FIELD OF THE INVENTION

The present invention is in the field of bacterial infections and relates to the use of self-assembling amphiphilic poly(amidoamine) amine (PAMAM) dendrimers, and particles thereof encapsulating antibacterial agents, as antibacterial agents.

BACKGROUND OF THE INVENTION

Antibacterial resistance has become a global threat, as drug resistance makes bacterial infection increasing and difficult to treat. This pressing public health problem is driving development of new antibacterial agents to overcome drug resistance, which includes modification of existing antibiotics, identification of active agents with novel targets against resistant bacteria, and elaboration of antimicrobial peptides with dual antibacterial and immunomodulatory activities, just to name a few. Among these, new antibacterial agents which are substantially different from conventional antibiotics are of peculiar interest and in urgent need in order to confront the increasingly alarming antibacterial resistance. In this context, amphiphilic antibacterial agents are particularly appealing because they mimic the antibacterial feature of natural antimicrobial peptides and antibacterial detergents. In addition, amphiphilic compounds may self-assemble into supramolecular structures which can also contribute to the antibacterial activity via cooperative and multivalent interaction. Consequently, a myriad of amphiphilic molecules has been established as antibacterial candidates.

Dendrimers are a unique family of synthetic molecules with precisely controlled radial architecture and special multivalent cooperativity confined within a small three-dimensional volume. Different dendrimers have been studied for their antimicrobial activity (Mintzer MA, et al., Mol Pharm. 2012, 9(3), 342-54; Castonguay A et al., New J. Chem., 2012, 36, 199-204. doi.org/10.1039/C 1NJ2048 IE; Alfei et al., Nanomaterials 2020 ;10(10):2022. doi: 10.3390/nano 10102022; Alfei and Caviglia Pharmaceutics 2022, 14(10), 2016). Among them, the poly(amidoamine) (PAMAM) dendrimers are of special interest by virtue of their excellent biocompatibility owing to peptide-mimicry (Lyu et al., in “Dendrimer Chemistry: Synthetic Approaches Towards Complex Architectures”, Eds: Michal Malkoch, Sandra Garcia Gallego, Royal Society Chemistry, 2020, 85-113) However, active dendrimers are often high-generation molecules bearing charged terminals associated with considerable cytotoxicity. Recently, small amphiphilic dendrimers composed of hydrophobic entities and hydrophilic dendrons have been explored for antibacterial activity, where their activity was frequently related to dendrimer generation, terminal charge, and hydrophilicity/hydrophobicity balance (Meyers et al., J Am Chem Soc 2008;130:14444-5; Kannan et al., ACS Appl Bio Mater 2019;2:3212-24).

There is still an unmet need for potent and safe antibacterial compounds having novel modes of action and capable of providing effective treatment of bacterial infections.

SUMMARY OF THE INVENTION

The present invention is based on an observation that certain amphiphilic dendrimers comprising a long hydrophobic chain and a small hydrophilic PAMAM dendron and having a positive zeta potential demonstrated an antibacterial activity. The observation demonstrates that not only the charge, but also the charge density and steric size associated with the terminal functionalities and self-assembling feature of the amphiphilic dendrimers, play a crucial role in their antibacterial activity. In particular, the amine-terminated dendrimer denoted la exhibits the most potent antibacterial activity against both Gram-positive and Gram-negative bacteria as well as drug-resistant bacteria, and shows biofilm eradication. Surprisingly, according to the present invention, dendrimers of low generation having a relatively low number of terminal amines provide a profound antibacterial activity. Such dendrimers are easy and inexpensive to manufacture and due to their relatively low molecular weight, they have a high chance to be effective upon administration. Interestingly, adding double-bound bonds to the lipophilic tail provided new antibacterial properties. Such, compounds having double bonds showed high activity against Borrelia bavariensis and Neisseria meningitidis, which was hard to obtain for by most of the dendrimers with a saturated hydrophobic tail. It is also demonstrated that aside antibacterial activity of the dendrimers of the present invention, they may be used for encapsulation of other antibacterial compounds and provide additive and even synergistic antibacterial effect. As an example, azithromycin (AZM), doxycycline (DOX), and ciprofloxacin (CIP) were encapsulated in micelles formed by dendrimer la. The resulted encapsulated structures provided an outstanding and even synergistic antibacterial activity.

According to one aspect, the present invention provides a composition comprising, as an active agent, a dendrimer selected from an amine-terminated, poly(amidoamine) (PAMAM) dendrimer comprising a hydrophobic tail and bola-amphiphilic amine- terminated PAMAM dendrimer, for use in treating a pathogenic infection. According to some examples, the pathogenic infection is a bacterial infection. According to some examples, the dendrimers are selected from generation 2, 3 or 4 dendrimers. According to certain examples, the terminal amine is a positively charged amine. According to some examples, the terminal amine is a primary amine. According to some examples, the hydrophobic tail comprises a C16-C32 alkyl. According to some examples, the dendrimer has a structure of Formula I:

X is a C16-C32 straight alkyl.

According to some examples, X is a C18 straight alkyl. According to some examples, R is o o . According to some examples, X is a C18 alkyl and R is

H H

According to some embodiments, the dendrimers have a structure selected from Formula II, III, IV, V, VI, VII, VIII, IX, X, and XI, as described below. According to some examples, the dendrimer is a bola-amphiphilic amine-terminated polyamidoamine dendrimer. According to other examples, the dendrimer has a structure selected from Formula XII, XIII, XIV, as described below

According to any one of the above examples, the composition is a pharmaceutical composition comprising a pharmaceutically acceptable carrier or excipient.

According to another aspect, the present invention provides a hydrophobic antipathogenic agent encapsulated within a dendrimer of the present invention. According to some examples the antipathogenic agent is an antibacterial agent. Thus, according to some examples, the present invention provides a hydrophobic antibacterial agent encapsulated within an amphiphilic poly(amidoamine) (PAMAM) amine-terminated dendrimers comprising a hydrophobic tail. According to some embodiments, the dendrimers and encapsulated hydrophobic antibacterial agent form particles, i.e., loaded particles. According to some embodiments, the hydrophobic antibacterial agent is selected from azithromycin, ciprofloxacin, doxycycline, erythromycin, vancomycin and cefuroxime. According to some embodiments, the dendrimer has a structure selected from Formula II, III, IV, V, VI, VII, VIII, IX, X and XI and the hydrophobic antibacterial agent is selected from azithromycin, ciprofloxacin, doxycycline, erythromycin, vancomycin and cefuroxime. according to some embodiments, the loaded particles of the present invention have a synergistic antibacterial activity of the dendrimer and the loaded agent. According to some embodiments, the pharmaceutical composition comprising loaded particles of the present invention comprises dendrimers and loaded agents having synergistic antibacterial activity.

According to some embodiments, the pharmaceutical composition of the present invention comprising the loaded particles is for use in treating a pathogenic, such as bacterial, infection.

According to another aspect, the present invention provides a method of treating a pathogenic, e.g. bacterial, infection in a subject in need thereof comprising administering to said subject an effective amount of amphiphilic poly(amidoamine) (PAMAM) amine- terminated dendrimers comprising a hydrophobic tail as described in any one of the above and below examples.

According to yet another aspect, the present invention provides an amine-terminated polyamidoamine dendrimer comprising a hydrophobic tail, wherein the hydrophobic tail comprises a C16-C32 alkenyl. According to some examples, the amine-terminated polyamidoamine dendrimer has a structure of Formula V, VI, VII or VIII, as described herein. According to some embodiments, the present invention provides particles formed by such dendrimers. According to some embodiments, the particles further encapsulate a hydrophobic active agent. According to some embodiments, the active agent is an antibacterial agent. According to some examples, the present invention provides a composition, such as a pharmaceutical composition, comprising said dendrimers or said particles. According to some embodiments, the composition is for use in treating a bacterial infection.

According to a further aspect, the present invention provides a bola-amphiphilic amine-terminated polyamidoamine dendrimer, wherein the terminal amine is a tertiary amine. According to some examples, the amine-terminated polyamidoamine dendrimer has a structure of Formula XII, XIII, or XIV, as described herein. According to some embodiments, the present invention provides particles formed by such dendrimers. According to some embodiments, the particles further encapsulate a hydrophobic active agent. According to some embodiments, the active agent is an antibacterial agent. According to some examples, the present invention provides a composition, such as a pharmaceutical composition, comprising said dendrimers or said particles. According to some embodiments, the composition is for use in treating a bacterial infection.

According to certain aspects, the present invention provides a method of treating a bacterial infection in a subject in need thereof comprising administering to said subject the pharmaceutical composition comprising loaded particles comprising the dendrimers of the present invention and a hydrophobic antibacterial agent.

BRIEF DESCRIPTION OF DRAWINGS

Figs. 1A-1D show the dendrimers la-d and their preparations. Fig. 1A - scheme of synthesis of the dendrimers la-d starting with the common dendrimer 1; Fig IB - general scheme of all 4 structures; Fig 1C - the structure of dendrimer la; Fig. ID - the structure of dendrimer lb; Fig. ID - the structure of 1 ^st generation NFL-terminated dendrimer; Fig. IE - the structure of 2 ^st generation NFL-terminated dendrimer; Fig. IF the structure of dendrimer DDC18-4TA; Fig. 1G - the structure of dendrimer DD4TA; Figs. 1H-1K the structure of dendrimer comprising one or more double bonds in the hydrophobic tail denoted as DD01e8A, DD01e8TA, DDLin8A and DDLin8TA, respectively; Fig. IL - a bola- amphiphilic dendrimer denoted as JCB4A; and Fig IM - a bola- amphiphilic dendrimer denoted as MBola-8T; and Fig. IN - a bola- amphiphilic dendrimer denoted as JCB8A.

Fig. 2 shows transmission electron microscopy (TEM) images of the supramolecular nanomicelles formed by dendrimers la (Fig. 2A), lb (Fig. 2B), 1c (Fig. 2C) and Id (Fig. 2D). Size distribution for the nanomicelles of dendrimers la-d measured using TEM. For each sample, at least 300 particles in different TEM images were randomly selected and measured by using ImageJ software to calculate the size distribution of the nanomicelles.

Fig. 3 shows cytotoxicity of dendrimers la-d in mouse fibroblast (L929) cells and human embryonic kidney 293 (HEK293) cells. Cell viability was measured in triplicate using the PrestoBlue assay.

Figs. 4A-4D show imaging of live and dead bacteria cells and their membrane integrity and morphology. Fluorescent microscopic imaging of live and dead cells of (Fig. 4A) E. coli and (Fig. 4B) S. m//' s7Mcthicillin -resistant S. aureus (MRS A) upon treatment with la. Live and dead cells were stained using SYTO9 (green) and propidium iodide (PI) (red), respectively. Fig. 4C and Fig. 4D show scanning electron microscopic imaging of E. coli and S. aureus MRS , respectively, upon treatment with la. Fig. 4E shows hemolysis data of dendrimers la-d in mouse red blood cells (RBC’s); Fig. 4E shows inhibition of biofilm formation of S. aureus JLA513 by dendrimer la. The biofilm viability after 24 hr incubation of the different treatments was measured by the tetrazolium dye MTT assay. Results present the mean (± SEM) of 3 independent experiments. Dendrimer 2 was used as negative control. Figs. 5A-5C show the permeability of the bacterial outer membrane (OM) in E. coli (Fig. 5A) and depolarization of the inner membrane (IM) in E. coli (Fig. 5B) and MRSA (Fig. 5C) after treatment with la was evaluated using the N-phenyl-1 -naphthylamine (NPN) dye and the 3,3 '-dipropylthiadicarbocyanine iodide (diSC3(5)) probe, respectively.

Fig. 6 shows the chemical structure of the dendron 2 having no hydrophobic alkyl chain. Figs. 7A and 7B show the chemical structure of 1 ^st (Fig. 7A) and 2 ^nd (Fig. 7B) generation of dendrimer la.

DETAILED DESCRIPTION OF THE INVENTION

The present application is based on an unexpected observation that low-generation amine-terminated dendrimer comprising a hydrophobic tail showed strong and broad antibacterial activity. As shown in the Examples, amine-terminated dendrimer shows high antibacterial activity and low cytotoxicity. In addition, it was shown that antibiotics encapsulated within nanoparticles formed by such dendrimers provided an extended and, in some cases, a synergistic antibacterial activity.

According to one aspect, the present invention provides a composition comprising, as an active agent, a dendrimer selected from a polyamidoamine (PAMAM) dendrimer comprising a hydrophobic tail or a bola- amphiphilic PAMAM dendrimer, for use in treating a pathogenic infection. According to some embodiments, the dendrimer is an amine- terminated dendrimer. According to some embodiments, the present invention provides a composition comprising, as an active agent, an amine-terminated poly(amidoamine) (PAMAM) dendrimer comprising a hydrophobic tail, for use in treating a pathogenic infection. According to any one of the embodiments and aspects of the present invention, the dendrimers are amphiphilic. According to some embodiments of the application, the pharmaceutical composition comprises a plurality of the dendrimers. According to some embodiments, the pathogenic infection is a bacterial infection and the dendrimers of the present invention act as antibacterial active agent. Thus, the present invention provides a composition comprising, as an active agent, an amine-terminated amphiphilic poly(amidoamine) (PAMAM) dendrimers comprising a hydrophobic tail, for use in treating a bacterial infection. The term “pathogenic infection” as used herein refers to a disease or infection caused by disease-causing microorganisms having a cell membrane and/or wall, their multiplication and the reaction of body tissues to these microorganisms and the toxins that they produce. The term “pathogenic infection” includes but are not limited to infections by bacteria, parasites, protozoans and fungi. According to some embodiments, the pathogenic infection is a bacterial infection. Thus, according to some embodiments, the present invention provides a composition comprising as an active agent an amphiphilic poly(amidoamine) (PAMAM) amine-terminated dendrimers comprising a hydrophobic tail, for use in treating a bacterial infection.

According to some embodiments, the pathogenic infection is a parasitic infection. According to some embodiments, the pathogenic infection is a fungal infection.

The term dendrimer refers to a molecule having a core and multiple shells of branched structures extending from the core. Dendron is a part of a dendrimer with branches coming from a central point and means a wedge-shaped dendrimer fraction having a plurality of surface functional groups and a central functional group. For both a dendrimer and a dendron, the branches can be connected to the core directly or through a linker. A dendrimer generally includes multiple shells or generations, such as Gl, G2, G3, G4, and so on. Repeated response chains are often used to add each generation to the dendrimer.

The PAMAM dendrimers of the present invention have amine terminations and can be any generation of dendrimers including, but not limited to, generation 1 PAMAM dendrimers, generation 2 PAMAM dendrimers, generation 3 PAMAM dendrimers, generation 4 PAMAM dendrimers, generation 5 PAMAM dendrimers. According to some embodiments, the dendrimers of the present invention are low-generation dendrimers.

According to some embodiments, the dendrimer is selected from a generation 2, 3 or 4 dendrimer. According to some embodiments, the dendrimer is G2 dendrimer. According to another embodiment, the dendrimer is G3 dendrimer. According to another embodiment, the dendrimer is G4 or G5 dendrimer. The generation number defines the number of terminal amines in the dendrimer. According to some embodiments, the dendrimer comprises 4 terminal amines. According to some embodiments, the dendrimer comprises 8 terminal amines. According to some embodiments, the dendrimer comprises 16 terminal amines. According to some embodiments, the dendrimer comprises 32 terminal amines. According to some embodiments, the dendrimer comprises 64 terminal amines. According to some embodiments, the dendrimer comprises 4, 8, 16 or 32 terminal amines. According to some embodiments, the amines of the dendrimer terminus are primary amines. According to another embodiment, the terminal amines are secondary amines. According to yet another embodiment, the terminal amines are tertiary amines. According to some embodiments, the terminal amines are positively charged at neutral and acidic pH. According to some embodiments, the terminal amine is a cyclic amine. According to some embodiment, the terminal amines are amidine.

According to some embodiments, the active agent is an amine-terminated polyamidoamine dendrimer comprising a hydrophobic tail. Thus, according to some embodiments, the present invention provides a pharmaceutical composition comprising, as an active agent, an amine-terminated polyamidoamine dendrimer comprising a hydrophobic tail, for use in treating a pathogenic infection. According to some embodiments, the dendrimers of the present invention have a positive surface charge, leading to charged zeta potential of the self-assembled nanoparticles. Specifically, zeta potential is the charge that develops at the interface between a solid surface and its liquid medium. According to some embodiments, the zeta potential of the dendrimers of the present invention is from +1 to +80 mV, from 5 to 60 mV, or from 10 to 50 mV. According to some embodiments, the zeta potential of the dendrimers is from 20 to 45 mV. Zeta potential may be measured e.g., as defined in Examples.

The term "hydrophobic tail" refers to any group having low water solubility and containing four or more carbon atoms. Examples of such hydrophobic tails are aliphatic and aromatic hydrocarbons. The term “hydrophobic tail” encompasses also a saturated or unsaturated, substituted or unsubstituted, straight or branched, cyclic or acyclic hydrocarbon group. As used herein, the term "hydrophobic tail" refers to an aliphatic tail bound to the core of the dendrimer.

According to some embodiments, the hydrophobic tail is an alkyl. As used herein, the term “alkyl”, “alkyl group”, or “alkylene” means a linear or branched, saturated hydrocarbon including one or more carbon atoms (e.g., one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, or more carbon atoms), which is optionally substituted. The notation “Cl 6-24 alkyl” means an optionally substituted linear or branched, saturated hydrocarbon including 16-24 carbon atoms. Unless otherwise specified, an alkyl group described herein refers to both unsubstituted and substituted alkyl groups. According to some embodiments, the hydrophobic tail comprises an alkyl having 10 to 60 carbon atoms. According to some embodiments, the tail comprises a straight alkyl comparing 12 to 40 carbon atoms. According to some embodiments, the hydrophobic tail comprises a C16-C32 alkyl. According to some embodiments, the hydrophobic tail comprises a C16-28 alkyl. According to some embodiments, the hydrophobic tail comprises a C16-C22 alkyl. According to some embodiments, the alkyl is a straight alkyl. According to some embodiments, the hydrophobic tail comprises a C16-C22 straight alkyl. According to some embodiments, the hydrophobic tail is a C16 alkyl. According to some embodiments, the hydrophobic tail is a C18 alkyl. According to some embodiments, the hydrophobic tail is a C20 alkyl. According to some embodiments, the hydrophobic tail is a C22 alkyl.

According to another embodiment, the alkyl is a branched alkyl. According to some embodiments, each chain branch of the branched alkyl comprises 10-20 carbons. According to some embodiments, at least one branch of the branched alky comprises 14 or more carbons. According to some embodiments, at least one branch of the branched alky comprises 16 or more carbons.

According to some embodiments, the hydrophobic tail is an alkenyl. As used herein, the term “alkenyl”, “alkenyl group”, or “alkenylene” means a linear or branched hydrocarbon including two or more carbon atoms (e.g., two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, or more carbon atoms) and at least one double bond, which is optionally substituted. The notation “C 16-22 alkenyl” means an optionally substituted linear or branched hydrocarbon including 16-22 carbon atoms and at least one carbon-carbon double bond. An alkenyl group may include one, two, three, four, or more carbon-carbon double bonds. For example, C18 alkenyl may include one or more double bonds. A C18 alkenyl group including two double bonds may be a linoleyl group. Unless otherwise specified, an alkenyl group described herein refers to both unsubstituted and substituted alkenyl groups.

According to some embodiments, the tail comprises a straight alkenyl comparing 12 to 40 carbon atoms. According to some embodiments, the hydrophobic tail comprises a C16- C32 alkenyl. According to some embodiments, the hydrophobic tail comprises a C16-C32 alkenyl. According to some embodiments, the hydrophobic tail comprises a C16-C28 alkenyl. According to some embodiments, the hydrophobic tail is a C16 alkenyl. According to some embodiments, the hydrophobic tail is a C18 alkenyl. According to some embodiments, the hydrophobic tail is a C20 alkenyl. According to some embodiments, the hydrophobic tail is a C22 alkenyl. According to some embodiments, the alkenyl is a straight alkenyl. According to some embodiments, the hydrophobic tail comprises a C16-C22 straight alkenyl. According to another embodiment, the alkenyl is a branched alkenyl. According to some embodiments, each chain branch of the branched alkenyl comprises 10- 20 carbons. According to some embodiments, at least one branch of the branched alkenyl comprises 14 or more carbons. According to some embodiments, at least one branch of the branched alkenyl comprises 16 or more carbons. According to some embodiments, at least one branch of the branched alkenyl comprises 18 or more carbons. According to some embodiments, at least one branch of the branched alkenyl comprises 20 carbons. According to some embodiments, one of the branches is an alkenyl and one or more other branches is an alkyl.

According to certain embodiments, the hydrophobic chain is a C16-C32 alkynyl. As used herein, the term “alkynyl”, “alkynyl group”, or “alkynylene” means a linear or branched hydrocarbon including two or more carbon atoms (e.g., two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, or more carbon atoms) and at least one carbon-carbon triple bond, which is optionally substituted.

According to some embodiments, the hydrophobic tail is bound to the dendrimer via a linker such as a cyclic, aromatic or non-aromatic compound. According to some embodiments, the linker comprises a triazole or a derivative thereof. According to some embodiments, the linker is an ester, amide, or disulfide. According to some embodiments, the hydrophobic tail is bound to the core of the dendrimer.

According to some embodiments, the present invention provides a composition comprising, as an active agent, an amphiphilic poly(amidoamine) (PAMAM) amine- terminated dendrimer comprising a hydrophobic tail, for use in treating a bacterial infection, wherein the dendrimers are selected from generation 2, 3 or 4 dendrimers comprising from 4 to 16 terminal amines and the hydrophobic tail is a moiety selected from a C16-C32 alkyl and a C16-C32 alkenyl. According to some embodiments, the alkyl or alkenyl are straight. According to some embodiments, the terminal amines of the dendrimers are primary amines. According to some embodiments, the terminal amines of the dendrimers are secondary amines. According to some embodiments, the terminal amines of the dendrimers are tertiary amines.

According to some embodiments, the dendrimer of the present invention has a structure of Formula I:

wherein R is selected from , and any

^{H H} H combination thereof, and X is a C12-C40 alkyl or alkenyl. According to some embodiment,

X is selected from C12, C14, C16, C18, C20, C22, C24, C26, C28, C30, C32, C34, C36 and

C38 alkyl. According to some embodiments, X is a C14-C32 alkyl. According to some embodiments, X is a C16-C24 alkyl. According to some embodiments, X is a C16-C24 alkenyl. According to some embodiments, X is a C16-C22 alkyl. According to some embodiments, X is C18 alkyl. According to some embodiments, X is a straight alkyl. According to some embodiments, X is a branched alkyl. According to some embodiment, X is selected from C12, C14, C16, C18, C20, C22, C24, C26, C28, C30, C32, C34, C36 and C38 alkenyl. According to some embodiments, X is a C14-C32 alkenyl. According to some embodiments, X is a C16-C24 alkenyl. According to some embodiments, X is a C16-C22 alkenyl. According to some embodiments, X is C18 alkenyl. According to some embodiments, the alkenyl comprises two double bonds, i.e., a diene. According to some embodiments, the double bonds are conjugated double bonds. According to some embodiments, the alkenyl comprises 3, 4 or 5 double bonds. According to some embodiments, X is a straight, unbranched alkenyl or alkyl.

According to some embodiments, X is C18 alkyl. According to other embodiments, X is C 18 alkenyl. ₀

According to some embodiments, R is . According to another embodiment, R is According to yet another embodiment, X is C18 straight

H alkyl and R is selected from

H H H

According to some embodiments, R does not comprise guanidine.

According to yet another embodiment, X is C 18 straight alkenyl and R is selected from

H H H According to one embodiment, the dendrimer has a structure of Formula II:

According to one embodiment, the dendrimer has a structure of Formula III:

According to one embodiment, the dendrimer of the present invention has a structure of Formula IV :

According to one embodiment, the dendrimer of the present invention has a structure of Formula V :

According to one embodiment, the dendrimer of the present invention has a structure of Formula VI:

According to one embodiment, the dendrimer of the present invention has a structure of Formula VII:

According to one embodiment, the dendrimer has a structure of Formula VIII:

According to one embodiment, the dendrimer has a structure of Formula IX: According to one embodiment, the dendrimer has a structure of Formula X:

According to one embodiment, the dendrimer of the present invention has a structure of Formula XI:

According to some embodiments, the present invention provides a composition comprising dendrimers having a structure selected from a structure of Formula I, II, III, IV, V, VI, VII, VIII, IX, X, XI, and any combination thereof, for use in treating a pathogenic infection. According to some embodiments, the present invention provides a composition comprising dendrimers having a structure selected from a structure of Formula I, II, III, IV, V, VI, VII, VIII, IX, X and XI, for use in treating a bacterial infection. According to some embodiments, the present invention provides a composition comprising dendrimers having a structure of Formula II, for use in treating a bacterial infection. According to some embodiments, the present invention provides a composition comprising dendrimers having a structure of Formula III, for use in treating a bacterial infection. According to some embodiments, the present invention provides a composition comprising dendrimers having a structure of Formula IV, for use in treating a bacterial infection. According to some embodiments, the present invention provides a composition comprising dendrimers having a structure of Formula V, for use in treating a bacterial infection. According to some embodiments, the present invention provides a composition comprising dendrimers having a structure of Formula VI, for use in treating a bacterial infection. According to some embodiments, the present invention provides a composition comprising dendrimers having a structure of Formula VII, for use in treating a bacterial infection. According to some embodiments, the present invention provides a composition comprising dendrimers having a structure of Formula VIII, for use in treating a bacterial infection. According to some embodiments, the present invention provides a composition comprising dendrimers having a structure of Formula IX, for use in treating a bacterial infection. According to some embodiments, the present invention provides a composition comprising dendrimers having a structure of Formula X, for use in treating a bacterial infection. According to some embodiments, the present invention provides a composition comprising dendrimers having a structure of Formula XI, for use in treating a bacterial infection. According to any one of the above embodiments, the use is for treating a parasitic infection. According to any one of the above embodiments, the use is for treating a fungal infection.

According to any one of the above embodiments, the dendrimers of the present invention are capable of self-assembling and forming particles. Thus, according to any one of the above embodiments, the present invention provides a composition comprising a plurality of particles formed by the dendrimers according to any one of the above embodiments, for use in treating bacterial infection. According to some embodiments, the size of the particles is from 5 to 100 nm, from 7 to 75, from 10 to 50 or from 5 to 50 nm. According to other embodiments, the size of the particles is from 7 to 35 nm. According to some embodiments, the nanoparticles have an average size of from 8 to 40 nm. According to some embodiments, the nanoparticles have an average size of from 10 to 35 nm. According to some embodiments, the nanoparticles have an average size of from 10 to 30 nm. According to some embodiments, the nanoparticles have an average size of from 8 to 30 nm. According to some embodiments, the nanoparticles have an average size of from 10 to 25 nm.

According to some embodiments, the dendrimer is bola- amphiphilic amine-terminated polyamidoamine dendrimer. Therefore, the present invention provides a pharmaceutical composition comprising, as an active agent, bola- amphiphilic amine-terminated polyamidoamine dendrimer, for use in treating a pathogenic infection.

The term "bola- amphiphilic dendrimer" refers to molecules that have two PAMAM dendrons connected via a long hydrophobic hydrocarbon chain. According to some embodiments, the bola- amphiphilic amine-terminated polyamidoamine dendrimer has a structure of Z-Y-Z, wherein Z has the structure of Formula I: wherein R is selected from ■ ■> ^an d any combination thereof, and X is a moiety selected from C8-C16 alkyl, Cl l alkyl, C8-C16 alkenyl and C 11 alkenyl and Y is a linker.

According to some embodiments, the linker has a structure L f J . According to some embodiments, the linker is a di-sulfide moiery.

According to some embodiments, the bola-amphiphilic amine-terminated poly amidoamine dendrimer has Formula XII According to some embodiments, the bola-amphiphilic amine-terminated poly amidoamine dendrimer has Formula XIII (Fig. IM): According to some embodiments, the bola-amphiphilic amine-terminated poly amidoamine dendrimer has Formula XIV (Fig. IN) According to any one of the above embodiments, the minimal minimum inhibitory concentration to inhibit 90 % of bacteria (MIC) of the dendrimers of the present invention or of particles formed by said dendrimers is from 0.1 to 100 pM, from 0.5 to 50 pM, from 1 to 50 pM or from 1 to 10 pM. According to any one of the above embodiments, the MIC of the dendrimers of the present invention or of particles formed by said dendrimers is from 20 to 70 pM or from 40 to 60 pM.

According to any one of the above embodiments, the pathogenic infection is a bacterial infection. According to some embodiments, the bacterial infection is caused by bacteria selected from Gram-positive and Gram-negative bacteria. According to some embodiments, the composition is for use in treating bacterial infection caused by Gram-positive bacteria. According to some embodiments, the composition is for use in treating bacterial infection caused by Gram-negative bacteria.

The term "infection" or "bacterial infection" as used herein refers to and includes the presence of bacteria, in or on a subject, which, if its growth were inhibited, would result in a benefit to the subject. As such, the term "infection" in addition to referring to the presence of bacteria also refers to normal flora, which is not desirable. The term "infection" includes infections caused by bacteria.

The term “Gram-positive” refers to bacteria that are stained dark blue or violet by Gram staining (crystal violet), in contrast to Gram-negative bacteria which cannot retain the crystal violet stain and instead take up the counterstain (safranin or fuchsine) thus appearing red or pink. Gram-positive organisms are able to retain the crystal violet stain because of a higher amount of peptidoglycan in the inner cell wall and typically lack the secondary or outer wall and lipopolysaccharide layer found in Gram-negative bacteria.

According to some embodiments, Gram-negative bacteria is selected from the group consisting of Actinobacillus, Aeromonas, Anaplasma, Arcobacter, Avibacterium, Bacteroides, Bartonella, Bordetella, Borrelia, Brachyspira, Brucella, Campylobacter, Capnocytophaga, Chlamydia, Chlamydophila, Chryseobacterium, Coxiella, Cytophaga, Dichelobacter, Edwardsiella, Ehrlichia, Escherichia, Flavobacterium, Francisella, Fusobacterium, Gallibacterium, Haemophilus, Histophilus, Klebsiella, Lawsonia, Leptospira, Mannheimia, Megasphaera, Moraxella, Neorickettsia, Nicoletella, Neisseria, Ornithobacterium, Pasteurella, Photobacterium, Piscichlamydia, Piscirickettsia, Porphyromonas, Prevotella, Proteus, Pseudomonas, Rickettsia, Riemerella, Salmonella, Streptobacillus, Tenacibaculum, Vibrio and Yersinia. According to some embodiments, the Gram-positive bacteria is selected from Staphylococcus spp, Streptococci, Enterococcus spp, Leuconostoc spp, Corynebacterium spp, Arcanobacteria spp, Trueperella spp, Rhodococcus spp, Bacillus spp, Anaerobic Cocci, Anaerobic Gram-Positive Nonsporulating Bacilli, Actinomyces spp, Clostridium spp, Nocardia spp, Erysipelothrix spp, Eisteria spp, Kytococcus spp, Mycoplasma spp, Ureaplasma spp, Borrelia spp and Mycobacterium spp.

Examples of infectious bacteria include Helicobacter pyloris, Borelia burgdorferi, Legionella pneumophilia, Mycobacteria spp. (e.g., M. tuberculosis, M. avium, M. intracellulare, M. kansasii, M. gordonae). Neisseria gonorrhoeae, Listeria monocytogenes, Streptococcus pyogenes (Group A Streptococcus), Streptococcus agalactiae (Group B Streptococcus), Streptococcus (viridans group), Streptococcus faecalis, Streptococcus Bovis, Streptococcus (anaerobic spp.), Streptococcus pneumoniae, pathogenic Campylobacter sp., Enterococcus sp., Haemophilus influenzae, Bacillus anthracis, Corynebacterium diphtheriae, Corynebacterium sp., Erysipelothrix rhusiopathiae, Clostridium perfringens, Clostridium tetani, Enterobacter aerogenes, Klebsiella pneumoniae, Pasturella multocida, Bacteroides sp., Fusobacterium nucleatum, Streptobacillus moniliformis, Treponema pallidum, Treponema pertenue, Leptospira, and Actinomyces israelii.

According to some embodiments, the composition of the present invention is for use in treating Pseudomonas bacteria, such as Pseudomonas aeruginosa. According to some embodiments, the composition of the present invention is for treating Staphylococcus bacteria, such as Staphylococcus aureus. According to some embodiments, the composition of the present invention is for treating Klebsiella bacteria, such as Klebsiella pneumoniae. According to some embodiments, the composition of the present invention is for treating Borrelia bacteria, such as Borrelia bavariensis. According to some embodiments, the composition of the present invention is for treating Neisseria bacteria, such as Neisseria meningitidis. According to some embodiments, the composition of the present invention is for treating drug resistant bacteria such as methicillin resistant Staphylococcus aureus (MRSA).

According to some embodiments, the composition of the present invention is for use in treating a bacterial infection, provided that the infection is not caused by Borrelia and/or Neisseria.

According to some embodiments, the present invention provides a dendrimer of

Formula II for use in treating a bacterial infection caused by Gram-positive bacteria. According to some embodiments, the present invention provides a dendrimer of Formula II for use in treating a bacterial infection caused by Gram-negative bacteria.

According to some embodiments, the present invention provides a dendrimer of Formula III for use in treating a bacterial infection caused by Gram-positive bacteria. According to some embodiments, the present invention provides a dendrimer of Formula III for use in treating a bacterial infection caused by Gram-negative bacteria.

According to some embodiments, the present invention provides a dendrimer of Formula IV for use in treating a bacterial infection caused by Gram-positive bacteria. According to some embodiments, the present invention provides a dendrimer of Formula IV for use in treating a bacterial infection caused by Gram-negative bacteria.

According to some embodiments, the present invention provides a dendrimer of Formula V for use in treating a bacterial infection caused by Gram-positive bacteria. According to some embodiments, the present invention provides a dendrimer of Formula V for use in treating a bacterial infection caused by Gram-negative bacteria.

According to some embodiments, the present invention provides a dendrimer of Formula VI for use in treating a bacterial infection caused by Gram-positive bacteria. According to some embodiments, the present invention provides a dendrimer of Formula VI for use in treating a bacterial infection caused by Gram-negative bacteria.

According to some embodiments, the present invention provides a dendrimer of Formula VII for use in treating a bacterial infection caused by Gram-positive bacteria. According to some embodiments, the present invention provides a dendrimer of Formula

VII for use in treating a bacterial infection caused by Gram-negative bacteria.

According to some embodiments, the present invention provides a dendrimer of Formula VIII for use in treating a bacterial infection caused by Gram-positive bacteria. According to some embodiments, the present invention provides a dendrimer of Formula

VIII for use in treating a bacterial infection caused by Gram-negative bacteria.

According to some embodiments, the present invention provides a dendrimer of Formula IX for use in treating a bacterial infection caused by Gram-positive bacteria. According to some embodiments, the present invention provides a dendrimer of Formula IX for use in treating a bacterial infection caused by Gram-negative bacteria.

According to some embodiments, the present invention provides a dendrimer of Formula X for use in treating a bacterial infection caused by Gram-positive bacteria. According to some embodiments, the present invention provides a dendrimer of Formula X for use in treating a bacterial infection caused by Gram-negative bacteria. According to some embodiments, the present invention provides a dendrimer of Formula XI for use in treating a bacterial infection caused by Gram-positive bacteria. According to some embodiments, the present invention provides a dendrimer of Formula XI for use in treating a bacterial infection caused by Gram-negative bacteria.

According to some embodiments, the present invention provides a dendrimer of Formula XII for use in treating a bacterial infection caused by Gram-positive bacteria. According to some embodiments, the present invention provides a dendrimer of Formula

XII for use in treating a bacterial infection caused by Gram-negative bacteria.

According to some embodiments, the present invention provides a dendrimer of Formula XIII for use in treating a bacterial infection caused by Gram-positive bacteria. According to some embodiments, the present invention provides a dendrimer of Formula

XIII for use in treating a bacterial infection caused by Gram-negative bacteria.

According to some embodiments, the present invention provides a dendrimer of Formula XIV for use in treating a bacterial infection caused by Gram-positive bacteria. According to some embodiments, the present invention provides a dendrimer of Formula

XIV for use in treating a bacterial infection caused by Gram-negative bacteria.

According to some embodiments, the composition of the present invention comprising the dendrimer of Formula II is for treating a bacterial infection caused by a bacteria selected from Pseudomonas bacteria, such as Pseudomonas aeruginosa, Staphylococcus bacteria, such as Staphylococcus aureus, Klebsiella bacteria, such as Klebsiella pneumoniae and drugresistant bacteria such as methicillin resistant Staphylococcus aureus (MRS A). According to some embodiments, the composition of the present invention comprising the dendrimer of Formula III is for treating a bacterial infection caused by a bacteria selected from Pseudomonas bacteria, such as Pseudomonas aeruginosa, Staphylococcus bacteria, such as Staphylococcus aureus, Klebsiella bacteria, such as Klebsiella pneumoniae and drugresistant bacteria such as methicillin resistant Staphylococcus aureus (MRS A). According to some embodiments, the composition of the present invention comprising the dendrimer of Formula IV is for treating a bacterial infection caused by a bacteria selected from Pseudomonas bacteria, such as Pseudomonas aeruginosa, Staphylococcus bacteria, such as Staphylococcus aureus, Klebsiella bacteria, such as Klebsiella pneumoniae, drug-resistant bacteria such as methicillin resistant Staphylococcus aureus (MRSA), Borrelia bacteria, such as Borrelia bavariensis and Neisseria bacteria, such as Neisseria meningitidis. According to some embodiments, the composition of the present invention comprising the dendrimer of Formula V is for treating a bacterial infection caused by a bacteria selected from Pseudomonas bacteria, such as Pseudomonas aeruginosa, Staphylococcus bacteria, such as Staphylococcus aureus, Klebsiella bacteria, such as Klebsiella pneumoniae, drugresistant bacteria such as methicillin resistant Staphylococcus aureus (MRSA), and Borrelia bacteria, such as Borrelia bavariensis. According to some embodiments, the composition of the present invention comprising the dendrimer of Formula VI is for treating a bacterial infection caused by a bacteria selected from Pseudomonas bacteria, such as Pseudomonas aeruginosa, Staphylococcus bacteria, such as Staphylococcus aureus, Klebsiella bacteria, such as Klebsiella pneumoniae, drug-resistant bacteria such as methicillin resistant Staphylococcus aureus (MRSA), and Borrelia bacteria, such as Borrelia bavariensis. According to some embodiments, the composition of the present invention comprising the dendrimer of Formula VII is for treating a bacterial infection caused by a bacteria selected from Pseudomonas bacteria, such as Pseudomonas aeruginosa, Staphylococcus bacteria, such as Staphylococcus aureus, Klebsiella bacteria, such as Klebsiella pneumoniae, drugresistant bacteria such as methicillin resistant Staphylococcus aureus (MRSA), and Borrelia bacteria, such as Borrelia bavariensis. According to some embodiments, the composition of the present invention comprising the dendrimer of Formula VIII is for treating a bacterial infection caused by a bacteria selected from Pseudomonas bacteria, such as Pseudomonas aeruginosa, Staphylococcus bacteria, such as Staphylococcus aureus, Klebsiella bacteria, such as Klebsiella pneumoniae, drug-resistant bacteria such as methicillin resistant Staphylococcus aureus (MRSA), and Borrelia bacteria, such as Borrelia bavariensis. According to some embodiments, the composition of the present invention comprising the dendrimer of Formula IX is for treating a bacterial infection caused by a bacteria selected from Pseudomonas bacteria, such as Pseudomonas aeruginosa, Staphylococcus bacteria, such as Staphylococcus aureus, Klebsiella bacteria, such as Klebsiella pneumoniae and drugresistant bacteria such as methicillin resistant Staphylococcus aureus (MRSA). According to some embodiments, the composition of the present invention comprising the dendrimer of Formula X is for treating a bacterial infection caused by a bacteria selected from Pseudomonas bacteria, such as Pseudomonas aeruginosa, Staphylococcus bacteria, such as Staphylococcus aureus, Klebsiella bacteria, such as Klebsiella pneumoniae and drugresistant bacteria such as methicillin resistant Staphylococcus aureus (MRSA). According to some embodiments, the composition of the present invention comprising the dendrimer of Formula XI is for treating a bacterial infection caused by a bacteria selected from Pseudomonas bacteria, such as Pseudomonas aeruginosa, Staphylococcus bacteria, such as Staphylococcus aureus, Klebsiella bacteria, such as Klebsiella pneumoniae, drug-resistant bacteria such as methicillin resistant Staphylococcus aureus (MRSA), Borrelia bacteria, such as Borrelia bavariensis and Neisseria bacteria, such as Neisseria meningitidis. According to some embodiments, the composition of the present invention comprising the dendrimer of Formula XII is for treating a bacterial infection caused by a bacteria selected from Pseudomonas bacteria, such as Pseudomonas aeruginosa, Staphylococcus bacteria, such as Staphylococcus aureus, Klebsiella bacteria, such as Klebsiella pneumoniae and drugresistant bacteria such as methicillin resistant Staphylococcus aureus (MRSA). According to some embodiments, the composition of the present invention comprising the dendrimer of Formula XIII is for treating a bacterial infection caused by a bacteria selected from Pseudomonas bacteria, such as Pseudomonas aeruginosa, Staphylococcus bacteria, such as Staphylococcus aureus, Klebsiella bacteria, such as Klebsiella pneumoniae and drugresistant bacteria such as methicillin resistant Staphylococcus aureus (MRSA). According to some embodiments, the composition of the present invention comprising the dendrimer of Formula XIV is for treating a bacterial infection caused by a bacteria selected from Pseudomonas bacteria, such as Pseudomonas aeruginosa, Staphylococcus bacteria, such as Staphylococcus aureus, Klebsiella bacteria, such as Klebsiella pneumoniae and drugresistant bacteria such as methicillin resistant Staphylococcus aureus (MRSA).

According to some embodiments, the composition of the present invention comprising the dendrimer of Formula V is for treating a bacterial infection caused by a bacteria selected from Borrelia bacteria, such as Borrelia bavariensis and Neisseria bacteria, such as Neisseria meningitidis. According to some embodiments, the composition of the present invention comprising the dendrimer of Formula VI is for treating a bacterial infection caused by a bacteria selected from Borrelia bacteria, such as Borrelia bavariensis and Neisseria bacteria, such as Neisseria meningitidis. According to some embodiments, the composition of the present invention comprising the dendrimer of Formula VII is for treating a bacterial infection caused by a bacteria selected from Borrelia bacteria, such as Borrelia bavariensis and Neisseria bacteria, such as Neisseria meningitidis. According to some embodiments, the composition of the present invention comprising the dendrimer of Formula VIII is for treating a bacterial infection caused by a bacteria selected from Borrelia bacteria, such as Borrelia bavariensis and Neisseria bacteria, such as Neisseria meningitidis. According to some embodiments, the composition of the present invention comprising the dendrimer of Formula IV is for treating a bacterial infection caused by a bacteria selected from Borrelia bacteria, such as Borrelia bavariensis and Neisseria bacteria, such as Neisseria meningitidis. According to some embodiments, the composition of the present invention comprising the dendrimer of Formula XII is for treating a bacterial infection caused by Borrelia bacteria, such as Borrelia bavariensis.

According to any one of the above embodiments, the composition of the present invention is for treating parasitic infections. According to any one of the above embodiments, the composition of the present invention is for treating fungal infections.

The term “treating” a condition or patient refers to taking steps to obtain beneficial or desired results, including clinical results. Beneficial or desired clinical results include, but are not limited to, or ameliorating, abrogating, substantially inhibiting, slowing or reversing the progression of a disease, condition or disorder, substantially ameliorating or alleviating clinical or esthetical symptoms of a condition, substantially preventing the appearance of clinical or esthetical symptoms of a disease, condition, or disorder, and protecting from harmful or annoying symptoms. Treating further refers to accomplishing one or more of the following: (a) reducing the severity of the disorder; (b) limiting development of symptoms characteristic of the disorder(s) being treated; (c) limiting worsening of symptoms characteristic of the disorder(s) being treated; (d) limiting recurrence of the disorder(s) in patients that have previously had the disorder(s); and/or (e) limiting recurrence of symptoms in patients that were previously asymptomatic for the disorder(s). The term "treat", "treating" or "treatment" includes both prophylactic and therapeutic purposes. The term "prophylactic treatment" refers to treating a subject who is not yet infected, but who is susceptible to, or otherwise at a risk of infection (preventing the bacterial infection).

According to any one of the above embodiments, the composition of the present invention comprises a carrier. The term “carrier” includes as a class any compound or composition useful in facilitating storage, stability, administration, cell targeting and/or delivery of the topical composition, including, without limitation, suitable vehicles, skin conditioning agents, skin protectants, diluents, emollients, solvents, excipients, pH modifiers, salts, colorants, rheology modifiers, thickeners, lubricants, humectants, antifoaming agents, erodeable polymers, hydrogels, surfactants, emulsifiers, emulsion stabilizers, adjuvants, surfactants, preservatives, chelating agents, fatty acids, mono-di- and tri-glycerides and derivates thereof, waxes, oils and water.

According to any one of the above embodiments, the composition of the present invention is a pharmaceutical composition and the carrier is a pharmaceutically acceptable carrier. Therefore, according to any one of the above embodiments, the term "composition" encompasses and may be replaced by the term "pharmaceutical composition". Thus, according to any one of the above embodiments, the present invention provides a pharmaceutical composition comprising as an active agent an amphiphilic PAMAM amine- terminated dendrimer comprising a hydrophobic tail and a pharmaceutically acceptable carrier, for use in treating a pathogenic infection such as a bacterial infection. According to any one of the above embodiments, the present invention provides a pharmaceutical composition comprising as an active agent a bola-amphiphilic amine-terminated PAMAM dendrimer a pharmaceutically acceptable carrier, for use in treating a pathogenic infection such as a bacterial infection. Any one of the above embodiments apply herein as well.

The term “pharmaceutical composition” as used herein refers to a composition comprising at least one active agent as disclosed herein formulated together with one or more pharmaceutically acceptable carriers.

Formulation of the pharmaceutical composition may be adjusted according to applications. In particular, the pharmaceutical composition may be formulated using a method known in the art so as to provide a rapid, continuous or delayed release of the active ingredient after administration to mammals. For example, the formulation may be any one selected from among plasters, granules, lotions, liniments, lemonades, aromatic waters, powders, syrups, ophthalmic ointments, liquids and solutions, aerosols, extracts, elixirs, ointments, fluidextracts, emulsions, suspensions, decoctions, infusions, ophthalmic solutions, tablets, suppositories, injections, spirits, capsules, creams, troches, tinctures, pastes, pills, and soft or hard gelatin capsules.

The term "pharmaceutically acceptable carrier" or "pharmaceutically acceptable excipient" as used herein refers to any and all solvents, dispersion media, preservatives, antioxidants, coatings, isotonic and absorption delaying agents, surfactants, fillers, disintegrants, binders, diluents, lubricants, glidants, pH adjusting agents, buffering agents, enhancers, wetting agents, solubilizing agents, surfactants, antioxidants the like, that are compatible with pharmaceutical administration. The use of such media and agents for pharmaceutically active substances is well known in the art. The compositions may contain other active compounds providing supplemental, additional, or enhanced therapeutic functions, solid carriers or excipients such as, for example, lactose, starch or talcum or liquid carriers such as, for example, water, fatty oils or liquid paraffins.

Solutions or suspensions used for parenteral, intradermal, or subcutaneous application typically include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol (or other synthetic solvents), antibacterial agents (e.g., benzyl alcohol, methyl parabens), antioxidants (e.g., ascorbic acid, sodium bisulfite), chelating agents (e.g., ethylenediaminetetraacetic acid), buffers (e.g., acetates, citrates, phosphates), and agents that adjust tonicity (e.g., sodium chloride, dextrose). The pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide, for example. The parenteral preparation can be enclosed in ampules, disposable syringes or multiple dose glass or plastic vials.

Pharmaceutical compositions adapted for parenteral administration include, but are not limited to, aqueous and non-aqueous sterile injectable solutions or suspensions, which can contain antioxidants, buffers, bacteriostats and solutes that render the compositions substantially isotonic with the blood of an intended recipient. Such compositions can also comprise water, alcohols, polyols, glycerine and vegetable oils, for example. Extemporaneous injection solutions and suspensions can be prepared from sterile powders, granules and tablets. Such compositions preferably comprise a therapeutically effective amount of a compound of the invention and/or other therapeutic agent(s), together with a suitable amount of carrier to provide the form for proper administration to the subject.

The composition for oral administration may be in a form of tablets, troches, lozenges, aqueous, or oily suspensions, dispersible powders or granules, emulsions, hard or soft capsules, or syrups or elixirs. Pharmaceutical compositions intended for oral use may be prepared according to any method known to the art for the manufacture of pharmaceutical compositions and may further comprise one or more agents selected from sweetening agents, flavoring agents, coloring agents and preserving agents in order to provide pharmaceutically elegant and palatable preparations. Tablets contain the active agent in admixture with nontoxic pharmaceutically acceptable excipients, which are suitable for the manufacture of tablets. These excipients may be, e.g., inert diluents such as calcium carbonate, sodium carbonate, lactose, calcium phosphate, or sodium phosphate; granulating and disintegrating agents, e.g., com starch or alginic acid; binders; and lubricating agents. The tablets are preferably coated utilizing known techniques to delay disintegration and absorption in the gastrointestinal tract and thereby provide an extended release of the drug over a longer period.

The composition of the present invention may be administered by any known method. The term "administering” or “administration of’ a substance, a compound or an agent to a subject can be carried out using one of a variety of methods known to those skilled in the art. For example, a compound or an agent can be administered, intravenously, arterially, intradermally, intramuscularly, intraperitonealy, intravenously, subcutaneously, ocularly, sublingually, orally (by ingestion), intranasally (by inhalation), intraspinally, intracerebrally, and transdermally (by absorption, e.g., through a skin duct). A compound or agent can also appropriately be introduced by rechargeable or biodegradable polymeric devices or other devices, e.g., patches and pumps, or formulations, which provide for the extended, slow or controlled release of the compound or agent. Administering can also be performed, for example, once, a plurality of times, and/or over one or more extended periods. According to some embodiments, the composition is administered 1, 2, 3, 4, 5 or 6 times a day. According to other embodiments, the composition is administered 1, 2, 3, 4, 5 or 6 times a month. In some embodiments, the administration includes both direct administration, including selfadministration, and indirect administration, including the act of prescribing a drug. For example, as used herein, a physician who instructs a patient to self-administer a drug, or to have the drug administered by another and/or who provides a patient with a prescription for a drug is administering the drug to the patient. According to some embodiments, the composition, e.g. a pharmaceutical composition, is administered orally. According to some embodiments, the composition, e.g. a pharmaceutical composition, is administered via injections, e.g. by IV infusion.

The composition of the present invention may be co-administered with any other anti- pathogenic agent. The term "co-administration" of the compounds is performed in a regimen selected from a single combined composition, separate individual compositions administered substantially at the same time, and separate individual compositions administered under separate schedules and include treatment regimens in which the agents are not necessarily administered by the same route of administration or at the same time. The compounds can be administered sequentially in either order or substantially simultaneously. The term “sequential manner” refers to an administration of two compounds at different times, and optionally in different modes of administration. The agents can be administered sequentially in either order.

The term “substantially simultaneous manner” refers to the administration of two compounds with only a short time interval between them. In some embodiments, the time interval is in the range of from 0.5 to 60 minutes.

In some embodiments, the anti-pathogenic agent is an antibacterial agent. The term "anti-pathogenic agent" as used herein refers to any substance, compound or a combination of substances or a combination of compounds capable of: (i) inhibiting, reducing or preventing the growth of a pathogen; (ii) inhibiting or reducing the ability of a pathogen to produce infection in a subject; or (iii) inhibiting or reducing the ability of a pathogen to multiply or remain infective in the environment. The composition of the present invention may be co-administered with any other antibacterial agent. The term "antibacterial agent" as used herein refers to any substance, compound or a combination of substances or a combination of compounds capable of: (i) inhibiting, reducing or preventing growth of bacteria; (ii) inhibiting or reducing the ability of a bacteria to produce infection in a subject; or (iii) inhibiting or reducing the ability of bacteria to multiply or remain infective in the environment. The term "antibacterial agent" also refers to a compound capable of decreasing infectivity or virulence of bacteria.

According to some embodiments, the present invention provides a composition for use in treating a bacterial infection as described in any one of the above embodiments, wherein the use comprises co-administering the composition with an additional antibacterial agent. According to some embodiments, the additional antibacterial agent is selected from aminoglycosides, carbacephems, carbapenems, cephalosporins, cephamycins, fluoroquinolones, glycopeptides, lincosamides, macrolides, monobactams, penicillins, quinolones, sulfonamides, and tetracyclines. According to some embodiments, the additional antibacterial agent is selected from azithromycin, ciprofloxacin, doxycycline, erythromycin, vancomycin and cefuroxime. According to some embodiments, the additional antibacterial agent is selected from rifampin, vancomycin, erythromycin, clarithromycin, azithromycin, tetracycline, doxycycline, chloramphenicol, clindamycin, linezolid, ciprofloxacin, levofloxacin, norfloxacin, moxifloxacin, gentamicin, amikacin, kanamycin, neomycin, streptomycin, polymyxin b, daptomycin, fosfomycin, imipenem, meropenem, nafcillin, oxacillin, penicillin g, piperacillin, ticarcillin, trimethoprim, sulfamethoxazole, rifabutin, colistin, teicoplanin, telithromycin, tigecycline, mupirocin, bacitracin, nitrofurantoin, and clavulanate. According to some embodiments, the co-administration provides a synergistic antibacterial effect.

According to another aspect, the present invention provides a method for treating a pathogenic infection in a subject in need thereof comprising administering to said subject a therapeutically effective amount of amphiphilic poly(amidoamine) (PAMAM) amine - terminated dendrimers comprising a hydrophobic tail or bola-amphiphilic amine-terminated polyamidoamine dendrimer, or a pharmaceutical composition thereof as described in any one of the above aspects and embodiments. All terms, embodiments and definitions disclosed in any one of the above aspects apply and are encompassed herein as well.

According to some embodiments, the pathogenic infection is a bacterial infection. The term “therapeutically effective amount” of a drug or agent is an amount of a drug or an agent that, when administered to a subject will have the intended therapeutic effect, e.g. antibacterial effect, such as bacteriostatic or bactericidic effect. The full therapeutic effect does not necessarily occur by administration of one dose, and may occur only after administration of a series of doses. Thus, a therapeutically effective amount may be administered in one or more administrations. The precise effective amount needed for a subject will depend upon, for example, the subject's size, health and age, the nature and extent of the cognitive impairment, and the therapeutics or combination of therapeutics selected for administration, and the mode of administration. The skilled person can readily determine the effective amount for a given situation by routine experimentation.

According to another aspect, the present invention provides use of poly(amidoamine) (PAMAM) amine-terminated dendrimers comprising a hydrophobic tail or of bola- amphiphilic amine-terminated polyamidoamine dendrimers in preparation of a medicament for treating a pathogenic, such as bacterial, infection. All terms, embodiments and definitions disclosed in any one of the above aspects apply and are encompassed herein as well.

According to another aspect, the present invention provides a pharmaceutical composition comprising a hydrophobic anti -pathogenic agent encapsulated within an amine- terminated poly(amidoamine) (PAMAM) dendrimer comprising a hydrophobic tail. According to any one of the above and below embodiments, the dendrimers are amphiphilic. All terms, embodiments and definitions disclosed in any one of the above aspects apply and are encompassed herein as well. The dendrimers of the present invention form particles that encapsulate/entrap the hydrophobic anti-pathogenic agent. Thus, the present invention provides a pharmaceutical composition comprising a plurality of particles, formed by an amine-terminated PAMAM dendrimer comprising a hydrophobic tail, encapsulating a hydrophobic anti-pathogenic agent. According to some embodiments, the anti -pathogenic agent is an antibacterial agent. Thus, according to some embodiments, the present invention provides a pharmaceutical composition comprising a hydrophobic antibacterial agent encapsulated within amphiphilic poly(amidoamine) (PAMAM) amine-terminated dendrimers comprising a hydrophobic tail. According to some embodiments, the antibacterial agent and dendrimers encapsulating said agent form particles, referred to as “loaded particles”. Thus, according to some embodiments, the present invention provides a pharmaceutical composition comprises a plurality of particles comprising a hydrophobic antibacterial agent encapsulated within amphiphilic poly (amidoamine) (PAMAM) amine- terminated dendrimers comprising a hydrophobic tail. In other words, the present invention provides a pharmaceutical composition comprising a plurality of particles formed by amphiphilic poly(amidoamine) (PAMAM) amine-terminated dendrimers comprising a hydrophobic tail loaded with/encapsulating a hydrophobic antibacterial agent. According to some embodiments, the anti -pathogenic agent is an anti-parasitic agent.

According to some embodiment, the dendrimers are as described in any one of the above aspects and embodiments. According to some embodiments, the dendrimers are selected from a generation 2, 3 or 4 dendrimers. According to some embodiments, the dendrimer comprises 4, 8, 16 or 32 terminal amines. According to some embodiments, the terminal amine is selected from a primary, secondary and tertiary amine. According to some embodiments, the amine is a positively charged amine. According to some embodiments, the wherein hydrophobic tail comprises a C16-C32 or C16-C24 alkyl or alkenyl.

According to some embodiments, the dendrimer has a structure of formula I: wherein R is selected from , and any n H > combination thereof, X is a C16-C32 alkyl or alkenyl. According to some embodiments, X is a straight alkyl. According to some embodiments, X is C16-C22 alkyl. According to some embodiment, X is selected from C12, C14, C16, C18, C20, C22, C24, C26, C28, C30, C32, C34, C36 and C38 alkyl. According to some embodiments, X is a C14-C32 alkyl. According to some embodiments, X is a C16-C24 alkyl. According to some embodiments, X is a C16- C24 alkenyl. According to some embodiments, X is a C16-C22 alkyl. According to some embodiments, X is C18 alkyl. According to some embodiments, X is a straight alkyl. According to some embodiments, X is a branched alkyl. According to some embodiment, X is selected from C12, C14, C16, C18, C20, C22, C24, C26, C28, C30, C32, C34, C36 and C38 alkenyl. According to some embodiments, X is a C14-C32 alkenyl. According to some embodiments, X is a C16-C24 alkenyl. According to some embodiments, X is a C16-C22 alkenyl. According to some embodiments, X is C18 alkenyl. According to some embodiments, the alkenyl comprises two double bonds, i.e., a diene. According to some embodiments, the double bonds are conjugated double bonds. According to some embodiments, the alkenyl comprises 3, 4 or 5 double bonds. According to some embodiments, X is a straight, unbranched alkenyl or alkyl.

According to some embodiments, X is C18 alkyl. According to some embodiments, X o is C 18 alkenyl According to some embodiments, R is According to another embodiments, X is C 18 straight alkyl and R is

H

According to some embodiments, R is selected from , and

According to some embodiments, the antipathogenic agent is an antibacterial agent.

According to some embodiments, the antibacterial agent is hydrophobic. According to some embodiments, the pharmaceutical composition comprises a hydrophobic antibacterial agent encapsulated within particles obtained upon self-assembling of the dendrimers of the present invention. According to some embodiments, the dendrimers are selected from dendrimers having the structure selected from Formula II, III, IV, V, VI, VII, VIII, IX, X, XI and any combination thereof. According to some embodiment, the dendrimer has a structure as defined in Fig. 1. According to some embodiments, the present invention provides a pharmaceutical composition comprising a hydrophobic anti-pathogenic agent, such as an antibacterial agent, encapsulated within particles formed by dendrimers having the structure of Formula II, and a pharmaceutically acceptable carrier. According to some embodiments, the present invention provides a pharmaceutical composition comprising a hydrophobic antipathogenic agent, such as an antibacterial agent, encapsulated within particles formed by dendrimers having the structure of Formula III, and a pharmaceutically acceptable carrier. According to some embodiments, the present invention provides a pharmaceutical composition comprising a hydrophobic anti -pathogenic agent, such as an antibacterial agent, encapsulated within particles formed by dendrimers having the structure of Formula IV, and a pharmaceutically acceptable carrier. According to some embodiments, the present invention provides a pharmaceutical composition comprising a hydrophobic anti-pathogenic agent, such as an antibacterial agent, encapsulated within particles formed by dendrimers having the structure of Formula V, and a pharmaceutically acceptable carrier. According to some embodiments, the present invention provides a pharmaceutical composition comprising a hydrophobic anti-pathogenic agent, such as an antibacterial agent, encapsulated within particles formed by dendrimers having the structure of Formula VI, and a pharmaceutically acceptable carrier. According to some embodiments, the present invention provides a pharmaceutical composition comprising a hydrophobic anti-pathogenic agent, such as an antibacterial agent, encapsulated within particles formed by dendrimers having the structure of Formula VII, and a pharmaceutically acceptable carrier. According to some embodiments, the present invention provides a pharmaceutical composition comprising a hydrophobic anti-pathogenic agent, such as an antibacterial agent, encapsulated within particles formed by dendrimers having the structure of Formula VIII, and a pharmaceutically acceptable carrier. According to some embodiments, the present invention provides a pharmaceutical composition comprising a hydrophobic anti-pathogenic agent, such as an antibacterial agent, encapsulated within particles formed by dendrimers having the structure of Formula IX, and a pharmaceutically acceptable carrier. According to some embodiments, the present invention provides a pharmaceutical composition comprising a hydrophobic anti-pathogenic agent, such as an antibacterial agent, encapsulated within particles formed by dendrimers having the structure of Formula X, and a pharmaceutically acceptable carrier. According to some embodiments, the present invention provides a pharmaceutical composition comprising a hydrophobic anti-pathogenic agent, such as an antibacterial agent, encapsulated within particles formed by dendrimers having the structure of Formula XI, and a pharmaceutically acceptable carrier.

According to some embodiments, the hydrophobic antibacterial agent is selected from azithromycin, ciprofloxacin, doxycycline, erythromycin, vancomycin and cefuroxime.

According to some embodiments, the hydrophobic antibacterial agent is selected from hydrophobic aminoglycosides, hydrophobic carbacephems, hydrophobic carbapenems, hydrophobic cephalosporins, hydrophobic cephamycins, hydrophobic fluoroquinolones, hydrophobic glycopeptides, hydrophobic lincosamides, hydrophobic macrolides, monobactams, hydrophobic penicillins, hydrophobic quinolones, hydrophobic sulfonamides, and hydrophobic tetracyclines. According to some embodiments, the hydrophobic antibacterial agent is selected from hydrophobic aminoglycosides, hydrophobic cephalosporins, hydrophobic fluoroquinolones, hydrophobic macrolides, hydrophobic penicillins, hydrophobic quinolones, and hydrophobic tetracyclines. In some examples, tetracycline is selected from doxycycline, minocycline, demeclocycline, oxytetracycline, lymecycline and methacycline. In some examples, the fluoroquinolone is selected ciprofloxacin, levofloxacin, levofloxacin, gatifloxacin, gatifloxacin and sparfloxacin. In some macrolide is selected from azithromycin, clarithromycin, dirithromycin, or roxithromycin. In some examples, cephalosporin is selected from cefuroxime, cefpodoxime, cefotaxime, ceftriaxone and ceftazidime.

Compositions comprising these particles are formulated and tested for their antibacterial activity.

According to some embodiments, the hydrophobic antibacterial agent is selected from rifampin, vancomycin, erythromycin, clarithromycin, azithromycin, tetracycline, doxycycline, chloramphenicol, clindamycin, linezolid, ciprofloxacin, levofloxacin, norfloxacin, moxifloxacin, gentamicin, amikacin, kanamycin, neomycin, streptomycin, polymyxin b, daptomycin, fosfomycin, imipenem, meropenem, nafcillin, oxacillin, penicillin g, piperacillin, ticarcillin, trimethoprim, sulfamethoxazole, rifabutin, colistin, teicoplanin, telithromycin, tigecycline, mupirocin, bacitracin, nitrofurantoin, and clavulanate.

According to some embodiments, the loaded particles comprise dendrimers of Formula II encapsulating an antibacterial agent selected from rifampin, vancomycin, erythromycin, clarithromycin, azithromycin, tetracycline, doxycycline, chloramphenicol, clindamycin, linezolid, ciprofloxacin, levofloxacin, norfloxacin, moxifloxacin, gentamicin, amikacin, kanamycin, neomycin, streptomycin, polymyxin b, daptomycin, fosfomycin, imipenem, meropenem, nafcillin, oxacillin, penicillin g, piperacillin, ticarcillin, trimethoprim, sulfamethoxazole, rifabutin, colistin, teicoplanin, telithromycin, tigecycline, mupirocin, bacitracin, nitrofurantoin, and clavulanate. According to some embodiments, the loaded particles comprise dendrimers of Formula II encapsulating azithromycin. According to some embodiments, the loaded particles comprise dendrimers of Formula II encapsulating ciprofloxacin. According to some embodiments, the loaded particles comprise dendrimers of Formula II encapsulating vancomycin. According to some embodiments, the loaded particles comprise dendrimers of Formula II encapsulating cefuroxime. According to some embodiments, the loaded particles comprise dendrimers of Formula II encapsulating azithromycin. According to some embodiments, the loaded particles comprise dendrimers of Formula II encapsulating doxycycline. According to some embodiments, the loaded particles comprise dendrimers of Formula II encapsulating erythromycin.

According to some embodiments, the loaded particles comprise dendrimers of Formula III encapsulating an antibacterial agent selected from azithromycin, ciprofloxacin, doxycycline, erythromycin, vancomycin and cefuroxime. According to some embodiments, the loaded particles comprise dendrimers of Formula III encapsulating an antibacterial agent selected from rifampin, vancomycin, erythromycin, clarithromycin, azithromycin, tetracycline, doxycycline, chloramphenicol, clindamycin, linezolid, ciprofloxacin, levofloxacin, norfloxacin, moxifloxacin, gentamicin, amikacin, kanamycin, neomycin, streptomycin, polymyxin b, daptomycin, fosfomycin, imipenem, meropenem, nafcillin, oxacillin, penicillin g, piperacillin, ticarcillin, trimethoprim, sulfamethoxazole, rifabutin, colistin, teicoplanin, telithromycin, tigecycline, mupirocin, bacitracin, nitrofurantoin, and clavulanate. According to some embodiments, the loaded particles comprise dendrimers of According to some embodiments, the loaded particles comprise dendrimers of Formula IV encapsulating an antibacterial agent selected from rifampin, vancomycin, erythromycin, clarithromycin, azithromycin, tetracycline, doxycycline, chloramphenicol, clindamycin, linezolid, ciprofloxacin, levofloxacin, norfloxacin, moxifloxacin, gentamicin, amikacin, kanamycin, neomycin, streptomycin, polymyxin b, daptomycin, fosfomycin, imipenem, meropenem, nafcillin, oxacillin, penicillin g, piperacillin, ticarcillin, trimethoprim, sulfamethoxazole, rifabutin, colistin, teicoplanin, telithromycin, tigecycline, mupirocin, bacitracin, nitrofurantoin, and clavulanate. According to some embodiments, the loaded particles comprise dendrimers of Formula IV encapsulating an antibacterial agent selected from azithromycin, ciprofloxacin, doxycycline, erythromycin, vancomycin and cefuroxime. According to some embodiments, the loaded particles comprise dendrimers of Formula IV encapsulating an antibacterial agent ciprofloxacin. According to some embodiments, the loaded particles comprise dendrimers of Formula IV encapsulating an antibacterial agent doxycycline. According to some embodiments, the loaded particles comprise dendrimers of Formula V encapsulating an antibacterial agent selected from azithromycin, ciprofloxacin, doxycycline, erythromycin, vancomycin and cefuroxime. According to some embodiments, the loaded particles comprise dendrimers of Formula VI encapsulating an antibacterial agent selected from azithromycin, ciprofloxacin, doxycycline, erythromycin, vancomycin and cefuroxime. According to some embodiments, the loaded particles comprise dendrimers of Formula VII encapsulating an antibacterial agent selected from azithromycin, ciprofloxacin, doxycycline, erythromycin, vancomycin and cefuroxime. According to some embodiments, the loaded particles comprise dendrimers of Formula VIII encapsulating an antibacterial agent selected from azithromycin, ciprofloxacin, doxycycline, erythromycin, vancomycin and cefuroxime. According to some embodiments, the loaded particles comprise dendrimers of Formula IX encapsulating an antibacterial agent selected from azithromycin, ciprofloxacin, doxycycline, erythromycin, vancomycin and cefuroxime. According to some embodiments, the loaded particles comprise dendrimers of Formula X encapsulating an antibacterial agent selected from azithromycin, ciprofloxacin, doxycycline, erythromycin, vancomycin and cefuroxime. According to some embodiments, the loaded particles comprise dendrimers of Formula XI encapsulating an antibacterial agent selected from azithromycin, ciprofloxacin, doxycycline, erythromycin, vancomycin and cefuroxime.

According to some embodiments, the loaded particles comprise dendrimers of Formula II, III, IV, V, VI, VII, VIII, IX, X, or XI encapsulating an antibacterial agent selected from rifampin, vancomycin, erythromycin, clarithromycin, azithromycin, tetracycline, doxycycline, chloramphenicol, clindamycin, linezolid, ciprofloxacin, levofloxacin, norfloxacin, moxifloxacin, gentamicin, amikacin, kanamycin, neomycin, streptomycin, polymyxin b, daptomycin, fosfomycin, imipenem, meropenem, nafcillin, oxacillin, penicillin g, piperacillin, ticarcillin, trimethoprim, sulfamethoxazole, rifabutin, colistin, teicoplanin, telithromycin, tigecycline, mupirocin, bacitracin, nitrofurantoin, and clavulanate.

According to some embodiments, the antipathogenic agent is an anti-parasitic agent. According to some embodiments, the antipathogenic agent is an anti-fungal agent.

According to any one of the above embodiments, the particles are nanoparticles (nanoformulations). According to some embodiment, the drug load of the loaded particles is from 20 to 70%. According to some embodiment, the drug load of the particles is from 25 to 65%, from 30 to 60%, from 35 to 55%, from 40 to 50%, or from 40 to 60%. The term “drug load” refers to the ratio between the amount of drug loaded into the particle to the amount of particles.

According to any one of the above embodiments, the particles are dry particles.

According to some embodiments, the loaded particles comprising an anti-pathogenic agent, such as antibacterial agent, encapsulated within the dendrimers of the present invention have a synergistic antipathogenic, such as a synergistic antibacterial, effect. According to some embodiments, the loaded particles comprising an antibacterial agent encapsulated within the dendrimers of the present invention have a synergistic antibacterial effect. According to some embodiments, the pharmaceutical composition is a synergistic pharmaceutical composition. As used herein, the term “synergistic” refers to a combination of a compound described herein, which, when taken together, is more effective than the additive effects of the individual therapies. A synergistic effect of a combination of therapies (e.g., a combination of therapeutic agents) permits the use of lower dosages of one or more of the therapeutic agent(s) and/or less frequent administration of the agent(s) to a subject with a disease or disorder, e.g., a proliferative disorder. The ability to utilize lower the dosage of one or more therapeutic agent and/or to administer the therapeutic agent less frequently reduces the toxicity associated with the administration of the agent to a subject without reducing the efficacy of the therapy in the treatment of a disease or disorder. In addition, a synergistic effect can result in improved efficacy of agents in the prevention, management or treatment of a disease or disorder, e.g. a proliferative disorder. Finally, a synergistic effect of a combination of therapies may avoid or reduce adverse or unwanted side effects associated with the use of either therapeutic agent alone. Thus, the antibacterial effect of dendrimer particles loaded with antibacterial agent have an effect which is more than additive antibacterial effect of dendrimers forming said particles and of entrapped antibacterial agent.

According to any one of the above embodiments, the loaded particles formed by dendrimers and loaded with an antibacterial agent have a size of from 10 to 1000 nm. According to some embodiments, the loaded particles have a size of from 10 to 500 nm or from 10 to 100 nm. According to some embodiments, the loaded particles have a size of from 10 to 80, from 15 to 70 nm. According to some embodiments, the loaded/encapsulating nanoparticles have an average size of from 8 to 40 nm. According to some embodiments, the nanoparticles have an average size of from 10 to 35 nm. According to some embodiments, the nanoparticles have an average size of from 10 to 30 nm. According to some embodiments, the nanoparticles have an average size of from 8 to 30 nm According to some embodiments, the nanoparticles have an average size of from 10 to 25 nm.

According to some embodiments, the present invention provides a pharmaceutical composition comprising particles consisting of a hydrophobic antibacterial agent encapsulated within amphiphilic poly(amidoamine) (PAMAM) amine-terminated dendrimers comprising a hydrophobic tail.

According to any one of the above embodiments, the pharmaceutical composition comprising a plurality of particles comprising a hydrophobic anti-pathogenic agent encapsulated within amphiphilic poly(amidoamine) (PAMAM) amine-terminated dendrimers comprising a hydrophobic tail is for use in treating a pathogenic infection. According to any one of the above embodiments, the pharmaceutical composition comprising particles comprising a hydrophobic antibacterial agent encapsulated within amphiphilic poly(amidoamine) (PAMAM) amine-terminated dendrimers comprising a hydrophobic tail is for use in treating a bacterial infection. Any one of the abovementioned embodiments related to treating bacterial infection applies herein as well. According to some embodiments, the present invention provides a pharmaceutical composition comprising a plurality of particles comprising dendrimers having a structure selected from Formula II, III, IV, V, VI, VII, VIII, IX, X, or XI encapsulating an actibacterial agent selected from azithromycin, ciprofloxacin, doxycycline, erythromycin, vancomycin and cefuroxime for use in treating a bacterial infection. According to some embodiments, the present invention provides a pharmaceutical composition comprising a plurality of particles comprising dendrimers of Formula II encapsulating an actibacterial agent selected from azithromycin, ciprofloxacin, doxycycline, erythromycin, vancomycin and cefuroxime for use in treating a bacterial infection. According to some embodiments, the particles comprising dendrimers of Formula II encapsulating ciprofloxacin. According to some embodiments, the particles comprise dendrimers of Formula II encapsulating vancomycin. According to some embodiments, the particles comprise dendrimers of Formula II encapsulating cefuroxime. According to some embodiments, the particles comprise dendrimers of Formula II encapsulating azithromycin. According to some embodiments, the particles comprise dendrimers of Formula II encapsulating doxycycline. According to some embodiments, the particles comprise dendrimers of Formula II encapsulating erythromycin. According to some embodiment, the pharmaceutical composition is for use in treating bacteria selected from Pseudomonas, Klebsiella, Staphylococcus, Borrelia and Neisseria. According to some embodiment, the pharmaceutical composition is for use in treating bacteria selected Pseudomonas aeruginosa, methicillin resistant Staphylococcus aureus (MRSA), Klebsiella pneumoniae, Staphylococcus aureus, Borrelia bavariensis and Neisseria meningitidis. According to some embodiment, the pharmaceutical composition is for use in treating bacteria selected Pseudomonas aeruginosa, methicillin resistant Staphylococcus aureus (MRSA), Klebsiella pneumoniae, and Staphylococcus aureus. In some alternative embodiments, the particles comprise dendrimers of Formula III and the encapsulated antibacterial agent. According to some embodiments, the present invention provides a pharmaceutical composition comprising a plurality of particles comprising dendrimers of Formula IV encapsulating an actibacterial agent selected from azithromycin, ciprofloxacin, doxycycline, erythromycin, vancomycin and cefuroxime for use in treating a bacterial infection. According to some embodiments, the particles comprising dendrimers of Formula IV encapsulating ciprofloxacin. In some alternative embodiments, the particles comprise dendrimers of Formula IV and the encapsulated antibacterial agent. In some alternative embodiments, the particles comprise dendrimers of Formula V and the encapsulated antibacterial agent. In some alternative embodiments, the particles comprise dendrimers of Formula VI and the encapsulated antibacterial agent. In some alternative embodiments, the particles comprise dendrimers of Formula VII and the encapsulated antibacterial agent. In some alternative embodiments, the particles comprise dendrimers of Formula VIII and the encapsulated antibacterial agent. In some alternative embodiments, the particles comprise dendrimers of Formula IX and the encapsulated antibacterial agent. In some alternative embodiments, the particles comprise dendrimers of Formula X and the encapsulated antibacterial agent. In some alternative embodiments, the particles comprise dendrimers of Formula XI and the encapsulated antibacterial agent. According to some embodiments, the treatment has/provides a synergistic effect. According to some embodiments, the pharmaceutical composition comprising particles comprising dendrimers of Formula II and doxycycline has a synergistic antibacterial effect, e.g. anti-MRSA effect. According to some embodiments, the pharmaceutical composition comprising particles comprising dendrimers of Formula VI and doxycycline has a synergistic antibacterial effect, e.g. anti-Borrelia effect. According to some embodiments, the pharmaceutical composition comprising particles comprising dendrimers of Formula IV and doxycycline has a synergistic antibacterial effect, e.g. anti-Neisseria effect.

According to some embodiments, the pharmaceutical composition may be administered by any known method, e.g. as described in any one of the above embodiments.

According to another aspect, the present invention provides a method of treating a pathogenic infection in a subject in need thereof comprising administering to said subject the pharmaceutical composition comprising particles comprising a hydrophobic antipathogenic agent encapsulated within amphiphilic poly(amidoamine) (PAMAM) amine- terminated dendrimers comprising a hydrophobic tail. According to another aspect, the present invention provides a method of treating a bacterial infection in a subject in need thereof comprising administering to said subject the pharmaceutical composition comprising particles comprising a hydrophobic antibacterial agent encapsulated within amphiphilic poly(amidoamine) (PAMAM) amine-terminated dendrimers comprising a hydrophobic tail. All terms, embodiments and definitions disclosed in any one of the above aspects apply and are encompassed herein as well.

According to some embodiments, the use comprises co-administering the pharmaceutical composition of the present invention as described in any one of the above embodiments with an additional anti-pathogenic agent. According to some embodiments, the use comprises co-administering the pharmaceutical composition particles comprising a hydrophobic antibacterial agent encapsulated within amphiphilic poly(amidoamine) (PAMAM) amine-terminated dendrimers comprising a hydrophobic tail with an additional antibacterial agent.

According to yet another aspect, the present invention provides a method of treating a pathogenic infection in a subject in need thereof comprising administering to said subject a plurality of particles comprising a hydrophobic antipathogenic agent encapsulated within amphiphilic poly(amidoamine) (PAMAM) amine-terminated dendrimers comprising a hydrophobic tail as described in any one of the above embodiments and aspects, or a pharmaceutical composition thereof. All terms, embodiments and definitions disclosed in any one of the above aspects apply and are encompassed herein as well.

According to yet another aspect, the present invention provides use of particles comprising a hydrophobic antipathogenic agent encapsulated within amphiphilic poly(amidoamine) (PAMAM) amine-terminated dendrimers comprising a hydrophobic tail as described in any one of the above embodiments and aspects, for preparation of a medicament for treating a pathogenic infection in a subject in need thereof comprising administering to said subject a plurality of or a pharmaceutical composition thereof. All terms, embodiments and definitions disclosed in any one of the above aspects apply and are encompassed herein as well.

According to yet another aspect, the present invention provides an amine-terminated poly(amidoamine) (PAMAM) dendrimer comprising a hydrophobic tail, wherein the hydrophobic tail is an aliphatic chain comprising at least one double bond. According to any embodiment, the dendrimer is an amphiphilic dendrimer. According to some embodiments, the present invention provides an PAMAM amine-terminated dendrimer comprising a hydrophobic tail being an aliphatic chain and comprising at least one double bond. According to some embodiments, the hydrophobic tail is an alkenyl. Thus, the present invention provides an amphiphilic poly(amidoamine) (PAMAM) amine-terminated dendrimer comprising a hydrophobic tail, wherein the hydrophobic tail comprises a C16- C32 alkenyl. All terms, embodiments and definitions disclosed in any one of the above aspects referring to such a composition apply and are encompassed herein as well.

According to some embodiments, the dendrimer is selected from generation 2, 3, 4 and 5 dendrimers. According to some embodiments, the dendrimer comprises 4 terminal amines. According to some embodiments, the dendrimer comprises 8 terminal amines. According to some embodiments, the dendrimer comprises 16 terminal amines. According to some embodiments, the dendrimer comprises 32 terminal amines. According to some embodiments, the dendrimer comprises 64 terminal amines. According to some embodiments, the dendrimer comprises 4, 8, 16 or 32 terminal amines. According to some embodiments, the dendrimer is selected from generation 2, 3, 4 and 5 dendrimers and comprising 4, 8, 16 or 32 terminal amines.

According to some embodiments, the amine of the dendrimer terminus is a primary amine. According to another embodiment, the terminal amines are secondary amines. According to yet another embodiment, the terminal amines are tertiary amine. According to some embodiments, the terminal amines are positively charged at neutral and acidic pH. According to some embodiments, the terminal amines are cyclic amines. According to some embodiment, the terminal amines are amidine.

According to some embodiments, the hydrophobic tail is an alkenyl. According to some embodiments, the hydrophobic tail is an alkenyl comparing 12 to 40 carbon atoms. According to some embodiments, the hydrophobic tail is a C16-C32 alkenyl. According to some embodiments, the hydrophobic tail is a C16-C22 alkenyl. According to some embodiments, the hydrophobic tail is a C16 alkenyl. According to some embodiments, the hydrophobic tail is a C18 alkenyl. According to some embodiments, the hydrophobic tail is a C20 alkenyl. According to some embodiments, the hydrophobic tail is a C22 alkenyl. According to some embodiments, the alkenyl is a straight alkyl. According to some embodiments, the hydrophobic tail comprises a C16-C22 straight alkenyl. According to another embodiment, the alkenyl is s branched alkenyl. According to some embodiments, each chain branch of the branched alkenyl comprises 10-20 carbons. According to some embodiments, at least one branch of the branched alkenyl comprises 14 carbons. According to some embodiments, at least one branch of the branched alkenyl comprises 16 carbons. According to some embodiments, at least one branch of the branched alkenyl comprises 18 carbons.

According to some embodiments, the alkenyl comprises two double bonds, i.e., a diene. According to some embodiments, the double bonds are conjugated double bonds. According to some embodiments, the alkenyl comprises 3, 4 or 5 double bonds.

According to some embodiments, the hydrophobic tail is bound to the dendrimer via a linker. According to some embodiments, the linker may be a cyclic, aromatic or nonaromatic compound. According to some embodiments, the linker comprises a triazole or a derivative thereof. According to some embodiments, the linker is an ester, amide, or disulfide. According to some embodiments, the hydrophobic tail is bound to the core of the dendrimer.

According to some embodiments, the dendrimer has a structure of Formula I:

wherein R is selected from J , and any combination thereof, X is a C16-C28 alkenyl. According to some embodiments, X is C16- C22 alkenyl. According to some embodiments, X is C18 alkenyl. According to some embodiments, X is C18 alkenyl. According to some embodiments, X is C20 alkenyl. According to some embodiments, X is a diene. According to some embodiments, X is a conjugated diene.

According to some embodiments, R is . According to another

O H embodiment, R is JI, /-\ .NH ₂ - According to yet another embodiment, X is C18 straight

H alkyl and R is selected from o

According to some embodiments, (i) X is C18 straight alkenyl; (ii) R is ' ^x ^N' ^x ^ ^/NH2 or (iii) both (i) and (ii).

According to some embodiments, wherein the dendrimers have a structure selected from a structure of Formula V, VI, VII, and VIII, as depicted hereinabove and in Figs. 1H, II, 1J and IK, respectively.

According to one aspect, the present invention provides particles comprising a plurality of amphiphilic poly(amidoamine) (PAMAM) amine-terminated dendrimers comprising a hydrophobic tail, wherein the hydrophobic tail comprises a C16-C32 alkenyl. According to some embodiments, the hydrophobic tail is a C16-C32 alkenyl. All terms, embodiments and definitions disclosed in any one of the above aspects referring to such a composition apply and are encompassed herein as well. According to some embodiments, the dendrimer comprises 4, 8, 16 or 32 terminal amines. According to some embodiments, the dendrimer is selected from generation 2, 3, 4 and 5 dendrimers.

According to some embodiments, the present invention provides particles comprising a plurality of particles comprising and formed by dendrimers having a structure of Formula I: wherein R is selected from , and any combination thereof, X is a C16-C28 alkenyl. According to some embodiments, X is C16- C22 alkenyl. According to some embodiments, X is C18 alkenyl. According to some embodiments, X is C18 alkenyl. According to some embodiments, X is C20 alkenyl. According to some embodiments, X is a diene. According to some embodiments, X is a conjugated diene.

According to some embodiments, the present invention provides particles comprising and formed by dendrimers having a structure selected from Formula V, VI, VII, and VIII.

According to some embodiment, the particles further comprise a biologically active agent. According to some embodiment, the particles encapsulate comprise a biologically active agent. In some embodiments, the biologically active agent is a nucleic acid. The term “nucleic acid” refers to a single- stranded or double- stranded sequence (polymer) of deoxyribonucleotides or ribonucleotides. In addition, the polynucleotide includes analogues of natural polynucleotides, unless specifically mentioned. According to an embodiment, the nucleic acid may be” selected from the group consisting of deoxyribonucleic acid (DNA), ribonucleic acid (RNA), peptide nucleic acid (PNA), locked nucleic acid (LN A), and analogues thereof, but is not limited thereto. The term encompasses DNA, RNA, single stranded or double stranded and chemical modifications thereof. According to some embodiments, the nucleic acid is DNA. According to some embodiments, the nucleic acid is RNA.

According to some embodiments, the biologically active agent is an active agent. The term "active agent" and “active moiety” are used herein interchangeable and refer to an agent that has biological activity, pharmacologic effects and/or therapeutic utility. According to some embodiments, the active agent is a small molecule. According to some embodiments, the active agent is an anti-pathogenic agent. The anti-pathogenic agent is as described and defined in any one of the above aspects and embodiments. According to some embodiments, the anti-pathogenic agent is an antibacterial agent. According to some embodiments, the anti-pathogenic agent is a hydrophobic antibacterial agent. According to some embodiments, the hydrophobic antibacterial agent is selected from rifampin, vancomycin, erythromycin, clarithromycin, azithromycin, tetracycline, doxycycline, chloramphenicol, clindamycin, linezolid, ciprofloxacin, levofloxacin, norfloxacin, moxifloxacin, gentamicin, amikacin, kanamycin, neomycin, streptomycin, polymyxin b, daptomycin, fosfomycin, imipenem, meropenem, nafcillin, oxacillin, penicillin g, piperacillin, ticarcillin, trimethoprim, sulfamethoxazole, rifabutin, colistin, teicoplanin, telithromycin, tigecycline, mupirocin, bacitracin, nitrofurantoin, and clavulanate.

According to some embodiments, the present invention provides a composition comprising a plurality of amphiphilic amine-terminated PAMAM dendrimers of the present invention comprising a hydrophobic tail, wherein the hydrophobic tail comprises a C16-C32 alkenyl or a plurality of particles comprising same, and a carrier. According to some embodiments, the composition is a pharmaceutical composition comprising a pharmaceutically acceptable carrier. According to some embodiment, the pharmaceutical composition comprising the dendrimers, the particles and/or the particles comprising an anti- pathogenic agent are for use in treating pathogenic infection. According to some embodiments, the pathogenic infection is bacterial infection. According to some embodiments, the particles comprising the dendrimer as described hereinabove and an antibacterial active agent provide a synergistic antibacterial effect.

According to another aspect, the present invention provides an amine-terminated polyamidoamine bola- amphiphilic dendrimer, wherein the terminal amine is a tertiary amine. According to some embodiments, the bola-amphiphilic amine-terminated PAMAM dendrimer is of 1 ^st , 2 ^nd , 3 ^rd , 4 ^th , 5 ^th or 6 ^th generation of PAMAM. According to some embodiments, the amine-terminated PAMAM bola- amphiphilic dendrimer has a structure of Z-Y-Z, wherein:

Z has the structure of Formula I: wherein R is selected from . _N ^NH , and any combination thereof, and X is a moiety selected from C8-C16 alkyl, Cl l alkyl, C8-C16 alkenyl and Cl l alkenyl and Y is a linker. According to some embodiments, X is C8-C14, C9-C13, CIO, Cl l, or C12 alkyl. According to some embodiments, X is C8-C14, C9-C13, CIO, Cl 1, or C 12 alkenyl. According to some embodiments, the linker has a structure

According to some embodiments, the linker is a disulfide linker. According to some embodiments, the linker is a moiety comprising a disulfide. According to some embodiments, the linker is a disulfide-comprising linker. According to some embodiments, the amine-terminated polyamidoamine bola-amphiphilic dendrimer has a structure of formula XIII as described above and provided in Fig. IM.

According to some embodiments, the present invention provides a composition comprising the bola-amphiphilic PAMAM amine-terminated dendrimer as described in the above embodiments of the aspect and a carrier. According to some embodiments, the composition is a pharmaceutical composition and the carrier is a pharmaceutically acceptable carrier. According to some embodiments, the composition is for use in treating a pathogenic infection, such as a bacterial infection.

The terms “a,” “an,” and “the” are used herein interchangeably and mean one or more. The term “and/or” is used to indicate one or both stated cases may occur, for example A and/or B includes, (A and B) and (A or B).

The term “or,” as used herein, denotes alternatives that may, where appropriate, be combined; that is, the term “or” includes each listed alternative separately as well as their combination if the combination is not mutually exclusive.

The terms “comprising”, "comprise(s)", "include(s)", "having", "has" and "contain(s)," are used herein interchangeably and have the meaning of “consisting at least in part of’. When interpreting each statement in this specification that includes the term “comprising”, features other than that or those prefaced by the term may also be present. Related terms such as “comprise” and “comprises” are to be interpreted in the same manner. The terms “have”, “has”, having” and “comprising” may also encompass the meaning of “consisting of’ and “consisting essentially of’, and may be substituted by these terms. The term “consisting of’ excludes any component, step or procedure not specifically delineated or listed. The term “consisting essentially of’ means that the composition or component may include additional ingredients, but only if the additional ingredients do not materially alter the basic and novel characteristics of the claimed compositions or methods.

As used herein, the term “about”, when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of +/- 10%, or +/-5%, +/-1%, or even +/-0.1% from the specified value.

Having now generally described the invention, the same will be more readily understood through reference to the following examples, which are provided by way of illustration and are not intended to be limiting of the present invention.

EXAMPLES

Materials and Methods

General/materials

The ester-terminating dendrimer 1 was synthesized according to the well-established protocol published in Yu et al., Angew Chem Int Ed Engl. 2012 Aug 20;51(34):8478-84. Synthesis of dendrimer la and lb was carried out by amidation of the ester-terminating dendrimer precursor 1 using ethylenediamine and A,A-dimethylethylenediamine respectively according to previously developed protocol (Chen et al., Gene Silencing. Small 2016;12:3667-76; Wu et al., 2005 Jan 21 ;(3):313-5). Whereas synthesis of dendrimer 1c was obtained by conjugation of la with the protected arginine followed by subsequent deprotection. The acid-terminated dendrimer Id was synthesized via hydrolysis of 1 using LiOH. Chemicals were purchased from Sigma Aldrich or Alfa Aesar. Methyl acrylate, ethylenediamine, N, A-Dimethylethylenediamine, dichloromethane and methanol were dried according to the described methods and distilled before use. The other chemicals were used without further purification. For inner/outer membrane assays N-phenyl-1 -naphthylamine (NPN) Arcos Organics (98%),,3'-Di-n-propylthiadicarbocyanine iodide [DiSC3(5)] Alfa Aesar (96%) were used. Dialysis tubing was purchased from Sigma Aldrich (St. Quentin Fallavier, France) and Yuanye Bio-Technology Co., Ltd. (Shanghai, China). Analytical thin layer chromatography (TLC) was performed using silica gel 60 F254 plates 0.2 mm thick with UV light (254 and 364 nm) as revelator. Chromatography was prepared on silica gel (Merck 200-300 mesh).

¹ H and ¹³ C NMR spectra were recorded on Bruker Avance III 400 (400 MHz, ¹ H; 100 MHz, ¹³ C) and Varian Mercury- VX600 (600 MHz, ^X H; 150 MHz, ¹³ C). Chemical shifts (<5) are expressed in parts per million (ppm). The HRMS analysis was carried out with a SYNAPT G2 HDMS (Waters) mass spectrometer equipped with a pneumatically assisted atmospheric pressure ionization (API) source. The sample was ionized in positive electrospray mode under the following conditions: electrospray voltage: 2.8 kV; orifice voltage: 20 V; Nebulization gas flow (nitrogen): 100 L / h. The high resolution mass spectrum (MS) was obtained with a flight time analyzer (TOF). The exact mass measurement was done in triplicate with an external calibration. The sample is dissolved in 300 pL of methanol and then diluted 1/10 in a 1.0% solution of methanol formic acid. The solution of the extract is introduced into the infusion ionization source at a flow rate of lOpL/min.

Synthesis of the amphiphilic 3 ^rd generation dendrimers la-d la: To a solution of 1 (98 mg, 0.057 mmol) in methanol (2.0 mL) was slowly added ethylenediamine (2.0 mL, 30 mmol) under ice bath. Then the reaction mixture was stirred for 3 days at 30 °C until IR showed the complete disappearance of the ester functions in 1. The reaction solution was evaporated, and the obtained residue was purified by dialysis (change dialysis water one hour per time for 6 times) and lyophilization. Repeating the operation cycles of dialysis and lyophilization for 3 times, the product was lyophilized to yield the corresponding la as a white solid (96 mg, yield: 86%)

’ H NMR (400 MHz, CDC1 ₃ /CD ₃ OD=3/1): 8 7.54 (s, 1H, CH), 4.17 (t, 2H, J = 7.4 Hz, CH ₂ ), 3.62 (s, 2H, CH ₂ ), 2.98-3.11 (m, 28H, CH ₂ ), 2.51-2.64 (m, 44H, CH ₂ ), 2.29-2.42 (m, 12H, CH ₂ ), 2.34-2.42 (m, 28H, CH ₂ ), 1.64-1.77 (m, 2H, CH ₂ ), 0.98-1.21 (m, 30H, CH ₂ ), 0.70 (t, 3H, J = 6.8 Hz, CH ₃ ); ¹³ C NMR (150 MHz, CD ₃ OD): 8 174.0, 173.6, 173.5,143.6, 124.0, 53.4, 52.3, 50.2, 49.9, 49.3, 41.7, 41.5, 40.8, 37.4, 33.6, 31.9, 30.2, 29.6, 29.4, 29.3, 28.9,

26.4, 22.5, 13.3; IR (cm’ ¹ ): u 1644.6; HRMS: calculated for C9iHi8 ₆ N3 ₂ Oi4 ⁴⁺ [M+4H] ⁴⁺ 488.1208, found 488.1294. lb: To a solution of 1 (100 mg, 0.058 mmol) in methanol (5.0 mL) was added N,N- dimethylethylenediamine (2.5 mL, 28 mmol). The reaction mixture was stirred for 5 days at 50 °C until the IR and NMR analysis showed the complete disappearance of the ester functions in 1. The reaction solution was evaporated, and the obtained residue was purified by precipitation with CH3OH/ECO for three times and followed by dialysis (change dialysis water one hour per time for 6 times) and lyophilization. Repeating the operation cycles of dialysis and lyophilization for 3 times, the product was lyophilized to yield the corresponding lb (108 mg, 86%) as a pale viscous oil.

¹ H NMR (400 MHz, CDC1 ₃ ): 6 7.90 (t, 2H, J = 5.3 Hz, NH), 7.73 (t, 4H, J = 5.2 Hz, NH), 7.57 (s, 1H, CH), 7.52 (t, 8H, J = 5.2 Hz, NH), 4.28 (t, 2H, J = 7.4 Hz, CH ₂ ), 3.81 (s, 2H, CH ₂ ), 3.24-3.32 (m, 28H, CH ₂ ), 2.72-2.75 (m, 28H, CH ₂ ), 2.51-2.62 (m, 12H, CH ₂ ), 2.40- 2.43 (m, 28H, CH ₂ ), 2.34-2.42 (m, 44H, CH ₂ ), 2.30 (br, 48H, CH ₂ ), 2.02 (br, 2H, CH ₂ ), 1.23 (br, 30H, CH ₂ ), 0.84 (t, 3H, J = 6.8 Hz, CH ₃ ); ¹³ C NMR (100 MHz, CDCI3): 6 172.7, 172.6, 172.4, 143.4, 122.9, 58.3, 52.6, 50.2, 50.1, 45.3, 45.3, 37.0, 34.5, 31.9, 29.7, 29.7, 29.5, 29.4, 26.6, 22.2, 14.2; IR (cm’ ¹ ): u 1648; HRMS: calculated for Cio7H _2i4 N ₃₂ Oi4 ³⁺ [M+3H] ³⁺ at 724.9079, found 724.9038.

1c: To a solution of Fmoc-Arg(pbf)-OH (726 mg, 1.12 mmol), HOBt (171 mg, 1.12 mmol), HBTU (424 mg, 1.12 mmol) and DIPEA (389 pL, 2.24 mmol) in 10.0 mL DMF stirring at 25 °C under argon, was added a solution of la (68 mg, 0.035 mmol) in 5.0 mL DMF. The resulting solution was stirred at 25 °C under argon for 2 days. Then DMF was removed under reduced pressure, and the residue was purified by precipitation with CH3OH/ECO (1.0 mL/20 mL) at 4.0 °C for 12 h (three times). The residual precipitate was collected, dried by rotavap and then dissolved in 8.0 mL 30% piperidine in DMF (v/v). The mixture was stirred at 25 °C under argon for 2.0 h, and DMF was then removed under reduced pressure. The residue was purified by precipitation with CH3OH/ECO (1.0 mL/12.0 mL) at 4.0 °C for 12 h (three times). A solution of 95% TFA, 2.5% triisopropylsilane (TIS) and 2.5% H ₂ O (v/v/v, 5.0 mL) was added to the residual precipitate and stirred at 25 °C under argon for 1 h. The crude product was dissolved in water (2.0 mL) and filtered, then purified by dialysis (change dialysis water one hour per time for 6 times) and lyophilization. Repeating the operation cycles of dialysis and lyophilization for 3 times, the product was lyophilized to yield the corresponding 1c as a white solid (125 mg, yield: 86%). 1c: ’ H NMR (600 MHz, D ₂ O): 8 7.87 (s, 1H, CH), 4.26 (t, 2H, J = 6.3 Hz, CH ₂ ), 3.76-3.81 (m, 10H, CH ₂ ), 3.11-3.28 (m, 28H, CH ₂ ), 3.05 (t, 16H, J = 6.9 Hz, CH ₂ ), 2.91- 2.96 (m, 28H, CH ₂ ), 2.66-2.80 (m, 28H, CH ₂ ), 2.37-2.43 (m, 28H, CH ₂ ), 1.71-1.79 (m, 18H, CH ₂ ), 1.46-1.48 (m, 16H, CH ₂ ), 1.00-1.05 (m, 30H, CH ₂ ), 0.65 (t, 3H, J = 6.6 Hz, CH ₃ ); ¹³ C NMR (150 MHz, D ₂ O): 8 173.8, 173.4, 170.2, 157.5, 156.9, 117.4, 115.5, 53.1, 53.0, 51.8, 49.4, 40.4, 39.2, 38.8, 35.6, 32.3, 29.1, 28.2, 23.7; IR (cm ^-1 ): v 1630.3; HRMS: calculated for Ci39H ₂ 83N64O ₂₂ ⁵⁺ [M + 5] ⁵⁺ 640.4598, found 640.4614.

Id: To a solution of ester dendrimer (300 mg, 0.17 mmol) in MeOH (5.0 mL) was added dropwise LiOH H ₂ O (117 mg, 2.8 mmol) in H ₂ O (5.0 mL) at 0 °C. The reaction solution was stirred at 0 °C for 10 min and then moved to 25 °C for 24 h. When the reaction was completed indicated by NMR monitoring, MeOH was evaporated and the pH of the aqueous phase was adjusted to 4.0 using 1.0 M HC1 solution, then purified by dialysis (change dialysis water one hour per time for 6 times) and lyophilization. Repeating the operation cycles of dialysis and lyophilization for 3 times, the product was lyophilized to yield the corresponding Id as a white solid (231 mg, yield: 83%).

’ H NMR (400 MHz, CD ₃ OD): 8 8.07 (s, 1H, CH), 4.41 (t, J = 7.1 Hz, 2H, CH ₂ ), 4.13 (s, 2H, CH ₂ ), 3.70-3.60 (m, 12H, CH ₂ ), 3.49-3.24 (m, 36H, CH ₂ ), 3.01 (t, J = 6.8 Hz, 4H, CH ₂ ), 2.81 (t, J = 6.6 Hz, 8H, CH ₂ ), 2.68-2.59 (m, 20H, CH ₂ ), 1.95-1.87 (s, 2H, CH ₂ ), 1.36-1.24 (m, 30H, CH ₂ ), 0.90 (t, J = 6.7 Hz, 3H, CH ₃ ); ¹³ C NMR (101 MHz, CD3OD): 3 177.2, 173.9,

173.3. 140.9. 126.8, 53.8, 53.0, 51.8, 51.4, 50.6, 50.1, 47.6, 35.6, 33.1, 32.5, 31.5, 31.3, 31.1,

30.8, 30.8, 30.7, 30.6, 30.5, 30.2, 27.6, 23.7, 14.5; IR (cm ^-1 ): v 1710.2; HRMS: calculated for C ₇₅ Hi37Ni6O ₂₂ ³⁺ [M+3H] ³⁺ 538.0026, found 538.0029.

Critical micelle concentration (CMC)

CMC was determined using pyrene as a fluorescence probe. Dendrimers stock solution was prepared in water and aliquots of the stock solutions were diluted by water to get desired concentration (1.0 pM to 1000 pM) in 1 mL solution. 1.0 pL of pyrene was added from its 1.0 mM stock solution prepared in ethanol and solutions were vortexed for 5 min and kept at room temperature for 24 h. Florescence emission was recorded at 373 nm and 384 nm using an excitation wavelength of 334 nm on CARY Eclipse fluorescence spectrophotometer at room temperature. Excitation and emission bandwidths were 5 nm. The fluorescence intensity ratio of I373/I383 was analyzed and a sigmoidal best fit analysis was applied to fluorescence intensity ratio data. The CMC was determined by plotting normalised fluorescence intensity ratio of pyrene I1/I3 against Log concentration of dendrimers. Transmission electron microscopy (TEM)

TEM was performed using JEOL 3010 transmission electron microscope (Tokyo, Japan) to characterize the size and morphology of the NPs at an accelerating voltage of 300 kV. The dendrimers were dispersed in milliQ water at a concentration of 1.0 mg/mL, and sonicated for 30 sec, then the solutions were diluted 100-fold and followed by depositing an aliquot (4.0 pL) onto a carbon-coated copper grid and dried at 37 °C. The grid was then stained with 3.0 pL uranyl acetate (2.0% in aqueous solution) for 4 sec, and the excess uranyl acetate was removed by filter paper before measurements. For each sample, at least 300 particles in different TEM images were randomly selected and measured by using ImageJ software to calculate the size and the size distribution of the nanoparticles.

Zeta potential measurement

Solutions of dendrimers were prepared in H2O with pH adjusted to 7.4 using 0.10 M NaOH/0.01 M HC1. After incubated at room temperature for 30 min, zeta potential measurement was performed using Zetasizer Nano-ZS (Malvern, Ltd. Malvern, U. K.) with a He-Ne ion laser of 633 nm. The experiments were done in triplicates.

Dru /dendrimer encapsulation

The film dispersion method was used to encapsulate the antibiotics azithromycin (AZM) and Ciprofloxacin (CIP) in the self-assembling dendrimer nanomicelles. Stock solution hydrophobic AZM and CIP as well as dendrimers were prepared in methanol. Antibiotic drugs solutions were mixed with the solution of la, resulting in different mass ratio of antibiotic s/dendrimer (from 0.2:1 to 1:1). The solvent of methanol was evaporated by vacuum rotary evaporation to form a dry film. The dry film was hydrated with water or PBS buffer for 5 min under constant stirring. Non-encapsulated antibiotic drugs were separated by filtration through a 0.45-pm polycarbonate membrane (Millipore Co.) and products were subsequently lyophilized. The amount of encapsulated antibiotic drugs in the micelles was measured using HPLC. The % encapsulation efficiency and % drug loading were calculated as below:

% drug loading: Wt/W _s x 100

% encapsulation efficiency: Wt/W ₀ x 100

Wt represents the amount of antibiotic drug that loaded into nanoparticles; Wo represents the initial amount of antibiotic drug fed; Ws represents the amount of nanoparticles after lyophilization. Cells

Human embryonic kidney 293 cells (HEK 293 cells) and mouse fibroblast cells (L929 cells) were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco, Invitrogen) containing 10% fetal bovine serum (FBS) (Biosera). Cells were maintained at 37°C with 5% CO2 in a humidified chamber.

Bacterial cells

Pseudomonas aeruginosa PAO1, Staphylococcus aureus RN4220, JLA513 and Methicillin-resistant staphylococcus aureus (MRSA) 1206 were used. The strains were grown in lysogeny broth (LB) or LB agar plates at 37°C. Bacteria were stored at -80 °C in 25% glycerol and at 4.0 °C on LB agar plate. PrestoBlue assay

L929 and HEK 293 cells were seeded at 1.0 x 10 ⁴ cells/well and 4.0 x 10 ³ cells/well in 96-well plates and allowed to grow overnight. Cells were then treated with various concentration (0.10 - 200 pM) of the amphiphilic dendrimers la-d for 48 h. Then 10 pL PrestoBlue reagent was added to each well containing 100 pL of blank, control, or treated cells in culture and incubated for another 3 h at 37 °C. The changes in cell viability were detected using fluorescence spectroscopy (excitation 570 nm; emission 610 nm). The cell viability was expressed as a percentage relative to the cells untreated with dendrimers. All samples were run in triplicate. Hemolysis assay

Red blood cells (RBCs) were isolated from the freshly collected whole blood of Swiss nude mice (with 1.0% heparin sodium solution). Blood was centrifuged at 7500 rpm for 10 min. several wash of PBS buffer given to RBCs until no color was seen in the supernatant. 2.0% RBCs concentration was achieved by diluting them in PBS (e.g. 20 pL of RBC suspension added to 0.98 mL PBS). 0.50 mL of 2.0% RBC solution was added into 1.5 mL Eppendorf tubes, and then 0.50 mL of dendrimer at different concentrations (2.0, 10.0, 20.0, 100, 200 and 500 pM) were added to make the final concentration 1.0, 5.0, 10.0, 50, 100 and 250 pM. 0.50 mL RBC suspension incubated with 0.50 mL PBS or 0.50 mL 1.0% TritonX-100 solution was used as the negative control and positive control, respectively. The samples were mixed gently, left at 37 °C for Ih, then centrifuged at 7500 rpm for 10 min. Then the supernatant was analyzed for the absorbance of hemoglobin at 540 nm on fluorescence spectrometer. The percentage of hemolysis was calculated as follows: Hemolysis% = [(sample absorbance - negative control) / (positive control - negative control)] xl00%. Minimum inhibitory concentration (MIC)

Minimal-inhibition concentration (MIC) assay was performed as described in Hayouka et al (J Am Chem Soc. 2013,135(32):11748-51. doi: 10.1021/ja406231b). Briefly, stock solutions of the tested compounds were diluted in LB broth and then diluted in 96-well plates (Coming 3650) with a 2-fold dilution, from 200ug/ml to3.1pg/ml. Bacteria were grown overnight (37°C, 200rpm), then diluted 1 : 100 and grown to OD600 of 0.1, subsequently lOOpL were added into the 96-well plate. Bacterial growth was determined after incubation of 24 h at 37°C by measurement of the optical density (OD600nm), using a Tecan infinite Pro Plate reader. MIC value defined as the lowest concentration at which there is complete inhibition of bacterial growth (no increase in OD over the course of the experiment). Each experiment was carried out at least three times, with three replicates per strain/concentration combination in each experiment.

Antibacterial activity assay against biofilms

Biofilm Inhibition assay

One colony of S. aureus JLA513 was inoculated from agar plate into 5.0 mL TSB media (50 mL tube) and grown overnight. The overnight suspension diluted 1:100 in fresh TSB supplemented with 1.0 % D-glucose (TSBG) and grown until ODs95 = 0.1. The bacterial suspension was then diluted 1 : 100 in TSBG and 100 pL were transferred into 96-well plate, which contained a serial concentrations of the dendrimer la in TSBG. The plate was then incubated for 48 hours at 37 °C. Then the supernatant liquid was discarded and the wells were washed with DDW 3 times. Dendrimer 2 was used as negative control. Biofilm quantification by MTT

For quantification of biofilm viability, the tetrazolium dye 3-[4,5-dimethyl-2- thiazolyl]-2, 5-diphenyl-2H-tetrazolium-bromide (MTT) was used as previously described with modifications. Briefly, after the 96-well plate washed from unattached cells and dried, 50 pL of MTT dissolved in PBS (0.50 pg/mL, pH 7.4) added to each well and incubated at 37 °C for 2 hours. Subsequently, 100 pL of DMSO were added to each well and incubated for 15 minutes. Then, the OD595 was measured using Tecan plate reader. The results were normalized to the control strain that was grown in the absence of antimicrobial agents. Crystal violet assay for biomass quantification:

The wells were treated with 125pL of 0.1% crystal violet and incubated for 15 min at 37 °C (G. A. O'Toole and R. Kolter, Mol Microbiol, 1998, 30, 295). Excess crystal violet was washed off thoroughly with milli-Q water three times. 125pL of 30% acetic acid was added to each well and the solution was transferred to a new 96-well plate, and absorbance was measured at 550 nm (Tecan plate reader) using 30% acetic acid in water as blank. The results were expressed as a percentage of biomass in the control biofilm, which was grown without any treatment.

Live/dead staining and fluorescence microscopy analysis

E. coli rp/MRSA culture was grown overnight in LB, and was then centrifuged and washed in PBS buffer for three times. The suspension was diluted to OD600 of 0.1. Incubation of 1 mL bacterial cells suspension was performed with shaking (200 RPM) with 25 pg/mL of tested compound (al) for 30 minutes (37°C). Incubation of the bacteria with PBS was used as control. After incubation, the bacterial cells were stained using LIVE/DEAD BacLight Bacterial Viability Kit (ThermoFisher). Each sample was incubated with 2.5 pL propidium iodide (red) and 2.5 pL SYTO9 (green) for 15 minutes in the dark. Afterwards fluorescent images were taken using EVOS M5000 imaging system (ThermoFischer). Images were analyzed using the software imageJ.

Outer membrane permeabilization assay

The outer membrane permeability was determined using the fluorescent probe biphenyl- 1 -naphthylamine (NPN). E. coli cells were grown overnight in LB media, then diluted 100-fold in LB and grown at 37°c to OD600 of 0.5. The cells were then washed with 5mM sodium HEPES buffer (pH 7.2), and resuspended in the same buffer to OD600 of 0.2. Subsequently, lOOuL of the suspended bacteria was added into 96-well plate (Nunc, Thermo Scientific, 167008) with 50 pl of NPN solution (lOuM). Fluorescence intensities were measured using Synergy Hl Biotek microplate reader (excitation: 350, emission: 420) for 5 minutes, then 50 pl of the tested compounds were added into each well and the fluorescence intenseties were measured for additionally 40 minutes. 8A molecule was used as negative control. The displayed results are representative of 3 independent experiments with 3 replicates for each treatment.

Inner membrane permeabilization assay

Depolarization of the inner membrane was assessed using the lipophilic potentiometric dye 3,3'-Dipropylthiadicarbocyanine Iodide [DiSC3(5)]. E. coli and MRSA bacterial cells were grown overnight in LB media (37°C, 200rpm), then diluted 1 :100 in LB and grown to OD600 of 0.5. Bacteria were then washed with 5mM HEPES, 20 mM glucose and 0.1M KC1, and resuspended in the same buffer to approximately 106 CFU/ml. The bacterial cells were then incubated with 0.4pM DiSC3(5) for 1 hr in 96-well plates (Nunc, Thermo Scientific, 167008), until a stable reduction of fluorescence was achieved. The tested compounds at the indicated concentrations, were then added to the bacterial sample and the changes in the fluorescence intensities were monitored using Synergy Hl Biotek microplate reader (excitation: 647, emission: 677). Triton-X 0.5% was used as positive control, 8A dendrimer (200 pg/ml final concentration) was used as a negative control. Results displayed are representative of 3 independent experiments with three replicates for each treatment. Scanning electron microscopy

E. coli rp and MRS A cultures were each grown overnight in LB, then centrifuged and washed in PBS buffer for three times. The suspension was diluted to OD600 of 0.1, to get -107 CFU/mL. Incubation of 1 mL bacterial suspension was incubated with shaking (200 RPM) with 6.3 or 50 pg/mL with the tested compound in PBS buffer at 37°C for 30 minutes. Incubation of the bacteria in PBS only, and dendrimer la in PBS were used as control. After incubation, the bacteria were centrifuged (4000 RPM, 5 minutes) and resuspended with 100 pL 4% glutaraldehyde in PBS was added to the samples for 1 hr at room temperature. The treated bacterial cells were dropped on a glass disk with poly-lysin, 30 pL at each side of the disk. Dehydration of the samples with the treated or non-treated bacteria was done in increasing concentrations of ethanol (25, 50, 75, 95, 100% ethanol in deionized water), 5 minutes twice for each concentration. Drying was performed with K850 critical point dryer (CPD BAL-TEC CPD-030). The samples were dried with ethanol and for 3 min in -10 °C and 20 min in 34 °C. The ethanol was replaced by liquid CO2 at ~40°C for 20 min. Then, iridium coating was performed with Q150T ES Spatter Coater. The microphotographs were recorded using imaging Jeol JSM 7800 SEM.

Example 1. Dendrimers

Synthesis

The amphiphilic dendrimers la-d were first synthesized starting with the common ester-terminating dendrimer 1 (Fig. 1A). Using our reported protocols, we prepared la and lb via the amidation of 1 with ethylenediamine and A,A-dimethylethylenediamine, respectively. Compound 1c was further obtained by conjugating la with the protected arginine followed by subsequent deprotection, whereas the carboxylic acid-terminated dendrimer Id was synthesized via hydrolysis of 1 using LiOH. The structural integrity and purity of all the synthesized dendrimers were confirmed using ¹ H- and ¹³ C-NMR as well as high-resolution mass spectroscopy. The resulted structures are presented in Fig. 1B-1D. Additionally, dendrimers of the first and second generation were prepared as shown in Figs. IE, Fig. IF (DDC18-4TA) and Fig. 1G (DD4TA). Further, dendrimers with a long C18 alkenyl hydrophobic tail (Figs. 1H-1K, denoted as DD01e8A, DD01e8TA, DDLin8A and DDLin8TA, respectively) were prepared.

Further, bola-amphiphilic PAMAM amine-terminated dendrimers (2 ^nd and 3 ^rd generation PAMAM dendrimers) were prepared and shown in Figs. IL, IM and IN (denoted as JCB4A, MBola-8T and JCB8A, respectively)

Water solubility

All the dendrimers la-d were soluble in water, with concentrations up to 20 mM. By virtue of the amphiphilic character and the dendritic structure, all the dendrimers selfassembled into small and spherical supramolecular nanomicelles with size ranging from 10 to 20 nm as demonstrated by transmission electronic microscopy (TEM) imaging (See Fig. 2). The nanomicelles formed by the dendrimers la-d had zeta potentials of +35 mV, +23 mV, +40 mV and -13 mV, respectively, in line with the distinct chemical entities at their terminals. The absolute values of the zeta potentials were all over 10 mV, highlighting the colloidal stability of these nanomicelles.

The self-assembly of the amphiphilic dendrimers la-ld were further studied by determining their critical micelle concentrations (CMC) using a fluorescence spectroscopic method based on pyrene. Id showed the lowest CMC value of 4.0 pM, whereas la and lb had similar CMC values of 15 and 17 pM, and 1c had the highest CMC value of 35 pM (Tables 1 and 2). The differing CMC values for la-ld can be likely ascribed to their structural feature: 1c has the largest hydrophilic entity and the greatest steric hindrance of the terminals among all the four dendrimers, hence the least packing and assembling ability therefore the highest CMC. On the other hand, dendrimer Id has the smallest hydrophilic entity, hence the highest packing ability and lowest CMC value.

Table 1: Self-assembly and toxicity of the dendrimers la-d. ^a CMC: critical micelle concentration; ^b Cytotoxicity IC50: concentration of a dendrimer required for 50% inhibition of cell growth; ^C L929: fibroblast cells; ^d HEK293: human kidney cell; ^e Hemolysis IC50: concentration to lysis 50 % of red blood cells. Toxicity

To evaluate the safety of these dendrimers, their cytotoxicity on fibroblast (L929) and kidney (HEK293) cell lines was determined using the PrestoBlue assay. No notable adverse effect was observed for dendrimers la, lb and Id at concentrations up to 200 pM, whereas dendrimer 1c showed considerably toxicity towards both cell types (Table 1 and Fig. 3). The hemolytic activity of the dendrimers on mouse red blood cell (RBC) was also examined. Similarly to the trend observed for cytotoxicity, 1c had the highest hemolytic activity, with IC50 value of 50 pM for 1c and 100 pM for la, lb, whereas Id did not show any notable hemolysis even at 250 pM (Table 1 and Fig. 4E). The observed toxicity of 1c may be ascribed to the arginine terminals, which interact and interfere strongly with the eukaryotic cell membrane via both electrostatic interaction and bivalent hydrogen bonds. Therefore, except dendrimer 1c, all the other dendrimers showed low cytotoxicity and good biocompatibility.

The cytotoxicity of all the synthesized dendrimers using label free high contrast microscopy was further evaluated. Among them, the dendrimers lb, Id, DDC18- 4TA, DDlin- 8TA, and DDole- 8TA showed no cytotoxicity to HEK293 cells up to 66 pM.

Example 2. Antibacterial activity of dendrimers

The antibacterial activity of dendrimers la-ld against the Gram-negative bacteria Escherichia coli (E. coli) and Pseudomonas aeruginosa (P. aeruginosa) as well as the Grampositive bacteria Staphylococcus aureus (S. aureus) and methicillin -resistant Staphylococcus aureus (MRSA) was then evaluated. The minimum inhibitory concentrations (MIC) of these dendrimers against the tested bacteria were obtained using microdilution broth assay (Table 2). Remarkably, the amine-terminated dendrimer la exhibited strong antibacterial activity against both Gram-negative and Gram-positive as well as drug-resistant MRSA, with MIC values of 3.1 pM. Interestingly, the tertiary amine- terminated dendrimer lb showed activity similar to that of la towards E. coli, but was not effective against all other tested bacteria, implying possible narrow -spectrum antibacterial activity specific to certain bacteria. Surprisingly, the arginine-terminated dendrimer 1c was not active against all the tested bacteria although 1c is the most toxic toward eukaryotic cells. Also, the carboxylic acid-terminated dendrimer Id was devoid of notable activity, in contrast to the amphiphilic dendrimers previously reported. Table. 2 Antibacterial activity of the dendrimers la-d.

It is to note that the distinct antibacterial activities of the dendrimers could be ascribed to their different terminal groups, which interact variably with bacteria. The inactive Id can be readily explained by its negatively charged carboxylate terminals, which generate repulsion when approaching the negatively charged bacteria membrane, hence preventing antibacterial activity. Although the dendrimers la, lb and 1c all have positively charged terminals at neutral pH under physiological conditions, the primary amine groups in la, by virtue of the small size and highly positive charge, could interact strongly with the bacterial membrane surface; whereas the arginine residues in 1c with the delocalized positive charges on the guanidine scaffolds, could not offer sufficient interaction with bacterial cell membrane, hence devoid of antibacterial activity. Meanwhile, the tertiary amine terminal, which is larger than the primary amine yet smaller than the guanidine moiety, has lower charge density than the primary amine but higher than the guanidine group, therefore the dendrimer lb bearing tertiary amine terminals generated rather susceptible and variable activity toward different bacteria. This finding might be interesting to be further exploited for developing selective and narrow- spectrum antibacterial agents.

The ability of the most potent dendrimer la to disrupt the bacterial membrane using SYTO9 and propidium iodide (PI) dyes for staining the live and dead cells, respectively was next assessed. SYTO9 is a universal dye that crosses intact membranes and stains nucleic acids of all cells, whereas PI can only cross compromised bacterial membranes, and emit green fluorescence when binding to nucleic acids. Therefore, SYTO9 stains live cells, while PI staining is an indicator of membrane integrity and cell death. Figs. 4A and Fig. 4B show that the live bacteria of both Gram-negative E. coli and Gram-positive S. aureus were damaged after treatment with dendrimer la, supporting the potent antibacterial activity of la. These findings also imply that la may kill bacterial cells by a mechanism that most likely involves membrane damage.

To visualize the bacterial membrane integrity and morphology upon treatment with la, scanning electron microscopy (SEM) studies were performed. E. coli and S. aureus cells upon treatment with la at MIC (6 pg/mL) clearly showed irregularities in cell surface with appearance of blebbing and surface deformations indicating loss of membrane integrity (Figs. 4C, 4D). At a higher la concentration (50 pg/mL) drastic cell lysis was observed with triggered release of ions and cellular materials. Untreated cells were used as a control and showed normal and smooth surface under the SEM.

To verify whether the antibacterial mechanism of la was related to the interaction with bacterial membrane, the permeability and depolarization of the bacterial outer membrane (OM) and inner membrane (IM) were evaluated. Following treatment with la, significant outer membrane permeation was observed in E. coli by monitoring the change in the fluorescent properties of the N-phenyl-1 -naphthylamine (NPN) dye (Fig. 5A), a probe which displays increased fluorescence upon binding to hydrophobic membrane regions. Furthermore, rapid inner membrane depolarization was observed in both E. coli and MRSA treated with la, as indicated by the rapid increase of the fluorescence intensity of the 3,3'- dipropylthiadicarbocyanine iodide (diSC3(5)) lipophilic potentiometric probe (Figs. 5 B and C, respectively). Both outer membrane permeation and inner membrane depolarization, match the bacterial cell death observed using the live/dead cell analysis (Fig. 4), suggesting that the bacterial death was associated with permeation and depolarization of both the outer and the inner membranes.

Encouraged by the excellent antibacterial activity alongside the safety profile of la, its activity against the biofilm formed by MRSA was further evaluated. Remarkably, la retained its antibacterial activity against MRSA biofilm with the MIC value remaining at 3.1 pM (Fig. 4F), confirming the promising potential of la as the effective antimicrobial candidate also against drug-resistant bacteria and bacteria within the biofilm matrix

Further, the antibacterial activity of several dendrimers against Borrelia bavariensis and Neisseria meningitidis was tested and the results are presented in Table 3. The MIC of all compounds for against Borrelia bavariensis (bacteriostatic effect) was about 50 pM. Borrelia spirochaete typically enters the skin after an infected tick bite and then spreads to various body parts such as the heart, joints, and central nervous system. High doses of antibiotics are needed and currently used to treat acute and chronic infection, and with no human vaccine available, Eyme arthritis (caused by this bacterim) and neuroborreliosis pose significant health risks. Most antibiotics are unable to reach optimal therapeutic levels in joints and the central nervous system, leaving Borrelia to thrive in those hidden niches, resulting in infection recurrence and serious neurologic sequelae. Moreover, in case of neuroborreliosis), the treatment is extremely difficult due to low biodistribution of antibiotics in brain. Neisserial meningitis typically sets very fast (1-2 days) and death can occur within 3-4 days (in case of toddlers and adolescents). Treatment is very difficult due to low biodistribution of antibiotics in the brain. Very high doses are required to achieve effect concentration of the antibiotics in the brain.

Table 3: Dendrimers with anti-borrelial and anti-neisserial activities

In case of Neisseria meningitidis, four non-cytotoxic dendrimers DDC18-4TA, lb, DD01e-8TA and MBola-8T showed a bactericidal effect. DD01e8TA and MBola-8T had MIC 50 pM, while lb and DDC18-4TA had MIC 6.25 pM. The minimum time required to kill N. meningitidis for lb was 3h.

Example 3. Hydrophobic chain

Further, it was tested how the hydrophobic chain affects the antibacterial activity of the dendrimers. In the present case, the dendrimer la, although at low generation, is endowed with strong antibacterial activity. The antibacterial activity of dendrimer 2, having no hydrophobic chain (see Fig. 6) was tested. Dendrimer 2, the structural analogue of la devoid of the hydrophobic chain has no antibacterial activity at all. This can be reasonably explained considering that the low -generation cationic dendrimer 2 alone is not sufficient to provide strong electrostatic interaction with the bacterial membrane for generating effective antibacterial activity. On the other hand, the presence of the long and hydrophobic chain C 18 in la, coupled with its amphiphilic character, is important for the observed antibacterial activity, as it leads to the formation of nano-assemblies for more effective and multivalent interaction with bacterial membrane via electrostatic interactions and, at the same time, via collective hydrophobic interactions between the alkyl chain of la and the hydrophobic region of bacterial membrane, hence leading to strong inhibition on bacterial growth. In addition, the influence of the generation on the antibacterial activity was tested. Dendrimers of 1 ^st and 2 ^nd generation (Fig. IE (first generation, Figs. IE, IF and 1G, second generation) have been generated. It was previously reported that cationic dendrimers of high generations may disrupt bacterial membrane by inducing pore formation via cooperative electrostatic interaction between the cationic dendrimer terminals and the anionic bacterial membrane surface. Surprisingly it was now shown that dendrimers of low generations are also active as antibacterial agents. These dendrimers showed lower but still significant antibacterial activity as shown in Table 4.

Table 4. Antibacterial activity of primary amine-terminated dendrimer of 1 ^st and 2 ^nd generation.

Example 4. Encapsulation of known antibiotics using the dendrimers.

As la is able to self-assemble into stable nanomicelles, we explored its ability to encapsulate the antibiotics Azithromycin (AZM), Doxycycline (DOX) and Ciprofloxacin (CIP) and tested the antibacterial activity of the complex, and in particular the ability of the nanomicelles to deliver the antibiotics across biofilm barriers. Both AZM and CIP are broadspectrum and effective against planktonic bacteria but fail to show potent activity against biofilms of MRSA 1206. Remarkably, AZM and CIP (separately) were loaded in the nanomicelles formed by la with excellent encapsulation efficiencies of > 90 % as well as loading efficiencies of 44 %, 55% and 24 %, respectively. In addition, the co-constructed nanosystems AZM/la, DOX/la and CIP/la were more effective than the individual AZM, CIP, DOX and la. The significantly enhanced activity of AZM, DOX and CIP demonstrates not only the antibacterial activity of la, but also the carrier feature of la which has good penetrability in bacteria biofilm membrane, offering new dimensions to antibacterial therapy against drug resistance bacteria and bacteria within biofilm matrix.

In an additional experiment, the capacity of the dendrimer lb for encapsulation of the antibacterial drug DOX was assessed and showed Encapsulation efficiency of 90% and drug loading of 57%. Further, doxycycline was encapsulated in DD01e-8TA to generate anti-borrelia nanoformulation (Borrelia bavariensis), whereas, ciprofloxacin was incorporated in lb to create anti-neis serial nanoformulation (Neisseria meningitidis).

DDole-8TA showed a synergistic anti-Borrelia effect with encapsulated doxycycline by reducing their individual MIC to > 8 times with a FICI value of < 0.5. Similarly, encapsulation of ciprofloxacin in lb showed a synergistic anti-Neisseria effect with a reduction in their individual MIC values by > 35 times for lb and > 104 time for ciprofloxacin with a FICI value of < 0.4.

When tested against MRSA, dendrimer la showed an additive effect with Ciprofloxacin with FICI >0.5 and additive to indifference effect with AZM, however, la showed synergistic effect with Doxycycline with FICI value of < 0.5.

FICI was calculated as follows (see e.g. Bellio et al., MethodsX. 2021; 8: 101543).

FICI ^;;; MIC a combination ,-i- MIC. h combination

MIC alone MIC _S , alone

Table 5. FICI Values meaning

These data support that the antibiotic s/dendrimer nanoformulations were much more effective than the individual antibiotics and dendrimer alone, highlight a synergic effect for antibacterial activity using the amphiphilic dendrimer-based drug delivery systems.

In addition, the following encapsulated particles are formed. Particles of dendrimers having a structure of Formula II encapsulating erythromycin are formed. Particles of dendrimers having a structure of Formula II encapsulating vancomycin are formed. Particles of dendrimers having a structure of Formula II encapsulating cefuroxime are formed. In addition, the following encapsulated particles are formed. Particles of dendrimers having a structure of Formula IV encapsulating erythromycin are formed. Particles of dendrimers having a structure of Formula IV encapsulating vancomycin are formed. Particles of dendrimers having a structure of Formula IV encapsulating cefuroxime are formed. In addition, the following encapsulated particles are formed. Particles of dendrimers having a structure of Formula IV encapsulating azithromycin are formed. Particles of dendrimers having a structure of Formula IV encapsulating ciprofloxacin are formed. Particles of dendrimers having a structure of Formula VI encapsulating erythromycin are formed. Particles of dendrimers having a structure of Formula VI encapsulating vancomycin are formed. Particles of dendrimers having a structure of Formula VI encapsulating cefuroxime are formed. In addition, the following encapsulated particles are formed. Particles of dendrimers having a structure of Formula VI encapsulating azithromycin are formed. Particles of dendrimers having a structure of Formula VI encapsulating ciprofloxacin are formed. Particles of dendrimers having a structure of Formula III encapsulating doxycycline are formed. Particles of dendrimers having a structure of Formula III encapsulating ciprofloxacin are formed. Particles of dendrimers having a structure of Formula III encapsulating erythromycin are formed. Particles of dendrimers having a structure of Formula III encapsulating vancomycin are formed. Particles of dendrimers having a structure of Formula III encapsulating cefuroxime are formed. In addition, the following encapsulated particles are formed. Particles of dendrimers having a structure of Formula III encapsulating azithromycin are formed. Particles of dendrimers having a structure of Formula III encapsulating ciprofloxacin are formed. Particles of dendrimers having a structure of Formula X encapsulating doxycycline are formed. Particles of dendrimers having a structure of Formula X encapsulating ciprofloxacin are formed. Particles of dendrimers having a structure of Formula X encapsulating erythromycin are formed. Particles of dendrimers having a structure of Formula X encapsulating vancomycin are formed. Particles of dendrimers having a structure of Formula X encapsulating cefuroxime are formed. In addition, the following encapsulated particles are formed. Particles of dendrimers having a structure of Formula X encapsulating azithromycin are formed. Particles of dendrimers having a structure of Formula X encapsulating ciprofloxacin are formed. In some examples, doxycycline is replaced by other tetracyclines minocycline, demeclocycline, oxy tetracycline, lymecycline or methacycline. In some examples, ciprofloxacin is replaced by other fluoroquinolones such as levofloxacin, levofloxacin, gatifloxacin, gatifloxacin or sparfloxacin. In some examples, azithromycin is replaced by other macrolides such as clarithromycin, dirithromycin, or roxithromycin. In some examples, particles of dendrimers having a structure as described above encapsulating a cephalosporin such as cefuroxime, cefpodoxime, cefotaxime, ceftriaxone or ceftazidime. Compositions comprising these particles are formulated and tested for their antibacterial activity.

In conclusion, amphiphilic dendrimers bearing distinct terminal functionalities were designed and synthesized and their antibacterial activity was evaluated. These dendrimers are all capable to self-assemble spontaneously into small and stable supramolecular nanomicelles in water. Among them, the positively charged amine-terminated dendrimer la showed the most powerful activity against both Gram-positive and Gram-negative bacteria as well as drug-resistant bacteria and biofilm eradication, whereas the negatively charged carboxylic acid-terminated dendrimer Id was devoid of activity. Although the positively charged tertiary amine-terminated dendrimer lb was effective against the Gram-negative bacteria Escherichia coli, it had a much weaker effect on the other bacteria tested; surprisingly, lb has a good antibacterial activity against Neisseria meningitidis. Interestingly, the guanidine-terminated dendrimer 1c didn’t exhibit any notable antibacterial activity. The distinct antibacterial activity observed with these dendrimers can be ascribed to the different charge and charge density associated with the varying terminal functionalities, leading to variable interaction with the negatively charged bacterial membranes, thereby divergent antibacterial activity. Also, the self-assembling feature of the amphiphilic dendrimer la is responsible and decisive for its potent antibacterial activity, as the hydrophilic dendrimer counterpart without the alkyl chain has no activity at all. By virtue of the unique self-assembling character and strong interaction with bacterial membrane, la is endowed with excellent antibacterial activity. It is also to note that, compared to the reported dendrimers with antibacterial activity, our amphiphilic dendrimers presented in this work are of low generation and easy to synthesize. In addition, the amphiphilic dendrimer la is able to form nanomicelle and co-deliver the antibacterial agents for more effective activity to overcome antimicrobial resistance of biofilm, offering a new perspective to develop potent and effective antibacterial agent. Moreover, some of the nanoparticles of the dendrimer encapsulating antibacterial agents provided a synergistic antibacterial activity.

Examples 5

In vivo safety and antibacterial activity of la was further assessed in different mouse models. CD-I mice that were exposed to 5 mg/kg la show indistinguishable serum biochemistry and haematology from the control cohort exposed to sterile saline. Additionally, no la treatment-associated toxicity or inflammatory changes were observed in the histopathological analysis of major organs (heart, lungs, liver, spleen, kidneys) when compared to the saline control.

Pharmacokinetic analysis exploiting live imaging of Cyanine7.5-labeled la using an IVIS SpectrumCT Imaging system revealed that the compound was stable for more than 72 hours with strong accumulation in infected organs. Importantly, la (5 mg/kg, twice daily) either outperformed or attained equal level of efficacy of commercially available antibiotics against ESKAPE pathogens, including acute pneumonia (5.05 log reduction compared to 4.63 log reduction by Tobramycin in lungs) and bacteraemia (2.99 log reduction compared to 2.16 log reduction by Tobramycin in spleens) models of infection by Pseudomonas aeruginosa; acute pneumonia (2.69 log reduction compared to 2.04 log reduction by Daptomycin in lungs) and bacteraemia (3.6 log reduction compared to 1.38 log reduction by Daptomycin in spleens) models of infection by MRS A; acute pneumonia (1.81 log reduction compared to 2.43 log reduction by Tigecycline in lungs) and bacteraemia (3.52 log reduction compared to 3.08 log reduction by Tigecycline in spleens) models of infection by Klebsiella pneumoniae; and immunosuppressed (neutropenic) soft tissue (thigh) infection by P. aeruginosa (4.17 log reduction compared to 4.88 log reduction by Tobramycin) and MRSA (4.47 log reduction compared to 4.95 log reduction by Daptomycin).

Finally, la (5 mg/kg, twice daily) showed great efficacy in a mouse model of bacteraemia mortality study. Mice infected with P. aeruginosa strain PAO1 and treated with saline showed 80% mortality. In contrast, no PAO1 -infected mice died in the la treated cohort. Collectively, our in vivo data indicate that la has strong safety, pharmacokinetics and efficacy in preclinical mouse models. In vivo antibacterial activity of lb and DDOle- 8TA (Formula IV and VI) are evaluated in mouse models of different infectious diseases.

Although the present invention has been described herein above by way of preferred embodiments thereof, it can be modified, without departing from the spirit and nature of the subject invention as defined in the appended claims