Delivery of Bioactive Gene Particles via Gelatin-Collagen-PEG-Based Electrospun Matrices.
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ABSTRACT:
SUBMITTER: Tsekoura EK
PROVIDER: S-EPMC8308744 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Electrospinning is a simple and efficient method of fabricating a non-woven polymeric nanofiber matrix. However, using fluorinated alcohols as a solvent for the electrospinning of proteins often results in protein denaturation. TEM and circular dichroism analysis indicated a massive loss of triple-helical collagen from an electrospun collagen (EC) matrix, and the random coils were similar to those found in gelatin. Nevertheless, from mechanical testing we found the Young's modulus and ultimate tensile stresses of EC matrices were significantly higher than electrospun gelatin (EG) matrices because matrix stiffness can affect many cell behaviors such as cell adhesion, proliferation and differentiation. We hypothesize that the difference of matrix stiffness between EC and EG will affect intracellular signaling through the mechano-transducers Rho kinase (ROCK) and focal adhesion kinase (FAK) and subsequently regulates the osteogenic phenotype of MG63 osteoblast-like cells. From the results, we found there was no significant difference between the EC and EG matrices with respect to either cell attachment or proliferation rate. However, the gene expression levels of OPN, type I collagen, ALP, and OCN were significantly higher in MG63 osteoblast-like cells grown on the EC than in those grown on the EG. In addition, the phosphorylation levels of Y397-FAK, ERK1/2, BSP, and OPN proteins, as well as ALP activity, were also higher on the EC than on the EG. We further inhibited ROCK activation with Y27632 during differentiation to investigate its effects on matrix-mediated osteogenic differentiation. Results showed the extent of mineralization was decreased with inhibition after induction. Moreover, there is no significant difference between EC and EG. From the results of the protein levels of phosphorylated Y397-FAK, ERK1/2, BSP and OPN, ALP activity and mineral deposition, we speculate that the mechanism that influences the osteogenic differentiation of MG63 osteoblast-like cells on EC and EG is matrix stiffness and via ROCK-FAK-ERK1/2.
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Project description:Since the first work by Laurencin and colleagues on the development of polymeric electrospinning for biomedical purposes, the use of electrospinning technology has found broad applications in such areas of tissue regeneration and drug delivery. More recently, coaxial electrospinning has emerged as an important technique to develop scaffolds for regenerative engineering incorporated with drug(s). However, the addition of a softer core layer leads to a reduction in mechanical properties. Here, novel robust tripolymeric triaxially electrospun fibrous scaffolds were developed with a polycaprolactone (PCL) (core layer), a 50:50 poly (lactic-co-glycolic acid) (PLGA) (sheath layer) and a gelatin (intermediate layer) with a dual drug delivery capability was developed through modified electrospinning. A sharp increase in elastic modulus after the incorporation of PCL in the core of the triaxial fibers in comparison with uniaxial PLGA (50:50) and coaxial PLGA (50:50) (sheath)-gelatin (core) fibers was observed. Thermal analysis of the fibrous scaffolds revealed an interaction between the core-intermediate and sheath-intermediate layers of the triaxial fibers contributing to the higher tensile modulus. A simultaneous dual release of model small molecule Rhodamine B (RhB) and model protein Fluorescein isothiocynate (FITC) Bovine Serum Albumin (BSA) conjugate incorporated in the sheath and intermediate layers of triaxial fibers was achieved. The tripolymeric, triaxial electrospun systems were seen to be ideal for the support of mesenchymal stem cell growth, as shrinkage of fibers normally found with conventional electrospun systems was minimized. These tripolymeric triaxial electrospun fibers that are biomechanically competent, biocompatible, and capable of dual drug release are designed for regenerative engineering and drug delivery applications.
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Project description:The determination of potential transplantable substrates and substitution cells for corneal endothelium transplantation may compensate for the shortage of cornea donors. Appropriate biodegradable and biocompatible tissue-engineered substratum with seed cells for endothelial keratoplasty has been increasingly studied. In the present study, electrospun gelatin/polycaprolactone (PCL) and collagen/PCL scaffolds were successfully established. Bone marrow endothelial progenitor cells (BEPCs) were cultured on these scaffolds to determine whether the scaffolds may promote the proliferation of BEPCs as well as maintain stem cell characteristics. Two variations of hybrid scaffolds, collagen/PCL (70% collagen and 30% PCL) and gelatin/PCL (70% gelatin and 30% PCL), were established via electrospinning. Microscopic structure, hydrophilicity and wettability of the two scaffolds were subsequently investigated. BEPCs were separately cultured on the scaffolds and were also seeded on glass slides to establish the control group. Furthermore, cell morphology; adherence, as determined by investigation of F-actin expression levels; proliferation, as determined via Cell Counting Kit-8 assays, Ki-67 staining and bromodeoxyuridine (BrdU) staining; and stem cell markers, as determined by cluster of differentiation (CD)-34 and CD-133 protein expression levels; were investigated. In addition, reverse transcription-quantitative polymerase chain reaction (RT-qPCR) was performed to determine gene expression. The two nanofiber scaffolds were established using electrospun techniques with expected hydrophilicity, wettability and biocompatibility. BEPCs were revealed to spread well on and strongly adhere to the collagen/PCL (70:30) and gelatin/PCL (70:30) scaffolds. Furthermore, Ki-67 and BrdU staining results revealed greater levels of positive dots on the two hybrid scaffolds compared with the control group. CD-34 and CD-133 protein staining demonstrated increased levels of fluorescence intensity on scaffolds compared with the control group. Furthermore, increased expression levels of differentiation markers, such as ATP binding cassette subfamily G member 2, leucine rich repeat containing G protein-coupled receptor 5 and CD166, were detected on both scaffolds. RT-qPCR results demonstrated that the expression of caspase-3, which is associated with apoptosis, was decreased on the two scaffolds compared with in the control group. The expression of inflammatory factors, including interleukin (IL)-1, exhibited a significant decrease on the gelatin/PCL scaffold compared with in the control group; whereas the difference between the expression level of IL-1 exhibited by the collagen/PCL group and the control group were not markedly different. Electrospun collagen/PCL and gelatin/PCL scaffolds exhibited the potential to enhance the adherence and proliferation of BEPCs. BEPCs cultured on the two scaffolds demonstrated increased stem cell characteristics and differentiation potential. Electrospun gelatin/PCL and collagen/PCL scaffolds may represent a promising substratum in tissue-engineered corneal endothelium.
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Project description:In the course of this study, a series of novel, biodegradable polyanhydrides based on betulin disuccinate and dicarboxylic derivatives of poly(ethylene glycol) were prepared by two-step polycondensation. These copolymers can be used as carriers in drug delivery systems, in the form of microspheres. Betulin and its derivatives exhibit a broad spectrum of biological activity, including cytotoxic activity, which makes them promising substances for use as therapeutic agents. Microspheres that were prepared from betulin based polyanhydrides show promising properties for use in application in drug delivery systems, including inhalation systems. The obtained copolymers release the active substance-betulin disuccinate-as a result of hydrolysis under physiological conditions. The use of a poly(ethylene glycol) derivative as a co-monomer increases the solubility and bioavailability of the obtained compounds. Microspheres with diameters in the range of 0.5-25 µm were prepared by emulsion solvent evaporation method and their physicochemical and aerodynamic properties were analyzed. The morphological characteristics of the microspheres depended on the presence of poly(ethylene glycol) (PEG) segment within the structure of polyanhydrides. The porosity of the particles depended on the amount and molecular weight of the PEG used and also on the speed of homogenization. The most porous particles were obtained from polyanhydrides containing 20% wt. of PEG 600 by using a homogenization speed of 18,000 rpm.
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Project description:Gemcitabine is a potent anticancer drug approved for the treatment of pancreatic, non-small-cell lung, breast, and ovarian cancers. The major deficiencies of current gemcitabine therapy, however, are its rapid metabolic inactivation and narrow therapeutic window. Herein, we employed polyethylene glycol-b-distearoylphosphatidylethanolamine (PEG-DSPE)/tocopheryl polyethylene glycol 1000 succinate (TPGS) mixed micelles as a delivery system, to improve the pharmacokinetic characteristics of gemcitabine and enhance its antitumor efficacy. By conjugating stearic acid to gemcitabine and subsequently encapsulating stearoyl gemcitabine (GemC18) within PEG-DSPE/TPGS mixed micelles, the deamination of gemcitabine was delayed in vitro and in vivo. Importantly, compared to free gemcitabine, GemC18-loaded micelles pronouncedly prolonged the circulation time of gemcitabine and elevated its concentration in the tumor by 3-fold, resulting in superior antitumor efficacy in mice bearing human pancreatic cancer BxPC-3 xenografts. Our findings demonstrate the promise of PEG-DSPE/TPGS mixed micelles as a nanocarrier system for the delivery of gemcitabine to achieve safer and more efficacious therapeutic outcomes.
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Project description:To examine the influence of substrate topology on the behavior of fibroblasts, tissue engineering scaffolds were electrospun from polycaprolactone (PCL) and a blend of PCL and gelatin (PCL+Gel) to produce matrices with both random and aligned nanofibrous orientations. The addition of gelatin to the scaffold was shown to increase the hydrophilicity of the PCL matrix and to increase the proliferation of NIH3T3 cells compared to scaffolds of PCL alone. The orientation of nanofibers within the matrix did not have an effect on the proliferation of adherent cells, but cells on aligned substrates were shown to elongate and align parallel to the direction of substrate fiber alignment. A microarray of cyotoskeleton regulators was probed to examine differences in gene expression between cells grown on an aligned and randomly oriented substrates. It was found that transcriptional expression of eight genes was statistically different between the two conditions, with all of them being upregulated in the aligned condition. The proteins encoded by these genes are linked to production and polymerization of actin microfilaments, as well as focal adhesion assembly. Taken together, the data indicates NIH3T3 fibroblasts on aligned substrates align themselves parallel with their substrate and increase production of actin and focal adhesion related genes.
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