ABSTRACT
The aim of the research was to check whether it is possible to use fragments of type IV collagen to obtain, as a result of self-assembling, stable spatial structures that could be used to prepare new materials useful in regenerative medicine. Collagen IV fragments were obtained by using DMT/NMM/TosO- as a coupling reagent. The ability to self-organize and form stable spatial structures was tested by the CD method and microscopic techniques. Biological studies covered: resazurin assay (cytotoxicity assessment) on BJ, BJ-5TA and C2C12 cell lines; an alkaline version of the comet assay (genotoxicity), Biolegend Legendplex human inflammation panel 1 assay (SC cell lines, assessment of the inflammation activity) and MTT test to determine the cytotoxicity of the porous materials based on collagen IV fragments. It was found that out of the pool of 37 fragments (peptides 1-33 and 2.1-2.4) reconstructing the outer sphere of collagen IV, nine fragments (peptides: 2, 4, 5, 6, 14, 15, 25, 26 and 30), as a result of self-assembling, form structures mimicking the structure of the triple helix of native collagens. The stability of spatial structures formed as a result of self-organization at temperatures of 4 °C, 20 °C, and 40 °C was found. The application of the MST method allowed us to determine the Kd of binding of selected fragments of collagen IV to ITGα1ß1. The stability of the spatial structures of selected peptides made it possible to obtain porous materials based on their equimolar mixture. The formation of the porous materials was found for cross-linked structures and the material stabilized only by weak interactions. All tested peptides are non-cytotoxic against all tested cell lines. Selected peptides also showed no genotoxicity and no induction of immune system responses. Research on the use of porous materials based on fragments of type IV collagen, able to form stable spatial structures as scaffolds useful in regenerative medicine, will be continued.
Subject(s)
Biocompatible Materials/metabolism , Collagen Type IV/metabolism , Peptides/metabolism , Animals , Biocompatible Materials/chemical synthesis , Biocompatible Materials/chemistry , Cell Line , Cell Survival/drug effects , Collagen Type IV/chemical synthesis , Collagen Type IV/chemistry , Humans , Integrins/metabolism , Materials Testing , Mice , Peptides/chemical synthesis , Peptides/chemistry , Regenerative MedicineABSTRACT
BACKGROUND: Vectors are essential for successful gene delivery. In the present study, a tumor-targeting cationic gene vector, known as the disulfide cross-linked arginine-aspartic acid peptide modified by HAIYPRH (T7) peptide (CRD-PEG-T7), was designed for targeted delivery of plasmid DNA (pDNA) for gene therapy of prostate cancer (PCa). METHODS: The structure of CRD-PEG-T7 was determined and the cellular uptake efficacy, gene transfection efficacy, cytotoxicity, and the targeting effect of the CRD-PEG-T7-plasmid DNA complex were examined. RESULTS: The results demonstrated that the CRD-PEG-T7-plasmid DNA complex was nanosized and had a positively charged surface, good cellular uptake efficacy, minimal cytotoxicity, and a dual-targeting effect as compared with the CRD-PEG-plasmid DNA complex. The peptide T7-modifed new delivery system was able to target the highly expressed transferrin receptor (TfR) on tumor cells with an efficiency four-fold higher than that of the non-modified system. CONCLUSION: The results above indicatd that the CRD-PEG-T7-plasmid DNA complex may prove to be a promising gene delivery system targeting bone-metastatic tumor.
Subject(s)
Collagen Type IV/chemistry , DNA/administration & dosage , DNA/genetics , Disulfides/chemistry , Gene Transfer Techniques , Genetic Therapy , Peptide Fragments/chemistry , Plasmids/administration & dosage , Prostatic Neoplasms/therapy , Animals , Benzoxazoles/chemistry , Cell Death , Cell Line, Tumor , Cell Movement , Collagen Type IV/chemical synthesis , Endocytosis , Humans , Male , Mice, Inbred BALB C , Mice, Nude , Particle Size , Peptide Fragments/chemical synthesis , Polyethylene Glycols/chemical synthesis , Polyethylene Glycols/chemistry , Prostatic Neoplasms/pathology , Proton Magnetic Resonance Spectroscopy , Quinolinium Compounds/chemistry , Static Electricity , Tissue Distribution , Transfection , Transferrin/chemistryABSTRACT
Nanofiber-based hydrogels (nanogels) with different, covalently bound peptides were used as an extracellular environment for lens epithelial cells (LECs) in order to modulate the capsular opacification (CO) response after lens surgery in a porcine eye model. Lenses were divided into 15 groups (n = 4 per group), the lens content was removed and the empty capsules were refilled with nanogel without peptides and nanogels with 13 combinations of 5 different peptides: two laminin-derived, two fibronectin-derived, and one collagen IV-derived peptide representing cell adhesion motifs. A control group of 4 lenses was refilled with hyaluronan. After refilling, lenses were extracted from the porcine eye and cultured for three weeks. LECs were assessed for morphology and alpha smooth muscle actin (αSMA) expression using confocal laser scanning microscopy. Compared to hyaluronan controls, lenses filled with nanogel had less CO formation, indicated by a lower αSMA expression (P = 0.004). Microscopy showed differences in morphological cell response within the nanogel refilled groups. αSMA expression in these groups was highest in lenses refilled with nanogel without peptides (9.54 ± 11.29%). Overall, LEC transformation is reduced by the presence of nanogels and the response is improved even further by incorporation of extracellular matrix peptides representing adhesion motifs. Thus, nanomaterials targeting biological pathways, in our case interactions with integrin signaling, are a promising avenue toward reduction of CO. Further research is needed to optimize nanogel-peptide combinations that fully prevent CO.
Subject(s)
Capsule Opacification/prevention & control , Epithelial Cells/cytology , Epithelial-Mesenchymal Transition/drug effects , Extracellular Matrix Proteins/administration & dosage , Hydrogels , Lens Capsule, Crystalline/cytology , Oligopeptides/administration & dosage , Actins/metabolism , Animals , Biomarkers/metabolism , Capsule Opacification/pathology , Collagen Type IV/administration & dosage , Collagen Type IV/chemical synthesis , Drug Delivery Systems , Extracellular Matrix Proteins/chemical synthesis , Fibronectins/administration & dosage , Fibronectins/chemical synthesis , Fluorescent Antibody Technique, Indirect , Laminin/administration & dosage , Laminin/chemical synthesis , Lens, Crystalline/cytology , Nanofibers , Oligopeptides/chemical synthesis , Organ Culture Techniques , Sus scrofaABSTRACT
Collagen type IV provides a biomechanically stable scaffold into which the other constituents of basement membranes are incorporated, but it also plays an important role in cell adhesion. This occurs with collagen type IV mainly via the alpha1beta1 integrin, and the proposed epitope involved in this type of collagen/integrin interaction corresponds to a non-sequential R/Xaa/D motif, where the arginine and aspartate residues are provided by the alpha2 and alpha1 chains of the collagen molecule, respectively. Since the stagger of the three alpha chains in native collagen type IV is still unknown and different alignments of the chains lead to different spatial epitopes, two heterotrimeric collagen peptides containing the natural 457-469 sequences of the cell adhesion site were synthesized in which the single chains were assembled via disulfide bonds into the two most plausible alpha1alpha2alpha1' and alpha2alpha1alpha1' registers. The differentiated triple-helical stabilities of the two heterotrimers suggest a significant structural role of the chain register in collagen, although the binding to alpha1beta1 integrin is apparently less affected as indicated by preliminary experiments.