Membranes for biohybrid liver support systems--investigations on hepatocyte attachment,morphology and growth

The biological properties of four different membranes were studied regarding their possible application in biohybrid liver support systems. Two of them, one made of polyetherimide (PEI), and a second based on polyacrylonitrile-N-vinylpyrollidone co-polymer (P(AN-NVP)), were recently developed in our lab and studied for the first time. Together with pure polyacrylonitrile (PAN) membranes, the three preparations were characterised as ultra-filtration membranes. Their ability to support cell attachment, morphology, proliferation and function of human hepatoblastoma C3A cells was studied. The role of surface morphology for the interaction with hepatocytes was highlighted using a commercial, moderately wettable polyvinylidendifluoride (PVDF) membrane with micro-filtration properties. Comparative investigations showed strongest interaction of C3A cells with PAN membranes, as the focal adhesion contacts were more expressed and cell growth was also high. However, the functional activity in terms of albumin synthesis was reduced. Very similar results were obtained with the most hydrophobic PEI membrane. In contrast, the most hydrophilic membrane P(AN-NVP) was found to provoke stronger homotypic adhesion (E-cadherin expression) of C3A cells and less substratum attachment (focal adhesions), but enhanced albumin secretion. However, proliferation of C3A cells was lowered. Micro-porous PVDF membrane showed very good initial attachment, but the resulting cell material and cell-cell interaction were relatively poor developed. Among four membranes tested, PEI seems to be the most attractive membrane for biohybrid liver devices, as it provides good surface properties for hepatocytes interaction, but in addition it is highly thermostable, which would permit steam sterilisation. No simple relationship, however, between the wettability of the membranes and their ability to support hepatocyte adhesion and function was found in this study.     

Interaction of human skin fibroblasts with moderate wettable polyacrylonitrile--copolymer membranes

The development of a bioartificial skin is a step toward the treatment of patients with deep burns or nonhealing skin ulcers. One possible approach is based on growing dermal cells on membranes to obtain appropriate living cellular stroma (sheets) to cover the wound. New membrane-forming copolymers were synthesized, based on acrylonitrile (AN) copolymerization with hydrophilic N-vinylpyrrolidone (NVP) monomer, in different percentage ratios, such as 5, 20, and 30% w/w, and with two other relatively high polar comonomers--namely, sodium 2-methyl-2-propene-1-sulfonic acid (NaMAS) and aminoethylmethacrylate (AeMA). All these copolymers were characterized for their bulk composition and number average molecular weight, and used to prepare ultrafiltration membranes. Water contact angles and water uptake were estimated to characterize the wettability and scanning force microscopy to visualize the morphology of the resulting polymer surface. Cytotoxicity was estimated according to the international standard regulations, and the materials were found to be nontoxic. The interaction of the membranes with human skin fibroblasts was investigated considering that these cells are among the first to colonize membranes upon implantation or with prolonged external contact. The overall cell morphology, formation of focal adhesion contacts, and cell proliferation were estimated to characterize the cell material interactions. It was found that the pure polyacrylonitrile homopolymer (PAN) membrane provides excellent conditions for seeding with fibroblasts, comparable only to a copolymer containing AeMA. In contrast, the presence of NaMAS with acidic ionic groups decreased both the attachment and proliferation of fibroblasts. Low content of NVP in the copolymer, up to about 5%, still enabled good attachment and spreading of cells, as well as subsequent proliferation of fibroblasts, but higher ratios of 20 and 30% resulted in a significant decrease of these cellular activities.     

Thrombogenicity and long-term cytokine removal capability of a novel asymmetric triacetate membrane hemofilter

Hemofilters applied in continuous renal replacement therapies (CRRTs) for the treatment of acute kidney injury must meet high standards in biocompatibility and permeability for middle and large molecules over extended treatment times. In general, cellulose-based membranes exhibit good biocompatibility and low fouling, and thus appear to be beneficial for CRRT. In this in vitro study, we compared a novel asymmetric cellulose triacetate (ATA) membrane with three synthetic membranes [polysulfone (PS), polyethersulfone (PES), and polyethylenimine-treated acrylonitrile/sodium methallyl sulfonate copolymer (AN69 ST)] regarding thrombogenicity and cytokine removal. For thrombogenicity assessment, we analyzed the thrombin–antithrombin complex (TAT) generation in human whole blood during 5 h recirculation and filtration. Sieving coefficients of interleukin-6 (IL-6), IL-8, IL-10, and tumor necrosis factor-alpha (TNF-α) were determined using human plasma as test fluid. ATA and AN69 ST membrane permeability were determined also during long-term experiments (48.5 h). ATA exhibited the lowest TAT generation (6.3 µg/L at 5 h), while AN69 ST induced a pronounced concentration increase (152.1 µg/L) and filter clogging during 4 out of 5 experiments. ATA (IL-8: 1.053; IL-6: 1.079; IL-10: 0.898; TNF-α: 0.493) and PES (0.973; 0.846; 0.468; 0.303) had the highest sieving coefficients, while PS (0.697; 0.100; 0.014; 0.012) and AN69 ST (N/A; 0.717; 0; 0.063) exhibited lower permeability. Long-term experiments revealed stronger fouling of the AN69 ST compared to the ATA membrane. We observed the highest permeability for the tested cytokines, the lowest thrombogenicity, and the lowest fouling with the ATA membrane. In CRRT, these factors may lead to increased therapy efficacy and lower incidence of coagulation-associated events.     

Feldionen- und Elektronenstoß-Massenspektrometrie von Polymeren und Copolymeren, 3. Copolymere des α-Methylstyrols mit Methylmethacrylat und Acrylnitril

Pyro-field ion mass spectrometry was used to study the primary fragments as well as the depolymerization behaviour of some α-methylstyrene (α-MS) copolymers. α-MS-methyl methacrylate (MMA) copolymers, when pyrolyzed, decompose mainly into the two monomers. The zip-depolymerization usually proceeds through the whole chain, disregarding the hetero-links between different monomeric units. Hetero-fragments are formed only with a small probability; still lower is the concentration of homo-dimers in the pyrolysate. Primary fragments of poly(acrylonitrile) (PAN) are acrylonitrile (AN), (AN)₂, (AN)₃, HCN, C₃H₅CN, C₄H₇CN, C₅H₉CN and higher nitriles. During pyrolysis of α-MS-AN copolymers, α-MS sequences zipdepolymerize with formation of the monomer. This retropolymerization stops at heterolinks, and fragments of the type α-MS-AN or α-MS-(AN)₂ are formed. Long AN sequences in the copolymer cause, as PAN itself, formation of AN oligomeres as well as higher alkene cyanides. Isolated AN units lead to the formation of monomeric AN.     

BSA-Modified Polyethersulfone Membrane: Preparation,Characterization and Biocompatibility

A polyethersulfone (PES) membrane was modified by blending with a co-polymer of acrylic acid (AA) and N-vinyl pyrrolidone (VP), followed by immobilization of bovine serum albumin (BSA) onto the surface. The scanning electron microscopy results showed that PES had good miscibility with the co-polymer. X-ray photoelectron spectroscopy confirmed the existence of P(VP-AA) co-polymer on the surface of the blended membrane and the existence of BSA after the immobilization process. The amount of BSA immobilized on the surface of the membranes was determined. It was found that the protein adsorption amounts from BSA, human plasma fibrinogen and diluted human plasma solutions decreased significantly after modification. According to the circular dichroism results, the proteins kept more α-helix conformation in the modified membranes than in the pure PES membrane. The number of the adhered platelets was reduced, and the morphology change for the adherent platelets was also suppressed by the modification with BSA. The SEM morphological observation of the cells and the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay demonstrated that the BSA-modified PES membrane surface promoted endothelial cell adhesion and proliferation.     

Multifunctional triblock co-polymer mP3/4HB-b-PEG-b-lPEI for efficient intracellular siRNA delivery and gene silencing

A non-viral siRNA carrier composed of mono-methoxy-poly (3-hydroxybutyrate-co-4-hydroxybutyrate)-block-polyethylene glycol-block-linear polyethyleneimine (mP3/4HB-b-PEG-b-lPEI) was synthesized using 1800 Da linear polyethyleneimine and evaluated for siRNA delivery. Our study demonstrated that siRNA could be efficiently combined with mP3/4HB-b-PEG-b-lPEI (mAG) co-polymer and was protected from nuclease degradation. The combined siRNA were released from the complexes easily under heparin competition. The particle size of the mAG/siRNA complexes was 158 nm, with a ζ-potential of around 28 mV. Atomic force microscopy images displayed spherical and homogeneously distributed complexes. The mAG block co-polymer displayed low cytotoxicity and efficient cellular uptake of Cy3–siRNA in A549 cells by flow cytometry and confocal microscopy. In vitro transfection efficiency of the block co-polymer was assessed using siRNA against luciferase in cultured A549-Luc, HeLa-Luc, HLF-Luc, A375-Luc and MCF-7-Luc cells. A higher transfection efficiency and lower cytotoxicity was obtained by mAG block co-polymer in five cell lines. Furthermore, a remarkable improvement in luciferase gene silencing efficiency of the mAG complex (up to 90–95%) over that of Lipofectamine? 2000 (70–82%) was observed in HLF-Luc and A375-Luc cells. Additionally, a mAG/p65-siRNA complex also showed a better capability than Lipofectamine? 2000/p65-siRNA complex to drastically reduce the p65 mRNA level down to 10–16% in HeLa, U251 and HUVEC cells at an N/P ratio of 70.     

Transmembranous transport and adsorption of beta-2-microglobulin during hemodialysis using polysulfone, polyacrylonitrile, polymethylmethacrylate and cuprammonium rayon membranes

Beta-2-microglobulin (b2M) was identified as a causative agent of amyloidosis associated with long-term hemodialysis (HD). Therefore, we examined handling of b2M during a 4-hour hemodialysis session. We compared b2M adsoprtion and diffusive/convective elimination between high-flux membranes such as polysulfone (PS; F 60, Fresenius), polyacrylonitrile (AN 69; Filtral, Hospal) and polyacrylonitrile (PAN, PAN 12CX2, Asahi) and less permeable membranes such as cuprammonium rayon (CR; AM 160 H, Asahi) and polymethylmethacrylate (PMMA; BK-1.6 U, Toray). To calculate total elimination, arterio-venous differences of b2M were measured at 0, 5, 20, 60 and 240 minutes; dialysate concentration was analyzed to evaluate diffusive/convective transport. Differences between recovery in dialysate and total removal were regarded as amount removed by adsorption. Total elimination per 4-hour hemodialysis session and per m2 membrane surface was 154.7 +/- 12.3 mg for the PS, 137.8 +/- 28.4 mg for the AN 69, 179.8 +/- 47.5 mg for the PAN, 130.8 +/- 11.8 mg for the PMMA and 14.4 +/- 16.0 mg for the CR membrane. Diffusive/convective transport was 128.0 +/- 18.1 mg for PS, 54.7 +/- 8.1 mg for AN 69 and 106.5 +/- 20.8 mg for PAN and insignificant for PMMA and CR. Adsorption was 26.7 +/- 4.3 mg for PS, 83.1 +/- 29.0 mg for AN 69 and 59.8 +/- 17.2 mg for PAN. Besides transmembranous transport sorption is an important mode of elimination. Weekly endogenous generation rate is about twice as high as b2M elimination.     

Morphological studies on the culture of kidney epithelial cells in a fiber-in-fiber bioreactor design with hollow fiber membranes

A hollow fiber-in-fiber-based bioreactor system was tested for the applicability to host kidney epithelial cells as a model system for a bioartificial kidney. Hollow fibers were prepared from polyacrylonitrile (PAN), polysulfone-polyvinylpyrollidinone (PVP) blend (PSU) and poly(acrylonitrile-N-vinylpyrollidinone) copolymer P(AN-NVP). Hollow fibers with smaller and larger diameters were prepared so that the smaller fitted into the larger, with a distance of 50-100 microm in between. The following material combinations as outer and inner fiber were applied: PAN-PAN; PSU-PSU, PSU-P(AN-NVP). Madin-Darby kidney epithelial cells (MDCK) were seeded in the interfiber space and cultured for a period up to 14 days. Light, scanning, and transmission electron microscopy were used to follow the adhesion and growth of cells, and to characterize their morphology. As a result, we found that MDCK cells were able to grow in the interfiber space in mono- and multilayers without signs of systemic degeneration. Comparison of the different materials showed that PAN and P(AN-NVP) provided the best growth conditions, indicated by a tight attachment of cells on hollow fiber membrane, and subsequent proliferation and development of structural elements of normal epithelia, such as tight junctions and microvilli. In conclusion, the fiber-in-fiber design seems to be an interesting system for the construction of a bioartificial kidney.     

Hybrid hydroxyapatite nanoparticles-loaded PCL/GE blend fibers for bone tissue engineering

In order to augment bone formation, a new biodegradable scaffold system was fabricated using different ratios of hydroxyapatite (HAp) blended with synthetic polymer polycaprolactone (PCL) and natural polymer gelatin (GE) followed by electrospinning method. Three different concentrations of HAp were used in PCL/GE to obtain a blend of 10, 30, and 50% (w/v) HAp–PCL/GE. These HAp-loaded PCL/GE blends were then compared with PCL/GE blends by different mechanical and biological in vitro and in vivo studies to understand the applicability of the system. Scanning electron microscopy, X-ray diffraction analysis, and tensile strength measurement were done to obtain physical properties. Fifty Percent HAp–PCL/GE blends possessed the highest mechanical strength. In vitro cytotoxicity and proliferation of osteoblast cells on the PCL/GE and HAp–PCL/GE scaffolds were examined and shown that addition of HAp in PCL/GE was beneficial by increasing cell viability (>85%) proliferation and cell-surface attachment. Expression of collagen and osteopontin was also found higher in 50% HAp–PCL/GE blends than the others. On the other hand, in vivo bone formation was examined using rat models and increased bone formation was observed in 50% HAp–PCL/GE blends within 6?weeks. Based on the combined results of this study, HAp–PCL/GE membranes were found to hold great promise for use in tissue engineering applications, especially in bone tissue engineering.     

Anaphylatoxins C3a and C5a adsorption on acrylonitrile membrane of hollow-fiber and plate dialyzer--an in vivo study

We studied the adsorption of anaphylatoxins C3a and C5a on acrylonitrile (AN69) hollow-fiber (AN69HF) and plate (AN69P) dialyzers in 8 patients during 4-hour hemodialyses (HD). Blood passed first through a cuprophan dialyzer and then through AN69 dialyzers that were not in contact with dialysis fluid. Plasma C3a and C5a were measured in samples taken from the afferent and efferent blood lines of the acrylonitrile dialyzers at 15, 60 and 240 min. Plasma C3a concentrations decreased significantly in blood that had passed through AN69 dialyzers. This decrease, indicating membrane adsorption, was maximal (by 65% in AN69HF and by 59% in AN69P) at 15 min and minimal (by 53% in AN69HF and by 18% in AN69P) at 240 min. The decrease in plasma C5a concentrations was smaller and significant throughout HD only with AN69HF. The amount of C3a adsorbed was at least 45,000 micrograms in AN69HF and 18,000 micrograms in AN69P. These findings demonstrate that acrylonitrile dialyzers adsorb more C3a and C5a than they produce. This membrane adsorption may explain why the increase of plasma C3a and C5a is inhibited during HD.     

Minimodule dialyser for quantitative ex vivo evaluation of membrane haemocompatibility in humans: comparison of acrylonitrile copolymer, cuprophan and polysulphone hollow fibres.

Minimodule hollow fibre dialysers, representing clinical dialysis modules on a scale of 1/25, enable quantitative evaluation of the haemocompatibility of hollow fibre membranes in an ex vivo flow system in humans. On line heparinization, adjusted for donor sensitivity, is maintained at a minimal level (approximately 0.14 units/ml). Blood samples collected at the minimodule exit over 30 min are analysed for heparin (anti-Xa activity), activated partial thromboplastin time, fibrinopeptide A, platelet count and beta-thromboglobulin, complement fragment C3a, leucocyte count and polymorphonuclear neutrophil elastase. Initial experiments were performed using well-characterized reference materials: acrylonitrile copolymer (AN 69 HF), polysulphone and cuprophan (CUP). Activation of coagulation and platelets was low for AN 69 HF, intermediate for CUP and greatest for polysulphone, while complement activation was negligible in the presence of AN 69 HF, moderate for polysulphone and most important for CUP. Future applications will be directed towards haemocompatibility screening of prototype membranes with the aim of developing clinical dialysers with improved biocompatibility.     

Physically and Chemically Cross‐Linked Poly{[(maleic anhydride)‐alt‐styrene]‐co‐(2‐acrylamido‐2‐methyl‐1‐propanesulfonic acid)}/Poly(ethylene glycol) Proton‐Exchange Membranes

Novel proton exchange membranes were solvent‐cast from DMF solutions of the terpolymers poly[(MA‐alt‐S)‐co‐AMPS], containing hydrophobic phenyl and reactive hydrophilic carboxylic and organo‐sulfonic acid fragments with different compositions, and PEGs with different molecular weights and amounts. These membranes were formed as a result of physical (via H‐bonding) and chemical (via PEG) cross‐linking. The structures of membranes were confirmed by FT‐IR and ¹H‐ and ¹³C NMR spectroscopy. Mechanical and thermal properties, swellability, and proton conductivity of these membranes were significantly affected both by the chemical composition of the terpolymers (mainly the AMPS content) and also the cross‐linker (PEG) molecular weight and content in the final form of the membranes. It was concluded that the membranes prepared by using the terpolymer with an AMPS content of 36.84 mol‐% and PEG with a molecular weight of 1 450 and with an initial PEG content of 30 wt.‐% are the most suitable ones for fuel cell applications.

     

Studies on the copolymerization of acrylonitrile with butyl acrylate,vinylidene dichloride,and styrene

Copolymer composition studies are reported, for different mole fractions of acrylonitrile (AN), butyl acrylate (BA), vinylidene dichloride (CV), and styrene (S) in the initial monomer mixture. A partial azeotropy of acrylonitrile was found.     

Isothermal Crystallization Kinetics of Poly(ε‐caprolactone) with Tetramethyl Polycarbonate and Poly(styrene‐co‐acrylonitrile) Blends Using Broadband Dielectric Spectroscopy

Summary: Phase behavior and isothermal crystallization kinetics of poly(ε‐caprolactone) (PCL) blends with tetramethyl polycarbonate (TMPC) and poly(styrene‐co‐acrylonitrile) with 27.5 wt.‐% acrylonitrile content have been investigated using broadband dielectric spectroscopy and differential scanning calorimeter. An LCST‐type phase diagram has been observed for PCL/SAN blend while all the different blend compositions of PCL/TMPC were optically clear without any phase separation structure even at high temperatures up to 300 °C. The composition dependence of T_gs for both blends has been well described by the Gordon‐Taylor equation. The phase diagram of PCL/SAN was theoretically calculated using the Flory‐Huggins equation considering that the interaction parameter is temperature and composition dependent. The equilibrium melting point of PCL depressed in the blend and the magnitude of the depression was found to be composition dependent. The interaction parameters of PCL with TMPC and SAN could not be calculated from the melting point depression based on Nishi‐Wang approach. The isothermal crystallization kinetics of PCL and in different blends was also investigated as a function of crystallization temperature using broadband dielectric spectroscopy. For pure PCL the rate of crystallization was found to be crystallization temperature (T_c) dependent, i.e., the higher the T_c, the lower the crystallization rate. The crystallization kinetics of PCL/TMPC blend was much slower than that of PCL/SAN at a constant crystallization temperature. This behavior was attributed to the fact that PCL is highly interacted with TMPC than SAN and consequently the stronger the interaction the higher the depression in the crystallization kinetics. It was also attributed to the different values of T_g of TMPC (191 °C) and SAN (100 °C); therefore, the tendency for crystallization decreases upon increasing the T_g of the amorphous component in the blend. The analysis of the isothermal crystallization kinetics was carried out using the theoretical approach of Avrami. The value of Avrami exponent was almost constant in the pure state and in the blends indicating that blending simply retarded the crystallization rate without affecting the crystallization mechanism.

Dielectric constant, ε′, of pure PCL, blends of PCL/TMPC = 80/20 and PCL/SAN = 80/20 as a function of crystallization time at 47 °C and 1 kHz.     

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.     

Hemocompatibility of polyacrylonitrile dialysis membrane immobilized with chitosan and heparin conjugate

Chitosan (CS)/heparin (HEP) polyelectrolyte complex (PEC) was covalently immobilized onto the surface of polyacrylonitrile (PAN) membrane. The effect of surface modification on the protein adsorption and platelet adhesion, metabolites permeation and anticoagulation activity of the resulting membrane was investigated. Surface characterization such as water contact angle, and X-ray photoelectron spectroscope were performed. The immobilization of PEC caused the water contact angle to reduce, thereby indicating the increase in the hydrophilicity. Protein adsorption, platelet adhesion, and thrombus formation were all reduced by the immobilization of HEP. Anticoagulant activity was evaluated with activated partial thrombin time (APTT), prothrombin time (PT), fibrinogen time, and thrombin time (TT). The results revealed that PEC-immobilizing membrane can improve antithrombogenicity of PAN membrane. In addition, the PEC-immobilized membranes can suppress the proliferation of Pseudomonas aeruginosa. In vitro cytotoxicity test showed leachable substance released was below cytotoxic level. The pure water permeability results show little variation due to PEC-immobilization. Thus PEC-immobilization can endow the PAN membrane hemocompatibility and antibacterial activity while retaining the original permeability.     

Copolymerization of acrylonitrile with ethyl methacrylate, 2

The copolymerization of acrylonitrile (AN) with ethyl methacrylate (EMA) was investigated in different solvents. The highest rate was observed in dimethylformamide and the lowest in benzene. The activation energy was determined in the temperature range of 60–80°C and was found to be 82 kJ/mol. The effects of temperature and monomer concentration on the intrinsic viscosity of the copolymers were investigated.     

Self‐assembled UCST‐Type Micelles as Potential Drug Carriers for Cancer Therapeutics

A methoxy‐poly(ethylene glycol)‐block‐poly(acrylamide‐co‐acrylonitrile) (mPEG‐b‐P(AAm‐co‐AN)) amphiphilic copolymer exhibiting upper critical solution temperature (UCST) behavior is synthesized, and micelles from this copolymer are fabricated. It is found that the thermal responses of these micelles are tunable through balancing the hydrophobic/hydrophilic blocks in the copolymer. The size of the doxorubicin (DOX)‐loaded micelles is dependent on the hydrophobic interaction as well as hydrogen bonding between polymer and drug molecules. As a proof of concept, the drug release behavior is studied in vitro, and the cumulative release of DOX increases at temperature above the UCST of blank micelles. 3‐(4,5‐dimethyl‐thiazol‐2‐yl)‐2,5‐diphenyl tetrazolium bromide (MTT) assays indicate that these polymers are non‐toxic towards human hepatic carcinoma cells (Bel 7402 cells) as well as human embryonic hepatocytes (L02 cells). DOX‐loaded micelles could effectively enter Bel 7402 cells in 2 h, and display much lower half inhibitory concentration compared with free DOX. These micelles may be exploited as a promising drug carrier for cancer therapeutics.

     

Porous copolymers of trimethylolpropane triacrylate and acrylonitrile

The homopolymer of trimethylolpropane triacrylate (TMPA) was synthesized by suspension polymerization using a thermodynamically good solvent (toluene) as a porogen (vol. ratio 1:1). This polymer possesses a high specific surface area (437 m² · g^?1) and porosity (50%). It was found by sorption of benzene and cyclohexane that the majority of the pores are in the range of 1–5 nm. Sorption of phenol, a standard sorbate, from a water solution is proportional to the specific surface area of the mesopores. This sorption is higher than that of nonspecific sorbents (like styrene/divinylbenzene copolymers) and smaller than the sorption of specific ones (like acrylonitrile/divinylbenzene copolymers). Two sets of reactive copolymers were obtained. In the first set, TMPA (20–40 vol.-%) was polymerized with acrylonitrile (AN) in the presence of a constant amount of toluene as an inert diluent (vol. ratio 1:1). In the second set of copolymers the cross-linking degree was kept constant (30 or 40 vol.-% of TMPA) but various solvent mixtures were used, namely: toluene/tetradecane or hexadecane, toluene/2-ethylhexanol, cyclohexanol/tetradecane or hexadecane, cyclohexanol/2-ethylhexanol (vol. ratio 9:1). It was found that all the copolymers are macroporous (volume of pores approx. 1,0 cm³ · g^?1) but those obtained in the presence of good solvents display a higher specific surface area and hence better sorption properties towards phenol than those obtained in the presence of poor solvents. The swelling of AN/TMPA copolymers is smaller than that of the TMPA homopolymer due to the presence of additional physical crosslinks formed by the strongly polar nitrile groups.     

Synthesis of Poly(ethylene glycol)-g-Chitosan-g-Poly(ethylene imine) Co-polymer and In Vitro Study of Its Suitability as a Gene-Delivery Vector

There are two main hindrances for the application of chitosan (CS) as a gene-delivery vector: poor water solubility and low transfection efficiency. To address these problems, we modified chitosan with poly(ethylene glycol) (PEG) and poly(ethylene imine) (PEI). As previously described, PEG was grafted onto CS by a reaction between the activated PEG and CS amine. This increased the solubility of CS in neutral or basic solution. Then, monomers of PEI (i.e., aziridine) were polymerized on the CS chain of the PEG(40k)-CS(50k) co-polymer obtained in the previous step. The resulting PEG-CS-PEI (PCP) co-polymer was characterized by ¹H-NMR, ¹³C-NMR and gel-permeation chromatography (GPC). It was found in the preliminary experiments that, amongst the series of PEG-CS-PEI co-polymers with various PEI molecular weights, PEG(40k)-CS(50k)-PEI(20k) was the most efficient one; therefore, it was chosen for the study. The PCP co-polymer showed lower cytotoxicity compared to PEI (25k) by MTT assay. Particle size and zeta potential of PCP/DNA complexes were measured by dynamic light scattering (DLS) and were shown to be predominantly affected by N/P ratios. PCP/DNA complexes at N/P ratio 20 were observed under a transmission electron microscope (TEM) as spherical particles with a mean diameter of about 50 nm. Plasmid DNA could be efficiently protected by PCP co-polymer from DNase I. The in vitro gene-transfection efficiency of PCP/pEGFP was higher than that of PEI(25k)/pEGFP and was markedly facilitated by serum.