K L DE ERQ S _.

The present invention relates to nanomaterials, in particular nanoniaterials having hybrid structures comprising a branched vinyl polymer scaffold together with dendritic components. The present invention is particularly, though not exclusively, concerned with such hybrid materials from the perspective of medical applications, for example the carrying and delivering of drugs and other medically useful materials, the enhancement of therapeutic and diagnostic properties, and improved or more efficient or cost-effective formulations,

Dendrimers have been extensively studied in this context, amongst many other contexts. The word "dendrimer" was coined in the early 1980s, following work on cascade chemistry and arborols, to describe polymers which contain dendrons. A "dendron" is a tree-like, repeatedly-branched, moiety. Thus, a dendron is a wedge- shaped dendritic fragment of a dendrimer. Typically, dendrimers have ordered, symmetrical architectures. A dendrimer comprises a core from which several dendrons branch outwards, to form a three-dimensional, usually spherical structure,

Dendrimers can be prepared by step-wise divergent or convergent growth. Divergent procedures start at the core of the dendrimer and grow outwards. Convergent procedures prepare dendrons first and then couple the dendrons together. In convergent procedures, the dendrons are typically coupled together at their focal points (i.e. at the base of the "tree", or the apex of the dendritic wedge) via chemically addressable groups,

For a nanomatcria! to carry and deliver a drug or other biologically useful material, it is necessary for it to exhibit suitable properties in aqueous media and to have suitable domains to encapsulate the drug (which, for most drags, need to be hydrophobic domains) and/or means of conjugating, bonding or otherwise associating with the drug. It is also advantageous for the nanomaierial to be able to carry a high

"payload" of drug, Dendrimers satisfy these requirements. Due to their repeatedly branched iterative nature, they are large compared to non-polymeric active molecules and contain a large number of surface groups, and can therefore encapsulate, and/or be conjugated to, a large amount of material. Whilst they can be made from all kinds of chemical building blocks, they commonly comprise organic chains which provide hydrophobic microenvironments for drugs or other organic molecules. At the same time they can be stable in aqueous media so that drugs or other hydrophobic materials can be delivered within the body.

Whilst dendrimers have many interesting properties and promising features, they also have significant disadvantages, Dendrimer syntheses are lengthy and costly. The production of ideally branched structures requires multiple repeated steps of synthesis, purification and characterisation. Maintaining a 100% degree of branching generates complexity and takes time and requires very controlled reaction conditions. Even with high levels of successful recovery between steps, the compound effect after several steps means that the overall mass recovery suffers significantly. Whilst convergent methods are better than divergent methods from the viewpoint of ease and speed of procedure, they are still arduous, and other problems beset convergent methods, for example steric difficulties hindering coupling.

Geometric realities of iterative branching mean that the crowding constraints at the surface of the dendrimer sphere limit the size of the nanomaterials. Therefore dendrimers typically have a maximum size of about 1 Onm, This limits the amount of material they can carry.

Further description of dendrimers and their structures, preparation and applications, can be found hi numerous articles including: S.M. Grayson and J.M. Frechet, Chenu Rev. 2001, 101, 3819-3867; H. Frauenrath, Prog, Polym. Sci 2005, 325-384; F. Aulenta, W, Hayes and S. Rannard, European Polymer Journal 2003, 39, 1741- 1771 ; E.R. Gillies and J.M.J, Frechet, Drug Discovery Today, 2005, 10, 1, 35-43: and S.H. Medina and M.E.H. El-Sayed, Chem. Rev, 2009, 109, 3141-3157. From a first aspect the present invention provides a method of preparing a pH responsive product, said product being a non-gelled branched vinyl polymer scaffold carrying dendrons, comprising the living or controlled polymerization of a mono functional vinyl monomer and a difunctional vinyl monomer, using a dendron initiator,

At least one dendron initiator is used in the present invention; optionally further initiators (selected from non-dendron initiators and/or other dendron initiators) may also be used in combination with the dendron initiator.

From a second aspect the present invention provides a pH responsive non-gelled branched vinyl polymer scaffold carrying a dendron moiety.

The vinyl polymer scaffold carries at least one type of dendron, and may optionally carry further moieties (selected from no -dendrons and/or other dendrons).

By "pH responsive" is meant that physical and/or chemical characteristics of the material change under different pH conditions. Such change encompasses for example: change in aggregation or so lubility of the material or particles or aggregates thereof; change in stability of particles or nanopariieles of the material; change in ability to undergo association or disassociation in particular environments, e.g. aqueous environments; change in solubility, hydrophilicity or hydrophobicity which allows an ac ive molecule, for example a drag or other pay!oad to be carried to a particular site, for example in the body, and then released under particular pH conditions; change which allows the release of carried or encapsulated matter to be triggered or controlled; and/or cleavage of one or more bonds, e.g. resulting in breakdown of the material and/or release of payload.

The present invention provides products which can be referred to as "polydendrons" because they contain a plurality of dendrons. The dendrons may be the same or different, Polydendrons retain the advantages of dendrimers without having their disadvantages of cost, complexity and arduous synthesis. Instead of the dendritic structure extending all the way to the centre, the core is a tuneable and cost-effective non-gelled branched vinyl polymer scaffold. The polydendrons typically take the form of units (which optionally are approximately spherical) with a large number of external surface dendron groups and with the vinyl scaffolds typically being present predominantly in the centre of the units,

Either the polymer scaffold or the dendrons or both may have the required pH responsive character.

One class of pH responsive polydendrons are those comprising functionality (e.g. amine or acidic functionality) within the polymer which can be protonated or deprotonated (particularly within pH ranges to be found in living systems) thereby exhibiting altered solubility. As exemplified herein, amine-cor.taining components (e.g. amine containing meth(acrylic) polymers) can be incorporated into the polydendrons, and the amines ca be protonated in acidic conditions to result in a carrier which is more liydrophilic and more soluble in aqueous systems under low pH conditions. This means that a material can be encapsulated within a polydendron nanopreeipitate, and then can be released in an acidic environment. Analogously, acid-containing components can be incorporated into polydendrons and exhibit the converse effect.

A second class of pH responsive polydendrons are those comprising functionality (e.g. amine or acidic functionality) within, or at the surface of. the dendron which can be protonated or deprotonated as described above (particularly within pH ranges to be found in living systems) thereby exhibiting altered solubility. As exemplified herein, amine-containing dendrons (e.g. tertiary amines) can be incorporated into the polydendrons, and the amines can be protonated in acidic conditions to result i a carrier with altered solubility hi aqueous systems under low pH conditions. This means that a polydendron may be assembled and triggered to disassemble and material can be encapsulated and then released in an acidic environment.

Analogously, acid-containing components can be incorporated into polydendrons and exhibit the converse effect.

The non-gelled branched vinyl polymer scaffolds of the present invention exhibit good solubility and low viscosity. They can be contrasted with polymer staictures which are insoluble and/or exhibit high viscosity, such as extensively crosslinked insoluble polymer networks, high molecular weight linear polymers, or micro gels..

The products can be made by, but are not limited to being made by, living polymerization, controlled polymerization or chain-growth polymerization. Several types of living and controlled polymerization are known in the art and suitable for use in the present invention. A preferred type of living polymerization is Atom Transfer Radical Polymerization ( AT I P), however other techniques such as

Reversible Addition-Fragmentation chain-Transfer (RAFT) and Nitroxide Mediated Polymerisation (NMP) or conventional free-radical polymerization controlled by the deliberate addition of chain-transfer agents are also suitable syntheses.

The skilled person is aware of techniques to provide branched but non-gelled vinyl polymer scaffolds. For example, suitable procedures are described in WO

2009/122220; N. O'Brien, A. McKee, D.C, Sherrington, A.T. Slark and A. Titterton, Polymer 2000, 41, 6027-6031 ; T. He, DJ. Adams, M.F, Butler, C.T. Yeoh, A.I. Cooper and S.P. Rannard, Ang w. Chem, int Ed, 2007, 46, 9243-9247; V, Βϋίϋη, I. Bannister, N.C, Billingham, D.C. Sherrington and S.P, Armes, Macromolecules 20Θ5, 38, 4977-4982; 1. Bannister, N.C. Billingham, S.P. Armes, S.P. Rarmard and P. Findlay, Macromehc les 2006, 39, 7483-7492; and R.A. Slater, T.O McDonald, DJ. Adams, E.R, Draper, J.V.M. Weaver and S. P. Rannard, Soft Matter 2012, 8, 9816-9827, The non-gelled and soluble products of the present invention are different to materials disclosed in L.A. Connal, R, Vestberg, CJ. Hawker and G.G. Qiao, Macromolecules 2007, 40, 7855- 7863 which comprise multiple cross-linking in a gelled network.

The polymerization of each vinyl polymer chain starts at an initiator. Polymerization of mono functional vinyl monomers leads to linear polymer chains.

Copolymerization with Afunctional vinyl monomers leads to branching between the chains. In order to control branching and prevent gelation there should be less than one effective brancher (dtfunctional vinyl monomer) per chain. Under certain conditions, this can be achieved by using a molar ratio of brancher to initiator of less than one: this assumes that the monomer (i.e. the mono&nctional vinyl monomer) and the branches: (i.e. the difunetional vinyl monomer) have the same reactivity, that there is no intramolecular reaction, that the two functionalities of the brancher have the same reactivity, and that reactivity remains the same even after part-reaclioa Of course, the systems and conditions may he different, but the skilled person understands how to control the reaction and determine without undue

experimentation how a non-gelled structure may be achieved. For example, under dilute conditions some branchers form intramolecular cycles which limit the number of branehers that branch between chains even if the molar ratio of brancher to initiator (i.e. polymer chain) is higher than 1 : 1 in the reaction.

In the present invention, dendrons are used as macromolecular initiators. In order to be able to initiate polymerization, the dendrons must bear suitable reactive functionality. For example, in ATRP, convenient and effective initiators include alky! halides (e.g. alkyl bromides), and so dendrons which carry halides at their focal points can act as initiators. In this scenario, propagation starts at the apex of the dendron '"wedge". The skilled person is well aware of the types of components and reagents which are used in ATRP and other living or controlled polymerizations, and hence the type of functionality which must be present on or introduced to dendrons for them to act as initiators.

One possible way of introducing bromo groups to dendrons is to functionalize dendron alcohols with alpha- bro mo isobutyryl bromide. There are however many other ways of functional iz.ing dendrons so that they can act as initiators and other types of functionality which will initiate polymerization. The concept of a. dendron. initiator is applicable to all suitable types of polymerization and the functionality can be varied as necessary.

There is no particular limitation regarding the type of dendron that can he used, or the chemistry used to prepare the dendrons. In some scenarios it is desirable to have particular groups present at the surface (i.e. at the tips of the "branches" of the dendron), and these may be incorporated during the synthesis of the dendron. The dendrons are preferably non- inyl Any suitable coupling chemistry may be used to build up the dendrons. In one example, amines and alcohols may be coupled together, for example using

carbonyldiimidazo le. This is, however, merely one example and numerous other coupling methods are possible.

5

If exclusively one type of dendron initiator is used then in the resultant hybrid branched product one end of each vinyl polymer chain bears that dendron.

Optionally, mixed initiators are used, in other words not only a dendron initiator but J O also at least one further initiator (which may be a different type of dendron initiator, or alternatively an initator other than a dendron initiator) may be used. This allows considerable advantages in terms of varying the composition and the properties of the resultant polydendron structure.

15 The pH responsiveness may reside in one or more component of the polydendron.

Either the vi yl polymer scaffold or the dendron or both [and/or other component(s) e.g. other initiator or substituent] can be tailored so as to be pH responsive. The experimental details below show the synthesis of various different polydendrons and components thereof some of which bring about pH responsive character, such that a

20 polydendron may comprise one or more component which brings about pH

responsive character and one of more component which is not.

Amine functionality has been found to be particularly useful in practice, in order to provide tuneable, pH-dependent response. This is due in part to the use of a

25 decreasing pH within varioiis cells, tissues and organs of the body to achieve

beneficial action (eg degradation of exogenous materials), leading to opportunities to trigger polydendron behaviour (eg releasing of payload drags).

The pH responsiveness can also be linked to the hydrophilicity hydrophobicity of 30 the dendron or the core. The non-gelled scaffold core represents a large volume of material within the confines of the polydendron. When hydrophobic, the scaffold may provide optimal conditions for encapsulation of hydrophobic active ingredient molecules (eg drug molecules). As such, the polydendron offers the ability to deliver such materials in hydrophilic solvent environmenis. Through the response to pH. the scaffold will become hydrophilic and result in the exclusion of the encapsulated hydrophobic molecules, Polydendrons may be nanoprecipitated to form aggregated structures. If the dendron is pH-responsive, and connected to a non-responsive scaffold core, the aggregated materials will be triggered to disassemble on modification of the pH, also leading to release of encapsulated material and/or a dramatic change in the physical size of the aggregate. The pH responsive

functionality of the polydendron may therefore be present exclusively at the dendron component of the polydendron, exclusively at the scaffold core of the polydendron or carried by both the core and the dendron of the polydendron.

As shown below the methodology can be tailored to make polydendrons of each of the following four types:

~ Hydrophilic dendron / hydrophobic core

- Hydrophobic dendron / hydrophilic core

Hydrophilic. dendron / hydrophilic core

- Hydrophobic dendron / hydrophobic core

Each of these four types have the capacity to be pH responsive.

A further way to provide pH responsiveness is to use linkers, moieties or substituents which are eleavable under particular pH conditions, e.g. in acid environments.

The pH responsiveness is particularly advantageous for drug delivery, drug transport and drag release applications. It also has applications when material or payload other than drug is carried.

Thus, for example, pH sensitive cores can be used which can release their drag or other material when the polydendron material enters a highly acidic cellular compartment.

In contrast conventional dendrimer polymers have internal chemistry which is redundant and merely serves as a scaffold. Multiple modes of responsivity can be used, e.g. a triple trigger release mechanism can be based on the use of a pH responsive polymer and acid cieavahle linker so that a drug may he released and the po!ydendron aggregate may break down and further degrade to low molecular weight components thai more easily leave the body.

Thus the present invention provides significant benefits in terms of improved drug delivery methods, improved drug release, targeting, administration, dose and breakdown of drug carriers to facilitate their removal from the body, Drugs that may be encapsulated include, but are not limited to, hydrophobic agents for chronic and acute, oral, topical, opthalmic and parenteral administration of anticancer, infectious disease, age-related disease, genetic, CNS, psychiatric, paed.iat.ric and parasitic therapies. Some non-limiting examples of functional groups which may bring about pH responsiveness include the following which may be present on the dendron(s), within the core, or both; amines (e.g. primary, secondary or tertiary amines, cyclic aliphatic and cyclic aromatic amine moieties); carboxylic acids including aromatic and aliphatic acids; non-carboxyiic acids such as sulphonic acids and organic sulphate groups; betaines, acetals, keials, t-butoxy carhonyl groups, acetates and acetoxy functionality.

Optionally, the drag or other payload is released at a pH of about 5 to about 8.5, e.g. about 5.5 to 8, or 6 to 7,5.

The present invention resides in the combination, of features which work well together. The branched vinyl polymer methodology is intermingled with the use of at least one dendron initiator. The way in which the living or controlled

polymerization occurs means that, if different initiators are used, these will he distributed statistically and evenly around the surface of the non-gelled branched vinyl polymer scaffold. Some polymer chains will have one type of initiator at one end whereas other polymer chains will have another type at their end. There may be one type of initiator, two types of initiator, or more, e.g. three or four or more, and therefore the multiplicity of types of end group may be one or more.

The vinyl polymer core is easily tuneable and very cost-effective. Different types of monomers, with different properties (e.g. differing solubility properties) may he used. The methodology allows a sizeable scaffold to be built, and the molecular weight and size can be controlled by choice of particular monomers (a wide range cart be used) and reaction conditions, for example the ratio of initiator to monomer. The material is non-gelled and therefore soluble. At the same time the option to use different types of initiator, or mixed initiators, allows further tuneabi!ity and flexibility. There are synergistic advantages: for example the use of dendrons and other moieties as initiators means that they do not need to he introduced separately hut instead are used as reagents within an already very efficient and convenient polymerization process. The process conveniently and cost-effectively results in the the initiators being distributed throughout the materials. The initiators themselves are relatively easy to synthesize. Regarding the need for the initiators to have suitable means and functionality to initiate polymerization, the considerations described above in relation to the dendron initiators apply mutatis mutandis to any further initiator(s) which may be used.

The living or controlled polymerization methodology inherently allows control in the synthesis of the polymeric scaffold. For example, ATRP and other techniq ues are robust and flexible in being suitable for use with a large variety of functional groups and in avoiding unwanted side reactions. The size and dispersity of the products can be controlled. The monomer units are usually homogeneously distributed between the initiator molecules and therefore the chain length, and hence the molecular weight, can be controlled. The conditions can be controlled to result in materials having low polydispersity indexes when forming linear polymers, i.e. mixtures wherein the individual components have approximately the same size. This is particularly useful in the present invention as the individual chains comprising the branched structure (i.e. the primary chains) have similar chain Iengtlis. The resulting branched polymers of the invention have a distribution of structures with varying numbers of linear chains connected to form the branched architectures. The optional use of at least one further initiator, in addition to the dendron initiator, within the living or controlled polymerization methodology, brings further advantages. The further initiator alters the properties of the polydendron, for example the solubility, hydrophilieity, hydrophobieity, aggregation, size, reactivity, stability, degradability, therapeutic, diagnostic, biological transport, plasma residence time, cell interaction, drug compatibility, stimulus response, targeting and/or imaging characteristics.

The optional further initiator may comprise or be derived from one or more of the following: a small molecule, a drug, an active pharmaceutical ingredient, a polymer, a peptide, a sugar, a dendron, a moiety which carries or can carry a drug, an anionic functional group, a cationic functional group, a moiety which enhances solubility (for example, of the polydendron within aqueous systems, or of a drug or other carried material), a moiety which prolongs residence time within the body, a moiety which enhances stability of a drug or other active material, a moiety which reduces macrophage uptake, a moiety which enhances controlled release, a moiety which enhances drug transport, or a moiety which enhances drug targeting,

The initiator may be a rnacromitiator, for example a macroimtiator prepared by synthesis from one or more monomer (e.g. a water soluble monofunctional monomer), or a macro initiator prepared by modification of a ore-synthesized polymer. The macroimtiator may be a copolymer, i.e. may comprise a polymer made from at least two monomers, e.g. monofunctional monomers. The rnacromitiator may further be selected from natural polymers, for example water soluble or partially soluble polymers, e.g. polysaccharides, polypeptides or proteins.

Each type of initiator may fall within one or more than one of the above definitions; for example the initiator may be a dendron and may also carry a drug. The initiator may also be a pro-drag, releasing a moiety that becomes pharmacologically active after a further process within the body.

The present inventors have been surprised at how effective the present invention is, in allowing a range of properties to be controlled and tuned. As described in more detail below, they have observed: that the surface chemistry can be varied widely across a hydrophobic - amphophilic - hydrophilic spectrum; that the encapsulation environment can be varied significantly; that the salt stability can be controlled; and that transcellular permeability (in an in vitro model) can be tuned and improved, in view of the drug delivery capabilities, from further aspects the present invention also provides pharmaceutical compositions comprising the products of the present invention, and allows enhancements in terms of medical administration possibilities. For example, the surprisingly effective way in which the polydendrons interact controllably with, and transport encapsulated materials through, model gut- epithelium, is relevant to oral delivery applications. Materials of this type are also useful within parenteral administration such as intravenous, subcutaneous and intramuscular injection.

Polyethylene glycol (PEG) groups are advantageous for use in the initiators of the present invention, in comparison to polydendrons which carry dendrons alone, polydendrons which carry not only dendrons but also PEG groups exhibit enhanced stability in aqueous systems, controlled interaction with cells, and prolonged systemic half-life. Non-limiting examples of suitable PEGs include those with end functionality such as methyl, hydroxy!, amine, acid etc, functionality, and/or those with molecular weights above 300 g mol, preferably those with hydroxy! and acid functional chains and/or with moleculai- weights >750 g mol. Panicularly preferred are hydro xyl compounds and/or those with molecular weights >1000 g/moi.

Alternatively, other chemical moieties which function in the same or similar way and which can advantageously be used in the present invention include acrylate and methacrylate moieties including water-soluble polymeric chains (e.g. less than 20000 g/mol), for example derived from vinyl or non vinyl monomers such as ethylene glycol methacylate, glycerol methacrylate, vinyl alcohol, acrylic acid, methacrylic acid, or hydroxyethyl methacrylate.

The initiators may include groups which allow post-fonetionaiization of the polydendrons. Thus, whilst various possible initiator structures and moieties have been discussed above, an alternative to them being present within the initiator at the start, of the reaction is to incorporate them later by reaction of the polydendron with suitable materials. Suitable functional groups in initiators which allow post-functionalization include thiols, hydroxyl groups, amines, acids or isoeyanaies, amongst others.

For example, N-hydroxysuccinimide functionalized initiators can be incorporated into po!ydendrons and post-funetionalized with materials containing amine groups.

The several means of flexibility and levels of control provided by the present invention reside in the ability to alter several variables including: the amount of initiator(s) relative to vinyl polymer, the ratio between dendron initiator(s) and non- dendron initiatoris) [or other dendron initiator(s)], the nature and properties of the dendron initiator(s), the nature and properties of non-dendron initiator(s), the extent of branching, the nature and properties of the monomer(s), the nature and properties of the branched s), and the capacity of the nano materials for drags or other materials.

A further advantage of the methods and products of the present invention is that they are compatible with the preparation of nanomaterials which are stable and of controllable and uniform size. Nanoprecipitation of branched vinyl polymers is disclosed in R.A. Slater, T.O McDonald, D.J, Adams, E. . Draper, J.V.M Weaver and S. P. Rannard, Soft Matter 2012, 8, 9816-9827. This technique has been successfully used on single and mixed init iator - carrying polydendrons of the present invention to prepare stable nanoparticles. The nanoparticles are prepared by the self assembly during precipitation with dispersity and size of these nanopaitides being effectively controlled by varying the nature of the solvents, precipitation method, concentration, and presence of other components. Uniform or near uniform assembled nanopartiele sizes with low polydispersities can be achieved.

Nanoparticles of uniform and controllable size are extremely useful in the field of drug encapsulation and delivery. The nanoparticles may for example be prepared by precipitation of the polydendron out of solution using a solvent which is a non-solvent for the vinyl polymer scaffold b t which is a good solvent for the dendrons or other surface groups. This nanoprecipitation using a solvent switch might have been expected to lead to collapse of the internal vinyl polymer core, but self-assembly of the individual polydendron particles is observed leading to very stable distributions of larger complex nanoparticles with a narrow size distribution. A preferred "no n- solvent" for the vinyl polymer, i.e. medium in which the nanoprecipitate particles are stable, is water.

By way of example, where the core is a polyHPMA-EGDMA material and the dendrons are selected from amine functional dendrons (eg G1A, G1D and G2D shown in the examples) , then the material can be first dissolved in THF and nanoprecipitated into water.,

The characteristics of the polydendron, including the electronic/ charge and steric nature, and the nature of the solvent, affect the way in which the material behaves in that solvent. Without wishing to be bound by theory, the particles generally increase in size until they reach a colloidally stable state during the nanoprecipitation process.

As exemplified below, the present invention allows the encapsulation and release of not only organic materials - e.g. ni!e red, simulating encapsulation of a drug - ^■ but also inorganic materials - e.g. magnetic particles. This expands the utility of the present invention to cover further therapeutic and targeting uses. The encapsulation of inorganic material (e.g. magnetic material, e.g. iron oxide) in olydendrons may also be considered as a standalone invention within this disclosure. The branches are typically distributed statistically throughout the connected linear polymer chains (rather than discretely in block polymerised monofdnctional vinyl monomers and Afunctional vinyl monomers). Each branch may be a glycol d tester branch, for example. The difunctional vinyl monomer acts as a brancher (or branching agent) and provides a branch between adjacent polymer chains. The branching agent may have two or more vinyl groups.

The monofunctional monomer utilised for the primary chain may comprise any carbon-carbon unsaturated compound which can be polymerised by an addition polymerisation mechanism, for example vinyl and ally! compounds . The monofunctional monomer may be hydrophilic, hydrophobic, amphophilic, anionic, cationic, neutral or z itterionie in. nature.

The monofunctional monomer may be selected from but is n t necessarily limited to monomers such as: vinyl acids and derivatives (including esters, amides and anhydrides), vinyl aryl compounds, vinyl ethers, vinyl amines and derivatives (including aryl amines), vinyl nitrites, vinyl ketones, and derivatives of the aforementioned compounds as well as corresponding allyl variants thereof.

Vinyl acids and derivatives thereof include: (meth)acrylic acid, fumaric acid, ma!eic acid, itaconic acid and acid halides thereof such as (nieth)acryloyi chloride.

Vinyl acid esters and derivatives thereof include: CI to C20 alkyl(meth)acrylates (linear and branched) such as for example methyl (meth)acrylate, stearyl

(meth)acrylate and 2~ethyl hexyl (meth)acrylate; ary!(meth)aerylates such as for example benzyl (meth)acrylate; tri(aIkyloxy)silylaIkyl(meth)acrylates such as trimethoxysilylpropyl(meih)acrylate; and activated esters of (meth)acrylic acid such as N-hydroxy succi amido (meth)acrylate.

Vinyl aryl compounds and derivatives thereof include: styrene, acetoxystyrene, styrene sulfonic acid, 2- and 4- inyl pyridine, vinyl naphthalene, vinylbenzy] chloride and vinyl benzoic acid.

Vinyl acid anhydrides and derivatives thereof include: maleic anhydride.

I S Vinyl amides and derivatives thereof include: (meth)acrylamide, N-(2- hydroxypropyl)methacrylamide, N-vinyl pyrrolidone, N-vinyl formamide, (meth)acrylamidopropyl trimethyl ammonium chloride, [3- ((meth)acrylamido)propyl]dimethyl ammonium chloride, 3-[N~(3- (meth)acrylamidopropyI)-N,N-dirnethyl]amiriopropane sulfonate, methyl

(meth)acrylamidoglyeolate methyl ether and N~isopropyl(meth)acrylamide,

Vinyl ethers and derivatives thereof include: methyl vinyl ether. Vinyl amines and derivatives thereof include: dmiethylaminoethyl (meth)acrylate, diethyl amino ethyl (meth)acrylate, diisopropylaminoeihyl (meth)acrylate, mono~t~ butylamino ethyl (meth)aerylate, morphoiinoethyl(meth)acrylate and monomers which can be post-reacted to form amine groups, such as N-vinyl formamide, Vinyl aryl amines and derivatives thereof include: vinyl aniline, 2 and 4- vinyl pyridine, N-vinyl carbazole and vinyl imidazole.

Vinyl nitriles and derivatives thereof include: (meth)acrylonitrile. Vinyl ketones or aldehydes and derivatives thereof include: acreolin.

Monomers based on styrene or those containing an aromatic functionality such as styrene, OHnethyl styrene, vinyl benzyl chloride, vinyl naphthalene, vinyl benzoic acid, N-vinyl carbazole, 2-, 3- or 4- vinyl pyridine, vinyl aniline, acetoxy styrene, styrene sulfonic acid, vinyl imidazole or derivatives thereof may also be used.

Other suitable mono functional monomers include: hydroxyl-containing monomers and monomers which can be post-reacted to form hydroxy! groups, acid-containing or acid- unctional monomers, zwitterionic monomers and quatemised amino monomers.

Hydroxyl-containing monomers include; vinyl hydroxy! monomers such as hydro xyethy! (meth)acrylate, 1- and 2-hydroxy propyl (raeth)acrylate, 2-hydroxy methacry!amide. glycerol mono(meth)acrylate and sugar raono(meih)acrylaies such as glucose mcmo(rneth)acrylate.

Monomers which can be post-reacted to form hydroxy! groups include: vinyl acetate. acetoxystyrene and glycidyl (meth)acrylate.

Acid-containing or acid functional monomers include: (meth)acrylic acid, styrene sulfonic acid, vinyl phosphonic acid, vinyl benzoic acid, maleic acid, fumarie acid, itaconie acid, 2~(meth)acry1amido 2- ethyl propanesulfeme acid, mono-2- ( (raeth) cr y lo y lo y) ethyl succinate and ammonium sulfatoethyl (meth)acrylate.

Zwitterionic monomers include: (rneth)acryloyl oxyethylphosphoryi choline and betaines, such as [2~((meth)acryloyloxy)ethyl] dimethy]~(3-suifopropyl)arnmomum hydroxide.

Quatemised amino monomers include: (meth)acryloy!oxyethyltri~

(alk/'aryl)ammonium halides such as (met h) acrylo ylo xyethylt r imethy ! ammonium chloride. Oligomeric, polymeric and di- or multi-funetiona!ised monomers may aiso be used, especially oligomeric or polymeric (meth)acrylic acid esters such as mono(alk/aryl) (meth)acryiic acid esters of poiyalkyleneglycol or polydimethylsiloxane or any other mono-vinyl or ally! adduct of a low molecular weight oligomer. Oligomeric and polymeric monomers include: oligomeric and polymeric

(meth)acrylic acid esters such as mono(alk/aryl)oxypolyalkyleneglycol

(meih)acrylaies and mono(a!k aryi)oxypolydiitiethyl-siioxane(meth)acrylates. These esters include for example: monomethoxy o ligo (ethy leneglyco 1)

mono(meth)aerylaie, monomethoxy oligo(propyleneglycoi) mono(meih)aerylaie, monohydroxy o ligo ( ethylenegl y co 1) mono(meth)acrylate, monohydroxy

oligo(propyleneglycol) mono(meth)acrylate, monomethoxy po ly(ethy leneglyco 1) mono(rneth)acrylate, monomethoxy poly(propyieneglycol) mono(meth)acrylate, onohydroxy poly(emyleneglyeol) mono(meth) acryl ate and raonohydroxy poly(propyleneglyco 1) mono (metis) acrylaie .

Vinyl acetate and derivatives thereof can also be utilised.

Further examples include: vinyl or ally! esters, amides or ethers of pre-formed oligomers or polymers formed via ring-opening polymerisation such as

oligo(caprolaclam), oligo(capro lactone), poly(eaprolactam) or poly(eapro lactone), or oligomers or polymers formed via a living polymerisation technique such as poly(l ,4-butadiene).

The corresponding allyl monomers to those listed above can also he used where appropriate. Specific examples of mo no functional monomers include: amide-containing monomers such as (rneth) acr ylam ide, N-(2-hydroxypropyl) methacrylamide, N,N'-dimethyl(metli)acrylamide ₅ N and/or '-di(alkyl or aryl) (meth)acrylamide, N- vinyl pyrroiidone, [3~((meth)aeryiamido)propyI] trimethyi ammonium chloride, 3-(dimethy!amino)propy[(meth)acryiamide, 3~[N-(3~

(meih)acrylamidopiOpyl)-N,N-dimethyl]ammopropane sui f nate, methyl

(meth)acrylamidoglycolate methyl ether and N~isopropyl(meth)acrylamide:

(meth)acrylic acid and derivatives thereof such as (meth)aerylic acid, (meth)acryloyl chloride (or any halide), (alky1/aryl)(meth)acrylate; vinyl amines such as aminoethyl (meth)acrylate, dimethylaminoethyl (meth)acrylate, dieihylaminoethyl (meth)acrylate, diisopropylaminoethyl (meth)acryiate, mono-t- butylamino (meth)acrylate, morpholino ethy i(met h) aerylate; vinyl aryl amines such as vinyl aniline, vinyl pyridine, N-vinyl carbazole, vinyl imidazole, and monomers which can be post-reacted to form amine groups, such as vinyl formamide; vinyl aryl monomers such as styrene, vinyl benzyl chloride, vinyl toluene, alpha- methyl styrene, styrene sulfonic acid, vinyl naphthalene and vinyl benzoic acid; vinyl hydroxy! monomers such as hydroxyethyl (meth)acrylate, hydroxy propyl (meth)acrylaie, glycerol mono(raeth)acrylate or monomers which can be post- functionahsed into hydroxyl groups such as vinyl acetate, acetoxy styrene and glycidyl (meth)acrylate; acid- containing monomers snch as (meth)acrylic acid, styrene sulfonic acid, inyl phosphonic acid, vinyl benzoic acid, ma!eie acid, fumark acid, itaconic acid, 2- (meth)acrylamido 2-ethyl propanesulfonic acid and mono-2- ((meth)acryl ylo xy) ethyl succinate or acid anhydrides such as maleic anhydride; zwiiterionic monomers such as (meth)acryloyl oxyethy!phosphoryl choline and beiaine- containing monomers, such as 2-((me†h)acryloyloxy)ethyl] dimethyl-(3- sulfopropyl)ammonium hydroxide; quatemised amino monomers such as (meth)aeryloyloxyethyltrimethyl ammonium chloride. vinyl acetate or vinyl butanoate or derivatives thereof.

The corresponding allyl monomer, where applicable, can also be used in each case, Mixtures of more than one monomer may also be used to give statistical, graft, gradient or alternating copolymers.

Some preferred nionoiunciionai vinyl monomers include methacrylate monomers or styrene. Some preferred hydrophobic methacrylate monomers include 2- hydroxypropyl methacrylate (HPMA), n-buty[ methacrylate (nBuMA), tert-butyl methacrylate (tBuMA), and o1igo(ethylene glycol) methyl ether methacrylate (OEGIVIA). BP MA is particularly preferred, arsd is readily available or synthesised as a mixture of (predominantly) 2-hydroxypropyl methacrylate and 2- hydroxyisopropyl methacrylate. A preferred hydrophilic methacrylaie monomers is diethyiarmnoethyl methacrylate (DEAEMA).

The polydendron also contains a brancher which is a nonfunctional (at least difunctional) vinyl containing molecule.

The multi&nctional monomer or brancher may comprise a molecule containing at least two vinyl groups which may be polymerised via addition polymerisation. The molecule may be hydrophilic, hydrophobic, amphophilic, neutral, cationic, zwitterionie, oligomeric or polymeric. Such molecules are often known as cross- linking agents in the art.

Examples include: di- or multivinyl esters, di~ or multivinyl amides, di~ or multivinyl aryl compounds, di- or multivinyl alk/aryl ethers. Typically, in the case of oligomeric or polymeric di~ or multifunctional branching agents, a linking reaction is used io attach a polymerisable moiety to a di- or multifunctional oligomer or polymer. The brancher may itself have more than one branching point, such as T- shaped divinylic oligomers or polymers. In some cases, more than one

multifunctional monomer may be used. The corresponding allyl monomers to those listed above can also be used where appropriate.

Preferred multi&nctional monomers or branchers include but are not limited to: divinyl aryl monomers such as divinyl benzene; (meth)aerylaie diesters such as ethylene glycol di(raeth)acrylate, propyleneglycol di(meth)acryiate and 1 ,3- butylenedi(meth)acrylate; polya!kylene oxide di(meth)acrylaies such as tetraethyleneglycol di(meth)acrylate, poly(e hylenegl col) di(meth)acrylate and polyCpropyleneglyco!) di(met )acrylate; divinyl (metli)acrylanaides such as methylene bisacrylamide; silicone-containing divinyl esters or amides such as (met )aciyloxypropyl erminated

poly(dimethylsiloxane); divinyl ethers such as poiy{ethyleneglycol)diviRyl ether; and. tetra- or tri~(meth)acrylaie esters such as pentaerythritol tetra(meth)acrylate, trimethyio Ipropane tri(meth)acry!ate or glucose di- to penta(rneth)acrylate.

Further examples include: vinyl or ally! esters, amides or ethers of pre --formed oligomers or polymers formed via ring-opening polymerisation such as

oligo(caprolactam), oligo(caprolactone), poly(eaprolactam) or poly(eapro lactone), or oligomers or polymers formed via a living polymerisation technique such as oligo- or poly(l ,4-butadiene). Some preferred types of difunctional vinyl monomers include diraethacrylate monomers, for example ethyleneglycol diniethacryiate (EGDMA).

The molar ratio of difunctional vinyl monomer to initiator is preferably no more than 2, more preferably no more than 1.5, and most preferably no more than 1 if conducted under appropriate conditions.

The amount of difunctional vinyl monomer relative to mono functional vinyl monomer is preferably 7.5 mol% or less, 2 mol or less, or 1.6moS% or less, more preferably between 1 and 7,5 moI%, for example between 1 and 2 mo! %

In a preferred embodiment, the method is a one-pot method. In this embodiment, the reaction of mo no functional vinyl monomer, difunctional vinyl monomer and initiators is carried out conveniently and cost-effectively, Preferably the method comprises preparing a mixture of the mono functional vinyl monomer, difunctional vinyl monomer and initiators under suitable conditions. The mixture may contain a catalyst (such as CuCl) or additional agents depending on the addition polymerisation technique being used. The mixture may also contain a !igand (such as 2,2'~bipyridine). The mixture may also contain a chain transfer agent,

Suitable ATRP initiators include isobutyrate esters, preferably haloisobutyrate esiers. most preferably bromoisobutyrate esters. Thus the initiator can for example have the following general formula I:

I wherein X denotes a chemically addressable group and is preferably a halide. for example CI or Br, most preferably Br; and wherein R is any suitable organic moiety. Where the initiator is a dendron initiator, R is branched into a dendritic wedge and X is the chemically addressable group at the apex of the dendritic wedge. Whilst isobutyryl esters are convenient and effective to use in this context, other chemistries are possible. it of course will be understood that part of the initiator (in this case the X group, usually bromide) is present in the initiator but reacts during the process so that it is not necessarily present in the product at the end of all primary chains, Where the initiator of general formula I is dendron initiator, R is a moiety which divides into two or more (preferably two) first generation branches (preferably identical first generation branches). Optionally each of those first generation branches then divides into two or more (preferably two) second generation branches (preferably identical second generation branches). Optionally each of those second generation branches then divides into two or more (preferably two) third generation branches (preferably identical third generation branches), There may analogously be further generations of branching. A dendron having only first generation branches is known as a generation 1 dendron; a Dendron having first and second generation branches is known as a generation 2 dendron.

The outermost branches of the dendron (the part most likely to end up on the surface of the polydendron) may comprise one or more of a variety of chemical groups, for example aromatic groups (e.g. benzene rings, e.g. of benzyloxy groups), amines (e.g. tertiary amines), alkyl groups (e.g. alky! chains or branched alkyi groups e.g. tertiary butyl groups), amide groups, xanfhates or carbamates (e.g terminating in a tertiary butyl group). These are however merely non-limiting examples: many chemistries are possible. One of the advantages of the present invention is that is compatible with a wide variety of different t pes of dendrons and other groups; the flexibility provided by the use of mixed initiators is considerable. The properties can be tuned by selecting dendrons with different chemical constituents find/or different surface groups, for example hydrophilic or hydrophobic groups, large or small moieties, groups of different polar or electronic character, groups which may allow further conjugation, etc..

Each segment may comprise one or more of an alkyi chain, ester, carbamate, or other linking group. Again these are merely no n- limiting examples and many chemistries are possible,

Within the dendron, the structure may divide at any suitable point, for example a carbon atom or a nitrogen atom, or a larger moiety such as a ring. For example the structure may comprise a Ν,Ν-bis-substituted amino component, e.g. esters of 1 - [N,N~bis~substituted ami no ] ·· 2 · pro pano 1.

Some specific and non-limiting examples of possible dendrons will now be described.

A first class of possible dendrons include those having benzyloxy surface groups. For example the surface gro tructure:

Optionally two of these moieties may be linked via carbamate chains to an amide branching point.

Examples in this class of dendrons include the Gl and G2 structures shown in Figure 1. A second class of possible dendrons include those having tertiary amine surface groups, for example where the end amines are dimethyl substituted. Optionally the branching may occur at tertiary amine centres and the segments may contain ester linkages.

Examples in this class of dendrons, and a suitable component thereof, are shown i Figure 2.

A third class of possible dendrons include those having carbamate surface functionality, for example tertiary butyl carbamates, and optionally carbamate functionality within the segment(s).

Examples in this class of dendrons are sho wn in Figure 3. A fourth class of possible dendrons include those having xanthate functionality, optionally with branches comprising esters.

Examples in this class of dendrons. and a suitable component thereof, are shown in Figure 4,

The dendrons may be prepared by known chemical techniques. Some possible methods of preparation include those described below.

The present invention will now be described in further non-limiting detail and with reference to the Examples and Figures in which:

Figures 1 to 4 show some examples of dendron initiators and components thereof which can be used in the present invention; Figure 5 shows, schematically, structural differences between dendrimers and polydendrons; Figure 6 and 7 show MTT assays ofCaco-2 cells following incubation with aqueous Nile Red and polyde.nd.rons;

Figure 8 and 9 show ATP assays of Caco-2 ceils following incubation with aqueous Nile Red and polydendrons;

Figure 10 shows results in relation to transcellular permeability of selected Nile Red polydendron materials across Caco-2 ceil monolayers Figure 1 1 shows, schematically, how using different dendron : polyethylene glycol initiator ratios can result in a spectrum of hydrophohicity, amphiphiliciiy and hydrophilieity;

Figure 12 is a photograph, corresponding to Figure 11 , and illustrates how using different dendron : polyethylene glycol initiator ratios can affect the response of encapsulated Nile Red;

Figure 13 shows, schematically, one method of nanoprecipitation of polydendrons; Figures 14a and I4b are SEM images of polydendron nanoprecipitates;

Figures 15 to 19 illustrate some effects of the polydendrons including pH responsive effects, The experimental details below relate to: preparative procedures for various dendron and non-dendron initiators used in the presen invention, including initiators containing polyethylene glycol (PEG) and sugar moieties; preparative procedures and properties of various polydendrons showing how hydrophilic or hydrophobic properties can be tailored and the effect of pH on these; nanoprecipitation methods and results; encapsulation experiments showing how molecules can be encapsulated and showing the effect of tailoring the encapsulation environment, as a model for drug encapsulation; cytotoxicity analysis using MTT and ATP assays in respect, of Caco-2 cells; transcellular permeability of polydendrons carrying Nile lied (to model *3 drag transfer across the intestinal epithelium); preparation of acid eleavable brancher; DEAEMA polydendron synthesis; hydrolysis of branched pDEAEMA; co- polydendron synthesis: nanoparticle formation, nile red encapsulation and fluoresceinamine encapsulation in respect of pH responsive polydendrons;

encapsulation of inorganic material (e.g. magnetic particles); and illustrations of encapsulation. pH responsive effects, and behaviour in transport buffer.

The experimental details below disclose initiators and polymer backbones of various functionality including those having amine groups, the behaviour of which depends on pH.

It is clear that the methodology can be tailored to make polydendrons of each of the following four types:

Hydrophilic dendron / hydrophobic core

- Hydrophobic dendron / hydrophilic core

Hydrophilic dendron / hydrophilic core

Hydrophobic dendron / hydrophobic core

Each of these four types have the capacity to be pH responsive.

Very positive results were obtained with regard to cytotoxicity and in the drug transport model. The experiments below show in particular that a material which would otherwise not pass effectively from gut to blood can be carried over by using polydendrons of the present invention.

Whereas a representation of an ideal dendriraer structure is sho wn in Figure 5a, the present invention is concerned with polydendrons which have dendrons and a polymer core as represented in Figure 5c, constituent parts of which include dendrons attached to polymer chains as represented in Figure 5b,

Polydendrons can be prepared by using mixed initiators, to end up with polydendron structures as represented for example in Figure 11. At the far left of Figure 1 1 is represented a hydrophilic polydendron made using 100% dendron initiator; at the far right of Figure 1 1 is represented a hydrophobic material made using 100% PEG, The hydrophobicity/ amphiphilieity/ hydrophiiicity can be tuned by varying the relative amounts of the different intiators. Figure 12 is a photograph of vials containing the seven different types of

polydendron shown schematically in Figure 11 (i.e. 100% dendron initiator with 0% PEG initiator on the left, through to 0% dendron initiator with 100% PEG initiator on the right) carrying Nile Red. in the original photograph, the darkest pink colour can he seen on the [eft, lighter pinks in the middle vials, and a very pale ink on the right, thereby showing that the hydrophobicity can be tuned in a discernible and

controllable manner.

The present invention is focused on pH responsive polydendrons and pH responsive polydendron panicles, aggregates and compositions, and methods of making them, Whilst some of the following examples disclose various components which are not in themselves pH responsive, nevertheless they may be used in combination with pH responsive components or features.

Novel products, components thereof, intermediates, methods or method steps, disclosed herein, also fall within the scope of the present invention

EXAMPLES

L Initiator syntheses

'-

LI Protected smgar Initiator

Lactose (4 g, 1 1 ,7 mmol) was weighed into a 100 mL round bottom flask equipped with a magnetic stirrer and dry h½ inlet. The flask was purged with nitrogen for 15 minutes. Acetic anhydride (30 mL) and odine (208 mg, 1.58 mmol) were added, instantly forming a brown coloured solution. Within 10 minutes the flask began to warm due to onset of aeety!ation. The solution was stirred overnight at room temperature under a positive flow of nitrogen. The solution was transferred to a 250 mL separating funnel containing dichloromethane (50 mL), sodium thiosulfate solution (30 mL) and crushed ice, and the product was extracted into the organic layer. The aqueous layer was further extracted with dichloromethane (2x50 mL), The organic phases were collected and washed with saturated sodium carbonate solution until neutral. The organic phase was collected, dried over anhydrous MgS0 ₄ , and concentrated in vacuo to give a white solid.

Lactose octa-acetate (5.1 g, 7.52 mmol) was weighed into a 250 mL round bottom flask equipped with a magnetic stirrer, and was dissolved in tetrahydrofuran (100 mL). Ethylene diamine (0.6 mL, 9.02 mmol) was added to the flask, followed by the slow addition of acetic acid (0.6 mL. 10.5 mmol), to give a white coloured turbid solution. A gas was evolved and the flask wamied slightly upon addition of the acid. The flask was lightly sealed with a rubber septum cap, and stirred overnight at room temperature, to give a cream coloured mixture. Distilled water (50 mL) was added to the flask, whereby the precipitate dissolved, leaving a slightly yellow coloured solution. The solution was transferred to a 500 mL separating funnel containing dichloromethane (100 mL), and the product was extracted into the organic solvent. A further extract ion of the aqueous layer was performed with dichloromethane (50 rnL). The organic layers were combined, washed with hydrochloric acid (80 mL, 2M), saturated sodium bicarbonate solution (80 mL) and distilled water (80 mL). The organic layer was dried over anhydrous MgSQ ₄ , filtered and concentrated in vacuo. The erode product was purified by flash column chromatography (silica, eluent hexane/acetone, 60/40) to give a white solid.

Lactose septa-acetate (3 g, 4,71 mmol) was added to a 50mL round bottom flask equipped with a magnetic stirrer and dry N ₂ inlet. The flask was then purged with nitrogen for 10 minutes. Anhydrous tetxahydrofuran (8 mL) was added to the flask, and N ₂ was bubbled through the mixture tor a further 10 minutes. Triethy!amine (0.99 mL, 7.07 mmol) was added to a vial, diluted with tetrahydrofuran (2 ml,), and then transferred to the reaction flask drop- wise. Following this, 2-bromoisobuiyryl bromide (0.87 mL, 7.07 mmol) was added to a vial, diluted with tetrahydrofuran (2 mL) and transferred to the reaction, flask drop-wise. Reaction mixture was left to stir overnight at room temperature under a positive flow of nitrogen. This gave a white coloured turbid mixture. The mixture was filtered by gravity filtration, the precipitate washed with tetrahydrofuran, and the solution concentrated in vacuo. The crude product was purified by flash column chromatography (silica, etuent hexane/ethyl acetate, 95/5) to give a white solid.

1.2 PEG initiators

1.2.1 750-FEG initiator

THF, RT 24hrs

Monomethoxy poly( ethyl ne glycol) (Mw—750 gmol ^"1 ) (23.0 g, 30.7 mmol) was dissolved in warm THF ( 40 °C), and the reaction was degassed with dry N ₂ . DMAF (37.5 mg. 0.3 mmol) and TEA (7.48 ml, 53.7 raraol) were added and the reaction was cooled to 0 °C in an ice bath, obromo isobutyryl bromide (5.69 ml, 46.0 mmol) was added dropwise over 30 minutes and a white precipitate appeared immediately; the Ε¾ΝΙ-Γ Br ^~ salt. After 24 hours the precipitate was filtered, THF removed in vacuo and the resulting crude product was precipitated from acetone into petroleum ether (30-40 °C) twice (72 %). Ή N R (400 MHz, D ₂ 0) δ ppm 4.31 (m, 2H), 3.77 (m, 2H), 3.70-3.59 (m, 60H), 3.55 (m, 2H) ₅ 3.31 (s, 3H) and 1.89 (s, 6H).

1.2.2 2K-PEG initiator

THF, RT 24hrs

Monomethoxy poly( ethylene glycol) (Mw «=2000 gmol ) (20,5 g, 10.25 mmol) was dissolved in warm THF ( 40 °C), and the reaction was degassed with DMA? (12,5 mg, 0.1 mmol) and TEA (3.14 ml, 22.5 mmol) were added and the reaction was cooled to 0 °C in an ice bath, tt-hrorao isobutyryl bromide (2.53 ml, 20.5 mmol) was added dropwise over 20 minutes and a white precipitate appeared immediately; the Et :N H¾r ^' salt. After 24 hours the precipitate was filtered, THF removed in vacuo and the resulting crude product was precipitated from acetone into petroleum ether (30-40 °C) twice (89 %). Ή NMR (400 MHz, D ₂ 0) δ ppm 4.34 (rn, 2H), 3.80-3.59 (m, 186H), 3.35 (s, 3H) and 1.93 (s, 6H).

13 , _n G0 _n £¾o _n d¾ _n ^I ^Q l) initiators

1 -dimethylamino~2-propartol (1.1207 g, 10.86 mniol, 1 eq.), TEA (1,5390 g, 15.2 mmol, 1.4 eq.) and DMAP (132.7 rng, 1.086 mrnol, 0.1 eq.) were added to a 250 mL 2 necked round-bottomed flask containing DCM (160 mL). The flask was deoxygenated under a positive N ₂ purge for 10 minutes, oi-bro o isohutyryl bromide (2.622 g, 1.4 mL, 1 1.4 mmol, 1.05 eq.) was added drop wise while the solution was stirring in an ice bath under a positive flow of N ₂ . The reaction mixture was allowed to warm to room temperature and left stirring overnight. The organic phase was washed with saturated sodium hydrogen carbonate (NaHCOs) solution (3 x 30 mL). The solution was dried with anhydrous Na ₂ S0 ₄ . Ή NMR (400 MHz, CDC1 ₃ ) δ 1.27 (d, 3H), 1.89 (m, 6H), 2.17-2.55 (m, 8H), 5.07 (m, 1H). rn/z (ES MS) 252 [M+H .

1,4 l, G2 dmdm initiators

L4.1 Gl -aromatic dendron initiator (Gl ft BOP Br)

13~Dibers2yloxy-2-propanoL 1, (9.80 g, 36.0 mmol) was weighed into a 2-neck round bottom flask which was equipped with magnetic stirrer and dry N? inlet.

Dichloro methane (DCM) (100 ml) was added followed by 4-

(dimethylamino)pyridine (DMAP) (0.44 g, 3.6 mmol) and triethylamine (TEA) (7.53 ml, 54.0 mmol). The reaction was cooled to 0 °C in an ice-bath and a- bromoisobutyryl bromide (5.34 ml, 43,2 rnmol) was added dropwise over 20 minutes. After complete addition the reaction was warmed to room temperature and left stirring overnight. Reaction could be observed by the formation of a white precipitate. After 24 hours the precipitate was removed by filtration, the resulting crude reaction medium was washed first with a saturated solution of NaHC0 ₃ (3 x 100 ml) followed by distilled water (3 x 100 ml). The organic layer was dried over Na ₂ S€>4 and concentrated in vacuo to give a pale yellow oil (81 ). Found, C. 59.55; H, 6.02 %. C ₂ - _: H ₂ sBr0 ₄ requires, C, 59.86; H, 5.98; Br, 18.96; O, 15.19 %. Ή NMR (400 MHz, CD(¾) δ ppm 7.35-7.20 (m, lOH), 5.26 (m, IB), 4.55 (m, 4H), 3.69 (d, 4H), 1.93 (s, 6H). »C NMR (100 MHz, CDC1 ₃ ) δ ppm 171 .2, 138.0, 128.4, 127.7, 127.6, 73.3, 68.5, 55.8, 30.7. m/z (ES MS) 443.1 [M+Naf, 461.1 [M+Kf , m/z required 420,1 [M ^' .

Ϊ , 1 '-Car oriyldiimidazole (GDI) (9.73 g, 60.0 mmol) was weighed into a 2-neck round bottom flask and equipped with magnetic stirring, condenser and dry ₂ in!

Si Anhydrous toluene (100 ml) was added, followed by OH (0.34 g, 6.0 mrnol) and 1 (12.35 ml, 50.0 mmol). The reaction was heated to 60 °C for 6 hours. Toluene was removed in vacuo, the crude mixture was dissolved in DCM (50 ml) and washed with distilled water (3 x 50 ml). The organic layer was dried over Na?SC>4 and concentrated in vacuo to give 3, a pale yellow oil (97 %). Found C, 68.64; H, 6,10; N, 7.85 ¾. C _2! H22N ₂ 0 ₄ requires C, 68.84; H, 6.05; N, 7.65; 0, 17,47 %. Ή NMR (400 MHz, CDC1 ₃ ) 6 ppm 8.1 1 (s, 1H), 7.41 (s, 1 H), 7.33-7.23 (m, 10H), 7.06 (s, 1H), 5,36 (qn, 1H), 4.53 (m, 411), 3,75 (m, 4H). ^{! J} C NMR (100 MHz, CDC1 ₃ ) 5 ppm

148.3, 137.5, 137.2, 130.6, 128.4, 127.9, 127.6, 117.2, 76.1, 73.3, 68. 1. m/z (ES MS) 367,2 [M -H] ^' \ 389.2 [M+Na , 405.1 [M+K] ⁺ , m/z required 366.2 [M] ⁺ .

3 (16.84 g, 46.0 mmol) was weighed into a 2 -neck round bottom flask which was equipped with magnetic stirring, condenser and dry N ₂ et. Anhydrous toluene (120 ml) was added followed by dieihylenetriamine (DETA) (2.48 ml, 23.0 mmol). The reaction was heated to 60 °C for 48 hours. Toluene was removed in vacuo, the resulting crude mixture was dissolved in DCM (100 mi) and washed with distilled water (3 x 100 ml). 'The organic layer was dried over N S€> and concentrated in vacuo to give 4, a yellow oil (93 %). Found C, 68.50; H, 7.13; , 6.00 %.

C40H ₄₉ N3O. _S requires, C, 68.65; H, 7.06; N, 6.00; O, 18.29 %. Ή NMR (400 MHz, CDC1 ₃ ) δ ppm 7.27-7.16 (m, 20H), 5.23 (s, br, NH), 5.03 (qn, 2H), 4.44 (m, 8H), 3.57 (d, 8H), 3.12 (m, 4H), 2.58 (m, 4H). ⁱ³ C NMR (100 MHz, CD£¾) 8 ppm 156.6,

138.4, 128.8, 128.1 , 73.7, 72.1 , 69,4, 49.0, 41 .2. m/z (ES MS) 700.4 [M+H] ⁺ , 722.3 [M+Na , 738.3 [M÷K] ⁺ , m/z required 699.4 [ΜΓ . 4 (15,01 g, 2.1 ,4 mmol) was weighed into a 2-neck round bottom flask, equipped with magnetic stirrer, condenser and dry N2 inlet. Anhydrous toluene (90 ml) was added lb Mowed by dropwise addition of jS-butyrolactone (2.62 ml, 32.2 mmol). The reaction was heated at reflux for 16 hours. Toluene was removed in vacuo, the resulting crude mixture was dissolved in DCM (50 ml) and washed with distilled water (3 x 50 ml). The organic layer was dried over Na _? .S0 ₄ and concentrated in vacuo to give a yellow oil. The crude product was purified by silica gel column chromatography with a mobile phase gradient of DCM:Me()H (100:0 - 95:5 - 90:10) to give 5, a pale yellow oil (45 %). Found C, 65.35; H, 6.72; N, 5.10 %. requires, C, 67.24; H, 7.05; N, 5.35; O, 20.36 %. Ή NMR (400 MHz, CDC1 ₃ ) θ ppm 7.34-7.25 (m, 20H), 5.35 (br, NH), 5.31 (br, NH), 5.11 (m, 2H), 4.50 (m, 8H), 4.14 (s, I H), 3.62 (m, 8H), 3.46-3.18 (m, br, 8H), 2.45-2.22 (m, 2H), 1.18-1.05 (m, 3H). ^I3 C NMR (100 MHz, CDCls) δ ppm 174.4, 156.8, 156.6, 138.4, 138.3, 128.8, 128.1, 128.0, 73.7, 73.6, 72.6, 72.4, 69.5, 69.3, 65.1 , 48.5, 46.5, 41 .2, 40.3, 39.9, 22.9. m/z (ES MS) 808.4 [M-!-Na ₅ m/z required 785.4 [Mf.

5 (9.31 g, 11 ,85 rrm ol) was dissolved in DCM (100 ml) and transferred to a round bottom flask which was equipped with magnetic stirring and a dry N2 inlet DM.A.P (0.14 g, 1.19 mmol). TEA (3.30 ml 23.7 mmol) were added and the reaction mistm-e was cooled to 0 °C in an ice bath followed by dropwise addition of a- bromoisobutyryl bromide (2.19 ml, 17.78 mmol). The reaction was warmed to room temperature for 24 hours. A colour change from pale orange to a dark orange/brown colour was observed over time. No precipitate was observed, the crude reaction mixture was washed with a saturated NaHC0 ₃ solution (3 x 100 ml) and distilled water (3 x 1.00 ml). The organic layer was dried over Na?.SC>4 and concentrated in vacuo to give 6, an orange oil (81 %). Found C, 59.50; H, 6.31 ; N, 4.39 %.

C ₄ sH ₆ oBrN ₃ O _H requires, C, 61.67; H, 6.47; Br, 8.55; N, 4.49; 0, 18.82 %. Ή NMR (400 MHz, CDCI ₃ ) δ ppm 7.35-7.23 (m, 20H), 5.33 (s, br, NH), 5.10 (m, 2H), 4.S2 (m _s 8H), 3.71-3.53 (s, 8H), 3.52-3.12 (m, br, 8H), 2.76 (d of d, I H), 2.47 (d of d, I H), 1.87 (s, 6H), 1.29 (d, 3H). ^!i C NMR (100 MHz, CDC¾) d ppm 192.5, 170.8, 156.3, 156.1, 137.9, 134.5, 128.4, 127.7, 127.6, 73.2, 73.1, 72.2, 71.8, 70.2, 69.1, 69.0, 68.8, 56.1, 48.3, 46.3, 39.6, 39.4, 38.9, 30.8, 30.7, 30.6, 19.7. m/z (ES MS) 958.3 [M+Ma , 974.3 [M+Kf, m/z required 933.3 [M] ⁺ .

1.4.3 Alternative G2 PBQ? Br synthesis

3 (14.03 g, 38.3 mmol) was added to a 2 -neck round bottom flask, which was equipped with magnetic stirring, condenser and a N ₂ inlet. Anhydrous toluene (100 ml) was added and the reaction was heated to 60 °C. The AB ₂ brancher (3.627 g, 1 .2 mmol) was dissolved in anhydrous toluene (5 ml) was added dropwise. After 18 hours the reaction was stopped, the toluene removed in vacuo, the crude mixture was dissolved in dichloro methane (100 ml) and washed with water (3 x 100 ml). The organic phase was dried over the solvent removed in vacuo and the resulting yellow oil was dried farther under high vacuum to give 7, as a pale yellow oil, (94 %). Ή NMR (400 MHz, CDC! ₃ ) 5 ppm 7,33-7.23 (m, 20H), 5.30 (s, br, NH), 5.09 (m, 2H), 4.51 (m, 8H), 3.73 (m, IH), 3.64 (d, 8H), 3.16 (m, 4H), 2,53 (m, 2H), 2.32 (m, 2H), 2.24 (rn, 2H), 1.59 (rn, 4H) _S 1.06 (d, 3H). m/z (ES MS) 786.4 [M+H , 808.4 [M+Naj \ m/z required 785.43 [M] ⁺ .

7, (13.381 g, 17.0 mmol) was dissolved in DCM ( 100 ml) and bubbled with N ₂ for 20 minutes. 4-(Dimethylamino)pyridine (DMAP) (21 mg, 0.17 mmol) and triethylamine (TEA) (3.56 ml, 26.0 mmol) were added and the reaction vessel was cooled to 0 °C. o-Bromo isohutyryl bromide (2.53 ml, 20.0 mmol) was added dropwise, then the reaction was warmed to room temperature for 24 hours. The organic phase was washed with a saturated solution of NaHC(¾ (3 x 150 ml) and distilled water (3 x 150 ml), dried over Na ₂ SC>4 and the solvent removed in vacuo to give an orange oil as the crude product. This was purified by column

chromatography with a silica stationary phase and mobile phase of ethyl

acetate:hexane (4: 1), to give 8 a yellow oil, (73 %). Found€, 63.24; H, 6.88; N, 4.44 %. OwHiwBrNaOto requires, C, 62.95; H. 6.90; N, 4.49 %. Ή NMR (400 MHz, CDC1 ₃ ) 6 ppm 7.33-7.24 (m, 20H), 5.36 (s, br, NH), 5.09 (m, 2H), 5.03 (m, I H), 4.51 (m, 8H), 3.64 (d, 8H), 3.16 (m, 4H), 2.64-2.35 (m, 6H), 1.89 (s, 6H) _; 1.60 (m, 4H), 1.22 (d, 30). "C NMR (100 MHz, CDC1 ₃ ) δ ppm 171.2, 1 56.0, 138.1 , 128.3, 127.60, 127.62, 73.2, 71.6, 70.4, 68.9, 59.1 , 56.1 , 52.2, 39.4, 30.6, 30.7, 27.2, 18.0. m/z (ES MS) 936.4 [M+H] ⁺ , 959.4 [M÷Na] ^÷ , m/z required 935.4 [M] \ 1.4.4

Scheme 4 - Synthesis of 20 (2~{Bis(3~aminopropyl)amino _J ^• opan~l~o!) using CDI chemistry

Synthesis of 18 - CD! (39. ! 37g, 0.241 mol) was added to an oven-dried 5G0mL 2~ neck RBF fitted with a reflux condenser, magnetic stirrer and a dry N ₂ inlet. Dry toluene (350mL) was added and the flask was purged with N ₂ for 10 minutes. The solution was stirred at 60°C and 17 (t~Butanol) (35.7g, 46mL, 0.483 mol) was added via a warm swinge. The mixture was left stirring at 60°C for 6 hours under a positive flow of nitrogen. Following this, BAPA (16.077g, 17.14ml,, 0.121 mol) was added dropwise. The reaction was leil stirring for a further 18 hours at 60"C under a positive flow of nitrogen, and then allowed to cool to room temperature. The pale yellow solution was filtered to remove any solid imidazole, and concentrated in vacuo. The remaining oil was dissolved in dichloromethane (250mL) washed with distilled water (3 x 250mL) and finally a saturated brine solution (150mL). The organic layer was dried with anhydrous filtered and concentrated in vacuo to give IS as a white solid powder, 38g, (95%) Found C _s 57.84; EL 10.45; N, 12.91%. Ct6¾N ₃ 0 requires, C, 57.98; H, 10,04; , 12.68%. ^¾ H NMR (400MHz, CDCU) 5.1 (s, hr, H - disappears on addition of D ₂ 0), 3.21 (t, 4H), 2,65 (t, 4H) ₅ 1,65 (q, 4H), 1.44 (s, 18H) °C NMR (100MHz, CDCI3) 156.48, 79.34, 47.77, 39.29, 30.11, 28.79. m/z (ES MS) 332.3 [M+H] ⁺

Synthesis of 19 - 18 (20g, 0.06 mol) was added to a SOOmL 2 -necked RBF fitted with a reflux condenser, magnetic stirrer and a dry N ₂ inlet. The ilask was degassed with dry nitrogen for 10 minutes, and dissolved in dry ethanol (200mL), Whilst stirring, and maintaining the temperature at 30°C, propylene oxide (10.51g, 11.21mL, 0.181 mol) was added dropwise over a period of 10 minutes. Under a positive flow of dry N , the reaction was left stirring at 30°C for 18 hows. After this time, the solvent and excess propylene oxide were removed in vacuo. The crude product was purified by liquid chromatography on silica gel, ekiting with EtGAc:MeOH, 4: 1 ₃ the solvent removed in vacuo to give 19 as a pale yellow viscous oil 19.90g, (85%) Found C, 58.50; H, 10,23; N, 10.82%. C19H39 3O5 requires, C, 58.58; H, 10.09; N, 10.79%. 1H NMR (400MHz, CDCI3) 4.93 (s, hr, NH), 3.76 (m, 1H), 3.15 (m, 4H), 2.61-2.88 (m, 6H), 1.62 (m, 4H), 1.44 (s, 18H), 1.11 (d, 3H). ⁿ C NMR (100MHz, CDCI3) 156.08, 79.1 8, 63.45, 62.55, 51.77, 38.75, 27.48, 20.14. m/z (ES MS) 390.3 [M÷Hf

Synthesis of 20 (Part 1} - in a 1L RBF, G i 011 (33.70g) was dissolved in ethyl acetate (330mL) and concentrated HC1 (35.03g, 30mL, d=1.18 36% active) was added very slowly. CQ ₂ began to evolve. The reaction vessel was left open and stirring for 6 hours. ^! H NMR (D ₂ 0) confirmed complete decarboxylation,

Synthesis of 21) (Part 2} - After removal of ethyl acetate, the crude oil was dissolved in 4M NaOH (300mL), and then reduced down by half (approx.) on the rotary evaporator (60°C). Following this, the oily mixture was extracted twice with CHCI ₃ (SOOmL). The organic layers were then combined, dried with anhydrous Na ₂ SC ^< 4, filtered and concentrated in vacuo to give the product as a pale yellow oil (15.27g, 94% yield) NMR (400MHz, CDCh) 3.79 (m, 1H), 2.68-2.40 (ddd, 2H), 2.31 (m, 4H), 1.89 (s, br, OH), 1.60 (m, 4H), 1.1 1 (d, 3H), ^ri C NMR (100MHz, CDCh) 63.95, 62.56, 52.10, 40.31, 30.80, 20.03

Preparation of t-BOC G2 dendron, 21

Scheme 5 - Synthesis of 21, l-BOC G2 dendron

Synthesis of 21 - 19 (5g, 12.8 nunol) was added to a 250mL 3 necked round bottom flask containing dry toluene (6()mL), which was fitted with a reflux condenser, magnetic stirrer and a dry Ni inlet. The flask was purged with N ₂ fer 10 minutes. The solution was stirred at room temperature and GDI (2.29g, 14.1 rnmole was added via a powder addition tunnel. The mixture was heated to 60°C with stirring for 6 hours. 2Θ (0.9 ImL, 6.4mmole) was added dropwise whilst the solution was stirring and the temperature was maintained at 60°C. The reaction was left overnight stirring for a further 12 hours at 60°C, and then allowed to cool to room temperature. The clear solution was filtered to remove any solid imidazole, and concentrated in vacuo. The crude product was purified by liquid chromatography, silica gel, eluting with EtOAc.'MeOHj 5: 1, the solvent removed in vacuo to give 21 as a pale yellow viscous oil (60%) Found C, 57.46; H, 9.83; K 12.17%, C19H39N3O5 requires, C, 57.68; H, 9.58; N, 12.35%. ^¾ H NMR (400MHz ₅ CDCI3) 4.92 (rn, br, 2H) ₅ 3.74 (m, 1H), 3.35· 2.93 (rn, 12H), 2.73-2.14 (m, 18H), 1.62 (m, 12H), 1.44 (s, 36H), 1.20 (m, 6H), 1.10 (d, 3H) °C NMR (100MHz, CDC ) 156.76, 156.15, 78.91 , 67.58, 63.51, 62.46, 59.36, 52.33, 51.75, 38.94, 28.50, 27.37, 20.13, 18.82, 14.20. (ES MS) 1020.7

Synthesis of t-BOC initiators 22 and 23

ii

Scheme 5 - Synthesis of 22 and 23 t~BOC ATRP initiators

General Procedure for focal point modification to ATRP initiator by acid bromide - 19 or 20 was added to a 50mL round bottom flask, which was equipped with a magnetic stirrer and purged with dry Nj. for 10 minutes. Following this,

dichloromethane (40mL), DMA? (0.2 eqv.) and TEA (2eqv.) were also added. The round bottom flask was then purged again with dry M ₂ , and placed into an ice bath, Dropwise, over a period of 10 minutes 2-Bromoisobutyryl bromide (L I eqv.) was added. The reaction was removed from the ice bath after 30 minutes and left for 24 hours at room temperature. A colour change from clear to yellow/orange was noted for ail reactions. After this time, the solution was filtered, washed with distilled water (3 x 40mL), washed with a saturated brine solution (40ml.) and the organic layer dried using anhydrous ₂ S04, The solvent was removed in vacuo, and the crude product purified by column chromatography

Synthesis of 22 - 19, Bromoisobutyryl bromide (1.1 eqv.), DMAP (0.2 eqv) and

TEA (2 eqv) were allowed to react according to the general esterifieation procedure above in 100 niL of dry for 24 h. The crude product was purified by liquid chromatography on silica gel, dating with 95/5 DCM/MeOH increasing to 90/10 DCM/MeOH to give 22 as a light yellow/brown viscous oil. (77%) ^l H NMR

(400MHz, CDCh; 5.06 (s, br _; NH), 3. 1 5 (m, 4H), 2.68-2.35 (m, 6ϊ I K 1.93 (s, 6H), 1.61 (q, 4H), 1.43 (s, 1811), 1.25 (d, 3H) °C NMR (IQOMHz, CDCh) 171,81, 156.05, 79.57, 70.78, 59.62, 56.36, 38.65, 31.14, 30.17, 27.36, 18.26. m/z (ES MS) 510.2 [M-i-H , 534.2 jM+Naf, 550.2 [M+K,

Synthesis of 23 - 20, Bromoisobutyry! bromide (1 ,1 eqv.), DMA? (0.2 eqv) and TEA (2 eqv) were allowed to react according to the general esterification procedure above in 100 mL of dry€¾C¾ for 24 k The crude product was purified by liquid chromatography on silica gel, eluting with 85:15 C(¾/MeOH to give 23 as a brown viscous oil. (54%) Ή NMR (400MHz, CDC¾) 4.92 (m, br, 2H), 3.63 (m, I H), 3.37- 2.94 (m, 12H), 2.77-2.12 (m, 18H), 1.91 (s, 6H), 1.62 (m, 12H), 1.44 (s, 36H), 1.20 (m, 9H) m/z (ES MS) 1 168.7 [M+H , 1192.7 [M+Na], 1208.7 [M+K

1.4.5 Gl Xanthate dendron initiator synthesis iXani~Gl)

Synthesis of Xa t h (sch m 5) - Potassium ethyl xanthogenate (40.1 g, 250.2 mmol) was transferred to a 500 mL two-necked round-bottomed flask, equipped with, a magnetic stirrer bar, dropping funnel and septa cap with outlet. Acetone (150 niL) was added to the flask. 3 -Brorno propionic acid (32.4 g, 21 1.8 mrnol) was dissolved in acetone (80 mL) and transferred to dropping runnel The acid was added to the flask dropwise with stirring. Once added, the reaction was left stirring at room temperature overnight, The initially yellow solid turns white as the reaction proceeds. The white solid is then filtered off and the solvent removed on the rotary evaporator. The resulting solid was dissolved in DCM (300 mL) and washed (1 x 200 nil., distilled water and 2 x 200 mL brine). The organic layer was dried over MgS0 ₄ , and the solid filtered off. The solvent was removed and placed in a vacuum oven to remove any residual solvent. Yield 59 %, ^¾ H NMR (400 MHz, CDCi ₃ ) δ: 1.42 (t, 3H) _S 2.85 (t, 2H), 3.38 (t 2H), 4.63 (q, 2H)

Synthesis of X&nt c (s heme 5) - Xanthate earboxylic acid, Xant b in (scheme 5) (15,0 g, 77,2 mrnol) was transferred to a 250 mL round-bottomed flask, equipped with a magnetic stirrer bar and septa cap containing outlet. DCM (100 mL) was added. 5 drops of DMF was added. Oxalyl chloride (19.6 g, 154.4 mrnol) was added dropwise via syringe with stirring. The reaction was left stirring for 2 hours. The reaction mixture changes from clear to a transparent orange as the reaction proceeds. The solvent was removed and washed twice with chloroform to remove any residual oxalyl chloride. Resulting viscous orange oil used as obtained. Yield quantitative. ^! H NMR (400 MHz, CDCI3) δ: 1.42 (t, 3H), 3.38 (m, 4H), 4.63 (q, 2H).

Synthesis of Xaat d (sefeeme 5) - Bis-MPA (4.1 g, 30.9 mrnol), TEA (12.9 mL, 101.2 mrnol) and DMAP (188.6 mg, 1 .6 mrnol) were transferred to a 250 rnL two- necked) round-bottomed flask equipped with a magnetic stirrer bar, dropping funnel and septa cap containing outlet. The flask was then deoxygenated using nitrogen. Dry DCM (60 mi..) was added via syringe under nitrogen. Xanthate acid chloride, Xant c in (scheme 5)... (16.4 g, 77.2 mmol) was degassed with nitrogen inside the sealed dropping funnel. Dry DCM (10 mL) was added to dissolve the acid chloride. The xanthate acid chloride was added dropwise and the reaction was left stirring under nitrogen overnight. The resulting solution was washed (1 x 200 mL distilled water and 2 x 200 mL brine). The organic layer was dried over MgS0 _s and the solid filtered off. The solvent was reduced and the product was ran through an automated flash column with a starting eluent of 95:5 hexane: ethyl acetate increasing to 20:80. Product fractions collected and solvent removed, The product was further washed with chloroform to remove residual ethyl acetate, and solvent removed again.

Resulting oily product was placed in vacuum oven to remove any residual solvent. Yield (35 %). ^! H NMR (400 MHz. CD€¾) 5: 1.30 (s, 3H), 1.42 (L 6H), 2.80 (t, 4H), 337 (t, 4H) _; 4.30 (in, 4H), 4.63 (q, 4H).

Synthesis of X t e (scheme S) - Xant d (scheme 5) (4.8 g, 9.9 mmoi) was transferred to a 100 mL round-bottomed flask equipped with a magnetic stirrer bar and septa cap containing outlet. DCM (30 mL) was added. 5 drops of DMF were added. Oxalyl chloride (2.5 g, 19.8 mmoi) was added dropwise via swinge. The reaction w&s left stirring for 3 hours. The solution, changed from pale yellow to dark orange as the reaction proceeds. The solvent was removed and the resulting oil was washed twice with chloroform to remove any residual oxalyl chloride. The product was in the form of viscous brown oil. Yield quantitative. Ή NMR (400 MHz, CDCI ₃ ) 0: 1.42 (m, 9H), 2.80 (t _s 4H), 3.38 (t 4H), 4.35 (m, 4H), 4.65 (q, 4H).

Synthesis of Xant-Gi (scheme 5)» Tertiary-bronioester alcohol (TBEA in scheme 5X1.8 g, 8.6 mmoi), TEA ( 1.8 mL, 12.9 mmoi) and DMAP (52.6 mg, 0.4 mmoi) were transferred to a 100 mL two-necked round -bottomed flask, equipped with a magnetic stirrer bar, dropping funnel and septa cap containing outlet. The flask was then deoxygenated using nitrogen. Dry DCM (30 mL) was added via syringe under nitrogen. Xant e (5.0 g, 9.9 mmoi) was deoxygenated using nitrogen inside the sealed dropping funnel. Dry DCM (10 mL) was added via syringe. Xant e was added dropwise. The flask was cooled in an ice bath during this addition. The reaction was left stirring overnight. The resulting brown solution was washed (1 x 80 mL distilled water and 2 x 80 mL brine). The organic layer was dried over Mg$€>4, and the solid filtered off. The solvent was reduced and the product was run through an automated flash column with a starting eluent of 100:0 hexane: ethyl acetate increasing to 20:80. Product fractions collected and solvent removed. The product was further washed with DCM to remove residual ethyl acetate, and solvent removed again. The resulting yellow/brown oil was left in a high vacuum vessel overnight to remove any residual solvent. Yield (40 %). 1H NMR (400 MHz, CDC1 ₃ ) δι 1.28 (s, 3H) _S 1.43 (t, 6H), 1.95 (s, 6H), 2.78 (t, 4H), 3.3? (t, 4H), 4.25 (ro, 4H), 4.42 (ra, 4H), 4,65 (q, 4H). Mass spec: m z ^:::: 703,0 [M+Naf ,

1 · 4.6 Gl, G2, G3 Xanthate ; dendren synthesis using bis MP A backbone For key references relating to the synthesis of bis-MPA dendrimers, refer to the following:

Macromolecules 2002, 35, 8307-8314

J. Am. Chem. Soc, 2001, 123, 5908-5917

J. Am. Chem. Soc, 2009. 131, 2906-2916

For preparation of benzylidene protected bis-MPA anhydride follow;

J. Am. Chem. Soc, 2001, 123, 5908-5917

For preparation of D ^' PTS 4-(Dimethyiamino)pyridini«m 4-toIuenesulfonate follow: J. S. Moore, S.I. Stupp, Macromolecuies, 1990, 23, 65

For preparation of 2-hydroxyethyl 2-bromo-2-methylpropanoate .follow:

J. Mater. Chem., 201 1 ,21, 18623-18629

Pre aration of Xanthate based carhoxyUc acid building block

Scheme 1 - Xanthate building block .1

Synthesis of 2-((Ethoxycarbonothioyi)thio)acetic acid 1 ·· A 500 ml. round-bottomed flask equipped with a dropping funnel was charged with a magnetic stirrer bar, potassium ethyl xanthogenate (26.77 g, 167 tnmol), and acetone (75 mL). A solution of 2-bromoacetic acid { 19,31 g. 103 mrnol) in acetone (40 mL) was added dropwise at room temperature over a period of 60 min. Stirring was continued overnight at room temperature. Solids were removed by filtration to afford a clear pale yeiiow solution. The solids on the funnel were washed with acetone (total of 50 mL), The combined washing and filtrate solutions were concentrated under vacuum to furnish a yellow viscous liquid that was dissolved in diehloromethane (150 mL). This solution was washed twice wiih brine (100 mL), and the organic phase was dried over MgS04 and evaporated to dryness to afford 18.75 g (75%) of a white solid. ^l H NMR (400 MHz, CDC1 ₃ ): δ - 1.43 (t, J === 7.32 Hz, 3H), 3.98 (s, 2H) 4.67 (q, J~ 7.2Ϊ Hz, 2H), 4.53 °C NMR (100 MHz, CDCk): 5 = 13.68, 37.60, 70.93, 174.30, 212.01

Scheme 2 - Preparation ofhis-mPA dendrons using anhydride chemistry

General procedure for dendon growth (2, 4 and 6} ~ To a SQOniL. oven-dried round- bottom flask equipped with a magnetic stirrer (under nitrogen atmosphere), the

I S benzylidene protected anhydride, the hydroxyl-termixiated dendron (generation 0 through to 3), and 4-dimethyiaminopyridine (DMAP) were all dissolved in a 1 :1 ratio of CH ₂ CI ₂ : pyridine (v/v). Alter stirring at room temperature for over 12 h, approximately 2 mL of water was added and the reaction was stirred for an additional 18 h in order to quench the excess anhydride. The product was isolated by diluting the mixture with€H >Ci? (150 ml.) and washing with 1 M NaHSCM (3 x 150 mL), saturated aqueous NaHCQj (2 x 150 mL), and brine (150 niL). The organic layer was dried over MgS04 and evaporated to dryness. Any residual solvent was removed under high vacuum overnight to yield a white foam with a typical yield greater than 95%.

General procedure for deprotection of henzyUdene by hydrogen tion (3, 5 and 7) · To a reactor suitable lor medium pressure hydrogenation fitted with a magnetic stirrer, the benzylidene protected dendriraer was dissolved in a 1 : 1 mixture of C¾Ch : MeOH (v/v). Pd(OH)i on carbon (20%) was added and the reactor was evacuated and back-filled with hydrogen three times (¾ pressure : 10 bar). After vigorous stirring tor 16 h, the reaction mixture was filtered through ceirte using a Buchner funnel and the filtrate was evaporated to dryness on a rotary evaporator under vacuo. The product was isolated as white foam in quantitative yields.

Scheme 3 - Preparation ofXanatkate dendrons and ATRP initiators

General procedure for surface group modification to Xanthaies (8, 9 and 10) - To a SQOrnL oven-dried round-bottom flask equipped with a magnetic stirrer (under nitrogen atmosphere), the hydroxyl-ter inated dendron (generation 0 through to 3), 2-((Ethoxyearbonothioyl)thio)aeetic acid I. and 4-(D imethylaniino)pyridmiura 4~ toluenesultonate (OPTS) were all dissolved in the minimum amount of CH ₂ CI ₂ . After the reaction flask was flushed with nitrogen, DCC was added. Stirring at room temperature was continued for 18 h under a nitrogen atmosphere. Once the reaction was complete the DCC- urea was filtered off and washed with a small volume of CH ₂ CI ₂ . The crude product was purified by liquid chromatography on silica gel, eluting with hexane gradually increasing to 40:60 ethyl aeetate/hexane to give a yellow viscous oil General procedure for deprotection of para-toluene sulfonyl ester (TSe) by DBU (11, 12 and 13) - To an oven-dried round-bottom flask equipped with a magnetic stirrer, the benzylidene protected dendrimer was dissolved in 50 mL of C¾Ck 1.4mL of 1,8- diazabicyclo[5.4.0]undec«7~ene (DBU) was added. The reaction was stirred under a nitrogen atmosphere for 3hrs and monitored until completion by TLC (60:40 hexane:ethyl acetate). The product was isolated by diluting the mixture with CH ₂ G2 ( 100 mi .) and washing with 1 M NaiiSO (2 x 100 mL), The organic layer was dried over MgS04 and evaporated to dryness. The product was then precipitated three times from hexanes, Any residual solvent was removed under high vacuum to yield a viscous oil with typical yields greater than 95%.

General procedure for focal point modification to an A TRP initiator by DCC/DPTS couplings (14, 15 and 16) ·· To a 500mL oven-dried round-bottom flask equipped with a magnetic stirrer (under nitrogen atmosphere), the carhoxyHc acid focal point xanthate dendron (generation 0 through to 3), 2-hydroxyethyl 2~brorno-2- methylpropano ate, and 4-(Dimethylamino)p> idhiium 4-toluenesulfonate (DPTS) were all dissolved in the minimum amount of CHiCk After the reaction flask was flushed with nitrogen, DCC was added. Stirring at room temperature was continued for 18 h under a nitrogen atmosphere. Once the reaction was complete the DCC- urea was filtered off and washed with a small volume of€¾¾. The crude product was purified by liquid chromatography on silica gel, eluting with hexane gradually increasing to 40:60 ethyl acetate/hexane to give a dark yellow viscous oil Synthesis of 2 ~ The dendron growth step was carried out as described above, using para-toluene suifonyl ethanol (lOg, 50 mmo!), benzylidene anhydride (42,65 g, 100 mmol. 2 equiv) and DMAP (2.57 g, 21 mmol)) dissolved in 220 ml... of dry {¾(¾ and 120 mL of pyridine, and stirred for 16 h at room temperature. Yield: 19.78 g. white foam (98%). ^l H NMR (400 MHz, CDChV b - 0.96 (s, 3H), 2.43 (s, 3H), 3.47 (t, J = 6.3 Hz, 2H), 3.60 (d, J = 11.6 Hz, 2H) 4.47 (t, 6.26 Hz, 2H) _; 4.53 (d, J = 11.54 Hz, 2H), 5.43 (s, ill), 7.33 (m ₅ 5H), 7.41 (m, 2H), 7.81 (d, J - 8.42 Hz, 2H). ¹³ C NM.R (100 MHz, CDC ): δ - 17.51, 21.64, 42.46, 55.13, 58.20, 73.32, 101.72, 126.15, 128.19, 128.23, 129.01 , 130.09, 136.01, 145.1 1, 149.86, 173.52.

Synthesis of 3 - Deproteciion of 2 (5.5g, 13.60 ramo!) in 210 ml... of CH ₂ Cl ₂ : MeOH (1 :1, v/v) was carried out as above for 16 h at room temperature under 10 bar ¾ atmosphere. 0.55g Pd(OH) ₂ was used. Yield: 4.3 g, white foam (99%). ^l E NMR (400 MHz. CD ₃ OD): δ - 1.03 (s, 3H), 2.45 (s, 3H), 3.50 (dd, J == 42.53, 10.95 Hz, 4H), 3.59 (t, I = 5.98 Hz, 2H) _S 4.39 (t, J - 5.85 Hz, 2H), 7.47 (d, 2H), 7.82 (d, 2H). ¹³ C NMR (100 MHz, CD ₃ OD): δ = 17.07, 21.61, 51.58, 55.90, 58.93, 65.66, 129.30, 131 .22, 137.76, 146.71 , 175.89.

Synthesis of 4 - The dendron growth step was carried out as described above, using 3 (4.10g, 12.96 mmol), benzylidene anhydride (16,58 g, 39 mrnoL 3 equiv) and DMAP (0.71 g, 5.38 mmoi)) all dissolved in 70 mL of dry C¾C and 35 mL of pyridine, and stirred for 16 h. at room temperature. Yield: 8,68 g, white foam (94%). "H NMR (400 MHz, CDC ): δ - 0.95 (s, 6H), 1.09 (s, 3H), 2.37 (s, 3H), 3.10 (t, J - 5.8 Hz, 2H), 3.60 (d, J = 12,45 Hz, 4H) 4.20 (m, 6H), 4.56 (t, J - 9 Hz, 4H), 5.42 (s, 2H), 7.30 (m, 8H), 7.39 (m, 4H), 7.68 (d, J = 8.43 Hz, 2H), ^{3 C NMR (100 MHz, CDCh): δ - 17.33, 17.72, 21.56, 42.60, 46.70, 54.65, 58.32, 65.20, 73.46, 73.53, 101.63, 126.12, 128.05, 128.16, 128.91, 130.00, 136.29, 137.78, 145.00, 172.00, 173.17. Accurate MS Calc'd for C^R^O^S [M + Naf == 747.2451. Found: [M + Naf = 742.2426, ES MS: [M + Na] ⁺ - 747.20, [M + K - 763.2 Synthesis of 5 ~ Deproieciion of 4 (7,90g, 10.90 mmol) in 190 mL of€¾¾ :

MeOil. (1 : 1, v/v) was carried out as above for 16 h at room temperature under 10 bar ¾ atmosphere. 0.40g Pd(OH) ₂ was used. Yield: 5.93 g, white foam (99%). ^¾ H NMR (400 MHz, CD3OD): 6 = 1.15 (s, 9H), 2.48 (s, 3H), 3.57-3.69 (m, 1QH), 4.1 1 (dd, J - 31.1 8, 9.37 Hz) 4H), 4.46 (t, J - 5.77 Hz, 2H), 7.49 (d, J == 8.81 Hz, 2H), 7.85 (d, J - 8.39 Hz, 2H). °C NMR (100 MHz, CD ₃ OD): b = 15.38, 15,94, 19.72, 45.76, 49.91, 53.92, 57.75, 63.95, 64.25, 127.40, 129,41 , 136.02, 144.82, 171.81, 173.94, Accurate MS Calc'd for C^HseO^S [M+Naf m/z - 571.1825, [M + Naf m/z - 571.1 821, Found ES MS: [M + Naf - 571 ,2, [M ÷ Kf - 587.2

Synthesis of 6 - The dendron growth step was carried out as described above, using 5 (2.5g, 4.56 mmol), benzyiidene anhydride (11.67 g, 27,36 mmol, 6 equiv) and DMA? (0.35 g, 2.83 mmol)) all dissolved in 46 mL of dry C¾C1 ₂ and 23 ml, of pyridine, and stirred for 16 h at room temperature. Yield: 6.23 g, white foam (94%). Ή NMR (400 MHz, CDCL): δ = 0,93 (m, 1 SH), 1.19 (s, 6H), 2.39 (s, 3H), 3.28 (t, J = 6,38 Hz, 2H), 3.58 (d, J ==== 1 1.82 Hz, 8H), 3.94 (dd, J - 30.95, 11.33 Hz, 4H), 4.33 (m, 10H), 4,56 (d, J - 12 Hz, 8H), 5.40 (s, 4H), 7.30 (m, 14H), 7.39 (m, 8B), 7.74 (d, J = 8.52 Hz, 2H), ^B C NMR (100 MHz, CDCI ₃ ): δ - 16.85, 17.66, 21,59, 42.59, 46.30, 46.87, 54.58, 58.22, 65.14, 65/70, 73.44, 73.52, 101.68, 126.20, 128.07,

128.13, 128.88, 1.30.04, 136.26, 137.82, 144,50, 171.63, 171.83, 173.20. ES MS: [M + Naf === 1387.5, [M + Kf - 1403.5

Synthesis of 7 - Deprotection of 6 (5.80g, 4.25 mmol) in 200 mL of CH ₂ C¾ ; MeOB (1 :1, v/v) was carried out. as above for 1 h at room temperature under 10 bar ¾ atmosphere. 0.29g Pd(OH) ₂ was used. Yield: 4.31 g, white foam (99%). ^l H NMR (400 MHz, CD ₃ OD): δ - 1.15 (m, 15H), 1.28 (s, 6H), 2.48 (5, 3H), 3,62 (m, 18H), 4.24 (m, 12H), 4.48 (t, J === ^: 6.14 Hz, 2H), 7.49 (d, J = 8.10 Hz, 2H), 7.85 (d, J - 8.19 Hz, 2H). ES MS: [M + Naf - 1035.4, [M + Kf == 1051 ,4

Synthesis of 8 - I, 4.65g (25,80 mmol), and 2.72g (8.60 mmol) of 3, 1.01 g (3.44 mmol) ofDPTS, and 5.86» (28.38 mmol) of DCC were allowed to react according to the general esterification procedure in 40 mL of dry CH2CI2 for 18 h. The crude product was purified by liquid chromatography on silica gel, e!uting with hexane gradually increasing to 40:60 ethyl acetate/hexane to give 6 as a yellow viscous oil 4,6g (84%). Ή NMR (400 MHz, CDC! ₃ ): δ = 1.16 (s, 3H), 1.42 (i, J - 7,15, 6H),

2.46 (s, 3H), 3.44 (t, J - 6.3 Hz, 2H), 3.91 (s, 4H), 4.18 (dd, J - 31.72, 1 1.36 Hz, 4H) 4.46 (t, J= 6,03 Hz, 2H), 4.64 (q, J ^■■■ 7.12 Hz, 4H), 7.39 (d, J - 8.23, 2H), 7.80 (d, J - 7.70, 2H), ^U C NMR (100 MHz, CDC1 ₃ ): h = 13.74, 17.56, 21.67, 37.70, 54.97, 58.36, 60,39, 66.21, 70.91, 128.12, 130.18, 136.18, 145.28, 167,33, 171.80, 212.57. ES MS: [M + Na - 663.0, [M + Kf = 679.0 Synthesis of 9 - 1, 9.97g (55.32 mmol), and 5.06g (9,22 mmol) of S, 2, 17g (7.38 mmol) of DPTS, and 12.56g (60.85 mmol) of DCC were allowed to react according to the general esteriiieation procedure in 170 ml, of dry CH ₂ CI2 for 18 h. The crude product was purified by liquid chromatography on silica gel, eluting with hexane gradually increasing to 50:50 ethyl acetate/hexane to give 6 as a orange viscous oil 9.65g (88%). ^S H NMR (400 MHz, CDCI3): 5 ==== 1.20 (s, 3H), 1.25 (s, 6H), 1.42 (t, J - 7.16, 12H), 2,47 (s, 3H), 3.44 (t, J - 5.97 Hz, 2H) _; 3.94 (s, 8H), 4.25 (ra, 12H) 4.46 (t, J= 5.90 Hz, 2H), 4.64 (q, J= 7.01 Hz, 8H), 7.40 (d, 1 - 8.51, 2H), 7.82 (d _s J === 8.31, 2H). Synthesis of 10 - See the genera! procedure

Synthesis of II -The removal of the para-toluene sv fonyl protecting group was carried out as described above, using 8 (4.60 g, 7.18 mmol, 1 .0 equiv), and DBU (I .40raL, 9.33 mmol, 1.3 equiv) dissolved in 80 mL of CH2CI2 and stirred tor 3 h. The reaction was monitored using TLC, 40:60 ethyl acetate/hexane. Yield: 3.2.9 g, orange viscous oil (99%), ⁵ H NMR (400 MHz, CDClj): δ - 1.32 {s, 3H), 1 ,42 (t, J - 7.05, 6H), 2.47 (s, 3H), 3.94 (s, 4H), 4.33 (dd, J = 39.96, 1 1.14 Hz, 2H), 4.64 (q, J- 7.14 Hz, 4H). ^{3 C NMR (100 MHz, CDC1 ₃ ): δ - 13.74, 17.86, 37.74, 46.06, 66.13, 70.87, 167.45, 177.80, 212.53. ES MS: [M + Naf - 481,0

For the synthesis of 12 and Ϊ3„ - see the general procedure

1.4.7 Gl Morpholine dendron initiator (Gl ML Br)

1J '-Carbonyldiimidazole (6.0994 g, 37.62 mmo!) was added to a 2-neek round bottom flask, which was equipped with magnetic stirring, condenser and a N ₂ inlet, Anhydrous toluene (60 ml) and "(2 iydroxypropyi)ni;orpholirie, I . (5.35 ml, 37.62 mmol) were added and the reaction was heated to 60 °C, The A¾ braneher (3.5603 g, 18.81 mmol) dissolved in anhydrous toluene (6.0 ml) was added after 3 hoars of reaction. After a further 16 hours the reaction was stopped, the toluene removed in vacuo, the crade mixture was dissolved in dichioromeiliane (100 ml) and washed with NaGH solution (pH 14) (3 x 100 ml). The organic phase was dried over Na ₂ S0 ₄ the solvent removed in vacuo and the resulting yellow oil was dried further under high vacuum to give 1, (75 %). Ή NMR (400 MHz, CDCi ₃ ): h 1.13 (d, 3H), 1,22 (d, 6H), 1.67 (m, 4H), 2.25-2.65 (br m, 18H), 3.22 (m, 4H), 3.68 (m, 8H), 3.79 (m, 1 H), 4.98 (m, 2H), 5.29 and 5.40 (br s, NH). ⁱ³ C NMR (100 MHz, CDC¾): δ 19.30, 20.83, 27.58, 27.76, 39.59, 52.28, 54.39, 62.86, 64.08, 67.36, 67.96, 68.12, 156.73. Caled.; [M] ⁺ m/z = 531 ,36, Found: ES-MS: [M÷Hf = 532.4, [M+Na - 554.4. Found, C, 56.58; H, 9.24; N, 13.23 %, C25H 9N5G7 requires, C, 56.47; H, 9.29; N, 13.17 %. 2, (7.546 g, 14.2 mmol) was dissolved in DCM (150 ml) and bubbled with N2 for 20 minutes. 4-(Diraethylamino)pyridine (D AP) (86.7 mg, 0,7 mmol) and

triethyiamine (TEA) (2.37 mi, 17.0 mmol) were added and the reaction vessel was cooled to 0 °C. o-Bromoisobutyryl bromide (1.93 nil, 15.6 mmol) was added dropwise, then the reaction was warmed to room, temperature for 16 hours. The reaction colour changed from pale yellow to a dark peach colour over this time period. The organic phase was washed with a saturated solution ofNaHC<¾ (3 x 150 ml) and distilled water (3 x 150 ml), dried over NaiSO^ and the solvent removed in vacuo to give a crude brown coloured oil, This was purified by silica column chromatography with a mobile phase ofEtOAc:MeOH (4:1 ), (Rf~ 0.49) to give a light brown coloured oil, 3, (49 %). Ή NMR (400 MHz, CDCI3): δ 1.24 (m, 9H), 1.65 (m, 4H), 1.92 (d, 6H), 2.26-2.70 (br m, 18B), 3.20 (m, 4H) ₅ 3.69 (m, 8H), 4.98 (m, 2H), 5.06 (rn, 1H) 5.36 (br s, NH). ⁾³ C NMR (100 MHz, CDCU r δ . Calcd.: [M m/2 = 679.32. Found: ES-MS: [M+Hf = 680.3, [ +Na] ⁺ - 702.3. Found, C, 50.87; H, 7.95; , 10.37 %. QgHj-j sQeBr requires, C, 51.17; H, 8.00; N, 10.29 %.

1 ,Γ-Carboaykiiimidazoie (9.729 g, 60,0 mxno!) was weighed into a ;

bottom flask fitted with a Na inlet, ^' magnetic stirrer and condenser. Anhydrous THF (120 mi) was added via double ended needle. The reaction was heated to 60 °C and iPbisMPA (10,4514 g, 60.0 ramoi) was added under a positive N2 Sow. Reaction could be observed by the evoiutioa of CO ₂ and the reaction became effervescent. To avoid too much effervescence the iPbisMPA was added slowly, approx. 2g at a time once the effervescence had died down. After 3 hours the reaction mixture was bubbled through with N ₂ to ensure any residual CO _? , had been removed from the reaction medium and flask. The AB ₂ brancher (5.949g, 30.0 mmo!) was added dropwise in anhydrous THF (20 ml), after a further 18 hours the reaction was stopped and THF removed in vacuo. The crude residue was dissolved in DCM (125 mi) and washed with NaOH solution (pH14) (3 x 125 ml) and distilled water (125 ml). The organic phase was dried over Na ₂ SC¾ and the DCM was removed in vacuo then under high vacuum, to give a pale yellow oil, 1, (78 %). Ή NMR (400 MHz, CDCI3): δ 1.02 (s, 61·-!), 1.10 (d, 3H) ₃ 1.42 (s _s 6H), 1.47 (s, 6H), 1.70 (m, 4H), 2.32 (d of d of d, 2H), 2.45 (m, 2H), 2.63 (m, 2H), 3.34 (q, 4H), 3.75 (m, 5H), 3.92 (d, 4PI). , ³ C NMS. (100 MHz, CDCI3): δ 18-30, 1 .1 1 , 20.38, 27.57, 29.08, 37.87, 40.59, 51.85, 63.00, 63.64, 67.54, 98.93, 175.24. Calcd,: [M wi/z - 501.34. Found: CI-MS: [M+H] ⁺ - 502.7. Found, C, 59,86: H, 9,41 ; N, 8.18 %. C25H47N3O7 requires, C, 59.86; FL 9.44; N. 8.38 %. Gl MP A OH dendron (5, 127 g, 10,2 mmol) was weighed into a round bottom flask and dissolved in DCM (70 mi) and degassed with dry nitrogen for 10 rain. DMAP ( 62 mg, 0.51 mmol) and TEA (1.71 ml, 123 mmol) were added, the vessel was maintained under a positive nitrogen flow and cooled to 0 °C. a-Bromoisobutyryl bromide (1.38 ml, 1 1.2 mmol) was added dropwise then was warmed to room temperature for 1 S hours. The reaction was a light yellow colour to begin with and changed to a slightly darker yellow over time, no precipitate was observed. The reaction mixture was washed with a saturated NaHCG ₃ solution (3 x 100 ml) and water (3 x 100 ml), dried over Na ₂ S0 and concentrated in vacuo to give Gl MP A Br, 2, (54 %) as a yellow viscous oil. Ή NMR (400 MHz, CDC¾): d 1.04 (s, 6H) _S 1.24 (d, 3H 1.42 (s, 6H), 1.46 (s, 6H), 1 .67 (m, 4H), 1.91 (s, 6H), 2.40-2.67 (m, 6H), 3.31 (m, 4H), 3.74 (d, 4H), 3.96 (d, 4H), 5.05 (m, I ). °C NMR (100 MHz, CDC!;): δ 18.35, 18.43, 27.58, 28.48, 31.16, 37.85, 40.69, 52.09, 56.54, 59.54, 67.44, 67.51, 70.94, 98.74, 171 .67, 175.12. Calcd.: [M] ⁺ m/z - 649.29. Found: ES-MS: requires, C, 53.53; H, 8.06; N, 6.46 %.

_■ QA-A Tertiary amine dendron initiator Synthesis of G1~A dendron

1 -dimethylamino-2~propanol (2,4758 g, 24 mmol, 4 eq.) was added to a 1 0 mL 2 necked round-bottomed flask containing anhydrous toluene (20 mL) and fitted with a reflux condenser, magnetic stirrer and a positive flow ofN ₂ . The solution was stirred at room temperature and CD ^' S (1.9458 g. 12 mmol, 2 eq.) was added. The mixture was heated to 60 ⁼ C with stirring for 6 hours. AB ₂ brancher (1.1358 g, 6 mmol, 1 eq.) dissolved in anhydrous toluene (5 mL) was deoxygenated using a N ₂ purge for 10 minutes and was added drop wise while the solution was stirred and the temperature was maintained at 6Q°C. The reaction was stirred for a further 18 hours at 60°C, and then allowed to cool to room temperature. The solution was concentrated in vacuo, and the remaining oil was dissolved in DCM (30 mL) and washed with 1M NaOH solution (3 x 30 mL), The solution was dried with anhydrous Na^SO, filtered and concentrated in vacuo to give Gl-A as a viscous liquid. Ή NMR (400 MHz, CD£¾) δ 1 ,25 (ra 9H), 1.64 (m, 3H), 2.05-2.67 (m, 22H), 3.20 (m, 3H), 3.78 (m, !H), 4.89 (m, 2H). m/z (ES MS) 448.4 [M+Hj-h 470.3 [M+Na]+. Synthesis of Gl-A dendron initiator

Gl-A (0.8944 g, 2 mmol, 1 eq.), TEA (0,2833 g, 2.8 mmol, 1 ,4 eq.) and DMA? (24.43 mg, 0/2 mmol, 0.1 eq.) were added to a 100 mL 2 necked round-bottomed flask containing DCM (40 mL-). The flask was deoxygenated under a positive N ₂ pisrge for 10 minutes, a-bromo isobutyry 1 bromide (0,4828, 0.26 niL, 2.7 mmoL 1.05 eq.) was added drop wise while the solution was stirring in an ice bath under a positive flow of N ₂ . The reaction mixture was allowed to warm to room temperature and left stirring overnight. The organic phase was washed with saturated sodium hydrogen carbonate (NaHCO-j) solution (3 x 30 mL). The solution was dried with anhydrous NaaSO-j, filtered and concentrated in vacuo to give initiator Gl-A as a viscous yellow liquid. ⁸ H NMR (400 MHz, CDCI3) δ 1 .24 im, 9H) ₅ 1.64 (ra, 4H), 1.92 (d of d, 8H), 2.05-2.05-2.67 (m, 22H), 3.21 (m, 4H), 4.89 (m, 2H), 5,06 (m, 1H). m/z (ES MS) 596.3 [M+H]+, 617,3 [M+Na]+, 639.2 [M+Kj-h

1.4.10 Gl-D Tertiary amine dendron initiator Synthesis oj ^' Gl-D dendron (L

2~(Dirnethylamino)ethyl acrylate (6.0 g, 42 rnmol, 6 eq.) was added to a 50 mL round 2 necked round-bottomed flask containing IP A (12 mL). The flask was deoxygenated under a positive purge for 10 minutes. 1 -amino-2-propanol (0.5246 g, 7.0 iranol, 1 eq.) dissolved in IPA (12 mL) was added drop wise while the solution was stirring in an ice bath under a positive flow of a- The final mixture was stirred for a fort her 10 minutes at 0°C before being allowed to warm to room temperature and left stirring ibr 48 firs. The solvent was removed and the product left to dry in vacuo overnight. Ή NMR (400 MHz, CDC1 ₃ ) 6 1.08 (d, 3H) _f 2.18-2.62 (m, 22H), 2.69 (m, 2H), 2,89 (m, 2H), 3.77 (m, 1 H), 4.16 (m, 4H). m/z (ES MS) 362.3 [M+H]+, 3S4.3 [M+Na]+. Synthesis of Gi-D dendron initiator (HR2-143)

Gl-D dendron (1 , 1207 g, 10.86 rnmol 1 eq.), TEA (1.5390 g, 15.2 nimoL 1.4 eq.) and DMAP ( 132.7 mg, 1.086 rnmol, 0.1 eq.) were added to a 250 mL 2 necked round-bottomed flask containing DCM ( 160 mL). The flask was deoxygenated under a positive N ₂ purge for 10 minutes, a-bromoisobutyryi bromide (2.622 g, 1.4 mL,

34 1 1 A mmol, 1.05 eq.) was added drop wise while the solution was stirring in an ice bath under a positive flow of . The reaction mixture was allowed to warm to room temperature and left stirring overnight. The organic phase was washed with saturated sodium hydrogen carbonate ( aHCOii) solution (3 x 160 ml,). The solution was dried with anliydrous Na ₂ S0 and the product left to dry in vacuo overnight. Ή NMR (400 MHz, CDC1 ₃ ) δ 1.22 (d, 3H), 1.89 (m, 6H), 2.24-2.69 (m, 22H), 2.83 (m, 4H), 4.20 (m, 4H), 5.0 (ni, 2H). m/z (ES MS) 510.2 [M÷H]+, 534.2 [M+Naj-K

Synthesis of G2-D dendron (HR2~H6)

—

\

2~(Dimethylarrnno)eth.y! acrylate (6.0 g, 42 mrnol, 6 eq.) was added to a 50 mL round 2 necked round-bottomed flask containing IPA (12 mL). The flask was deoxygenated under a positive N ₂ purge for 10 minutes. Bis(3- arainopropyi)amino)propan-2~ol (1 ,3221 g, 6.984 mmol, 1 eq.) dissolved in IPA (12 mL.) was added drop wise while the solution was stirring in an ice hath under a positive flow ofNi-. The final mixture was stirred for a further 10 minutes at 0°C, allowed to warm to room temperature and left stirring for 48 hrs. The solvent was removed and the product left to dry in vacuo overnight. *H NMR (400 MHz, CDCI ₃ ) 6 1.13 (d 3H) ₅ 1.67 (m, 4H), 2.26-2.65 (m, 50H) ₅ 2.77 (m, 8H), 3.87 (m,lH), 4.17 (m, 8H). m/z (ES MS) 762.6 [M+H]+, 784.6 [M+Na]+.

1.4.1 1 G2-P Tertiary amine dendroa initiator

Synthesis of G2-D dendron initiator (HR2-121)

G2-dendron (5.1431 g, 6.749 mmol, 1 eq.), TEA (0.9561 g, 9.449 mmol, 1.4 eq.) and DMA? (82.5 mg, 0.6749 ramol, 0.1 eq.) were added to a 250 mL 2 necked round- bottomed flask containing DCM (160 nil.,). The flask was deoxygenated under a positive N ₂ purge for 10 minutes, obromoisobutyryl bromide (1.629 g, 0,88 mL, 7.087 mmol, 1.05 eq.) was added drop wise while the solution was stirring in an ice hath under a positive flow ofNj. The reaction mixture was allowed to warm to room temperature and left stirring overnight. The organic phase was washed with saturated sodium hydrogen carbonate (NaHCCh) solution (3 x 160 mL). The solution was dried with anhydrous Na ₂ SQ and the product left to dry in vacuo overnight. Ή NMR (400 MHz, CDCfe) δ 1.26 (d, 3H), 1.56 (m, 4H), 1.91 (m ₅ 6H), 2.22-2.67 (m, 50H), 2.76 (m, 8H), 4.19 (a 8H), 5.04 (m, 1H). m/z (ES MS) 912.5 [M÷H]÷, 934.5 [ - Na - 950.5 [M+K]+. 2, P lydendmns · 1θϋ% dendron initiated branched polymers

2.1.1.1 Aromatic dendrons Gl and G2 DBOF Br In a typical experiment, Gl DBOF Br (0.291 g, 0.69 mmol) or G2 DBOF Br (0.648 g, 0.69 mmol) and HPMA (targeted DP = 50) (5.0 g, 34.7 mmol) were weighed into a round bottom flask. EGDMA (105 μ\, 0.55 mmol) was added and the flask was equipped with magnetic stirrer bar, sealed and degassed by bubbling with N ₂ for 20 minutes and maintained under N ? at 30 °C. Anhydrous methanol was degassed separately and subsequently added to the mono mer/init iator/brancher mixture via syringe to give a 50 % v/v mixture with respect to the monomer. The catalytic system; Cu(I)Cl (0.069 g, 0.69 mmol) and 2,2'-bipyridyi (bpy) (0.217 g, 1.39 mmol), were added under a positive nitrogen flow in order to initiate the reaction. The polymerisations were stopped when conversions had reached over 98 %. The polymerisations were stopped by diluting with a large excess 0 f tetrahydro furan

(THF), which caused a colour change from dark brown to a bright green colour. The catalytic system was removed using Dowex ^® Marathon ^{1 M} MSG (hydrogen form) ion exchange resin beads and basic alumina. The resulting polymer was isolated by precipitation from the minimum amount of THF into cold hexane. The

[initiator] : [CuCi] : [bpy] molar ratios in all polymerizations were 1 : 1 :2. Other DPs targeted were DP20 and DPI 00 with both Gl and G2 DBOP initiators.

2.1.1.2 BOC dendrons Gl ¾OC Er The Gl BOC Dendron initiator ( lOOmg, 0.186ramol) was added to a 25 ml, round bottom flask equipped with a magnetic stirrer bar, followed by the addition of 2,2- bipyridyl (58.1mg, 0.372mrnol), EGDMA (35, 1 nig, 0J 77mmoi) and HPMA (1.34j 9.28rnmol). The reaction mixture was bubbled with N ₂ for 15 minutes. Degassed anhydrous methanol (3.45mL) was added to the flask, and its contents stirred and bubbled with N ₂ for a further 5 minutes. Copper (1) chloride (18.4mg, 0.186mmol) was quickly weighed out and added to the flask, instantly forming a brown coloured mixture, which was stirred and bubbled with N ₂ for a further 5 minutes. A N? pressure was then built up within the flask, then Ni inlet removed, and the flask stirred for 24 hours at 40 °C. Once the polymerisation was complete. THF was added to the reaction flask to poison the Cu (I) catalyst, forming a green coloured solution. The solution was passed through an alumina (neutral) column to remove the catalytic system, concentrated in vacuo, and precipitated into hexane. The supernatant was decanted off, and the remaining white solid dried overnight in a vac- oven.

2.1.1,3 Xanthate dendron Xant-Gl Xant-Gl initiator (578.0 rag, 0.868 mmol), BIPY (272.2 mg, 1.743 rnrnol), HPMA

(6.3 g, 43.6 mmol,) and EGDMA (146.8 rag, 0.741 mmol) was transferred to 25 ml, round-bottomed flask equipped with stirrer bar and septa cap. The .flask was deoxygenated using nitrogen. Separately deoxygenated MeOH (12.9 ml,, 38% w/v based on HPMA) added via syringe. Once all reactants had dissolved, nitrogen was bubbled through solution for 5 rnins. Cn (I) CI (86.3 mg. 0.868 mmol,) quickly measured out and added to round-bottomed flask. Reaction mixture went from clear solution to deep red/brown, Nitrogen was bubbled through solution tbr an additional 10 mins. The reaction was then left to stir overnight under nitrogen. Reaction mixture forms a deep red/brown viscous liquid on completion. THF (20 ml,) added to kill reaction. Once solution turned a bright green colour, solution passed through a short alumina c lumn to remove copper catalyst, yielding a translucent pale green solution. Solvent removed and resulting oily liquid precipitated into cold hexane (approx, 50 ruL, cooled in dry ice bath). The resulting pale green crystals were filtered off and washed with cold hexane. The sample was placed in a vacuum oven to remove any residual solvent.

2.1.2 Hvdropkilic dendrons 2.1.2.1 Gl-A Tertiaxy _. an ineMtiaigr

In a typical synthesis, targeting a number average degree of polymerisation (DP _» ) - 50 monomer units = poiy( ϊΡ Α)^ nDEAF.MA njnit ator: 50), bpy (173.3 mg, 1 .1096 mmol, 2 eq.), HPMA. (4 g, 27.7 mmol, 50 eq.), EGDMA (77,0 mg, 0.3883 mmol, 0.7 eq) and isopropanol (IP A) (38,9% v/v based on HPMA) were placed into a 25 ml,- round-bottomed flask. The solution was stirred and deoxygenated using a nitrogen ( ) purge for 15 minutes. Cu(i)Cl (54.9 mg, 0.5548 mrnol, 1 eq.) was added to the flask and left to purge for a further 5 minutes. G l-A dendron initiator (0.3310 g, 0.5548 mrnol, 1 eq.) was added to the flask under a positive flow of N ₂ , and the solution was left to polymerise at 4G°C, Reactions were terminated when >99% conversion was reached, as judged by Ή NMR, by exposure to oxygen and addition of THF. The catalyst residues were removed by passing the mixture over a basic alumina column. THF was removed under vacuum to concentrate the sample before precipitation into hexane and drying in the vacuum oven overnight.

2.1.2.2 Gl morphoUne initiator (Gl ML Br)

Gl ML Br (0.378 g, 0.55 mmol) and HPMA (4.0 g, 27.7 mmol) were weighed into a round bottom flask. EGDMA (73.2 μί, 0.39 mrnol) was added and the flask was equipped with magnetic stirrer bar, sealed and degassed by bubbling with N ₂ for 20 minutes and maintained under N ₂ at 30 °C Isopropanol was degassed separately and subsequently added to the monomer/initiator brancher mixture via syringe to give a 50 wt/wt% mixture with respect to the monomer. The catalytic system; Cu(i)Ci (0.055 g, 0,55 mmol) and 2,2'-bipyridyl (bpy) (0.1 73 g, 1.1 mmol), were added under a positive nitrogen flow in order to initiate the reaction. The polymerisations were stopped when conversions had reached over 98 %. The polymerisations were stopped by diluting with a large excess of tetrahydrofuran (THF), which caused a colour change from dark brown to a bright green colour. The catalytic system was removed using Dowex® Marathon ^{1 M} MSG (hydrogen form) ion exchange resin beads and basic alumina. The resulting polymer was isolated by precipitation from the minimum amount of THF into cold hexane. The [initiator] : [CuCl] : [bpy] molar ratios in all polymerizations were 1 : 1 :2 2.1.2.3 Gl bisMPA initiator (Gl MP A Br)

CM MFA Br (0.451 g, 0,69 rnmol) and HPMA (5.0 g, 34,7 mmol) were weighed into a round bottom flask. EGDMA (105 μΐ, 0.55 rnmol) was added and the flask was equipped with magnetic stirrer bar, sealed and degassed by bubbling with N ₂ for 20 minutes and maintained under ₂ at 30 °C. Isopropanol was degassed separately and subsequently added to the monomer/initiator/branchet mixture via syringe to give a 50 wt/wt% mixture with respect to the monomer. The catalytic system; Cu(I)Cl (0.0687 g, 0.69 mmol) and 2,2'-bipyridyl (bpy) (0.217 g, 1.39 mmol), were added under a positive nitrogen flow in order to initiate the reaction. The polymerisations were stopped when conversions had reached over 98 %. The polymerisations were stopped by diluting with a large excess of tetrahydrofuran (THF), which caused a colour change from dark brown to a bright green colour. The catalytic system was removed using Do ex ^® Marathon ^{5 M} MSC (hydrogen form) ion exchange resin beads and basic alumina. The resulting polymer was isolated by precipitation from the minimum amount of THF into cold hexane. The [initiator] : [CuCl] : [bpy] molar ratios in all polymerizations were 1 : 1 :2.

2- tB¾MA (hydrophobic core)

2.2.1 Ct-A Tertiary amine dendron initiator

In a typical synthesis, targeting a number average degree of polymerisation (DP„) - 50 monomer units (poIy(iBuMA)s!j; ι¾ΕΑΒ Α ^η ω««ο!·: 50), bpy (175.7 mg, 1.1252 mmol, 2 eq.), .BuMA (4 g, 28.13 mmol, 50 eq.), EGDMA (105.9 rag, 0.5345 mmol, 0.95 eq) and aqueous isopropanol (7.5% water by volume) (33.3% v/v based on ?BuMA) were placed into a 25 mL round- bottomed flask. The solution was stirred and deoxygenated using a nitrogen (Nj) purge for I S minutes. CuQCl (55.7 mg, 0.5626 mmol, 1 eq.) was added to the flask and left to purge for a further 5 minutes. Gl-A dendron initiator (0.3356 g, 0.5626 rnmol, 1 eq.) was added to the flask under a positive flow of N _∑ , and the solution was left to polymerise at 40°C. Reactions were terminated when >99% conversion was reached, as judged by ^l H MR, by exposure to oxygen and addition of THF. The catalyst residues were removed by passing the mixture over a basic alumina column. THF was removed under vacuum to concentrate the sample before precipitation into hexane and drying in ihc vacuum oven overnight.

2,3

2.3.1 Gl-A Tertiary amine dendron initiator in a typical synthesis, targeting a number average degree of polymerisation (DP,.) ^::: 50 monomer units (poly(DEAEMA)so; DEAEN ni tiator: 50), bpy (134.9 rng. 0.8637 mmol, 2 eq.), DEAEM (4 g, 21.59 mmol, 50 eq.), EGDMA (77.0 mg, 0.3886 mmol, 0.9 eq) and IP A ^{3 ,} (38.9% v/v based on DEAEMA) were placed into a 25 mL round-bottomed flask. The solution was stirred and deoxygenated using a N? purge for 15 minutes. Cu(i)Cl (42.8 n g, 0.4318 mmol, 1 eq.) was added to the flask and left to purge for a further 5 minutes, G1 ~A dendron initiator (0.2576 g, 0.4318 rnmol, 1 eq.) was added to the flask under a positive flow of N ₂ , and the solution was l ft to polymerise at 40°C. Reactions were terminated when >99% conversion was reached. as judged by ^! H NMR, by exposure to oxygen and addition of acetone, The catalyst residues were removed by passing the mixture over a basic alumina column. Acetone was removed under vacuum to concentrate the sample before precipitation into cold petroleum ether (40°C - 60°C). The polymerisation conditions and procedure is identical to those described for linear polymers above and drying in the vacuum oven overmght

2.3.2 G0-D Tertiary amine dendron initiator

In a typical synthesis, targeting a number average degree ofpolymerisation (DP„) - 50 monomer units (poly(DEAEMA)so; nDEAi nimttaor: 50), bpy (134.9 r g, 0,8637 mmol, 2 eq.), DEAEMA (4 g, 21.59 mmol, 50 eq.), EGDMA (77.0 mg, 0.3886 mmol 0.9 eq) and IPA ⁱ⁷ (38.9% v/v based on DEAEMA) were placed into a 25 mL round -bottomed flask. The solution was stirred and deoxygenated using a N ₂ purge for 15 minutes. CuQCl (42.8 mg, 0.4318 mmo!, 1 eq.) was added to the flask and left to purge for a further 5 minutes. GO-D dendron initiator (0,1089 g, 0.4318 mrnol, I eq.) was added to the flask under a positive flow of N ₂ , and Che solution was left to polymerise at 40°C, Reactions were temnnated when >99% conversion was reached, as judged by ^l E NMR, by exposisre to oxygen and addition of acetone. The catalyst residues were removed by passing the mixture over a basic alumina column. Acetone was removed under vacuum to concentrate the sample before precipitation into cold petroleum ether (40°C - 60°C) and drying in the vacuum oven overnight. The polymerisation conditions and procedure is identical to those described for linear polymers above.

2.3.3 Gl-D Tertiary amine dendron initiator

In a typical synthesis, targeting a number average degree of polymerisation (DP,,) ^::: 50 monomer units (poly(DEAEMA)5o; nDEAEMA/ni tiator'. 50), bpy (134.9 mg, 0.8637 mrnol 2 eq.), DEAEMA (4 g, 21.59 mrnol, 50 eq.), EGDMA (77.0 mg, 03886 mrnol, 0.9 eq) and IPA ³ ' (38.9% v/v based on DEAEMA) were placed into a 25 mi, round-bottomed flask. The solution was stirred and deoxygenated using a N ₂ purge for 15 minutes. CuQCl (42,8 mg, 0.4318 mrnol, 1 eq.) was added to the flask and left to purge for a further 5 minutes. G l -D dendron initiator (0.2204 g, 0.4318 mrnol, 1 eq.) was added to the flask under a positive flow of M?, and the solution was left to polymerise at 40°€. Reactions were terminated when >99% conversion was reached, as judged by ^l H NMR, by exposure to oxygen and addition of acetone. The catalyst residues were removed by passing the mixture over a basic alumina column. Acetone was removed under vacuum to concentrate the sample before precipitation into cold petroleum ether (40°C ~ 60°C) and drying in the vacuum oven overnight. The polymerisation conditions and procedure is identical to those described for linear polymers above,

2.3.4 G2-I) Tertiary amine dendron initiator

in a typical synthesis, targeting a number average degree of polymerisation (DP _n ) - 50 monomer units (poly( DEAE Α)$β; ^Di- MA^imiisior- 50), bpy (134.9 mg, 0,8637 mrnol, 2 eq.), DEAEMA (4 g, 21.59 mrnol, 50 eq.), EGDMA (77.0 mg, 03886 mmo i, 0.9 eq) and IPA ³⁷ (38,9% v/v based on DEAEMA) were placed into a 25 mL round-bottomed flask. The solution was stirred and deoxygenated using a N ₂ purge for 15 minutes.€u(s)Cl (42.8 rag, 0.4318 mmol, 1 eq.) was added to the flask and left to purge for a further 5 minutes. G2-D dendron initiator (0.3934 g, 0,431 8 mmol, 1 eq.) was added to the flask under a positive flow of N?, and the solution was left to polymerise at 40°C. Reactions were terminated when >99% eonversion was reached, as judged by Ή NMR, by exposure to oxygen and addition of acetone. The catalyst residues were removed by passing the mixture over a basic alumina column. Acetone was removed under vacuum to concentrate the sample before precipitation into cold petroleum ether (40°C - 60°C) and drying in the vacuum oven overnight. The polymerisation conditions and procedure is identical to those described for linear polymers above.

2.4 OEGMA ( ydrophiiic core) 2.4.1 Gl-A Tertiary amine dendron initiator

In a typical synthesis, targeting a number average degree of polymeria don (DP,,) ^::: 50 monomer units (poiy(OEG!Vf A) ₅ -o; ηοΕΑΕ Α<¼η!ίΐ3¾>ι·: 50), bpy (83.3 mg, 0.5333 mmol, 2 eq.), OEGMA (4 g, 13.3 mmol, 50 eq.), EGDMA (50.2 mg, 0.2533 mmol, 0.95 eq) and aqueous isopropanol (7.5% water by volume) (33.3% v/v based on

OEGMA) were placed into a 25 mL round-bottomed flask. The solution was stirred and deoxygenated using a nitrogen (N ₂ ) purge for 15 minutes. CuQCl (26.4 mg, 0.2667 mmol, 1 eq.) was added to the flask and left to purge for a further 5 minutes. Gl-A dendron initiator (0, 1591 g, 0.2667 mmol, 1 eq.) was added to the flask under a positive flow of N ₂ , and the solution was left to polymerise at 40°C. Reactions were terminated when >99% conversion was reached, as judged by Ή NMR. by exposure to oxygen and addition of THF. The catalyst residues were removed by passing the mixture over a basic alumina column. THF was removed under vacuum to concentrate the sample before precipitation into cold hexane and drying in the vacuum oven overnight.

2.5 Co olymer sy hesis 2.5.1 G2-D Tertiary amine initiator. pDEAEIv!A^-b-ptB¾M.½-st.-

In a typical synthesis, targeting a number average degree of polymerisation (DP _f! ) ^::: 50 monomer units (poly(DEAEMA)5o; nDEAEMA/ninitiatof: 50), bpy (134.9 mg, 0.8637 mmoL 2 eq.) ₅ DEAEMA (4 g, 21.59 mmol, 50 eq,) and isopropanol (ΪΡΑ) (37.7% v/v based on DEAEMA) were placed into a 50 mL round- bottomed flask, The solution was stirred and deoxygenated using a nitrogen (N?) purge for 15 minutes, Cu(i)Cl (42.8 mg, 0.4318 mmol, 1. eq.) was added to the flask and left to purge for a further 5 mkustes. G2-D dendron initiator (0.3934 g, 0.4318 mmol, 1 eq,) was added to the flask under a positive flow of N ₂ , and the solution was left to polymerise at 40°C. In another 25 ml. round-bottomed flask, bpy (134.9 mg, 0.8637 mmol), rBuMA (4,0 g, 28,1 mmol, 65 eq.), EGDMA (77.0 mg, 0.3886 mmol, 0,9 eq) and aqueous

isopropanol (23.8% v/v based on iBuMA) were added. The solution was stirred and deoxygenated using a nitrogen (N?) purge for 15 minutes, Cu(s)Cl (42,8 mg, 0,4318 mmo!, 1 eq.) was added to the flask and left to purge for a further 5 minutes. After the conversion of DEAEMA reached around 85%, the mixture from the second flask was added into the first flask rapidly using a syringe and taking care not to admit any air into the vessel. A sample was taken immediately after the addition of the iBuMA monomer solution for Ή N!vlR analysis. The block copolymerization reaction was carried out at ambient temperature and samples were taken periodically from the reaction mixture for ^l H NMR analysis, Reactions were terminated when >99% conversion was reached, as judged by li NMR, by exposure to oxygen and addition of acetone. The catalyst residues were removed by passing the mixture over a basic alumina column. Acetone was removed under vacuum to concentrate the sample before precipitation into cold petroleum ether (40°C - 60°C) and drying in the vacuum oven overnight.

2¾6!e i - OO % . Dendron initiated poiydendrons

Generatio Initiator Polymer EGDMA

Mn (gmol ^"1 ) Mw (gmor) PDI n Functionality Core (mol%)

Gl DBOP pHPMA20 0,8 52 800 545 000 10.32

Gi DBOP pHPMASO 0.8 47 200 1 169 000 24,74 Gl DROP pHPMAl OO 0.8 69 300 1 354 500 19.54

G2 DBOP pHPMA:20 0.8 153 000 1 565 000 10.23

G2 DBOP pHPMASO 0,8 59978 739440 12,33

G2 DBOP pHPMAlOO 0,8 164 200 2 227 500 13,58

G l tBOC pHPMASO 0.95 177 7 45539 3.71

Gl Xanthate pHPMASO 0.85 63800 1070000 15

Gl Morpholine pHPMASO 0.7 76687 454746 5.93

Gl bisMPA pHPMASO 0.8 77745 436461 5.61

Gi-A /-amine pHPMASO 0,7 661 180 966552 1 ,50

Gl -A i-amine pfBuMASO 0.95 150264 284002 1.90 pDEAEMAS

GI -A /-amine 0.9 201497 244622 1.20

0

Gl-A i-amine pOEGMASO 0.95 97082 216813 2.20 pDEAEMAS

GO-D i-amine 0,9

0

pDEAEMAS

G l-D /•-amine 0.9

0

pDEAEMAS

G2-D / ^•• amine 0,9 125652 302557 2.40

0

pDEAEMAS

0-6-

G2-D /-amine 0.9 129737 374192 2,90 st-EGDMA

he Gl ¾OC Dendron initiator (67.9mg _s 0, !26ramoi) and G2 ¾OC Dendron litiator (63.1 rag, 0.054mmol) was added to a 25 mL round bottom flask equipped. with a magnetic stirrer bar, followed by the addition of 2.2~bipyridyl (56.2mg _s G,360mmol), EGDMA (28.5mg, 0J 44r;unol} and HFMA (i .3g, 9.0 n ou The reaction mixture was bubbled with N ₂ for 15 minutes. Degassed anhydrous methanol (3.3mL) was added to the flask, and its contents stirred and bubbled with N ₂ for a further 5 minutes. Copper (I) chloride (17. Smg, 0.180mmol) was quickly weighed out and added to the flask, instantly forming a brown coloured mixture, which was stirred and bubbled with M? for a further 5 minutes, A N ₂ pressure was built up within the flask, then N ₂ inlet then removed, and the flask stirred for 24 hours at 40 °C. Once the polymerisation was complete, THF was added to the reaction flask to poison the Cu (Ϊ) catalyst, forming a green coloured solution. The solution was passed through an alumina (neutral) column to remove the catalytic system, concentrated in vacuo, and precipitated into hexane. The supernatant was decanted off, and the remaining white solid dried overnight in a vac-oven. 3,2 Mixed deftdro with ¾oii-dei_dron toitiator

3,2.1 G2 DBOP Br and 750 PEG initiated pHPMA core

In a typical reaction, G2 DBOP Br (0.259 g, 0.28 mmo!) and 750 PEG initiator (0.250 g, 0.28 mmol) (for a targeted ratio of G2 dendron:750 PEG of 50:50 mol%) were weighed into a round bottom flask, followed by HPMA (4.0 g, 27.7 mmol), EGDMA (84 μΐ, 0.44 mmol) was added and the ilask was equipped with magnetic stirrer bar, sealed and degassed by bubbling with N ₂ for 20 minutes and maintained under N ₂ at 30 °C. Anhydrous methanol was degassed separately and subsequently added to the monomer/initiator brancher mixture via syringe to give a 50 wt/wt% mixture with respect to the monomer. The catalytic system; Cu(I)Cl (0.055 g, 0.55 mmol) and 2,2'-bipyridyl (bpy) (0.173 g, 1 , 1 mmol), were added under a positive nitrogen flow in order to initiate the reaction. The polymerisations were stopped when conversions had reached over 98 %, The polymerisations were stopped by diluting with a large excess of tetrahydrofuran (THF), which caused a colour change from dark brown to a bright green colour. The catalytic system was removed using Dowex ^® Marathon™ MSG (hydrogen form) ion exchange resin beads and basic alumina. The resulting polymer was isolated by precipitation from the minimum amount of THF into cold hexane. The [initiator] : [CuClj : [bpy] molar ratios in all polymerizations were 1 :1 :2.

3.2.2 G2 DBOF Br and 2K PEG initiated pHFMA core

In a typical reaction, G2 DBOP Br (0.324 g. 0.35 mmol) and 2K PEG initiator (0.745 g, 0.35 mmol) (for a targeted ratio of G2 dendron:750 FEG of 50:50 raol%) were weighed into a round bottom flask, followed by HPMA (5.0 g, 34.7 mmol). EGDMA (112 μ\, 0.59 mmol) was added and the flask was equipped with magnetic stirrer bar, sealed and degassed by bubbling with N ₂ for 20 minutes and maintained under N? at 30 °C, Anhydrous methanol was degassed separately and subsequently added to the monomer/initiator/braneher mixture via syringe to give a 50 % v/v mixture with respect to the monomer. The catalytic system; Cu(I)C! (0.069 g, 0.69 mmol) and 2,2'~bipyridyl (bpy) (0.217 g, 1.39 mmol), were added under a positive nitrogen flow in order to initiate ihe reaction. The polymerisations were stopped when conversions had reached over 98 %. The polymerisations were stopped by diluting with a large excess of tetrahydroi ran (THF), which caused a colour change f om dark brown to a bright green colour. The catalytic system was removed using Dowex* Marathon™ MSG (hydrogen form) ion exchange resin beads and basic alumina. The resulting polymer was isolated by precipitation from the minimum amount of THF into cold hexane. The [initiator] : [CuCl] : [bpy] molar ratios in all polymerizations were 1 : 1 :2.

3.2.3 Gl tBOC dendron and Lactose initiated pHFMA core The Gi BOC Dendron initiator (48.5mg, 0,09mmol) and Lactose ATRP

initiator (7G,7mg, 0.09mmo!) was added to a 25 mL round bottom flask equipped with a magnetic stirrer bar, followed by the addition of 2,2-bipyridyl (56.2rng, G.36Qmniol), EGDMA (28.5rag, 0.144mmol) and HPMA (1.3g, 9.0mmol). The reaction mixture was bubbled with N ₂ for 15 raimrtes. Degassed anhydrous methanol (3.3mL) was added to the flask, and its contents stirred and bubbled with N ₂ for a further 5 minutes. Copper (Ϊ) chloride (17.8mg, O. l SOmmol) was quickly weighed out and added to the flask, instantly forming a brown coloured mixture, which was stirred and bubbled with N ₂ for a further 5 minutes. A 2 pressure was built up within the flask, then N2 inlet then removed, and the flask stirred for 24 hours at 40 ¹ C. Once the polymerisation was complete, THF was added to the reaction flask to poison the Cu (I) catalyst, forming a green coloured solution. The solution was passed through air alumina (neutral) column to remove the catalytic system, concentrated in vacuo, and precipitated into hexane. The supernatant was decanted off, and the remaining white solid dried overnight in a vac-oven,

3.2.4 Gl tBOC dendron.and bifunetional initiator pI PMA dumbbell

The Gl ^! BOC Dendron initiator (181mg, 0.330mraoi) and bi- functional initiator (36.6mg, 0.084mmoi) was added to a 25 ml, round bottom flask equipped with a magnetic stirrer bar, followed by the addition of 2,2-bipyridyl (157,4mg, LOlmmol), EGDMA (79- l mg, G.399mmol) and HPMA (3.63g, 25.2mmo!). The reaction mixture was then bubbled with N ₂ for 15 minutes. Degassed anhydrous methanol (lOmL) was added to the flask, and its contents stirred and bubbled with N ₂ for a further 5 minutes. Copper (I) chloride (49.9mg, 0.504mmol) was quickly weighed out and added to the flask, instantly forming a brown coloured mixture, which was stirred and bubbled with N ₂ for a further 5 minutes. A N ₂ pressure was built up within the flask, then N ₂ inlet then removed, and the flask stirred for 24 hours at 40 °C. Once the polymerisation was complete, TBF was added to the reaction flask to poison the Cu (I) catalyst, forming a green coloured solution, The solution was passed through an alumina (neutral) column to remove the catalytic system, concentrated in vacuo, and precipitated into hexane. The supernatant was decanted off, and the remaining white solid dried overnight in a vac-oven,

3.2.5 G2 tBOC dendron and bifunctional initiator pHFMA. dumbbell cor The G2 ¾Ο0 Dendron initiator ( 197mg, 0.168mrn l) and bi-functional initiator (18.3rag, 0.042mmol) was added to a 25 mL round bottom flask equipped with a magnetic stirrer bar, followed by the addition of 2,2-bipyridyl (78.7rag, 0.504mmol), EGDMA (33.3mg, 0.168mmol) and HPMA (3.63g, I2.6mmo!). The reaction mixture was bubbled with N ₂ for 15 minutes. Degassed anhydrous methanol

(4.65mL) was added to the flask, and its contents stirred and bubbled with h½ for a further 5 minutes. Copper (I) chloride (24,9mg, Q,252mmol) was quickly weighed out and added to the flask, instantly forming a brown coloured mixture, which was stirred and bubbled with N ₂ for a further 5 minutes. A N ₂ pressure was built up within the flask, then N ₂ inlet then removed, and the flask siiiTed for 24 hours at 40 : ^;' C. Once the polymerisation was complete, THF was added to the reaction flask to poison the Cu (I) catalyst, forming a green coloured solution. The solution was passed through an alumina (neutral) column to remove the catalytic system,

concentrated in vacuo, and precipitated into hexane. The supernatant was decanted off, and the remaining white solid dried overnight in a vac-oven. fable 2 - Mixed initiator polydendrons

EG DM

Polymer

Imitator 1 Initiator 2 A M (gujoF ¹ ) Mw (gmoF ¹ ) PDI

Core

(raol%)

Gl tBOC G2 tBOC pHPMASO 0.8 61500 153500 2.49

Gl tBOC Lactose pHPMASO 0.8 102000 216000 2.1 1

Gl tBOC Afunctional pHPMASO 0.95 47000 227000 4.83

G2 tBOC bifunctional pHPMASO 0.8 177500 555500 3.13

G2 DBOP 750 PEG

100 0 pHPMASO 0.8 90 500 1 304 000 9,67

90 10 pHPMASO 0.8 68457 1495000 21.84

75 25 pHPMASO 0.8 52431 987762 18.88 so 50 pHPMASO 0.8 39447 480638 12.19

25 75 pHPMASO 0.8 36157 315320 8.73

10 90 pHPMASO 0.8 37672 286049 7.61

0 100 pHPMASO 0.8 68133 296179 4.35

25 75 pHPMASO 0.9 60738 6751 19 1 1 , 13

0 100 pHPMASO 0,95 74740 642728 8.60

G2 DBOP 2K PEG

100 0 pHPMASO 0..8 193576 2225000 11.49

90 10 pHPMASO 0.8 348067 2464000 7.08 25 pHPMASO 0,8 55050 1 067000 IS ⁽ .38

50 pHPMASO 0.85 29372 709209 24 , 1 5

75 pHPMASO 0.95 141272 1862000 13 M 8

90 pHPMASO 0.95 40195 795274 IS >.79

100 pHPMASO 0,95 32246 476990 14 k79

50 pHPMAl OO 0.8 79448 516794 6. 51

4, Nanoprecipitation of Polydendrons

4. Nanoparticle formation (slow addition) - HR method in a typical procedure, 10 mg of sample was completely dissolved in 2 ml, of acetone at room temperature the resulting solution (5 rng ml/') was added drop wise to 10 mL of distilled water under vigoro us stirring tbr ca. 15 mm using a glass pipette. The solution was stirred vigorously for 24 at room temperature, until the acetone was completely evaporated as determined by *H NM analysis, where no peak at b 2.22 corresponding to acetone was observed.

4.2 Nanpprecipitation (fast addition) Polydendrons were dissolved in THF tbr a minimum of 6 hours at various

concentrations. Once rally dissolved polymer in THF (I ml. 5 mg ml) was added quickly to a vial of water (5 ml) stirring at 30 °C. The solvent was allowed to evaporate overnight in a fume cupboard to give a final concentration of 1 mg/ml polymer in water. By adjusting the starting concentration and the volume of water used, the size of the corresponding nanoparticles can be controlled to an extent. The nanopartieles formed were analysed by dynamic light scattering (DLS) and

fluorimetry.

Table 3 - DLS data tbr 100% Dendron initiated polydendrons

G l DBOP pHPMA20 0.8 61.72 0.1 17 Gl DBOP pHPMASO 0.8 63.9 0.130

Gl DBGP pHPMAlOO 0.8 69.89 0.070

G2 DBOP P.HPMA20 0.8 si , 33 0.076

G2 DBOP pHPMASO 0.8 80.78 0.083

G2 DBOP pHPMAlQO 0.8 80.56 0.119

Gl -A famine pHPMASO 0.7 70.6 0.366

Gl-A famine p.BuMA50 0.95 45.98 0.217

Gl-A /amine pDEAEMASO 0.9 136.2 0.148

Gl-A famine pOEGMASO 0.95 44.98 0.519

GG-D ramine pDEAEMASO 0.9

Gl -D /amine pDEAEMASO 0.9

G2-D famine pDEAEMASi) 0.9 115.9 0.158 pDEAEMA50~Moc&-

G2-B famine 0.9 162.9 0.082

t vMAst-EGDMA

Xani Gl - post modified

with;

benzyl pHPMASO 0.85 141.1 0.238 n~morpfoolmo pHPMASO 0.85 159.3 0.166

PEG480 pHPMASO 0.85 106.9 0.257

PEG5000 pHPMASO 0.85 156.8 0.427

Table 4 - DLS data for mixed initiator polydendrons

-Oiymer bifunciiona

Gl tBOC pHPMA50 0.95 73.78 0.109

1

bi&nctioi a

G2 tBOC pHPMASO 0.8 27.33 0.116

1

G2 DBOP 750 PEG

100 0 pHPMASO 0.8 80 78 0.083

90 10 pHPMASO 0.8 1 15,6 0.069

75 25 pHPMASO 0.8 109.8 0,073

50 50 pHPMASO 0.8 1 14.6 0.067 25 75 pHPMASO 0,8 Q? 57 0.078

10 90 pHPMASO 0.8 94,26 0,091

0 100 pHPMASO 0.8 87.8 0.0 /6

0 100 pHPMASO 0,95 89.53 0.083

G2 E SOP 2K PEG

100 0 pHPMASO 0.8 62,15 0.391

90 10 pHPMASO 0.8 144.4 0.036

75 pHPMASO 0.8 214.6 0,085

50 50 pHPMASO 0.85 105.5 0.058

25 75 pHPMASO 0.95 52.17 0.277

10 90 pHPMASO 0.95 37,81 0.207

0 100 pHPMASO 0,95 36.18 0.24

50 50 pHPMA20 0.85 54.9 0,296

pHPMA!O

50 50 0.8 232,2 0.133

0

5. Encapsulation of fluorescent molecules

5.1 Nile Red encapsulation - HR method n a typical procedure, 1 mg of sample and. 0, 1 mg Nile R ed was dissolved completely in 2 mL of acetone at. room temperature; the resulting solution (5.05 rng mL ^" *) was added drop wise to 10 mL of distilled water under vigorous stirring for ca. 15 min using a glass pipette. The solution was stirred vigorously for 24 h at room temperature, until the acetone was completely evaporated as determined by "H MR analysis, where no peak at δ 2.22 corresponding to acetone was observed.

5.2 Fluoresceinamine encapsulation - HR method

In a typical procedure, 10 mg of sample and 1 mg of fluorescemamine was dissolved completely in 2 mL of acetone at room temperature; the resulting solution (5.5 mg mL ^"1 ) was added drop wise to 10 mL of distilled water under vigorous stirring for ca. 15 min using a glass pipette. The solution was stirred vigorously for 24 h at room temperature, until the acetone was completely evaporated as determined by H NMR analysis, where no peak at δ 2,22 corresponding to acetone was observed.

5.3 Encapsulation of idle red or pyrene using mixed initator polydendrons

Stock solutions of nile red in THF at 0.2 mg/ml and pyrene in THF at 0.5 mg/ml were made. In a typical experiment the desired amount of nile red or pyrene was added to a vial using a pipette (e.g for a stock solution at 0,2 mg/ml 100 μΐ would be used if 0.02 mg was required). The vial was left in the fomecupboard for 20 min to allo w evaporation of the THF, A pre~dissolved sample of polymer in THF ( 1 ml, 5 mg ml) was added to the vial. The vial was shaken gently to allow dissolution of the fluorescent molecule in the THF containing polymer. Once the desired amount of polymer and fluorescent molecule was dissolved in the 1 ml of THF, this was added quickly to a vial of water (5 ml) stirring at 30 °C. The solvent was allowed to evaporate in a fume cupboard overnight, giving a final concentration of 1 mg ml polymer in water. The nanoparticles formed were analysed by dynamic light scattering (DLS) and Imorimetry,

Table 5 shows data for polymer nanoparticles with a final concentration of 1 mg/ml polymer with 0.1 w/w% nile red or pyrene encapsulated (1 μ ηϊ)

Table 5 - Fluorimetry of nanoparticles with nile red and pyrene encapsulated

Nile red

Pyrene

Fc fiymer EGDMA e capsulation

Initiator 1 Initiator 2 eueaps re (mol%) (mas intensity

11/13 r. 630 smj

G2 DBOP 750 PEG

100 0 pi IPMA50 0.8 702.1693 1.42

90 10 ≠ 1PMA50 0.8 625.9234 1 ,4458

75 25 ΙΡΜΆ50 0.8 574.7425 1 ,4666

50 50 pi IPMA50 0,8 548.357 1.4685

25 75 ρϊ 1PMA50 0,8 243.2502 1 ,479

10 90 pi 1PMA50 0.8 404.1123 1.5208

0 100 pi: ΪΡΜΑ50 0.8 285,757 1.5315 100 pHPMASG 0 226.2446

Pharmacology

L Materials & Methods

1. Materials

Dulbecco's Modified Eagles Medi m (DMEM), Hanks buffered saline solution (HBSS), Trypsin-EDTA, bovine seram albumin (BSA), Nile red, 3 -(4, 5- D imethylthiaz.o 1- 2-yl)- 2 , 5— diphenyltetrazolium bromide (MTT reagent), aeetonitrile (ACN) and ail general laboratory reagents were purchased from Sigma (Poole, UK). Foetal bovine seram (FBS) was purchased from Gibco (Paisley, UK). The CellTiter- Glo® Luminescent Cell Viability Assay kit was from Pro mega (UK). The 24-well HTS transwell plates were obtained from Comh g (New York, USA). The 96-well black walled, flat bottomed plates were from Sterilin (Newport. UK).

1,1 Rowtine cell cii!ture/cel! m&m eE&Eee

Caeo-2 cells were purchased from American Tvpe Culture Collection (ATCC, USA) and maintained in Dulbecco's Modified Eagles Medium (DMEM) supplemented with 15% filtered sterile foetal bovine serum. Cells were incubated at 37°C and 5% CC¾ and were routinely sub-cultured every 4 days when 90% confluent. Ceil count and viability was determined using a Countess automated cell counter (Invitrogen).

Caeo-2 cells were seeded at a density of 1.0 x 10 ⁴ cells / 100 μΐ in DMEM

supplemented with 15% FBS into each well of a 96 well plate (N aclon, Denmark) and incubated at 37°C and 5% C<¾. Ceils from 4 separate flasks of biological replicates of each ceil type were used (ΝΠ -4) to improve statistical power. Media was then aspirated from, column 1 and replaced with media containing each polydeadron or aqueous Nile Red solution at an equivalent 1 μΜ Nile Red concentration then diluted 1 :1 in media across the plate up to column 11. Column 12 served as a negative control and consisted of media and untreated cells. Following polydendron addition, the plates were incubated for 24 hours or 120 hours at 37°C, 5% C<¾ prior to assessment of cytotoxicity.

1.3 MTT assay

Following incubation of treated plates for 24 h or 120 h, 20 μ! of 5 mg nil ^" ' MTT reagent was added to each well and incubated for 2 hours. Subsequently, ΙΟΟμΙ, MTT lysis buffer (50% N-N-Dimethylfon¾amide in water containing 20% SDS, 2.5% glacial acetic acid and 2.5% hydrochloric acid, pH 4.7) was added to each well to iyse overnight at 37°C, 5% C0 ₂ . Following incubation the absorbance of each well was read using a Tecan Genosis plate reader at. 560nm (Tecan Magellan, Austria),

1.4 ATP assa

Following incubation of treated plates for 24 h or 120 h, cells were equilibrated, to room temperature for approximately 30 minutes. All but 20 μΐ of media was removed from each well and 20 id CelITiter~Glo®(Promega, UK) reagent was added. All reagents were made fresh and in accordance with the manufacturer's instructions. Plates were put on an orbital shaker for 10 minutes to mix contents and allow for stabilisation of luminescence signal. Luminescence was then measured using a Tecan Genios plate reader (Tecan Magellan, Austria).

2, TraiisceH !ar permeability of Nile Red across Caco-2 mo!ioiayers

2,1 Setting up and treating trsmsweil plates

Transwells were seeded with 3.5 x 10 ⁴ cells per well and propagated to a monolayer over a 21 day period, during which media in the apical and basolateral wells was changed every other day. Trans-epithelial electrical resistance (TEER) values were monitored until they were >1300 . 1 μΜ of Nile Red polydendron or 1 μΜ aqueous Nile Red was added to the apical chamber of 4 wells and the basolateral chamber of 4 wells to quantify transport in both Apical, to Basolateral (A>B) and Basolateral to Apical (B>A) direction and sampled on an hourly basis over a 4 h time period.

Apparent permeability coefficient was then determined by the amo unt of compound transported over time using the equation:

Papp = (dQ/dt) ( l /AC ₀ ) where (dQ/άή is the amount per time (nmol. see ^'1 ), A is the surface area of the filter and C is the starting concentration of the donor chamber (1 μΜ).

23 Extraction and quseiMcatloffi of Nile Red

100 μΐ of each collected sample was mixed with 900 μΐ acetone, vortexed, sonicated for 6 minutes and centrifuged at 13300 rprn for 3 minutes. The supernatant was completely dried in a vacuum centrifuge at 30°C until the dry solid sample was left. This was reconstituted in 150 μΐ acetonitrile, transferred to a 96- ell black walled, flat bottomed plate and measured for fluorescence intensity excitation wavelength 480 nm. emission wavelength 560 nm using a Tecan Genios plate reader (Tecan Magellan, Austria).

3. Results 3.1 Cytotoxicity ···· MTT assa s

Following 24 hour incubation of Caco-2 cells with each polydendron, analysis of cytotoxicity by MTT assay (Figure 6) showed that aqueous Nile Red and each polydendron did not affect metabolic turnover of Caco-2 cells compared to untreated cells at the range of concentrations investigated. It can be inferred that metabolic turnover correlates to cell viability in which case each material was not cytotoxic.

Figure 6: MTT assay of Caco-2 cells following 24 hour incubation with aqueous Nile Red and each polydendron. A ^::i aqueous Nile Red, ECso 1, 160, B ^:::: 0:100 ₅ ECso 2.509. C - 10:90, ECso 1.410. D = 25:75, EC ₅ o 1.567. E = 50:50, EC ₅₀ 1.083. F = 75:25, ECso 1.565, G = 90:10, ECso 1.607. H - 100:0, EC ₅₀ 2.678.

Following 120 hour incubation of Caco-2 cells with each polydendron, analysis of cytotoxicity by MTT assay (Figure 7) showed that aqueous NR. and each

polydendron at the range of concentrations investigated did not affect the viability of

Caco-2 cells. Figure 7: MTT assay of Caco-2 cells following 120 hour incubation with aqueous Nile Red and each polydendron. A = aqueous Nile Red, EC50 No ECJQ. B = 0:100, ECso 1.528. C ^« 10:90, EC50 No EC50. » - 25:75, EC ₅₀ 6.166. E = 50:50, EC _SC 0.7856. F = 75:25, ECso No ECso, G = 90:10, EC _5C 0.2176. H = 100:0, EC50 No ECso-

33 ATP assay

Following 24 hour incubation of Caco-2 cells with each polydendron, analysis of cytotoxicity by ATP assay using a CellTiter-Glo® kit (Promega, UK) (Figure 8} indicated that ATP presence was not affected in cells treated with aqueous Nile Red solution and polydendron formulated Nile Red at the range of concentrations investigated compared to untreated cells. It can be inferred that the presence of ATP correlates to cell viability in which case each material was not cytotoxic.

Figure $1 ATP assay of Caco-2 eelis following 24 hour incubation with aqueous Nile Red and each polydendron. A = aqueous Nile Red, EC 50 1.946, B = 0:100, EC50 2.855. C = 10:90, EC ₅₀ No EC ₅ Q. D === 25:75, EC ₅₀ No EC ₅₀ . E = 50:50, EC50 No ECso. F === 75:25, EC _S0 No EC50, G - 90: 10, EC ₅₀ 2.848. H = 100:0, EC _S0 0.1961.

Following 120 hour incubation of Caco~2 cells with each polydendron, analysis of cytotoxicity by ATP assay using a CellTiter-GlG® kit (Promega, UK) (Figure 9) indicated viability was not affected in cells treated with aqueous Nile Red solution and each polydendron material at the range of concentrations investigated compared to untreated cells.

Figure 9: ATP assay of€aco~2 cells following 120 hour incubation with aqueous Nile Red and each polydendron. A = aqueous Nile Red, ECso No EC50. B = 0:100, ECso No ECso. C = 10:90, EC ₅₀ 3.168. D = 25:75, EC ₅₀ 2.565. E = 50:50, EC ₅₀ No ECso. F === 75:25, EC _S0 3.032, G = 90:10, EC50 No EC50. H - 100:0, EC50 No EC ₅₀ .

4. Traasceilukr permeability of selected Nile Red polydendron materials across Caeo-2 cell monolayers. Transcelliilar pentseability of Nile Red through Caeo~2 cell monolayers (to model the intestinal epithelium) was significantly higher in the apical to basolateral (A>8, gut to blood) direction for the polydendron preparation ! 0G2: 0PEG compared to an aqueous solution of Nile Red (Figure 10 A&B). All the polydendron materials produced a greater apical to basolateral (A>B, gut to blood), basolateral to apical (B>A, blood to gut) ratio than an aqueous preparation of Nile Red following 1 hour incubation (Table 1, Figure 10€). A statistically significant correlation (P ^::: ().05) between the ratio of dendron and PEG used in the polydendron formulation and the ratio of apical to basolateral (A>B, gut to blood), basolateral to apical ( B>A, blood to gut) movement of Nile Red across the Caco-2 monolayer was observed (Figure 10

Figure 10, (A&B) Transcelliilar permeability across Caco2 cell monolayers of polydendron formulated Nile Red relative to an aqueous solution of Nile Red. Data are given as the mean of experiments conducted in biological triplicate, (C)

Con-elation between polydendron formulation and the ratio of Nile Red transported (A>B/B>A) across Caco2 cell monolayers (r ² 0,784). Data were normally distributed, statistical analysis was conducted using a Pearson correlation (P ^::: <0.05) a two-tailed P value was used to reduce the chance of a type Ϊ error.

Table L Apparent permeability (i¾pp) of Nile Red polydendrons and aqueous Nile Red across Caco2 cell monolayers following 1 hour incubation. Data are given as tl mean of experiments conducted in biological tripKcs

10 BDVE (5,6 ml, 35.21 mmol) was added to a two-necked 250 mi round bottomed flask equipped with a condenser, a magnetic stirrer and a positive flow of nitrogen. A small amount of radical iiiliibitor 4~ter/-butylcatecho 1 (end of a spatula) was added and the mixture deoxygenated using a nitrogen purge for 15 minutes. Once dissolved, the temperature was raised to 70°C. MAA (14.9 n l, 175.8 mn ol) was

15 added dropwise over 10 minutes through a sepia. The reaction was allowed to

proceed at 70°C for a further 6 hours with stirring. After this time, the reaction was stopped by cooling and exposing to the air. The crude product was dissolved m chloroform ( 100 ml) and washed with basic ¾Q ( pill 2, 3 x 100 nil). The combined washings were collected and dried over aS€>4 and the solvent removed

20 by rotary evaporation.

(Found: C 61.45; H 8.28%. C _i6 ¾ _& G ₆ requires C 61.15; H 8.28%); ^f H NMR (400 MHz; CDC ; Me ₄ Si) δ 1.44 (611 d, C/¾CH), 1.65 (4H, m, CH ₂ C¾CH ₂ ), 1.95 (611 s, G¾C= H2), 3.50-3.69 (4H, m, OCi¾CH ₂ ), 5.60 and 6.15 (4H, 2s, Ci¾=CCH ₃ ) and 5.95-5.99 (2H, q, CffC¾). ^!3 C NMR (400 MHz; CDC1 ₃ ; M<¾Si) δ 18.27 (s),

25 20.83 (s), 26.29 (s) 68.85 (s), 96.93 (s), 125.90 (s), 136.37 (s) and 167.01 (s). n/z (EI) 314.2 (M* - C _{i 6} H ₂₆ 0 ₆ requires 314).

8. BEAEMA Poiydendr&n Synthesis

30 8.1 Polymerisation of G1 -A dendron initiated DEAEMAgn In a typical synthesis, targeting a number average degree of polymerisation (DP„) =

50 monomer units (poly(DEAEMA)5o; I¾)EAE . ninkiawv _* SO), bpy (134.9 mg, 0,8637 mmol, 2 eq.), DEAEMA (4 g, 21,59 mmol, 50 eq.) and isopropanol (IP A) (56% v/v based on DEAEMA) were placed into a 25 nil, round-bottomed flask. The solution was stirred and deoxygenated using a nitrogen (N?) purge for 15 minutes, Cu(s)Cl (42.8 mg, 0,4318 rnmol, 1 eq.) was added to the flask and left to purge for a further 5 minutes. Gl -A dendron initiator (0.2576 g, 0.4318 mmol, 1 eq.) was added to the flask under a positive flow of Na, and the solution was left to polymerise at. 40°C. Reactions were terminated when >99% conversion was reached, as judged by ^! H NM , by exposure to oxygen and addition of acetone. The catalyst residues were removed by passing the mixture over a basic alumina column. Acetone was removed under vacuum to concentrate the sample before precipitation into cold petroleum ether (40°C - 60°C) and drying in the vacuum oven overnight.

In a typical synthesis, targeting a number average degree of polymerisation (DP„) = 50 monomer units (poly(DE.AEMA)5o; ο£Αε. Α ^η ι 50), bpy (134,9 mg, 0,8637 mmol, 2 eq.), DEAEMA (4 g, 21.59 mmol, 50 eq.), EGDMA (77.0 mg, 0.3886 mmol, 0.9 eq) and IP A ³ ' (38,9% v/v based on DEAEMA) were placed into a 25 mL round-bottomed flask. The solution was stirred and deoxygenated using a N? purge for 15 minutes. Cu(i)Cl (42.8 mg, 0,4318 mmol, 1 eq.) was added to the flask and left to purge for a further 5 minutes, Gl-A dendron initiator (0.2576 g, 0.4318 mmol, 1 eq.) was added to the flask under a positive How of ί¾ and the solution was left to polymerise at 40°C. Reactions were terminated when >99% conversion was reached, as judged by 1H NMR, by exposure to oxygen and addition of acetone. The catalyst residues were removed by passing the mixture over a basic alumina colurrm. Acetone was removed under vacuum to concentrate the sample before precipitation into cold petroleum ether (40°C - 60°C). The polymerisation conditions and procedure is identical to those described for linear polymers above and drying in the vacuum oven overnight. /Da Mn/Da PDI Z-Ave/s m Pdi

DEAEMA50- 244,622 201,497 136.2 0.148

EGDMAso

In a typical synthesis, targeting a degree of polymerisation (DP) ^:::: 50 monomer units (pDEAEMAjo), bipy (160.8mg, 0.8637 mmol), BDME (271.2mg, 0.864 mraol) and DEAEMA (4,0g, 21.59 mmol) in IPA (4 ml, 56 v/v% based on DEAEMA) were added to a 25ml round bottomed flask. A branches- to initiator ratio of 1 : 2 was applied for the branching polymerisation The solution was stirred and deoxygenated for 15 minutes using a nitrogen purge. Cu(l)Ci (42.8mg, 0.4318 mmol) was quickly added to the flask under a positive flow of nitrogen and the mixture deoxygenated for a further 5 minutes. A dark brown solution resulted. Gl-A (0,2576 g, 0.4318 mmol) - was added through the septa and the solution was left to polymerise at 40°C for 24 hours. Alter this time, the reaction was terminated by exposure to oxygen and vigorously stirring in acetone (100 ml). The mixture turned green alter 20 minutes. The catalyst residues were removed from the reaction mixture by passing the mixture over a basic alumina column. The solvent was removed under reduced pressure and the crude polymer redissoived in a minimum volume of acetone ( 10 ml) before precipitation into cold petroleum ether. The resulting polymer was dried overnight at 40°C in a vacuum oven. For determining monomer conversion (%), the reaction was sampled and diluted into€Di¾ for ^S H. NMR. analysis,

Mw ba Tv /^ \ Bl j Z-Ave/nm Ράί

8,4 Polymerisation of GO-D dendron initiated DEAEMi In a typical synthesis, targeting a number average degree of polymerisation (DP ) - 50 monomer units (poly(DEAEMA)so: ηοΕΑΕΜΑ ¾ώΐ_.ω«·: 50), bpy (134,9 mg, 0.8637 mmol, 2 eq.), DEAEMA (4 g, 21.59 mmol, 50 eq.) and isopropanol (IP A) (56% v/v based on DEAEMA) were placed into a 25 mL round-bottomed flask. The solution was stirred and deoxygenated using a nitrogen (Ni) purge for 15 minutes, Cu({)Q (42.8 mg, 0.4318 mmol, 1 eq,) was added to the flask and left to purge for a further 5 minutes. G0-D dendron initiator (0.1089 g, 0.4318 mmol, 1 eq.) was added to the flask under a positive flow of N?, and the solution was left to polymerise at 40°C. Reactions were terminated when >99% conversion was reached, as judged by ^! H NM , by exposure to oxygen and addition of acetone. The catalyst residues were removed by passing the mixture over a basic alumina column. Acetone was re under vacuum to concentrate the sample before precipitation into cold petroleum ether (40°C - 60°C) and drying in the vacuum oven overnight.

8.5 Polymerisation of G0-D dendron initiated DEAEMAsn-EGDMAn 3.9

I n a typical synthesis, t argeting a number average degree of polymerisation (DP„) - 50 monomer units (poly(DEAEMA)so; noEAEMA/niniiiator: 50), bpy (134.9 mg, 0.8637 mmol, 2 eq.), DEAEMA (4 g, 21 ,59 mmol, 50 eq.), EGDMA (77.0 mg, 0.3886 mmol, 0.9 eq) and iPA ³⁷ (38,9% v/v based on DEAEMA) were placed into a 25 mL round -bottomed flask. The solution was stirred and deoxygenated using a _∑ purge for 15 minutes. Cu(;)Cl (42.8 mg, 0,4318 mmol, 1 eq.) was added to the flask and left to purge for a ftirther 5 minutes. G0-D dendron initiator (0.1089 g, 0.4318 mmol, 1 eq.) was added to the flask under a positive flow of _; ? ₅ and the solution was left to polymerise at 40 ^W C. Reactions were terminated when >99% conversion was reached, as judged by H NMR, by exposure to oxygen and addition of acetone. The catalyst residues were removed by passing the mixture over a basic alumina column. Acetone was removed under vacuum to concentrate the sample before precipitation into cold petroleum ether (40°C - 60°C) and drying in the vacuum oven overnight. The polymerisation conditions and procedure is identical to those described for linear polymers above.

8.6 Polymerisation of Gl-D dendron initiated DEAEMA¾s

In a typical synthesis, targeting a number average degree of polymerisation (DP _« ) - 50 monomer units (poly(DEAEMA) ₅ ; ηοΕΑΕ Α/¾«ίώ«»·' 50), bpy (134.9 mg, 0,8637 mmol, 2 eq.), DEAEMA (4 g, 21.59 mmol, 50 eq.) and isopropanol (IP A) (56% v/v based on DEAEMA) were placed into a 25 niL round-bottomed flask. The solution was stirred, and deoxygenated using a nitrogen { ½) purge for 15 minutes. Cu(t)Cl (42.8 mg, 0,4318 mmol, 1 eq.) was added to the flask and left to purge for a farther 5 minutes. Gl -D dendron initiator (0.2204 g, 0,431 8 mmol, 1 eq.) was added to the flask under a positive flow of N?. and the solution was left to polymerise at 40°C. Reactions were terminated when >99% conversion was reached, as judged by ⁵ H NMR, by exposure to oxygen and addition of acetone. The catalyst residues were removed by passing the mixture over a basic alumina column. Acetone was removed under vacuum to concentrate the sample before precipitation into cold petroleum ether (40°C - 60°C) and drying in the vacuum oven overnight.

! ,

DEAE Aso 59,416 In a typical synthesis, targeting a number average degree of polymerisation (DP„) ^::: 50 monomer units (poly(DEAEM.A)5c; ηοΕΑΕΜΑ/»3πΗϊ«ι _< *·: 50), bpy (134,9 mg, 0.8637 mmol, 2 eq.), DEAEMA (4 g, 21.59 mmol, 50 eq.), EGDMA (77.0 rag, 0.3886 mmoi, 0,9 eq) and IP A: ^{5 '} ' (38,9% v/v based on DEAEMA) were placed into a 25 ml.. round-bottomed flask. The solution was stirred and deoxygenated using a ? purge for 15 minutes. Cu(j)Cl (42,8 mg, 0,4318 mmol, I eq.) was added to the flask and left to purge for a further 5 minutes. GI-D dendron initiator (0,2204 g, 0.4318 mmol, 1 eq.) was added to the flask under a positive flow of s, and the solution was left to polymerise at 40°C. Reactions were terminated when >99% conversion was reached, as judged by Ή NMR. by exposure to oxygen and addition of acetone. The catalyst residues were removed by passing the mixture over a basic alumina column. Acetone was removed under vacuum to concentrate die sample before precipitation into cold petroleum ether (40°C - 60°C) and drying in the vacuum oven overnight. The polymerisation conditions and procedure is identical to those described tor linear polymers above.

8.8 Polymerisation of G2-D dendron initiated DEAEMAgn In a typical synthesis, targeting a number average degree of polymerisation (DP„) - 50 monomer units (poly(DEAEMA)5c; nDEAEMA ¾mtiator: 50), bpy (134,9 mg, 0.8637 mmol, 2 eq.), DEAEMA (4 g, 21,59 mmol, 50 eq.) and isopropanol (ΪΡΑ) (56% v/v based on DEAEMA) were placed into a 25 mL round-bottomed flask. The solution was stirred and deoxygenated using a nitrogen (N ₂ ) purge for I S minutes, CuQCl (42.8 mg, 0.4318 mmol, 1 eq.) was added to the flask and left to purge for a further 5 minutes. G2~D dendron initiator (0,3934 g, 0.4318 mmol, 1 eq.) was added . to the flask under a positive flow of N ?, and the solution was left to polymerise at 40°C. Reactions were terminated when >99% conversion was reached, as judged by ⁵ H NMR, by exposure to oxygen and addition of acetone. The catalyst residues were removed by passing the mixture over a basic alumina column. Acetone was removed under vacuum to concentrate the sample before precipitation into cold petroleum ether (4()°C ·- 60 ^U C) and drying in the vacuum oven overnight.

Mw/Da Mn/Da [ POI Z-Ave/nm Pdi

DEAE Aso 34,386 21,553 1.6 110.9 0.123 SJUiojvmms^

In a typical synthesis, targeting a number average degree of polymerisation (DP _« ) - 50 monomer units Cpo!y(DEAEMA)<o; RDEAEM A/ ftinitntor ^* 50), bpy (134.9 mg, 0.8637 mmol, 2 eq.), DEAEMA (4 g, 21.59 mmol, 50 eq.), EGDMA (77,0 mg, 0.3886 mmol, 0.9 eq) and IPA ³⁷ (38.9% v/v based on DEAEMA) were placed into a 25 ml, round-bottomed flask. The solution was stirred and deoxygenated rising a N ₂ purge for 15 minutes. CuQCl (42,8 mg, 0.4318 mmol 1 eq.) was added to the flask and left to purge for a further 5 minutes. G2-D dendron initiator (0.3934 g, 0.4318 mmoL 1 eq.) was added to the flask under a positive flow of N _? ., and the solution was left to polymerise at 40°C. Reactions were terminated when >99% conversion was reached, as judged by ¾ NMR, by exposure to oxygen and addition of acetone. The catalyst residues were removed by passing the mixture over a basic alumina column. Acetone was removed under vacuum to concentrate the sample before precipitation into cold petroleum ether (40°C - 60 ^U C) and drying in the vacuum oven overnight. The polymerisation conditions and procedure is identical to those described for linear polymers above.

9, Hydrolysis of branched pDEAEMAx

Hydrolysis of branched. pDE AEM A50 was carried out in acetone at room temperature h the presence of a small amount of aqueous HQ, with magnetic stirring. Solutions of each branched polymer in acetone were prepared (40 mg mi, 9 ml). HCl (6M, 300 μϊ) was added dropwise to each of the polymer solutions. The solutions were stirred vigorously at room temperature for 20 minutes, resulting in a cloudy solutio with solid precipitate. Distilled water (9 ml) was added to each of the acidic polymer solutions. The solutions were allowed to stir ovenught in a sealed vial. Each of the hydro Sysed polymer solutions were frozen in liquid nitrogen and iyophilised for 72 hours, then dissolved in a THF/2 v/v% TEA eluent system and analysed by GPC.

Co-Poiydeitdr n Synthesis In a typical synthesis, targeting a number average degree of polymerisation (DP„) = 50 monomer units (poly{DEAEMA)$o; nDEAEMA ¾ tiator.' 50), bpy (134.9 nig, 0.8637 mmol, ^' 2 eq.), DEAEMA (4 g, 21.59 mmol, 50 eq.) and isopropanol (IP A) (37,7% v/v based on DEAEMA) were placed into a 50 mL round-bottomed flask. The solution was stirred and deoxygenated using a nitrogen (N ₂ ) purge for 15 minutes, Cu(j)Cl (42.8 nig, 0,4318 mmol, 1 eq.) was added to the flask and left to purge for a further 5 minutes. G2-D dendron initiator (0.3934 g, 0.4318 mmol, 1 eq.) was added to the flask under a positive Sow of N ₂ , and the solution was left to polymerise at 40°C. In another 25 mL round-bottomed flask, bpy (134,9 mg, 0.8637 mmol), rBuMA (4.0 g, 21.59 mmol) and aqueous isopropanol (23.8% v/v based on rBuMA) were added. The solution was stirred and deoxygenated using a nitrogen (N ₂ ) purge for 15 minutes. Cu({)Cl (42.8 mg, 0.4318 mmol, 1 eq.) was added to the flask and left, to purge for a further 5 minutes. After the conversion of DEAEMA reached around 85%, the mixture from the second flask was added into the first lask rapidly using a syringe and taking care not to admit any air into the vessel. A. sample was taken

immediately after the addition of the iBuMA monomer solution for ^! H NMR

analysis. The block copolymer ization reaction was carried out at ambient

temperature and samples were taken periodically from the reaction mixture for ^! H

NMR analysis. Reactions were terminated when >99¾ conversion was reached, as

judged by ^f H NMR, by exposure to oxygen and addition of acetone. The catalyst

residues were removed by passing the mixture over a basic alumina column. Acetone was removed under vacuum to concentrate the sample before precipitation into cold petroleum ether (40°C - 60°C) and drying in the vacuum oven overnight.

I Mw/Da n/Da PDI Z-Ave/nm | Pdi j t DEAEMAso 92,61? 80,687 1.1 38.42 10.244 !

EGDMAo. _? } In a typical synthesis, targeting a number average degree of polymerisation (DP„) - 50 monomer units (poly(DEAE A)5o; ηοΕΑΕ .Α. πΐηί 50), bpy ( 134.9 mg, 0,8637 mmol, 2 eq.), DEAEMA (4 g, 21.59 mmol, 50 eq.) and isopropanol ( PA) (37.7% v/v based on DEAEMA) were placed into a 50 niL round-bottomed flask. The solution was stirred and deoxygenated using a nitrogen (N ) purge for 15 minutes. Cu(j)Cl

(42.8 mg, 0,4318 mmol, 1 eq,) was added to the flask and left to purge for a further 5 minutes. G2-D dendron initiator (0.3934 g, 0.4318 mmol, 1 eq.) was added to the

flask under a positive flow of N^, and the solution was left to polymerise at 40°C. In another 25 mL round-bottomed flask, bpy (134.9 mg, 0.8637 mmoi), iBuMA (4.0 g,

28.1 mmoi, 65 eq.), EGDMA (77,0 mg, 0.3886 mmol 0.9 eq) and aqueous

isopropanol (23.8% v/v based on iSu A) were added. The solution was stirred and deoxygenated using a nitrogen (N ₂ ) purge for 15 minutes. Cu(i)Cl (42,8 mg, 0.4318 mmol 1 eq.) was added to the flask and left to purge tor a further 5 minutes. After the conversion ofDEAEMA reached around 85%, the mixture from the second flask was added into the first flask rapidly using a syringe and taking care not to admit any air into the vessel. A sample was taken immediately after the addition of the iBuMA monomer solution for H NMR analysis. The block copolymerization reaction was carried out at ambient temperature and samples were taken periodically from the reaction mixture for ^! H NMR analysis. Reactions were terminated when >99% conversion was reached, as judged by ^{ H NMR, by exposure to oxygen and addition of acetone. The catalyst residues were removed by passing the mixture over a basic alumina column. Acetone was removed under vacuum to concentrate the sample before precipitation into cold petroleum ether (40°C - 60°C) and drying in the vacuum, oven overnight.

1L N nop rticle formation in respect of polydendrons of sections 8 to 10

In a typical procedure, 10 mg of sample was completely dissolved in 2 mL of acetone at room temperature; the resulting solution (5 mg mL ^" ') was added drop wise to 10 mL o f distilled water under vigorous stirring for ca. 15 min using a glass pipette. The solution was stirred vigorously for 2.4 h at room temperature, until the acetone was completely evaporated as determined by ^! H NMR analysis, where no peak at δ 2.22 corresponding to acetone was observed.

12. Nile Red encapsulation in respect of polydendrons of sections 8 to 10

In a typical procedure, 10 mg of sample and 0.1 mg Nile Red was dissolved completely in 2 mL of acetone at room temperature; the resulting solution (5.05 mg mL ^" ') was added drop wise to 10 mL of distilled water under vigorous stirring for ca. 15 min using a glass pipette. The solution was stirred vigorously for 24 h at room temperature, until the acetone was completely evaporated as determined by Ή NMR analysis, where no peak at δ 2.22 corresponding to acetone was observed. 13. Fluorescemamine encapsulation in respect of polydetxdrons of sections 8 to 10

In typical procedure, 10 mg of sample and 1 mg of Fluorescein amine was dissolved completely in 2 ml, of acetone at room temperature; the resulting solution (5,5 mg ml.. ^' ') was added drop wise to 10 mL of distilled water under vigorous stirring for ca. 15 min using a glass pipette, The solution was stirred vigorously for 24 h at room temperature, until the acetone was completely evaporated as determined by Ή N R analysis, where no peak at δ 2,22 corresponding to acetone was observed.

14, Example of nanoprecipitation to encapsulate norganic magnetic

nanoparticles

Polydendron (G2i2 . PEG(50:50)--pHPMA ₅ o-EGDMAo.8) was dissolved in THF for a minimum of 6 hours. Once fully dissolved the polymer i THF (0,2 ml, 25 mg/ml) was mixed with Fs^O,*. 1 nm paiticies in THF (0,5 ml, 5 mg ml) and this mixture of polymer and Fe ₃ C>4 was added quickly to a viai of water (1 mi) stirring at 30 °C. The solvent was allowed to evaporate overnight in a fume cupboard to give a final concentration of 5 mg/ml polymer, 2.5 mg ml Fe ₃ G ₄ in water, The nanoparticles formed were analysed by dynamic light scattering (DLS). scanning electron microscopy (SEM) and transmission electron microscopy (TEM).

SEM imaging showed spherical nanoparticles of size range varying from

approximately 150 to 250 am while TEM imaging showed the majority of nanoparticles to have encapsulated Fe ₃ G4 with no free Fe- h observed,

DLS (2.5 mg/ml in. water) determined the Z-Ave hydrodynamic diameter to be 182 nm with PDI to he 0.01. In the presence of a magnetic field (i.e. with a magnetic suspended above, just touching the surface of the dispersion) DLS measurements showed a 50% reduction in derived count rate after 12 hours and a 40% reduction in derived count rate alter 8 hours, with Z-Ave diameter remaining constant throughout. The reduction in derived count rate is intrinsic to a decrease in concentration of nanopaiticles within the dispersion and demonstrates the effect of the magnetic field on directing the behaviour of the nanoprecipitate. In the absence of a magnetic field there is no drop in derived count rate.

25. Ehisirati ns of some effects of the polydendrons including pH responsive effects

Figures 15 to 1 illustrate some effects of the polydendrons including pH responsive effects. Figure 15 is a photograph showing the encapsulation of oil red into aniine containing polydendron nanoprecipitates. In the vial on the left, oil red is not dissolved in water due to the inherent hydrophobicity of the dye (in the original photograph, the fluid in the vial is almost colourless). In the vial on the right, oil red is encapsulated in polydendron nanoprecipitate (in the original photograph, the fluid in the vial is dark red).

Figure 16 contains photographs showing fluoresceinamine encapsulated within poly(DEAEMA) polydendron in a dialysis bag. The photograph on the left shows the hydrophobic dye encapsulated in the polydendron nanoprecipitate after standing in an aqueous solution at neutral pH for 24 hours (in the original photograph, a yellow colour is confined to the dialysis bag). The photograph on the right shows the release of the dye into the dialysis sink water after addition of HCl to the sink water thereby triggering release from the polydendron nanoprecipitates (in the original photograph, a yellow colour is visible throughout the fluid in the beaker, not just confined to the dialysis bag).

Figure 17 shows a photograph of two vials. The vial on the left contains an amine containing dendron initiated polydendron nanoprecipiate in water after the addition of transport buffer. The vial on the right shows a branched polymer nanoprecipitate (without amine containing dendron end groups) after the addition of transport buffer. This shows that the presence of the dendron prevents precipitation. Figure 18 is a photograph showing nanoprecipitated amine containing polydendron at neutral pH (left) and after addition of HO (right). The clarity of the vial on the right (compared to the cloudiness of the vial of the left) indicates solvation and lack of nanoprecipitated particles after HQ addition.

Figure 1 shows a dynamic light scattering (DLS) trace from amine containing polydendron nanoprecipiates at neutral pH (sharp peak to the right: approx z~ average 136 nm, PDI 0.14) and the same sample after addition of HC! (broad peak to the left: appro z-average 28 nm, PDI 0.38). The change in particle size and increased PDI shows solvation and disassembly of the nanoprecipitaie.

9!