35458 Synthesis of FG0-G2-LYS-OH-P:

FG0-G1.5-LYS (0.048 g, 0.020 mmol) was dissolved in 3 mL of methanol. Freshly distilled ethanolamine (0.771 g, 0.013 mol) was dissolved in 1 mL methanol and cooled to 0 °C, using an ice bath. The FG0-G1.5-LYS solution was added to this solution dropwise over 1 hour under nitrogen atmosphere. The ice bath was kept for another 3 hours, and afterwards, the reaction was stirred for 6 days at room temperature. Workup was done by removing methanol using a rotary evaporator. After that, the product/ethanolamine solution was triturated into cold dioxane. The product settled and stuck to the bottom of the beaker, and the dioxane/ethanolamine solution was decanted. The beaker was rinsed/decanted twice more with dioxane. The product was then dissolved in methanol followed by solvent removal using a rotary evaporator and high vacuum. This gave the hygroscopic hydroxyl -terminated product at 78 % recovery (0.041 g, 0.016 mmol) and 20 % purity as confirmed by LCMS TIC chromatogram. The final product was confirmed with 1 H, 13 C NMR and LCMS. 1H-NMR (400 MHz; MeOD): d 4.84 (s, H 2 0), 4.21 (q, 4H), 3.60-3.55 (q, 34H), 3.31 (s, MeOD), 3.26 (m, MeOD), 2.99 (t, 12H), 2.75 (m, 44H), 2.53 (s, 4H), 2.36 (m, 36H), 1.74 (m, 4H), 1.62 (m, 4H), 1.45 (m, 4H), 1.39 (s, 36H), 1.32 (m, 4H). 13 C-NMR (100 MHz; MeOD): d 175.5, 175.1, 174.9, 174.8, 158.5, 79.9, 62.9, 61.6, 55.0, 51.6, 51.1, 51.0, 50.6, 50.5, 49.9, 49.0 (MeOD), 44.1, 43.0, 41.1, 40.2, 39.8, 36.7, 36.6, 36.0, 34.6, 34.5, 34.3, 32.9, 30.6, 28.9, 24.4.

HRMS (ESI-TOF) Calc. for [C 118 H 220 N 30 O 36 ] [M+2H] 2+ = 1317.82260, found = 1317.81851, DM (ppm) = -3.109.

Synthesis of FG0-G2-LY S-OH (BOC deprotection of FG0-G2-LYS-OH-P)

FG0-G2-LYS-OH-P (0.020 g, 0.008 mmol) was purified using preparative liquid chromatography. The fractions were analyzed for purity with LCMS, and the purest fractions were combined and neutralized to pH 7 with ammonium hydroxide. The combined fractions were placed under high vacuum for 24 hours to evaporate the mobile phase. To deprotect the BOC groups, the remaining solid was dissolved in 50% TFA/DCM and stirred for 3 hours at room temperature. The TFA and DCM were evaporated with the rotary evaporator and placed on the high vacuum overnight. The remaining solid was dissolved in 5 mL of deionized water and was dialyzed with 500-1000 MWCO dialysis tubing for 4 days against deionized water. The solution inside the dialysis tubing was removed and was evaporated under high vacuum for 24 hours. The product was confirmed with LCMS. The product was recovered (4 mg, 0.0018 mmol) at 96 % purity by LCMS TIC chromatogram.

HRMS (ESI-TOF) Calc for [C 98 H 188 N 30 O 28 ] [M+4H] 4+ = 559.36523, found = 559.36876, DM (ppm) = - 6.31073.

Synthesis of FGl-Gl-GLU-P

Under a nitrogen atmosphere, 2.240 g (3.8 mmol) of Fmoc-Glu(OtBu)-OPfp was dissolved in 8 mL of DMSO and stirred at room temperature in a round bottom flask. G1 PAMAM dendrimer, 0.270 g (0.19 mmol), was dissolved in 8 mL of DMSO and added dropwise to the stirring amino acid solution over 2 hours. The reaction was stirred for 72 hours at room temperature. The crude reaction mixture was triturated into 300 mL of 1 % sodium chloride DI water, and this solution was centrifuged at 4000 rpm for 30 minutes. The supernatant was decanted leaving a yellow pellet, and 50 mL of fresh DI water was added to each centrifuge tube. The pellet and water were centrifuged once more at 3000 rpm for 2 minutes, and the water was decanted leaving a white pellet. The sample was placed on a high-vacuum for 24 h, and subsequently suspended in 20 mL of DCM. The crude suspension was triturated into cold ether, and the ether solution was centrifuged at 3000 rpm for 3 minutes. The supernatant was decanted leaving a waxy pellet, 20 mL of ether was added to each centrifuge tube, the sample was centrifuged at 3000 rpm for 3 minutes, and the supernatant was decanted leaving a waxy pellet. The pellet was suspended in DCM, transferred to a round bottom flask, and the DCM was removed via rotary evaporation and high vacuum overnight. The final product was confirmed with 1 H and 19 F NMR at a 73 % recovery (0.549 g, 0.117 mmol).

1 H-NMR (400 MHz; DMSO-d 6 ): d 7.97 (br, 8H), 7.87-7.85 (br, 24H), 7.71 (t, 16H), 7.50-7.48 (d, 8H), 7.39 (t, 16H), 7.30 (t, 26H), 5.75 (DCM), 4.29 (br, 8H), 4.20 (br, 16H), 3.97-3.91 (q,

8H), 3.34 (s, water), 3.07 (br, 36H), 2.65 (br, 16H), 2.50 (s, DMSO-d 6 ), 2.14 (br, 8H), 2.29 (br, 8H), 2.18 (br, 30H), 1.87 (m, 8H), 1.73 (m, 8H), 1.35 (s, 72H).

19 F-NMR (400MHZ; DMSO-d 6 ): no peaks found. FMOC Deprotection of FGl-Gl-GLU-P:

Under a nitrogen atmosphere, 0.548 g (0.12 mmol) of FG1-G1-GLU was dissolved in a 10 mL stirred 20 % piperidine/DMF solution at room temperature. This solution was allowed to stir for 20 minutes. To remove piperidine, DMF, and the fluorenyl group, the crude reaction mixture was triturated into cold ether, and the ether solution was centrifuged at 3000 rpm for 3 minutes. After the supernatant was removed, 20 mL of ether was added to each centrifuge tube, the sample was centrifuged at 3000 rpm for 3 minutes, and the supernatant was decanted. The remaining oily pellet was dissolved in methanol and transferred to a round bottom flask. The solvent was removed using a rotary evaporator and high vacuum, and the final product was confirmed with 1 H, 13 C NMR and LCMS at a 68 % recovery and 69 % purity (0.231 g, 0.079 mmol).

1 H-NMR (400 MHz; CDCl 3 ): d 7.87 (br, 16H), 7.26 (s, CDCl 3 ), 3.42 (s, MeOH), 3.31 (br, 32H), 3.20 (br, 8H), 2.69 (br, 20H), 2.50 (br, 12H), 2.30 (m, 40H), 2.01 (m, 8H), 1.75 (m, 8H), 1.40 (s, 72H). 13 C-NMR (100 MHz; CDCl 3 ): d 175.7, 173.4, 172.8, 80.6, 77.16 (CDCl 3 ), 54.9, 52.5, 50.6, 39.4, 39.35, 37.8, 34.3, 33.7, 32.0, 31.0, 30.5, 28.2.

HRMS (ESI-TOF) Calc. for [C 134 H 248 N 34 O 36 ] [M+4H] 4+ = 727.46552, found = 727.62095.

Synthesis of FG1-G2-GLU-ALA-P

Under a nitrogen atmosphere, 0.608 g (0.0013 mol) of Fmoc-Ala-OPfp was dissolved in 5 mL of DMSO and stirred at room temperature in a round bottom flask. FG1-G1-GLU, 0.186 g (0.064 mmol), was dissolved in 10 mL of DMSO and added dropwise to the stirring amino acid solution over 2 hours. The reaction was stirred for 96 hours at room temperature. The crude reaction mixture was triturated into 300 mL of 1 % sodium chloride DI water, and this solution was centrifuged at 4000 rpm for 30 minutes. The supernatant was decanted leaving a yellow pellet, and 50 mL of fresh DI water was added to each centrifuge tube. The pellet and water were centrifuged once more at 3000 rpm for 2 minutes, and the water was decanted leaving a white pellet. The sample was placed on a high vacuum for 24 h and was subsequently suspended in 20 mL of DCM. The crude suspension was triturated into cold ether, and the ether solution was centrifuged at 3000 rpm for 3 minutes. The supernatant was decanted leaving a waxy pellet, 20 mL of ether was added to each centrifuge tube, the sample was centrifuged at 3000 rpm for 3 minutes, and the supernatant was decanted. The pellet was suspended in DCM, transferred to a round bottom flask, the DCM was removed via rotary evaporation and high vacuum overnight, and the product was immediately deprotected with piperidine and DMF.

FMOC Deprotection of FG1-G2-GLU-ALA-P:

Under a nitrogen atmosphere FG1-G2-GLU-ALA-P was dissolved in a 10 mL stirred 20 % piperidine/DMF solution at room temperature. This solution was allowed to stir for 20 minutes. To remove piperidine, DMF, and the fluorenyl group, the crude reaction mixture was triturated into cold ether, and the ether solution was centrifuged at 3000 rpm for 3 minutes. After the supernatant was removed, 20 mL of ether was added to each centrifuge tube, the sample was centrifuged at 3000 rpm for 3 minutes, and the supernatant was decanted. The pellet was dissolved in methanol and transferred to a round bottom flask. The solvent was removed using a rotary evaporator and high vacuum, and the final product was confirmed with 1 H NMR and LCMS at a 97% recovery and 65% purity (0.183g, 0.053mmol).

1 H-NMR (400 MHz; CDCl 3 ): d 8.07 (br, 24H), 7.26 (s, CDCl 3 ), 4.39 (br, 8H), 3.42 (s, MeOH), 3.27 (br, 5 OH), 3.02 (br, 16H), 2.70 (br, 20H), 2.49-2.31 (br, 60H), 2.11 (m, 8H), 1.89 (m, 8H), 1.40 (s, 72H). HRMS (ESI-TOF) Calc. for [C 150 H 288 N 42 O 44 ] [M+2H] 2+ = 869.53975, found = 869.59415.

Synthesis of FG1-G2.5-GLU:

Freshly distilled methyl acrylate 0.171 g (0.002 mol) was dissolved in 1 ml of methanol and cooled with an ice bath to 0 °C. Under nitrogen atmosphere, FG1-G2-GLU-ALA (0.121 g, 0.042 mmol) dissolved in 5 mL methanol was added dropwise over 10 minutes. The ice bath was kept for another 3 hours, and afterwards, the reaction was stirred for 48 hours at room temperature. The solvent and excess methyl acrylate was removed on a rotary evaporator and high vacuum. This gave the half-generation FG1-G2.5-GLU in the form of slightly yellowish oil at 70 % recovery (0.143 g, 0.029 mmol). The final product was confirmed with 1 H, 13 C NMR and LCMS at 23 % purity. 1 H-NMR (400 MHz; CDCl 3 ): d 7.81 (br, 16H), 7.26 (s, CDCl 3 ), 4.39 (q, 8H), 3.68 (s, 14H), 3.65 (s, 32H), 3.32 (br, 26H), 3.23 (br, 12H), 2.89 (t, 2.89H), 2.78 (m, 54H), 2.58-2.24 (m, 88H), 2.08 (m, 8H), 1.89 (m, 8H), 1.41 (s, 72H). 13 C-NMR (100 MHz; CDCl 3 ): d 173.5, 173.1, 172.8, 172. 5, 172.2, 80.7, 77.4, 77.16, 52.9, 52.1, 51.8, 50.6, 50.2, 49.7, 48.7, 48.6, 39.9, 38.9, 34.3, 33.9, 31.9, 31.9, 31.5, 28.2, 27.7.

HRMS (ESI-TOF) Calc. for [C 222 H 384 N 42 O 26 ] [M-7H] 7+ = 694.53535, found = 694.54590 Synthesis of FG1-G3-GLU-OH:

FG1-G2.5-GLU (0.074 g, 0.015 mmol) was dissolved in 3 mL of methanol. Freshly distilled ethanolamine 0.540 g, 0.009 mol was dissolved in 3 mL methanol and cooled to 0 °C, using an ice bath. The FG1-G2.5-GLU solution was added to this solution dropwise over 1 hour under nitrogen atmosphere. The ice bath was kept for another 3 hours, and afterwards, the reaction was stirred for 6 days at room temperature. Workup was done by removing methanol using a rotary evaporator. After that, the product/ethanolamine solution was triturated into cold dioxane. The product settled and stuck to the bottom of the beaker, and the dioxane/ethanolamine solution was decanted. The beaker was rinsed/decanted twice more with dioxane. The product was then dissolved in methanol followed by solvent removal using a rotary evaporator and high vacuum. This gave the hygroscopic hydroxyl -terminated product at 99 % recovery. The final product was confirmed with 1 H, 13 C NMR and LCMS. 1 H-NMR (400 MHz; MeOD): d 4.83 (s, water), 4.20 (q, 8H), 3.63 (s, 4H), 3.31 (s, MeOD), 3.27 (m, MeOD), 2.99 (m, 28H), 2.73 (br, 104H), 2.53 (br, 8H), 2.35 (br, 62H), 1.74 (m, 8H), 1.62 (m, 8H), 1.45 (s, 72H), 1.30 (br, 8H), 1.14 (br, 8H).

13 C-NMR (100 MHz; MeOD): d 175.7, 175.2, 174.9, 174.7, 158.5, 79.8, 75.3, 55.0, 52.2, 51.0, 50.5, 49.0 (MeOD), 46.7, 46.5, 45.7, 43.59, 42.50, 41.9, 41.6, 41.4, 41.2, 40.2, 39.8, 38.5, 36.9,

36.7, 36.0, 35.1, 34.7, 34.5, 34.4, 33.0, 30.7, 28.9, 24.4.

HRMS (ESI-TOF) Calc. for [C 238 H 432 N 58 O 76 ] [M+7H] 7+ = 760.88902, found = 760.88746, DM (ppm) = -2.057. Synthesis of FG1-G3-GLU-OH (OtBu deprotection of FG1-G3-GLU-OH-P)

FG1-G3-GLU-0H-P (90 mg, 0.017 mmol) was purified using preparative liquid chromatography. The fractions were analyzed for purity with LCMS, and the purest fractions were combined and neutralized to pH 7 with ammonium hydroxide. The combined fractions were placed under high vacuum for 24 hours to evaporate the mobile phase. To deprotect the OtBu groups, the remaining solid was dissolved in 90 % TFA/DCM and stirred for 5 hours at room temperature. The TFA and DCM were evaporated with the rotary evaporator and placed on the high vacuum overnight. The remaining solid was dissolved in 5 mL of deionized water and was dialyzed with 500-1000 MWCO dialysis tubing for 4 days against deionized water. The solution inside the dialysis tubing was removed and was evaporated under high vacuum for 24 hours. The product was confirmed with LCMS. The product was recovered (20 mg, 0.004 mmol) at 96 % purity by LCMS TIC chromatogram. HRMS (ESI-TOF) Calc. for [C 206 H 368 N 58 O 76 ] [M+7H] 7+ = 696.81120, found = 696.81199, DM (ppm) = 1.1337.

Synthesis of FG0-GO-GLU-P

Under a nitrogen atmosphere, 2.623 g (4.4 mmol) of Fmoc-Glu(OtBu)-OPfp was dissolved in 8 mL of DMSO and stirred at room temperature in a round bottom flask. GO PAMAM dendrimer, 0.229 g (0.44 mmol), was dissolved in 8 mL of DMSO and added dropwise to the stirring amino acid solution over 2 hours. The reaction was stirred for 72 hours at room temperature. The crude reaction mixture was triturated into 300 mL of 1 % sodium chloride DI water, and this solution was centrifuged at 4000 rpm for 30 minutes. The supernatant was decanted leaving a yellow pellet, and 50 mL of fresh DI water was added to each centrifuge tube. The pellet and water were centrifuged once more at 3000 rpm for 2 minutes, and the water was decanted leaving a white pellet. The sample was placed on a high-vacuum for 24 h, and subsequently suspended in 20 mL of DCM. The crude suspension was triturated into cold ether, and the ether solution was centrifuged at 3000 rpm for 3 minutes. The supernatant was decanted leaving a waxy pellet, 20 mL of ether was added to each centrifuge tube, the sample was centrifuged at 3000 rpm for 3 minutes, and the supernatant was decanted leaving a waxy pellet. The pellet was suspended in DCM, transferred to a round bottom flask, and the DCM was removed via rotary evaporation and high vacuum overnight. The final product was confirmed with 1 H and 19 F NMR at a 93 % recovery (0.883 g, 0.411 mmol).

1 H-NMR (400 MHz; DMSO-d 6 ): d 7.93 (br, 8H), 7.88-7.86 (d, 8H), 7.71 (t, 8H), 7.49-7.37 (d, 4H), 7.40 (t, 8H), 7.31 (t, 8H), 5.75 (s, DCM), 4.30-4.16 (m, 12H), 3.97-3.92 (q, 4H), 3.43-3.24 (b, H 2 0/ether), 3.09 (br, 12H), 2.66 (br, 8H), 2.50 (s, DMSO-d 6 ), 2.20 (t, 12H), 1.89 (m, 4H), 1.73 (br, 4H), 1.37 (s, 36H), 1.09 (t, ether).

19 F-NMR (400MHZ; DMSO-d 6 ): no peaks found.

Under a nitrogen atmosphere, 0.882 g (0.41 mmol) of FG0-G0-GLU was dissolved in a 10 mL stirred 20 % piperidine/DMF solution at room temperature. This solution was allowed to stir for 20 minutes. To remove piperidine, DMF, and the fluorenyl group, the crude reaction mixture was triturated into cold ether, and the ether solution was centrifuged at 3000 rpm for 3 minutes. After the supernatant was removed, 20 mL of ether was added to each centrifuge tube, the sample was centrifuged at 3000 rpm for 3 minutes, and the supernatant was decanted. The remaining oily pellet was dissolved in methanol and transferred to a round bottom flask. The solvent was removed using a rotary evaporator and high vacuum, and the final product was confirmed with 1 H, 13 C NMR and LCMS at a 66 % recovery and 78 % purity (0.341 g, 0.272 mmol).

1 H-NMR (400 MHz; CDCl 3 ): d 7.79 (br, 8H), 7.26 (s, CDCl 3 ), 3.40 (s, MeOH), 3.30 (br, 16H), 2.91 (s, DMF), 2.83 (s, DMF), 2.64 (br, 8H), 2.42 (s, 4H), 2.29 (br, 16H), 2.03 (br, 12H), 1.73 (m, 8H), 1.38 (s, 36H). 1 3 C-NMR (100 MHz; CDCl 3 ): d 175.7, 173.3, 172.7, 80.6, 77.16 (CDCl 3 ), 54.8, 51.2, 50.4, 50.3, 39.4, 39.3, 34.0, 32.0, 30.4, 28.1.

HRMS (ESI-TOF) Calc. for [C 58 H 108 N 14 6 ] [M+2H] 2+ =628.4034, found = 628.40924 Synthesis of FG0-Gl-GLU-ALA-P

Under a nitrogen atmosphere, 0.367 g (0.00077 mol) of Fmoc-Ala-OPfp was dissolved in 5 mL of DMSO and stirred at room temperature in a round bottom flask. FG0-G0-GLU, 0.097 g (0.077 mmol), was dissolved in 10 mL of DMSO and added dropwise to the stirring amino acid solution over 2 hours. The reaction was stirred for 96 hours at room temperature. The crude reaction mixture was triturated into 300 mL of 1 % sodium chloride DI water, and this solution was centrifuged at 4000 rpm for 30 minutes. The supernatant was decanted leaving a yellow pellet, and 50 mL of fresh DI water was added to each centrifuge tube. The pellet and water were centrifuged once more at 3000 rpm for 2 minutes, and the water was decanted leaving a white pellet. The sample was placed on a high vacuum for 24 h and was subsequently suspended in 20 mL of DCM. The crude suspension was triturated into cold ether, and the ether solution was centrifuged at 3000 rpm for 3 minutes. The supernatant was decanted leaving a waxy pellet, 20 mL of ether was added to each centrifuge tube, the sample was centrifuged at 3000 rpm for 3 minutes, and the supernatant was decanted. The pellet was suspended in DCM, transferred to a round bottom flask, and the DCM was removed via rotary evaporation and high vacuum overnight. The final product was confirmed with 1 H and 19 F NMR at a 91 % recovery (0.171 g, 0.070 mmol).

1 H-NMR (400 MHz; DMSO-d 6 ): d 7.99-7.94 (br, 8H), 7.89-7.87 (d, 18H), 7.69 (t, 18H), 7.41 (t, 16H), 7.32 (t, 21H), 6.72 (m, 4H), 5.75 (s, DCM), 4.27 (t, 17H), 4.20 (t, 8H), 4.12 (q, 4H), 3.33

(br, water), 3.22-3.09 (br, 40H), 2.87 (br, 12H), 2.72 (br, 8H), 2.50 (s, DMSO-d 6 ) 2.38 (t, 12H), 2.33 (t, 8H), 2.23 (br, 8H), 1.61 (br, 4H), 1.47 (br, 4H), 1.34 (s, 36H) 1.19 (s, 8H).

19 F-NMR (400MHZ; DMSO-de): 161.9 (d, 2F), 165.5, (t, 2F), 172.1, (t, 1H)

FMOC Deprotection of FG0-Gl-GLU-ALA-P:

Under a nitrogen atmosphere 0.170 g (0.000070 mol) FG0-G1-GLU-ALA was dissolved in a 10 mL stirred 20 % piperidine/DMF solution at room temperature. This solution was allowed to stir for 20 minutes. To remove piperidine, DMF, and the fluorenyl group, the crude reaction mixture was triturated into cold ether, and the ether solution was centrifuged at 3000 rpm for 3 minutes. After the supernatant was removed, 20 mL of ether was added to each centrifuge tube, the sample was centrifuged at 3000 rpm for 3 minutes, and the supernatant was decanted. The pellet was dissolved in methanol and transferred to a round bottom flask. The solvent was removed using a rotary evaporator and high vacuum, and the final product was confirmed LCMS at a 79 % recovery and 78 % purity.

FIRMS (ESI-TOF) Calc. for [C 70 H 128 N 18 20 ] [M+2H] 2+ = 770.4776, found = 770.48071.

Synthesis of FG0-G1.5-GLU:

Freshly distilled methyl acrylate 0.078 g (0.00090 mol) was dissolved in 1 ml of methanol and cooled with an ice bath to 0 °C. Under nitrogen atmosphere, FG0-G1-GLU-ALA (0.058 g, 0.038 mmol) dissolved in 5 mL methanol was added dropwise over 10 minutes. The ice bath was kept for another 3 hours, and afterwards, the reaction was stirred for 48 hours at room temperature. The solvent and excess methyl acrylate was removed on a rotary evaporator and high vacuum. This gave the half-generation FG0-G1.5-GLU in the form of slightly yellowish oil with 85 % recovery. The final product was confirmed with 1 H, 13 C NMR and LCMS at 51 % purity.

1 H-NMR (400 MHz; CDCl 3 ): d 7.80 (br, 8H), 7.66 (br, 4H), 7.26 (s, CDCl 3 ), 4.41-4.35 (q, 4H), 3.70 (s, 4H), 3.66 (s, 19H), 3.34 (br, 16H), 2.92 (t, 6H), 2.79 (m, 32H), 2.57 (t, 8H), 2.50 (t, 16H), 2.30 (m, 12H), 2.30 (t, 8H), 2.09 (m, 4H), 1.89 (m, 4H), 1.42 (s, 36H).

13 C-NMR (100 MHz; CDCl 3 ): d 173.1, 172.9, 172.6, 172.2, 172.1, 80.7, 77.4, 53.0, 52.2, 51.9, 50.2, 49.6, 48.7, 48.5, 40.0, 39.0, 33.9, 31.9, 31.3, 30.8, 29.8, 28.2, 27.7.

HRMS (ESI-TOF) Calc. for [C 102 H N 18 O 36 ] [M-4H] 4+ = 558.31238, found = 558.32147.

Synthesis of FG0-G2-GLU-OH:

FG0-G1.5-GLU (0.041 g, 0.018 mmol) was dissolved in 2 mL of methanol. Freshly distilled ethanolamine (0.268 g, 0.0044 mol) was dissolved in 1 mL methanol and cooled to 0 °C, using an ice bath. The FG0-G1.5-GLU solution was added to this solution dropwise over 1 hour under nitrogen atmosphere. The ice bath was kept for another 3 hours, and afterwards, the reaction was stirred for 6 days at room temperature. Workup was done by removing methanol using a rotary evaporator. After that, the product/ethanolamine solution was triturated into cold dioxane. The product settled and stuck to the bottom of the beaker, and the dioxane/ethanolamine solution was decanted. The beaker was rinsed/decanted twice more with dioxane. The product was then dissolved in methanol followed by solvent removal using a rotary evaporator and high vacuum. This gave the hygroscopic hydroxyl -terminated product at 34 % recovery and 61 % purity as confirmed by LCMS TIC chromatogram. The final product was confirmed with 1 H, 13 C NMR and LC-MS. 1 H-NMR (400 MHz; MeOD): d 4.88 (s, water), 4.32 (q, 4H), 3.62 (m, 160H), 3.31 (s, MeOD), 3.02 (br, 5 OH), 2.94 (t, 24H), 2.81 (br, 130H), 2.50 (t, 24H), 2.43 (m, 94H), 2.08 (m, 4H), 1.89 (m, 4H), 1.45 (s, 36H) 1.33 (br, 8H). 13 C-NMR (100 MHz; MeOD): d 175.5, 175.0, 174.2, 173.8, 62.8, 61.6, 51.6, 51.0, 49.0 (MeOD), 44.0, 43.0, 38.4, 36.7, 35.8, 34.9, 34.6, 28.4.

HRMS (ESI-TOF) Calc. for [C 110 H 200 N 26 O 36 ] [M+2H] 2+ = 1231.73820, found = 1231.73305, DM (ppm) = -4.187.

Synthesis of FG0-G2-GLU-OH (OtBu deprotection of FG0-G2-GLU-OH-P)

FG0-G2-GLU-OH-P (75 mg, 0.030 mmol) was purified using preparative liquid chromatography. The fractions were analyzed for purity with LCMS, and the purest fractions were combined and neutralized to pH 7 with ammonium hydroxide. The combined fractions were placed under high vacuum for 24 hours to evaporate the mobile phase. To deprotect the OtBu groups, the remaining solid was dissolved in 90 % TFA/DCM and stirred for 5 hours at room temperature. The TFA and DCM were evaporated with the rotary evaporator and placed on the high vacuum overnight. The remaining solid was dissolved in 5 mL of deionized water and was dialyzed with 500-1000 MWCO dialysis tubing for 4 days against deionized water. The solution inside the dialysis tubing was removed and was evaporated under high vacuum for 24 hours. The product was confirmed with LCMS. The product was recovered (15 mg, 0.0067 mmol) at 90 % purity by LCMS TIC chromatogram.

HRMS (ESI-TOF) Calc. for [C 94 H 168 N 26 O 36 ] [M+4H] 4+ = 560.31288, found = 560.30969, DM (ppm) = -5.69325.

References Cited in Example 2

(1) Maingi, V.; Jain, V.; Bharatam, P. V.; Maiti, P. K. Dendrimer Building Toolkit: Model Building and Characterization of Various Dendrimer Architectures. J. Comput. Chem. 2012, 33, 1997-2011.

(2) James J. P. Stewart. MOPAC2016 http://openmopac.net (accessed Jun 24, 2019).

(3) Gaussian 09, Revision C.01, M. J. Frisch, G. W. Trucks, H. B. Schlegel, G. E. Scuseria, M. A. Robb, J. R. Cheeseman, G. Scalmani, V. Barone, B. Mennucci, G. A. Petersson, H. Nakatsuji, M. Caricato, X. Li, H. P. Hratchian, A. F. Izmaylov, J. Bloino, G. Zheng, J. L. Sonnenberg, M. Hada, M. Ehara, K. Toyota, R. Fukuda, J. Hasegawa, M. Ishida, T. Nakajima, Y. Honda, O. Kitao, H. Nakai, T. Vreven, J. A. Montgomery, Jr., J. E. Peralta, F. Ogliaro, M. Bearpark, J. J. Heyd, E. Brothers, K. N. Kudin, V. N. Staroverov, T. Keith, R. Kobayashi, J. Normand, K. Raghavachari, A. Rendell, J. C. Burant, S. S. Iyengar, J. Tomasi, M. Cossi, N. Rega, J. M. Millam, M. Klene, J. E. Knox, J. B. Cross, V. Bakken, C. Adamo, J. Jaramillo, R. Gomperts, R. E. Stratmann, O. Yazyev, A. J. Austin, R. Cammi, C. Pomelli, J. W. Ochterski, R. L. Martin, K. Morokuma, V. G. Zakrzewski, G. A. Voth, P. Salvador, J. J. Dannenberg, S. Dapprich, A. D. Daniels, O. Farkas, J. B. Foresman, J. V. Ortiz, J. Cioslowski, and D. J. Fox, Gaussian, Inc., Wallingford CT, 2010.} via DFT using the B3LYP functional and the 6-31g(d) basis set. Gaussian 09 was also used to calculate ESP charges which were further used to assign RESP charges in Amber 14. (ref: D.A. Case, V. Babin, J.T. Berryman, R.M. Betz, Q. Cai, D.S. Cerutti, T.E. Cheatham, III, T.A. Darden, R.E. Duke, H. Gohlke, A.W. Goetz, S. Gusarov, N. Homeyer, P. Janowski, J. Kaus, I. Kolossvary, A. Kovalenko, T.S. Lee, S. LeGrand, T. Luchko, R. Luo, B. Madej, K.M. Merz, F. Paesani, D.R. Roe, A. Roitberg, C. Sagui, R. Salomon-Ferrer, G. Seabra, C.L. Simmerling, W. Smith, J. Swails, R.C. Walker, J. Wang, R.M. Wolf, X. Wu and P.A. Kollman (2014), AMBER 14, University of California, San Francisco.

Example 3. DendriPep-Drug Conjugates (DDCs)

Antibody-drug conjugates (ADCs) are a major class of oncology therapeutics. Since 2013, more than 30 new, 2 nd generation (G.2) ADCs have entered clinical development, and more than 60 are in clinical trials. G.2 ADCs are more stable and potent than prior ADCs, owing to novel ADC-tailored antibodies that feature enhanced biorecognition activity for a wide variety of antigens and chemical handles for linking the drug warheads at defined positions. ADCs are now being developed for indications such as triple-negative breast cancer (TNBC) and ovarian cancer, which have very poor prognosis and no approved naked antibodies. Despite the promising clinical trials, G2 ADCs present critical limitations. Their primary cause of clinical failure is the limited amount of drug effectively delivered by ADCs, which is related to the small number of antigens on tumor cells and the low drug-to-antibody ratio. These issues can be mitigated using warheads with extremely high potency (e.g., emtansine), although the widely used ones are hydrophobic and tend to induce ADC aggregation, thus shortening shelf-life and causing immunogenic responses and rapid clearance rates. Further, exposure of the conjugation sites to aqueous environments results in drug de-conjugation and off-target toxicity. Concerns on the feasibility of ADCs are also posed by the high manufacturing costs and variability related to drug conjugation protocols and isolation of ADCs.

Cancer-targeting DendriPep-Drug Conjugates (DDCs) can be used as synthetic mimetics of ADCs, featuring synergistic drug warheads conjugated inside their dendritic structure at stoichiometrically precise ratios. DendriPeps have been developed as a novel class of protein- like macromolecules consisting of a PAMAM dendrimer framework whose branches are hybridized with amino acids. The functional group of the amino acids, together with their number and position within the PAMAM framework has been demonstrated to determine the size, shape, and z-potential of DendriPeps. In previous work, the synergistic activity of Doxorubicin (DOX) and Gemcitabine (GEM) against TNBC has been demonstrated. Further, microfluidic models of mammalian cells have been developed, which are ideal for evaluating the cancer cell targeting and cytotoxicity of DDCs, and have shown that in vitro microfluidic results correlate with in vivo performance.

The central hypothesis is that optimal design parameters exist, i.e., amino acid-mediated size, z-potential, and drug payload of DDCs, that afford combined (i) higher therapeutic efficacy, (ii) higher safety, and (iii) comparable targeted delivery compared to G.2 ADCs.

Utilize DendriPeps to develop an ensemble of select DDCs targeting MDA-MB-231 cells

DendriPeps have been developed of define properties (i.e. size, z-potential, and stability in solution) different structure, as well as functionality (amine and carboxyl), number (4 and 8), and position of amino acids. Guided by a Design-of-Experiment approach, an ensemble of DDCs can be developed by conjugating DOX and GEM in different amounts and synergistic molar combinations onto different base DendriPeps via acid-labile linkages (i.e., imine and ester). DDCs can be characterized and selected in terms of stability, safety, and amount and ratio of drug release at physiological (pH 7.4) and tumor-like (pH 5.0) conditions, for subsequent testing in vitro.

Evaluation of DDC efficacy and targeting in a novel microfluidic model of triple-negative breast cancer (TNBC)

The cytotoxicity of the DDCs developed against TNBC cells (MDA-MB-231) can be evaluated using standard in vitro assays, to select champion DDCs for in-depth evaluation using our microfluidic model of TNBC. The device features MDA-MB-231 cancer cells and MFC 10A breast epithelial cells co-cultured in a single system, which enables real-time analysis of DDC cancer-targeting and cytotoxic activity in a physiologically representative model. To enable targeted delivery, the selected DCCs can be functionalized with the peptide PRWAVSP, which specifically binds MDA-MB-231 cells. MDA-MB-231 -targeting DDCs can be evaluated in the microfluidic model of TNBC, in presence of circulatory immune cells (i.e., monocytes). Specifically, the following can be measured: (i) combination (Chou-Talalay) index, (ii) MDA- MB-231 vs. MFC 10A cell viability, (iii) kinetics of cell death under physiological flow conditions, and (iv) intracellular trafficking of DDCs. These data can be used to derive correlations between DDC design (drug loading and GEM:DOX ratio, size and z-potential, and peptide-to-DDC ratio) and therapeutic efficacy (rate of cancer vs. healthy cell death, therapeutic synergism, localization of DDCs on cancer cells by DOX self fluorescence).

Background and Significance

Antibody-Drug Conjugates (ADCs) consist of monoclonal antibodies (mAbs) conjugated with cytotoxic drugs (“warheads”) by synthetic linkers. Second (G.2) and third-generation (G.3) ADCs under current development are more stable and potent compared to the initial G.l ADCs [9] Despite their tremendous therapeutic promise, ADCs present a number of limitations including:

Low number of antigens on cancer cells and drugs-per-antibody ratio. The surface density of antigens on cancer cells is generally low (10 3 - 10 4 ), and the average drugs-per- antibody ratio cannot exceed 3.5 - 4 to avoid structural alteration of the mAb by chemical conjugation [10] Thus, to ensure therapeutic activity, ADCs must carry warheads with very high toxicity (IC 50 < 10 -10 M). These, however, can cause other issues such as:

Severe off-target cytotoxicity resulting from unwanted drug de-conjugation. Cleavable linkers are used to release the warhead at the target site. Recent studies on pharmacokinetics and metabolism of G.2 ADCs show that the linkers can undergo undesired cleavage, thus releasing the drug payload in the systemic circulation [11]

Drug-induced aggregation. Current drug warheads are hydrophobic and tend to reduce the stability of ADCs in solution during both storage and circulation [12]

ADCs also present engineering-related issues including:

1) Availability of antigen-targeting mAbs. The laborious development of mAbs towards selectivity and safety limits the limited number of known therapeutic mAbs compared to the pool of suitable antigens [13,14]

2) Heterogeneity of ADCs. The biochemical diversity of mAbs and conjugation chemistries, and the complexity of separating conjugated vs. naked mAbs result in batch variability of conjugation sites and drug-to-mAb ratios [15]

To overcome these issues, DendriPep-Drug Conjugates (DDCs) can be used as synthetic mimetics of, and alternatives to, ADCs. DendriPeps are a new class of hyperbranched molecules consisting of a PAMAM dendrimer framework whose branches are hybridized with amino acids. It is hypothesize that, by tuning the design parameters of DDCs, these advantages can be achieved compared to ADCs:

A) Safety, stability, permeability. DendriPeps feature a polyamide scaffold with globular structure that is protein-like, yet devoid of immunogenic sites. Their hydrodynamic radius (size) and z-potential can be tuned by varying the functionality, number, and position of the amino acids within the PAMAM branches, to ensure colloidal stability on-shelf and in circulation, slow clearance rates, and no/negligible hemolysis;

B) Combined drug loading. Unlike ADCs and PAMAM-drug conjugates, DDCs feature the conjugation of the drug warheads directly to the amino acids hybridized in the hyperbranched structure. Drug concealment prevents the off-target effects related to hydrophobic aggregation and release in circulation. Site-specific drug conjugation to the side-chain functional groups of the amino acids ensures reproducible conjugation of drugs at defined molar ratios and higher loading than ADCs, thus achieving drug synergism and product homogeneity. Synergism ensures therapeutic efficacy while allowing the use of drugs that are individually less toxic than those utilized in ADCs, thus providing for higher safety and the use of a broader arsenal of drug warheads.

C) “Smart” drug release. Like ADCs, DDCs feature a drug-amino acid linkage that is cleaved by the acidic tumor environment (e.g., imine, ester). Unlike encapsulating vectors (e.g., liposomes and nanoparticles), however, DendriPeps do not pose any transport limitation to drug release upon linker cleavage.

D) Tumor targeting. Like the hyper-variable region of ADCs, the outer layer (corona) of DDCs displays antigen-binding peptides that ensure selective targeting of the cancer cells. A myriad of peptide ligands for antigens (e.g., CD19, CD20, CD22, CD79b) are available in the literature.

To validate, DendriPeps can be developed loaded with a synergistic combination of DOX and GEM, and validated in a customized microfluidic model of triple-negative breast cancer (TNBC). TNBC is a model of interest, due to its poor prognosis and lack of approved naked mAbs [11]

DendriPeps represent a paradigm shift in the design of dendrimers for biomedical use. A wide variety of dendrimers with different composition, size and shape, and terminal groups have been developed [16] Yet, poor solution stability, leakage of the drug payload, and basal cytotoxicity have posed safety and efficacy concerns, and prevented clinical translation [17,18] With their novel design, DendriPeps show true promise to:

Improve biomedical applications (e.g., drug- and gene- delivery) by enabling superior control over physicochemical properties and drug loading, thus collectively enhancing stability in solution, safety, product homogeneity, therapeutic efficacy, and targeting. This is not possible with traditional ADCs and PAMAM-drug conjugates, where drug conjugation inevitably (and detrimentally) compromises stability and off-target effects.

Innovate the science of “soft” functional materials. DendriPeps represent an ideal model system to discover new fundamental correlations between the solution behavior of hyperbranched macromolecules and their molecular design parameters, and generate new knowledge on protein-like macromolecules in solution, coherently with the mission of the Material Genome Initiative (https://www.mgi.gov).

Construct a rationally designed ensemble of DOX/GEM-DDCs targeting TNBC cells.

Guided by a Design-of-Experiment approach, DendriPeps can be used to construct an initial ensemble of DDCs loaded with a synergistic ratio DOX and GEM. To this end, the base OH-terminated DendriPeps will comprise 2 - 4 lysine and 4 - 8 aspartic acid residues to enable direct conjugation of DOX and GEM by acid-labile imine and ester bonds, respectively. Auxiliary amino acids and PAMAM dendritic layers surrounding the drug-conjugated core region of the DDCs can be used to achieve a protein-like hydrodynamic radius (4 - 6 nm), a values of z-potential and amphiphilicity that ensure long-term stability in solution. The first ensemble of DDCs can be characterized in terms of colloidal stability in PBS and human serum (i.e., formation rate and amount of DDC and DDC-protein aggregates), and drug release in solutions at physiological (pH 7.4) and tumor-like (pH 5.0) conditions. Response Surface Metodology in JMP ® can be employed to correlate solution stability and GEM:DOX release ratio to the design parameters (global generation or size, number and position of amino acids, number of conjugated DOX and GEM), and design a second ensemble of optimized DDCs with predicted high stability and GEM:DOX release ratio to be evaluated.

DendriPeps can be constructed with different global dimension, number and position of amino acids, and evaluated their size, z-potential and stability in solution. Drug-polymer conjugates can also be developed featuring synergistic drug combinations (DOX/GEM and DOX/camptothecin), and evaluated for their therapeutic efficacy against breast cancer, including TNBC, both in vitro and in vivo [1-4]. Finally, Response Surface Metodology can be utilized in JMP ® to design the initial ensemble of DOX/GEM-DDCs.

An ensemble of ~ 10 - 15 DDCs are identified with tailored physicochemical properties that ensure safety and therapeutic efficacy, as detailed.

DOE-guided design of DDCs.

The design parameters of DendriPeps are (D.l) global size (amino acid layers + PAMAM layers), (D.2) size of the core PAMAM dendrimer, and (D.3 and D. 4) position and number of the amino acids. The amino acids utilized for drug conjugation are lysine (Lys) and aspartic acid (Asp). The imine bond formed by the e-amino group of Lys and the ketone group of DOX, and the ester bond formed by the b-carboxyl group of Asp and the hydroxyl group of GEM are cleavable by the acidic tumor environment. The auxiliary amino acids for controlling the z-potential of DDCs are histidine (His) and g-carboxyglutamic acid (Gla). To rationally sample this design space (D.l -D.4), a Design-of-Experiment (DOE) approach can be utilized to design the initial and subsequently optimized ensembles of candidate DendriPeps.

Optimization of DDCs by Response Surface Methodology.

The resulting properties and their goal values (response values R.1 - R.6) of DendriPeps and DDCs are:

R.l. DDC size: from - 2 nm (G.2 DendriPep)to - 5 nm (G.4 DendriPep);

R.2. z-potential of -10 mV to +5 mV, as dendrimers with z-potential < 5 mV are cytocompatible [19-22];

R.3. Hemolytic activity of DendriPeps < 2% (over 24 h) and MFC 10A epithelial cell viability > 98% (over 72 h); R.4. Drug/Dendripep ratio in DDCs: 4 - 8 (note: average drug load on ADCs is ~ 3.5 - 4);

R.5. Released vs. loaded drug ratio > 90%;

R.6. Molar GEMDOX release ratio of 1.5 - 3, which is documented as synergistic against TNBC

[3];

The response values (R.1 - R.6) can be correlated to the design space (D.l - D.4) by Response Surface Methodology in JMP ® and design an optimized set of DDCs that are predicted to return the above-listed goal values Experimental evaluation of DDCs DendriPep Synthesis and analytics.

The following has been constructed: (i) a Generation 2 (G.2) Dendripep with 4 lysines at layer 0; (ii) a G.3 Dendripep with 8 lysines at layer 1, (iii) a G.3 Dendripep with 4 aspartic acids at layer 1, and (iv) a G.4 Dendripep with 4 lysines at layer 1 and 8 aspartic acids at layer 1. The DendriPep synthesis was confirmed by mass spectrometry (LC-ESI-MS).

DDC size and stability in aqueous buffer and plasma.

The size of DDCs can be measured by dynamic light scattering (DLS) at different concentrations (0.01 - 0.1 mg/mL) in PBS at pH 7.4 using a Malvern ZetaSizer Nano ZS. To evaluate the long-term storage stability of the DDCs, the formation of aggregates in solutions at different concentrations of DDCs (0.01, 0.1, 1, and 10 mg/mL) in PBS, stored at different temperatures (4°C and 37°C) and for different times (6h, 24h, 2 days, 7 days, and 1 month) can be investigated. To evaluate the stability of DDCs in circulation, the DDCs can be incubated in platelet-rich plasma at a total DDC concentration between 0.1 - 0.5 mg/mL, at 37°C for 15 min and lh. Following the work by Dobrovolskaia et al. [21], light transmission aggregometry and flow cytometry can be used to quantify platelet-DDC aggregates in plasma.

Hemolytic and cytotoxic activity of base DendriPeps.

The hemolytic activity of DendriPeps on suspensions of red blood cells can be measured over 24 hours, as described by Bhadra et al. [23] The cytotoxicity of DendriPeps on healthy MFC 10A epithelial cells over 72 hours can be measured as described by El-Hammadi et al. [24]

Drug loading on DDCs.

The loading of DOX and GEM on the DendriPeps can be measured by UV-vis spectroscopy, as done in prior work [2] Briefly, the amount of DOX can be measured, and subsequently that of GEM by subtractive absorbance, to obtain average drug load and conjugated GEM/DOX ratio. Drug release in buffer and human plasma.

As done in prior work [2], the drug release kinetics can be measured from DDCs at either pH 7.4 or pH 5.0. The DOX and GEM released at lh, 24h, 48h, 62h, and 96h can be measured by UV-vis spectroscopy, as described. The stability of DDCs in human plasma can then be tested. After incubation in plasma, all proteins can be precipitated, and intact and deconjugated DDCs using organic solvents, and quantify DOX and GEM in the supernatant by liquid chromatography (C18 HPLC).

Approximately 10 - 15 DDCs are identified with optimal z-potential and cytocompatibility of base DendriPeps, to ensure solution stability and safety, as well as high drug release and synergistic molar GEM:DOX release ratio, to ensure therapeutic efficacy. Evaluation of DDC efficacy and targeting in a novel microfluidic model of TNBC.

The cytotoxicity of the DDCs can be initially evaluated against selected against TNBC cells (MDA-MB-231) using standard in vitro assays, to select 3 - 4 candidate DDCs for in-depth studies in the microfluidic TNBC model. This device features MDA-MB-231 cancer cells and MFC 10A breast epithelial cells co-cultured in a single chamber, and enables real-time analysis of DDC cancer-targeting and cytotoxic activity in a physiologically representative model. For targeted delivery, the DCCs can be functionalized with the peptide PRWAVSP, which specifically bind MDA-MB-231 cells. The peptide-DDCs can be evaluated in the TNBC model in presence of circulatory immune cells (monocytes), by measuring (i) combination index, (ii) MDA-MB-231 vs. MFC 10A cell viability, and (iii) kinetics of cell death under physiological flow conditions. Correlations can be derived between DDC design parameters (size, z-potential, GEM:DOX loading and ratio, peptide:DDC ratio) to therapeutic outcome (cancer vs. healthy cell death, drug synergism, DDC trafficking in cancer cells).

Polymer-drug conjugates have been developed featuring synergistic drug combinations and evaluated them against 4T1 breast cancer (DOX/CMT) and TNBC (DOX/GEM) [1,2]. Cell internalization and trafficking of the conjugates has been tracked and the resulting cell viability and drug combination index measured in vitro, and therapeutic efficacy in vivo. Microfluidic devices have also been developed for testing targeted drug-delivery formulations, and demonstrated correlation with in vivo outputs for numerous ligand-receptor-cell types [6]. In one instance, we cultured ICAM-expressing lung endothelial cells in a synthetic microvascular network that mimics physiological forces, geometry and flow rates of lungs (Fig. 7, panels A-B) [6]. Flow experiments of devices containing endothelial cells via particle tracking software show that anti-ICAM nanoparticles evaluated in our microfluidic chips were predictive of anti-ICAM targeting results in vivo (Fig. 7, panels C-E).

The design of a DDC can be finalized that (i) target cancer cells selectively in presence of healthy epithelial and circulatory immune cells under physiological flow conditions; (ii) afford a combination index 0.01 - 0.1, indicating strong synergism; (iii) high post-treatment MFC 10A cellular viability (> 90%).

Design and construction of the microfluidic model of TNBC Fabrication.

The microfluidic devices can be fabricated by standard photolithographic approaches. A photomask (CAD/ Art Services) can be utilized to create a master that will serve to mold the PDMS in the shape of the microfluidic channel (Fig. 8a). The PDMS can then be bonded onto glass slides, and the device can finally be biopsy punched at the inlet and outlets to enable flow through the device.

Co-culture of MDA-MB-231 and MFC 10A cells.

The glass bottom of the devices can be modified with fibrinogen/fibronectin to enable adhesion and proliferation of cells. GFP-expressing MDA-MB-231 cancer cells will be introduced into the device at low concentrations (lOVmL), followed by MFC 10A cells at high concentration (10 6 /ml). This staggered introduction can be expected to afford a mixed culture surface featuring 95% coverage by MFC 10A cells and 5% coverage by MDA-MB-231 cells (Fig. 8b) to enable evaluation of DDC targeting.

Conjugation of MDA-MB-231 -binding peptides to DDCs.

The MDA-MB-231 -binding peptide PRWAVSP [8] can be conjugated at the last step of DendriPep synthesis. Specifically, the amine-derivatized peptide Ac-PRWAVSP-(CH2)2-NH2 can be produced, mixed with ethanolamine in 1:1 - 1:10 ratios, and reacted with the methyl ester- terminated G-2.5 and G-3.5 DDCs by Michael addition, as done in traditional PAMAM synthesis [25], to obtain the final G-3 and G-4 DDC products. The resulting PRWAVSP-DDCs can be analyzed by mass spectrometry (ESI-MS) to quantify peptide conjugation and calculate the peptide/DDC ratios.

Evaluation of targeting vs. non-targeting DDCs Initial DDC cytotoxicity study on TNBC cells.

The activity of the DDCs selected against TNBC MDA-MB-231 cells can be evaluated via MTT cytotoxicity assay, as done previously [1, 2] Confluent MDA-MB-231 cells can be incubated with the different DDCs for 72 hours at 37°C, and analyzed by MTT assay to measure the fractional cell inhibition; pure DOX, pure GEM, and DOX and GEM combined at the same molar ratio of tested DDCs can be utilized as controls. Based on the data of fractional cell inhibition as function of drug concentration, the Combination Index (Cl) of the DDCs can be calculated using the Chou and Talalay model [26] The DDCs giving a Cl < 0.1 (strong synergism) can be selected for testing in the microfluidic device.

Evaluation of targeting and cytotoxicity of peptide-DDCs on cancer vs. healthy cells.

The DDCs selected can be functionalized with the PRWAVSP peptides and evaluated in the microfluidic model of TNBC. PRWAVSP-GEM/DOX-DDCs at a concentration of ~ 10 8 DDCs/mL can be injected in the microfluidic device at a flow rate of ~ 0.3 mL/h for 1 h, followed by saline at the same flow rate for 1 hour to remove unbound DDCs. The cytotoxicity against GFP-expressing MDA-MB-231 cells can be quantified by monitoring the reduction of GFP intensity in the cell culture chamber over 72 hours. Likewise, to determine non-target toxicity, analogous studies can be performed with GFP-expressing MFC 10A cells.

To track the intracellular trafficking of DDCs, Rhodamine (instead of DOX) can be conjugated on base DendriPeps and PRWAVSP-DendriPeps via acid-resistant amide bonds, and incubate them with the GFP-expressing MDA-MB-231 cells; following incubation, cells will be stained with Hoechst nuclear dye, and imaged by high-resolution confocal fluorescence microscopy at the UNC Imaging Core Facilities.

To evaluate potential DDC toxicity to monocytes, selected PRWAVSP-GEM/DOX- DDCs at ~ 10 8 DDCs/mL will be injected in the microfluidic device alongside monocytes at ~ 10 5 cells/mL (standard blood concentration [27]). The monocytes can be collected in the outlet, separated from free DDCs by centrifugation, and analyzed to determine whether interactions with DDCs in simulated blood-flow lead to DDC phagocytosis and death upon release of the drug payload. Internalization of DDCs by monocytes can be quantified, relying on the fluorescence of DOX. GEM/DOX-DDCs without peptide functionalization and free DOX/GEM at the same molar ratio of tested DDCs can be used as controls.

Finally, following exposure to PRWAVSP-GEM/DOX-DDCs, the cancer epithelial cells (MDA-MB-231) and healthy epithelial cells (MFC 10A) can be evaluated by imaging loss of GFP signal from both cell types. The ratio of residual viability (RRV) for each cell population can be measured. Two sets of experiments, where only MFC 10A or MDA-MB-231 cells can be GFP labeled, can be performed to independently evaluate both cell types.

Statistical Analysis. Statistical significance for cytotoxicity and cell viability can be assessed using a two-tailed, unpaired Student’s t-test. Annexin V/Svtox Green Apoptosis Assay. Cell apoptosis can be assessed via Annexin V and Sytox Green counterstaining, as done in prior work [1] Briefly, MDA-MB-231 cancer cells and MFC 10A healthy cells can be extracted from the device using Annexin V binding buffer, and analyzed using the Apoptosis Assay protocol (Life Technologies). Cells can be analyzed by flow cytometry (UNC Lineberger Flow Cytometry Core Facility) to quantify Annexin V-647 and Sytox Green fluorescence. Cells gated as -AV/-SG are live, cells with +AV/-SG are pre- apoptotic, and cells gated as +AV/+SG are either late apoptotic or necrotic.

These examps can be used to (1) finalize the design of optimal PRWAVSP-DDCs that (i) targets cancer cells in presence of healthy epithelial and circulatory immune cells under physiological flow conditions; (ii) enable high drug synergism; (iii) ensures high post-treatment viability of healthy cells. Should PRWAVSP-DDCs fail to target MDA-MB-231 cells, PRWAVSP can be replaced with other MDA-MB-231 -targeting peptides [8], such as CRGDSP [28]; other targeting moieties are (3-Aminomethylphenyl)boronic acid, bombesin peptide, AP-1 peptide [8] Achieving synergism with DOX/GEM (Cl < 0.1) is strongly supported by our prior work [2]; alternatively, we will try other drug pairs with proven synergistic against MDA-MB- 231, such as doxorubicin and gamitrinib [29], or methotrexate and genistein [30]

It can also be expected (2) develop a novel microfluidic system to evaluate targeted cancer therapeutics. If the staggered introduction of cancer cells and epithelial cells does not provide a suitable co-culture, a backup microfluidic design can be employed where cross-hatch (i.e. #) patterns can be used to facilitate perpendicular flow of cancer cells to healthy epithelial cells, and create cancer-cell patches where the perpendicular flow occurs. If GFP cannot be used to quantify cell death in the microfluidic devices bioluminescent reporters can be trialed in mammalian cells; alternatively, time-lapse microscopy with a heated stage to track individual cells in microfluidic channels can be used to visually determine when cell death occurs.

References Cited in Example 3

1. Camacho, K.M., et al. Synergistic antitumor activity of camptothecin doxorubicin combinations and their conjugates with hyaluronic acid. Journal of Controlled Release, 2015. 210: p. 198-207.

2. Vogus, D.R., et al. A hyaluronic acid conjugate engineered to synergistically and sequentially deliver gemcitabine and doxorubicin to treat triple negative breast cancer. Journal of Controlled Release, 2017. 267: p. 191-202. 3. Camacho, K.M., S. Menegatti, and S. Mitragotri, Low-molecular-weight polymer drug conjugates for synergistic anticancer activity of camptothecin and doxorubicin combinations. Nanomedicine, 2016. 11(9): p. 1139-1151.

4. Camacho, K.M., et al., DAFODIL: a novel liposome-encapsulated synergistic combination of doxorubicin and 5FU for low dose chemotherapy. Journal of Controlled Release, 2016. 229: p. 154-162.

5. Anselmo, A.C., et al., Platelet-like nanoparticles: mimicking shape, flexibility, and surface biology of platelets to target vascular injuries. ACS nano, 2014. 8(11): p. 11243-11253.

6. Kolhar, P., et al., Using shape effects to target antibody-coated nanoparticles to lung and brain endothelium. Proceedings of the National Academy of Sciences, 2013. 110(26): p. 10753- 10758.

7. Anselmo, A.C., et al., Elasticity of nanoparticles influences their blood circulation, phagocytosis, endocytosis, and targeting. ACS nano, 2015. 9(3): p. 3169-3177.

8. Nobrega, F.L., et al., Screening and characterization of novel specific peptides targeting MDA-MB-231 claudin-low breast carcinoma by computer-aided phage display methodologies. BMC cancer, 2016. 16(1): p. 881.

9. Alley, S.C., N.M. Okeley, and P.D. Senter, Antibody drug conjugates: targeted drug delivery for cancer. Current opinion in chemical biology, 2010. 14(4): p. 529-537.

10. Sievers, E.L. and P.D. Senter, Antibody-drug conjugates in cancer therapy. Annual review of medicine, 2013. 64.

11. Beck, A., et al., Strategies and challenges for the next generation of antibody drug conjugates. Nature reviews Drug discovery, 2017. 16(5): p. 315.

12. Mohamed, H.E., et al., Stability assessment of antibody-drug conjugate Trastuzumab emtansine in comparison to parent monoclonal antibody using orthogonal testing protocol. Journal of pharmaceutical and biomedical analysis, 2018. 150: p. 268-277.

13. Kozlowski, S. and P. Swann, Current and future issues in the manufacturing and development of monoclonal antibodies. Advanced drug delivery reviews, 2006. 58(5-6): p. 707- 722.

14. An, Z., Monoclonal antibodies a proven and rapidly expanding therapeutic modality for human diseases. Protein & cell, 2010. 1(4): p. 319-330.

15. Panowski, S., et al. Site-specific antibody drug conjugates for cancer therapy in MAbs. 2014. Taylor & Francis. 16. Noriega-Luna, B., et al., Applications of dendrimers in drug delivery agents, diagnosis, therapy, and detection. Journal of Nanomaterials, 2014. 2014: p. 39.

17. Madaan, K., et al., Dendrimers in drug delivery and targeting: Drug-dendrimer interactions and toxicity issues. Journal of pharmacy & bioallied sciences, 2014. 6(3): p. 139.

18. Svenson, S., The dendrimer paradox high medical expectations but poor clinical translation. Chemical Society Reviews, 2015. 44(12): p. 4131-4144.

19. Tawfik, M.A., M.I. Tadros, and M.I. Mohamed, Polyamidoamine (PAMAM) dendrimers as potential release modulators and oral bioavailability enhancers of Vardenafil hydrochloride. Pharmaceutical development and technology, 2018: p. 1-10.

20. Bharatwaj, B., et al., Dendrimer nanocarriers for transport modulation across models of the pulmonary epithelium. Molecular pharmaceutics, 2015. 12(3): p. 826-838.

21. Dobrovolskaia, M.A., et al., Nanoparticle size and surface charge determine effects of PAMAM dendrimers on human platelets in vitro. Molecular pharmaceutics, 2011. 9(3): p. 382- 393.

22. Jones, D.E., H. Ghandehari, and J.C. Facelli, Predicting cytotoxicity of PAMAM dendrimers using molecular descriptors. Beil stein journal of nanotechnology, 2015. 6: p. 1886.

23. Bhadra, D., S. Bhadra, and N. Jain, PEGylated peptide-based dendritic nanoparticulate systems for delivery of artemether. Journal of Drug Delivery Science and Technology, 2005. 15(1): p. 65-73.

24. El-Hammadi, M.M., et al., Folic acid-decorated and PEGylated PLGA nanoparticles for improving the antitumour activity of 5-fluorouracil. International journal of pharmaceutics, 2017. 516(1-2): p. 61-70.

25. Lee, J.W., et al., Convergent synthesis of symmetrical and unsymmetrical PAMAM dendrimers. Macromolecules, 2006. 39(6): p. 2418-2422.

26. Chou, T.-C., Drug combination studies and their synergy quantification using the Chou- Talalay method. Cancer research, 2010: p. 0008-5472. CAN-09-1947.

27. Heil, M., et al., Blood monocyte concentration is critical for enhancement of collateral artery growth. American Journal of Physiology -Heart and Circulatory Physiology, 2002. 283(6): p. H2411-H2419.

28. Shroff, K. and E. Kokkoli, PEGylated liposomal doxorubicin targeted to a5b1- expressingMDA-MB-231 breast cancer cells. Langmuir, 2012. 28(10): p. 4729-4736. 29. Park, H.-K., et al., Combination treatment with doxorubicin and gamitrinib synergistically augments anticancer activity through enhanced activation of Bim. BMC cancer, 2014. 14(1): p. 431.

30. Kaushik, S., et al., Genistein synergizes centchroman action in human breast cancer cells. Indian journal of pharmacology, 2016. 48(6): p. 637.

Example 4. Exploring the Physicochemical and Morphological Properties of Peptide- Hybridized Dendrimers (Dendripeps) and Their Aggregates

Introduction

Dendrimer aggregates are a widely studied class of supramolecular constructs. [1,2] Their morphology and physicochemical properties in solution resemble those of large biopolymers, such as proteins and DNA-protein complexes. This feature makes them an ideal model to study or manipulate the mechanisms of tissue morphogenesis and pathogenesis. [3] Traditionally, dendrimer aggregates are produced by either physical or chemical crosslinking. [4-9] The former is achieved by mixing multiple dendrimers or dendrimers and linear polymers that feature opposite charge and/or amphiphilic behavior. [7] Chemical crosslinking is frequently utilized to construct hydrogels/networks and relies on “click” or light-activated reactions. [10] Although widely utilized in a myriad of fundamental and applied studies, these methods present some limitations. Physical crosslinking, while allowing reconfiguration of the supramolecular structure in solution, requires the optimization of composition, ionic strength, and pH of the solvent. [5] Chemical crosslinking, on the other hand, is less dependent on the solvent, but results in structures whose morphology is less responsive to external stimulation.

In this example, the study of DendriPeps as units that form large supramolecular aggregates in an aqueous environment is presented. Unlike recent examples of peptide-modified dendrimers that exhibit branch terminating peptide segments for tailored biologic or catalytic activity, [11-20] DendriPeps feature a hyperbranched poly (amidoamine) (PAMAM) backbone hybridized with (di) peptide segments. [21] The PAMAM core and outer segments are constructed following the conventional sequence of reactions between methyl acrylate and diaminoethane, while the peptide inserts are constructed via Fmoc/tBu chemistry. Our group has previously described the construction of a set of four hydroxylterminated DendriPeps, namely two Generation 2 (G.2) DendriPeps comprising either 4 lysine (Lys) or 4 glutamic acid (Glu) residues, and two G.3 DendriPeps comprising either 8 Lys or 8 Glu residues. [21] DendriPeps can assemble and disassemble reversibly and rapidly in a broad range of solution conditions. The integration of amino acids displaying titratable groups within the dendritic scaffold endows DendriPeps with a functional behavior in solution that is drastically different compared with that of their conventional PAMAM counterparts. DLS and TEM showed, in fact, that DendriPeps comprising free primary amino (G.2-Lys4-OH and G.3-Lys8- OH) and carboxylic acid groups (G.2-Glu4-OH, and G.3-Glu8-OH) aggregate over a wide range of pH in aqueous environments; further testing by rheology demonstrated the ability of DendriPeps to assemble and disassemble reversibly upon application and removal of mechanical shear. Interestingly, aggregation of DendriPeps occurs despite their large z-potential values (positive for Lys-containing DendriPeps and negative for Glu-containing DendriPeps), indicating the contribution of noncovalent (e.g., van der Waals and hydrophobic) interactions other than ionic interactions. Most notably, titrimetry experiments revealed that the values of pKa of the tertiary amines on the DendriPep backbone are significantly altered by the charge of the “guest” amino acids. The values of pKa were employed in modeling the structure of the above-listed DendriPeps at different charged states via atomistic molecular dynamics (MD) simulations. The resulting structures illustrate the importance of intramolecular electrostatic interactions and hydrogen bonding between electron-rich/electron-poor groups. By extension, these models suggest that the same interactions, when occurring inter-molecularly, are implicated in the formation of DendriPep aggregates.

This example demonstrates the versatility of DendriPeps as a new class of self- assembling macromolecules, whose behavior in solution can lead to a myriad of fundamental studies and technologically relevant applications, including carriers for drug delivery with improved balance of residence time and tissue permeation, soft materials for cells and tissue engineering, and mimetics of protein aggregates.

Experimental

The Dendripeps as used in the present example were synthesized as described in Example 2.

DLS and z-potential of aqueous DendriPep solutions

An ensemble of DendriPep solutions was prepared by dissolving lyophilized DendriPeps at either 0.5 and 1 mg/ml in Milli-Q water, 1 mg/ml in dry methanol, 1 mg/ml in 100 mM citrate buffer at pH 5; 1 mg/ml in 100 mM phosphate buffer at pH 7; or 1 mg/ml in 100 mM Tris buffer at pH 9. All solutions were vortexed for 30 s, sonicated in a water bath for 30 s using a Branson 3800 ultrasonic cleaner, and filtered using a 0.45 mm nylon syringe filter (VWR, PA). The solutions were analyzed at 25 °C in a low volume quartz cuvette (path length of 10 mm) using a Zetasizer Nano ZS instrument (Malvern, MA) equipped with a 4-mW helium-neon laser operating at a 633 nm; the (back) scattered light was detected at an angle of 173°. Three measurements per sample were performed. The hydrodynamic radius (R h ) was obtained as numberweighted average. Every sample was then transferred into a pre-rinsed zeta cell (ZEN1002, Malvern) and measured at 25 °C with automatic voltage selection and three measurements per sample.

DendriPep imaging via TEM

Aliquots of 10 pi of solutions of G.3-Lys8-OH, G.3-Glu8-OH, G.3-Glu(OtBu)8-OH, and G.3 PAMAM-OH (0.2 mg/ml) in 9:1 water:methanol were drop-casted on a Ted Pella-Carbon Film only on 300-mesh Formvar-free copper grids. The grids were then inverted on a drop of an aqueous sodium phosphotungstate (NaPTA) solution for staining. NaPTA solution is a 2% wt solution of phosphotungstic acid with its pH modified to pH 7 using sodium hydroxide. TEM images were obtained at 200 kV using a FEI Talos F200x at different magnifications. Images were acquired and analyzed using the Velox software.

Isothermal shear rate sweep analysis of DendriPep solutions

All measurements were performed at ambient temperature using a TA Instruments Discovery Series Hybrid Rheometer HR-3, outfitted with a cone-and-plate geometry (20 mm diameter, 1° cone angle, 26 mm gap). Solutions of G.3-Lys8-OH, G.3-Glu8-OH, G.3 PAMAM- OH, and G.l xAMAMNH2 at 100 mg/ml in Milli-Q water were analyzed by shear rate (g) in the range of 0.1 to 1,000 s -1 . Steady state data were collected at a 15 s sampling period within ±5% SE up to 20 min equilibration time.

Titrimetry of DendriPeps and PAMAM

Dendrimer samples (G.3-Lys8-OH, G.2-Lys4-OH, G.3-Glu8-OH, G.2-Glu4-OH, and G.3-PAMAM-OH) were dissolved in Milli-Q water at a concentration of 0.02 M. pH was measured using a Mettler Toledo SevenCompact pH meter. The G.3-Lys8-OH solution was titrated with 50 equivalents (eq.) of NaOH; the G.2-Lys4-OH solution was titrated with 28 eq. of NaOH; the G.3-Glu8-OH solution was titrated with 52 eq. of NaOH; the G.2-Glu4-OH solution was titrated with 16 eq. of NaOH; the G.3-PAMAM-OH solution was titrated with 36 eq. of HC1. Two sets of curves were obtained for each experiment, namely (a) pH versus the number of equivalents (N) of added acid/base and (b) the derivative d pH / d N versus N. The values of pKa were identified on every curve (a) based on the values of N producing relative maxima on the corresponding curve (b).

Atomistic molecular dynamic modeling and structural evaluation of PAMAMs and DendriPeps The structures of G.3 PAMAM-OH, G.3-Lys8-OH, G3-Glu8-OH, G3-Lys(Boc)8-OH, and G.3-Glu(OtBu)8-OH were initially designed using the molecular editor Avogadro. The editor allows building the protonated structures of the dendrimers by assigning a formal charge of +1 to the tertiary amine groups of the PAMAM backbone or the primary e-amine group of the Lys residues or a charge of -1 to the g-carboxyl group of the Glu residues. Counterions Cl- and Na+ were used, and GAFF was the force field used to describe their interaction. The dendrimers were initially equilibrated by MD in the GROMACS simulation package[22-24] using the OPLS all-atom force field[25,26] and periodic boundary conditions. [27-29] The production run was performed in the NPT ensemble, at constant T = 300 K using the Nose-Hoover thermostat[30- 32] and constant p = 1 atm using the Parrinello-Rahman barostat. [33,34] The coordinates of atoms were saved every 2 ps. The leap-frog algorithm was used to integrate the equations of motion, with integration steps of 2 fs, and all of the covalent bonds were constrained by means of the Linear Constraint Solver algorithm. [35-37] The short-range electrostatic and Lennard- Jones interactions were calculated within a cutoff of 1.0 nm and 1.4 nm, respectively, whereas the particle-mesh Ewald method was utilized to treat the long-range electrostatic interactions. [36,37] The nonbonded interaction pair-list was updated every 5 fs, using a cutoff of 1.4 nm. The variation of the radius of gyration (Rg) along the simulations during the final part of the trajectories showed stable oscillations around their mean values. Based on the spatial coordinates of the atoms, (a) the Rg of the dendrimers was calculated as described by Kanchi et ah, [38] (b) the atomic distance between a titratable nitrogen atom (i.e., a tertiary amine in the PAMAM backbone or an e-amine of a lysine residue) and the oxygen atom of the closest hydroxyl group, (c) the atomic distance between the carbon atom in the g-carboxyl group of a glutamic acid residue and the nitrogen atom of the closest tertiary amine, (d) the atomic distance between the center of mass of a Boc-protected e-amine of lysine and the oxygen atom of the closest hydroxyl group, and (e) the atomic distance between the center of mass of a OtBu protected g-carboxyl group of a glutamic acid residue and the nitrogen atom of the closest tertiary amine in the PAMAM backbone were calculated.

Results and Discussion

Evaluation ofDendriPep self-assembly in aqueous environment by DLS In the synthesis of the disclosed Dendripeps, four hydroxyl-terminated DendriPeps were constructed: two Generation 2 DendriPeps comprising either four lysines or four glutamic acid residues (G.2-Lys4-OH and G.2-Glu4-OH), and two Generation 3 DendriPeps comprising either eight lysines or eight glutamic acid residues (G.3-Lys8-OH and G.3-Glu8-OH).

Like PAMAMs, hydroxyl-terminated DendriPeps contain tertiary amines as titratable groups. The pKa values of internal tertiary amines in various types of dendrimers are known to be influenced by their chemical composition and architecture. [39,40] In one example, Tomalia et al. showed that tertiary amines in primary amine-terminated PAMAM featured a pKa of -3.86, much lower than the values typical of triethylamine (10.75) and other small tertiary amines. [41,42] In another example, Diallo et al. demonstrated that the pKa of tertiary amines of PAMAMs varies significantly with the size and type of terminal groups. [39]

DendriPeps feature a unique additional parameter of physicochemical tuning in the titratable moieties introduced by the (di)peptide segments. In the model DendriPeps, lysine displays an e-amino group and glutamic acid displays a g-carboxyl group, whose native pKa values are 10.3 and 4.5, respectively. In the absence of electronic interplay between tertiary amines and amino acid residues, only a small fraction of tertiary amines is expected to be positively charged (protonated) at pH7. [39,43] Accordingly, DendriPeps hybridized with lysines or glutamic acids were expected to carry a net positive or negative charge at neutral pH, respectively, thereby maintaining a monomeric state in solution due to mutual ionic repulsion.

The characterization of aqueous solutions of DendriPeps by DLS, however, contradicted these expectations. As shown in Table 1, DendriPeps dissolved in Milli-Q water exhibited R h values varying between -50 and 110 nm. In contrast, G.l PAMAM-NH2 and G.3 PAMAM-OH dendrimers, whose molecular weight is close to that of G.2 and G.3 DendriPeps, feature values of R h of 1.8 and 2.7 nm (Table 1), respectively, consistent with the literature on PAMAM dendrimers. [44] Such difference indicates that DendriPeps form supramolecular aggregates. It was also noted that the size of the aggregates does not feature a defined dependence on the type (lysine vs. glutamic acid), number, and position of the guest amino acids within the dendritic scaffold. The larger difference is observed between G.3-Lys8-OH and G.3-Glu8-OH DendriPeps at low concentration. The size of the aggregates was further confirmed by imaging self- assembled G.3-Lys8-OH and G.3-Glu8-OH DendriPeps by TEM. It is interesting to note that although the exact morphology of the aggregates was generally challenging to discern, G.3- Glu8-OH displayed excellent resolution to the degree that dendritic “clusters” were observed to comprise the globular aggregates. Table 1. Values of hydrodynamic radius (R h ) on G.2-Lys 4 -OH, G.2-Glu 4 -OH, G.3-Lys 8 -OH, G.3-Glu 3 -OH, G.l PAMAM-NH2, and G.3 PAMAM-OH dendrimers measured by DLS alanysis of DendriPep solutions at 0.5 and 1 mg/ml in Milli-Q water using a Malvern ZetaSizer Nano

The combined effect of amino acid make-up and solution pH on the aggregation of DendriPeps was subsequently investigated. Because DendriPeps are rich in titratable groups, buffered aqueous solutions were employed to control the pH of the environment. Specifically, 100 mM citrate buffer at pH 5, 100 mM phosphate buffer at pH 7, and 100 mM Tris-HCl buffer at pH 9 were utilized. The values of R h of the four DendriPeps versus solution pH are reported in FIG. 9. The size of the aggregates formed by G.2-Lys4-OH and G.3-Lys8-OH DendriPeps consistently increased with pH. In an acidic environment, where a fraction of both tertiary and primary amines are protonated, aggregation is inhibited — although not prevented — by the strong net positive charge of individual DendriPeps; as the pH of solution increased to pH 7 and 9, the fraction of protonated amines decreases, thereby reducing the ionic (i.e., Coulombic) repulsion and enabling the formation of larger aggregates. The opposite trend is observed with G.2-Glu4- OH and G.3-Glu8-OH DendriPeps. As the solution pH increases, in fact, the g-carboxyl groups become progressively more deprotonated, thus increasing the net charge of DendriPeps and effectively inhibiting their aggregation by ionic repulsion. As observed before, both G.l and G.3 PAMAM dendrimers maintained a monomeric form under all solution conditions. Further analysis and discussion of the pH- and charge-dependent mechanisms of aggregation is presented below.

Based on these data, it was hypothesized that the propensity of DendriPeps to aggregate may stem from the ability of the solvent to act as a proton donor/acceptor to/from DendriPeps. To evaluate this aspect, additional DLS measurements were conducted on a solution of the four DendriPeps and PAMAMs in methanol at 1 mg/ml; with a pKa of 15.5, in fact, methanol has a lower ability to deprotonate compared to water. [45] No aggregation was observed in methanolic conditions with either DendriPeps or PAMAM.[46]

A second control experiment was performed by conducting DLS measurements on protected DendriPeps G.2-Lys(Boc)4-OH, G.3-Lys(Boc)8-OH, G.2-Glu(OtBu)4-OH, and G.3- Glu(OtBu)8-OH dissolved in the aforementioned aqueous buffers. Protected DendriPeps resemble the intramolecular charge distribution of PAMAMs, in that their only titratable groups are the nodal tertiary amines; at the same time; however, the [(tertbutoxycarbonyl)amino]butyl side chain group of Lys(Boc) and the 3-(tertbutoxy)-3-oxopropyl of Glu(OtBu) impart some hydrophobic character to protected DendriPeps compared to their cognate PAMAMs. The values of Rh reported in Table 2 show that protected DendriPeps have a much less propensity to aggregate, especially at pH 5 and 7. In these environments, a fraction of tertiary amines are expected to be positively charged and prevent DendriPep aggregation by ionic repulsion. At pH 9, instead, the lack of protonation of tertiary amines (i.e., lower ionic repulsion) combined with the added hydrophobicity of Lys(Boc) and Glu(OtBu) promote aggregation. When dissolved in an anhydrous organic solvent (methanol), however, all protected DendriPeps were once again found to be monomeric (Table 2).

Table 2. Value of R h of G2-Lys(Boc) 4 -OH, G.2-Glu(tBu) 4 -OH, G.3-Lys(Boc) 4 -OH, G.3- Glu(tBu) 8 -OH, G.3 PAMAM-OH, G.l PAMAM-NH2, and corresponding SEs measured by DLS analysis of solutions at 1 mg/ml in aqueous buffers of 100 mM citrate buffer at pH 5, 100 mM phosphate buffer at pH 7, and 100 mM Tris-HCl buffer at pH 9 and in methanol using a Malvern ZetaSizer Nano

Evaluation of the charge of DendriPep aggregates and their titratable groups by titrimetry and z-potential measurements The surface charge (z-potential) of DendriPep aggregates was subsequently examined using a Zetasizer Nano ZS instrument (Table 3). Consistent with the literature, the hydroxyl- terminated G.3 PAMAM dendrimer displayed a relatively neutral z-potential (-0.4 mV), while the G.1 PAMAM-NH2 dendrimer displayed a positive z-potential (26.3 mV) in Milli-Q water. [44] The aggregates formed by G.2-Lys4-OH and G.3-Lys8-OH feature positive z- potential (46.9 and 54.0 mV), consistent with the presence of guest primary e-amine residues that are positively charged in Milli-Q water (pH ~ 6) (vide infra). Likewise, the aggregates formed by G.2-Glu4-OH and G.3-Glu8-OH have negative z-potential (-136.7 and -183.0 mV), imparted by the guest g-carboxylic acid residues, which are negatively charged in Milli-Q water (pH ~ 6) (vide infra).

Table 3. z-potential of PAMAMs and DendriPeps at 1 mg/ml in Milli-Q water

Titration curves of G.3 PAMAM-OH, G.2-Lys4-OH, G.3-Lys8-OH, G.2-Glu4-OH, and G.3-Glu8-OH were constructed to determine the pKa of the titratable groups in DendriPeps. Most notably, the pKa values of the tertiary amines varied depending on the guest amino acid. Acid-like behavior was observed for the basic amino acid (Lys), and basic-like behavior was observed for the acidic amino acid (Glu) (Table 4). The tunable electrophilicity of tertiary amines, while known in PAMAM literature, [48] becomes particularly remarkable in DendriPeps, where it spans an interval of 7.9 units of pH. Table 4. Values of pK a of the titratable groups in nonprotected DendriPeps G.3-Lys 8 -OH, G.2- Lys 4 -OH, G.3-Glu 8 -OH, G.2-Glu 4 -OH, and G.3-PAMAM-OH measured by titrimeric tests of 1 mM dendrimer solutions in Milli-Q water at 25 °C reported in the literature pK a values of the ionizable groups of proteins Study of DendriPep self-assembly versus shear During the experimental work presented above, a key timing element was noted for the aggregation of the DendriPep compounds. With DLS analysis immediately following the filtration of the DendriPeps, monomeric Rh values were observed. With a certain amount of time following filtration, however, the DendriPeps were observed to reform into aggregates. The use of syringe filters with large pore diameter (0.45 mm) suggests that filtration did not simply remove the DendriPep aggregates by size exclusion, but rather caused their disassembly by mechanical shear. The values of Rh of the DendriPeps sheared immediately prior to DLS measurements (Table 5), in fact, are on par with those of their cognate PAMAMs. It was further noted that, if left at rest following shear, the DendriPep aggregates would reform until recovering the equilibrium size listed in Table 1. As an example, the size of G3-Lys4-OH DendriPep was monitored at regular time intervals (240 s), starting immediately after shearing, indicating that the DendriPep aggregates were reformed within approximately 16 min.

Table 5. Values of R h of G.2-Lys 4 -OH, G.2-Glu 4 -OH, G.3-Lys 8 -OH, G.3-Glu 8 -OH, and corresponding SEs measured by DLS analysis in pH 5, 100 mM citrate buffer DendriPep solutions at 1 mg/ml using a Malvern ZetaSizer Nano immediately after shearing by filtration with a 0.45 mm syringe filter To investigate systematically the correlation between DendriPep self-assembly and mechanical stimuli, a rheological characterization of solutions of DendriPep aggregates in Milli- Q water was conducted. Shear rate sweep tests were performed by gradually decreasing the shear rate from 1,000 to 0.1 s _1 . The profiles of viscosity (h) versus shear rate obtained with aqueous solutions of G.3-Lys8-OH and G.3-Glu8-OH at 100 mg/ml are reported in FIG. 10. The observed transition region can be ascribed to the deformation of the DendriPep aggregates followed by their disassembling into smaller entities, and ultimately monomeric DendriPeps, as the shear rate reaches 100-250 s _1 . The controls G.3 PAMAM-OH and G.1 PAMAM-NH2 were evaluated under identical shear rates (1000-0.1 s _1 ), concentration (100 mg/ml), and solvent. The shear-thinning behavior exhibited by the DendriPeps was anticipated, since the aggregation of DendriPeps was observed by DLS at significantly lower concentrations in Milli-Q water (1 mg/ml).

Like DendriPeps, both G.3 PAMAM-OH and G.1 PAMAM-NH2 displayed shear- thinning behavior. The relatively scant literature on rheological characterization of concentrated PAMAM solutions indicates Newtonian behavior; these tests, however, were conducted in organic solvents. [49] Small-amplitude oscillatory shear tests on bulk PAMAM dendrimers, on the other hand, have shown a shear-thinning behavior at high shear rates[50] indicating the presence of intermolecular interactions. Uppuluri et al. theorized that lower generation PAMAM dendrimers (having a less dense and more “open” structure compared to the more congested structure of higher generations) are more susceptible to intermolecular interactions through interpenetration of neighboring dendrimer branches. [39] Interestingly, these intermolecular interactions were thought to be enhanced by hydrogen bonding between the primary amines of one dendrimer and amide carbonyl oxygens of a second dendrimer. [49]

The DLS measurements of aqueous PAMAM solutions have revealed that, while monomeric in a broad range of concentrations, PAMAMs form aggregates at 100 mg/ml, which corresponds to the concentration utilized in rheological work presented herein. The use of concentrated dendrimer solutions in this work was imposed by the limit of sensitivity of the rheometer. The shear rate of sweep tests could not generate reliable data of h at intermediate values of shear rate for either DendriPeps or PAMAMs, despite equilibration times of 20 min/data point; higher equilibration times were not possible due to water evaporation, which occurred despite the use of a chamber at controlled humidity. This phenomenon was attributed to the small size of the plate (holding a volume of ~ 40 pi) utilized in these tests, which was dictated by the limited availability of the DendriPep samples. Ramzi et al. corroborated the finding that PAMAM dendrimers form aggregates at high concentrations in Milli-Q water, as they discovered that polypropylene amine) (PPI) dendrimers (similar to PAMAM dendrimers and DendriPeps in that PPI dendrimers contain both tertiary and primary amines) exhibit interm olecular interactions at 10 wt. % in water, as evidenced by the results of small-angle neutron scattering experiments. [51]

Nonetheless, rheology proved a useful method to probe the unique aggregation characteristics of DendriPeps. These tests corroborated observations that DendriPep aggregates can be disassembled into smaller constituents — and ultimately monomers — as shear is applied and reform as shear is reduced. Most notably, this behavior is observed in DendriPeps independently of concentration, whereas PAMAMs only form aggregates (and thus exhibit shear-thinning behavior) at high concentrations in Milli-Q water.

In silico evaluation of the structure of charged variants of cationic and anionic DendriPeps

Atomistic MD simulations of G.3-Lys8-OH and G.3-Glu8-OH DendriPeps were performed to investigate the behavior of DendriPeps at the molecular level in comparison with their G.3 PAMAM-OH counterpart. Based on the values of pKa obtained from potentiometric titrations, the three dendrimers were modeled in their different charged states (i.e., 2 for PAMAM, 3 for G.3-Lys8-OH, and 3 for G.3-Glu8-OH) and protected versions, and specifically:

1. G.3 PAMAM-OH with (1.i) tertiary amines of the PAMAM backbone protonated, representing the condition in an aqueous environment at pH < 4.5, and (l.ii) tertiary amines deprotonated, representing the condition in an aqueous environment at pH > 6.5;

2. G.3-Lys8-OH with (2.i) both the tertiary amines of the PAMAM backbone and the e-amine groups of the lysine residues protonated, representing the condition in an aqueous environment at pH < 3.6; (2.ii) tertiary amines deprotonated and e-amines protonated, representing the condition in an aqueous environment at 3.6 < pH < 8.4; and (2.iii) all amines deprotonated, representing the condition in aqueous environment at pH > 8.4;

3. G.3-Glu8-OH with (3.i) tertiary amines of the PAMAM backbone protonated, and the g-carboxyl group of glutamic acid residues protonated, representing the condition in an environment at pH < 5.1; (3.ii) tertiary amines protonated and g-carboxyl groups deprotonated, representing the condition in an aqueous environment at 5.1 < pH < 6.1; and (3.iii) all tertiary amines and g-carboxyl groups deprotonated, representing the condition in an aqueous environment at pH > 10.6;

4. G.3-Lys(Boc)8-OH and G.3-Glu(OtBu)8-OH with the tertiary amines of the PAMAM backbone either (4.i and 4.ii) protonated or (4.iii and 4.iv) deprotonated. The above-listed charged variants were individually modeled by atomistic MD simulations to obtain representative DendriPep conformations. These were in turn utilized to calculate a host of geometrical parameters that portray the effect of protonation/deprotonation of the amino acid residues and/or the effect of protecting groups on the intramolecular interactions and thus the internal structure. Given that these molecules have a propensity to aggregate, the results of these simulations were scrutinized to determine whether the attractive interactions that form intramolecularly can also form intermolecularly, potentially favoring aggregation between the DendriPep molecules.

Lee et al. previously reported that the R g of PAMAM dendrimers decreased with increasing pH using MD simulations. [52] This decrease in R g was attributed to the fraction of protonated tertiary amines decreasing with increasing pH, which translates in lower repulsive electrostatic interactions and ultimately in an expansion of the dendrimer's structure. [52] To evaluate whether this phenomenon exists in DendriPeps, R g values obtained for the molecules in their various protonation states (l)-(4) are collated in Table 6. As anticipated, the protonation of the amines — initially of e-amines only (corresponding to 3.6 < pH < 8.4) and then all amines (corresponding to pH < 3.6) — increases the value of R g of G.3-Lys8-OH from 1.57 to 1.87 nm as a result of the ionic repulsion between cationic ammonium groups; analogously, the R g of neutral to charged G.3-Lys(Boc)8-OH increases for the same reason. A similar behavior is observed in G.3 PAMAM-OH, whose Rg increases from 1.67 to 1.84 nm as the tertiary amines become protonated. The R g of G.3-Glu8-OH, on the other hand, fluctuates between the two maxima and a minimum as the charge states of the ionizable groups vary. The charged variants corresponding to acidic (corresponding to pH < 5.1) and alkaline (corresponding to pH > 10.6) environments possess only one family of charged groups, for example, tertiary ammonium (R g of 1.67 nm) and g-carboxylate groups (Rg of 1.62 nm), which expand the DendriPep by ionic repulsion. At intermediate pH (corresponding to 5.1-6.1), where tertiary ammonium and g-carboxylate groups are oppositely charged, the DendriPep contracts as a result of the network of intramolecular electrostatic (including ionic) attractions, reaching a minimum R g of 1.42 nm. Another intermediate condition is achieved by G.3-Glu(OtBu)8-OH in an alkaline environment, which represent the only noncharged variant of glutamic acid containing DendriPeps and whose R g is 1.62 nm. These results are in agreement with the values of R h for monomeric DendriPeps and PAMAMs obtained from DLS.

Table 6. Values of R g of the charged variants of dendrimers G.3-Ly s8 -OH, G.3-Glu 8 -OH, G.3- Lys(Boc) 8 -OH, G.3-Glu(tBu) 8 -OH, and G.3 PAMAM-OH

Note: Charged status of the titratable groups: tN, tertiary amine not protonated; tN + , tertiary amine protonated; e-Lys, e-amino group of lysine residues not protonated; e-Lys + , e-amino group of lysine residues protonated; g-Glu, g-carboxyl group of glutamic acid residues protonated; g- Glu-, g-carboxyl group of glutamic acid residues deprotonated.

The second structural parameter investigated was the distance between titratable nitrogen atoms (either of a e-amine of a lysine residue ( e-Lys and e-Lys+, Table 7) or a tertiary amine in the PAMAM backbone (tN and tN+) and the oxygen atom of the closest hydroxyl group. The results indicate that these groups form an intramolecular network of electrostatic interactions (i.e., van der Waals forces and hydrogen bonds). For example, in the charged variant G.3-Lys8- OH (e-Lys+, tN), four e-Lys+/OH close pairings are formed, characterized by an average atomic distance of 0.29 nm (FIG. 11 A); the other four e-Lys+ groups, on the other hand, are unpaired and protrude outward, maintaining an average distance of 1.23 nm from the closest hydroxyl groups. These e-Lys+ could thus form intermolecular interactions with the OH termini of other DendriPep molecules. On the other hand, when the e-Lys residues are deprotonated, in G.3- Lys8-OH at pH > 8.4, their average distance with the closest hydroxyl groups increased by ~0.5 nm. Similarly, tN+ groups pair with terminal hydroxyl groups, as observed in the charged variant of G.3-Lys8-OH at pH < 3.6, maintaining average distances of 0.72 nm. In so doing, they compete with e-Lys residues, whose average distance to the closest hydroxyl groups is consistently higher when tertiary amines are protonated. On the other hand, when all amine groups are deprotonated (pH > 8.4), the intramolecular nitrogen/oxygen pairing is not observed, with both e-Lys+/OH and tN/OH distances averaging at -0.8 nm. The magnitude of these distances indicates that intramolecular hydrogen bonding is not the sole or dominant interaction governing the conformation of these molecules. Other phenomena, such as steric hindrance and hydrophobic interactions, likely contribute in addition to hydrogen bonding in determining structure. Conversely, the amine groups, especially when charged, do have a strong hydrogen bonding interaction with the terminal hydroxyl groups. Thus, there is a propensity for them to “seek” to pair, which cannot be completely satisfied intramolecularly.

Table 7. Values of atomic distance between (a) a free e-amine group of a lysine residue ( e-Lys or e-Lys ) or (b) a Boc-protected lysine residue (Lys(Boc)) and the oxygen atom of the closest terminal hydroxyl group in G.3-Lys 8 -OH or G.3-Lys(Boc) 8 -OH Dendripeps in their different charged variants

The third structural parameter examined was the atomic distance between the carbon atom of a g-carboxyl group of glutamic acid residues, either free (i.e., g-Glu or g-Glu-) or protected, and the closest nitrogen atom of a tertiary amine in the PAMAM backbone of G.3- Glu8-OH or G.3-Glu(OtBu)8-OH DendriPeps in their different charged variants (Table 8). When the g-carboxyl groups of G.3-Glu8-OH are negatively charged (pH > 5.1), they form an intramolecular network of close electrostatic pairings with eight tN+ groups; in this case, owing to the contribution of the ionic component to the electrostatic interactions, the contacts are particularly close, featuring an average distance of 0.34 nm and resulting in a contraction of the DendriPep structure (FIG. 1 IB). In all other cases, when either the carboxylic acid groups or amine groups are not charged, the carbon-nitrogen distance increases to 0.65-0.75 nm. Table 8. Values of atomic distance between g-carboxyl groups of glutamic acid residues, either free or protected, and the closest nitrogen atom of a tertiary amine in the PAMAM backbone in G.3.-Glu 8 -OH or G.3.-Glu(OtBu) 8 -OH DendriPeps in their different charged variants The formation of tN+/OH pairs, described earlier for G.3-Lys8-OH, is also found in the charged variants of G.3-Glu8-OH and G.3-Glu(OtBu)8-OH DendriPeps (corresponding to pH < 6.1), where several close contacts averaging a distance of 0.3-0.5 nm were formed. Once again, terminal hydroxyl groups competed with carboxyl groups, whether free or protected, in forming hydrogen bonds with tN+ groups: as the average carbon-nitrogen distance increases in the order g-Glu-/tN+ (0.35 nm), g-Glu/tN+ (0.76 nm), and g-Glu(OtBu)/tN+ (0.79 nm) (Table 8), and the oxygen-nitrogen distance in the tN+/OH contacts in the corresponding DendriPeps decreases from 0.83 nm in g-Glu- to 0.73 nm in g-Glu and to 0.71 nm in g-Glu(OtBu) (Table S4). With the exception of g-Glu-/tN+ average distance, the remaining average distances are too large for all of the possible hydrogen bond donor/acceptor interactions to be satisfied intramolecularly. Thus, as earlier, this behavior potentially facilitates intermolecular interactions.

The key to understanding DendriPep aggregation compared with monomeric behavior of PAMAMs is the observation that the terminal hydroxyl groups of DendriPeps show a much stronger propensity to form close intramolecular pairing with internal tN+ groups compared with the OH termini of their PAMAM cognates. The comparison of the tN+/OH distances indicates that terminal hydroxyl groups in DendriPeps can form close contacts with G.1 tN+ groups and even the core G.O tN+ groups, as in the case of G.3-Lys(Boc)8-OH. In contrast, the terminal hydroxyl groups in G.3 PAMAM-OH can form close contacts only with the closest tN+ groups (those found in the third generation of the structure). This difference arguably results from the steric density of G.3 PAMAM-OH, whose Rg is comparable to that of G.3 DendriPeps (Table 6), but whose molecular weight is considerably higher (i.e., 6,940.5 Da against 4,866.1 of G.3-Lys8- OH and 4,870.7 Da of G.3-Glu8-OH due to the termination of an “arm” upon amino-acid incorporation). The effect of steric density in DendriPeps is exemplified by G.3-Glu(OtBu)8- OH, wherein the steric hindrance of eight protected glutamic acid residues likely renders the terminal hydroxyl groups unable to form close pairings with tN+ groups, resulting in higher N-0 distances. The observation that terminal hydroxyl groups in G.3 PAMAM-OH resist significant backfolding to facilitate hydrogen bonding suggest that intermolecular penetration of these groups is likely disfavored.

The structural analysis presented herein suggests a reasonable hypothesis to describe the propensity of DendriPeps to form aggregates despite their net charge. In the analysis of the charged variants of G.3-Lys8-OH, only four of eight e-Lys+ residues are paired with terminal hydroxyl groups, whereas the others protrude outward to maximize the distance, thus minimizing the ionic repulsion from the other protonated amines. At the same time, six to seven terminal hydroxyl groups are completely unpaired, and five to six are paired with each other via hydrogen bonding (FIG. 11 A). Although intramolecularly unpaired, it is suggested that these e- Lys+ residues and terminal hydroxyl groups form intermolecular contacts via nonionic electrostatic interactions as outlined earlier. The formation of a network of intermolecular contacts would manage to (a) establish attractive interactions between DendriPeps and (b) soften their ionic repulsion. It is hypothesized that, as incoming DendriPeps join the growing aggregate network, all the DendriPeps already therein rearrange their relative orientation in order for the attractive electrostatic (i.e., van der Waals and hydrogen bonding) and hydrophobic interactions to offset the ionic repulsion. This results in low growth rates and produces aggregates with the internal structure of a weak 3D lattice with a high net positive charge (Table 3). This is coherent with the observations presented herein — that DendriPep aggregates can be disrupted by applying mild shearing and reform slowly only in the absence of mechanical stimuli. A similar mechanism is expected for G.3-Glu8-OH DendriPeps, where g-Glu-/tN+ and tN+/ OH pairings can occur both intramolecularly and intermolecularly. These pairings offset the ionic repulsion between glutamic acid residues, resulting in a 3D lattice formed by partially compensated DendriPeps and bearing a strong net negative charge (Table 3).

Although not captured by MD simulations of a single DendriPep, a broad spectrum of noncovalent interactions arguably play a role in driving DendriPep aggregation. Although all nonprotected DendriPeps aggregate at all values of pH (Table 1), their protected counterparts aggregate less readily, except at a higher pH (Table 2). Since both the [(tertbutoxycarbonyl)amino]butyl and 3-(tertbutoxy)-3-oxopropyl side chain groups of Lys(Boc) and Glu(OtBu) can form hydrogen bonds, the lower propensity of protected DendriPeps to aggregate can be imputed to the absence of ion-dipole interactions caused by the chemical protection of the e-amine and g-carboxyl groups.

Overall, these results support the unanticipated experimental behavior that DendriPeps exhibit, most notably the propensity to aggregate, resulting from the integration of amino acids in the PAMAM scaffold. The introduction of anionic or cationic groups on flexible side chains imparts unique properties to the physicochemical properties of other titratable groups (e.g., pKa of tertiary amines) and a remarkable decrease in molecular density of the hyperbranched structure. This modification results in the formation of intramolecular interactions that stabilize the structure of the single and, potentially, of an intermolecular network that enables the formation of large, soft, and charged aggregates.

Conclusion

In summary, what can be viewed as a small perturbation (amino acid backbone insertion) to the PAMAM structure produces striking changes to the physicochemical properties of DendriPeps. DendriPeps form relatively monodisperse aggregates in aqueous conditions over a wide range of pH values despite a net negative or positive charge. The tertiary amines along the DendriPep backbone have pKa values that are affected significantly by the type of amino acid inserted. Rheology experiments demonstrated that DendriPep aggregates can be reversibly assembled and -disassembled into their monomeric constituents as a response to mechanical shear, independent of concentration. Finally, molecular dynamic simulations illustrated possible structural features leading to aggregation, and in particular, suggested that intramolecular “pairing” occurs between a fraction of hydroxyl termini/primary amines or carboxylic acids/tertiary amines, respectively, through hydrogen bonding. It was hypothesized that the intramolecularly “unpaired” fraction of these groups would be available for intermolecular “pairing” leading to aggregation between DendriPeps. This balance between intermolecular and intramolecular interactions is affected by the charge state of the hydrogen bond donating and accepting groups as evidenced by the pH dependence of the phenomenon. These results of DendriPep characterization may lead to exciting implications for future applications.

DendriPeps challenge the fundamental understanding that charged nanoparticles strictly resist aggregation and are demonstrated as excellent subjects to study this behavior due to the unparalleled control over the type, position, and number of internal functionalities that the DendriPeps possess. It is anticipated that this new understanding of aggregate formation will significantly contribute to the breadth of knowledge regarding mechanisms of nanoparticle aggregation and inform future nanoparticle design.

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Example 5. Synthesis of Dendripep-Microgel Systems

Microgel Synthesis via Precipitation Polymerization of N-Isopropylacrylamide (NIP AM) and Acrylic Acid (AA) in Water

NIP AM (1.27 g), AA (0.811 g), and BIS (684. Mg) were initially dissolved in 200 mL MilliQ water. The solution was placed in a three-neck flask fit with a temperature probe, condenser, and nitrogen line, and purged with N2 for one hour at 70 °C. Following degassine, APS (102.7 mg) was added to initiate polymerization. The reaction was allowed to proceed at 70 °C for 5 hours, after which the solution was cooled overnight. The resulting suspension of PNIPAm-co-AA microgels were filtered through glass wool to remove any aggregates and dialyzed against MilliQ. The microgels were finally lyophylized and stored at room temperature.

The dendrimer is conjugated to fluorescein by reacting the dendrimer with fluorescein- NHS in methanol at room temperature for 24 hours.

The dendrimer-fluorescein conjugate is adhered to the microgel by mixing the microgel and dedrimer-fluorescein conjugate in MilliQ water for 24 hours at a wt/wt ratio of either 1/0.01, 1/0.1, 1/1, 1/10, 1/100, 1/1000, 1/10000, 1/100000, 1/1000000 or any ratio that is between these ratios using slow stirring. Shear force is applied to the dendrimer/microgel system by fast stirring, vortexing, rapid passage through a cannula, rapid passage through a syringe filter, sonication, or exposure to ultrasonic sound waves. Publications cited herein are hereby specifically incorporated by reference in their entireties and at least for the material for which they are cited.

While it should be understood that while the present disclosure has been provided in detail with respect to certain illustrative and specific aspects thereof, it should be considered limited to such, as numerous modifications are possible without departing from the broad spirit and scope of the present disclosure as defined in the appended claims. It is, therefore, intended that the appended claims cover all such equivalent variations as fall within the true spirit and scope of the invention.