Direct Cardiac Epigenetic Reprogramming through Codelivery of 5'Azacytidine and miR-133a Nanoformulation.

Direct reprogramming of cardiac fibroblasts to induced cardiomyocytes (iCMs) is a promising approach to cardiac regeneration. However, the low yield of reprogrammed cells and the underlying epigenetic barriers limit its potential. Epigenetic control of gene regulation is a primary factor in maintaining cellular identities. For instance, DNA methylation controls cell differentiation in adults, establishing that epigenetic factors are crucial for sustaining altered gene expression patterns with subsequent rounds of cell division. This study attempts to demonstrate that 5'AZA and miR-133a encapsulated in PLGA-PEI nanocarriers induce direct epigenetic reprogramming of cardiac fibroblasts to cardiomyocyte-like cells. The results present a cardiomyocyte-like phenotype following seven days of the co-delivery of 5'AZA and miR-133a nanoformulation into human cardiac fibroblasts. Further evaluation of the global DNA methylation showed a decreased global 5-methylcytosine (5-medCyd) levels in the 5'AZA and 5'AZA/miR-133a treatment group compared to the untreated group and cells with void nanocarriers. These results suggest that the co-delivery of 5'AZA and miR-133a nanoformulation can induce the direct reprogramming of cardiac fibroblasts to cardiomyocyte-like cells in-vitro, in addition to demonstrating the influence of miR-133a and 5'AZA as epigenetic regulators in dictating cell fate.

Biological Synthesis of Bioactive Gold Nanoparticles from Inonotus obliquus for Dual Chemo-Photothermal Effects against Human Brain Cancer Cells.

The possibility for an ecologically friendly and simple production of gold nanoparticles (AuNPs) with Chaga mushroom (Inonotus obliquus) (Ch-AuNPs) is presented in this study. Chaga extract's reducing potential was evaluated at varied concentrations and temperatures. The nanoparticles synthesized were all under 20 nm in size, as measured by TEM, which is a commendable result for a spontaneous synthesis method utilizing a biological source. The Ch-AuNPs showed anti-cancer chemotherapeutic effects on human brain cancer cells which is attributed to the biofunctionalization of the AuNPs with Chaga bioactive components during the synthesis process. Further, the photothermal ablation capability of the as-prepared gold nanoparticles on human brain cancer cells was investigated. It was found that the NIR-laser induced thermal ablation of cancer cells was effective in eliminating over 80% of the cells. This research projects the Ch-AuNPs as promising, dual modal (chemo-photothermal) therapeutic candidates for anti-cancer applications.

GANT61 and curcumin-loaded PLGA nanoparticles for GLI1 and PI3K/Akt-mediated inhibition in breast adenocarcinoma.

Current conventional mono and combination therapeutic strategies often fail to target breast cancer tissue effectively due to tumor heterogeneity comprising cancer stem cells (CSCs) and bulk tumor cells. This is further associated with drug toxicity and resistivity in the long run. A nanomedicine platform incorporating combination anti-cancer treatment might overcome these challenges and generate synergistic anti-cancer effects and also reduce drug toxicity. GANT61 and curcumin were co-delivered via polymeric nanoparticles (NPs) for the first time to elicit enhanced anti-tumor activity against heterogeneous breast cancer cell line MCF-7. We adopted the single-emulsion-solvent evaporation method for the preparation of the therapeutic NPs. The GANT61-curcumin PLGA NPs were characterized for their size, shape and chemical properties, and anti-cancer cell studies were undertaken for the plausible explanation of our hypothesis. The synthesized GANT61-curcumin PLGA NPs had a spherical, smooth surface morphology, and an average size of 347.4 d. nm. The NPs induced cytotoxic effects in breast cancer cells at a mid-minimal dosage followed by cell death via autophagy and apoptosis, reduction in their target protein expression along with compromising the self-renewal property of CSCs as revealed by their in vitro cell studies. The dual-drug NPs thus provide a novel perspective on aiding existing anti-cancer nanomedicine therapies to target a heterogeneous tumor mass effectively.

Collagen Functionalization of Polymeric Electrospun Scaffolds to Improve Integration into Full-Thickness Wounds.

BACKGROUND: Electrospun fibers are widely studied in regenerative medicine for their ability to mimic the extracellular matrix (ECM) and provide mechanical support. In vitro studies indicated that cell adhesion and migration is superior on smooth poly(L-lactic acid) (PLLA) electrospun scaffolds and porous scaffolds once biofunctionalized with collagen. METHODS: The in vivo performance of PLLA scaffolds with modified topology and collagen biofunctionalization in full-thickness mouse wounds was assessed by cellular infiltration, wound closure and re-epithelialization and ECM deposition. RESULTS: Early indications suggested unmodified, smooth PLLA scaffolds perform poorly, with limited cellular infiltration and matrix deposition around the scaffold, the largest wound area, a significantly larger panniculus gape, and lowest re-epithelialization; however, by day 14, no significant differences were observed. Collagen biofunctionalization may improve healing, as collagen-functionalized smooth scaffolds were smallest overall, and collagen-functionalized porous scaffolds were smaller than non-functionalized porous scaffolds; the highest re-epithelialization was observed in wounds treated with collagen-functionalized scaffolds. CONCLUSION: Our results suggest that limited incorporation of smooth PLLA scaffolds into the healing wound occurs, and that altering surface topology, particularly by utilizing collagen biofunctionalization, may improve healing. The differing performance of the unmodified scaffolds in the in vitro versus in vivo studies demonstrates the importance of preclinical testing.

Scaffold mediated delivery of dual miRNAs to transdifferentiate cardiac fibroblasts.

The standard scaffold-mediated delivery of drugs/biomolecules has been successful due to the unique attributes of scaffolds, specifically the electrospun polymeric scaffolds that mimic ECM are well suited for advanced regenerative applications. Cardiac tissue engineering includes the interpretation of cellular and molecular mechanisms concerning heart regeneration and identifying an efficient reprogramming strategy to overcome the limitation of delivery systems and enhance the reprogramming efficiency. This study is a step towards developing a functional scaffold through a parallel interpretation of electrospun PLLA scaffolds with two distinct topologies to achieve sustained delivery of two muscle-specific microRNAs (miR-1 and miR-133a) to directly reprogram the adult human cardiac fibroblasts into cardiomyocyte-like cells. Polyethyleneimine was used to form stable PEI-miRNA complexes through electrostatic interactions. These complexes were immobilized on the electrospun smooth and porous scaffolds, where a loading efficiency of ~96% for the fibronectin modified and ~38% for unmodified surfaces was observed, regardless of their surface topology. The in-vitro release experiment exhibited a biphasic release pattern of PEI-miRNA polyplexes from the scaffolds. These dual miRNA scaffold systems proved to be an excellent formulation since their combinatorial effect involving the topographic cues of electrospun fibers, and dual miRNAs helped control the cardiac fibroblast cell fate precisely.

Direct Cardiac Reprogramming with Engineered miRNA Scaffolds.

Ischemic heart disease is a predominant cause of death worldwide. The loss or death of cardiomyocytes due to restricted blood flow often results in a cardiac injury. Myofibroblasts replace these injured cardiomyocytes to preserve structural integrity. However, the depleted cardiomyocytes lead to cardiac dysfunction such as pathological cardiac dilation, reduced cardiac contraction, and fibrosis. Repair and regeneration of myocardium are the best possible therapy for end-stage heart failure patients because the current cardiomyocytes restoration therapies are limited to heart transplantation only. The emergence of interests to directly reprogram a mammalian heart with minimal regenerative capacity holds a promising future in the field of cardiovascular regenerative medicine. Repair and regeneration become the two crucial factors in the field of cardiovascular regenerative medicine since heart muscles have no substitutes, like heart valves or blood vessels. Cardiac regeneration includes strategies to reprogram with diverse factors like small molecules, genetic and epigenetic regulators. However, there are some constraints like low efficacy, immunogenic problems, and unsafe delivery systems that pose a daunting challenge in human trial translations. Hence, there is a need for a holistic nanoscale approach in regulating cell fate effectively and efficiently with a safer delivery and a suitable microenvironment that mimics the extracellular matrix. In this review, we have discussed the current state-of-the-art techniques, challenges in direct reprogramming of fibroblasts to cardiac muscle, and prospects of biomaterials in miRNA delivery and cardiac regeneration predominantly during the past decade (2008-2019).

Collagen-functionalized electrospun smooth and porous polymeric scaffolds for the development of human skin-equivalent.

Electrospun polymer fibers have garnered substantial importance in regenerative medicine owing to their intrinsic 3D topography, extracellular matrix microenvironment, biochemical flexibility, and mechanical support. In particular, a material's nano-topography can have a significant effect on cellular responses, including adhesion, proliferation, differentiation, and migration. In this study, poly(l-lactic acid) (PLLA), a biodegradable polymer with excellent biocompatibility was electrospun into fibers with either smooth or porous topologies. The scaffolds were further modified and biofunctionalized with 0.01% and 0.1% collagen to enhance bioactivity and improve cellular interactions. Human keratinocytes (HaCaTs) and fibroblasts (human foreskin fibroblasts-HFF) were cultured on the scaffolds using a modified co-culture technique, where keratinocytes were grown on the dorsal plane for 5 days, followed by flipping, seeding with fibroblasts on the ventral plane and culturing for a further 5 days. Following this, cellular adhesion of the skin cells on both the unmodified and collagen-modified scaffolds (smooth and porous) was performed using scanning electron microscopy (SEM) and immunofluorescence. Distinct outcomes were observed with the unmodified smooth scaffolds showing superior cell adhesion than the porous scaffolds. Modification of the porous and smooth scaffolds with 0.1% collagen enhanced the adhesion and migration of both keratinocytes and fibroblasts to these scaffolds. Further, the collagen-modified scaffolds (both porous and smooth) produced confluent and uniform epidermal sheets of keratinocytes on one plane with healthy fibroblasts populated within the scaffolds. Thus, presenting a vast potential to serve as a self-organized skin substitute this may be a promising biomaterial for development as a dressing for patients suffering from wounds.

ECM Mimetic Electrospun Porous Poly (L-lactic acid) (PLLA) Scaffolds as Potential Substrates for Cardiac Tissue Engineering.

Cardiac tissue engineering (CTE) aims to generate potential scaffolds to mimic extracellular matrix (ECM) for recreating the injured myocardium. Highly porous scaffolds with properties that aid cell adhesion, migration and proliferation are critical in CTE. In this study, electrospun porous poly (l-lactic acid) (PLLA) porous scaffolds were fabricated and modified with different ECM derived proteins such as collagen, gelatin, fibronectin and poly-L-lysine. Subsequently, adult human cardiac fibroblasts (AHCF) were cultured on the protein modified and unmodified fibers to study the cell behavior and guidance. Further, the cytotoxicity and reactive oxygen species (ROS) assessments of the respective fibers were performed to determine their biocompatibility. Excellent cell adhesion and proliferation of the cardiac fibroblasts was observed on the PLLA porous fibers regardless of the surface modifications. The metabolic rate of cells was on par with the conventional cell culture ware while the proliferation rate surpassed the latter by nearly two-folds. Proteome profiling revealed that apart from being an anchorage platform for cells, the surface topography has modulated significant expression of the cellular proteome with many crucial proteins responsible for cardiac fibroblast growth and proliferation.

Bioactive bacterial cellulose sulfate electrospun nanofibers for tissue engineering applications.

Cellulosic materials have been of tremendous importance to mankind since its discovery due to its superior properties and its abundance in nature. Recently, an increase in demand for alternate green materials has rekindled the interest for cellulosic materials. Here, bacterial cellulose has been functionalized with sulfate groups through acetosulfation to gain solubility in aqueous media, which provides access to several applications. The cell viability, antioxidant, and hemocompatibility assays have verified the biocompatible and antioxidant characteristics of bacterial cellulose sulfate (BCS) in both in vitro and ex vivo conditions. Further, novel BCS/polyvinyl alcohol nanofibers were fabricated by simple electrospinning route to engineer ultrafine nanoscale fibers. The biological evaluation of BCS/polyvinyl alcohol nanofiber scaffolds was done using L929 mouse fibroblast cells, which confirmed that these nanofibers are excellent matrices for cell adhesion and proliferation.

Extremophilic polysaccharide nanoparticles for cancer nanotherapy and evaluation of antioxidant properties.

Polysaccharides that show finest bioactivities and physicochemical properties are always promising for bionanoscience applications. Mauran is such a macromolecule extracted from halophilic bacterium, Halomonas maura for biotechnology and nanoscience applications. Antioxidant properties of MR/CH nanoparticles were studied using biochemical assays to prove the versatility of these test nanoparticles for biomedical applications. Here, we demonstrate the prospects of extremophilic polysaccharide, mauran based nanoparticles for scavenging reactive oxygen species in both in vitro and ex vivo conditions. 5-fluorouracil loaded MR/CH nanoparticles were tested for anticancer proliferation and compared their therapeutic efficiency using breast adenocarcinoma and glioma cells. Fluorescently labeled nanoparticles were employed to show the cellular uptake of these nanocarriers using confocal microscopic imaging and flow cytometry.

In vitro evaluation of antioxidant defense mechanism and hemocompatibility of mauran.

Mauran (MR), a highly polyanionic sulfated exopolysaccharide was extracted from moderately halophilic bacterium; Halomonas maura and characterized using X-ray photoelectron spectroscopy and Fourier transform infrared spectroscopy. Purified MR was evaluated for antioxidant defense mechanisms under in vitro conditions using L929, mouse fibroblast cell line and mice liver homogenate. It was demonstrated that MR could impart protective effect against oxidative stress in both cells and tissue up to a concentration of 500 µg, which is found to be safe under laboratory conditions. Various enzymatic and non-enzymatic parameters of antioxidant mechanisms were evaluated and concluded that MR has the tendency to maintain a balance of antioxidative enzymes with in the test systems studied. Also, hemocompatibility assay performed revealed that MR has a lesser hemolytic index and exhibited a prolonged clotting time, which shows both antihemolytic, and antithrombogenic nature respectively. Furthermore, absorption studies performed using fluorescent-labeled MR confirmed that MR accumulated within the cell cytoplasm neither induced cellular lysis nor affected the cell integrity.