Role of inorganic ions in wound healing: an insight into the various approaches for localized delivery.

Recently, the role of inorganic ions has been explored for its wound-healing applications. Ions do play key role in the normal functioning of the skin, including the epidermal barrier property, maintaining redox balance, enzymatic activities, tissue remodeling, etc. The care of chronic wounds is a concern and new cost-effective therapeutic strategies that modulate the wound microenvironment and cell behaviour are needed. First, this review illustrates the ions that play a role in wound healing and their molecular mechanisms that are accountable for modifying the wound. Further, the emerging strategies using metal ions to modulate the healing will be discussed. In this direction, localized delivery of inorganic ions of importance using advanced wound care biomaterials for wound healing applications is discussed.

The wound repair and regeneration process is regarded as a highly complex biological process that involves multiple phases. Chronic wounds are those that get stalled at the initial phases of wound healing. This review discusses the role of inorganic ions in wound healing and its therapeutic potential that could improve healing outcomes in chronic wounds. Utilizing inorganic ions in innovative ways to modulate the wound-healing processes is an emerging field. In this regard, the review paper also encompasses a comprehensive overview of numerous delivery approaches for the localized release of metal ions to the wound site.

Ascorbic acid-loaded gellan-g-poly(ethylene glycol) methacrylate matrix as a wound-healing material.

Ascorbic acid (AA) is one of the important biomolecules involved in all phases of wound healing. The aim of this study was to develop a new hydrogel system that offers topical delivery of ascorbic acid to wounds during wound care management. In this work, we grafted poly (ethylene glycol) methacrylate onto a renewable biopolymer gellan, and the graft copolymer (GPMA) formed was crosslinked covalently and ionically, and used as a matrix for delivering AA to the wounds. By the processes of grafting and crosslinking, the mechanical properties of the gellan increased several fold compared to mechanically weak native gellan. In vitro cytotoxicity evaluation showed that GPMA was non-cytotoxic to fibroblast cells. GPMA hydrogel matrix allowed the sustained release of AA. When AA was incorporated in GPMA, a significant improvement in wound closure was observed in scratch wound assay performed with keratinocytes. Since AA acts as a cofactor in collagen synthesis, the controlled delivery of AA to the wound microenvironment favors the up-regulation of colα1 gene expression. This study revealed that ascorbic acid, at a concentration of 150 µM, has a favorable impact on wound healing when tested in vitro. Overall results indicate that the GPMA matrix could be a promising material for wound healing applications.

Proline conjugated chitosan as wound healing material: In vitro studies on the influence of the scaffold on collagen production and wound healing.

The present study reports the development of L-proline conjugated chitosan scaffold for wound healing application. Proline plays a beneficial role in collagen synthesis, and as a biochemical, it has the potential to modulate wound healing. In this regard, amino acid L-proline was conjugated onto chitosan, and the scaffolds were synthesised. FTIR and NMR analysis confirmed amino acid conjugation. The prepared scaffold was characterized by studies such as swelling, dissolution, tensile strength, porosity, water-vapor transmission rate and in-vitro healing properties. Cell viability assay showed that the scaffold has no cytotoxicity against the L929 and HaCaT cells. The in-vitro wound healing potential of the scaffold by scratch wound assay on the L929 cell line showed 53.35 ± 2.3 %, 72.96 ± 2.2 %, and 50.89 ± 0.3 % wound closure for CS-P 200, CS-P 400 and CS-P 600, respectively when compared to native CS scaffold (38.86 ± 1.6 %). A similar observation was found with HaCaT cells too. The studies showed that the modified scaffold promotes collagen deposition from fibroblast cells. These findings suggest that scaffold cues remodel the wound microenvironment for a better wound-healing state, and the L-proline conjugated scaffold may have considerable potential as a wound dressing to improve wound healing.

Synthesis and evaluation of an alginate-methacrylate xerogel for insulin delivery towards wound healing applications.

Background: Alginate is one of the most widely used biopolymer for wound healing. But poor mechanical strength and degradability limits its application especially as a drug-delivery matrix. The aim of this study was to develop stable alginate based scaffold for insulin delivery toward wound care. Materials & methods: The xerogel alginate-g-poly (methacrylic acid; AGM2S) was characterized by various analytical techniques. Results: AGM2S xerogel showed improved physical stability, low degradation, good swelling and water vapour transmission rate (WVTR). About 70% of insulin was released from loaded xerogel over a period of 48 h and favorably modulated the healing response in in vitro scratch wound assay. Conclusion: Grafting improved the strength and stability of alginate xerogel and the results suggest the application of insulin loaded AGM2S xerogels as a potential wound healing material.

Histidine and arginine conjugated starch-PEI and its corresponding gold nanoparticles for gene delivery.

The high surface energy of gold nanoparticles (GNPs) along with its unique physical and chemical properties delivers it as an effective nonviral gene delivery platform for anticancer treatments. GNPs were synthesized by using starch-PEI in which PEI act as reducing agent and starch as a stabilizer. Cytocompatibility studies carried out in C6 cells revealed that gold modification significantly improved the percentage cell viability even at higher polymer concentration. Irrespective of excellent cellular internalization, the transfection efficiency studied with p53 plasmid was found to be compromised with gold modification. For better transfection efficiency, we further modified starch-PEI with amino acids (l-Arginine, l-Histidine) and synthesized the corresponding GNPs. Though starch-PEI gold nanoparticles exhibited low transfection, its amino acid modified counterparts were found to have good transfection efficiency along with low cytotoxicity. It was found that the GNPs containing l-Arginine showed improved transfection efficiency. Hence, it can be inferred that selective amino acid modification is beneficial for improving the transfection efficiency.

Efficacy of vinyl imidazole grafted cationized pullulan and dextran as gene delivery vectors: A comparative study.

The present study deals with efficacy evaluation of two cationised polysaccharides - Pullulan PEI and Dextran PEI as gene delivery vector after grafting with vinyl imidazole. Chemical modifications of these polysaccharides were confirmed by FTIR and NMR. Nanoplex formed from dextran PEI imidazole (DPI) were of smaller size and possess greater positive charge as compared to that of pullulan PEI imidazole (PPI). MTT assay carried out in C6 and HeLa cell lines revealed that both PPI and DPI are cytocompatible and the percentage cell viability is higher for former. Though DPI and PPI derivatives showed similar cellular uptake, the cell morphology was maintained with later and this may due to the high molecular flexibility of pullulan that masks the cytotoxicity of PEI. Similarly transfection studies carried out with p53 plasmid also revealed that PPI derivatives promote more apoptosis in both C6 and HeLa cell lines as compared to that of DPI derivatives. Based on cytocompatibility and transfection efficiency, PPI was found to be a better gene delivery vector that can be further explored for in vivo applications.

Alginate stabilized gold nanoparticle as multidrug carrier: Evaluation of cellular interactions and hemolytic potential.

This work delineates the synthesis of curcumin (Ccm) and methotrexate (MTX) conjugated biopolymer stabilized AuNPs (MP@Alg-Ccm AuNPs). The dual drug conjugated nano-vector was characterized by FTIR, (1)H NMR and UV-vis spectroscopic techniques. Hydrodynamic diameter and surface charge of the AuNPs were determined by DLS analysis and the spherical particles were visualized by TEM. MP@Alg-Ccm AuNPs exhibited improved cytotoxic potential against C6 glioma and MCF-7 cancer cell lines and was found to be highly hemocompatible. MP@Alg-Ccm AuNPs also exhibited active targeting efficiency against MCF-7 cancer cells due to the presence of "antifolate" drug MTX. Thus MP@Alg-Ccm AuNPs may find potential application in targeted combination chemotherapy for the treatment of cancer. The study is also interesting from the synthetic point of view because, here generation of AuNPs was done using "green chemical" alginate and dual drug conjugated AuNPs were created in two simple reaction steps using "green solvent" water.

Simultaneous Effect of Thiolation and Carboxylation of Chitosan Particles Towards Mucoadhesive Oral Insulin Delivery Applications: An In Vitro and In Vivo Evaluation.

Thiomalyl chitosan (TCS), a pH sensitive thiolated chitosan derivative, was developed and investigated towards oral protein delivery application. Particles of z-average 364 ± 5.6 nm with a negative zeta potential of 14.4 mV was obtained by tripolyphosphate cross linking of TCS. The release of insulin from TCS particles was significantly restricted at pH 1.2 minimizing up to about < 10% in 3 hours. The permeation enhancement ratio was found to 13 times higher than the FD4 alone and was 1.6 times higher than the unmodified chitosan particles. The protein protective properties of the matrix were established in presence of pepsin and pancreatic enzymes. Confocal microscopy studies proved the tight junction opening of Caco-2 cells by these thiolated chitosan particles and the in vivo studies on diabetic rats established its potential towards oral peptide delivery with pharmacological availability (PA) of 1.5%. The significance of this work is to establish that, the presence of multiple functional groups having similar property in the same matrix can improve its suitability as a promising candidate for oral peptide delivery with improved release characteristics, mucoadhesion as well as protecting the insulin activity and enhancing the permeability across the intestinal wall.

Collagen synthesis promoting pullulan-PEI-ascorbic acid conjugate as an efficient anti-cancer gene delivery vector.

Cationized pullulan (pullulan-PEI; PP) was synthesized and further modified with an anti-oxidant molecule, ascorbic acid (PPAA) at various ratios. The nanoplexes formed at an optimum ratio of 4:1 was within a size of 150nm and had a zeta potential of 9-14mV. The nanoplexes at this ratio was used for further investigations. The cell internalization and transfection efficiency of these nanoplexes were determined in presence of serum. The internalization and transfection efficiency were found to be unaffected by the presence of fetal bovine serum. Another interesting observation was that this polymer was found to have collagen synthesis promoting property. The collagen synthesis effect of these polymers was quantified and observed that PPAA3 promoted the highest. Transfection efficiency was evaluated by assessing the p53 gene expression in C6 rat glioma cells and cell death was quantified to be 96% by flow cytometry, thus establishing the high efficacy of this polymer.

Betaine conjugated cationic pullulan as effective gene carrier.

Polyethyleneimne (PEI) is a very efficient transfecting agent but is toxic due to high charge density. To generate a vector which is efficient and less cytotoxic, PEI was conjugated with pullulan (PPEI). Further conjugation was done on PPEI with zwitter ionic betaine which possess antifouling property. PEI of molecular weight 1.2, 2, and 10 kDa were used in the study. Buffering capacity of pullulan-PEI-betaine (PPB) conjugates was found to be sufficient enough for the polymers to make endosomal escape. The polymers proved to be less cytotoxic and highly hemocompatible than PEI. Nuclear localization of YOYO tagged DNA was observed with the nanoplexes developed using PPEI and PPBs of PEI 10 kDa. Transfection efficiency was evaluated using p53 expressing gene and the live dead assay demonstrated very high transfection efficiency with PPB conjugates of PEI 10 kDa.

Pullulan-protamine as efficient haemocompatible gene delivery vector: synthesis and in vitro characterization.

Biodegradable non-viral vectors with good transfection efficiency is essential for successful gene delivery. The purpose of this study was to design a non-viral vector by conjugating protamine to pullulan and elucidate the potential use of pullulan protamine conjugate (PPA) as an effective, non toxic and haemocompatible gene delivery system. The particle size and surface charge were measured using Nanosizer. Derivatization was confirmed by NMR, FTIR and DSC analyses. Acid base titration revealed the buffering behaviour of the conjugate. The protection of DNA from nuclease enzyme and interaction of plasma components on the stability of nanoplexes were also analysed. The uptake studies confirmed the plasmid delivery into the nucleus and the inhibitor studies determined the uptake mechanism. Transfection experiments revealed the capability of PPA to cellular uptake in C6 cells and facilitate high gene expression. Thus, PPA proves to be a promising non-viral vector.

Multifunctional polymeric nanoplexes for anticancer co-delivery of p53 and mitoxantrone.

Co-delivery of the anticancer drug, mitoxantrone (MTO) and the gene encoding tumor suppressor protein p53 was evaluated towards anticancer combinatorial therapy. The nanoplatforms developed herein are assembled by coupling ß-cyclodextrin and the cationic polymer, polyethyleneimine to a hydrophilic polymer, pullulan (PPEICD). The ß-cyclodextrin serves as a nanocontainer for the drug MTO, while the cationic moiety can condense pDNA. Acid base titrations provided insight into the buffering capacity of the PPEICD conjugate. Cytotoxicity studies by MTT assay in HepG2 and C6 cell lines and hemocompatibility studies confirmed the conjugate to be nontoxic and hemocompatible. In vitro release studies of MTO in phosphate buffered saline pH 7.4 showed an initial burst effect followed by a slow drug release. The released data fitted with the Korsmeyer-Peppas model and their diffusional exponents suggest that the drug release from the polymeric system followed diffusion and non-Fickian transport. Combined drug and gene loaded nanoplexes have a more apoptotic effect than either the drug or gene individually as confirmed by MTT assay and live dead assay. This indicated the significance of the combined drug and gene delivery system and the ability of the nanoplatform to overcome the multidrug resistance (MDR) of MTO. Moreover, preference of asialoglycoprotein receptor (ASGPR) mediated internalization for nanoparticle cellular uptake in HepG2 cell lines was identified by treating with the inhibitor asialofetuin. Cell studies in both HepG2 and C6 cell lines demonstrated that the nanoplatform PPEICD can efficiently and selectively deliver both p53 and MTO to cancer cells inducing high cell death.

Biomimetic mucin modified PLGA nanoparticles for enhanced blood compatibility.

Efforts to develop long circulating polymeric nanoparticles have propelled many strategies in nanoparticle surface modification to bypass immune surveillance and systemic clearance. In this context, our present study reports on the preparation and evaluation of mucin functionalized poly lactic-co-glycolic acid (PLGA) nanoparticles as hemocompatible, cell penetrating nanoparticulate drug delivery system. Amino groups of mucin were conjugated to the terminal carboxylic acid groups on PLGA to be followed by nanoparticle synthesis via standard solvent evaporation technique. Detailed in vitro experiments were performed to illustrate the significance of alternating copolymer structured mucin modified PLGA nanoparticles in terms of enhanced hemocompatibility and cellular uptake. Mucylation proved promising in controlling PLGA nanoparticle- interaction with plasma proteins (opsonins) and blood components via hemolysis, thrombogenecity and complement activation. Besides hemocompatibility, the modified and unmodified nanoparticles were also found to be cytocompatible with L929 and C6 cell lines. The fluorescent and confocal image analysis evaluated the extent of cellular uptake of nanoparticles into C6 cells. Specifically the combination of stealth properties and cellular internalization capacity of mucin modified PLGA nanoparticle (PLGA-Mucin) lead us to propose it as a safe, efficient and multifunctional nanoplatform for disease specific intravenous drug delivery applications as far as in vitro experiments are concerned.

Pullulan-histone antibody nanoconjugates for the removal of chromatin fragments from systemic circulation.

The billions of cells that die in the adult human body daily release considerable amounts of fragmented chromatin in the form of mono- and oligonucleosomes into the circulation in normal individuals, and in higher quantities in many disease conditions. Recent results suggest that circulating chromatin fragments (Cfs) especially from abnormal cells can spontaneously enter into healthy cells to damage their DNA and induce genomic instability. Furthermore, Cfs isolated from cancer patients may induce oncogenic transformation in the recipients' cells. Thus, it follows that if such Cfs emanating from apoptotic cells could be prevented from reaching other cells, it could potentially inhibit pathological conditions, including cancer. Here we have developed pullulan based histone antibody nanoconjugates for the removal of Cfs. Nanoconjugates were developed and various physico-chemical characterizations were carried out. The efficacy of these nanoconjugates on removing Cfs was evaluated both in vitro and in vivo. Our results indicate that nanoconjugates may have therapeutic value in the efficient removal of Cfs, reducing inflammation and fatality in a mouse model of sepsis, and in preventing neutropenia following treatment with Adriamycin.

Oral delivery of therapeutic protein/peptide for diabetes--future perspectives.

Diabetes is a metabolic disease and is a major cause of mortality and morbidity in epidemic proportions. A type I diabetic patient is dependent on daily injections of insulin, for survival and also to maintain a normal life, which is uncomfortable, painful and also has deleterious effects. Extensive efforts are being made worldwide for developing noninvasive drug delivery systems, especially via oral route. Oral route is the most widely accepted means of administration. However it is not feasible for direct delivery of peptide and protein drugs. To overcome the gastro-intestinal barriers various types of formulations such as polymeric micro/nanoparticles, liposomes, etc. are investigated. In the recent years lot of advances have taken place in developing and understanding the oral peptide delivery systems. Simultaneously, the development and usage of other peptides having anti-diabetic potentials are also considered for diabetes therapy. In this review we are focusing on the advances reported during the past decade in the field of oral insulin delivery along with the possibility of other peptidic incretin hormones such as GLP-1, exendin-4, for diabetes therapy.

Polymers for gene delivery: current status and future perspectives.

Gene therapy is a hope for curing many diseases and pathological conditions which are relatively difficult to treat. However lack of proper gene delivery vehicle is the main limiting step in this direction. Though viral vectors still lead as the major vehicle used in gene therapy clinical trials, their immunogenicity and low capacity restrict their wide use. Hence there is a need for developing non-viral vectors which can really be used for clinical applications. Polymers are a versatile group of molecules which can be modified and designed or engineered according to the end needs of the applications. The objective of this review is to summarize the recent advances in the development of polymeric vectors for gene delivery applications reported in patents and scientific journals.

Unraveling the intracellular efficacy of dextran-histidine polycation as an efficient nonviral gene delivery system.

In this study, we attempted to elucidate the capability of a natural polymer dextran, by modification with histidine, to be an efficient, safe and promising nucleic acid delivery system in gene therapy. Physicochemical characterizations were performed to get an insight into the derivative. The efficiency of the derivative as a gene delivery vehicle was also studied in depth using fluorescence microscopy. Extensive efforts were made to have a better understanding of the cellular dynamics involved. The derivative proved itself to be 6.7-fold more excelling than PEI in its transfecting capability. Mechanisms underlying cellular internalization, vector unpacking, intranuclear localization and transgene expression were also investigated. The possibility of recruiting intracellular histone to promote the entry of the gene into the nucleus seemed promising. Our findings also explored the links that mediate the correlation between the uptake of the derivative and various endocytic pathways. The results thus obtained reflect the success of the entire journey of the synthesized delivery vehicle.

Spermine grafted galactosylated chitosan for improved nanoparticle mediated gene delivery.

Despite multitude of beneficial features, chitosan has poor water solubility and transfection ability which affect its gene delivery efficacy. The two features are improved when certain chemical modifications are incorporated into the chitosan parent backbone. This strategy is adopted here, by coupling galactose and spermine into the chitosan backbone. The conjugation was determined with FTIR and (1)H NMR and nanoparticle morphology was assessed by TEM and AFM techniques. Particle size, zeta potential, buffering capacity and DNA binding ability gave encouraging result of enhanced solubility and stability. In vitro studies of GCSM in HepG2 cell lines displayed low cytotoxicity and improved transfection. We also identified the preference of receptor mediated internalization for nanoparticles cellular uptake by treating with cellular uptake inhibitors. The results evidently led us to comprehend that galactosylated chitosan-g-spermine could be considered as a promising chitosan derivative for conducting nanoparticle mediated gene delivery.

Hemocompatible pullulan-polyethyleneimine conjugates for liver cell gene delivery: In vitro evaluation of cellular uptake, intracellular trafficking and transfection efficiency.

Polyethyleneimine (PEI; 25 kDa)-conjugated pullulans (PPE1, PPE2 and PPE3) were developed and investigated for possible use in gene delivery applications. The cytotoxicity, blood component interactions such as red blood cell/white blood cell aggregation, platelet and complement activation, and protein interaction of the pullulan-conjugated PEI was drastically reduced in comparison to PEI-based nanocomplexes. Based on the blood compatibility studies, PPE1 was selected for further study. The buffering capacity of this derivative was similar to that of PEI, which plays an important role in efficient gene transfection. The particle size, zeta potential, stability in the presence of plasma and resistance to nuclease degradation were evaluated. In addition, cellular uptake and localization of plasmid, as well as transgene expression, were evaluated following in vitro transfection of HepG2 cells. Endocytosis inhibitors, confocal laser scanning microscopy and fluorescent labeling techniques were used to visualize the nanoplex uptake mechanism, cellular distribution and nuclear localization. The results from inhibitor experiments in the presence of asialofetuin indicated that the asialoglycoprotein receptor is involved in transfection of hepatocytes with pullulan-PEI complexes. The conjugation of pullulan with PEI did not hinder the plasmid nuclear localization ability of PEI. The transfection efficiency of pullulan conjugate was similar to PEI, with the added advantage of hemocompatibility and non-cytotoxicity. The transfection efficiency of PEI and PPE1 was 1.6- and 2-fold more, respectively, in the presence of serum than in the absence of serum. Therefore, the pullulan-PEI conjugate seems to be a promising gene delivery vector with good hemocompatibility and low toxicity but without compromising the transfection efficacy of PEI.

Dextran-protamine polycation: an efficient nonviral and haemocompatible gene delivery system.

Despite the remarkable progress in the field of gene therapy with viral vectors, nonviral vectors have attracted great interests due to their unique properties. Imparting desired characteristics to nonviral gene delivery systems requires the development of cationic polymers. The purpose of this work was to design a cationic derivative (Dex-P) of dextran using protamine in order to assert target specific cellular binding. Our objective was to elucidate the potential use of Dex-P as a haemocompatible, nontoxic and efficient nonviral candidate for gene therapy. Nanoplexes were prepared with calf thymus DNA and Dex-P. Derivatization was confirmed by FTIR, gel permeation chromatography and TNBS assay. Dynamic light scattering and TEM studies determined the size and morphology of the nanoplex. The buffering behaviour was assessed by acid base titration. Complexation stability was evaluated using agarose gel electrophoresis and EtBr displacement assay. The protection of ctDNA from nuclear digestion and the effect of plasma components towards stability of the nanoplexes were also analyzed. Various haemocompatible studies were performed to check haemolysis, aggregation, clotting time, and complement activation. Transfection and cytotoxicity experiments were performed in vitro. The nanosize, spherical shape and stability of nanoplexes were affirmed. Various experiments conducted confirmed Dex-P to be nontoxic and haemocompatible. Transfection experiments revealed the capability of Dex-P to facilitate high gene expression and cellular uptake in HepG2 cells. With the improved physicochemical, biological and transfection properties, Dex-P seems to be a promising gene delivery system.