Shujun Gao

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.

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.

Co-delivery of thioridazine and doxorubicin using polymeric micelles for targeting both cancer cells and cancer stem cells

In this study, thioridazine (THZ), which was reported to kill cancer stem cells, was used in a combination therapy with doxorubicin (DOX) to eradicate both cancer cells and DOX-resistant cancer stem cells to mitigate the reoccurrence of the disease. Both THZ and DOX were loaded into micelles with sizes below 100 nm, narrow size distribution and high drug content. The micelles were self-assembled from a mixture of acid-functionalized poly(carbonate) and poly(ethylene glycol) diblock copolymer (PEG-PAC) and urea-functionalized poly(carbonate) (PUC) and PEG diblock copolymer (PEG-PUC). The drug-loaded mixed micelles (MM) were used to target both cancer cells and stem cells via co-delivery. Cancer stem cells were sorted by a side population assay from BT-474 and MCF-7 human breast cancer cell lines, and identified by CD44+/CD24- phenotype. The cytotoxicity of various formulations was evaluated on the sorted cancer stem cells (side population SP cells), sorted non-stem-like cancer cells (non-side population NSP cells) and unsorted cancer cells. Antitumor activity was also evaluated on BT-474 xenografts in nude mice. As compared with NSP cells, DOX suppressed SP cell growth less effectively, while THZ and THZ-MM were more effective in the inhibition of SP cells. A stronger inhibitory effect was observed on SP cells with the co-delivery of free DOX and THZ or DOX-MM and THZ-MM as compared to free DOX or DOX-MM. THZ and THZ-MM were capable of lowering the population of SP cells in unsorted cells. In the BT-474 xenografts, the co-delivery of DOX-MM and THZ-MM produced the strongest antitumor efficacy, and both THZ and THZ-MM showed strong activity against cancer stem cells. This combination therapy may provide a promising strategy for breast cancer treatment by targeting both cancer cells and cancer stem cells.

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.

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.

Diaminododecane-based cationic bolaamphiphile as a non-viral gene delivery carrier

The advancement in gene therapy relies upon the discovery of safe and efficient delivery agents and methods. In this study, we report the design and synthesis of a cationic bolaamphiphile as a non-viral gene delivery agent. The bolaamphiphile is composed of 1,12-diaminododecane as the central hydrophobic unit linked to the hydrophilic pentaethylenehexamine via thioether-based glycidyl units. This bolaamphiphile condensed DNA efficiently into nanoparticles of sizes around 150-200 nm with positive zeta potential of 30-35 mV. In vitro luciferase expression levels and percentage of GFP expressing cells induced by the bolaamphiphile/DNA complexes were higher than those mediated by the often used golden standard of non-viral systems, polyethyleneimine (PEI, branched, 25 kDa) at its optimal N/P ratio in HEK293, HepG2, NIH3T3, HeLa and 4T1 cells. In vitro cytotoxicity testing revealed that the DNA complexes fabricated from this cationic bolaamphiphile displayed marginal toxicity towards all the cell lines tested. In addition, in vivo transfection studies carried out in a 4T1 mouse breast cancer model showed that the cationic bolaamphiphile delivered DNA more efficiently than PEI. This cationic bolaamphiphile may make a promising gene delivery vector for future gene therapy.

Phenformin-loaded polymeric micelles for targeting both cancer cells and cancer stem cells in vitro and in vivo

Conventional cancer chemotherapy often fails as most anti-cancer drugs are not effective against drug-resistant cancer stem cells. These surviving cancer stem cells lead to relapse and metastasis. In this study, an anti-diabetic drug, phenformin, capable of eliminating cancer stem cells was loaded into micelles via self-assembly using a mixture of a diblock copolymer of poly(ethylene glycol) (PEG) and urea-functionalized polycarbonate and a diblock copolymer of PEG and acid-functionalized polycarbonate through hydrogen bonding. The phenformin-loaded micelles, having an average diameter of 102xa0nm with narrow size distribution, were stable in serum-containing solution over 48xa0h and non-cytotoxic towards non-cancerous cells. More than 90% of phenformin was released from the micelles over 96xa0h. Lung cancer stem cells (side population cells, i.e. SP cells) and non-SP cells were sorted from H460 human lung cancer cell line, and treated with free phenformin and phenformin-loaded micelles. The results showed that the drug-loaded micelles were more effective in inhibiting the growth of both SP and non-SP cells. Inxa0vivo studies conducted in an H460 human lung cancer mouse model demonstrated that the drug-loaded micelles had greater anti-tumor efficacy, and reduced the population of SP cells in the tumor tissues more effectively than free phenformin. Liver function analysis was performed following drug treatments, and the results indicated that the drug-loaded micelles did not cause liver damage, a harmful side-effect of phenformin when used clinically. These phenformin-loaded micelles may be used to target both cancer cells and cancer stem cells in chemotherapy for the prevention of relapse and metastasis.

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.

pH and redox dual-responsive biodegradable polymeric micelles with high drug loading for effective anticancer drug delivery

Diblock copolymers of poly(ethylene glycol) (PEG) and biodegradable polycarbonate functionalized with GSH-sensitive disulfide bonds and pH-responsive carboxylic acid groups were synthesized via organocatalytic ring-opening polymerization of functional cyclic carbonates with PEG having different molecular weights as macroinitiators. These narrowly-dispersed polymers had predictable molecular weights, and were used to load doxorubicin (DOX) into micelles primarily through ionic interactions. The DOX-loaded micelles exhibited the requisite small particle size (<100 nm), narrow size distribution and high drug loading capacity. When exposed to endolysosomal pH of 5.0, drug release was accelerated by at least two-fold. The introduction of GSH further expedited DOX release. Effective DOX release enhanced cytotoxicity against cancer cells. More importantly, the DOX-loaded micelles with the optimized composition showed excellent antitumor efficacy in nude mice bearing BT-474 xenografts without inducing toxicity. These pH and redox dual-responsive micelles have the potential as delivery carriers to maximize the therapeutic effect of anticancer drugs.

Biodegradable functional polycarbonate micelles for controlled release of amphotericin B

Amphotericin B (AmB), a poorly soluble and toxic antifungal drug, was encapsulated into polymeric micelles self-assembled from phenylboronic acid-functionalized polycarbonate/PEG (PEG-PBC) and urea-functionalized polycarbonate/PEG (PEG-PUC) diblock copolymers via hydrogen-bonding, boronate ester bond, and/or ionic interactions between the boronic acid group in the micellar core and amine group in AmB. Three micellar formulations were prepared: AmB/B micelles using PEG-PBC, AmB/U micelles using PEG-PUC and AmB/B+U mixed micelles using 1:1molar ratio of PEG-PBC and PEG-PUC. The average particle sizes of the micelles were in the range of 54.4-84.8nm with narrow size distribution and zeta potentials close to neutral. UV-Vis absorption analysis indicated that AmB/B micelles significantly reduced AmB aggregation status due to the interactions between AmB and the micellar core, while Fungizone® and AmB/U micelles had no effect. AmB/B+U mixed micelles exerted an intermediate effect. Both AmB/B micelles and AmB/B+U mixed micelles showed sustained drug release, with 48.6±2.1% and 59.2±1.8% AmB released respectively after 24hunder sink conditions, while AmB/U micelles displayed a burst release profile. All AmB-loaded micelles showed comparable antifungal activity to free AmB or Fungizone®, while AmB/B micelles and AmB/B+U mixed micelles were much less hemolytic than other formulations. Histological examination showed that AmB/B and AmB/B+U micelles led to a significantly lower number of apoptotic cells in the kidneys compared to Fungizone®, suggesting reduced nephrotoxicity of the micellar formulations in vivo. These phenylboronic acid-functionalized polymeric micelle systems are promising drug carriers for AmB to reduce non-specific toxicities without compromise in antifungal activity.nnnSTATEMENT OF SIGNIFICANCEnThere is a pressing need for a novel and cost-effective delivery system to reduce the toxicity induced by the antifungal agent, amphotericin B (AmB). In this study, phenylboronic acid-functionalized polycarbonate/PEG diblock copolymers were used to fabricate micelles for improved AmB-micelle interaction via the manipulation of hydrogen-bonding, boronate ester bond, ionic and hydrophobic interactions. Compared to free AmB and Fungizone®, the resultant micellar systems displayed improved stability while reducing non-specific toxicities without a compromise in antifungal activity. These findings demonstrate the potential of biodegradable functional polycarbonate micellar systems as promising carriers of AmB for the treatment of systemic fungal infections.