Jeremy P. K. Tan

Biodegradable nanostructures with selective lysis of microbial membranes

Macromolecular antimicrobial agents such as cationic polymers and peptides have recently been under an increased level of scrutiny because they can combat multi-drug-resistant microbes. Most of these polymers are non-biodegradable and are designed to mimic the facially amphiphilic structure of peptides so that they may form a secondary structure on interaction with negatively charged microbial membranes. The resulting secondary structure can insert into and disintegrate the cell membrane after recruiting additional polymer molecules. Here, we report the first biodegradable and in vivo applicable antimicrobial polymer nanoparticles synthesized by metal-free organocatalytic ring-opening polymerization of functional cyclic carbonate. We demonstrate that the nanoparticles disrupt microbial walls/membranes selectively and efficiently, thus inhibiting the growth of Gram-positive bacteria, methicillin-resistant Staphylococcus aureus (MRSA) and fungi, without inducing significant haemolysis over a wide range of concentrations. These biodegradable nanoparticles, which can be synthesized in large quantities and at low cost, are promising as antimicrobial drugs, and can be used to treat various infectious diseases such as MRSA-associated infections, which are often linked with high mortality.

Hydrogen bonding-enhanced micelle assemblies for drug delivery.

Ring-opening polymerization (ROP) of functionalized cyclic carbonates derived from 2,2-bis(methylol)propionic acid (bis-MPA) allows for incorporation of H-bonding urea-functional groups into block copolymers with a potential application of supramolecular drug-delivery systems. The strong H-bonding functionalities of poly(ethylene glycol)-block-poly(ethyl-random-urea carbonate) (PEG-P(E(1-x)-U(x))C) block copolymers not only lowered critical micelles concentration (cmc) of the block copolymer (to 1/4x) in aqueous environment compared to conventional PEG-poly(trimethylene carbonate) (PEG-PTMC) block copolymer without the non-covalent stabilization, but also improved kinetic stability of micelles and Dox-loaded micelles in the presence of a destabilizing agent. It was observed that the incorporation of anticancer drug doxorubicin affected the micellization process of block copolymers in water and caused a sudden increase in sizes of drug-loaded micelles above 200 nm. This phenomenon that can be a significant drawback in drug delivery applications was considerably mitigated in urea-bearing block copolymer/Dox micelles with simultaneously accompanying a significant improvement in drug loading. In vitro drug release profile showed that the increase in urea content led to a slight decrease in Dox release rate. Block copolymer did not have any significant cytotoxicity against HEK293 and HepG2 cells up to 400 mg/L. Importantly, Dox-loaded micelles exerted cytotoxic effect against HepG2 cells.

Synthesis of a family of amphiphilic glycopolymers via controlled ring-opening polymerization of functionalized cyclic carbonates and their application in drug delivery

Polymers bearing pendant carbohydrates have a variety of biomedical applications especially in the area of targeted drug delivery. Here we report the synthesis of a family of amphiphilic block glycopolymers containing d glucose, d galactose and d mannose via metal-free organocatalyzed ring-opening polymerization of functional cyclic carbonates generating narrowly dispersed products of controlled molecular weight and end-group fidelity, and their application in drug delivery. These glycopolymers self-assemble into micelles having a high density of sugar molecules in the shell, a size less than 100 nm with narrow size distribution even after drug loading, and little cytotoxicity, which are important for drug delivery. Using galactose-containing micelles as an example, we demonstrate their strong targeting ability towards ASGP-R positive HepG2 liver cancer cells in comparison with ASGP-R negative HEK293 cells although the galactose is attached to the carbonate monomer at 6-position. The enhanced uptake of DOX-loaded galactose-containing micelles by HepG2 cells significantly increases cytotoxicity of DOX as compared to HEK293. This new family of amphiphilic block glycopolymers has great potential as carriers for targeted drug delivery.

Simple Approach to Stabilized Micelles Employing Miktoarm Terpolymers and Stereocomplexes with Application in Paclitaxel Delivery

A simple and versatile approach to miktoarm co- and terpolymers from carbonate functional oligomers is described. The key building block employed is a carboxylic acid functional cyclic carbonate, derived from 2,2-bis(methylol)propionic acid, that was readily coupled to a hydroxyl functional monomethylether poly(ethylene glycol) oligomer. Ring-opening of the cyclic carbonate using functional amines generates a carbamate linkage bearing a functional group capable of initiating either controlled radical or ring-opening polymerization, together with a primary hydroxyl group for ring-opening polymerization. Two tandem polymerization steps were possible which add the second two arms, thus generating the targeted ABC miktoarm terpolymer. The resulting amphiphilic miktoarm terpolymers containing poly(D- and L-lactide) formed polylactide stereocomplexes in the bulk. In aqueous solution, the stereocomplex mixture of Y-shaped miktoarm copolymers, poly(ethylene glycol)-poly(D-lactide)-poly(D-lactide) and poly(ethylene glycol)-poly(L-lactide)-poly(L-lactide), or the stereoblock miktoarm poly(ethylene glycol)-poly(D-lactide)-poly(L-lactide) form stabilized micelles with a significantly lower critical micelle concentration than those derived from conventional stereo regular linear or Y-shaped amphiphiles. This simple and versatile approach provides a useful synthetic route to complex macromolecular architectures that can assemble into stable micelles. These micelles provide high capacity for loading of the anticancer drug paclitaxel and possess narrow size distribution as well as unique structure, leading to sustained and near zero-ordered release of drug without significant initial burst.

The role of non-covalent interactions in anticancer drug loading and kinetic stability of polymeric micelles.

A new series of acid- and urea-functionalized polycarbonate block copolymers were synthesized via organocatalytic living ring-opening polymerization using methoxy poly(ethylene glycol) (PEG) as a macroinitiator to form micelles as drug delivery carriers. The micelles were characterized for critical micelle concentration, particle size and size distribution, kinetic stability and loading capacity for a model anticancer drug, doxorubicin (DOX) having an amine group. The acid/urea groups were placed in block forms (i.e. acid as the middle block or the end block) or randomly distributed in the polycarbonate block to investigate molecular structure effect. The micelles formed from the polymers in both random and block forms provided high drug loading capacity due to strong ionic interaction between the acid in the polymer and the amine in DOX. However, the polymers with acid and urea groups placed in the block forms formed micelles with wider size distribution (two size populations), and their DOX-loaded micelles were less stable. The number of acid/urea groups in the random form was further varied from 5 to 8, 13 and 19 to study its effects on self-assembly behaviors and DOX loading. An increased number of acid/urea groups yielded DOX-loaded micelles with smaller size and enhanced kinetic stability because of improved inter-molecular polycarbonate-polycarbonate (urea-urea and urea-acid) hydrogen-bonding and polycarbonate-DOX (acid-amine) ionic interactions. However, when the number of acid/urea groups was 13 or higher, micelles aggregated in a serum-containing medium, and freeze-dried DOX-loaded micelles were unable to re-disperse in an aqueous solution. Among all the polymers synthesized in this study, 1b with 8 acid/urea groups in the random form had the optimum properties. In vitro release studies showed that DOX release from 1b micelles was sustained over 7 h without significant initial burst release. MTT assays demonstrated that the polymer was not toxic towards HepG2 and HEK293 cells. Importantly, DOX-loaded micelles were potent against HepG2 cells with IC(50) of 0.26 mg/L, comparable to that of free DOX (IC(50): 0.20 mg/L). In addition, DOX-loaded 1b micelles yielded lower DOX content in the heart tissue of the tested mice as compared to free DOX formulation after i.v. injection. These findings signify that 1b micelles may be a promising carrier for delivery of anticancer drugs that contain amine groups.

Organocatalytic Approach to Amphiphilic Comb-Block Copolymers Capable of Stereocomplexation and Self-Assembly

Biocompatible amphiphilic block copolymers comprised of poly(ethylene glycol) (PEG) as the hydrophilic component and a poly(methylcarboxytrimethylene carbonate) (PMTC) as a hydrophobic backbone having either poly(L-lactide) (L-PLA) or poly(D-lactide) (D-PLA) branches were prepared by organocatalytic ring-opening polymerization (ROP). The polycarbonate backbone was prepared by copolymerization of two different MTC-type monomers (MTCs) including a tetrahydropyranyloxy protected hydroxyl group, a masked initiator for a subsequent ROP step. Interestingly, the organic catalyst used in the ROP of MTCs was also effective for acetylation of the hydroxyl end-groups by the addition of acetic anhydride added after polymerization. Acidic deprotection of the tetrahydropyranyloxy (THP) protecting group on the carbonate chain generated hydroxyl functional groups that served as initiators for the ROP of either D- or L-lactide. Comb-shaped block copolymers of predictable molecular weights and narrow polydispersities (approximately 1.3) were prepared with up to 8-PLA branches. Mixtures of the D- and L-lactide based copolymers were studied to understand the effect of noncovalent interactions or stereocomplexation on the properties.

Broad-spectrum antimicrobial and biofilm-disrupting hydrogels: stereocomplex-driven supramolecular assemblies.

Fighting the resistance: biodegradable and injectable/moldable hydrogels with hierarchical nanostructures were made with broad-spectrum antimicrobial activities and biofilm-disruption capability. They demonstrate no cytotoxicity in vitro, and show excellent skin biocompatibility in animals. These hydrogels have great potential for clinical use in prevention and treatment of various multidrug-resistant infections.

The efficacy of self-assembled cationic antimicrobial peptide nanoparticles against Cryptococcus neoformans for the treatment of meningitis.

Cationic antimicrobial peptides have received considerable interest as new therapeutics with the potential for treatment of multiple-drug resistant infections. We recently reported that cholesterol-conjugated G(3)R(6)TAT (CG(3)R(6)TAT) formed cationic nanoparticles via self-assembly, which demonstrated strong antimicrobial activities against various types of microbes in vitro. In this study, the possibility of using these nanoparticles for treatment of Cryptococcus neoformans (yeast)-induced brain infections was studied. The antimicrobial activity of the nanoparticles was tested against 12 clinical isolates of C. neoformans in comparison with conventional antifungal agents amphotericin B and fluconazole. Minimum inhibitory concentrations (MICs) of the nanoparticles were determined to be much lower than those of fluconazole in all the isolates, but slightly higher than those of amphotericin B in some isolates. At a concentration three times higher than the MIC, the nanoparticles completely sterilized C. neoformans after 3.5 h. Cell wall disruption and release of cytoplasmic content were observed under TEM. The biodistribution studies of FITC-loaded nanoparticles in rabbits revealed that the nanoparticles were able to cross the blood-brain barrier (BBB). The efficacy of nanoparticles was further evaluated in a C. neoformans meningitis rabbit model. The nanoparticles crossed the BBB and suppressed the yeast growth in the brain tissues with similar efficiency as amphotericin B did. In addition, unlike amphotericin B, they neither caused significant damage to the liver and kidney functions nor interfered with the balance of electrolytes in the blood. CG(3)R(6)TAT nanoparticles can be a promising antimicrobial agent for treatment of brain infections caused by C. neoformans.

The effect of kinetic stability on biodistribution and anti-tumor efficacy of drug-loaded biodegradable polymeric micelles

This study was aimed to investigate the effect of kinetic stability on biodistribution and antitumor efficacy of drug-loaded biodegradable polymeric micelles. Four diblock copolymers of acid- and urea-functionalized polycarbonate (i.e. PAC and PUC) and poly(ethylene glycol) (PEG) with the same polycarbonate length and two different PEG molecular weights (Mn: 5 kDa and 10 kDa), i.e. 5K PEG-PAC, 10K PEG-PAC, 5K PEG-PUC and 10K PEG-PUC, were synthesized via organocatalytic living ring-opening polymerization using methoxy PEG as a macroinitiator. These polymers were employed to prepare 5K PEG-PAC/5K PEG-PUC and 10K PEG-PAC/10K PEG-PAC mixed micelles via urea-acid hydrogen bonding. An amine group-containing anticancer drug, doxorubicin (DOX) was loaded into the mixed micelles via a self-assembly process. DOX-loaded 5K and 10K PEG mixed micelles had particle sizes of 66 and 87 nm respectively with narrow size distribution (polydispersity index: 0.12), and DOX loading levels were 28.9 and 22.8% in weight. DOX-loaded 5K PEG mixed micelles had greater kinetic stability than DOX-loaded 10K PEG mixed micelles due to stronger hydrophobicity of 5K PEG block copolymers. The results of in vitro release studies showed that DOX release was sustained without obvious initial burst release. The DOX-loaded mixed micelles effectively suppressed the proliferation of HepG2 and 4T1 cells. The in vivo studies conducted in a 4T1 mouse breast cancer model demonstrated that the mixed micelles were preferably transported to the tumor with the 5K PEG mixed micelles accumulating in the tumor more rapidly to a larger extent than 10K PEG mixed micelles, and DOX-loaded 5K PEG mixed micelles with greater kinetic stability inhibited tumor growth more effectively than free DOX and DOX-loaded 10K PEG mixed micelles without causing significant body weight loss or cardiotoxicity. The 5K PEG mixed micelles with sizes below 100 nm and narrow size distribution as well as excellent kinetic stability holds great potential as a delivery carrier for amine group-containing anticancer drugs.

Computational studies on self-assembled paclitaxel structures: templates for hierarchical block copolymer assemblies and sustained drug release.

Paclitaxel-loaded poly(ethylene oxide)-b-poly(lactide) (PEO-b-PLA) systems have been observed to assemble into fiber structures with remarkably different properties using different chirality and molecular weight of PLA segments. In this study, dissipative particle dynamics (DPD) simulations were carried out to elaborate the microstructures and properties of pure paclitaxel and paclitaxel-loaded PEO-b-PLA systems. Paclitaxel molecules formed ribbon or fiber like structures in water. With the addition of PEO-b-PDLA, PEO-b-PLLA and their stereocomplex, paclitaxel acted as a template and polymer molecules assembled around the paclitaxel structure to form core/shell structured fibers having a PEO shell. For PEO19-b-PDLA27 and PEO19-b-PLLA27 systems, PLA segments and paclitaxel molecules were distributed homogeneously in the core of fibers based on the hydrophobic interactions. In the stereocomplex formulation, paclitaxel molecules were more concentrated in the inner PLA stereocomplex core, which led to slower release of paclitaxel. By increasing the length of PLA segments (e.g. 8,16,22 and 27), the crystalline structure of paclitaxel was gradually weakened and destroyed, which was further proved by X-ray diffraction studies. All the simulation results agreed well with experimental data, suggesting that the DPD simulations may provide a powerful tool for designing drug delivery systems.