Insoluble ionic complexes of polyacrylic acid with a cationic drug for use as a mucoadhesive, ophthalmic drug delivery system

We have developed a new mucoadhesive drug delivery formulation based on an ionic complex of partially neutralized poly(acrylic acid) (PAA) and a highly potent beta blocker drug, levobetaxolol x hydrochloride (LB x HCl), for use in the treatment of glaucoma. PAA was neutralized with sodium hydroxide to varying degrees of neutralization. Aqueous solutions containing concentrations of LB x HCl equivalent to the degree of PAA neutralization were added to the PAA solutions and formed insoluble complexes, which were isolated. The complex formation was followed by turbidimetric titration, and the complexes were characterized by IR and 1H NMR spectroscopy. Complexes were prepared with varying degrees of drug loading, such that the same PAA chain would have free -COOH groups for mucoadhesion along with ionic complexes of LB x H+ with COO- groups. Thin films of the complexes dissociated to release the drug by ion exchange with synthetic tear fluid. The films shrunk continuously during release of the drug and dissolved completely in 1 h. Solid inserts of these films could be useful as a mucoadhesive ophthalmic drug delivery system.     

Pilocarpine-loaded chitosan-PAA nanosuspension for ophthalmic delivery

Chitosan-poly(acrylic acid) (CS-PAA) nanoparticles, to be used as ophthalmic drug carrier, were successfully prepared using template polymerization of acrylic acid (AA) in a chitosan solution. When the polymerization was done at 70°C for 45 min with a CS/AA weight ratio of 1:1, the surface structure of the prepared nanoparticles was most stable with the smallest mean diameter (92.0±7.5 nm) and a stable zeta potential (25.5±2.6 mV) in a buffer solution (pH 4.5). The size of the nanoparticles dramatically increased with the pH value of the medium. Both in vitro and in vivo studies revealed that the prepared nanoparticle suspension was better at sustaining the release of pilocarpine than either simulated tear fluid or commercial eye drops.     

A polymeric micelle system with a hydrolysable segment for drug delivery

A potential anti-cancer drug-delivery polymeric micelle system with an in vitro degradation half-life of about 48 h that releases its drug upon application of ultrasound was synthesized. This vehicle was composed of an amphiphilic co-polymer, poly(ethylene oxide)-b-poly(N-isopropylacrylamide-co-2-hydroxyethyl methacrylate-lactate _n ). The degree of polymerization of the lactate side group, n, was 0, 3 or 5. The molar ratio of NIPAAm to HEMA-lactate _n to PEO in polymerization was optimized to produce an in vitro polymeric micelle half-life of about 48 h at 40°C. 1,6-Diphenyl-1,3,5-hexatriene (DPH) was used as a fluorescent probe to study the hydrophobicity of the cores of the polymeric micelles. The results showed that the cores of the polymeric micelles were hydrophobic enough to sequester DPH and the anti-cancer drug doxorubicin (Dox). Dox was encapsulated into the polymeric micelles having a molar feed ratio of NIPAAm to HEMA-lactate₃ to PEO equal to 20 : 5 : 1; this drug was released upon the application of low-frequency ultrasound. The Dox release was about 2% at room temperature and 4% at body temperature, and the drug returned to the polymeric micelles when insonation ceased.     

A Novel GAP460 Biopolymer for Use as a Carrier in Drug-Delivery Applications

We synthesized a new non-toxic biopolymer (GAP460) containing γ,L-glutamic acid and aspartate (Asp). Conjugates of GAP460 and cisplatin exhibited a drug-carrying capacity of nearly 40%, 3-times higher than γ-PGA and dramatically decreasing the amount of biopolymer required for high-dose delivery. Treatment with GAP460-cisplatin conjugate (PACC) not only effectively inhibited tumor growth in nude mice, but also resulted in extended survival and lower nephrotoxicity, suggesting that GAP460 could be used as an effective carrier for drug delivery and that PACC may have potential therapeutic applications in the clinical treatment of cancer.     

Polymeric complexes of lidocaine hydrochloride with poly(acrylic acid) and poly(2-hydroxyethyl vinyl ether)

The specific interactions of local anesthetic lidocaine hydrochloride with poly(acrylic acid) and poly(2-hydroxyethyl vinyl ether), as well as in a triple system composed of the drug and both polymers, have been studied in aqueous solutions by viscometric, turbidimetric, potentiometric, and FTIR spectroscopic methods. The mechanism of the drug binding to the polymers and the structures of the polycomplexes formed are clarified.     

Poly(ethylenimine)-grafted-poly[(aspartic acid)-co-lysine], a potential non-viral vector for DNA delivery

A potential non-viral gene-transfer vector, poly(ethylenimine)-grafted-poly[(aspartic acid)-co-lysine] (PSL), has been developed by thermal polycondensation of aspartic acid and lysine under reduced pressure. Low-molecular-mass branch poly(ethylenimine) (PEI600) was conjugated to the backbone. The chemical structure of the resulting co-polymer was identified by H-NMR, FT-IR, TGA and X-ray diffraction. The results of the MTT assay showed that at concentration up to 4000 nmol/l of the vector cell viability was over 80% and showed low toxicity. Electrophoretic retardation and ethidum bromide assay showed that at N/P ratios 12–15 (w/w) the DNA could be condensed and neutralized. Using the zeta potential assay we discovered that it had a high positive charge on its surface of the particle (over 30 mV). The particle sizes of the co-polymer/DNA complexes were 150–170 nm, as measured by DLS and AFM. Compared with PEI600, co-polymer/DNA complexes showed a significant enhancement of transfection activity in the absence and presence of serum in NT2 and COS7 cell lines. This means that the PEI600-PSL co-polymer is a promising candidate for gene delivery.     

Poly(acrylic acid)-grafted Poly(N-isopropyl acrylamide) Networks: Preparation,Characterization and Hydrogel Behavior

Poly(acrylic acid)-grafted poly(N-isopropylacrylamide) co-polymer networks (PNIPAAm-g-PAA) were prepared via the reversible addition-fragmentation transfer (RAFT) polymerization of N-isopropyl- acrylamide (NIPAAm) with trithiocarbonate-terminated PAA as a macromolecular chain-transfer agent in the presence of N,N-methylenebisacrylamide. The PNIPAAm-g-PAA co-polymer networks were characterized by means of Fourier transform infrared spectroscopy, differential scanning calorimetry and small-angle X-ray scattering. It is found that the PNIPAAm-g-PAA co-polymer networks were microphase-separated, in which the microdomains of PNIPAAm-PAA interpolymer complexes were dispersed into the PNIPAAm matrix. The PNIPAAm-g-PAA hydrogels displayed a dual response to temperature and pH values. The thermoresponsive properties of PNIPAAm-g-PAA networks were investigated. Below the volume phase transition temperatures, the PNIPAAm-g-PAA hydrogels possessed much higher swelling ratios than control PNIPAAm hydrogel. In terms of swelling, deswelling and reswelling tests, it is judged that the PNIPAAm-g-PAA hydrogels displayed faster response to the external temperature changes than control PNIPAAm hydrogel. The improved thermoresponsive properties of hydrogels are ascribed to the formation of PAA-grafted PNIPAAm networks, in which the water-soluble PAA chains behave as the hydrophiphilic tunnels and allow water molecules to go through and, thus, to accelerate the diffusion of water molecules.     

Hydrophilic interpenetrating polymer networks of poly(dimethyl siloxane) (PDMS) as biomaterial for cochlear implants

Poly(dimethyl siloxane) (PDMS) was bulk-modified to develop a new intra-cochlear electrode that can closely hug the inner wall of scala tympani (ST). The hydrophilicity of bulk and surface of PDMS was changed using a sequential method for preparation of interpenetrating polymer networks (IPNs). A series of IPNs, based on PDMS and poly(acrylic acid) (PAAc), was synthesized and characterized by means of attenuated total reflectance Fourier transform infrared spectroscopy, water contact-angle measurement, dynamic mechanical thermal analysis and peel strength tests. The performances of actual-sized fabricated electrodes were assessed inside a transparent model of ST, which was filled with saline. The cell behavior of L929 fibroblasts on materials was studied in vitro.     

Chitosan-poly(acrylic acid) polyelectrolyte complex membranes: preparation,characterization and permeability studies

Polyelectrolyte complex (PEC) membranes were obtained by mixing solutions of two polymers of opposite charges, chitosan (Chi) and poly(acrylic acid) PAA. Three membranes were obtained: one made of pure chitosan and two membranes with chitosan mixed with PAA at a ratio of 95:5 (one prepared using PAA solution in 3.5% formic acid, named ChiPAA3.5, and another one using a PAA solution in 10% formic acid, named ChiPAA10). The membranes were characterized by swelling experiments, FT-IR spectroscopy, scanning electron microscopy (SEM), atomic force microscopy (AFM), mechanical properties and permeability studies in relation to a drug model (sodium sulphamerazine). The calculation of degree of ionization showed that the lower the formic acid concentration was, the higher the PAA dissociation degree. Polyelectrolyte complex formation was characterized by FT-IR. Water uptake results showed that PEC membranes were more hydrophilic than pure chitosan, ChiPAA3.5 being the most. Morphological analysis by SEM and AFM showed that PAA addition changed the membranes morphology, especially for ChiPAA3.5. Mechanical properties indicated that PEC membranes were more rigid than pure chitosan membranes and that the morphology has an influence on tensile strength values. Permeability values decreased with complex formation and were lower for ChiPAA10 than ChiPAA3.5. However, as drug concentration was increased, the difference between the two complex membranes disappeared. The results were discussed considering the drug–membrane interactions. Diffusion coefficient values indicated that ChiPAA3.5 had a higher drug retention capacity than ChiPAA10.     

PEG- and PDMAEG-Graft-Modified Branched PEI as Novel Gene Vector: Synthesis,Characterization and Gene Transfection

The cytotoxicity of polyethylenimine (PEI) was a dominating obstacle to its application. Introduction of poly(ethylene glycol) (PEG) blocks to PEI is one of the strategies to alleviate the cytotoxocity of PEI. However, it is well known that the transfection efficiency of PEGylated PEI is decreased to some extent compared to the corresponding PEI. Thus, the aim of our study was to enhance the transfection efficiency of PEGylated PEI. A series of tri-block co-polymers, PEG-g-PEI-g-poly(dimethylaminoethyl L-glutamine) (PEG-g-PEI-g-PDMAEG), as novel vectors for gene therapy was synthesized and evaluated. PEG-g-PEI was first obtained by linking PEG and PEI using isophorone diisocyanate (IPDI) as coupling reagent. The anionic co-polymerization of γ-benzyl-L-glutamate N-carboxyanhydride (BLG-NCA) using PEG-g-PEI as a macro-initiator was carried out, followed by aminolysis with 2-dimethylaminoethylamine to obtain the target water-soluble tri-block co-polymer. The structures of the polymers were confirmed by FT-IR and ¹H-NMR. The influence of the molecular weight of PEI and the length of the PDMAEG chain on the physicochemical properties and transfection activity of polymer/DNA was evaluated. All PEI derivates were revealed to compact plasmid DNA effectively to give polyplexes with suitable size (approx. 100 nm) and moderate zeta potentials (10–15 mV) at N/P ratios over 10. The PEG-g-PEI-g-PDMAEG tri-block co-polymers displayed particularly low cytotoxicity, even at high concentration, reflecting an improved safety profile compared to PEI 25k. Gene transfection efficiency of PEG-g-PEI-g-PDMAEG on HeLa in the presence and absence of serum was determined. Remarkably, the transfection activity of PEG-g-PEI (10k)-g-PDMAEG (PPP-4)/DNA polyplex formulations was nearly twofold higher than PEI 25k/DNA formulations in vitro, and the transfection efficiency was less affected by the presence of serum. These results indicated that the synthesized PEG-g-PEI-g-PDMAEG tri-block co-polymers are promising candidates as carriers for gene delivery.     

Synthesis,characterization, biodegradation,and drug delivery application of biodegradable lactic/glycolic acid polymers. Part II: Biodegradation

A series of previously-synthesized lactic/glycolic acid polymers (PLGA) with various molar ratios of lactic to glycolic acid and various molecular weights were further studied with regard to their biodegradation behavior, and in particular, the factors affecting the biodegradation rate. The biodegradation of PLGA is affected by many factors including polymer composition, molecular weight, and nature of the incubating media. The biodegradation rate of PLGA containing higher content of lactic acid moiety is lower than those containing a lower content of lactic acid moiety. PLGAs with a higher molecular weight, degrade faster than those with a lower molecular weight, i.e. the molecular weight decreases more rapidly for higher molecular weight PLGAs than their lower molecular weight counterparts. Nature or properties of the hydrolysis/incubating media may have an effect on the biodegradation of PLGAs. A basic medium may slow down the biodegradation of PLGA in comparison with samples in an acidic medium. The rate of pH reduction for the incubating medium can be divided into three deferent phases, giving an inverted S-type pH profile for the non-buffered incubating media.     

Differential degradation rates in vivo and in vitro of biocompatible poly(lactic acid) and poly(glycolic acid) homo- and co-polymers for a polymeric drug-delivery microchip

The biocompatibility and biodegradation rate of component materials are critical when designing a drug-delivery device. The degradation products and rate of degradation may play important roles in determining the local cellular response to the implanted material. In this study, we investigated the biocompatibility and relative biodegradation rates of PLA, PGA and two poly(lactic-co-glycolic acid) (PLGA) polymers of 50 : 50 mol ratio, thin-film component materials of a drug-delivery microchip developed in our laboratory. The in vivo biocompatibility and both in vivo and in vitro degradation of these materials were characterized using several techniques. Total leukocyte concentration measurements showed normal acute and chronic inflammatory responses to the PGA and low-molecular-weight PLGA that resolved by 21 days, while the normal inflammatory responses to the PLA and high-molecular-weight PLGA were resolved but at slower rates up to 21 days. These results were paralleled by thickness measurements of fibrous capsules surrounding the implants, which showed greater maturation of the capsules for the more rapidly degrading materials after 21 days, but less mature capsules of sustained thicknesses for the PLA and high-molecular-weight PLGA up to 49 days. Gel-permeation chromatography of residual polymer samples confirmed classification of the materials as rapidly or slowly degrading. These materials showed thinner fibrous capsules than have been reported for other materials by our laboratory and have suitable biocompatibility and biodegradation rates for an implantable drug-delivery device.     

A novel construct as a cell carrier for tissue engineering

A 3D scaffold, in the form of a foam, with the top surface carrying a micropattern, was constructed from biodegradable polyesters poly(3-hydroxybutyric acid-co-3-hydroxyvaleric acid) (PHBV) and poly(L-lactide-co-D,L-lactide) (P(L/DL)LA) to serve as a substitute for the extracellular matrix (ECM) of tissues with more than one cell type. The construct was tested in vitro for engineering of such tissues using fibroblasts (3T3) and epithelial cells (retinal pigment epithelial cells, D407). The patterned surface was seeded with D407 cells and the foam was seeded with 3T3 cells to represent a tissue with two different cell types. To improve cell adhesion, the construct was treated with fibronectin. The cells were seeded on the construct in a sequence allowing each type time for adhesion. Cell proliferation, studied by MTS assay, was significantly higher than that of tissue culture polystyrene control by day 14. Scanning electron and fluorescence microscopy showed that the foam side of the construct was highly porous and the pores were interconnected and this allowed cell mobility and proliferation. Immunostaining showed collagen deposition, indicating the secretion of the new ECM by the cells. On the film side of the construct D407 cells formed piles in the grooves and covered the surface completely. It was concluded that the 3D P(L/DL)LA-PHBV construct with one micropatterned surface has a serious potential for use as a tissue engineering carrier in the reconstruction of complex tissues with layered organization and different types of cells in each region.     

Haemocompatibility of vitamin-E-enriched poly(D,L-lactic acid)

Poly(D,L-lactic acid) (P(D,L)LA) is a biocompatible and biodegradable polymer whose use is limited to orthopaedic applications. In fact, the mechanical properties of P(D,L)LA are not usually utilized for cardiovascular applications, as the polymer has been proven to activate both granulocyte- and platelet-causing inflammation. In order to improve P(D,L)LA haemocompatibility vitamin E (α-tocoferol, 10–30% (w/w)), a natural biological anti-oxidant and anti-inflammatory agent, was added during the solvent casting of P(D,L)LA film. The P(D,L)LA films obtained were then analysed using FT-IR analysis to assess vitamin E presence; polymer surface wettability and human plasma protein adsorption were measured by sessile drop test, spectrophotometric protein quantification and Western blot, respectively, and polymer haemocompatibility was assessed measuring platelet and granulocyte adhesion and whole blood coagulation. Vitamin E presence caused an increase in polymer surface wettability and human plasma protein adsorption. The combination of both effects may account for the decrease in platelet and granulocyte adhesion and for the doubling of whole blood clotting time measured onto vitamin-E-enriched P(D,L)LA compared to control P(D,L)LA. Our results indicate that vitamin E addition improves P(D,L)LA haemocompatibility, making this polymer suitable for cardiovascular application.     

Amoxicillin-Loaded Sponges Made of Collagen and Poly[(methyl vinyl ether)-co-(maleic anhydride)] for Root Canal Treatment: Preparation,Characterization and In Vitro Cell Compatibility

The difficulty of eliminating Enterococcus faecalis and other bacteria infecting dental root canals makes it desirable to develop formulations capable of sustained release of antibiotics within the canal. With this function in view, in this work we compared the mechanical, drug release and biocompatibility properties of amoxicillin-loaded collagen (CL) and CL complexed with poly[(methyl vinyl ether)-co-(maleic anhydride)] (PVMMA), with or without glutaraldehyde (GTA) or the natural product genipin (GN) as cross-linker. Collagen was not denatured by complexation with PVMMA. Only CL–PVMMA–GN sponges did not disintegrate during 7 days exposure to cell culture medium (un-cross-linked CL disintegrated within 24 h and un-cross-linked CL–PVMMA within 4 days), and CL–PVMMA–GN sponges also exhibited the most appropriate combination of mechanical properties (hardness, modulus of deformability and plasticity). CL–PVMMA–GN sponges absorbed aqueous medium faster than other cross-linked formulations, but their maximum uptake was less; and drug release from CL–PVMMA–GN sponges tended to be faster than from any other, except un-cross-linked CL–PVMMA, maximum release taking about 4 days. No formulation significantly altered the viability of L929 fibroblast-like mouse connective tissue cells, but cells growing on sponges showed signs of non-adherence. It is concluded that genipin-cross-linked CL–PVMMA sponges merit further investigation as antibiotics vehicles and aids to tissue regeneration in the dental root canal.     

Spontaneous Formation of a Hydrogel Composed of Water-Soluble Phospholipid Polymers Grafted with Enantiomeric Oligo(lactic acid) Chains

We designed and synthesized water-soluble biocompatible and biodegradable polymers composed of 2-methacryroyloxyethyl phosphorylcholine and oligo(L- or D-lactic acid) macromonomers to develop an injectable hydrogel matrix. Aqueous solutions containing the polymers with oligo(L-lactic acid) (OLLA) and oligo(D-lactic acid) (ODLA) chains underwent spontaneous gelation when mixed together. This was due to the formation of a stereocomplex between the OLLA and ODLA side-chains, which act as cross-linking components in the hydrogel. Therefore, the hydrogel could be re-dissolved in a buffer solution by hydrolysis of the oligo(lactic acid) chains. We obtained an injectable, biocompatible and degradable hydrogel, and we anticipate that it will be used in applications involving the controlled release of bioactive molecules and cell-based tissue engineering.     

Synthesis and In Vitro Biocompatibility Assessment of a Poly(amic acid) Derived from Ethylenediaminetetraacetic Dianhydride

Poly(amic acid) (PAA) derived from ethylenediaminetetracetic dianhydride shows great potential as a biomaterial suitable for biomedical applications. To evaluate this polymer class further, in vitro cell toxicity (WST-1/ECS, ELISA based) and cell compatibility (cell adhesion and cell proliferation) tests were conducted to establish structure–toxicity relationships. PAAs with a number-average molecular weight ranging between 100 to 200 kg/mol were synthesized at 37°C after 24 h. Porcine radial artery cells (RACs) and descending aorta endothelial cells (ECs) were seeded independently in a 96-well cell culture plate at a cell density of 5000 cells/cm² to observe toxic effects. Similarly, RACs and ECs were seeded independently onto PAA coated and uncoated cover slips at a cell density of 7000 cells/cm² to observe growth patterns. Our results showed no toxicity after 96 h of incubation and in addition, both RACs and ECs adhered and proliferated on the PAA films, preserving their phenotype during this time. The tested synthetic material seems promising as a future biomaterial and should elicit a desired cellular response upon implantation.     

Preparation and Characterization of a Porous Scaffold Based on Poly(D,L-Lactide) and N-Hydroxyapatite by Phase Separation

In this study, a series of porous scaffolds were prepared from poly(D,L-lactide) (PLA) and nanohydroxyapatite (HA) using the phase separation method. HA/PLA composite membranes and PLA membranes with a microporous structure (pore size around 10–20 μm) were observed by scanning electron microscopy and these micropores were well distributed throughout the PLA membranes. The surface morphology of HA/PLA composite membranes was significantly improved compared to pure PLA membrane. Also, the mechanical property and contact angle of composite membranes were different from that of pure PLA films. The immortalized rat osteoblastic ROS 17/2.8 cell line was used in this research to study the cell adhesion and proliferation behavior, and the results indicated that composite membranes had great cell affinity and good biocompatibility.     

Doxorubicin-loaded amphiphilic polypeptide-based nanoparticles as an efficient drug delivery system for cancer therapy

An amphiphilic anionic copolymer, methoxy poly(ethylene glycol)-b-poly(l-glutamic acid-co-l-phenylalanine) (mPEG-b-P(Glu-co-Phe)), with three functionalized domains, was synthesized and used as a nanovehicle for cationic anticancer drug doxorubicin hydrochloride (DOX·HCl) delivery via electrostatic interactions for cancer treatment. The three domains displayed distinct functions: PEG block chain for prolonged circulation; poly(phenylalanine) domain for stabilizing the nanoparticle construct through hydrophobic/aromatic interactions; and the poly(glutamic acid) domain for providing electrostatic interactions with the cationic drug to be loaded. The copolymer could self-assemble into micellar-type nanoparticles, and DOX was successfully loaded into the interior of nanoparticles by simple mixing of DOX·HCl and the copolymer in the aqueous phase. DOX-loaded mPEG-b-P(Glu-co-Phe) nanoparticles (DOX-NP) had a superior drug-loading content (DLC) (21.7%), a high loading efficiency (almost 98%) and a pH-triggered release of DOX. The size of DOX-NP was ～140 nm, as determined by dynamic light scattering measurements and transmission electron microscopy. In vitro assays showed that DOX-NP exhibited higher cell proliferation inhibition and higher cell uptake in A549 cell lines compared with free DOX·HCl. Maximum tolerated dose (MTD) studies showed that DOX-NP demonstrated an excellent safety profile with a significantly higher MTD (15 mg DOX kg^?1) than that of free DOX·HCl (5 mg DOX kg^?1). The in vivo studies on the subcutaneous non-small cell lung cancer (A549) xenograft nude mice model confirmed that DOX-NP showed significant antitumor activity and reduced side effects, and then enhanced tumor accumulation as a result of the prolonged circulation in blood and the enhanced permeation and retention effect, compared with free DOX, indicating its great potential for cancer therapy.