A novel Y-shaped photoiniferter used for the construction of polydimethylsiloxane surfaces with antibacterial and antifouling properties.

The simultaneous introduction of two new functionalities into the same polymeric substrate under mild reaction conditions is an interesting and important topic. Herein, dual-functional polydimethylsiloxane (PDMS) surfaces with antibacterial and antifouling properties were conveniently developed via a novel Y-shaped asymmetric dual-functional photoiniferter (Y-iniferter). The Y-iniferter was initially immobilized onto the PDMS surface by radical coupling under visible light irradiation. Afterwards, poly(2-hydroxyethyl methacrylate) (PHEMA) brushes and antibacterial ionic liquid (IL) fragments were simultaneously immobilized on the Y-iniferter-modified PDMS surfaces by combining the sulfur(VI)-fluoride exchange (SuFEx) click reaction and UV-photoinitiated polymerization. Experiments using E. coli as a model bacterium demonstrated that the modified PDMS surfaces had both the expected antibacterial properties of the IL fragments and the excellent antifouling properties of PHEMA brushes. Furthermore, the cytotoxicity of the modified PDMS surfaces to L929 cells was examined in vitro with a CCK-8 assay, which showed that the modified surfaces maintained excellent cytocompatibility. Briefly, this strategy of constructing an antibacterial and antifouling PDMS surface has the advantages of simplicity and convenience and might inspire the construction of diverse dual-functional surfaces by utilizing PDMS more effectively.

Polymer-Coated NH2-UiO-66 for the Codelivery of DOX/pCRISPR.

Herein, the NH2-UiO-66 metal organic framework (MOF) has been green synthesized with the assistance of high gravity to provide a suitable and safe platform for drug loading. The NH2-UiO-66 MOF was characterized using a field-emission scanning electron microscope, transmission electron microscope (TEM), X-ray diffraction, and zeta potential analysis. Doxorubicin was then encapsulated physically on the porosity of the green MOF. Two different stimulus polymers, p(HEMA) and p(NIPAM), were used as the coating agents of the MOFs. Doxorubicin was loaded onto the polymer-coated MOFs as well, and a drug payload of more than 51% was obtained, which is a record by itself. In the next step, pCRISPR was successfully tagged on the surface of the modified MOFs, and the performance of the final nanosystems were evaluated by the GFP expression. In addition, successful loadings and internalizations of doxorubicin were investigated via confocal laser scanning microscopy. Cellular images from the HeLa cell line for the UiO-66@DOX@pCRISPR and GMA-UiO-66@DOX@pCRISPR do not show any promising and successful gene transfections, with a maximum EGFP of 1.6%; however, the results for the p(HEMA)-GMA-UiO-66@DOX@pCRISPR show up to 4.3% transfection efficiency. Also, the results for the p(NIPAM)-GMA-UiO-66@DOX@pCRISPR showed up to 6.4% transfection efficiency, which is the first and superior report of a MOF-based nanocarrier for the delivery of pCRISPR. Furthermore, the MTT assay does not shown any critical cytotoxicity, which is a promising result for further biomedical applications. At the end of the study, the morphologies of all of the nanomaterials were screened after drug and gene delivery procedures and showed partial degradation of the nanomaterial. However, the cubic structure of the MOFs has been shown in TEM, and this is further proof of the stability of these green MOFs for biomedical applications.

Toxicity and photosensitizing assessment of gelatin methacryloyl-based hydrogels photoinitiated with lithium phenyl-2,4,6-trimethylbenzoylphosphinate in human primary renal proximal tubule epithelial cells.

Gelatin methacryloyl (GelMA) and lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP) photoinitiator are commonly used in combination to produce a photosensitive polymer but there are concerns that must be addressed: the presence of unreacted monomer is well known to be cytotoxic, and lithium salts are known to cause acute kidney injury. In this study, acellular 10% GelMA hydrogels cross-linked with different LAP concentrations and cross-linking illumination times were evaluated for their cytotoxicity, photosensitizing potential, and elastic moduli. Alamar Blue and CyQuant Direct Cell viability assays were performed on human primary renal proximal tubule epithelial cells (hRPTECs) exposed to extracts of each formulation. UV exposure during cross-linking was not found to affect extract cytotoxicity in either assay. LAP concentration did not affect extract cytotoxicity as determined by the Alamar Blue assay but reduced hRPTEC viability in the CyQuant Direct cell assay. Photocatalytic activity of formulation extracts toward NADH oxidation was used as a screening method for photosensitizing potential; longer UV exposure durations yielded extracts with less photocatalytic activity. Finally, elastic moduli determined using nanoindentation was found to plateau to approximately 20-25 kPa after exposure to 342 mJ/cm2 at 2.87 mW of UV-A exposure regardless of LAP concentration. LAP at concentrations commonly used in bioprinting (<0.5% w/w) was not found to be cytotoxic although the differences in cytotoxicity evaluation determined from the two viability assays imply cell membrane damage and should be investigated further. Complete cross-linking of all formulations decreased photocatalytic activity while maintaining predictable final elastic moduli.

Cationic polymers for non-viral gene delivery to human T cells.

The clinical success of chimeric antigen receptor (CAR) T cell immunotherapy in treating multiple blood cancers has created a need for efficient methods of ex vivo gene delivery to primary human T cells for cell engineering. Here, we synthesize and evaluate a panel of cationic polymers for gene delivery to both cultured and primary human T cells. We show that a subset of comb- and sunflower-shaped pHEMA-g-pDMAEMA polymers can mediate transfection with efficiencies up to 50% in the Jurkat human T cell line with minimal concomitant toxicity (>90% viability). We then optimize primary human T cell transfection conditions including activation time, cell density, DNA dose, culture media, and cytokine treatment. We demonstrate transfection of both CD4+ and CD8+ primary human T cells with messenger RNA and plasmid DNA at efficiencies up to 25 and 18%, respectively, with similarly high viability.

Theranostic Gold Nanomicelles made from Biocompatible Comb-like Polymers for Thermochemotherapy and Multifunctional Imaging with Rapid Clearance.

A new generation of photothermal theranostic agents based on assembling 6 nm gold nanoparticles (AuNPs) is developed by using a novel comb-like amphipathic polymer as the template. The small AuNPs are assembled into DOX@gold nanomicelles, which show strong absorbance in the near-infrared region, for multimodal bioimaging and highly effective in vivo chemotherapy and photothermal therapy.

Pre-clinical evaluation of a new coral-based bone scaffold.

Coral is used worldwide for bone reconstruction. The favorable characteristics that make this material desirable for implantation are (i) osteoinduction, (ii) and osteoconduction. These proprieties have been demonstrated by in vivo studies with animal models and clinical trials over a twenty-year period. Also poly(2-hydroxyethylmethacrylate) [poly(HEMA)] is a widely used biomaterial. By using coral and poly(HEMA), a scaffold for bone reconstruction application has been recently synthesized. Cytological, histological and genetic analyses were performed to characterize this new alloplastic material. Four samples were analyzed: (a) white coral (WC), (b) red coral (RC), (c) WC plus polymer (WCP) and (d) RC plus polymer (RCP). Quantification of mitochondrial dehydrogenase activity by MTT assay was performed as indirect detector of cytotoxicity. In vivo effects were revealed by implanting corals and coral-based polymers in rabbit tibia. Samples were collected after 4 weeks and subjected to histological analysis. To evaluate the genetic response of cells to corals and coral-derived polymers an osteoblastlike cell line (i.e. MG63) was cultured in wells containing (a) medium, (b) medium plus corals and (c) medium plus two types of scaffolds (RCP or WCP). RNAs extracted from cells were retro-transcribed and hybridized on DNA 19.2K microarrays. No cytotoxicity was detected in corals and coral-based biopolymers. No inflammation or adverse effect was revealed by histological examination. By microarray analysis 154 clones were differentially expressed between RC and WC (81 up and 73 down regulated) whereas only 15 clones were repressed by the polymer. Histological evaluation not only confirmed that coral is a biocompatible material, but also that the polymer has no adverse effect. Microarray results were in agreement with cytological and histological analyses and provided further data regarding the genetic effects of RC, WC and the new polymer.

Repair of the injured spinal cord by implantation of a synthetic degradable block copolymer in rat.

The present study is designed to assess the properties of a new degradable PLA-b-PHEMA block copolymer hydrogel and its therapeutic effectiveness after implantation following a thoracic spinal cord hemisection on rats. Degradable characteristics and porous aspect of the scaffold are respectively analyzed by the evaluation of its mass loss and by electron microscopy. The biomaterial toxicity is measured through in vitro tests based on motoneuron survival and neurite growth on copolymer substrate. Functional measurements are assessed by the Basso, Beattie and Bresnahan (BBB) and the Dynamic Weight Bearing (DWB) tests during 8 weeks post-surgery. Histological analyses are achieved to evaluate the presence of blood vessels and axons, the density of the glial scar, the inflammatory reaction and the myelination at the lesion site and around it. The results indicate that the synthetic PLA-b-PHEMA block copolymer is a non-toxic and degradable biomaterial that provides support for regenerating axons and seems to limit scar tissue formation. Additionally, the implantation of the porous PLA-b-PHEMA scaffold enhances locomotor improvement. The observed functional recovery highlights the potential benefits of plain tissue engineering material, which can further be optimized by bioactive molecule functionalization or transplanted cell encapsulation.

Preparation of novel biodegradable pHEMA hydrogel for a tissue engineering scaffold by microwave-assisted polymerization.

OBJECTIVE: To prepare a novel biodegradable poly(2-hydroxyethylmethacrilate) (pHEMA) hydrogel as tissue engineering scaffold. METHODS: The pHEMA hydrogel was synthesized by microwave-assisted polymerization using 2-hydroxyethyl methacrylate (HEMA) as the raw material, potassium persulfate as the initiator, and PCLX as the cross-linking additive. The hydrogels was characterized with FTIR and NMR spectroscopy. The physical and chemical properties of the prepared hydrogel were evaluated, and its degradation performance was tested. The cytotoxicity of the optimum composite hydrogel was measured by an MTT assay to confirm the feasibility of its use in tissue engineering. RESULTS: The optimum conditions under which the hydrogel was prepared by microwave-assisted polymerization are as follows: 1.5 g cross-linking additive, 0.3 g initiator, reaction temperature of 80 °C, and microwave power of 800 W. Degradation studies showed good degradation profiles with 75% in 17 days. Additionally, the hydrogels did not elicit any cytotoxic response in in vitro cytotoxic assays. CONCLUSION: A biodegradable pHEMA hydrogel was successfully prepared by microwave-assisted polymerization, as confirmed from FTIR and NMR results. The hydrogel shows promising applications in tissue engineering, and its healing ability and biocompatibility will be evaluated in detail in the future.

In vitro and in vivo demonstration of physically and chemically in situ gelling NIPAAm-based copolymer system.

Poly(NIPAAm-co-hydroxyethylmethacarylate (HEMA)) acrylate and poly(NIPAAm-co-cysteine ethyl ester (CysOEt)) were synthesized and characterized by GPC(gel permeation chromatography), rheology, NMR (nuclear magnetic resonance), and Ellman's method. Upon mixing of these materials in aqueous solution, they formed gels immediately at body temperature owing to temperature-driven physical gelling, and gradually cured by chemical cross-linking through Michael-type addition reactions between thiols and acrylates. The rate of nucleophilic attack in the Michael-type addition reaction was shown to be highly dependent on the mole ratio of thiol to acrylate at neutral pH. Physical and chemical gelation improved the mechanical properties of the materials compared to purely physical gels. In vitro and in vivo results revealed that chemical and physical gels formed stiffer less viscoelastic materials compared to purely physical gels. Physical and chemical gel systems using thermosensitive polymer with acrylates and thermosensitive polymer with thiols showed minimum toxicity.

The morphological and biomechanical changes of keratocytes cultured on modified p (HEMA-MMA) hydrogel studied by AFM.

The poor integration with host cornea tissue and the low mechanical properties of pHEMA hydrogel for artificial cornea remains a difficult problem to solve. A modified pHEMA hydrogel, MMA copolymerized and type-I collagen and bFGF immobilized, was previously prepared in an attempt to solve the problems. In this study, the cytotoxicity of Col/bFGF-p (HEMA-MMA) and p (HEMA-MMA) was studied by cell adhesion assay and atomic force microscopy (AFM). The results of cell adhesion assay show that the attachment of keratocytes on the modified membrane is much higher than that of the unmodified membrane. This indicates that the material after modification have better cell-material interaction. The AFM images reveal that the morphology of keratocytes cultured on different substrate is obviously different. The cell cultured on modified membrane presented a completely elongated and spindle-shape morphology. The force-distance indicates that the biomechanical of keratocytes changes significantly after culturing on different substrates. The adhesion force (2328+/-523 pN) and Young's modulus (0.51+/-0.125 kPa) of the cell cultured on modified membrane are much higher, and the stiffness (0.08+/-0.022 mN/m) is lower than those of the cell cultured on unmodified membrane. These results show that the cytotoxicity of Col/bFGF-p (HEMA-MMA) for keratocytes is much improved.

Polyurethane/poly(hydroxyethyl methacrylate) semi-interpenetrating polymer networks for biomedical applications.

The thermodynamic miscibility, morphology, phase distribution, mechanical properties, surface properties, water sorption, bacterial adhesion and cytotoxicity of semi-interpenetrating polymer networks (semi-IPNs) based on crosslinked polyurethane (PU) and poly(hydroxyethylmethacrylate) (PHEMA) were studied to give an insight into their structure and properties. The free energies of mixing of the two polymers in semi-IPNs have been determined and it was shown that the values are positive and depend on the amount of PHEMA. This demonstrates that the components are immiscible, the extent of which is dependent upon variations in composition. The morphology of the semi-IPNs was analyzed with scanning electron microscopy and tapping mode atomic force microscopy (TMAFM). The micrographs of the semi-IPNs and TMAFM phase images indicated that distinct phase separation at the nanometer scale is observed. The mechanical properties reflect the changes in structure of semi-IPNs with composition. The stress at break increases from 3.4 MPa to 23.9 MPa, and the Young's modulus from 12.7 MPa up to 658.5 MPa with increasing amounts of PHEMA, but strain at break has a maximum at 40.4% PHEMA. The bacterial adhesion and cytotoxicity data suggest that semi-IPNs with PHEMA content above 22% may be used for biomedical material applications.

HEMA/MMMA microcapsule implants in hemiparkinsonian rat brain: biocompatibility assessment using [3H]PK11195 as a marker for gliosis.

Microencapsulation of dopamine-secreting cells in biocompatible, semi-permeable polymer membranes has been proposed as an alternative strategy for dopamine replacement for Parkinson's disease. In order to assess the viability of this proposal, dopamine-secreting PC12 cells were immunoisolated via microencapsulation in a 75:25 2-hydroxyethyl methacrylate/methyl methacrylate (HEMA/MMA) copolymer. A submerged nozzle-liquid jet method was used to produce small diameter (400 microm) microcapsules, which were stereotaxically implanted in the denervated striatum of hemi-Parkinsonian rats. A 96% survival rate was associated with the implantation surgery and no deleterious side effects were apparent. Light microscopy revealed good biocompatibility between the HEMA/MMA copolymer and the host brain, as evidenced by the absence of gross tissue damage at the neuronal tissue/capsule interface. Autoradiographic analyses using [3H]PK11195 as marker for reactive astrocytes revealed a moderate inflammatory response, confined to the immediate vicinity of the injection tract. Quantitative analyses indicated that the local tissue response did not differ significantly between brains implanted with PC12-containing capsules and those implanted with vehicle-containing capsules. Taken together, these results support the biocompatibility of HEMA/MMA copolymer as well as the feasibility and safety of stereotaxic implantation of microcapsules.

Hydrogels in endovascular embolization. VI. Toxicity tests of poly(2-hydroxyethyl methacrylate) particles on cell cultures.

Cytotoxicity of poly(2-hydroxyethyl methacrylate) [poly(HEMA)] hydrogel spherical particles, prepared by radical suspension polymerization and designed for endovascular occlusion, was studied in vitro on cell cultures. Testing methods included a direct contact test and extraction test. No inhibition of growth of cells surrounding the poly(HEMA) beads and a very low inhibition of cell viability, only in concentrated extracts in long-term contact, were observed. As a result, poly(HEMA) beads can be considered non-toxic.

Nontoxic embolic liquids for treatment of arteriovenous malformations.

Interventional radiology is becoming one of the standard treatments of arteriovenous malformation (AVM). Cyanoacrylate derivatives and polymer solutions are widely used to occlude the AVM nidus by their injection through a catheter, but they are far from satisfactory embolic liquids. For instance, cyanoacrylate derivatives sometimes glue the catheter to the artery, resulting in serious complications; in addition, the organic solvents used to dissolve polymers cause damage to the surrounding brain tissue of the AVM. Therefore, we attempted to develop embolic liquids by dissolving poly(2-hydroxyethyl methacrylate-co-methyl methacrylate) in Iopamiron with an addition of a small amount of ethyl alcohol. This new embolic liquid is not cytotoxic and is easily injected into the AVM through a thin, long catheter to effectively occlude the AVM.

Preliminary in vitro cytotoxicity screening of a bead-formed macroporous hydrophilic polymer matrix.

A prescreen of the in vitro cytotoxicity of both the primary fabrication components and potential leachables from a bead-formed macroporous poly(2-hydroxyethyl methacrylate), (pHEMA) matrix has been carried out using INVITTOX Neutral red and Kenacid blue R dye binding methods. Of the eluants obtained from 24, 48, and 72-h incubated beads, only the 72-h eluant produced a greater than 20% (ID20) inhibition of 3T3-L1 cell proliferation with values of 20.98 +/- 2.33% and 21.41 +/- 1.37% inhibition for the Neutral red and Kenacid blue R binding methods, respectively. ID50 values for the fabrication components obtained using the Kenacid blue R method were generally higher than those obtained by the Neutral red assay, although the ranking of the chemicals in terms of their relative cytotoxicities was identical by both methods, i.e. ethylene glycol dimethacrylate > uranyl nitrate > purified HEMA > n-hexane > ethylene glycol (mmol 1(-1)). Whilst extended washing of finished PHEMA beads in water will reduce their acute in vitro cytotoxicity, this will only be achieved with some loss of previously encapsulated water soluble macromolecules.

Chemical sorbents: official guidelines and experimental approach.

To evaluate the biocompatibility of an anionic exchange resin with active group trimethylammonium and charged with chloride ions an experimental study was performed with different tests to determine cytocompatibility, acute systemic toxicity, intracutaneous reactivity, allergic sensibilization, haemocompatibility and release of acetone, dichloromethane, quaternary amines and heavy metals (Pb, Cu, Cd) in eluate. Thus an anionic exchange resin can link all anionic substances; an ex vivo study on the participation of the dissociated sodic heparin in the anionic exchange was also conducted. In fact every time an extracorporeal circuit is made an anticoagulant must be administered to the patient and the removal of the drug could cause serious haemocoagulative problems. The results of the study showed a high biocompatibility of the resin that did not cause different significant reactions in comparison with negative control substances. The coating of the resin with poly-hydroxy-ethyl methacrilate with a separation phase technique avoided heparin removal from blood. Therefore adjustment of the dosage may not be necessary during further in vivo experiments. Future researches aiming to evaluate the the effectiveness and the biocompatibility of the sorbent in a living being will be performed in the next steps of the study.

Preparation of poly(2-hydroxyethyl methacrylate)-collagen composites.

Linear and three-dimensional polymer composites were prepared on the basis of poly(2-hydroxyethyl methacrylate)--pHEMA--and collagen. Their biological properties were tested by in vitro as well as in vivo methods. The composite material (unlike pure pHEMA) supported myoblast adhesion as well as their fusion into multinuclear myotubes in vitro. The three-dimensional polymer composites stimulated a new bone formation after their intraosseal implantation in dogs and pigs. The biomaterial itself was degraded in the host organism, in contrast to stable pHEMA.

Tissue responses to Hydron, assessed by intraosseous implantation.

Teflon tubes containing freshly mixed, polymerized Hydron root canal filling material, fully set AH26 or Teflon were implanted into the mandible of guinea pigs and assessed histologically at 2 days, 1, 2, 4, 12 and 26 weeks. None of the materials tested elicited signs of overt or significant tissue damage, and polymerized Hydron was assessed to be as biocompatible as fully set AH26 and Teflon. Bone formed in very close apposition to the polymerized Hydron, whereas a soft tissue capsule separated the regenerated bone from implants of AH26 and Teflon.

Tissue responses to Hydron, assessed by intramuscular implantation.

Freshly mixed polymerized Hydron root canal filling material, fully set AH26 and Teflon were implanted in the quadriceps muscle of guinea pigs and assessed histologically at 2 days, 1, 2, 3, 12 and 26 weeks after implantation. All materials were characterized by peri-implant fibrous connective tissue capsule formation. Von Kossa-positive calcific material was observed at the implant-tissue interface of Hydron implants. The amount of apparently calcified material increased with time. Inflammation was not a prominent tissue response for any of the test materials, nor was a foreign body giant cell response.

Cell responses to Hydron by a new in-vitro method.

An in-vitro biotoxicity test system, suitable for the assessment of endodontic filling materials, has been developed and used to test cell responses to Hydron, AH26 and Tubliseal. A robust, well-characterized and stable cell line (L-cells) which was grown as uniform cultures on Millipore filters, has been used as indicator cells. As they approached confluence they were exposed to test substances for 24 h and biosynthetic activities were measured. The test system is a modification of that described by Wennberg et al. (1979). By inverting the cultures on organ-culture rafts, cells were separated from the test material, which was placed on top of the Millipore filters. Freshly mixed polymerizing Hydron and prepolymerized Hydron were tested. The cell responses were compared with those of cultures exposed to freshly mixed AH26 and Tubliseal. Polymerizing and prepolymerized Hydron depressed both cell division, assessed by 3H-thymidine incorporation, and the synthesis and secretion of matrix material as measured by precipitable 35S-sulphate. Prepolymerized Hydron decreased cell functions by 59% and 56% of live cell controls, respectively, while the freshly mixed polymerizing Hydron inhibited biosynthesis by 89% and 94%, respectively. The data for polymerizing Hydron were compared with results for other root-filling materials and showed similar values to those for Tubliseal (92% and 95%), but greater inhibition of biosynthesis than for AH26 (53% and 50%). The AH26 values were similar to those obtained from cultures exposed to the prepolymerized Hydron. Recovery of biosynthetic capacity by these cultures after removal of all endodontic material was also assessed. Partial biosynthetic recovery of cell cultures was observed 24 h after removal of preopolymerized Hydron.(ABSTRACT TRUNCATED AT 250 WORDS)