Degradable disulfide core-cross-linked micelles as a drug delivery system prepared from vinyl functionalized nucleosides via the RAFT process

A nucleosides containing block copolymer, poly(polyethylene glycol methyl ether methacrylate)- block-poly(5'-O-methacryloyluridine) (PPEGMEMA 30- b-PMAU 80) was self-assembled in aqueous medium and cross-linked via RAFT polymerization at 60 degrees C to afford core-cross-linked micelles exhibiting a PPEGMEMA corona and a polynucleotide core. A disulfide cross-linking agent, bis(2-methacryloyloxyethyl)disulfide, was employed to cross-link the structure via the RAFT process resulting in core-shell nanoparticles, which can degrade under reductive conditions. The resulting core-cross-linked micelles readily hydrolyzed into free block copolymers in the presence of dithiothreitol (DTT) in less than 1 h, depending on the concentration of the reducing agent and the amount of cross-linker in the micelle. A small fraction of permanently cross-linked micelle was found as the result of conventional chain transfer to disulfide containing compounds. A model drug, vitamin B 2, was loaded into the micelle. The loading capacity increased with increasing cross-linking degree. The amount of drug released reached 60-70% after 7 h in the presence of DTT (0.65 mM), while the cross-linked micelle in the absence of dithiothreitol shows only a delayed drug release. Cytotoxicity tests confirmed the biocompatibility of the polymers and the residues after reduction.     

Acid-labile core cross-linked micelles for pH-triggered release of antitumor drugs

Micelles of a model amphiphilic block copolymer, poly(hydroxyethyl acrylate)-block-poly(n-butyl acrylate) (PHEA-b-PBA), synthesized via the RAFT polymerization were cross-linked by copolymerization of a degradable cross-linker from the living RAFT-end groups of PBA chains, yielding a cross-linked core without affecting significantly the original micelle size. The cross-linker incorporation into the micelles was evidenced via physicochemical analysis of the copolymer unimers formed upon acidic cleavage of the cross-linked micelles. High doxorubicin loading capacities (60 wt %) were obtained. Hydrolysis of less than half of the cross-links in the core was found to be sufficient to release doxorubicin faster at acidic pH compared to neutral pH. The system represents the first example of core-cross-linked micelles that can be destabilized (potentially both above and below CMC) by the pH-dependent cleavage of the cross-links and the subsequent polarity change in the core to enable the release of hydrophobic drugs entrapped inside the micelle.     

Enhanced stability of core-surface cross-linked micelles fabricated from amphiphilic brush copolymers

"Stealth" nanoparticles made from polymer micelles have been widely explored as drug carriers for targeted drug delivery. High stability (i.e., low critical micelle concentration (CMC)) is required for their intravenous applications. Herein, we present a "core-surface cross-linking" concept to greatly enhance nanoparticle's stability: amphiphilic brush copolymers form core-surface cross-linked micelles (nanoparticles) (SCNs). The amphiphilic brush copolymers consisted of hydrophobic poly(epsilon-caprolactone) (PCL) and hydrophilic poly(ethylene glycol) (PEG) or poly(2-(N,N-dimethylamino)ethyl methacrylate) (PDMA) chains were synthesized by macromonomer copolymerization method and used to demonstrate this concept. The resulting SCNs were about 100 times more stable than micelles from corresponding amphiphilic block copolymers. The size and surface properties of the SCNs could be easily tailored by the copolymer's compositions.     

Effect of cross-linking on the performance of micelles as drug delivery carriers: a cell uptake study

Poly(polyethylene glycol methyl ether methacrylate-co-methacrylic acid)-block-poly(methyl methacrylate) P(PEGMEMA-co-MAA)-b-PMMA block copolymer were prepared via RAFT (reversible addition-fragmentation chain transfer) polymerization and subsequently self-assembled into micelles as a drug delivery carrier for albendazole (ABZ). For comparison, the micelles were additionally cross-linked to study the effect of shell-cross-linking on the biological activity. The hydrodynamic diameter of cross-linked and un-cross-linked micelles was approximately 40 nm in both cases. While the cross-linked micelle was stable even in good solvents for both blocks, the un-cross-linked micelle was found to lose its integrity in cell growth media. Crosslinking had a major effect on the rate of drug release reducing it dramatically from 50% (uncrosslinked) to around 20% (crosslinked) over a 30 h incubation period. Both drug delivery systems were tested on human prostate cancer cells (PC-3, DU-145) and human ovarian cancer cells (OVCAR-3, A-2780). No toxic effects were measured with the unloaded micelle while the ABZ loaded un-cross-linked micelle lead to IC(50) values between 0.2 and 0.9 μM depending on the cell line. The IC(50) dropped to values between 0.006 and 0.06 μM, depending on cell line, once the micelles were stabilized by cross-linking. Three treatment cycles with ABZ for one day, followed by two days incubation in media using ABZ-loaded drug carriers led to complete cell death even at low concentrations in the case of the cross-linked micelle only. Cellular uptake has been studied using fluorescently labeled micelles and Nile red as model drug, showing cell uptake above the CMC but no micelle uptake below the CMC. Additional biological studies, such as colony formation assay and tubulin disorganization tests, were also performed to gain more insight into the effect of cross-linking of the shell of the micelle. In conclusion, shell-cross-linking is highly recommended, even for glassy micelles, for an efficient cellular uptake at low concentrations.     

Biodegradable amphiphilic block copolymers bearing protected hydroxyl groups: synthesis and characterization

A new biodegradable amphiphilic block copolymer, poly(ethylene glycol)-b-poly(L-lactide-co-9-phenyl-2,4,8,10-tetraoxaspiro[5,5]undecan-3-one) [PEG-b-P(LA-co-PTO)], was successfully prepared by ring-opening polymerization (ROP) of L-lactide (LA) and functionalized carbonate monomer 9-phenyl-2,4,8,10-tetraozaspiro[5,5]undecan-3-one (PTO) in the presence of monohydroxyl poly(ethylene glycol) as macroinitiator using Sn(Oct)2 as catalyst. NMR, FT-IR, and GPC studies confirmed the copolymer structure. It could self-assemble into micelles in aqueous solution with critical micelle concentration (CMC) in the magnitude of mg/L, which changed with the composition of the copolymer. After catalytic hydrogenation, copolymers with active hydroxyl groups were obtained. Adhesion and proliferation of Vero cells on the copolymer films showed that the synthesized copolymers were good biocompatible materials. In vitro degradation of the copolymer before and after deprotection was investigated in the presence of proteinase K. The free hydroxyl groups on the copolymers were capable of further modification with biotin. This new amphiphilic block copolymer has great potential for both drug encapsulation and conjugate because of its low CMC and the presence of active hydroxyl groups.     

Core-cross-linked polymeric micelles as paclitaxel carriers

Cross-linkable di- and triblock copolymers of poly(epsilon-caprolactone) (PCL) and monomethoxyl poly(ethylene glycol) (MPEG) were synthesized. These amphiphilic copolymers self-assembled into nanoscale micelles capable of encapsulating hydrophobic paclitaxel in their hydrophobic cores in aqueous solutions. To further enhance their thermodynamic stability, the micelles were cross-linked by radical polymerization of the double bonds introduced into the PCL blocks. Reaction conditions were found to significantly affect both the cross-linking efficiency and the micelle size. The encapsulation of paclitaxel into the micelles was confirmed by the proton nuclear magnetic resonance (1H NMR) spectroscopy. Encouragingly, paclitaxel-loading efficiency of micelles was enhanced significantly upon micelle core-cross-linking. Both the micelle size and the drug loading efficiency increased markedly with increasing the PCL block lengths, no matter if the micelles were core-cross-linked or not. However, paclitaxel-loading did not obviously affect the micelle size or size distribution. The cross-linked micelles exhibited a significantly enhanced thermodynamic stability against dilution with aqueous solvents. The efficient cellular uptake of paclitaxel loaded in the nanomicelles was demonstrated by confocal laser scanning microscopy (CLSM) imaging. This new biodegradable nanoscale carrier system merits further investigations for parenteral drug delivery.     

Thiol-yne and thiol-ene "click" chemistry as a tool for a variety of platinum drug delivery carriers, from statistical copolymers to crosslinked micelles

Statistical and block copolymers based on poly(2-hydroxyethyl methacrylate) (PHEMA) and poly[oligo(ethylene glycol) methylether methacrylate] (POEGMEMA) were modified with 4-pentenoic anhydride or 4-oxo-4-(prop-2-ynyloxy)butanoic anhydride to generate polymers with pendant vinyl or acetylene, respectively. Subsequent thiol-ene or thiol-yne reaction with thioglycolic acid or 2-mercaptosuccinic acid leads to polymers with carboxylate functionalities, which were conjugated with cisplatin (cis-diamminedichloroplatinum(II) (CDDP)) to generate a drug carrier for Pt-drugs. Only the polymers modified with 2-mercaptosuccinic acid resulted in the formation of soluble well-defined polymers with gel formation being prevented. Due to the hydrophobicity of the drug, the block copolymers took on amphiphilic character leading to micelle formation. The micelles were in addition crosslinked to further stabilize their structure. Pt-containing statistical copolymer, micelles, and crosslinked micelles were then tested regarding their cellular uptake by the A549 lung cancer cell line to show a superior uptake of crosslinked micelles. However, due to the better Pt release of the statistical copolymer, the highest cytotoxicity was observed with this type of polymer architecture.     

Fine tuning micellar core-forming block of poly(ethylene glycol)-block-poly(ε-caprolactone) amphiphilic copolymers based on chemical modification for the solubilization and delivery of doxorubicin

This study aimed to optimize poly(ethylene glycol)-b-poly(ε-caprolactone) (PEG-b-PCL)-based amphiphilic block copolymers for achieving a better micellar drug delivery system (DDS) with improved solubilization and delivery of doxorubicin (DOX). First, the Flory-Huggins interaction parameters between DOX and the core-forming segments [i.e., poly(ε-caprolactone) (PCL) and poly[(ε-caprolactone-co-γ-(carbamic acid benzyl ester)-ε-caprolactone] (P(CL-co-CABCL))] was calculated to assess the drug-polymer compatibility. The results indicated a better compatibility between DOX and P(CL-co-CABCL) than that between DOX and PCL, motivating the synthesis of monomethoxy-poly(ethylene glycol)-b-poly[(ε-caprolactone-co-γ-(carbamic acid benzyl ester)-ε-caprolactone] (mPEG-b-P(CL-co-CABCL)) block copolymer. Second, two novel block copolymers of mPEG-b-P(CL-co-CABCL) with different compositions were prepared via ring-opening polymerization of CL and CABCL using mPEG as a macroinitiator and characterized by (1)H NMR, FT-IR, GPC, WAXD, and DSC techniques. It was found that the introduction of CABCL decreased the crystallinity of mPEG-b-PCL copolymer. Micellar formation of the copolymers in aqueous solution was investigated with fluorescence spectroscopy, DLS and TEM. mPEG-b-P(CL-co-CABCL) copolymers had a lower critical micelle concentration (CMC) than mPEG-b-PCL and subsequently led to an improved stability of prepared micelles. Furthermore, both higher loading capacity and slower in vitro release of DOX were observed for micelles of copolymers with increased content of CABCL, attributed to both improved drug-core compatibility and favorable amorphous core structure. Meanwhile, DOX-loaded micelles facilitated better uptake of DOX by HepG2 cells and were mainly retained in the cytosol, whereas free DOX accumulated more in the nuclei. However, possibly because of the slower intracellular release of DOX, DOX-loaded micelles were less potent in inhibiting cell proliferation than free DOX in vitro. Taken together, the introduction of CABCL in the core-forming block of mPEG-b-PCL resulted in micelles with superior properties, which hold great promise for drug delivery applications.     

Enhanced stability of polymeric micelles based on postfunctionalized poly(ethylene glycol)-b-poly(γ-propargyl L-glutamate): the substituent effect

One of the major obstacles that delay the clinical translation of polymeric micelle drug delivery systems is whether these self-assembled micelles can retain their integrity in blood following intravenous (IV) injection. The objective of this study was to evaluate the impact of core functionalization on the thermodynamic and kinetic stability of polymeric micelles. The combination of ring-opening polymerization of N-carboxyanhydride (NCA) with highly efficient "click" coupling has enabled easy and quick access to a family of poly(ethylene glycol)-block-poly(γ-R-glutamate)s with exactly the same block lengths, for which the substituent "R" is tuned. The structures of these copolymers were carefully characterized by (1)H NMR, FT-IR, and GPC. When pyrene is used as the fluorescence probe, the critical micelle concentrations (CMCs) of these polymers were found to be in the range of 10(-7)-10(-6) M, which indicates good thermodynamic stability for the self-assembled micelles. The incorporation of polar side groups in the micelle core leads to high CMC values; however, micelles prepared from these copolymers are kinetically more stable in the presence of serum and upon SDS disturbance. It was also observed that these polymers could effectively encapsulate paclitaxel (PTX) as a model anticancer drug, and the micelles possessing better kinetic stability showed better suppression of the initial "burst" release and exhibited more sustained release of PTX. These PTX-loaded micelles exerted comparable cytotoxicity against HeLa cells as the clinically approved Cremophor PTX formulation, while the block copolymers showed much lower toxicity compared to the cremophor-ethanol mixture. The present work demonstrated that the PEG-b-PPLG can be a uniform block copolymer platform toward development of polymeric micelle delivery systems for different drugs through the facile modification of the PPLG block.     

Block copolymer micelles with pendant bifunctional chelator for platinum drugs: effect of spacer length on the viability of tumor cells

Three monomers with 1,3-dicarboxylate functional groups but varying spacer lengths were synthesized via carbon Michael addition using malonate esters and ethylene- (MAETC), butylene- (MABTC), and hexylene (MAHTC) glycol dimethacrylate, respectively. Poly[oligo-(ethylene glycol) methylether methacrylate] (POEGMEMA) was prepared in the presence of a RAFT (reversible addition-fragmentation chain transfer) agent, followed by chain extension with the prepared monomers to generate three different block copolymers (BP-E80, BP-B82, and BP-H79) with similar numbers of repeating units, but various spacer lengths as distinguishing features. Conjugation with platinum drugs created macromolecular platinum drugs resembling carboplatin. The amphiphilic natures of these Pt-containing block copolymers led to the formation micelles in solution. The rate of drug release of all micelles was similar, but a noticeable difference was the increasing stability of the micelle against dissociation with increasing spacer length. The platinum conjugated polymer showed high activity against A549, OVCAR3, and SKOV3 cancer cell lines exceeding the activity of carboplatin, but only the micelle based on the longest spacer had IC(50) values as low as cisplatin. Cellular uptake studies identified a better micelle uptake with increasing micelle stability as a possible reason for lower IC(50) values. The clonogenic assay revealed that micelles loaded with platinum drugs, in contrast to low molecular weight carboplatin, have not only better activity within the frame of a 72 h cell viability study, but also display a longer lasting effect by preventing the colony formation A549 for more than 10 days.     

Reduction-Responsive Disassemblable Core-Cross-Linked Micelles Based on Poly(ethylene glycol)-b-poly(N-2-hydroxypropyl methacrylamide)-Lipoic Acid Conjugates for Triggered Intracellular Anticancer Drug Release

Reduction-sensitive reversibly core-cross-linked micelles were developed based on poly(ethylene glycol)-b-poly(N-2-hydroxypropyl methacrylamide)-lipoic acid (PEG-b-PHPMA-LA) conjugates and investigated for triggered doxorubicin (DOX) release. Water-soluble PEG-b-PHPMA block copolymers were obtained with M(n,PEG) of 5.0 kg/mol and M(n,HPMA) varying from 1.7 and 4.1 to 7.0 kg/mol by reversible addition-fragmentation chain transfer (RAFT) polymerization. The esterification of the hydroxyl groups in the PEG-b-PHPMA copolymers with lipoic acid (LA) gave amphiphilic PEG-b-PHPMA-LA conjugates with degrees of substitution (DS) of 71-86%, which formed monodispersed micelles with average sizes ranging from 85.3 to 142.5 nm, depending on PHPMA molecular weights, in phosphate buffer (PB, 10 mM, pH 7.4). These micelles were readily cross-linked with a catalytic amount of dithiothreitol (DTT). Notably, PEG-b-PHPMA(7.0k)-LA micelles displayed superior DOX loading content (21.3 wt %) and loading efficiency (90%). The in vitro release studies showed that only about 23.0% of DOX was released in 12 h from cross-linked micelles at 37 °C at a low micelle concentration of 40 μg/mL, whereas about 87.0% of DOX was released in the presence of 10 mM DTT under otherwise the same conditions. MTT assays showed that DOX-loaded core-cross-linked PEG-b-PHPMA-LA micelles exhibited high antitumor activity in HeLa and HepG2 cells with low IC(50) (half inhibitory concentration) of 6.7 and 12.8 μg DOX equiv/mL, respectively, following 48 h incubation, while blank micelles were practically nontoxic up to a tested concentration of 1.0 mg/mL. Confocal laser scanning microscope (CLSM) studies showed that DOX-loaded core-cross-linked micelles released DOX into the cell nuclei of HeLa cells in 12 h. These reduction-sensitive disassemblable core-cross-linked micelles with excellent biocompatibility, superior drug loading, high extracellular stability, and triggered intracellular drug release are promising for tumor-targeted anticancer drug delivery.     

Preparation and Investigation of Sustained Drug Delivery Systems Using an Injectable, Thermosensitive, In Situ Forming Hydrogel Composed of PLGA–PEG–PLGA

In situ gelling systems are very attractive for pharmaceutical applications due to their biodegradability and simple manufacturing processes. The synthesis and characterization of thermosensitive poly(D,L-lactic-co-glycolic acid) (PLGA)-polyethylene glycol (PEG)-PLGA triblock copolymers as in situ gelling matrices were investigated in this study as a drug delivery system. Ring-opening polymerization using microwave irradiation was utilized as a novel technique, and the results were compared with those using a conventional method of polymerization. The phase transition temperature and the critical micelle concentration (CMC) of the copolymer solutions were determined by differential scanning calorimetry and spectrophotometry, respectively. The size of the micelles was determined with a light scattering method. In vitro drug release studies were carried out using naltrexone hydrochloride and vitamin B12 as model drugs. The rate and yield of the copolymerization process via microwave irradiation were higher than those of the conventional method. The copolymer structure and concentration played critical roles in controlling the sol-gel transition temperature, the CMC, and the size of the nanomicelles in the copolymer solutions. The rate of drug release could be modulated by the molecular weight of the drugs, the concentration of the copolymers, and their structures in the formulations. The amount of release versus time followed zero-order release kinetics for vitamin B12 over 25 days, in contrast to the Higuchi modeling for naltrexone hydrochloride over a period of 17 days. In conclusion, PLGA-PEG1500-PLGA with a lactide-to-glycolide ratio of 5:1 is an ideal system for the long-acting, controlled release of naltrexone hydrochloride and vitamin B12.     

Preparation of bionanoreactor based on core-shell structured polyion complex micelles entrapping trypsin in the core cross-linked with glutaraldehyde

Recently, the polyion complex (PIC) micelle has been suggested as a promising carrier system for peptide and proteins. However, its utilities are limited by its sensitivity to the environment such as dilution and ionic strength of the solution. In this study, to overcome these obstructions, PIC micelles prepared from an anionic block copolymer, poly(ethylene glycol)-poly(alpha,beta-aspartic acid), and a cationic protein, trypsin, were cross-linked with glutaraldehyde through the Schiff base formation. On the basis of a light scattering technique, the results revealed an efficient resistance of the cross-linked PIC micelle to a high salt concentration, which was a key parameter controlling the structure of the PIC micelles. Moreover, the stability of trypsin after cross-linking was remarkably improved. Evidently, as a bionanoreactor and/or bionanoreservoir, the PIC micelles entrapping protein molecules in the cross-linked core reveal an improved stability, allowing their wide application in the fields of biotechnology and pharmaceutical sciences.     

Micellization phenomena of amphiphilic block copolymers based on methoxy poly(ethylene glycol) and either crystalline or amorphous poly(caprolactone-b-lactide)

This study focuses on the aggregation behavior of the biodegradable amphiphilic block copolymers based on methoxy poly(ethylene glycol) (mPEG) as a hydrophilic block and either crystalline poly(caprolactone-b-l-lactide) (P(CL-LLA)) or amorphous poly(caprolactone-b-D,L-lactide) (P(CL-DLLA)) as a hydrophobic block. These block copolymers have a strong tendency to form micelles in aqueous medium, with very low critical micelle concentrations (CMCs). The CMC of P(CL-LLA)-b-mPEG is higher than that of P(CL-DLLA)-b-mPEG when the mPEG block has the same molecular weight. Furthermore, the partition equilibrium coefficient (K(v)) of pyrene in the micellar solution of P(CL-LLA)-b-mPEG copolymer was lower than that of P(CL-DLLA)-b-mPEG copolymer when the mPEG block was the same length. These differences were believed to be related to the physical state of the core-forming blocks, i.e., the crystalline P(CL-LLA) block and the amorphous P(CL-DLLA) block. The TEM images showed that micelles formed by P(CL-LLA)-b-mPEG assembled in a cylindrical morphology, whereas those formed by P(CL-DLLA)-b-mPEG took a classical spherical shape. In addition, with differential scanning calorimetry (DSC) and wide-angle X-ray diffraction (WAXD) analyses, it is believed that the crystallization tendency of the core-forming blocks is the main factor governing the morphology of micelles in water. A possible mechanism for the cylindrical assembly morphology was discussed.     

Synthesis of well-defined amphiphilic block copolymers having phospholipid polymer sequences as a novel biocompatible polymer micelle reagent

To realize safer and effective drug administration, novel well-defined and biocompatible amphiphilic block copolymers containing phospholipid polymer sequences were synthesized. At first, the homopolymer of 2-methacryloyloxyethylphosphorylcholine (MPC) was synthesized in water by reversible addition-fragmentation chain transfer (RAFT) controlled radical polymerization. The "living" polymerization was confirmed by the fact that the number-average molecular weight increased linearly with monomer conversion while the molecular weight distribution remained narrow independent of the conversion. The poly(MPC) thus prepared is end-capped with a dithioester moiety. Using the dithioester-capped poly(MPC) as a macro chain transfer agent, AB diblock copolymers of MPC and n-butyl methacrylate (BMA) were synthesized. Associative properties of the amphiphilic block copolymer (pMPC(m)-BMA(n)) with varying poly(BMA) block lengths were investigated using NMR, fluorescence probe, static light scattering (SLS), and quasi-elastic light scattering (QELS) techniques. Proton NMR data in D2O indicated highly restricted motions of the n-butyl moieties, arising from hydrophobic associations of poly(BMA) blocks. Fluorescence spectra of N-phenyl-1-naphthylamine indicated that the probes were solubilized in the polymer micelles in water. The formation of polymer micelles comprising a core with poly(BMA) blocks and shell with hydrophilic poly(MPC) blocks was suggested by SLS and QELS data. The size and mass of the micelle increased with increasing poly(BMA) block length. With an expectation of a pharmaceutical application of pMPC(m)-BMA(n), solubilization of a poorly water-soluble anticancer agent, paclitaxel (PTX), was investigated. PTX dissolved well in aqueous solutions of pMPC(m)-BMA(n) as compared with pure water, implying that PTX is incorporated into the hydrophobic core of the polymer micelle. Since excellent biocompatible poly(MPC) sequences form an outer shell of the micelle, pMPC(m)-BMA(n) may find application as a promising reagent to make a good formulation with a hydrophobic drug.     

Phosphonium-containing diblock copolymers for enhanced colloidal stability and efficient nucleic Acid delivery

RAFT polymerization successfully controlled the synthesis of phosphonium-based AB diblock copolymers for nonviral gene delivery. A stabilizing block of either oligo(ethylene glycol(9)) methyl ether methacrylate or 2-(methacryloxy)ethyl phosphorylcholine provided colloidal stability, and the phosphonium-containing cationic block of 4-vinylbenzyltributylphosphonium chloride induced electrostatic nucleic acid complexation. RAFT polymerization generated well-defined stabilizing blocks (M(n) = 25000 g/mol) and subsequent chain extension synthesized diblock copolymers with DPs of 25, 50, and 75 for the phosphonium-containing block. All diblock copolymers bound DNA efficiently at ± ratios of 1.0 in H(2)O, and polyplexes generated at ± ratios of 2.0 displayed hydrodynamic diameters between 100 and 200 nm. The resulting polyplexes exhibited excellent colloidal stability under physiological salt or serum conditions, and they maintained constant hydrodynamic diameters over 24 h. Cellular uptake studies using Cy5-labeled DNA confirmed reduced cellular uptake in COS-7 and HeLa cells and, consequently, resulted in low transfection in these cell lines. Serum transfection in HepaRG cells, which are a predictive cell line for in vivo transfection studies, showed successful transfection using all diblock copolymers with luciferase expression on the same order of magnitude as Jet-PEI. All diblock copolymers exhibited low cytotoxicity (>80% cell viability). Promising in vitro transfection and cytotoxicity results suggest future studies involving the in vivo applicability of these phosphonium-based diblock copolymer delivery vehicles.     

Solubilization and controlled release of a hydrophobic drug using novel micelle-forming ABC triblock copolymers

Amphiphilic ABC triblock copolymers composed of monomethoxy-capped poly(ethylene glycol) (MPEG), poly(2-(dimethylamino)ethyl methacrylate) (DMA), and poly(2-(diethylamino)ethyl methacrylate) (DEA) have been synthesized by atom transfer radical polymerization (ATRP). These copolymers dissolve molecularly in acidic aqueous media at room temperature due to protonation of the tertiary amine groups on the DMA and DEA residues. On adjusting the pH with base, micellization occurred at pH 8, with the water-insoluble, deprotonated DEA block forming the hydrophobic cores and the MPEG and DMA blocks forming the hydrophilic micellar coronas and inner shells, respectively. This pH-induced micellization has been exploited to develop a solvent-free protocol for drug loading. A model hydrophobic drug, dipyridamole (DIP), which dissolves in acid but is insoluble above pH 5.8, was incorporated into the micelles by increasing the pH of an aqueous drug/copolymer mixture to 9. Both the empty and the drug-loaded micelles were characterized by dynamic light scattering and fluorescence studies. The interaction of both pyrene and DIP with the MPEG-DMA-DEA micelles was studied by fluorescence; both compounds had relatively high partition coefficients into the micelles, 4.5 x 10(5) and 1.5 x 10(4), respectively. Intensity-average micelle diameters ranged from 20 to 90 nm, depending on the polymer composition and concentration. Shorter MPEG blocks (Mn = 2000) produced larger micelles than longer MPEG blocks (Mn = 5000) due to the shift in the hydrophilic-hydrophobic balance of the copolymer. Transmission electron microscopy studies of the drug-loaded micelles indicated spherical morphologies and reasonably uniform particle size distributions, which is in marked contrast to the needlelike morphology observed for pure DIP in the absence of the copolymer. Experiments on controlled release demonstrated that DIP-loaded MPEG-DMA-DEA micelles act as a drug carrier, giving slow release to the surrounding solution over a period of days. Rapid release can be triggered by reducing the pH to reverse the micellization.     

Fabrication of multiresponsive shell cross-linked micelles possessing pH-controllable core swellability and thermo-tunable corona permeability

A double hydrophilic ABC triblock copolymer, poly(2-(diethylamino)ethyl methacrylate)-b-poly(2-(dimethylamino)ethyl methacrylate)-b-poly(N-isopropylacrylamide) (PDEA-b-PDMA-b-PNIPAM), containing the well-known pH-responsive PDEA block and thermoresponsive PNIPAM block, was synthesized by atom transfer radical polymerization via sequential monomer addition using ethyl 2-chloropropionate as the initiator. The obtained triblock copolymer exhibits interesting "schizophrenic" micellization behavior in aqueous solution, and supramolecularly self-assembles into three-layer "onion-like" PNIPAM-core micelles at acidic pH's and elevated temperatures and PDEA-core micelles with "inverted" structures at alkaline pH's and room temperature. In both cases, dynamic laser light scattering (LLS) and optical transmittance reveal the presence of near-monodisperse micelles, and the micelle formation/inversion process is fully reversible. Novel shell cross-linked (SCL) micelles with pH-responsive PDEA cores and thermoresponsive PNIPAM coronas were then facilely fabricated from the PDEA-b-PDMA-b-PNIPAM triblock copolymer by cross-linking the PDMA inner shells with 1,2-bis(2-iodoethoxy)ethane. The reversible pH-dependent swelling/shrinking of PDEA cores and thermosensitive collapse/aggregation of PNIPAM coronas of the obtained SCL micelles were investigated in detail by dynamic LLS, optical transmittance, and transmission electron microscopy. As the structurally stable SCL micelles possess pH-controllable core swellability and thermo-tunable corona permeability, the release profile of a model hydrophobic drug, dipyridamole, initially loaded within the hydrophobic PDEA core, can be dually controlled by both the solution pH and the temperature. This represents the first report of SCL micelles with multiresponsive cores and coronas, which may find practical applications in fields such as drug delivery and smart release.     

Effects of the incorporation of a hydrophobic middle block into a PEG-polycation diblock copolymer on the physicochemical and cell interaction properties of the polymer-DNA complexes

One-component homopolymers of cationic monomers (polycations) and diblock copolymers comprising poly(ethylene glycol) (PEG) and a polycation block have been the most widely used types of polymers for the formulation of polymer-based gene delivery systems. In this study, we incorporate a hydrophobic middle block into the conventional PEG-polycation architecture and investigate the effects of this hydrophobic modification on the physicochemical and cell-level biological properties of the polymer-DNA complexes that are relevant to gene delivery applications. The ABC-type triblock copolymer used in this study consists of (A) PEG, (B) hydrophobic poly( n-butyl acrylate) (PnBA), and (C) cationic poly(2-(dimethylamino)ethyl methacrylate) (PDMAEMA) component polymers. The properties of the triblock copolymer/DNA complexes are compared with those of two other more conventional DNA carriers derived, respectively, using a PDMAEMA homopolymer and a PEG-PDMAEMA diblock copolymer that had comparable molecular weights for individual blocks. In aqueous solution, the PEG-PnBA-PDMAEMA polymer forms positively charged spherical micelles. The electrostatic complexation of these micelles with plasmid DNA molecules results in the formation of stable small-sized DNA particles that are coated with a micelle monolayer, as confirmed by agarose gel electrophoresis, dynamic light scattering (DLS), and cryogenic transmission electron microscopy (cryo-TEM). Proton nuclear magnetic resonance ( (1)H NMR) spectroscopy measurements indicate that the whole micelle-DNA assembly (named "micelleplex" for convenience) is shielded predominantly by the PEG chains. DLS and optical microscopy imaging measurements indicate that compared with PDMAEMA-DNA polyplexes, the micelleplexes have a significantly lower tendency to aggregate under physiological salt concentrations and show reduced interactions with negatively charged components in serum such as albumin and erythrocytes. While the micelleplexes are comparable to the PEG-PDMAEMA-based DNA polyplexes in terms of their stability against aggregation under high salt concentrations and in the presence of the albumin protein, they have a slightly higher tendency to interact with erythrocytes than the diblock copolymer polyplexes. Agarose gel electrophoresis measurements indicate that relative to the PEG-PDMAEMA polyplexes, the micelleplexes provide better protection of the encapsulated DNA from enzymatic degradation and also exhibit greater stability against disintegration induced by polyanionic additives; in these respects, the PDMAEMA homopolymer-based polyplexes show the best performance. In vitro studies in HeLa cells indicate that the PDMAEMA polyplexes show the highest gene transfection efficiency among the three different gene delivery systems. Between the micelleplexes and the PEG-PDMAEMA polyplexes, a higher gene transfection efficiency is observed with the latter system. All three formulations show comparable levels of cytotoxicity in HeLa cells.     

Synthesis of amphiphilic star block copolymers and their evaluation as transdermal carriers

Amphiphilic star polymers offer substantial promise for a range of drug delivery applications owing to their ability to encapsulate guest molecules. One appealing but underexplored application is transdermal drug delivery using star block copolymer reverse micelles as an alternative to the more common oral and intravenous routes. We prepared 6- and 12-arm amphiphilic star copolymers via atom transfer radical polymerization (ATRP) of sequential blocks of polar oligo (ethylene glycol)methacrylate and nonpolar lauryl methacrylate from brominated dendritic macroinitiators based on 2,2-bis(hydroxymethyl) propionic acid. These star block copolymers demonstrate the ability to encapsulate polar dyes such as rhodamine B and FITC-BSA in nonpolar media via UV/vis spectroscopic studies and exhibit substantially improved encapsulation efficiencies, relative to self-assembled "1-arm" linear block copolymer analogs. Furthermore, their transdermal carrier capabilities were demonstrated in multiple dye diffusion studies using porcine skin, verifying penetration of the carriers into the stratum corneum.