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.     

Block Co-polymer Nanoparticles with Degradable Cross-Linked Core and Low-Molecular-Weight PEG Corona for Anti-tumour Drug Delivery

Biodegradable/bioeliminable, core-cross-linked, block co-polymer nanoparticles have been synthesized as a potential anti-tumour drug-delivery system. Methacrylate-modified poly(ethylene glycol)-b-poly(D,L-lactide) (PEG-b-PDLLA) composed of low-molecular-weight polymer blocks (<5 kg/mol) were synthesized by ring-opening polymerization and post-polymerization chemical modifications. Nanoparticles with a diameter of 110 ± 20 nm were formed from methacrylate-modified PEG₄₅-b-PDLLA₄₁ in a THF/water mixture (1:3). The particles were then core-cross-linked using a new, highly acid-labile ketal cross-linker. The cross-linked particles had a hydrodynamic diameter of 104 ± 20 nm (in THF/water, 1:3), as determined by DLS. The particles in THF exhibited a similar hydrodynamic diameter. Doxorubicin as a model anti-tumour drug was loaded into the nanoparticles (25–31 wt%). The particles released 50% of the loaded drug slowly approximately in 2 days at pH 5.5 and in 5 days at pH 7.4. The particles degraded to bioeliminable polymer fragments (<40 kg/mol) after the hydrolysis of the ketal cross-links at pH 5.5 in seven days, as determined by GPC. Doxorubicin-loaded cross-linked particles (9.3 μM doxorubicin/2.5 μM polymer) inhibited the viability of human neuroblastoma SH-EP cells, whilst the particles without drug at the same concentration were non-toxic, as determined by an Alamar Blue assay. Flow cytometry experiments revealed that the doxorubicin-loaded cross-linked particles were taken up by SH-EP cells in quantities comparable with free doxorubicin. Overall the results support the value of the cross-linked particles for further investigation as a carrier for anti-tumour drugs.     

Preparation of poly(ethylene glycol)-introduced cationized gelatin as a non-viral gene carrier

The objective of this study was to prepare cationized gelatins grafted with poly(ethylene glycol) (PEG) (PEG-cationized gelatin) and evaluate the in vivo efficiency as a non-viral gene carrier. Cationized gelatin was prepared by chemical introduction of ethylenediamine to the carboxyl groups of gelatin. PEG with one terminal of active ester group was coupled to the amino groups of cationized gelatin to prepare PEG-cationized gelatins. Electrophoretic experiments revealed that the PEG-cationized gelatin with low PEGylation degrees was complexed with a plasmid DNA of luciferase, in remarked contrast to that with high PEGylation degrees. When the plasmid DNA complexed with the cationized gelatin or PEG-cationized gelatin was mixed with deoxyribonuclease I (DNase I) in solution to evaluate the resistance to enzymatic degradation, stronger protection effect of the PEG-cationized gelatin was observed than that of the cationized gelatin. The complex of plasmid DNA and PEG-cationized gelatin had an apparent molecular size of about 300 nm and almost zero surface charge. These findings indicate that the PEG-cationized gelatin–plasmid DNA complex has a nano-order structure where the plasmid DNA is covered with PEG molecules. When the PEG-cationized gelatin–plasmid DNA complex was intramuscularly injected, the level of gene expression was significantly increased compared with the injection of plasmid DNA solution. It is concluded that the PEG-cationized gelatin was a promising non-viral gene carrier to enhance gene expression in vivo.     

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.     

Characterization of the Differentiation and Leptin Secretion Profile of Adult Stem Cells on Patterned Polylactide Films

Several issues need to be better understood before breast tissue engineering becomes a clinically viable option. One of the most important aspects is the interaction between cells and the microtopography of the implant surface. The aim of this study was to evaluate the efficacy of D1 cells, multipotent mouse bone marrow stromal precursors, in differentiating to adipocytes and to characterize their metabolic activity (lactic acid released and glucose consumed), leptin secretion and lipid production when cultured on patterned poly(L-lactide) (PLLA) films. It was determined that, by appropriate stimulation, the D1 cells displayed morphological characteristics of adipocytes and produced lipid. The results showed that a patterned surface did affect the rate of lipid production. Polynomial models were proposed to predict the amount of leptin secreted by the cells over a period of time.     

Preparation of PVA/PEI ultra-fine fibers and their composite membrane with PLA by electrospinning

Ultra-fine fibers of poly(vinyl alcohol)/polyethylenimine (PVA/PEI) were prepared by electrospinning of their blend solutions in water. Effects of PVA/PEI mass ratio and the polymer concentration on the fiber morphology were discussed by analysis of scanning electron micrographs. Results showed that uniform ultra-fine fibers could be obtained from an 8% PVA/PEI solution with 75:25 mass ratio. It was supposed that the introduction of PVA could promote electrospinning of PEI by weakening the intermolecular interaction and increasing solution viscosity. A composite membrane of PVA/PEI with poly(D,L-lactide) (PLA) was produced by co-electrospinning simultaneously from the aqueous 8% PVA/PEI (75:25) solution and a 20% PLA solution in N,N-dimethylformamide in two separated syringes. Fourier transform infrared spectroscopy, X-ray diffraction and X-ray photoelectron spectroscopy verified the existence of PVA/PEI and PLA in the fibrous membrane. We attempted to incorporate PEI with PLA as ultra-fine fibers to diminish the acidic inflammation caused by biodegradation of PLA. The fibrous composite membrane of PVA/PEI-PLA could provide better biocompatibility and would be used as drug-delivery carriers or tissue-engineering scaffolds.     

Biocompatibility of Poly(L-lactide) Films Modified with Poly(ethylene imine) and Polyelectrolyte Multilayers

Poly(L-lactide) (PLLA) films were modified with poly(ethylene imine) (PEI) either by adsorption or covalent binding to prepare the material for immobilization of polyelectrolyte multilayers (PEM). Two different PEI, low- and high-molecular-weight (LMW or HMW, respectively) PEI, were used. The PEI modification efficiency was monitored via surface amino group density, water contact angle and X-ray photoelectron spectroscopy (XPS) measurements. Covalent binding of HMW PEI by a two-step-activation method produced the highest amino group density and the lowest water contact angle. On the other hand, the adsorption method resulted in moderate amounts of immobilized PEI on the surface. Subsequently sulphated hyaluronan and chitosan were used to form PEM on PLLA that was covalently modified with HMW PEI. Regular formation of PEM was achieved, which was demonstrated by change of water contact angles and mass increase measured with quartz crystal microbalance. An osteoblast-like cell line, MG 63, was used to test the effects of modifications on biocompatibility. Contrarily to earlier reports showing that particularly HMW PEI had certain cytotoxicity, it was found that all modifications including PEM resulted in a better biocompatibility than plain PLLA indicated by a more spread phenotype of cells, their increased growth and metabolic activity.     

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 · hydrochloride (LB · 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 · 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 · 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.     

A vanadium/aspirin complex controlled release using a poly(β - propiolactone) film. Effects on osteosarcoma cells

A delivery system for vanadium was developed using poly(β-propiolactone) (PβPL)films. The release kinetics of a complex of vanadium (IV) with aspirin (VOAspi) was evaluated with films prepared from polymers of different molecular weights, as well as with variable drug load. A sustained release of vanadium over 7 days was achieved. The drug release kinetics depends on contributions from two factors: (a) diffusion of the drug; and (b) erosion of the PβPL film. The experimental data at an early stage of release were fitted with a diffusion model, which allowed determination of the diffusion coefficient of the drug. VOAspi does not show strong interaction with the polymer, as demonstrated by the low apparent partition coefficient (approximately 10-2). UMR106 osteosarcoma cells were used as a model to evaluate the anticarcinogenic effects of the VOAspi released from the PβPL film. VOAspi-PβPL film inhibited cell proliferation in a dose-response manner and induced formation of approximately half of the thiobarbituric acid reactive substances (TBARS), an index of lipid peroxidation, compared to that with free VOAspi in solution. The unloaded PβPL film did not generate cytotoxicity, as evaluated by cell growth and TBARS. Thus, the polymer-embedded VOAspi retained the antiproliferative effects showing lower cytotoxicity than the free drug. Results with VOAspi-PβPL films suggest that this delivery system may have promising biomedical and therapeutic applications.     

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.     

Apatite nano-crystalline surface modification of poly(lactide-co-glycolide) sintered microsphere scaffolds for bone tissue engineering: implications for protein adsorption

A number of bone tissue engineering approaches are aimed at (i) increasing the osteconductivity and osteoinductivity of matrices, and (ii) incorporating bioactive molecules within the scaffolds. In this study we examined the growth of a nano-crystalline mineral layer on poly(lactide-co-glycolide) (PLAGA) sintered microsphere scaffolds for tissue engineering. In addition, the influence of the mineral precipitate layer on protein adsorption on the scaffolds was studied. Scaffolds were mineralized by incubation in simulated body fluid (SBF). Scanning electron microscopy (SEM) analysis revealed that mineralized scaffolds possess a rough surface with a plate-like nanostructure covering the surface of microspheres. The results of protein adsorption and release studies showed that while the protein release pattern was similar for PLAGA and mineralized PLAGA scaffolds, precipitation of the mineral layer on PLAGA led to enhanced protein adsorption and slower protein release. Mineralization of tissue-engineered surfaces provides a method for both imparting bioactivity and controlling levels of protein adsorption and release.     

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 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.     

Synthesis,Characterization and Cytocompatibility of a Poly(diol-tricarballylate) Visible Light Photo-Cross-Linked Biodegradable Elastomer

The synthesis, characterization and in vitro cytocompatibility of a new family of photo-cross-linked amorphous poly(diol-tricarballylate) (PDT) biodegradable elastomeric polyesters are reported. The synthesis was based on the polycondensation reaction between tricarballylic acid and alkylene diols, followed by acrylation. The prepared and acrylated poly(diol-tricarballylate) (APDT) was characterized by means of FT-IR, ¹H-NMR, GPC and DSC. Liquid-to-solid photo-curing was carried out by exposing the APDT to visible light in the presence of camphorquinone as a photoinitiator. The thermal properties, mechanical characteristics, sol content, long-term in vitro degradation and cytocompatibility of the prepared PDT elastomers were also reported. The mechanical and degradation properties of this new photocurable elastomer can be precisely controlled by varying the density of acrylate moieties in the matrix of the polymer, and through changes in the pre-polymer chain length. The use of visible light cross-linking, possibility of solventless drug loading, controllable mechanical properties and cytocompatibility of these new elastomers make them excellent candidates for use in controlled implantable drug-delivery systems of protein drugs and other biomedical applications.     

Sustained Release of VEGF by Coaxial Electrospun Dextran/PLGA Fibrous Membranes in Vascular Tissue Engineering

VEGF-loaded core/shell fibrous membranes were prepared by coaxial electrospinning with dextran (DEX) as the core component and poly(lactide-co-glycolide) (PLGA) as the shell polymer, respectively. The electrospun DEX/PLGA fibers were observed by scanning electron microscopy, transmission electron microscopy and confocal microscopy to identify the core/shell fiber structure and the protein distribution. The results of tensile tests showed that the DEX/PLGA membranes possessed lower tensile strength and higher Young's modulus than PLGA one. The release profiles demonstrated that vascular endothelial growth factor (VEGF) release sustained for more than 28 days. Studies on cell viability and spreading demonstrated that the DEX(VEGF)/PLGA membranes positively promoted cell proliferation and cell–membrane interaction, which further testified that the processed VEGF remained bioactivities. Furthermore, the detections for the up-regulation of intercellular adhesion molecular-1 and the release of von Willebrand factor under pathological stimuli, which are related to inflammation process and thrombus formation, exhibited a normal immune response for the DEX(VEGF)/PLGA membrane. These data suggested that the VEGF-loaded fibers could be feasible in vascular tissue engineering.     

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.     

Measurements of Movement and Diffusion Coefficients of Single Cells on Polymeric Surface from Image Analysis

Time-lapse digital images were taken every 30 s of PC12 cells cultured on polystyrene dishes, collagen-coated dishes and poly(L-lysine)-coated dishes in high-serum medium, low-serum medium and neurite outgrowth factor (NGF)-containing medium to investigate their diffusion coefficients (i.e., self-diffusion coefficients), D and specific movement (i.e., specific frequent movement) using image analysis and Fast Fourier Transform (FFT) analysis. D for these cells was found to fluctuate as a function of time, D varying between 0 and 0.08 μm²/s. The trend observed upon examination of average D values was: D in high-serum medium ≥ D in low-serum medium ≥ D in NGF-containing medium. Image analysis and FFT analysis of single cells cultured on polymeric dishes in these three media did not have any specific frequency of cell movement between 0 and 0.0167 Hz. The high diffusion coefficient and high amplitude of power spectra of PC12 cells in high-serum medium might be attributed to the high energy necessary for their continual suppression of the mitogen-activated protein kinase (MAPK) cascade and for them to maintain their undifferentiated state.     

Poly(ether imide) membranes: studies on the effect of surface modification and protein pre-adsorption on endothelial cell adhesion,growth and function

Poly(ether imide) (PEI) membranes of which the surface was modified with carboxylic groups were tested in comparison to pure PEI and poly(ethylene terephtalate) (PET) for their ability to support attachment, growth and function of human umbilical vein endothelial cells (HUVEC) with respect to endothelization of the above materials. Flat sheet PEI membranes were modified by covalent binding of iminodiacetic acid (IDA) for different periods of time (1 to 30 min) to obtain surfaces with various content of carboxylic groups. In addition, fibronectin (FN) and fibrinogen (FNG) pre-adsorption on the various membranes were studied for their effect on HUVEC behaviour. The results show a decreased protein adsorption and HUVEC adhesion, growth and function in terms of prostacyclin production with an increase in carboxylic groups. Pre-adsorption of the membranes with FN or FNG promoted activity of HUVEC, which became superior to cells on PET. FN-coated membranes were found to be a better substrate for HUVEC adhesion and prostacyclin production, while on FNG-coated membranes cells grew better. Overall it can be concluded that PEI is a promising materials for endothelial cells immobilization as it is needed for improving the haemocompatibility of cardiovascular devices.     

Synthesis,Characterization and Biocompatibility of Novel Biodegradable Cross-linked Co-polymers Based on Poly(propylene oxide) Diglycidylether and Polyethylenimine

Novel biodegradable cross-linked co-polymers were prepared from poly(propylene glycol) diglycidylether (PPGDGE) and poly(ethylene imine) (PEI). PPGDGE and PEI were mixed at ambient temperature with varying PEI concentrations of 10, 15, 18.5, 25, 30, 40 and 50 wt%; the homogenous PPGDGE/PEI mixtures obtained were cured at elevated temperatures, resulting in formation of PPG–PEI cross-linked co-polymers via ring-opening reaction of PPGDGE with PEI. The physicochemical and biological properties of these co-polymers were dependent on the PEI content and the extent of curing reaction. The glass transition temperature of PPG–PEI cross-linked co-polymers varied in the range from ?14 to +42°C, while the co-polymers displayed composition-dependent mechanical behavior, from brittle to ductile with increasing PEI content from 18.5 wt% to 40 wt%. Chinese hamster ovary (CHO) cells were cultured on the PPG–PEI co-polymers; the MTT assay was used to measure cell viability and determine the cytotoxicity. The cell viability rate, relative to tissue-culture polystyrene (TCPS), increased from 49% to 125% with increasing PEI content from 18.5 wt% to 40 wt%. Although epoxy monomers usually exhibit cytotoxicity, the epoxy groups were exhausted via curing reaction in the fully cross-linked co-polymers. The PEI-cured PPG epoxy resin, i.e., PPG–PEI cross-linked co-polymers obtained in this study, showed excellent biocompatibility.     

Rat osteoblast functions on the o-carboxymethyl chitosan-modified poly(D,L-lactic acid) surface

In this study, the functions of rat osteoblasts on o-carboxymethyl chitosan-modified poly(D,L-lactic acid) (PDLLA) films were investigated in vitro. The surface characterization was measured by contact angle and electron spectroscopy for chemical analysis (ESCA). Cell adhesion and proliferation were used to assess cell behavior on the modified surface and control. The MTT assay was used to determined cell viability and alkaline phosphatase (ALP) activity was performed to evaluate differentiated cell function. Compared to the control films, cell adhesion of osteoblasts on o-carboxymethyl chitosan-modified PDLLA films was significantly higher (p < 0.05) after 6 and 8 h culture, and osteoblast proliferation was also significantly hlgher (p < 0.01) between 4 and 7 days. The MTT assay suggested cell viability of osteoblasts cultured on o-carboxymethyl chitosan modified PDLLA films was significantly greater (p < 0.05) than that seeded on control one, and the ALP activity of cells cultured on modified PDLLA films was significantly higher (p < 0.01) than that found on control. These results give the first evidence that o-carboxymethyl chitosan could be used to modify PDLLA surface for improving biocompatibility.