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Photothermal-responsive (PTR) and anti-oxidative silk fibroin/dopamine nanoparticles (SD NPs) mediated by tyrosinase were produced, and decorated either by curcumin or albumin (BSA) to produce SD/curcumin or SD/BSA NPs as drug delivery vehicles, respectively. Both drug loaded NPs were further blended into SF solutions to produce SD films, as a depot-based drug delivery. The reaction mechanisms for producing new SD NPs were proposed. Anti-oxidative activities for SD NPs were examined by H2O2 scavenge capacities of NPs. NPs were not cytotoxic at concentration of 1000μg/mL. Moreover, heparin was coated to SD films to produce SDH films for temporary implants. Cumulative release profiles for drugs loaded SDH films showed fast releases and then sustained releases stages. Furthermore, the releases of curcumin in sustained stages for varying SD/curcumin NPs loaded into SDH films were dependent on amounts of NPs. BSA releases profiles for SD/BSA NPs loaded into SDH films were similar to those profiles for the films carried with SD/curcumin NPs but release periods of BSA were short. Degrees of PTR effects with irradiation of near infrared on the releases of two drugs loaded films were different. Blood clot at wound areas of rats with SDH films implantations was not found for 24 h study.

Background Magnetic nanoparticles (MNPs) can be localized against hemodynamic forces in blood vessels with the application of an external magnetic field. In addition, PEGylation of nanoparticles may increase the half-life of nanocomposites in circulation. In this work, we examined the effect of PEGylation on the magnetic capture of MNPs in vivo. Methods Laser speckle contrast imaging and capillaroscopy were used to assess the magnetic capture of dextran-coated MNPs and red blood cell (RBC) flow in cremaster microvessels of anesthetized rats. Magnetic capture of MNPs in serum flow was visualized with an in vitro circulating system. The effect of PEGylation on MNP-endothelial cell interaction was studied in cultured cells using an iron assay. Results In microcirculation through cremaster muscle, magnet-induced retention of 250 nm MNPs was associated with a variable reduction in RBC flow, suggesting a dynamic coupling of hemodynamic and magnetic forces. After magnet removal, faster restoration of flow was observed in PEG(+) than PEG(-) group, which may be attributed to a reduced interaction with vascular endothelium. However, PEGylation appears to be required for magnetic capture of 50 nm MNPs in microvessels, which was associated with increased hydrodynamic diameter to 130±6 nm in serum, but independent of the ς-potential. Conclusion These results suggest that PEGylation may enhance magnetic capture of smaller MNPs and dispersion of larger MNPs after magnet removal, which may potentially affect the targeting, pharmacokinetics and therapeutic efficacy.

Background Ischemia/reperfusion (I/R) injury causes the generation of many ROS such as H2O2 and leads to vascular thrombosis, which causes tissue damage. Purpose In this investigation, poly (lactideco-glycolide) (PLGA)-based nanoparticles are used for their anticoagulant and antioxidant properties in vascular therapy. Methods Both heparin and glutathione are entrapped on PLGA-stearylamine nanoparticles by layer-by-layer interactions. Results The drug release rate is successfully controlled with only 10.3% of the heparin released after 96 hours. An H2O2-responsive platform is also developed by combining silk fibroin and horse peroxidase to detect H2O2 in this drug delivery system. Besides, hyaluronic acid was decorated on the surface of nanoparticles to target the human bone marrow mesenchymal stem cells (hBMSCs) for cell therapy. The results of an in vitro study indicate that the nanoparticles could be taken up by hBMSCs within 2 hours and exocytosis occurred 6 hours after cellular uptake. Conclusion We propose that the multifunctional nanoparticles that are formed herein can be effectively delivered to the site of an I/R injury via the hBMSC homing effect. The proposed approach can potentially be used to treat vascular diseases, providing a platform for hBMSCs for the controlled delivery of a wide range of drugs.

Bone is a flexible and electro active tissue that is vulnerable to various traumatic injuries. The self-healing of damaged bone tissue towards reconstruction is limited due to the lack of proper niche compliances. Nevertheless, the classical grafting techniques like autograft/allograft for bone repair pose challenges like bacterial infections and donor-site morbidity with unsatisfactory outcomes. The use of appropriate biomaterial with osteogenic potential can meet these challenges. In this regard, bioactive glass ceramics is widely used as a bone filler or graft material because of its bonding affinity to bone leading towards bone reconstruction applications without the challenge of post implant infections. Hence, the current study is aimed at addressing this potentiality of zinc (Zn) for doped the bioglass at nano-scale advantages for bone tissue repair. Since, Zn has been demonstrated to have not only antibacterial property but also the stimulatory effect on osteoblasts differentiation, mineralization by enhancing the osteogenic genes expression. In view of these, the present study is focused on sol–gel synthesis and pysico-chemical characterization of Zinc-doped bioglass nanoparticles (Zn-nBGC) and also analyzing its biological implications. The surface morphological and physiochemical characterizations using SEM, EDX, FT-IR and XRD analysis has shown the increased surface area of Zn-nBGC particles providing a great platform for biomolecular interaction, cytocompatibility, cell proliferation and osteogenic differentiation. The obtaining hydroxy apatite groups have initiated in vitro mineralization towards osteogenic lineage formation. Zn has not only involved in enhancing cellular actions but also strengthen the ceramic nanoparticles towards antibacterial application. Hence the finding suggests a biomaterial synthesis of better biomaterial for bone tissue engineering application by preventing post-operative bacterial infection.

This study spots light on combined Wound healing process conjoining blood coagulation, inflammation reduction, proliferation and remodeling of the cells. The objective is to overcome the drawbacks of conventional clinically applied wound dressings such as poor rigidity, porosity, mechanical potency and bactericidal activity. As nosocomial infection is a very common condition at the wound site, bio-adhesive materials with intrinsic antibacterial properties are used in clinical applications. Considering the provenability of Wollastonite [Calcium silicate (CaSiO3)] to regenerate the soft tissues by inducing vascularization and regeneration of fibroblast cells And the antibacterial potentiality of zinc in clinical applications, the present study focuses on synthesis of Zn-Ws particles and evaluation of its antimicrobial and wound healing potentialities towards skin tissue engineering applications. The compositional characterization by EDAS and FT-IR spectral analysis have substantiated the presence of major elements and corresponding band stretching associated with the synthesized particles whereas the particles morphology by SEM images have shown the size of the Ws and Zn-Ws to be 370 nm and 530 nm respectively. From the in vitro studies, skin regenerative potential of Zn-Ws was determined on promoting fibroblast cell (NIH3T3) proliferation by providing better adhesiveness, biocompatibility and cytocompatibility. The antibacterial property of Zn-Ws evaluation by minimum inhibitory concentration (MIC) and zone of inhibition (ZOI) methods against clinical isolates of Gram +Ve and Gram –Ve bacterial strains have confirmed that the addition of Zn has diminished the bacterial growth and also helped in degrading the bacterial biofilms. Thus it is summed up that the process of wound healing is expected to occur with reduced risk of post-injury infections by the presence of zinc-doping on wollastonite for skin tissue application.

In the tissue engineering research field, nanobiomaterials highlight the impact of novel bioactive materials in both current applications and their potentials in future progress for tissue engineering and regenerative medicine. Tissue engineering is a well-investigated and challenging biomedical field, with promising perspectives to improve and support quality of life for the patient. To assess the response of those extracellular matrices (ECMs), induced by biomedical materials, this review will focus on cell response to natural biomaterials for biocompatibility.

A silk fibroin composite film that can simultaneously scavenge and probe H2O2 in situ was developed for possibly examining local concentrations of H2O2 for biomedical applications. A multi-functional composite film (GDES) that consists of graphene oxide (G), a photothermally responsive element that was blended with polydopamine (PDA, D)/horseradish peroxidase (HRP, E) (or DE complex), and then GDE microaggregates were coated with silk fibroin (SF, S), a tyrosine-containing protein. At 37 &deg, C, the H2O2-scavenging ability of a GDES film in solution at approximately 7.5 &times, 10&minus, 3 &mu, mol H2O2/mg film was the highest compared with those of S and GS films. The intensities of UV-excitable blue fluorescence of a GDES film linearly increased with increasing H2O2 concentrations from 4.0 &mu, M to 80 &mu, M at 37 &deg, C. Interestingly, after a GDES film scavenged H2O2, the UV-excitable blue fluorescent film could be qualitatively monitored by eye, making the film an eye-probe H2O2 sensor. A GDES film enabled to heat H2O2-containing samples to 37 &deg, C or higher by the absorption of near-IR irradiation at 808 nm. The good biocompatibility of a GDES film was examined according to the requirements of ISO-10993-5. Accordingly, a GDES film was developed herein to scavenge and eye-probe H2O2 in situ and so it has potential for biomedical applications.

In the tissue engineering research field, nanobiomaterials highlight the impact of novel bioactive materials in both current applications and their potentials in future progress for tissue engineering and regenerative medicine. Tissue engineering is a well-investigated and challenging biomedical field, with promising perspectives to improve and support quality of life for the patient. To assess the response of those extracellular matrices (ECMs), induced by biomedical materials, this review will focus on cell response to natural biomaterials for biocompatibility.

A silk fibroin composite film that can simultaneously scavenge and probe H2O2 in situ was developed for possibly examining local concentrations of H2O2 for biomedical applications. A multi-functional composite film (GDES) that consists of graphene oxide (G), a photothermally responsive element that was blended with polydopamine (PDA, D)/horseradish peroxidase (HRP, E) (or DE complex), and then GDE microaggregates were coated with silk fibroin (SF, S), a tyrosine-containing protein. At 37 °C, the H2O2-scavenging ability of a GDES film in solution at approximately 7.5 × 10−3 μmol H2O2/mg film was the highest compared with those of S and GS films. The intensities of UV-excitable blue fluorescence of a GDES film linearly increased with increasing H2O2 concentrations from 4.0 μM to 80 μM at 37 °C. Interestingly, after a GDES film scavenged H2O2, the UV-excitable blue fluorescent film could be qualitatively monitored by eye, making the film an eye-probe H2O2 sensor. A GDES film enabled to heat H2O2-containing samples to 37 °C or higher by the absorption of near-IR irradiation at 808 nm. The good biocompatibility of a GDES film was examined according to the requirements of ISO-10993-5. Accordingly, a GDES film was developed herein to scavenge and eye-probe H2O2 in situ and so it has potential for biomedical applications.

The effects of the stiffness of substrates on the cell behaviours of human bone marrow-derived mesenchymal stem cells (hBMSC) have been investigated, but the effects of the secondary structures of proteins in the substrates on the morphological transformation and differentiation of hBMSC have yet been elucidated. To investigate these issues, silk fibroin-poly(e-caprolactone) SP cardiac patches of poly(e-caprolactone; P), on which is grafted by silk fibroin (SF) with various β-sheet contents (or crystallinity) to provide various degrees of stiffness, were produced to examine the in vitro behaviours of hBMSC during proliferation, and cardiomyogenesis on the SP patches. β-sheet contents of SF from 20% to 44% (SP20 to SP44, respectively) were induced on patches, which were examined by attenuated total reflection Fourier-transform infrared (ATR-FTIR) spectroscopy, and analysed using the Fourier self-deconvolution method. The stiffness of the SP patches, quantified by their Young's moduli and elasticities, increased with the crystallinity of the SF. During 3 days of proliferation, hBMSC migrated and morphologically transformed into 3D microtissues with diameters of approximately 150-200 μm on low-stiffness SP20 and SP30 patches, whereas 2D monolayers were observed on the SP37 and SP44 patches. The 3D microtissues/patch yielded more extensive in vitro cardiomyogenesis of hBMSC than the 2D cell monolayer with significantly higher expressions of all examined cardiac-specific proteins after induction by 5-aza. Notably, in vivo subcutaneously growing 3D microtissues on SP20 patches and a 2D monolayer on SP44 patches were preliminarily demonstrated in a rat model. Morphological transformations of hBMSC from a 2D monolayer to a 3D microtissue by low-stiffness SP cardiac patches, promoting cardiomyogenesis, provide a new opportunity for cardiac tissue engineering.

Cardiovascular disease (CVD), leading to myocardial infarction and heart failure, is one of the major causes of death worldwide. The physiological system cannot significantly regenerate the capabilities of a damaged heart. The current treatment involves pharmacological and surgical interventions; however, less invasive and more cost-effective approaches are sought. Such new approaches are developed to induce tissue regeneration following injury. Hence, regenerative medicine plays a key role in treating CVD. Recently, the extrinsic stimulation of cardiac regeneration has involved the use of potential polymers to stimulate stem cells toward the differentiation of cardiomyocytes as a new therapeutic intervention in cardiac tissue engineering (CTE). The therapeutic potentiality of natural or synthetic polymers and cell surface interactive factors/polymer surface modifications for cardiac repair has been demonstrated in vitro and in vivo. This review will discuss the recent advances in CTE using polymers and cell surface interactive factors that interact strongly with stem cells to trigger the molecular aspects of the differentiation or formulation of cardiomyocytes for the functional repair of heart injuries or cardiac defects.

Erythrocyte deformability for suspensions of washed cells was tested by measuring the initial pressure drop during constant volumetric flow through 3-μm pore size polycarbonate membranes. 1. Effect of white cell concentration - 2. Assessing erythrocyte deformability : a) Cell number density vs. hematocrit, b) the shear thinning of erythrocyte suspensions

Pacemaker cells can be differentiated from stem cells or transdifferentiated from quiescent mature cardiac cells via genetic manipulation. Here we show that the exposure of rat quiescent ventricular cardiomyocytes to a silk-fibroin hydrogel activates the direct conversion of the quiescent cardiomyocytes to pacemaker cardiomyocytes by inducing the ectopic expression of the vascular endothelial cell-adhesion glycoprotein cadherin. The silk-fibroin-induced pacemaker cells exhibited functional and morphological features of genuine sinoatrial-node cardiomyocytes in vitro, and pacemaker cells generated via the injection of silk fibroin in the left ventricles of rats functioned as a surrogate in situ sinoatrial node. Biomaterials with suitable surface structure, mechanics and biochemistry could facilitate the scalable production of biological pacemakers for human use.

Background Magnetic nanoparticles (MNPs) can be localized against hemodynamic forces in blood vessels with the application of an external magnetic field. In addition, PEGylation of nanoparticles may increase the half-life of nanocomposites in circulation. In this work, we examined the effect of PEGylation on the magnetic capture of MNPs in vivo. Methods Laser speckle contrast imaging and capillaroscopy were used to assess the magnetic capture of dextran-coated MNPs and red blood cell (RBC) flow in cremaster microvessels of anesthetized rats. Magnetic capture of MNPs in serum flow was visualized with an in vitro circulating system. The effect of PEGylation on MNP-endothelial cell interaction was studied in cultured cells using an iron assay. Results In microcirculation through cremaster muscle, magnet-induced retention of 250 nm MNPs was associated with a variable reduction in RBC flow, suggesting a dynamic coupling of hemodynamic and magnetic forces. After magnet removal, faster restoration of flow was observed in PEG(+) than PEG(–) group, which may be attributed to a reduced interaction with vascular endothelium. However, PEGylation appears to be required for magnetic capture of 50 nm MNPs in microvessels, which was associated with increased hydrodynamic diameter to 130±6 nm in serum, but independent of the ς-potential. Conclusion These results suggest that PEGylation may enhance magnetic capture of smaller MNPs and dispersion of larger MNPs after magnet removal, which may potentially affect the targeting, pharmacokinetics and therapeutic efficacy.

The goal of this study was to examine the effects of lipid composition on physicochemical characteristics of hydrogenated soybean lecithin (HL)/d-α-tocopheryl polyethylene glycol 1000 succinate (TPGS) dispersions using dynamic light scattering, transmission electron microscopy, differential scanning calorimetry, and fluorescence polarization spectroscopy. Our results showed that HL-rich nano-dispersions exhibited larger size, narrower size distribution, higher negative zeta potential, more obvious phase transition regions, and lower membrane fluidity than did the TPGS-rich ones. The membrane packing order, intermolecular attraction interaction, and molecular cooperativity in the phase transition region of the HL dispersions could be reduced by the incorporation of a large amount of TPGS. Furthermore, chemical stability, a drug release profile, and free radical-scavenging capacity of HL/TPGS nano-dispersions encapsulating black soybean seed coat extract (BE) were analyzed. HL/TPGS nano-dispersions improved the stability and radical-scavenging capacity of phytochemicals, such as BE. Nano-dispersions with the molar ratio of HL/TPGS = 9/1 exhibited good storage stability, a slow BE release rate, good free radical-scavenging capacity, and high in vitro biocompatibility; thus, they could be a high-potential vehicle for carrying active herbal agents.

Gelatin (Gtn) is a biodegradable and non-immunogenic biopolymer. However, Gtn materials have limited mechanical properties, especially in aqueous solutions. Additionally, since Gtn materials have high hydrophilicity, delivering a high load with a sustained releases of hydrophobic drugs is difficult. To overcome these drawbacks, Gtn-based hydrogel films are incorporated into a low concentration of PLGA and aminolyzed PLGA (e.g., PLGA-NH2) for the delivery of hydrophobic drugs; indomethacin (IDM), as a model, is crosslinked with different concentrations of 1,6-hexamethylene diisocyanate (HMDI) in a DMSO solution. Water contact angles were increased by incorporating PLGA (e.g., 53.9° ± 2.2° and 61.0° ± 0.9° for Gtn and Gtn/PLGA, respectively). The glass transition temperature values for Gtn, Gtn/PLGA and Gtn/PLGA-NH2 films were decreased by incorporating PLGA. The compressional stress of Gtn, Gtn/ PLGA and Gtn/ PLGA-NH2 hydrogel films was 8.9 ± 0.7, 15.5 ± 2.2 and 11.2 ± 0.8 kPa, respectively. The biocompatible Gtn/PLGA-NH2 hydrogel films had high IDM encapsulation efficiencies (e.g., roughly 84.8%) with low burst releases, sustained IDM release up to 96 h and the highest accumulative releases (e.g., 76.6 ± 3.7%) among the hydrogel films while concentrations of HMDI highly influenced the release characteristics of IDM in the films.

Silk fibroin (SF) is a protein with bulky hydrophobic domains and can be easily purified as sericin-free silk-based biomaterial. Silk fibroin modified chitosan nanoparticle (SF-CSNP), a biocompatible material, has been widely used as a potential drug delivery system. Our current investigation studied the bio-effects of the SF-CSNP uptake by liver cells. In this experiment, the characterizations of SF-CSNPs were measured by particle size analysis and protein assay. The average size of the SF-CSNP was 311.9 ± 10.7 nm, and the average zeta potential was +13.33 ± 0.3 mV. The SF coating on the SF-CSNP was 6.27 ± 0.17 μg/mL. Moreover, using proteomic approaches, several proteins involved in the ubiquitin proteasome pathway were identified by analysis of differential protein expressions of HepG2 cell uptake the SF-CSNP. Our experimental results have demonstrated that the SF-CSNP may be involved in liver cancer cell survival and proliferation.

The effects of the stiffness of substrates on the cell behaviours of human bone marrow-derived mesenchymal stem cells (hBMSC) have been investigated, but the effects of the secondary structures of proteins in the substrates on the morphological transformation and differentiation of hBMSC have yet been elucidated. To investigate these issues, silk fibroin-poly(ε-caprolactone) SP cardiac patches of poly(ε-caprolactone; P), on which is grafted by silk fibroin (SF) with various β-sheet contents (or crystallinity) to provide various degrees of stiffness, were produced to examine the in vitro behaviours of hBMSC during proliferation, and cardiomyogenesis on the SP patches. β-sheet contents of SF from 20% to 44% (SP20 to SP44, respectively) were induced on patches, which were examined by attenuated total reflection Fourier-transform infrared (ATR-FTIR) spectroscopy, and analysed using the Fourier self-deconvolution method. The stiffness of the SP patches, quantified by their Young's moduli and elasticities, increased with the crystallinity of the SF. During 3 days of proliferation, hBMSC migrated and morphologically transformed into 3D microtissues with diameters of approximately 150-200 μm on low-stiffness SP20 and SP30 patches, whereas 2D monolayers were observed on the SP37 and SP44 patches. The 3D microtissues/patch yielded more extensive in vitro cardiomyogenesis of hBMSC than the 2D cell monolayer with significantly higher expressions of all examined cardiac-specific proteins after induction by 5-aza. Notably, in vivo subcutaneously growing 3D microtissues on SP20 patches and a 2D monolayer on SP44 patches were preliminarily demonstrated in a rat model. Morphological transformations of hBMSC from a 2D monolayer to a 3D microtissue by low-stiffness SP cardiac patches, promoting cardiomyogenesis, provide a new opportunity for cardiac tissue engineering.

Micelles, with the structure of amphiphilic molecules including a hydrophilic head and a hydrophobic tail, are recently developed as nanocarriers for the delivery of drugs with poor solubility. In addition, micelles have shown many advantages, such as enhanced permeation and retention (EPR) effects, prolonged circulation times, and increased endocytosis through surface modification. In this study, we measured the critical micelle concentrations, diameters, stability, and cytotoxicity and the cell uptake of micelles against hepatic cells with two kinds of hydrophilic materials: PEG-PCL and HA-g-PCL. We used 131I as a radioactive tracer to evaluate the stability, drug delivery, and cell uptake activity of the micelles. The results showed that HA-g-PCL micelles exhibited higher drug encapsulation efficiency and stability in aqueous solutions. In addition, the 131I-lipiodol loaded HA-g-PCL micelles had better affinity and higher cytotoxicity compared to HepG2 cells.

The purpose of this study was to develop the pathway of silk fibroin (SF) biopolymer surface induced cell membrane protein activation. Fibroblasts were used as an experimental model to evaluate the responses of cellular proteins induced by biopolymer material using a mass spectrometry-based profiling system. The surface was covered by multiwalled carbon nanotubes (CNTs) and SF to increase the surface area, enhance the adhesion of biopolymer, and promote the rate of cell proliferation. The amount of adhered fibroblasts on CNTs/SF electrodes of quartz crystal microbalance (QCM) greatly exceeded those on other surfaces. Moreover, analyzing differential protein expressions of adhered fibroblasts on the biopolymer surface by proteomic approaches indicated that CD44 may be a key protein. Through this study, utilization of mass spectrometry-based proteomics in evaluation of cell adhesion on biopolymer was proposed.

Poly(e-caprolactone) (PCL) scaffolds functionalized by grafting nerve growth factor (NGF) and Asp-Arg-Gly-Asp (GRGD)(PCL-NGF/GRGD) for neural tissue engineering. The influences of PCL-NGF/GRGD scaffolds on the growth and differentiation of PC12 cells were investigated. The successfully grafting NGF and GRGD into PCL-CS scaffold were verified by FTIR spectra. The densities of GRGD and NGF in the scaffolds were about 2.10×10(-1) μmol/cm(2) and 1.51×10(-3) nmol/cm(2) . Growths of PC12 cells in PCL-GRGD and PCL/NGF-GRGD scaffolds via MTS measurements were significantly higher (p < 0.01, n = 4) than that in PCL-CS or PCL-NGF ones for three days of cultivation that was consistent with SEM observations. Moreover, the differentiation of PC12 cells, induced by NGF at 50 ng/mL for four days, in PCL-NGF/GRGD scaffolds were qualitatively more numbers and longer outgrowth of neurites than those in PCL-CS, PCL-GRGD, and PCL-NGF ones by SEM observations. The synergistic effects of grafting both NGF and GRGD ligands to PCL-CS scaffolds on the growth and differentiation of PC12 cells provide a new biomaterial for neural tissue engineering.

Background The thyroid hormone, thyroxine (T4), is a tyrosine-based hormone produced by the thyroid gland, which is essential in regulating a number of biological processes, including growth, neurodevelopment, carbohydrate metabolism, oxygen consumption and protein synthesis. Data on human thyroid hormone metabolism were gathered since the middle of the 1970s mainly by the use of radioactive iodinated (125I or 131I) hormones. Methods We describe an isotope dilution-matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) method for the simultaneous determination of endogenous thyroid hormone and its 13C6-labeled analogue in serum. The 13C6-thyroxine (13C6-T4) was used as an internal standard; T4 and its isotopically labeled analogue were measured in the selected reaction monitoring mode for the transitions from m/z 777.8 to 732.1 and from m/z 784.2 to 738.1, respectively. Results Serum samples were prepared and concentrated by a solid-phase extraction chromatography method. The recovery rate was measured by 125I-T4 and can be up to 82.8 ± 2.8%. The detection limit and linear range for T4 were 5 ng/ml and 5–400 ng/ml, respectively. The correlation coefficient (R2) between radioimmunoassay (RIA) and isotope dilution-MALDI-TOF MS for detection of serum T4 was 0.982. Conclusion This assay has a good relationship against a commercial RIA and the isotope dilution-mass spectrometry method and may serve as a reference method for quantitative analysis of T4.

The microenvironment of neuron cells plays a crucial role in regulating neural development and regeneration. Hyaluronic acid (HA) biomaterial has been applied in a wide range of medical and biological fields and plays important roles in neural regeneration. PC12 cells have been reported to be capable of endogenous NGF synthesis and secretion. The purpose of this research was to assess the effect of HA biomaterial combining with PC12 cells conditioned media (PC12 CM) in neural regeneration. Using SH-SY5Y cells as an experimental model, we found that supporting with PC12 CM enhanced HA function in SH-SY5Y cell proliferation and adhesion. Through RP-nano-UPLC-ESI-MS/MS analyses, we identified increased expression of HSP60 and RanBP2 in SH-SY5Y cells grown on HA-modified surface with cotreatment of PC12 CM. Moreover, we also identified factors that were secreted from PC12 cells and may promote SH-SY5Y cell proliferation and adhesion. Here, we proposed a biomaterial surface enriched with neurotrophic factors for nerve regeneration application.

Bone marrow mesenchymal stem cells/silk fibroin/hyaluronic acid (BMSC/SH) patches were implanted into myocardial infarction (MI) rat hearts to investigate the efficacies of them on enhancing left ventricular (LV) remodeling and cardiac repair. 45 rats were divided into four groups: Sham, MI (MI hearts, induced by a cryo-injury technique), SH and BMSC/SH (MI hearts with implantations of SH and BMSC/SH patches, respectively). After eight weeks of post-implantation, the patches for the SH and BMSC/SH groups were intact and well adhered on the MI zones with no and minor immunological responses, respectively, examined by a CD68 marker, while severe inflammation on the zones was observed for the MI group. The SH group showed the efficacy of cardiac repair on MI zones. Moreover, BMSC/SH group significantly improved the wall thickness of LV, assessed by echocardiography, and had high viability of delivery BMSC, largely reduced apoptosis, significantly promoted neo-vascularization and stimulated the secretions of various paracrine factors such as VEGF, examined by real-time PCR, in MI zones compared with those of the SH and MI groups. In conclusion, the therapeutic efficacies of using BMSC/SH patches for repairing MI hearts were demonstrated by showing the advantages of both bioactive SH patches and BMSC-based therapy.

The early responses of mesenchymal stem cells (MSCs) to various biopolymers are important to their proliferation and differentiation. Here, the adhesions of rat MSCs on the electrode of quartz crystal microbalance (QCM) decorated with multi-walled carbon nanotubes (CNT)/chitosan (CS), CNT/CS/hyaluronic acid (HA) and CNT/CS/fibronectin (FN) layers were investigated and compared with other techniques at a serum-free condition for 12-h incubation. The numbers of adhered rMSCs on CNT/CS/FN and CNT electrodes were 76 and 26% of the initial values, respectively, determined by cell-count technique while MTS assay could not detect the differences. The weights of adhered rMSCs on CNT/CS/FN electrodes of QCM were 50% higher than those on other surfaces that were consistent with cell-count technique. The morphology of adhered rMSCs on the CNT/CS/FN surfaces markedly differed from those on other surfaces. In conclusion, the electrodes of QCM decorated with CNT/CS/FN or CNT/CS/carbohydrate polymers could be quantified the early adhesion of rMSCs on their surfaces.

In this investigation, drug delivery characteristics of incorporating CS and anhydrous tri-polyphosphate (TPP) into SF in fabricating microparticles by a spray-dried technique were examined. Among various formulas, SF/CS microparticles at 10/4.0 (weight ratio) had the highest positive zeta potential (23.0 ± 3.60 mV ( n = 3)) while SF ones showed a negative zeta potential (−14.7 ± 1.5 mV ( n = 3)). The equilibrium swelling ratio of SF/CS (10/4.0) microparticles was 9.80 ± 0.77 times of themselves in a dry state, which was higher than those of SF and SF/CS (10/4.0)–TPP (0.05%) ones ( e.g. , 5.50 ± 0.58 and 7.43 ± 1.43 times, respectively). The release period for a model hydrophilic drug, tetracycline·Hcl (Tc), from SF was two days which prolonged to four and ten days for SF/CS (10/4.0) and SF/CS (10/4.0)–TPP (0.05%) microparticles, respectively. Through this investigation, new microparticulate SF/CS (10/4.0)–TPP (0.05%) is highly promising for drug delivery applications.

This study investigates in vitro the protein drug delivery characteristics of new thermal sensitive gels, poloxamer (P)–chitosan (CS)/hyaluronic acid (HA) gels (P–CS/HA), in which a CS solution is interacting with various concentrations of HA that interpenetrates P gels. The polyion complexation occurs between CS and HA that can protect drugs from proteolysis. The results indicate that the swelling ratios of all P–CS/HA gels are markedly superior to those of nonswelling P and P–CS gels. For example, P–CS/HA (0.5% (w/w)) gels have swelling ratios of 48.3 ± 2.7% (w/w), which are maintained for approximately 0.5 h in water at 37°C. In vitro releases of insulin from P–CS/HA (0.5% (w/w)) gels had significantly lowered initial burst release (P < 0.01) and lasted much longer than those from gels without a CS network. The duration of insulin release was in a significantly sustained manner for up to 3.5 h, which was about two times or longer than the period of delivery using P or P–CS gels.

This investigation examines the in vitro release of insulin in new thermo-response P-CS/GA/Gly gels, in which a chitosan (CS), crosslinked using glutaraldehyde (GA), interpenetrates Poloxamer (P) gels, and glycine (Gly) is further added to inhibit the crosslinking reactions of GA. The delivery of insulin by nasally administering P-CS/GA/Gly gels in diabetic rats was also examined. The release of insulin from P-CS/GA/Gly gels was significantly sustained ( P C min , 55.5 ± 2.8% of original values, n = 6) for 3 h, with a pharmacological efficiency of 18.0 ± 1.8% ( n = 6)) that significantly exceeded those achieved using other gels.

RGD-PCP copolymers were fabricated by grafting Arg-Gly-Asp (RGD) peptide to poly(epsilon-caprolactone)-b-chitooligosaccharide-b-poly(ethylene glycol) (PCP) copolymers and the rate of internalization of RGD-PCP micelles by PC 12 cells were examined. Increasing intensity of the absorbance of amine groups in FT-IR spectra of RGD-PCP copolymers compared with those of PCP copolymers indicated the presence of RGD in new copolymers. Moreover, the grafting efficiency and molar ratio of RGD peptides to PCP copolymers were 88.2% and 0.45, respectively, analysed with HPLC. The RGD-PCP copolymers self-assemble to micelles at the critical micelle concentration (CMC) of 0.018 wt% (178 mg L(-1)) and with a mean diameter of 90 nm using a dynamic light-scattering (DLS) analyser. Interestingly, the internalization of DPH-loaded RGD-PCP micelles into PC 12 cells is much faster (e.g. within 5 min) than that of PCP micelles. The new RGD-PCP micelles may potentially be used in cellular drug delivery.

This study investigates in vitro the drug delivery characteristics of new thermo-sensitive gels, P-CS/GA gels, in which a chitosan (CS) network is crosslinked with various concentrations of glutaraldehyde (GA) that interpenetrates Poloxamer (P) gels. The results indicate that the swelling ratios of all P-CS/GA gels are markedly superior to those of non-swelling P and P-CS gels. For example, P-CS/GA (0.1 wt.%) gels have swelling ratios of 13.2+/-1.0, which are maintained for approximately 18 h in water at 37 degrees C. In vitro releases of 5-FU from P-CS/GA (0.1 wt.%) gels had significantly lower initial burst release (P

In order to potentiate the low immunogenicity of the inactivated Newcastle disease virus immunized into chickens by mucosal route, liposomes as a drug delivery system and LPS (lipopolysaccharide) as an immuno-stimulator were evaluated. Here, we report a new nasal delivery system of inactivated Newcastle disease virus (NDV) vaccine. The intranasal vaccine was based on different lipids to form MLV (multi-lamellar vehicles) liposomes. The liposomes had combined carrier and adjuvant activities, which induced strong systemic (serum) and local (lung and nasal) humoral responses in SPF (specific-pathogen-free) chickens, and provided protective immunity. PC-Lip (phosphatidylcholine-liposome) elicited significant mucosal secretary immunoglobulin A (s-IgA) levels (p < 0.05) in tracheal lavage fluid and serum IgG levels (p < 0.05). In response to virulent viral challenge, birds treated with PBS (phosphate buffered saline) as control group died, whereas 80% of chickens which received PC-Lip, PC-Lip-LPS, PS-Lip (phosphatidylserine-liposome), and PS-Lip-LPS survived. HAI titers were 1:2560 in the PS-Lip-LPS group and 1:1280 in the PC-Lip, PC-Lip-LPS, and PS-Lip groups after two vaccinations. The results suggest that PC-Lip or PS-Lip might thus be suitable as a potential adjuvant for mucosal vaccination against NDV in chickens.

The aim of this study was to characterize a nasally delivered bioadhesive liposome using an inactivated H5N3 virus as a model antigen. Bioadhesive liposomes were developed using tremella (T) or xanthan gum (XG) as the bioadhesive polysaccharide. Using chickens as the target animal, we evaluated whether delivery of a bioadhesive liposomal influenza vaccine via a mucosal site of infection could improve vaccine effectiveness. 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) cytotoxicity assays demonstrated that T, XG and liposomes were non toxic to chicken spleen macrophages. Enzyme-linked immunosorbent assay (ELISA) was used to determine the adjuvant effect of the bioadhesive liposomal-vaccines. Chickens immunized with a low dose (200 microL) of bioadhesive liposomal influenza vaccine had significantly higher mucosal and serum antibody levels (P

Polysaccharides and proteins profoundly impact the development and growth of tissues in the natural extra-cellular matrix (ECM). To mimic a natural ECM, polysaccharides were incorporated to/or co-sprayed with silk fibroin (SF) to produce SF/chitosan (CS) or SF/CS-hyaluronic acid (SF/CS-HA) microparticles that were further processed by mechanical pressing and genipin cross-linking to produce hybrid cardiac patches. The ATR-FTIR spectra confirm the co-existence of CS or CS-HA and SF in microparticles and patches. For evaluating the cellular responses of rMSCs to the SF/CS and SF/CS-HA cardiac patches, the growth of rMSCs and cardiomyogenic differentiation of 5-aza inducing rMSCs cultured on patches was examined. First, the isolated rMSCs were identified with various positive and negative surface markers such as CD 44 and CD 31 by a flow cytometric technique, respectively. For examining the growth of rMSCs on the patches, MTT viability assay was performed, and the results demonstrated that the growth of rMSCs on SF and SF-hybrid patches significantly exceeded (P

A new liposomes/chitosan scaffold/human fibrin gel composite system (LCSHFG), as a depot drug delivery system, was developed to deliver low-molecular weight hydrophilic drugs. An antithrombosis drug, Tirofiban, was used as a model drug. Human fibrin gels encapsulated Tirofiban loaded liposomes were formed within chitosan scaffolds to configure the LCSHFG. The in vitro release behaviors of Tirofiban from LCSHFG were studied by characterizing the constituents of LCSHFG. The results show that the release periods of Tirofiban from LCSHFG with 50 microm pores in the chitosan scaffolds are generally 20% or longer more than those with 200 microm pores. The following results were obtained for the system that comprised 50 microm pores. The release periods of Tirofiban from LCSHFG loaded with stearylamine (SA)-liposomes can sustain 20% longer and significantly less burst release (p < 0.01, n = 3) than with liposomes. The release profiles of Tirofiban from LCSHFG change markedly when 0.5 and 2.5% glutaraldehyde is used to cross-link the system. Additionally, for all liposomes, the release periods of Tirofiban from cross-linked LCSHFG with 2.5% glutaraldehyde are 40% or more longer time (e.g., 19 days) with significantly less burst release (p < 0.01, n = 3) than those of noncrosslinked LCSHFG. Notably, the bioactivity of released Tirofiban from LCSHFG that is crosslinked by 2.5% glutaraldehyde effectively inhibits adenosine diphosphate inducing platelet aggregation. The work also suggests that LCSHFG may have potential as a depot drug delivery system for low-molecular-weight hydrophilic drugs.

Accelerating thrombolysis using plasminogen activators (PAs) encapsulated liposomes or PEG microparticles by pressure-driven permeation have been demonstrated in vitro and in vivo in animal models. However, designing and delivering PA-encapsulated nanoparticles (NPs) to enhance thrombolysis by applying electrostatic forces or ligand-receptor interactions between the NPs and blood clots has not been proposed. Therefore, without a pressure-driving factor, tissue-plasminogen activator (t-PA) encapsulated in PLGA NPs with chitosan (CS) and CS-GRGD coating and their thrombolysis capabilities in a blood clot-occluded tube model were evaluated by determining clot lysis times and the masses of the digested clots. The characteristics and release profiles of t-PA-encapsulated PLGA, PLGA/CS and PLGA/CS-GRGD NPs are determined by FT-IR, a laser particle/zeta potential analyzer and HPLC. Additionally, the permeation capacities of the NPs into flat blood clots were examined. For example, the mean particle sizes and encapsulation efficacies of t-PA for the NPs are in the ranges 260-320 nm and 65.5-70.5%, respectively. The results reveal that the NPs for the shortest clot lysis time and the highest weight percentages of digested clot are PLGA/CS (20.7 +/- 0.7 min) and PLGA/CS-GRGD (25.7 +/- 1.3 wt%), respectively. Compared with t-PA solution, the NPs can significantly shorten clot lysis times in the following order: PLGA/CS NPs (38.8 +/- 1.5%) > PLGA/CS-GRGD NPs (16.3 +/- 1.0%) > PLGA NPs (7.7 +/- 1.2%). Compared with t-PA solution, the NPs significantly increase the weight of digested clots in the order, PLGA/CS-GRGD (40.9 +/- 1.5%) > PLGA/CS (27.8 +/- 1.2%) > PLGA (8.6 +/- 0.6%). The highest release rate of t-PA in the fast release phase and the highest permeability into intra-clots of PLGA/CS and PLGA/CS-GRGD NPs, respectively, correspond to the shortest clot lysis time and the largest increase in weight of the digested clots among the NP system. In conclusion, the NPs designed based on new concepts significantly accelerate thrombolysis in vitro in this model, and may be useful in clinical study.

A new PCP tri-block copolymer consisting of poly (e-caprolactone)-b-chitooligosaccharide-b-poly(ethylene glycol) (PCL-b-COS-b-PEG, PCP) was synthesized and characterized. The potential for delivering doxorubicin (DOX), a model drug, with or without genipin crosslinking was evaluated. The PCP copolymers were analyzed by Fourier-transform infrared spectrometry (FT-IR) to confirm the amine and ester groups of the COS and the PCL of the copolymer, respectively. 1 H nuclear magnetic resonance ( 1 H NMR) was performed to determine the structure of the PCP copolymer for demonstrating both PCL and PEG blocks grafted onto the COS block. Moreover, gel permeation chromatography (GPC) was applied to determine the number average molecular weight of the tri-block copolymer ( M n ), which was 11,340 Da/mole. The PCP form polymeric micelles at the critical micelle concentration (CMC) of 0.0107 wt% (or 1.0 μM) with the mean diameter 90 nm, as determined by a dynamic light-scattering (DLS) analyzer. Since PCP micelles contain COS, the zeta potentials of the micelles are changed from a neutral (e.g., −3.2 ± 1.3 mV at pH 7.4) to a cationic state (13.9 ± 4.4 mV at pH 3.0) when pH values of the suspension medium are varied. This change is a unique property of the micelles. In addition, drug delivery behavior of the PCP micelles is influenced by genipin crosslinking COS. The DOX release period of crosslinked micelles is significantly longer than that of the non-crosslinked ones (e.g., 8 days vs. 4 days, respectively) while the burst release of DOX of crosslinked ones is significantly reduced compared with that of non-crosslinked ones. In conclusion, a new tri-block COS-containing PCP copolymer/polymeric micelle has been synthesized and characterized. Moreover, the unique properties of COS-containing PCP micelles are demonstrated by varying zeta potentials via changing pH of medium and by influencing DOX delivering behaviors after genipin crosslinking.

New amine-groups containing tri-block copolymers and micelles that consisting of poly(ε-caprolactone)-b-chitooligosaccharide-b-poly(ethylene glycol) (PCL-b-COS-b-PEG, PCP), were synthesized, characterized, and evaluated for delivering doxorubicin (DOX) with or without crosslinked amine groups by genipin. The characteristics of the PCP copolymers of Fourier-transform infrared spectrometry (FT-IR) verify the amine and ester groups of the COS and the PCL of the copolymers, respectively. 1H nuclear magnetic resonance (1H NMR) spectra verify the structures of the PCP copolymers consisting two PCL and PEG polymers reacted onto the COS block. In addition, gel permeation chromatography (GPC) determines the number average molecular weight of the triblock copolymers (Mn) of approximately 11340 Da/mole. The PCP copolymers can self-assemble to form polymeric micelles at the critical micelle concentration (CMC) of 1.0 μM as determined by the UV-VIS absorption spectra. The mean diameter of the PCP micelles is 90 nm, as determined using a dynamic light-scattering (DLS) analyzer. Moreover, the zeta potentials of PCP micelles change from neutral to cationic state when pH of suspension mediums varied from 7.4 to 3.0. For evaluating delivery characteristics of hydrophobic DOX, it was loaded into PCP micelles with or without crosslinked by genipin. The burst release and release period of DOX for the crosslinked micelles are significantly reduced (P < 0.003, n = 3, for pH = 7.4) and sustained (e.g., 8 days), respectively, than those non-crosslinked ones (e.g., 4 days). In conclusion, new tri-block amine groups containing PCP copolymers are synthesized that can self-assemble as PCP micelles. After post-crosslinked amine groups of DOX loaded the micelles, they can effectively reduce the burst release and sustain the release of DOX at different pH dissolution mediums. Further applications of PCP copolymers and micelles for drug delivery can be explored in future.

A depot drug delivery system, fibrin encapsulated liposome-in-chitosan matrix (FLCM), has been developed to deliver a water-soluble drug which is configured by a porous chitosan matrix containing a bovine fibrin network encapsulated different surface properties of liposomes. Quinacrine (QR), a water-soluble, low-molecular weight fluorescent marker, is used as a model drug to evaluate the delivery characteristics of the system. The SEM photographs show that the fibrin network adheres to the surfaces and pores of the chitosan matrix of a FLCM system. The QR release periods of the FLCM are sustained for about four times longer than those of QR encapsulated into the liposomes. However, the QR release periods and profiles of the FLCM are influenced by the surface properties of liposomes. The release of QR from FLCM is sustained for 9 days for neutral liposomes and only 5 days for PEG modified liposomes (PEG-liposome). After crosslinking the fibrin network of the FLCM with 0.5% of glutaldehyde, the release of QR is further sustained for 17 days with good linear profiles (e.g., 13 days) and with 50% of reduced burst release compared with those of without crosslinking, indicating that the stability of the fibrin network plays an important role on QR release of the system. More interestingly, the release periods and profiles of QR of the FLCM system are highly similar to those of Tirofiban, low-molecular weight of a water-soluble clinical cardiovascular drug, although the study has been done by human platelet poor plasma instead of bovine fibrinogen as a source of fibrin network. It suggests that the QR is a suitable model for investigating the drug delivery behaviors for water-soluble, low-molecular weight drugs of the FLCM. In conclusion, with QR as a model drug, FLCM with crosslinked fibrin network can effectively sustain the release of QR for 17 days but the release profiles are influenced by the surface properties of encapsulated liposomes. This study suggests that FLCM may have the potential as a depot drug delivery system for water-soluble drugs.

The effects of alginate coated on tetracycline (Tc) loaded poly (D, L-lactic-co-glycolic acid) (PLGA) microspheres fabricated by double emulsion solvent evaporation technique for local delivery to periodontal pocket were investigated. Alginate coated PLGA microspheres showed smoother surface but enlarged their particle sizes compared with those of uncoated ones. In addition, alginate coated microspheres enhanced Tc encapsulation efficiency (E.E.) from 11.5 +/- 0.5% of uncoated ones to 17.9 +/- 0.5%. Moreover, all of the coated PLGA microspheres even fabricated at different conditions could prolong Tc release from 9-12 days with 50% or higher in cumulative release of Tc compared with those of uncoated ones. The swelling ratios of PLGA microspheres for alginate coated or uncoated ones, one of the possible mechanisms for enhancing Tc release for the coated ones, were measured. The results showed that 20% or higher in swelling ratio for the coated microspheres at the earlier stage of hydration (e.g. < or = 24 h) could be an important factor to result in high Tc release compared to the uncoated ones. In conclusion, alginate coated Tc loaded PLGA microspheres could enhance Tc delivery to periodontal pocket by enhancing drug encapsulated efficiency, released quantities and sustained release period compared with uncoated ones.

Immobilization of β-cyclodextrin to chitosan beads (Ch-BCD) by cross-linking with 1,6-hexamethylene diisocyanate (HMDI) reagent has been successfully developed and demonstrated that it possessed excellent capacity for cholesterol adsorption from egg yolk. Under optimum conditions, a maximum β-cyclodextrin (BCD) loading of 0.43 g/g-chitosan was obtained. The experimental data on cholesterol adsorption fitted well in the Langmuir isotherm equation with a maximum adsorption capacity ( N m ) of cholesterol 0.33 g cholesterol/g-adsorbent. 92% of the cholesterol was removed from a 30-fold diluted yolk solution at 25 °C using 1% (w/v) Ch-BCD in 2 h. On the other hand, 96% of adsorbed cholesterol could be dissociated from the Ch-BCD using 95% ethanol at 50 °C. In addition, the Ch-BCD retained 84% adsorption capacity after 12 reuses.

This study investigated whether a nanometer scale of surface roughness could improve the adhesion and growth of human endothelial cells on a biomaterial surface. Different molecular weights or chain lengths of polyethylene glycol (PEG) were mixed and then grafted to a polyurethane (PU) surface, a model smooth surface, to form a nanometer (nm) scale of roughness for PU-PEG surfaces (PU-PEG(mix)) while PEG with a molecular weight of 2000 was also grafted to PU to form PU-PEG(2000) for comparison. In addition, the concept was tested on cell-adhesive peptide Gly-Arg-Gly-Asp (GRGD) that was photochemically grafted to PU-PEG(mix) and PU-PEG(2000) surfaces (e.g., PU-PEG(mix)-GRGD and PU-PEG(2000)-GRGD surfaces, respectively). To prepare GRGD-grafted PU-PEG(mix) and PU-PEG(2000) surface, 0.025M of GRGD-SANPAH (N-Succinimidyl-6-[4'-azido-2'-nitrophenylamino]-hexanoate) solutions was grafted to PU-PEG(mix) and PU-PEG(2000) by surface adsorption of the peptide and subsequent ultraviolet (UV) irradiation for photoreaction. The grafting efficiencies for GRGD to PU-PEG(mix) and PU-PEG(2000) surfaces were about 67% for both surfaces, semi-quantitatively analyzed by an HPLC. The surface roughness, presented with a roughness parameter, R(a), and the topography of the tested surfaces were both measured and imaged by an atomic force microscope (AFM). Among the R(a) values of the films, PU was the smoothest (e.g., R(a)=1.53+/-0.20 nm, n=3) while PU-PEG(mix) was the roughest (e.g., R(a)=39.79+/-10.48 nm, n=4). Moreover, R(a) values for PU-PEG(mix) and PU-PEG(mix)-GRGD surfaces were about 20 nm larger than those for PU-PEG(2000) and PU-PEG(2000)-GRGD, respectively, which were consistent with the topographies of the films. Human umbilical vein endothelial cells (HUVECs) were adhered and grown on the tested surfaces after 36 h of incubation. Among the films, HUVEC's adhesion on the surface of PU-PEG(mix)-GRGD was the densest while that on the surface of PU-PEG(2000) was the sparsest. Also, the adhesion and growth of HUVECs for the roughness surfaces were statistically significantly better than that of smooth surface for both GRGD grafted and un-grafted surfaces, respectively. The viability for the growth of HUVECs on the tested surfaces analyzed by MTT assay also confirmed the efficacy of the increased surface roughness. In conclusion, increased surface roughness of biomaterial surfaces even at 10-10(2) nm scale could enhance the adhesion and growth of HUVECs on roughness surfaces that could be useful for applications of tissue engineering.