Ashlynn L. Z. Lee

The use of cholesterol-containing biodegradable block copolymers to exploit hydrophobic interactions for the delivery of anticancer drugs

A series of biodegradable amphiphilic block copolymers with controlled composition and relatively low polydispersity index were synthesized from monomethoxy polyethylene glycol (mPEG-OH, 5 kDa) via organocatalytic ring opening polymerization of aliphatic cyclic carbonate monomers - trimethylene carbonate (TMC) or cholesteryl 2-(5-methyl-2-oxo-1,3-dioxane-5-carboxyloyloxy)ethyl carbamate (MTC-Chol) or a copolymer of both the monomers (TMC and MTC-Chol): mPEG(113)-b-PTMC(67), mPEG(113)-b-P(MTC-Chol(11)) and mPEG(113)-b-P(MTC-Chol(x)-co-TMC(y))(x+y). These well-defined polymers were employed to study the role of molecular weight and composition of the hydrophobic block of the polymers in loading paclitaxel (PTX), an extremely hydrophobic anticancer drug with rigid structure and strong tendency of self-association to form long fibers. The PTX-loaded micelles were fabricated by simple self-assembly without sonication or homogenization procedures. The results demonstrated that the presence of both MTC-Chol and TMC in the hydrophobic block significantly increased PTX loading levels, and the micelles formed from the polymer with the optimized composition (i.e. mPEG(113)-b-P(MTC-Chol(11)-co-TMC(30))) were in nanosize (36 nm) with narrow size distribution (PDI: 0.07) and high PTX loading capacity (15 wt.%). In vitro treatment of human liver hepatocellular carcinoma HepG2 cells with blank micelles showed that these polymeric carriers were non-cytotoxic with cell viability greater than 90% at ~2400 mg/L. Importantly, PTX-loaded micelles were able to kill cancer cells much more effectively compared to free PTX. In addition, these nanocarriers also possessed exceptional kinetic stability. The results from non-invasive near-infrared fluorescence (NIRF) imaging studies showed that these micelles allowed effective passive targeting, and were preferably accumulated in tumor tissue with limited distribution to healthy organs.

The co-delivery of paclitaxel and Herceptin using cationic micellar nanoparticles

We have recently reported micellar nanoparticles self-assembled from a biodegradable and amphiphilic copolymer poly{(N-methyldietheneamine sebacate)-co-[(cholesteryl oxocarbonylamido ethyl) methyl bis(ethylene) ammonium bromide] sebacate}, P(MDS-co-CES), which were able to deliver small molecular drugs and biomacromolecules such as genes and functional proteins individually or simultaneously into various types of cells. In this study, these cationic micellar nanoparticles were employed as carriers to co-deliver paclitaxel and Herceptin for achieving targeted delivery of paclitaxel to human epidermal growth factor receptor-2 (HER2/neu)-overexpressing human breast cancer cells, and enhanced cytotoxicity through synergistic activities. Paclitaxel-loaded nanoparticles have an average size less than 120 nm and a zeta potential of about 60 mV. Herceptin was complexed onto the surface of the nanoparticles. The drug-loaded nanoparticle/Herceptin complexes remained stable under physiologically-simulating conditions with sizes at around 200 nm. The nanoparticles delivered Herceptin much more efficiently than BioPorter, a commercially available lipid-based protein carrier, and displayed a much higher anti-cancer effectiveness. Twice-repeated daily treatment with Herceptin showed significantly higher cytotoxicity especially in HER2-overexpressing breast cancer cells when compared to single treatment. Anti-cancer effects of this co-delivery system was investigated in human breast cancer cell lines with varying degrees of HER2 expression level, namely, MCF7, T47D and BT474. The co-delivery of Herceptin increased the cytotoxicity of paclitaxel and this enhancement showed a dependency on their HER2 expression levels. Targeting ability of this co-delivery system was demonstrated through confocal images, which showed significantly higher cellular uptake in HER2-overexpressing BT474 cells as compared to HER2-negative HEK293 cells. This co-delivery system may have important clinical implications against HER2-overexpressing breast cancers.

Synergistic Co‐Delivery of Membrane‐Disrupting Polymers with Commercial Antibiotics against Highly Opportunistic Bacteria

A series of vitamin E-containing biodegradable antimicrobial cationic polycarbonates is designed and synthesized via controlled organocatalytic ring-opening polymerization. The incorporation of vitamin E significantly enhances antimicrobial activity. These polymers demonstrate broad-spectrum antimicrobial activity against various microbes, e.g., S. aureus (Gram-positive), E-coli (Gram-negative) and C. albicans (fungi). More importantly, the co-delivery of such polymers with selected antibiotics (e.g., doxycycline) shows high synergism towards difficult-to-kill bacteria P. aeruginosa. These findings suggest that these vitamin E-functionalized polycarbonates are potentially useful antimicrobial agents against challenging bacterial/fungal infections.

Synergistic Anticancer Effects Achieved by Co-Delivery of TRAIL and Paclitaxel Using Cationic Polymeric Micelles

Cationic micellar nanoparticles self-assembled from a biodegradable amphiphilic copolymer have been used to deliver human TRAIL and paclitaxel simultaneously. Polyplexes formed between paclitaxel-loaded nanoparticles and TRAIL are stable with a size of ≈180 nm and a zeta potential at ≈75 mV. Anticancer effects and apoptotic pathway mechanisms of this drug-and-protein co-delivery system are investigated in various human breast cancer cell lines with different TRAIL sensitivity. The co-delivery nanoparticulate system induces synergistic anti-cancer activities with limited toxicity in non-cancerous cells. An advantage of this co-delivery is a significantly higher anti-cancer effect as compared to free drug and protein formulations.

Injectable Biodegradable Hydrogels from Vitamin D-Functionalized Polycarbonates for the Delivery of Avastin with Enhanced Therapeutic Efficiency against Metastatic Colorectal Cancer

Humanized vascular endothelial growth factor (VEGF) antibody (bevacizumab; Avastin) is a highly effective monoclonal antibody against metastatic colorectal cancer and several other advanced late stage cancers. However, limited aqueous solubility and short circulation half-life of the antibody result in long infusion time (30-90 min) and frequent injections. Such direful medical procedures often cause considerable patient inconvenience and prolonged pharmacy preparation. Subcutaneous delivery of Avastin using injectable hydrogels can continuously provide Avastin to treat the malignancy and mitigate antibody degradation. In this study, ABA triblock copolymers of vitamin D-functionalized polycarbonate and poly(ethylene glycol), that is, VDm-PEG-VDm were synthesized and employed to form physically cross-linked injectable hydrogels for encapsulation and subcutaneous delivery of Avastin in a sustained fashion. Antitumor studies were performed using two different HCT116 xenograft mouse models: a subcutaneous and an intraperitoneal metastatic tumor models. The therapeutic efficacy of Avastin-loaded hydrogel injected subcutaneously (s.c.) was compared to an Avastin solution injected via either intravenous (i.v.) or intraperitoneal (i.p.) route. In the subcutaneous tumor model, the Avastin-loaded hydrogel resulted in greater tumor suppression as compared to i.v. and i.p. administration of Avastin solution. The biodistribution pattern of the hydrogel delivery system was also different from the other formulations as there was significantly higher accumulation in the tumor tissue and lesser accumulation within the liver and kidneys as compared to Avastin delivered through i.v. and i.p. administration. Furthermore, in vivo studies carried out on mice with peritoneal metastasis demonstrated that Avastin-loaded hydrogel and weekly administration of Avastin solution resulted in higher survival (87 and 77% over 62 days, respectively) when compared to the control, blank hydrogel and bolus Avastin solution (i.v.; 50-60%). The antimetastatic activity of Avastin delivered using a one-time injection of the hydrogel was as effective as that of 4× weekly injections (i.v.) of Avastin. The reduced injection frequency provided by the subcutaneous formulation may enhance patient convenience and compliance for metastatic cancer therapy.

Synergistic anti-cancer effects via co-delivery of TNF-related apoptosis-inducing ligand (TRAIL/Apo2L) and doxorubicin using micellar nanoparticles

The use of small molecule drugs in cancer chemotherapy has mostly been limited by dose-dependent toxicity and development of drug resistance resulting from repeated administrations. To overcome such problems, efforts have been made to develop drug delivery systems that can bear multiple therapeutic agents in one system. The purpose of this study is to deliver human tumor necrosis factor (TNF)-related apoptosis-inducing ligand (Apo2L/TRAIL) and doxorubicin (Dox, an anti-cancer drug) with micellar nanoparticles self-assembled from a biodegradable cationic copolymer P(MDS-co-CES) to achieve synergistic cytotoxic effects in cancer cells. Exogenously expressed TRAIL using recombinant methods shows great potential in cancer therapy as it induces cell death selectively in cancer cells with limited toxicity to normal tissues. Dox-loaded nanoparticles and TRAIL formed stable nanocomplexes with a size of ∼ 225 nm and zeta potential of ∼ 70 mV. Effects of nanocomplexes on both wild type and TRAIL-resistant SW480 colorectal carcinoma cells were investigated. The assemblies of Dox and TRAIL with P(MDS-co-CES) nanoparticles were efficiently delivered to cancer cells. Receptor-blocking studies showed that the nanocomplexes entered cells via death receptor-mediated endocytosis. Synergism in cell death induction was analysed by the isobologram method to study drug interactions. Cytotoxicity of the nanocomplexes to non-cancerous cells was significantly lower than cancerous cells. Anti-proliferative effects of nanocomplexes were retained in remaining cancer cells in long-term cultures after treatment with the nanocomplexes. In summary, this Dox and TRAIL co-delivery system can be a promising candidate for cancer treatment.

Access to different nanostructures via self-assembly of thiourea-containing PEGylated amphiphiles.

Readily water-soluble PEGylated amphiphiles containing bis-thiourea-based molecular recognition units at the interface of hydrophobic and hydrophilic blocks are developed. Self-assembly of these amphiphiles is found to be dependent on the exact chemical composition of the hydrophobic component. Elongated, spherical, and disk-like micelles are formed with the change in hydrophobic group from stearyl (2A), oleyl (2B), and dodecanol (2C), respectively. The length of the rod-like elongated micelles formed by 2A could be tuned by thermal treatment as well. Synthesis and detailed structural characterization of these amphiphiles by TEM, DSC, synchrotron SAXS techniques are reported. Organic solvent-free direct aqueous encapsulation of doxorubicin, an anticancer drug into these nanostructures is demonstrated.

A macromolecular approach to eradicate multidrug resistant bacterial infections while mitigating drug resistance onset

Polymyxins remain the last line treatment for multidrug-resistant (MDR) infections. As polymyxins resistance emerges, there is an urgent need to develop effective antimicrobial agents capable of mitigating MDR. Here, we report biodegradable guanidinium-functionalized polycarbonates with a distinctive mechanism that does not induce drug resistance. Unlike conventional antibiotics, repeated use of the polymers does not lead to drug resistance. Transcriptomic analysis of bacteria further supports development of resistance to antibiotics but not to the macromolecules after 30 treatments. Importantly, high in vivo treatment efficacy of the macromolecules is achieved in MDR A. baumannii-, E. coli-, K. pneumoniae-, methicillin-resistant S. aureus-, cecal ligation and puncture-induced polymicrobial peritonitis, and P. aeruginosa lung infection mouse models while remaining non-toxic (e.g., therapeutic index—ED50/LD50: 1473 for A. baumannii infection). These biodegradable synthetic macromolecules have been demonstrated to have broad spectrum in vivo antimicrobial activity, and have excellent potential as systemic antimicrobials against MDR infections.Antibiotic resistance is a major threat across the whole healthcare spectrum. Here, the authors report on the development of biodegradable guanidinium functionalized polycarbonates and demonstrate antimicrobial activity against drug resistant infections.

Modular composite hydrogels from cholesterol-functionalized polycarbonates for antimicrobial applications

Micellar composite hydrogel systems represent a promising class of materials for biomolecule and drug delivery applications. In this work a system combining micellar drug delivery with supramolecular hydrogel assemblies is developed, representing an elegant marriage of aqueous hydrophobic drug delivery and next-generation injectable viscoelastic materials. Novel shear thinning and injectable micellar composite hydrogels were prepared using an amphiphilic ABA-type triblock copolymer consisting of a hydrophilic middle block and cholesterol-functionalized polycarbonates as terminal hydrophobic blocks. Varying the concentration and relative hydrophobic-hydrophilic content of the amphiphilic species conferred the ability to tune the storage moduli of these gels from 200 Pa to 3500 Pa. This tunable system was used to encapsulate drug-loaded polymeric micelles, demonstrating a straightforward and modular approach to developing micellar viscoelastic materials for a variety of applications such as delivery of hydrophobic drugs. These hydrogels were also mixed with cholesterol-containing cationic polycarbonates to render antimicrobial activity and capability for anionic drug delivery. Additionally, small-angle X-ray scattering (SAXS) and electron microscopy (EM) results probed the mesoscale structure of these micellar composite materials, lending molecular level insight into the self-assembly properties of these gels. The antimicrobial composite hydrogels demonstrated strong microbicidal activity against Gram-negative and Gram-positive bacteria, and C. albicans fungus. Amphotericin B (AmB, an antifungal drug)-loaded micelles embedded within the hydrogel demonstrated sustained drug release over 4 days and effective eradication of fungi. Our findings demonstrate that materials of different nature (i.e. small molecule drugs or charged macromolecules) can be physically combined with ABA-type triblock copolymer gelators to form hydrogels for potential pharmaceutical applications.

Co-delivery of antiviral and antifungal therapeutics for the treatment of sexually transmitted infections using a moldable, supramolecular hydrogel.

In this investigation, a therapeutic co-delivery hydrogel system is developed to provide effective HIV prophylaxis, alongside the prevention and/or treatment of candidiasis. Two components-a HIV reverse transcriptase inhibitor, tenofovir, and a cationic macromolecular antifungal agent derived from a vitamin D-functionalized polycarbonate (VD/BnCl (1:30))-are formulated into biodegradable vitamin D-functionalized polycarbonate/PEG-based supramolecular hydrogels. The hydrogels exhibit thixotropic properties and can be easily spread across surfaces for efficient drug absorption. Sustained release of tenofovir from the hydrogel is observed, where approximately 85% tenofovir is released within 3 h. VD/BnCl (1:30) does not impede drug diffusion from the hydrogel as the drug release profiles are similar with and without the polycation. Antimicrobial efficacy studies indicate that the hydrogels kill C. albicans efficiently with a minimum bactericidal concentration (MBC) of 0.25-0.5 g L(-1) . These hydrogels also eradicate C. albicans biofilm effectively at 4× MBC. When human dermal fibroblasts (as model mammalian cells) are treated with these hydrogels, cell viability remains high at above 80%, demonstrating excellent biocompatibility. When applied topically, this dual-functional hydrogel can potentially prevent HIV transmission and eliminate microbes that cause infections in the vulvovagina region.