Substrate Engineering in Lipase-Catalyzed Selective Polymerization of d-/l-Aspartates and Diols to Prepare Helical Chiral Polyester.

The synthesis of optically pure polymers is one of the most challenging tasks in polymer chemistry. Herein, Novozym 435 (Lipase B from Candida antarctica, immobilized on Lewatit VP OC 1600)-catalyzed polycondensation between d-/l-aspartic acid (Asp) diester and diols for the preparation of helical chiral polyesters was reported. Compared with d-Asp diesters, the fast-reacting l-Asp diesters easily reacted with diols to provide a series of chiral polyesters containing N-substitutional l-Asp repeating units. Besides amino acid configuration, N-substituent side chains and the chain length of diols were also investigated and optimized. It was found that bulky acyl N-substitutional groups like N-Boc and N-Cbz were more favorable for this polymerization than small ones probably due to competitively binding of these small acyl groups into the active site of Novozym 435. The highest molecular weight can reach up to 39.5 × 103 g/mol (Mw, D = 1.64). Moreover, the slow-reacting d-Asp diesters were also successfully polymerized by modifying the substrate structure to create a "nonchiral" condensation environment artificially. These enantiocomplementary chiral polyesters are thermally stable and have specific helical structures, which was confirmed by circular dichroism (CD) spectra, scanning electron microscope (SEM), and molecular calculation.

Enzymatic Synthesis and Stereocomplex Formation of Chiral Polyester Containing Long-Chain Aliphatic Alcohol Backbone.

Herein we demonstrated a novel lipase-catalyzed synthesis of isotactic D-/L-poly(aspartate-octanediol) ester containing long chain alcohols backbone and discovered their stereocomplex feature with an increased Tm for the first time. Simple design of monomer structures not only overcomes the inherent selectivity limitation of enzyme used, but also achieves totally isotactic polyester products. By crystallizing the mixed enantiopure isotactic polyesters in different solvents, the formation of amorphous mixture, homocrystallites or stereocomplex crystallites were observed, respectively. This study is expected to open up a new way to prepare various stereocomplex polyesters containing a long-chain aliphatic alcohol backbone and a wide variety of functional groups.

Solvent-Free Lipase-Catalyzed Synthesis: Unique Properties of Enantiopure D- and L- Polyaspartates and Their Complexation.

Amino acids are attractive monomers for the large-scale preparation of chiral polyamides. For enzymatic polymerization of amino acids using protease in aqueous environment as the catalysis system, one main restriction is oligomer formation, usually along with other displayed advantages. Herein we developed an efficient solvent-free lipase-catalyzed polymerization of diethyl D- or L-aspartate, providing chiral D- and L-polyaspartates with an average degree of polymerization (DPavg) up to 60 and having about 96% ß-linkages. Additionally, their distinct chemical and physical properties were characterized by circular dichroism (CD) spectra, X-ray powder diffraction (XRD), microscopic observation, and thermal analysis. Poly(ß-D-AspEt) and Poly(ß-L-AspEt) showed vertically mirrored negative and positive CD signals, high crystallinity, and entirely different microscopic morphology. They are thermal stable while having different decomposition (Td), melting (Tm), and cold crystallization temperatures (Tcc), respectively. Our results also showed that the complexation of enantiopure D- and L-polyaspartates was not stereocomplex but homocomplex.

High performance liquid chromatography enantioseparation of the novel designed mexiletine derivatives and its analogs.

A series of novel designed mexiletine derivatives and its analogs were prepared, the structures were confirmed by Nuclear Magnetic Resonance (NMR), Fourier Transform Infrared Spectroscopy (FTIR), and Electrospray Ionization-Mass Spectrometry (ESI-MS), and the enantioseparations were performed on polysaccharide-based chiral stationary phase (CSP), Chiralcel OD-H, and Chiralcel OJ-H, under normal-phase mode. The effects of the concentration of isopropanol in the mobile phase were studied, seven of the eight enantiomers got baseline separation on Chiralcel OD-H, and five of the eight enantiomers got successfully separation on Chiralcel OJ-H. The effects of structural features were also discussed.

Hepatic-targeting microcapsules construction by self-assembly of bioactive galactose-branched polyelectrolyte for controlled drug release system.

We describe the construction of hepatic-targeting microcapsules by self-assembly of chemo-enzymatic synthesized poly(vinyl galactose ester-co-methacryloxyethyl trimethylammonium chloride) (PGEDMC) containing galactose branches, which can be specifically recognized by membrane bound galactose receptors (ASGPR), for acyclovir (ACV) controlled release system. Alternate deposition of PGEDMC and poly(sodium 4-styrenesulfonate) (PSS) was carried out on ACV microcrystals. It was revealed that the drug release rate decreases with the increase of coated layer number and a microcapsule-drying treatment would enhance the sustained release effect probably because of a multilayer shrink and tightness during the process. The complete release of ACV yielded a hollow PGEDMC/PSS multilayered network with favorable integrity and nano-thickness by TEM and SEM. The potential targetability of the system was proved in vitro by PNA lectin recognition. Lectin hardly adsorbed on the film where the outmost layer was a polyanion or a polycation without galactose component. Whilst the galactose-containing layer (PGEDMC) was the outmost layer, a significant lectin combination was observed. This technique could provide a promising way to encapsulate and deliver various target substances in biological and pharmaceutical applications.

Novel nylon-supported organic-inorganic hybrid membrane with hierarchical pores as a potential immobilized metal affinity adsorbent.

Chitosan-based porous organic-inorganic hybrid membranes supported by microfiltration nylon membranes were prepared, in which gamma-glycidoxypropyltrimethoxysilane (GPTMS) was used as an inorganic source as well as crosslinking reagent. Polyethylene glycol (PEG) with different molecular weight and content was used as imprinting molecule for morphology control. In situ crosslinking of chitosan and simultaneous polymerization of GPTMS in PEG template environment endowed the hybrid membrane with specific characteristics. Distinct hybrid effect between chitosan (CS) and GPTMS was revealed by shifting in X-ray diffraction (XRD) pattern, decomposition in simultaneous thermogravimetry and differential scanning calorimetry (TG/DSC) testing. As manifested by scanning electron microscopy (SEM), the molecular weight and content of PEG had remarkable effect on the resulting surface morphology of the hybrid membrane and a given surface morphology could be obtained by extracting of the imprinted PEG molecular. Among three types of porogen used: PEG 400, PEG 4000 and PEG 20000, only PEG 20000 could result in a porous surface. Moreover, a special porous surface with three-dimensional (3D) hierarchical structure-in-structure pore fashion was obtained when content of PEG 20000 was controlled at 15%. Experimental results also showed that the hybrid membrane had low swelling ratio and high stability in acidic solution. After conveniently coordinated with copper ions, the porous metal chelating hybrid membrane could effectively adsorb the model protein, bovine serum albumin (BSA). As expected, the hybrid membrane imprinted with 15% PEG 20000 had remarkably high copper ion binding and BSA adsorption capacity, which might result from the large surface area, high ligand density and suitable interconnected 3D hierarchical porous surface.

Chemo-enzymatic synthesis of raffinose-branched polyelectrolytes and self-assembly application in microcapsules.

A novel biocompatible polyelectrolyte poly(vinyl raffinose-co-acrylic acid) (PRCA) containing a raffinose branch was prepared via redox polymerization using Fe(2+)/K(2)S(2)O(8)/H(2)O(2) starting from enzymatically-synthesized monomer: 1-O-vinyldecanedioyl raffinose. Copolymers with different monomer feed ratios were prepared and characterized with IR, NMR, and GPC. PRCA can be alternated with polycation to form microcapsules on a crystals template by electrostatic layer-by-layer technique. The multilayers of PRCA/poly(methacryloyloxyethyl dimethylbenzyl ammonium chloride) (PMBA) on quartz slides and PRCA/poly(dimethyldiallyl ammonium chloride) (PDDA) on acyclovir crystals template were fabricated and characterized with UV-Vis spectra, the microelectrophoretic measurement, and TEM. Hollow capsules can be formed after the removal of acyclovir crystals template in a buffer solution. The nano-capsule-carrying galactose residue is a potential targeting drug-controlled delivery systems.

Antitumor gemcitabine conjugated micelles from amphiphilic comb-like random copolymers.

Gemcitabine is an important pyrimidine antimetabolite that inhibits cellular DNA synthesis. However, the therapeutic efficacy and clinical benefit of gemcitabine are severely compromised due to its rapid plasma metabolism and low selectivity towards tumor tissues. To overcome these limitations, we prepared novel PEGylated gemcitabine-contained comb-like copolymers poly(monomethoxyl PEG350 methylacrylate -co- 5'-O-vinyladipyl- gemcitabine) (poly(mPEG350MA-co-VAG) and (poly(mPEG1000MA-co-VAG), which could self-assemble into micelles and displayed enhanced antitumor activity. The copolymers and the formed micelles were well characterized for their structure, critical aggregation concentration (CAC), morphology, cellular uptake, cell cytotoxicity, and controlled drug release. Cellular uptake and in vitro cytotoxicity assays against human lung cancerous cells (A549) demonstrated that these micelles could be effectively internalized and induced cell apoptosis. These micelles efficiently inhibited tumor growth when injected intravenously into A549 cell derived xenograft tumor bearing Balb/C nude mice using a dose of 10mg/kg in terms of reduced tumor volume compared to free gemcitabine. In conclusion, PEGylated micelles could protect gemcitabine from rapid plasma metabolism, provided a sustained release and showed enhanced antitumor activity, thus have the potential to be used as novel anticancer drug delivery vehicle.

Glucose-functionalized multidrug-conjugating nanoparticles based on amphiphilic terpolymer with enhanced anti-tumorous cell cytotoxicity.

It is well known that combination therapy can significantly enhance the cytotoxicity and bypass some resistance mechanisms. However, the different solubility and pharmacokinetics of drugs limit the applications of combination therapy. In this study, novel glucose-functionalized polymeric micelle nanoparticles containing multidrugs were successfully fabricated and characterized. Two chemotherapeutic agents, cytarabine (Ara-C) and fluorodeoxyuridine (FUDR), were conjugated to a glucose-functionalized amphiphilic random terpolymer to create a novel nanocarrier for the delivery of multiple drugs simultaneously with an identical pharmacokinetic profile. The incorporation of d-glucose markedly increased the dispersity and biocompatibility of the novel polymeric micelles. In vitro drug release studies showed the two anticancer agents could be simultaneously released from multidrug-conjugating nanoparticles. Cellular uptake assay observed by confocal laser scanning microscopy and cytotoxicity tests performed by MTT assay against hepG2 human hepatoma cells indicated that glucose-functionalized multidrug-conjugating nanoparticles could be effectively internalized by HepG2 cells and showed much more effective growth-inhibitory activity than two single-drug-conjugating polymer aggregates or free drugs. This finding, therefore, illustrated that the d-glucose functionalized nanoparticles could be used as a novel potential multidrug delivery vehicle.

A novel immunosensor based on an alternate strategy of electrodeposition and self-assembly.

A novel amperometric immunosensor for the determination of carcinoembryonic antigens (CEA) was developed. Firstly, ordered multilayer films of Prussian blue (PB) and multiwalled-carbon nanotube/polyethylenimine/Au (MWNT-PEI-Au) nanocomposite were fabricated onto the surface of a glassy carbon electrode via alternate electrodeposition and self-assembly. Then a layer of chitosan mixed with gold nanoparticles was cast onto the surface of the electrode. Subsequently, the electrode was coated with antibody (Ab(1)) and blocked with BSA. The morphology of the MWNT-PEI-Au nanocomposite was characterized by transmission electron microscopy (TEM). The fabrication process of the ordered multilayer structure and immunosensor were characterized by scanning electron microscopy (SEM) and electrochemical measurements, respectively. The proposed fabrication strategy effectively ensured the stability of the Prussian blue as electron mediator. Under optimal conditions, the fabricated immunosensor exhibited a good response to CEA, with a detection range from 0.5 to 160 ng/mL and a detection limit of 0.08 ng/mL at 3δ. The current fabricated immunosensor exhibited good sensitivity, selectivity, and long-term stability. Furthermore, current study demonstrated the promising application of the alternate strategy based on electrodeposition and self-assembly for the construction of biosensor.

Multidrug nanoparticles based on novel random copolymer containing cytarabine and fluorodeoxyuridine.

Novel multidrug nanoparticles were self-assembled from the random copolymer containing cytarabine and fluorodeoxyuridine. The multidrug copolymer carrying 28.7wt.% of cytarabine and 29.1wt.% of fluorodeoxyuridine was prepared by radical polymerization combined with enzymatic selective transesterification. Homopolymers of the two drugs were also synthesized by the same method. And the polymers were characterized by FTIR, (1)H NMR, and gel permeation chromatography (GPC). Self-assembly of the multidrug copolymer was verified by UV-vis and fluorescence spectroscopy. The morphology of nanoparticles formed from the copolymer was investigated by transmission electron microscopy (TEM) and dynamic light scattering (DLS), which indicated that the nanoparticles were regular spheres with a diameter of 133+/-28nm. In vitro drug release studies illustrated that the two synergistic anticancer agents could be simultaneously released from the multidrug nanoparticles.

Controllable synthesis of polymerizable ester and amide prodrugs of acyclovir by enzyme in organic solvent.

A facile control of the acylation position at the primary hydroxyl and amino of acyclovir, respectively, was achieved and five polymerizable acyclovir prodrugs were synthesized. Various reaction conditions were studied in detail. Thus, lipase acrylic resin from Candida antarctica (CAL-B) in pyridine or acetone showed high chemo-selectivity toward the primary hydroxyl of acyclovir. However, lipase PS 'Amano' (PS) in DMSO selectively acylated the amino group. The selectivity of PS could be adjusted by changing reaction solvents. The acyclovir vinyl derivatives obtained would be important monomers used for the preparation of macromolecular nucleoside drugs.

Bioactive galactose-branched polyelectrolyte multilayers and microcapsules: self-assembly, characterization, and biospecific lectin adsorption.

We describe the fabrication of multilayers and microcapsules with biologically designed targeting activity using chemoenzymatic synthesized carbohydrate-branched polyelectrolytes. A novel cationic d-galactose-branched copolymer [poly(vinyl galactose ester-co-methacryloxyethyl trimethylammonium chloride), PGEDMC] is alternated with poly(styrene sulfonate) (PSS) to form thin multifilms by the layer-by-layer (LbL) technique on such different solid surfaces as quartz slides, poly(ethylene terephthalate) (PET) films, silicon wafers, and polystyrene (PS) microparticles. The experimental protocols were first optimized on flat, smooth silica substrates using UV-vis, contact angle, and atomic force microscopy (AFM) measurements. The film properties of PGEDMC/PSS multilayers are modified by varying polyelectrolyte concentration, ionic strength, and counteranion types. Hollow capsules were formed after the removal of colloidal templates; transmission (TEM) and scanning (SEM) electron microscopy were used to verify the LbL process integrity. PGEDMC/PSS planar films and capsules carrying beta-galactose as recognition signals have specific recognition abilities with peanut agglutinin (PNA) lectin rather than concanavalin A (Con A) lectin observed by fluorescence spectroscopy.

Controllable regioselective enzymatic synthesis of polymerizable 5'-O-vinyl- and 3'-O-vinyl-nucleoside analogues in acetone.

An efficient synthesis of polymerizable 3'- and 5'-O-acyl-nucleoside derivatives has been developed from inosine and 2'-deoxyuridine by enzyme-catalyzed regioselective acylation with divinyl dicarboxylates. In acetone, Lipozyme (immobilized lipase from Mucor miehei) gave 5'-O-acyl-nucleoside products, and PPL (lipase from porcine pancreas) provided 3'-O-acyl-nucleoside products.