"Schizophrenic" micellization associated with coil-to-helix transitions based on polypeptide hybrid double hydrophilic rod-coil diblock copolymer

A polypeptide hybrid double hydrophilic diblock copolymer (DHBC), poly( N-isopropylacrylamide)- b-poly( l-glutamic acid) (PNIPAM- b-PLGA), was synthesized via the ring-opening polymerization of gamma-benzyl- l-glutamate N-carboxyanhydride (BLG-NCA) using monoamino-terminated PNIPAM as the macroinitiator, followed by deprotection of benzyl groups under alkaline conditions. Containing a thermoresponsive PNIPAM block and a pH-responsive PLGA block, the obtained polypeptide hybrid diblock copolymer molecularly dissolves in aqueous solution at alkaline pH and room temperature but supramolecularly self-assembles into PNIPAM-core micelles at alkaline pH and elevated temperatures and PLGA-core micelles at acidic pH and room temperature accompanied with coil-to-helix transition of the PLGA sequence. The pH- and thermoresponsive "schizophrenic" micellization behavior of PNIPAM- b-PLGA diblock copolymer has been investigated by (1)H NMR, optical transmittance, fluorescence probe measurement, transmission electron microscopy (TEM), dynamic and static laser light scattering (LLS), and circular dichroism (CD) spectroscopy. Moreover, the micellization process was investigated employing stopped-flow light scattering technique. The pH-induced micelle growth of PNIPAM- b-PLGA in aqueous solution exhibits drastically different kinetics compared to that of conventional pH-responsive DHBCs, probably due to the stabilization effects exerted by the formed alpha-helix secondary structures within the PLGA core at low pH. Exhibiting "schizophrenic" micellization, the polypeptide sequence of PNIPAM- b-PLGA can either locate within micelle cores or stabilizing coronas. The incorporation of polypeptide block into DHBCs can endow them with structural versatility, tunable spatial arrangement of chain segments within self-assembled nanostructures, and broader applications in the field of biomedicines.     

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.     

Synthesis of amphiphilic tadpole-shaped linear-cyclic diblock copolymers via ring-opening polymerization directly initiating from cyclic precursors and their application as drug nanocarriers

We report on the facile synthesis of well-defined amphiphilic and thermoresponsive tadpole-shaped linear-cyclic diblock copolymers via ring-opening polymerization (ROP) directly initiating from cyclic precursors, their self-assembling behavior in aqueous solution, and the application of micellar assemblies as controlled release drug nanocarriers. Starting from a trifunctional core molecule containing alkynyl, hydroxyl, and bromine moieties, alkynyl-(OH)-Br, macrocyclic poly(N-isopropylacrylamide) (c-PNIPAM) bearing a single hydroxyl functionality was prepared by atom transfer radical polymerization (ATRP), the subsequent end group transformation into azide functionality, and finally the intramacromolecular ring closure reaction via click chemistry. The target amphiphilic tadpole-shaped linear-cyclic diblock copolymer, (c-PNIPAM)-b-PCL, was then synthesized via the ROP of ε-caprolactone (CL) by directly initiating from the cyclic precursor. In aqueous solution at 20 °C, (c-PNIPAM)-b-PCL self-assembles into spherical micelles consisting of hydrophobic PCL cores and well-solvated coronas of cyclic PNIPAM segments. For comparison, linear diblock copolymer with comparable molecular weight and composition, (l-PNIPAM)-b-PCL, was also synthesized. It was found that the thermoresponsive coronas of micelles self-assembled from (c-PNIPAM)-b-PCL exhibit thermoinduced collapse and aggregation at a lower critical thermal phase transition temperature (T(c)) compared with those of (l-PNIPAM)-b-PCL. Temperature-dependent drug release profiles from the two types of micelles of (c-PNIPAM)-b-PCL and (l-PNIPAM)-b-PCL loaded with doxorubicin (Dox) were measured, and the underlying mechanism for the observed difference in releasing properties was proposed. Moreover, MTT assays revealed that micelles of (c-PNIPAM)-b-PCL are almost noncytotoxic up to a concentration of 1.0 g/L, whereas at the same polymer concentration, micelles loaded with Dox lead to ～60% cell death. Overall, chain topologies of thermoresponsive block copolymers, that is, (c-PNIPAM)-b-PCL versus (l-PNIPAM)-b-PCL, play considerable effects on the self-assembling and thermal phase transition properties and their functions as controlled release drug nanocarriers.     

Shell-cross-linked micelles containing cationic polymers synthesized via the RAFT process: toward a more biocompatible gene delivery system

Block copolymers poly(2-(dimethylamino) ethyl methacrylate)-b-poly(polyethylene glycol methacrylate) (PDMAEMA-b-P(PEGMA)) were prepared via reversible addition fragmentation chain transfer polymerization (RAFT). The polymerization was found to proceed with the expected living behavior resulting in block copolymers with varying block sizes of low polydispersity (PDI <1.3). The resulting block copolymer was self-assembled in an aqueous environment, leading to the formation of pH-responsive micelles. Further stabilization of the micellar system was performed in water using ethylene glycol dimethacrylate and the RAFT process to cross-link the shell. The cross-linked micelle was found to have properties significantly different from those of the uncross-linked block copolymer micelle. While a distinct critical micelle concentration (CMC) was observed using block copolymers, the CMC was absent in the cross-linked system. In addition, a better stability against disintegration was observed when altering the ionic strength such as the absence of changes of the hydrodynamic diameter with increasing NaCl concentration. Both cross-linked and uncross-linked micelles displayed good binding ability for genes. However, the cross-linked system exhibited a slightly superior tendency to bind oligonucleotides. Cytotoxicity tests confirmed a significant improvement of the biocompatibility of the synthesized cross-linked micelle compared to that of the highly toxic PDMAEMA. The cross-linked micelles were taken up by cells without causing any signs of cell damage, while the PDMAEMA homopolymer clearly led to cell death.     

Destruction of Pseudomonas aeruginosa pre-formed biofilms by cationic polymer micelles bearing silver nanoparticles

Abstract

Pseudomonas aeruginosa is an opportunistic Gram-negative pathogen often associated with biofilm infections. This study evaluated the capacity for biofilm destruction of a novel combination of cationic polymer micelles formed from poly(2-(dimethylamino)ethyl methacrylate)-b-poly(ε-caprolactone)-b-poly(2-(dimethylamino)ethyl methacrylate) (PDMAEMA-PCL-PDMAEMA) triblock copolymer either alone, or loaded with silver nanoparticles (M_AgNPs). Pre-formed P. aeruginosa biofilms were incubated with either blank micelles, AgNO₃, or M_AgNPs. Biofilm biomass (crystal violet assay), metabolic activity (Alamar blue reduction), structure (SEM) and viability (CLSM after Live/Dead staining, or plating for CFU) were checked. The results showed that the micelles alone loosened the biofilm matrix, and caused some alterations in the bacterial surface. AgNO₃ killed the bacteria in situ leaving dead biofilm bacteria on the surface. M_AgNPs combined the two types of activities causing significant biofilm reduction, and alteration and death of biofilm bacteria. Therefore, the applied PDMAEMA-based micelles appear to be a successful candidate for the treatment of P. aeruginosa biofilm infections.     

Self-assembled micelles of biodegradable triblock copolymers based on poly(ethyl ethylene phosphate) and poly(-caprolactone) as drug carriers

A series of novel amphiphilic triblock copolymers of poly(ethyl ethylene phosphate) and poly(-caprolactone) (PEEP-PCL-PEEP) with various PEEP and PCL block lengths were synthesized and characterized. These triblock copolymers formed micelles composed of a hydrophobic core of poly(-caprolactone) (PCL) and a hydrophilic shell of poly(ethyl ethylene phosphate) (PEEP) in aqueous solution. The micelle morphology was spherical, determined by transmission electron microscopy. It was found that the size and critical micelle concentration values of the micelles depended on both hydrophobic PCL block length and PEEP hydrophilic block length. The in vitro degradation characteristics of the triblock copolymers were investigated in micellar form, showing that these copolymers were completely biodegradable under enzymatic catalysis of Pseudomonas lipase and phosphodiesterase I. These triblock copolymers were used for paclitaxel (PTX) encapsulation to demonstrate the potential in drug delivery. PTX was successfully loaded into the micelles, and the in vitro release profile was found to be correlative to the polymer composition. These biodegradable triblock copolymer micelles are potential as novel carriers for hydrophobic drug delivery.     

Functionalized thermoresponsive micelles self-assembled from biotin-PEG-b-P(NIPAAm-co-HMAAm)-b-PMMA for tumor cell target

Novel micelles, comprising hydrophilic PEG shells, hydrophobic PMMA cores, and thermosensitive P(NIPAAm-co-HMAAm) segments were self-assembled from the biotin-PEG-b-P(NIPAAm-co-HMAAm)-b-PMMA triblock copolymer. The thermosensitive micelles exhibited superior stability and showed thermotriggered drug release behavior upon temperature alterations. The fluorescence spectroscopy and confocal microscopy studies confirmed that the self-assembled biotinylated micelles can be specifically and efficiently bonded to cancer cells with the administration of biotin-transferrin, suggesting that the multifunctional micelles have great potential as drug carriers for tumor targeting chemotherapy.     

pH-responsive three-layered PEGylated polyplex micelle based on a lactosylated ABC triblock copolymer as a targetable and endosome-disruptive nonviral gene vector

Nonviral vectors for gene therapy have recently received an increased impetus because of the inherent safety problems of the viral vectors, while their transfection efficiency is generally low compared to the viral vectors. The lack of the ability to escape from the endosomal compartments is believed to be one of the critical barriers to the intracellular delivery of noviral gene vectors. This study was devoted to the design and preparation of a novel ABC triblock copolymer for constructing a pH-responsive and targetable nonviral gene vector. The copolymer, lactosylated poly(ethylene glycol)-block-poly(silamine)-block-poly[2-(N,N-dimethylamino)ethyl methacrylate] (Lac-PEG-PSAO-PAMA), consists of lactosylated poly(ethylene glycol) (A-segment), a pH-responsive polyamine segment (B-segment), and a DNA-condensing polyamine segment (C-segment). The Lac-PEG-PSAO-PAMA spontaneously associated with plasmid DNA (pDNA) to form three-layered polyplex micelles with a PAMA/pDNA polyion complex (PIC) core, an uncomplexed PSAO inner shell, and a lactosylated PEG outer shell, as confirmed by 1H NMR spectroscopy. Under physiological conditions, the Lac-PEG-PSAO-PAMA/pDNA polyplex micelles prepared at an N/P (number of amino groups in the copolymer/number of phosphate groups in pDNA) ratio above 3 were found to be able to condense pDNA, thus adopting a relatively small size (< 150 nm) and an almost neutral surface charge (zeta approximately +5 mV). The micelle underwent a pH-induced size variation (pH = 7.4, 132.6 nm --> pH = 4.0, 181.8 nm) presumably due to the conformational changes (globule-rod transition) of the uncomplexed PSAO chain in response to pH, leading to swelling of the free PSAO inner shell at lowered pH while retaining the condensed pDNA in the PAMA/pDNA PIC core. Furthermore, the micelles exhibited a specific cellular uptake into HuH-7 cells (hepatocytes) through asialoglycoprotein (ASGP) receptor-mediated endocytosis and achieved a far more efficient transfection ability of a reporter gene compared to the Lac-PEG-PSAO/pDNA and Lac-PEG-PAMA/pDNA polyplex micelles composed of the diblock copolymers and pDNA. The effect of hydroxychloroquine as an endosomolytic agent on the transfection efficiency was not observed for the Lac-PEG-PSAO-PAMA/pDNA polyplex micelles, whereas the nigericin treatment of the cell as an inhibitor for the endosomal acidification induced a substantial decrease in the transfection efficiency, suggesting that the protonation of the free PSAO inner shell in response to a pH decrease in the endosome might lead to the disruption of the endosome through buffering of the endosomal cavity. Therefore, the polyplex micelle composed of ABC (ligand-PEG/pH-responsive segment/DNA-condensing segment) triblock copolymer would be a promising approach to a targetable and endosome disruptive nonviral gene vector.     

Synthesis of biocompatible,stimuli-responsive,physical gels based on ABA triblock copolymers

ABA triblock copolymers [A = 2-(diisopropylamino)ethyl methacrylate), DPA or 2-(diethylamino)ethyl methacrylate), DEA; B = 2-methacryloyloxyethyl phosphorylcholine, MPC] prepared using atom transfer radical polymerization dissolve in acidic solution but form biocompatible free-standing gels at around neutral pH in moderately concentrated aqueous solution (above approximately 10 w/v % copolymer). Proton NMR studies indicate that physical gelation occurs because the deprotonated outer DPA (or DEA) blocks become hydrophobic, which leads to attractive interactions between the chains: addition of acid leads to immediate dissolution of the micellar gel. Release studies using dipyridamole as a model hydrophobic drug indicate that sustained release profiles can be obtained from these gels under physiologically relevant conditions. More concentrated DPA-MPC-DPA gels give slower release profiles, as expected. At lower pH, fast, triggered release can also be achieved, because gel dissolution occurs under these conditions. Furthermore, the nature of the outer block also plays a role; the more hydrophobic DPA-MPC-DPA triblock gels are formed at lower copolymer concentrations and retain the drug longer than the DEA-MPC-DEA triblock gels.     

Anticancer efficacies of cisplatin-releasing pH-responsive nanoparticles

The objective of these investigations was to test the hypothesis that a rapid cytoplasmic release profile from nanoparticles would potentiate the anticancer activity of cisplatin. Cisplatin-loaded nanoparticles with pH-responsive poly[2-(N,N-diethylamino)ethyl methacrylate] (PDEA) cores were synthesized from PDEA-block-poly(ethylene glycol) (PDEA-PEG) copolymer by using a solvent-displacement (acetone-water) method. Nanoparticles with pH-nonresponsive poly(epsilon-caprolactone) (PCL) cores made from PCL-block-PEG (PCL-PEG) were used for comparison. Nanoparticle sizes, zeta potentials, drug-loading capacities, and pH responsiveness were characterized. The cellular uptakes and localization in lysosomes were visualized by using confocal fluorescence microscopy. Cytostatic effects of free and encapsulated cis-diammineplatinum(II) dichloride (cisplatin) toward human SKOV-3 epithelial ovarian cancer cells were estimated by using the MTT assay. Intraperitoneal tumor responses to cisplatin and cisplatin/PDEA-PEG were evaluated in athymic mice at 4-6 weeks postinoculation of SKOV-3 cells. PDEA-PEG nanoparticles dissolved at pH < 6 and rapidly internalized and transferred to lysosomes; it therefore was predicted that the PDEA nanoparticles would rapidly release cisplatin into cytoplasm upon integration into acidic lysosomes and thereby overwhelm the chemoresistant properties of SKOV-3 cells. Indeed, relative proportions of viable cells were diminished to a greater extent by exposure in vitro to fast-releasing nanoparticles compared to slow-releasing nanoparticles or an equivalent dose of free cisplatin. Incidences of cellular pyknosis (a morphological indicator of apoptosis) were most evident within intestinal/mesentery tumors of mice treated with cisplatin/PDEA-PEG; tumor burdens were correspondingly reduced.     

Degradation behavior of poly(epsilon-caprolactone)-b-poly(ethylene glycol)-b-poly(epsilon-caprolactone) micelles in aqueous solution

Poly(epsilon-caprolactone)-b-poly(ethylene glycol)-b-poly(epsilon-caprolactone) triblock copolymers were synthesized by the ring-opening polymerization of epsilon-caprolactone in the presence of hydroxyl-terminated poly(ethylene glycol) with different molecular weights, using stannous octoate catalyst. Micelles prepared by the precipitation method with these triblock copolymers exhibit a core-shell structure. The degradation behaviors of these core-shell micelles in aqueous solution were investigated by FT-IR, 1H NMR, GPC, DLS, TEM, and AFM. It was found that the degradation behavior of micelles in aqueous solution was quite different from that of bulk materials. The size of the micelles increased in the initial degradation stages and decreased gradually when the degradation period was extended. The caprolactone/ethylene oxide (CL/EO) ratio in micelles measured by NMR also shows an increase at the initial degradation stage and a decrease at later stages. The morphology of these micelles became more and more irregular during the degradation period. We explain the observed behavior by a two-stage degradation mechanism with interfacial erosion between the cores and the shells followed by core erosion.     

Oxime linkage: a robust tool for the design of pH-sensitive polymeric drug carriers

Oxime bonds dispersed in the backbones of the synthetic polymers, while young in the current spectrum of the biomedical application, are rapidly extending into their own niche. In the present work, oxime linkages were confirmed to be a robust tool for the design of pH-sensitive polymeric drug delivery systems. The triblock copolymer (PEG-OPCL-PEG) consisting of hydrophilic poly(ethylene glycol) (PEG) and hydrophobic oxime-tethered polycaprolactone (OPCL) was successfully prepared by aminooxy terminals of OPCL ligating with aldehyde-terminated PEG (PEG-CHO). Owing to its amphiphilic architecture, PEG-OPCL-PEG self-assembled into the micelles in aqueous media, validated by the measurement of critical micelle concentration (CMC). The MTT assay showed that PEG-OPCL-PEG exhibited low cytotoxicity against NIH/3T3 normal cells. Doxorubicin (DOX) as a model drug was encapsulated into the PEG-OPCL-PEG micelles. Drug release study revealed that the DOX release from micelles was significantly accelerated at mildly acid pH of 5.0 compared to physiological pH of 7.4, suggesting the pH-responsive feature of the drug delivery systems with oxime linkages. Flow cytometry and confocal laser scanning microscopy (CLSM) measurements indicated that these DOX-loaded micelles were easily internalized by living cells. MTT assay against HeLa cancer cells showed DOX-loaded PEG-OPCL-PEG micelles had a high anticancer efficacy. All of these results demonstrate that these polymeric micelles self-assembled from oxime-tethered block copolymers are promising carriers for the pH-triggered intracellular delivery of hydrophobic anticancer drugs.     

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.     

Polymeric micelle-based bioassay with femtomolar sensitivity

Target-specific polymeric micelles loaded with fluorescence dye molecules in their hydrophobic cores were made from block copolymer of poly(caprolactones)23-b-poly(ethylene oxide)45. It was found that the micelles are stable against pH changes from pH 2 to 12 and temperature variation up to 65 degrees C. The dye molecules can be released to the solution on exposing the micelles to organic solvents or ultrasound. A rapid and highly sensitive immunoassay based on the above micelles was developed, and the assay can detect specific target proteins in the femtomolar range from complex biological samples such as serum mimics and cell lysate. For example, less than 0.15 U/ml of ovarian cancer-specific antigen 125, equivalent to 7.5 x 10(-15)M, can be reliably detected in solution. We also demonstrated that the assay can detect a cell surface biomarker, stage-specific embryonic antigen 4, from a single human embryonic stem cell.     

Polyolefin matrixes with permanent antibacterial activity: preparation, antibacterial activity, and action mode of the active species

Poly[2-(tert-butylamino)ethyl methacrylate] (PTBAEMA) belongs to a novel class of water-insoluble biocides. Dispersion of a poly(ethylene-co-butylene)-b-poly[2-(tert-butylamino)ethyl methacrylate] diblock copolymer (PEB-b-PTBAEMA) within low-density polyethylene (LDPE) imparts antimicrobial properties to the polyolefin as assessed by the viable cell counting method against Escherichia coli (E. coli). This diblock copolymer has been synthesized by atom transfer radical polymerization (ATRP) with a poly(ethylene-co-butylene) (PEB) oligomer end-capped by an activated bromide as a macroinitiator for the polymerization of 2-(tert-butylamino)ethyl methacrylate (TBAEMA). Morphological changes of E. coli bacteria in contact with modified LDPE have been observed by transmission and scanning electron microscopy and indicate that the diblock copolymer is bactericide rather than bacteriostatic. Finally, the action mode of the PEB-b-PTBAEMA copolymer more likely relies on the displacement of the Ca(2+) and/or Mg(2+) ions of the outer membrane of the bacteria, which is disorganized and finally disrupted.     

Facile preparation of well-defined AB2 Y-shaped miktoarm star polypeptide copolymer via the combination of ring-opening polymerization and click chemistry

Well-defined AB2 Y-shaped miktoarm star polypeptide copolymer, PZLL-b-(PBLG)2, was synthesized via a combination of ring-opening polymerization (ROP) of alpha-amino acid N-carboxyanhydride (NCA) and click chemistry, where PZLL is poly(epsilon-benzyloxycarbonyl-L-lysine) and PBLG is poly(gamma-benzyl-L-glutamate). First, two types of primary-amine-containing initiators, N-aminoethyl 3,5-bis(propargyloxyl)-benzamide and 3-azidopropylamine, were synthesized and employed for the ROP of NCA, leading to the formation of dialkynyl-terminated PZLL and azide-terminated PBLG, dialkynyl-PZLL and PBLG-N3, respectively. The subsequent copper(I)-catalyzed cycloaddition reaction between dialkynyl-PZLL and slightly excess PBLG-N3 led to facile preparation of PZLL-b-(PBLG)2 Y-shaped miktoarm star polypeptide copolymer. The excess PBLG-N3 was scavenged off by reacting with alkynyl-functionalized Wang resin. The obtained Y-shaped miktoarm star polypeptide copolymer was characterized by gel permeation chromatograph (GPC), Fourier transform-infrared spectroscopy (FT-IR), and (1)H NMR. Moreover, after the hydrolysis of protecting benzyl and benzyloxycarbonyl groups of PZLL-b-(PBLG)2, water-soluble pH-responsive Y-shaped miktoarm star polypeptide copolymer, PLL-b-(PLGA)2, was obtained, where PLL is poly(L-lysine) and PLGA is poly(L-glutamic acid). It can self-assemble into PLGA-core micelles at acidic pH and PLL-core micelles at alkaline pH, accompanied with the coil-to-helix transition of PLGA and PLL sequences, respectively. The spontaneous pH-responsive supramolecular assembly of PLL-b-(PLGA)2 miktoarm star polypeptide copolymer has been investigated via a combination of (1)H NMR, laser light scattering (LLS), transmission electron microscopy (TEM), and circular dichroism (CD) spectroscopy.     

Degradable thermoresponsive nanogels for protein encapsulation and controlled release

Reversible addition-fragmentation chain transfer (RAFT) polymerization technique was used for the fabrication of stable core cross-linked micelles (CCL) with thermoresponsive and degradable cores. Well-defined poly(2-methacryloyloxyethyl phosphorylcholine), poly(MPC) macroRAFT agent, was first synthesized with narrow molecular weight distribution via the RAFT process. These CCL micelles (termed as nanogels) with hydrophilic poly(MPC) shell and thermoresponsive core consisting of poly(methoxydiethylene glycol methacrylate) (poly(MeODEGM) and poly(2-aminoethyl methacrylamide hydrochloride) (poly(AEMA) were then obtained in a one-pot process by RAFT polymerization in the presence of an acid degradable cross-linker. These acid degradable nanogels were efficiently synthesized with tunable sizes and low polydispersities. The encapsulation efficiencies of the nanogels with different proteins such as insulin, BSA, and β-galactosidase were studied and found to be dependent of the cross-linker concentration, size of protein, and the cationic character of the nanogels imparted by the presence of AEMA in the core. The thermoresponsive nature of the synthesized nanogels plays a vital role in protein encapsulation: the hydrophilic core and shell of the nanogels at low temperature allow easy diffusion of the proteins inside out and, with an increase in temperature, the core becomes hydrophobic and the nanogels are easily separated out with entrapped protein. The release profile of insulin from nanogels at low pH was studied and results were analyzed using bicinchoninic assay (BCA). Controlled release of protein was observed over 48 h.     

Thermoreversible hydrogels from RAFT-synthesized BAB triblock copolymers: steps toward biomimetic matrices for tissue regeneration

Narrowly dispersed, temperature-responsive BAB block copolymers capable of forming physical gels under physiological conditions were synthesized via aqueous reversible addition fragmentation chain transfer (RAFT) polymerization. The use of a difunctional trithiocarbonate facilitates the two-step synthesis of BAB copolymers with symmetrical outer blocks. The outer B blocks of the triblock copolymers consist of poly(N-isopropylacrylamide) (PNIPAM) and the inner A block consists of poly(N,N-dimethylacrylamide). The copolymers form reversible physical gels above the phase transition temperature of PNIPAM at concentrations as low as 7.5 wt % copolymer. Mechanical properties similar to collagen, a naturally occurring polypeptide used as a three-dimensional in vitro cell growth scaffold, have been achieved. Herein, we report the mechanical properties of the gels as a function of solvent, polymer concentration, and inner block length. Structural information about the gels was obtained through pulsed field gradient NMR experiments and confocal microscopy.     

Biodegradable block copolymer micelles with thiol-responsive sheddable coronas

Novel sheddable micelles having hydrophilic coronas capable of being shed from biodegradable polylactide (PLA) cores by the cleavage of disulfide linkages in response to thiols were prepared by aqueous micellization of PLA-based amphiphilic block copolymers functionalized with disulfides at block junctions. These well-defined copolymers were synthesized by a combination of ring-opening polymerization and atom transfer radical polymerization in the presence of a new disulfide-functionalized double-head initiator having both terminal OH and Br groups. (1)H NMR and GPC results indicate that both polymerizations were well-controlled with molecular weight distribution as low as M(w)/M(n) < 1.2. Aqueous micellization to form core/shell micelles with disulfides at the interface of PLA cores and hydrophilic coronas and their thiol-responsive degradation were investigated. In the presence of water-soluble thiols, disulfide linkages in the micelles were cleaved and hydrophilic coronas were lost, causing PLA cores to precipitate due to the loss of colloidal stability. In a biomedical perspective, the new sheddable micelles were not cytotoxic and hence biocompatible.     

Phosphorylcholine-based pH-responsive diblock copolymer micelles as drug delivery vehicles: light scattering, electron microscopy, and fluorescence experiments

The micellization behavior of a diblock copolymer comprising a highly hydrophilic and biocompatible poly(2-methacryloyloxyethyl phosphorylcholine) (PMPC) corona-forming block and a pH-sensitive poly(2-(diisopropylamino)ethyl methacrylate) (PDPA) core-forming block (PMPC-b-PDPA) has been studied by static and dynamic light scattering (SDLS), transmission electron microscopy (TEM), and potentiometry. Self-assembly of PMPC-b-PDPA copolymers with two different DPA volume fractions (phi(DPA)) leads to narrowly distributed and structurally distinct spherical micelles, as evidenced by their molecular weight (M(w,mic)), aggregation number (N(agg)), hydrodynamic radius (R(H)), corona width (W), and core radius (R(c)). The excellent potential of these pH-responsive micelles as nanosized drug delivery vehicles was illustrated by the encapsulation of dipyridamole (DIP), a model hydrophobic drug that dissolves in acid solutions and becomes insoluble above pH 5.8, which is comparable to the pK(a) of the PDPA block. The influence of micelle structure (namely M(w,mic), N(agg), R(H), W, and R(c)) on drug loading content, drug loading efficiency, partition coefficient, and release kinetics was investigated and confirmed by fluorescence spectroscopy studies. The maximum dipyridamole loadings within PMPC(30)-b-PDPA(30) (R(H) = 14.0 nm; W = 4.8 nm; R(c) = 9.2 nm) and PMPC(30)-b-PDPA(60) (R(H) = 27.1 nm; W = 11.0 nm; R(c) = 16.1 nm) micelles were 7 and 12% w/w(p), respectively. This preferential solubilization of DIP into micelles formed by copolymer chains having longer core-forming blocks (i.e., possessing larger core volumes) reflects the larger partition coefficient (K(V)) of DIP between the aqueous phase and PMPC(30)-b-PDPA(60) micelles (K(V) = 5.7 x 10(4)) compared to PMPC(30)-b-PDPA(30) micelles (K(V) = 1.1 x 10(4)). This enhanced ability of PMPC(30)-b-PDPA(60) aggregates to entrap/stabilize small hydrophobic molecules also produces slower release kinetics. Rapid release can be triggered by lowering the pH to induce micellar dissociation.