Preparation of micelle-forming polymer-drug conjugates.

Adriamycin, a hydrophobic anticancer drug, was conjugated with poly(ethylene oxide)-poly(aspartic acid) block copolymers composed of various lengths of each block copolymer segment ranging from 1000 to 12,000 in molecular weight and from 10 to 80 units, respectively. Conjugation was achieved without precipitation by adjusting the ratio of adriamycin to aspartic acid residues of the block copolymer and the quantity of DMF used for the reaction. Thus obtained conjugates showed high water solubility irrespective of a large amount of the conjugated adriamycin. Furthermore, these conjugates were found to form micellar structures with a hydrophobic inner core and a hydrophilic outer shell. This micellar architecture may be utilized for effective drug targeting.     

Modular construction of multifunctional bioresponsive cell-targeted nanoparticles for gene delivery

Multifunctional and modular block copolymers prepared from biocompatible monomers and linked by a bioreducible disulfide linkage have been prepared using a combination of ring-opening and atom-transfer radical polymerizations (ATRP). The presence of terminal functionality via ATRP allowed cell-targeting folic acid groups to be attached in a controllable manner, while the block copolymer architecture enabled well-defined nanoparticles to be prepared by a water-oil-water double emulsion procedure to encapsulate DNA with high efficiency. Gene delivery assays in a Calu-3 cell line indicated specific folate-receptor-mediated uptake of the nanoparticles, and triggered release of the DNA payload via cleavage of the disulfide link resulted in enhanced transgene expression compared to nonbioreducible analogues. These materials offer a promising and generic means to deliver a wide variety of therapeutic payloads to cells in a selective and tunable way.     

Biocompatible wound dressings based on chemically degradable triblock copolymer hydrogels

The synthesis of a series of thermo-responsive ABA triblock copolymers in which the outer A blocks comprise poly(2-hydroxypropyl methacrylate) and the central B block is poly(2-(methacryloyloxy)ethyl phosphorylcholine) is achieved using atom transfer radical polymerization. These novel triblock copolymers form thermo-reversible physical gels with critical gelation temperatures and mechanical properties that are highly dependent on the copolymer composition and concentration. TEM studies on dried dilute copolymer solutions indicate the presence of colloidal aggregates, which is consistent with micellar gel structures. This hypothesis is consistent with the observation that incorporating a central disulfide bond within the B block leads to thermo-responsive gels that can be efficiently degraded using mild reductants such as dithiothreitol (DTT) over time scales of minutes at 37 degrees C. Moreover, the rate of gel dissolution increases at higher DTT/disulfide molar ratios. Finally, these copolymer gels are shown to be highly biocompatible. Only a modest reduction in proliferation was observed for monolayers of primary human dermal fibroblasts, with no evidence for cytotoxicity. Moreover, when placed directly on 3D tissue-engineered skin, these gels had no significant effect on cell viability. Thus, we suggest that these thermo-responsive biodegradable copolymer gels may have potential applications as wound dressings.     

Selective and effective cleavage of RNAs by vinyladenine-vinylamine copolymer.

Poly(uridylic acid) (poly[U]) and poly(adenylic acid) (poly[A]) are efficiently cleaved, via phosphodiester linkage hydrolysis, at pH 8, 50 degrees C by use of poly(vinyladenine-co-vinylamine) as catalyst. The catalytic activity of the copolymer surpasses the value of poly(vinylamine), indicating a significant role of the adenine residue in the copolymer for the effective catalysis.     

Thermogelling poly(ethylene oxide-b-propylene oxide-b-ethylene oxide) disulfide multiblock copolymer as a thiol-sensitive degradable polymer

We report a reverse thermogelling poly(ethylene oxide-b-propylene oxide-b-ethylene oxide) disulfide multiblock copolymer as a thiol-sensitive biodegradable polymer. The poly(ethylene oxide-b-propylene oxide-b-ethylene oxide) aqueous solutions studied in this research underwent sol-gel-sol or sol-gel-sol-gel transition depending on the molecular weight and concentration of the polymer, whereas the corresponding disulfide multiblock copolymer aqueous solutions underwent sol-gel transition as the temperature increased in a range of 0-60 degrees C. The hydrophobic dye solubilization and dynamic light scattering of the polymer aqueous solution suggest that the poly(ethylene oxide-b-propylene oxide-b-ethylene oxide)s undergo unimer (3 nm) to micelle (12 nm) transition, whereas the disulfide multiblock copolymers undergo unimer (6 nm) to aggregated polymer (600 nm) transition as the temperature increases. The gel duration increased from 6 h (poly(ethylene oxide-b-propylene oxide-b-ethylene oxide)) to more than 12 days (the corresponding disulfide multiblock copolymer) in phosphate buffer saline, and the gel duration of the latter depended on the glutathione concentration of the medium. The model drug, paclitaxel, was released from the in-situ-formed poly(ethylene oxide-b-propylene oxide-b-ethylene oxide) disulfide multiblock copolymer gel in a glutathione concentration-sensitive manner.     

Molecular stability of chicken and rabbit immunoglobulin G.

Molecular stability of chicken egg yolk immunoglobulin G (IgY) and that of rabbit IgG were compared by measuring antibody activities and conformational changes. Stability of rabbit IgG to acid denaturation was much higher than that of IgY. Conformation of the IgY molecule was readily changed in acidic conditions, resulting in a rapid loss of antibody activity. Much less stable natures of IgY to heat-treatment and guanidine-HCl denaturation than rabbit IgG were also observed. Differences in the structure between the two immunoglobulins that might participate in their different stability were inferred from their amino acid sequence data. Importance of the intramolecular disulfide linkage in the rabbit light chain and some other structural differences were suggested.     

Synthesis of poly(epsilon-caprolactone)-b-poly(gamma-benzyl-L-glutamic acid) block copolymer using amino organic calcium catalyst

A biodegradable two block copolymer, poly(epsilon-caprolactone)-b- poly(gamma-benzyl-L-glutamic acid) (PCL-PBLG) was synthesized successfully by ring-opening polymerization of N-carboxyanhydride of gamma-benzyl-L-glutamate (BLG-NCA) with aminophenyl-terminated PCL as a macroinitiator. The aminophenethoxyl-terminated PCL was prepared via hydrogenation of a 4-nitrophenethoxyl-terminated PCL, which was novelly obtained from the polymerization of epsilon-caprolactone (CL) initiated by amino calcium 4-nitrobenzoxide. The structures of the block copolymer and its precursors from the initial step of PCL were confirmed and investigated by 1H NMR, FT-IR, GPC, and FT-ICRMS analyses and DSC measurements.     

Quaternized chitosan (QCS)/poly (aspartic acid) nanoparticles as a protein drug-delivery system

The aim of this study was to generate a new type of nanoparticles made of quaternized chitosan (QCS) and poly (aspartic acid) and to evaluate their potential for the association and delivery of protein drugs. QCS and poly (aspartic acid) were processed to nanoparticles via the ionotropic gelation technique. The size, polydispersity, zeta potential, and morphology of the nanoparticles were characterized. Entrapment studies of the nanoparticles were conducted using bovine serum albumin (BSA) as a model protein. The effects of the pH value of nanoparticles with different QCS/poly (aspartic acid) ratios, QCS molecular weight (MW), poly (aspartic acid) concentration, and BSA concentration on the nanoparticle size, the nanoparticle yield, and BSA encapsulation were studied in detail. Suitably pH value of nanoparticles with different QCS/poly (aspartic acid) ratios, moderate QCS MW, optimal concentration ratio of poly (aspartic acid), and QCS favored more nanoparticles formed and higher BSA encapsulation efficiency. The release of BSA from nanoparticles was pH-dependent. Fast release occurred in 0.1 M phosphate buffer solution (PBS, pH 7.4), while the release was slow in 0.1 M HCl (pH 1.2). The results showed that the new QCS/poly (aspartic acid) nanoparticles have a promising potential in protein delivery system.     

Poly(Aspartic Acid) Degradation by a Sphingomonas sp. Isolated from Freshwater

A poly(aspartic acid) degrading bacterium (strain KT-1 [JCM10459]) was isolated from river water and identified as a member of the genus Sphingomonas. The isolate degraded only poly(aspartic acid)s of low molecular masses (<5 kDa), while the cell extract hydrolyzed high-molecular-mass poly(aspartic acid)s of 5 to 150 kDa to yield aspartic acid monomer.     

Block copolymerization of vinyl compounds by the terminal-group activation of poly(α-amino acids) and the characterization of block copolymers

The terminal amino groups of polysarcosine, poly(γ-benzyl l-glutamate), and poly(ε-benzyloxycarbonyl-l-lysine) were haloacetylated. The mixture of the terminally haloacetylated poly(α-amino acid) and styrene or methyl methacrylate was photoirradiated in the presence of Mn₂(CO)₁₀, or heated with Mo(CO)₆, yielding A-B-A-type block copolymers consisting of poly(α-amino acid) (the A component) and vinyl polymer (the B component). The block copolymers were characterized, and the present investigation revealed that the thermally initiated polymerization of vinyl compounds by the trichloroacetyl poly(α-amino acid)/Mo(CO)₆ system was the most suitable for the synthesis of the α-amino acid/vinyl compound block copolymers. The A-B-A type block copolymers showed higher antithrombogenicity than the corresponding homopolymers. In particular, a film of the A-B-A-type block copolymer of poly[Glu(OBzl)] and polystyrene possessed a microphase-separated structure and did not induce a conformational change of fibrinogen adsorbed, leading to a high antithrombogenicity.     

Controlling the aggregation of conjugates of streptavidin with smart block copolymers prepared via the RAFT copolymerization technique

Block copolymers containing stimuli-responsive segments provide important new opportunities for controlling the activity and aggregation properties of protein-polymer conjugates. We have prepared a RAFT block copolymer of a biotin-terminated poly(N-isopropylacrylamide) (PNIPAAm)-b-poly(acrylic acid) (PAA). The number-average molecular weight (M(n)) of the (PNIPAAm)-b-(PAA) copolymer was determined to be 17.4 kDa (M(w)/M(n) = 1.09). The PNIPAAm block had an M(n) of 9.5 kDa and the poly(acrylic acid) (PAA) block had an M(n) of 7.9 kDa. We conjugated this block copolymer to streptavidin (SA) via the terminal biotin on the PNIPAAm block. We found that the usual aggregation and phase separation of PNIPAAm-SA conjugates that follow the thermally induced collapse and dehydration of PNIPAAm (the lower critical solution temperature (LCST) of PNIPAAm is 32 degrees C in water) is prevented through the shielding action of the PAA block. In addition, we show that the cloud point and aggregation properties (as measured by loss in light transmission) of the [(PNIPAAm)-b-(PAA)]-SA conjugate also depended on pH. At pH 7.0 and at temperatures above the LCST, the block copolymer alone was found to form particles of ca. 60 nm in diameter, while the bioconjugate exhibited very little aggregation. At pH 5.5 and 20 degrees C, the copolymer alone was found to form large aggregates (ca. 218 nm), presumably driven by hydrogen bonding between the -COOH groups of PAA with other -COOH groups and also with the -CONH- groups of PNIPAAm. In comparison, the conjugate formed much smaller particles (ca. 27 nm) at these conditions. At pH 4.0, however, large particles were formed from the conjugate both above and below the LCST (ca. 700 and 540 nm, respectively). These results demonstrate that the aggregation properties of the block copolymer-SA conjugate are very different from those of the free block copolymer, and that the outer-oriented hydrophilic block of PAA shields the intermolecular aggregation of the block copolymer-SA bioconjugate at pH values where the -COOH groups of PAA are significantly ionized.     

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.     

An acid-labile block copolymer of PDMAEMA and PEG as potential carrier for intelligent gene delivery systems

Intelligent gene delivery systems based on physiologically triggered reversible shielding technology have evinced enormous interest due to their potential in vivo applications. In the present work, an acid-labile block copolymer consisting of poly(ethylene glycol) and poly(2-(dimethylamino)ethyl methacrylate) segments connected through a cyclic ortho ester linkage (PEG- a-PDMAEMA) was synthesized by atom transfer radical polymerization of DMAEMA using a PEG macroinitiator with an acid-cleavable end group. PEG- a-PDMAEMA condensed with plasmid DNA formed polyplex nanoparticles with an acid-triggered reversible PEG shield. The pH-dependent shielding/deshielding effect of PEG chains on the polyplex particles were evaluated by zeta potential and size measurements. At pH 7.4, polyplexes generated from PEG- a-PDMAEMA exhibited smaller particle size, lower surface charge, reduced interaction with erythrocytes, and less cytotoxicity compared to PDMAEMA-derived polyplexes. At pH 5.0, zeta potential of polyplexes formed from PEG- a-PDMAEMA increased, leveled up after 2 h of incubation and gradual aggregation occurred in the presence of bovine serum albumin (BSA). In contrast, the stably shielded polyplexes formed by DNA and an acid-stable block copolymer, PEG- b-PDMAEMA, did not change in size and zeta potential in 6 h. In vitro transfection efficiency of the acid-labile copolymer greatly increased after 6 h incubation at pH 5.0, approaching the same level of PDMAEMA, whereas there was only slight increase in efficiency for the stable copolymer, PEG- b-PDMAEMA.     

In vitro poly(ADP-ribosyl)ation of seminal ribonuclease

The site of in vitro ADP-ribosylation of seminal ribonuclease was determined. Seminal enzyme was found to be a good receptor of [14C]ADP-ribose residues under the reaction conditions used. The recovery of [14C]ADP-ribosylated RNase was about 65% after purification. After tryptic digestion of modified enzyme, a fraction containing [14C]ADP-ribosylated peptides was separated from the others by ion-exchange chromatography on M82 resin. Radioactive peptides were then purified by affinity chromatography on anti-poly(ADP-ribose)IgG-Sepharose. High performance liquid chromatography of a mixture obtained after pronase digestion of purified ADP-ribosylated peptides revealed only one radioactive peptide whose amino acid composition corresponded to a peptide that has equimolar quantities of aspartic acid, serine, and glycine. Carboxypeptidase Y digestion of this peptide showed that its amino acid sequence was Asp-Ser-Gly. Only position 14-16 of seminal RNase corresponded to this sequence. The chemical stability of the ADP-ribose/enzyme linkage indicated that aspartic acid 14 is the modification site in seminal RNase.     

Reduction/pH dual-sensitive PEGylated hyaluronan nanoparticles for targeted doxorubicin delivery

To minimize the side effect of chemotherapy, a novel reduction/pH dual-sensitive drug nanocarrier, based on PEGylated dithiodipropionate dihydrazide (TPH)-modified hyaluronic acid (PEG-SS-HA copolymer), was developed for targeted delivery of doxorubicin (DOX) to hepatocellular carcinoma. The copolymer was synthesized by reductive amination via Schiff's base formation between TPH-modified HA and galactosamine-conjugated poly(ethylene glycol) aldehyde/methoxy poly(ethylene glycol) aldehyde. Conjugation of DOX to PEG-SS-HA copolymer was accomplished through the hydrazone linkage formed between DOX and PEG-SS-HA, and confirmed by FTIR and ¹H NMR spectra. The polymer–DOX conjugate could self-assemble into spherical nanoparticles (∼150 nm), as indicated by TEM and DLS. In vitro release studies showed that the DOX-loaded nanoparticles could release DOX rapidly under the intracellular levels of pH and glutathiose. Cellular uptake experiments demonstrated that the nanoparticles could be efficiently internalized by HepG2 cells. These results indicate that the PEG-SS-HA copolymer holds great potential for targeted intracellular delivery of DOX.     

Synthesis and characterization of a head-tail type polycation block copolymer as a nonviral gene vector

A head-tail type polycation block copolymer, which is composed of the polyamidoamine (PAMAM) dendron and poly(L-lysine) (PLL) blocks, was newly designed as a nonviral gene vector in this study. This block copolymer (PAMAM dendron-PLL) was successfully synthesized in two steps: the synthesis of the PAMAM dendron block and the polymerization of the PLL block from the PAMAM dendron block. PAMAM dendron and PLL blocks in block copolymer showed independent deprotonation behavior, and their pK(a) were determined to be 6.8 and 9.0, respectively. The complexation with pDNA was evaluated by gel retardation assay and dye exclusion assay, and both assays indicated that pDNA was selectively complexed with PLL block of block copolymer. Also, the PAMAM dendron-PLL poplyplexes showed 10(2) fold higher transfection efficiency to HeLa cells as that for PLL polyplexes. This might be due to the buffering effect of the PAMAM dendron block. This block copolymer could produce a function share in each block, i.e., tail block complexed with pDNA and head block showed a buffering effect. This molecular design of the head-tail type block copolymer might provide a new approach for realizing in vivo gene therapy.     

Supramolecular assemblies for the cytoplasmic delivery of antisense oligodeoxynucleotide: polyion complex (PIC) micelles based on poly(ethylene glycol)-SS-oligodeoxynucleotide conjugate

A novel cytoplasmic delivery system of antisense oligodeoxynucleotide (asODN) was developed by assembling a PEG-asODN conjugate with disulfide linkage (smart linkage) (PEG-SS-asODN) into polyion complex (PIC) micelles through the complexation with branched poly(ethylenimine) (B-PEI). The PIC micelle thus prepared showed a significant antisense effect against luciferase gene expression in HuH-7 cells, far more efficient than nonmicelle systems (asODN and PEG-SS-asODN in free form) and PIC micelle encapsulating the conjugate without the disulfide linkage. Use of poly(l-lysine) (PLL) instead of the B-PEI for PIC micellization led to a substantial decrease in the antisense effect. These results indicate that the PIC micelles formulated from PEG-SS-asODN conjugate and B-PEI is successfully transported from the endosomal compartment into the cytoplasm by the buffering effect of the B-PEI, releasing hundreds of active asODN molecules via cleavage of the disulfide linkage into the cellular interior, responding to a high glutathione concentration in the cytoplasmic compartment. Furthermore, the type of smart linkage (glutathione-sensitive SS linkage vs pH-sensitive linkage) in the conjugates substantially affected the antisense effect of the PIC micelles, depending on the nature of the counter polycation (B-PEI vs PLL).     

Synthesis and evaluation of a star amphiphilic block copolymer from poly(epsilon-caprolactone) and poly(ethylene glycol) as a potential drug delivery carrier

A star polymer composed of amphiphilic block copolymer arms has been synthesized and characterized. The core of the star polymer is polyamidoamine (PAMAM) dendrimer, the inner block in the arm is lipophilic poly(epsilon-caprolactone) (PCL), and the outer block in the arm is hydrophilic poly(ethylene glycol) (PEG). The star-PCL polymer was synthesized first by ring-opening polymerization of epsilon-caprolactone with a PAMAM-OH dendrimer as initiator. The PEG polymer was then attached to the PCL terminus by an ester-forming reaction. Characterization with SEC, (1)H NMR, FTIR, TGA, and DSC confirmed the star structure of the polymers. The micelle formation of the star copolymer (star-PCL-PEG) was studied by fluorescence spectroscopy. Hydrophobic dyes and drugs can be encapsulated in the micelles. A loading capacity of up to 22% (w/w) was achieved with etoposide, a hydrophobic anticancer drug. A cytotoxicity assay demonstrated that the star-PCL-PEG copolymer is nontoxic in cell culture. This type of block copolymer can be used as a drug delivery carrier.     

Biodegradable amphiphilic triblock copolymer bearing pendant glucose residues: preparation and specific interaction with Concanavalin A molecules

A novel biodegradable amphiphilic block copolymer PLGG-PEG-PLGG bearing pendant glucose residues is successfully prepared by the coupling reaction of 3-(2-aminoethylthio)propyl-alpha-D-glucopyranoside with the pendant carboxyl groups of PLGG-PEG-PLGG in the presence of N,N'-carbonyldiimidazole. The polymer PLGG-PEG-PLGG, i.e., poly{(lactic acid)-co-[(glycolic acid)-alt-(L-glutamic acid)]}-block-poly(ethylene glycol)-block- poly{(lactic acid)-co-[(glycolic acid)-alt-(L-glutamic acid)]}, is prepared by ring-opening copolymerization of L-lactide (LLA) with (3s)-benzoxylcarbonylethylmorpholine-2,5-dione (BEMD) in the presence of dihydroxyl PEG with molecular weight of 2000 as macroinitiator and Sn(Oct)2 as catalyst, and then by catalytic hydrogenation. The glucose-grafted copolymer shows a lower degree of cytotoxicity to ECV-304 cells and improved specific recognition and binding with Concanavalin A (Con A). Therefore, this kind of glucose-grafted copolymer may find biomedical applications.     

Design of polypeptide-functionalized polystyrene microspheres

In this contribution, the principle of spontaneous surface segregation has been applied for the preparation of polypeptide-functionalized polystyrene microspheres. For that purpose, an amphiphilic diblock copolymer was introduced in the mixture styrene/divinylbenzene and polymerized using AIBN as initiator. During the polymerization, cross-linked particles were obtained in which the diblock copolymer was encapsulated. The amphiphilic diblock copolymers used throughout this study contain a hydrophilic polypeptide segment, either poly(L-lysine) or poly(L-glutamic acid) and a hydrophobic polystyrene block. After 4 h of polymerization, rather monodisperse particles with sizes of approximately 3-4 microm were obtained. Upon annealing in hot water, the hydrophilic polypeptides migrate to the interface, hence, either positively charged or neutral particles were obtained when poly(L-lysine) is revealed at the surface and exposed to acidic or basic pH, respectively. On the opposite, negatively charged particles were achieved in basic pH water by using poly(L-glutamic acid) as additive. The surface chemical composition was modified by changing the environment of the particles. Thus, exposure in toluene provoked a surface rearrangement, and due to its affinity, the polystyrene block reorients toward the interface.