Designing and enhancing the antifungal activity of corneal specific cell penetrating peptide using gelatin hydrogel delivery system.
ABSTRACT: Background:Fungal keratitis is a major cause of corneal blindness accounting for more than one-third of microbiologically proven cases. The management of fungal keratitis is through topical or systemic antifungal medications alone or in combination with surgical treatment. Topical medications such as natamycin and voriconazole pose major challenges due to poor penetration across the corneal epithelium. To address the issue various carrier molecules like nanoparticles, lipid vesicles, and cell penetrating peptides were explored. But the major drawback such as non-specificity and lack of bioavailability remains. Purpose:In this study, we have attempted to design corneal specific cell penetrating peptide using subtractive proteomic approach from the published literature and tried to improve its bioavailability through gelatin hydrogel delivery system. Material and Methods:Using subtractive proteomic approach two peptides VRF005 and VRF007 were identified on the basis of solubility, cell permeability and amphipathicity. The peptides were modeled for three-dimensional structure and simulated for membrane penetration. The peptides were characterized using circular dichroism spectroscopy, dynamic light scattering and native polyacrylamide gel electrophoresis. Further uptake studies were performed on primary corneal epithelial cells and the stability was analyzed in corneal epithelial tissue lysates. Insilico prediction of peptides showed it to have antifungal activity which was further validated using colony forming assay and time killing kinetics. The duration of antifungal activity of peptide was improved using gelatin hydrogel through sustained delivery. Results:VRF005 and VRF007 showed ?-helical structure and was within the allowed region of Ramachandran plot. The simulation study showed their membrane penetration. The peptide uptake was found to be specific to corneal epithelial cells and also showed intracellular localization in Candida albicans and Fusarium solani. Peptides were found to be stable up to 2 hours when incubated with corneal epithelial tissue lysate. Dynamic light scattering, and native polyacrylamide gel electrophoresis revealed aggregation of peptides. VRF007 showed antifungal activity up to 24 hour whereas VRF005 showed activity up to 4 hours. Hence gelatin hydrogel-based delivery system was used to improve the activity. Actin staining of corneal epithelial cells showed that the cells were attached on gelatin hydrogel. Conclusion:We have designed corneal specific cell penetrating peptides using subtractive proteomic approach. Bioavailability and delivery of peptide was enhanced using gelatin hydrogel system.
Project description:In addition to endocytosis-mediated cellular uptake, hydrophilic cell-penetrating peptides are able to traverse biological membranes in a non-endocytic mode termed transduction, resulting in immediate bioavailability. Here we analysed structural requirements for the non-endocytic uptake mode of arginine-rich cell-penetrating peptides, by a combination of live-cell microscopy, molecular dynamics simulations and analytical ultracentrifugation. We demonstrate that the transduction efficiency of arginine-rich peptides increases with higher peptide structural rigidity. Consequently, cyclic arginine-rich cell-penetrating peptides showed enhanced cellular uptake kinetics relative to their linear and more flexible counterpart. We propose that guanidinium groups are forced into maximally distant positions by cyclization. This orientation increases membrane contacts leading to enhanced cell penetration.
Project description:<h4>Purpose</h4>Elastin-like polypeptide (ELP) is a bioengineered protein widely applied as a drug carrier due to its biocompatibility and amenability to modification with cell-penetrating peptides (CPPs) and therapeutic agents. The purpose of this study was to determine whether topically applied ELP or CPP-fused ELPs penetrate the corneal barrier.<h4>Methods</h4>In vitro binding and cytotoxicity to human corneal epithelial (HCE) cells were determined for ELP or CPP-ELPs. Corneal binding, clearance, and penetration were assessed in a rabbit model following topical application of the fluorescently labeled proteins by quantitative fluorescence imaging and histology.<h4>Results</h4>ELP bound to HCE cells in vitro, and binding/uptake was enhanced 2- to 3-fold by the addition of CPPs. When applied topically to rabbit eyes, ELP accumulated in the cornea at levels 7.4-fold higher than did an equivalent dose of immunoglobulin G. Both ELP and a CPP-ELP penetrated the corneal epithelium and were detectable in the stroma. Addition of CPPs to ELP, however, did not significantly enhance corneal uptake or penetration in vivo relative to ELP alone. The polypeptides cleared from the cornea over a period of 20-30?min after application, after which cornea levels reached a steady state of 15-30??g/mL for up to 3?h.<h4>Conclusions</h4>The ELP drug carrier can penetrate the corneal epithelium and accumulate in the stroma. Given its amenability for fusion to multiple types of therapeutic agents, ELP has the potential to serve as a drug carrier for topical ocular applications.
Project description:Fungal keratitis (FK) remains a severe eye disease, and effective therapies are limited by drug shortages and critical ocular barriers. Despite the high antifungal potency and broad spectrum of econazole, its strong irritant and insolubility in water hinder its ocular application. We designed and fabricated a new drug delivery system based on a polymeric vector for the ocular antifungal application of econazole. This novel system integrates the advantages of its constituent units and exhibits superior comprehensive performance. Using the new system, drug content was significantly increased more than 600 folds. The results of in vivo and in vitro experiments demonstrated that the econazole-loaded formulation exhibited significantly enhanced corneal penetration after a single topical ocular administration, excellent antifungal activity, and good tolerance in rabbits. Drug concentrations and ocular relative bioavailability in the cornea were 59- and 29-time greater than those in the control group, respectively. Following the topical administration of one eye drop (50??L of 0.3% w/v econazole) in fungus-infected rabbits, a high concentration of antimycotic drugs in the cornea and aqueous humor was sustained and effective for 4?h. The mechanism of corneal penetration was also explored using dual fluorescent labeling. This novel drug delivery system is a promising therapeutic approach for oculomycosis and could serve as a candidate strategy for use with various hydrophobic drugs to overcome barriers in the treatment of many other ocular diseases.
Project description:The basement membrane is a specialized extracellular matrix substrate responsible for support and maintenance of epithelial and endothelial structures. Engineered basement membrane-like hydrogel systems have the potential to advance understanding of cell-cell and cell-matrix interactions by allowing precise tuning of the substrate or matrix biochemical and biophysical properties. In this investigation, we developed tunable hydrogel substrates with conjugated bioactive peptides to modulate cell binding and growth factor signaling by endothelial cells. Hydrogels were formed by employing a poly(ethylene glycol) crosslinker to covalently crosslink gelatin polymers and simultaneously conjugate laminin-derived YIGSR peptides or vascular endothelial growth factor (VEGF)-mimetic QK peptides to the gelatin. Rheological characterization revealed rapid formation of hydrogels with similar stiffnesses across tested formulations, and swelling analysis demonstrated dependency on peptide and crosslinker concentrations in hydrogels. Levels of phosphorylated VEGF Receptor 2 in cells cultured on hydrogel substrates revealed that while human umbilical vein endothelial cells (HUVECs) responded to both soluble and conjugated forms of the QK peptide, conditionally-immortalized human glomerular endothelial cells (GEnCs) only responded to the conjugated presentation of the peptide. Furthermore, whereas HUVECs exhibited greatest upregulation in gene expression when cultured on YIGSR- and QK-conjugated hydrogel substrates after 5 days, GEnCs exhibited greatest upregulation when cultured on Matrigel control substrates at the same time point. These results indicate that conjugation of bioactive peptides to these hydrogel substrates significantly influenced endothelial cell behavior in cultures but with differential responses between HUVECs and GEnCs.
Project description:Among enhancing strategies proposed in ocular drug delivery, a rising interest is directed to cell penetrating peptides (CPPs), amino acid short sequences primarily known for their intrinsic ability to cell internalization and, by extension, to cross biological barriers. In fact, CPPs may be considered as carrier for delivering therapeutic agents across biological membranes, including ocular tissues. Several CPPs have been proposed in ophthalmic delivery, and, among them, penetratin (PNT), a 16-amino acids natural peptide, stands out. Therefore, we describe the synthesis via the mimotopic approach of short fluorescently labeled analogues of both PNT and its reversed sequence PNT-R. Their ability to cross ocular membranes was checked ex vivo using freshly explanted porcine cornea. Furthermore, some sequences were studied by circular dichroism. Despite the hydrophilic nature and the relatively high molecular weight (approx. 1.6 kDa), all analogues showed a not negligible trans-corneal diffusion, indicating a partial preservation of penetration activity, even if no sequences reached the noteworthy ability of PNT. It was not possible to find a correlation between structure and corneal penetration ability, and further studies, exploring peptides distribution within corneal layers, for example using imaging techniques, deserve to be performed to figure out a possible difference in intracellular delivery.
Project description:Generally, topically applied eye drops have low bioavailability due to short residence time and low penetration of the drug. The aim of the present study was to incorporate dexamethasone (DXM) into nano lipid carriers (NLC), which contain mucoadhesive polymer, in order to increase the bioavailability of the drug. A 23 factorial experimental design was applied, in which the three factors were the polymer, the DXM, and the emulsifier concentrations. The samples were analyzed for particle size, zeta potential, polydispersity index, and Span value. The significant factors were identified. The biocompatibility of the formulations was evaluated with human corneal toxicity tests and immunoassay analysis. The possible increase in bioavailability was analyzed by means of mucoadhesivity, in vitro drug diffusion, and different penetration tests, such as in vitro cornea PAMPA model, human corneal cell penetration, and ex vivo porcine corneal penetration using Raman mapping. The results indicated that DXM can be incorporated in stable mucoadhesive NLC systems, which are non-toxic and do not have any harmful effect on cell junctions. Mucoadhesive NLCs can create a depot on the surface of the cornea, which can predict improved bioavailability.
Project description:To optimally penetrate biological hydrogels such as mucus and the tumor interstitial matrix, nanoparticles (NPs) require physicochemical properties that would typically preclude cellular uptake, resulting in inefficient drug delivery. Here, we demonstrate that (poly(lactic-co-glycolic acid) (PLGA) core)-(lipid shell) NPs with moderate rigidity display enhanced diffusivity through mucus compared with some synthetic mucus penetration particles (MPPs), achieving a mucosal and tumor penetrating capability superior to that of both their soft and hard counterparts. Orally administered semi-elastic NPs efficiently overcome multiple intestinal barriers, and result in increased bioavailability of doxorubicin (Dox) (up to 8 fold) compared to Dox solution. Molecular dynamics simulations and super-resolution microscopy reveal that the semi-elastic NPs deform into ellipsoids, which enables rotation-facilitated penetration. In contrast, rigid NPs cannot deform, and overly soft NPs are impeded by interactions with the hydrogel network. Modifying particle rigidity may improve the efficacy of NP-based drugs, and can be applicable to other barriers.
Project description:Alginate hydrogel is a popular biologically inert material that is widely used in 3D bioprinting, especially in extrusion-based printing. However, the printed cells in this hydrogel could not degrade the surrounding alginate gel matrix, causing them to remain in a poorly proliferating and non-differentiating state. Here, we report a novel study of the 3D printing of human corneal epithelial cells (HCECs)/collagen/gelatin/alginate hydrogel incubated with a medium containing sodium citrate to obtain degradation-controllable cell-laden tissue constructs. The 3D-printed hydrogel network with interconnected channels and a macroporous structure was stable and achieved high cell viability (over 90%). By altering the mole ratio of sodium citrate/sodium alginate, the degradation time of the bioprinting constructs can be controlled. Cell proliferation and specific marker protein expression results also revealed that with the help of sodium citrate degradation, the printed HCECs showed a higher proliferation rate and greater cytokeratin 3(CK3) expression, indicating that this newly developed method may help to improve the alginate bioink system for the application of 3D bioprinting in tissue engineering.
Project description:Regeneration of corneal stroma has always been a challenge due to its sophisticated structure and keratocyte-fibroblast transformation. In this study, we fabricate grid poly (?-caprolactone)-poly (ethylene glycol) microfibrous scaffold and infuse the scaffold with gelatin methacrylate (GelMA) hydrogel to obtain a 3 D fiber hydrogel construct; the fiber spacing is adjusted to fabricate optimal construct that simulates the stromal structure with properties most similar to the native cornea. The topological structure (3 D fiber hydrogel, 3 D GelMA hydrogel, and 2 D culture dish) and chemical factors (serum, ascorbic acid, insulin, and ?-FGF) are examined to study their effects on the differentiation of limbal stromal stem cells to keratocytes or fibroblasts and the phenotype maintenance, in vitro and in vivo tissue regeneration. The results demonstrate that fiber hydrogel and serum-free media synergize to provide an optimal environment for the maintenance of keratocyte phenotype and the regeneration of damaged corneal stroma.
Project description:Biomaterial vascularization remains a major focus in the field of tissue engineering. Biomaterial culture of endometrial cells is described as a platform to inform the design of proangiogenic biomaterials. The endometrium undergoes rapid growth and shedding of dense vascular networks during each menstrual cycle mediated via estradiol and progesterone in vivo. Cocultures of endometrial epithelial and stromal cells encapsulated within a methacrylamide-functionalized gelatin hydrogel are employed. It is reported that proangiogenic gene expression profiles and vascular endothelial growth factor production are hormone dependent in endometrial epithelial cells, but that hormone signals have no effect on human telomerase reverse transcriptase (hTERT)-immortalized endometrial stromal cells. This study subsequently examines whether the magnitude of epithelial cell response is sufficient to induce changes in human umbilical vein endothelial cell network formation. Incorporation of endometrial stromal cells improves vessel formation, but co-culture with endometrial epithelial cells leads to a decrease in vascular formation, suggesting the need for stratified cocultures of endometrial epithelial and stromal cells with endothelial cells. Given the transience of hormonal signals within 3D biomaterials, the inclusion of sex hormone binding globulin (SHBG) to alter the bioavailability of estradiol within the hydrogel is reported, demonstrating a strategy to reduce diffusive losses via SHBG-mediated estradiol sequestration.