Optimizing protein delivery rate from silk fibroin hydrogel using silk fibroin-mimetic peptides conjugation.

Controlled release of proteins, such as growth factors, from biocompatible silk fibroin (SF) hydrogel is valuable for its use in tissue engineering, drug delivery, and other biological systems. To achieve this, we introduced silk fibroin-mimetic peptides (SFMPs) with the repeating unit (GAGAGS)n. Using green fluorescent protein (GFP) as a model protein, our results showed that SFMPs did not affect the GFP function when conjugated to it. The SFMP-GFP conjugates incorporated into SF hydrogel did not change the gelation time and allowed for controlled release of the GFP. By varying the length of SFMPs, we were able to modulate the release rate, with longer SFMPs resulting in a slower release, both in water at room temperature and PBS at 37 °C. Furthermore, the SF hydrogel with the SFMPs showed greater strength and stiffness. The increased ß-sheet fraction of the SF hydrogel, as revealed by FTIR analysis, explained the gel properties and protein release behavior. Our results suggest that the SFMPs effectively control protein release from SF hydrogel, with the potential to enhance its mechanical stability. The ability to modulate release rates by varying the SFMP length will benefit personalized and controlled protein delivery in various systems.

Hyaluronic Acid Nanogels: A Promising Platform for Therapeutic and Theranostic Applications.

Hyaluronic acid (HA) nanogels are a versatile class of nanomaterials with specific properties, such as biocompatibility, hygroscopicity, and biodegradability. HA nanogels exhibit excellent colloidal stability and high encapsulation capacity, making them promising tools for a wide range of biomedical applications. HA nanogels can be fabricated using various methods, including polyelectrolyte complexation, self-assembly, and chemical crosslinking. The fabrication parameters can be tailored to control the physicochemical properties of HA nanogels, such as size, shape, surface charge, and porosity, enabling the rational design of HA nanogels for specific applications. Stimulus-responsive nanogels are a type of HA nanogels that can respond to external stimuli, such as pH, temperature, enzyme, and redox potential. This property allows the controlled release of encapsulated therapeutic agents in response to specific physiological conditions. HA nanogels can be engineered to encapsulate a variety of therapeutic agents, such as conventional drugs, genes, and proteins. They can then be delivered to target tissues with high efficiency. HA nanogels are still under development, but they have the potential to become powerful tools for a wide range of theranostic or solely therapeutic applications, including anticancer therapy, gene therapy, drug delivery, and bioimaging.

From Linear to Nets: Multiconfiguration Polymer Structure Generation with PolyFlin.

Molecular modeling and simulations are essential tools in polymer science and engineering, enabling researchers to predict and understand the properties of macromolecules, including their structure, dynamics, thermodynamics, and overall material characteristics. However, one of the key challenges in polymer simulation and modeling lies in the initial topology design, as existing programs often lack the capability to generate all types of polymer forms. In this study, we present PolyFlin, a powerful Python module that addresses this limitation by allowing the generation of a wide range of polymer structures, from simple homopolymers to complex copolymers, including grafts, cyclic, star, dendrimers, and nets. PolyFlin offers a versatile and efficient tool for exploring and creating diverse polymer architectures, facilitating advancements in various fields that require precise polymer modeling and simulation.

Structure-based computational screening of 470 natural quercetin derivatives for identification of SARS-CoV-2 Mpro inhibitor.

Coronavirus disease 2019 (COVID-19) is a global pandemic infecting the respiratory system through a notorious virus known as the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Due to viral mutations and the risk of drug resistance, it is crucial to identify new molecules having potential prophylactic or therapeutic effect against SARS-CoV-2 infection. In the present study, we aimed to identify a potential inhibitor of SARS-CoV-2 through virtual screening of a compound library of 470 quercetin derivatives by targeting the main protease-Mpro (PDB ID: 6LU7). The study was carried out with computational techniques such as molecular docking simulation studies (MDSS), molecular dynamics (MD) simulations, and molecular mechanics generalized Born surface area (MMGBSA) techniques. Among the natural derivatives, compound 382 (PubChem CID 65604) showed the best binding affinity to Mpro (-11.1 kcal/mol). Compound 382 interacted with LYS5, TYR126, GLN127, LYS137, ASP289, PHE291, ARG131, SER139, GLU288, and GLU290 of the Mpro protein. The SARS-CoV-2 Mpro-382 complex showed acceptable stability during the 100 ns MD simulations. The SARS-CoV-2 Mpro-382 complex also showed an MM-GBSA binding free energy value of -54.0 kcal/mol. The binding affinity, stability, and free energy results for 382 and Mpro were better than those of the native ligand and the standard inhibitors ledipasvir and cobicistat. The conclusion of our study was that compound 382 has the potential to inhibit SARS-Cov-2 Mpro. However, further investigations such as in-vitro assays are recommended to confirm its in-silico potency.

Potential roles of hyaluronic acid in in vivo CAR T cell reprogramming for cancer immunotherapy.

Chimeric antigen receptor (CAR) T cell therapy has recently shown unprecedented clinical efficacy for cancer treatment, particularly of hematological malignancies. However, the complex manufacturing processes that involve ex vivo genetic modification of autologous T cells limits its therapeutic application. CAR T cells generated in vivo provide a valid alternative immunotherapy, "off-the-shelf", for cancer treatment. This approach requires carriers for the delivery of CAR-encoding constructs, which are plasmid DNA or messenger RNA, to T cells for CAR expression to help eradicate the tumor. As such, there are a growing number of studies reporting gene delivery systems for in vivo CAR T cell therapy based on viral vectors and polymeric nanoparticles. Hyaluronic acid (HA) is a natural biopolymer that can serve for gene delivery, because of its inherent properties of cell recognition and internalization, as well as its biodegradability, biocompatibility, and presence of functional groups for the chemical conjugation of targeting ligands. In this review, the potential of HA in the delivery of CAR constructs is discussed on the basis of previous experience of HA-based nanoparticles for gene therapy. Furthermore, current studies on CAR carriers for in vivo-generated CAR T cells are included, giving an idea of a rational design of HA-based systems for the more efficient delivery of CAR to circulating T cells.

Complexed Polymer Film-Forming Spray: An Optimal Delivery System for Secretome of Mesenchymal Stem Cell as Diabetic Wound Dressing?

Diabetes-related wounds have physiological factors that make healing more complicated. High sugar levels can increase microbial infection risk while limiting nutrition and oxygen transfer to the wound area. The secretome of mesenchymal stem cells has been widely known for its efficacy in regenerative therapy. However, applying the secretome directly to the wound can reduce its effectiveness. In this review, we examined the literature on synthesizing the combinations of carboxymethyl chitosan, hyaluronic acid, and collagen tripeptides, as well as the possibility of physicochemical properties enhancement of the hydrogel matrix, which could potentially be used as an optimal delivery system of stem cell's secretome for diabetic wound healing.

In vitro biological activities of the flexible and virus nanoparticle-decorated silk fibroin-based films.

Flexible films were prepared from silk fibroin (SF) and gelatin (GA) with a presence of glycerol (Gly), followed by water vapor annealing to achieve water-insoluble matrices. The blended SF/GA/Gly films were chemically conjugated with tobacco mosaic virus (TMV), either native (TMV-wt) or genetically modified with Arg-Gly-Asp (RGD) sequences (TMV-rgd), to improve cellular responses. The attachment and proliferation of L929 cells on TMV-decorated films were improved, possibly due to enhanced surface roughness. The cellular responses were pronounced with TMV-rgd, due to the proper decoration of RGD, which is an integrin recognition motif supporting cell binding. However, the biological results were inconclusive for human primary cells because of an innate slow growth kinetic of the cells. Additionally, the cells on SF/GA/Gly films were greater populated in S and G2/M phase, and the cell cycle arrest was notably increased in the TMV-conjugated group. Our findings revealed that the films modified with TMV were cytocompatible and the cellular responses were significantly enhanced when conjugated with its RGD mutants. The biological analysis on the cellular mechanisms in response to TMV is further required to ensure the safety concern of the biomaterials toward clinical translation.

Biocompatibility study of tobacco mosaic virus nanoparticles on human alveolar bone cells.

One of the most important factors in a dental implant's success is an adequate quantity of supporting bone. However, there are still some limitations for the bone substitution material. Previous studies found that tobacco mosaic virus (TMV) had the potential for bone formation induction. The aim of this study was to evaluate the biocompatibility of TMV with primary human alveolar bone cells. Primary human alveolar bone cells were cultured on TMV coated substrates. Cell viability, alkaline phosphatase activity, calcium matrix mineralization forming ability, immunofluorescence staining for osteocalcin synthesis and cell morphology were assessed. The results showed that primary human alveolar bone cells cultured on the TMV coated substrates had a higher metabolic rate than the non-TMV coated control group at days 1, 3, 7 and 14. Moreover, the calcium deposition was positive and the alkaline phosphatase activity assay was found significantly greater than the control group at day 14 (p < 0.05). The osteocalcin protein synthesis was found in both the TMV coated substrates and the control group. The immunofluorescence study revealed that in the TMV coated substrates group, the cell morphology changed into a polygonal shape and aggregated more quickly than the control group. The present findings conclude that TMV is biocompatible with primary human alveolar bone cells and also shows osteoinduction potential.

Liposome-polymer complex for drug delivery system and vaccine stabilization.

Liposomes have been used extensively as micro- and nanocarriers for hydrophobic or hydrophilic molecules. However, conventional liposomes are biodegradable and quickly eliminated, making it difficult to be used for delivery in specific routes, such as the oral and systemic routes. One way to overcome this problem is through complexation with polymers, which is referred to as a liposome complex. The use of polymers can increase the stability of liposome with regard to pH, chemicals, enzymes, and the immune system. In some cases, specific polymers can condition the properties of liposomes to be explicitly used in drug delivery, such as targeted delivery and controlled release. These properties are influenced by the type of polymer, crosslinker, interaction, and bond in the complexation process. Therefore, it is crucial to study and review these parameters for the development of more optimal forms and properties of the liposome complex. This article discusses the use of natural and synthetic polymers, ways of interaction between polymers and liposomes (on the surface, incorporation in lamellar chains, and within liposomes), types of bonds, evaluation standards, and their effects on the stability and pharmacokinetic profile of the liposome complex, drugs, and vaccines. This article concludes that both natural and synthetic polymers can be used in modifying the structure and physicochemical properties of liposomes to specify their use in targeted delivery, controlled release, and stabilizing drugs and vaccines.

Osteogenic differentiation of encapsulated cells in dexamethasone-loaded phospholipid-induced silk fibroin hydrogels.

The tissue engineering triad comprises the combination of cells, scaffolds and biological factors. Therefore, we prepared cell- and drug-loaded hydrogels using in situ silk fibroin (SF) hydrogels induced by dimyristoyl glycerophosphoglycerol (DMPG). DMPG is reported to induce rapid hydrogel formation by SF, facilitating cell encapsulation in the hydrogel matrix while maintaining high cell viability and proliferative capacity. In addition, DMPG can be used for liposome formulations in entrapping drug molecules. Dexamethasone (Dex) was loaded into the DMPG-induced SF hydrogels together with human osteoblast-like SaOS-2 cells, then the osteogenic differentiation of the entrapped cells was evaluated in vitro and compared to cells cultured under standard conditions. Calcium production by cells cultured in DMPG/Dex-SF hydrogels with Dex-depleted osteogenic medium was equivalent to that of cells cultured in conventional osteogenic medium containing Dex. The extended-release of the entrapped Dex by the hydrogels was able to provide a sufficient drug amount for osteogenic induction. The controlled release of Dex was also advantageous for cell viability even though its dose in the hydrogels was far higher than that in osteogenic medium. The results confirmed the possibility of using DMPG-induced SF hydrogels to enable dual cell and drug encapsulation to fulfil the practical applications of tissue-engineered constructs.

The Dual Modification of PNIPAM and ß-Cyclodextrin Grafted on Hyaluronic Acid as Self-Assembled Nanogel for Curcumin Delivery.

Curcumin is an extract of turmeric (Curcuma longa) which possesses anti-inflammatory, anti-cancer and wound-healing effects and has been used as an active compound in biomedical research for many years. However, its poor solubility presents challenges for its use in drug delivery systems. A modified nanogel delivery system, with PNIPAM and ß-cyclodextrin grafted onto hyaluronic acid (PNCDHA), was utilized to enhance the solubility. The polymer was characterized by NMR, and the inclusion complex between curcumin and ß-cyclodextrin was confirmed by FTIR. The potential of this PNCDHA polymer complex as a drug delivery vehicle was supported by a curcumin encapsulation efficiency of 93.14 ± 5.6% and the release of encapsulated curcumin at 37 °C. At a concentration of 0.5% w/v in water, PNCDHA nanogels were biocompatible with fibroblast cell line (L929) up to a curcumin concentration of 50 µM. There was a direct concentration between curcumin loading and cellular internalization. A more detailed study of the cellular internalization of PNCDHA nanogel should be considered in order to clarify cellular delivery mechanisms and to assess how its viability as a carrier may be optimized.

In Vivo Biocompatible Self-Assembled Nanogel Based on Hyaluronic Acid for Aqueous Solubility and Stability Enhancement of Asiatic Acid.

Asiatic acid (AA), a natural triterpene found in Centalla asiatica, possesses polypharmacological properties that can contribute to the treatment and prophylaxis of various diseases. However, its hydrophobic nature and rapid metabolic rate lead to poor bioavailability. The aim of this research was to develop a thermoresponsive nanogel from hyaluronic acid (HA) for solubility and stability enhancement of AA. Poly(N-isopropylacrylamide) (pNIPAM) was conjugated onto HA using a carbodiimide reaction followed by 1H NMR characterization. pNIPAM-grafted HA (HA-pNIPAM) nanogels were prepared with three concentrations of polymer, 0.1, 0.15 and 0.25% w/v, in water by the sonication method. AA was loaded into the nanogel by the incubation method. Size, morphology, AA loading capacity and encapsulation efficiency (EE) were analyzed. In vitro cytocompatibility was evaluated in fibroblast L-929 cells using the PrestoBlue assay. Single-dose toxicity was studied using rats. HA-pNIPAM nanogels at a 4.88% grafting degree showed reversible thermo-responsive behavior. All nanogel formulations could significantly increase AA water solubility and the stability was higher in nanogels prepared with high polymer concentrations over 180 days. The cell culture study showed that 12.5 µM AA in nanogel formulations was considered non-toxic to the L-929 cells; however, a dose-dependent cytotoxic effect was observed at higher AA-loaded concentrations. In vivo study proved the non-toxic effect of AA loaded in HA-pNIPAM nanogels compared with the control. Taken together, HA-pNIPAM nanogel is a promising biocompatible delivery system both in vitro and in vivo for hydrophobic AA molecules.

Impacts of Blended Bombyx mori Silk Fibroin and Recombinant Spider Silk Fibroin Hydrogels on Cell Growth.

Binary-blended hydrogels fabricated from Bombyx mori silk fibroin (SF) and recombinant spider silk protein eADF4(C16) were developed and investigated concerning gelation and cellular interactions in vitro. With an increasing concentration of eADF4(C16), the gelation time of SF was shortened from typically one week to less than 48 h depending on the blending ratio. The biological tests with primary cells and two cell lines revealed that the cells cannot adhere and preferably formed cell aggregates on eADF4(C16) hydrogels, due to the polyanionic properties of eADF4(C16). Mixing SF in the blends ameliorated the cellular activities, as the proliferation of L929 fibroblasts and SaOS-2 osteoblast-like cells increased with an increase of SF content. The blended SF:eADF4(C16) hydrogels attained the advantages as well as overcame the limitations of each individual material, underlining the utilization of the hydrogels in several biomedical applications.

Multifunctional Polymeric Nanogels for Biomedical Applications.

Currently, research in nanoparticles as a drug delivery system has broadened to include their use as a delivery system for bioactive substances and a diagnostic or theranostic system. Nanogels, nanoparticles containing a high amount of water, have gained attention due to their advantages of colloidal stability, core-shell structure, and adjustable structural components. These advantages provide the potential to design and fabricate multifunctional nanosystems for various biomedical applications. Modified or functionalized polymers and some metals are components that markedly enhance the features of the nanogels, such as tunable amphiphilicity, biocompatibility, stimuli-responsiveness, or sensing moieties, leading to specificity, stability, and tracking abilities. Here, we review the diverse designs of core-shell structure nanogels along with studies on the fabrication and demonstration of the responsiveness of nanogels to different stimuli, temperature, pH, reductive environment, or radiation. Furthermore, additional biomedical applications are presented to illustrate the versatility of the nanogels.

Self-Assembled Thermoresponsive Nanogel from Grafted Hyaluronic Acid as a Biocompatible Delivery Platform for Curcumin with Enhanced Drug Loading and Biological Activities.

A hyaluronic acid-grafted poly(N-isopropylacrylamide) (HA-pNIPAM) was synthesized as a polymeric nanogel platform for encapsulation and delivery of hydrophobic bioactive compounds using curcumin as a model drug. As demonstrated by transmission electron microscopy and dynamic light scattering techniques, the HA-pNIPAM was simply assembled into spherical nano-sized particles with the thermoresponsive behavior. The success of curcumin aqueous solubilization was confirmed by fluorescent spectroscopy. The resulting nanogel formulation enhanced the aqueous solubility and uptake into NIH-3T3 cells of curcumin. This nanogel formulation also demonstrates cytocompatibility against NIH-3T3 cells, which deems it safe as a delivery vehicle. Moreover, the formulation has a slight skin-protection effect using an artificial skin equivalence model. The curcumin-loaded HA-pNIPAM nanogel showed an anti-proliferative activity against MDA-MB-231, Caco-2, HepG2, HT-29, and TNF-α-induced hyperproliferation of keratinocyte (HaCaT) cells. The thermoresponsive HA-pNIPAM nanogel reported here could be further optimized as a platform for controlled-release systems to encapsulate pharmaceuticals for therapeutic applications.

Biocompatibility study of modified injectable hyaluronic acid hydrogel with mannitol/BSA to alveolar bone cells.

The quality and quantity of bone are crucial to the success of dental implant treatment. Recently, bone grafting materials have reached some limitations. This study aimed to evaluate the biocompatibility of novel drug delivery material, injectable methacrylated hyaluronic acid hydrogel incorporated with different ratios of mannitol and BSA (Man/BSA MeHA), to human alveolar bone cells. The three-dimensionally encapsulated cell culture was evaluated with the resazurin cell viability test, alkaline phosphatase activity assay, immunohistochemistry test for collagen type-I synthesis, and cell morphology. The results showed that the encapsulated cells were viable in all four ratios of Man/BSA MeHA hydrogel and the average metabolic rate was not less than the control group. The morphology test showed round shape cells at the upper portion of the hydrogel and fibroblast-like or polygonal shape at the lower portion of hydrogel next to the culture plate. All four groups could express enzyme alkaline phosphatase and collagen type-I. In conclusion, four ratios of Man/BSA MeHA hydrogel were biocompatible with primary human alveolar bone cells.

Development of in situ gel containing asiaticoside/cyclodextrin complexes. Evaluation in culture human periodontal ligament cells (HPLDCs).

Asiaticoside (AS), an active herbal compound isolated from Centella asiatica, has the potential benefit in promoting type I collagen (COL I) synthesis and osteogenic differentiation in human periodontal ligament cells (HPDLCs). However, it has low aqueous solubility which may hamper the bioavailability. Thus, the aim of this study was to develop thermoresponsive in situ gel containing AS/cyclodextrin (CD) complexes. The non-encapsulated formulations consisted of AS/hydroxypropyl ß-CD (HPßCD) complexes and encapsulated formulations containing AS loaded sulfobutylether ß-CD/chitosan nanoparticles (SBEßCD/CS NPs) were prepared. The appearance, pH and viscosity of all formulations were within the acceptable range. All formulations formed relatively rapid sol-to-gel transition when contacted with simulated salivary fluid at body temperature. Compared to non-encapsulated formulations, in vitro gelation and rheological studies of encapsulated formulations displayed gel formation that remained longer with high mechanical strength. In vitro mucoadhesion and in vitro release studies revealed that nanoencapsulated in situ gel had excellent mucoadhesive property and could release AS in a sustained manner. These formulations exhibited no cytotoxic effects to HPDCLs. The SBEßCD/CS NPs containing low AS content could express the COL I synthesis. Thus, nanoencapsulated platform could serve as a promising carrier to deliver AS for periodontal tissue regeneration.

A hydrogen sulfide-releasing alginate dressing for effective wound healing.

For wounds with heavy exudate levels, a dressing that can help to absorb wound exudate and improve the wound healing process is highly desired. Hydrogen sulfide (H2S) has been recognized as an important gasotransmitter that can improve angiogenesis which is crucial for wound healing. In this study, a functional sodium alginate (SA) dressing with H2S-releasing property (SA/JK-1) was fabricated by incorporating JK-1 molecule, a pH-dependent H2S donor, into SA sponge. The resultant SA/JK-1 sponge provided a moist and protective healing environment and was capable of releasing H2S consistently under acidic pH condition by absorbing exudate at the wound interface. The H2S release of JK-1 donor was prolonged by the SA sponge compared with JK-1 in solution. Cell study in vitro indicated that SA/JK-1 not only exhibited good cyto-compatibility, but also improved fibroblast proliferation and migration. In addition, the effects of the SA/JK-1 dressing on wound healing was evaluated using an in vivo full thickness dermal defect model, which revealed that SA/JK-1 can significantly improve wound healing process with enhanced granulation tissue formation, re-epithelialization, collagen deposition and angiogenesis, due to the H2S released from JK-1. Taken together, our results showed that SA dressing doped with H2S donor could potentially serves as an effective wound healing strategy. STATEMENT OF SIGNIFICANCE: The gasotransmitter H2S has been proven to improve the wound healing process in nanofibrous dressing due to its biological functions on angiogenesis. However, for non-healing wounds with heavy exudates, a wound dressing that can absorb wound exudates and controlled gasotransmitter release to improve the wound healing process is still in urgent need. Here we fabricated a sodium alginate (SA) sponge incorporated with H2S donor JK-1 (SA/JK-1), which showed strong water uptake capability, and released H2S under acidic condition. The SA/JK-1 sponge exhibited biocompatibility to fibroblasts and promoted cell migration in vitro, and exhibited obviously positive influence on wound healing in vivo. This H2S donor doped alginate wound dressing represents a promising strategy for treatment of non-healing wound.

Hyaluronic acid-based hydrogels with tobacco mosaic virus containing cell adhesive peptide induce bone repair in normal and osteoporotic rats.

Tobacco mosaic virus (TMV) has been studied as a multi-functional agent for bone tissue engineering. An osteo-inductive effect of wild-type TMV has been reported, as it can significantly enhance the bone differentiation potential of bone marrow stromal cells both on a two-dimensional substrate and in a three-dimensional (3D) hydrogel system. A TMV mutant (TMV-RGD1) was created which featured the adhesion peptide arginyl-glycyl-aspartic acid (RGD), the most common peptide motif responsible for cell adhesion to the extracellular matrix, on the surface of the virus particle to enhance the bio-functionality of the scaffold material. We hypothesised that the incorporation of either wild-type TMV or TMV-RGD1 in the 3D hydrogel scaffold would induce bone healing in critical size defects of the cranial segmental bone. We have previously tested the virus-functionalised scaffolds, in vitro, with a hyaluronic acid-based system as an in-situ hydrogel platform for 3D cell encapsulation, culture, and differentiation. The results of these experiments suggested the potential of the virus-functionalised hydrogel to promote in vitro stem cell differentiation. The hydrogel-forming system we employed was shown to be safe and biocompatible in vivo. Here, we further explored the physiological responses regarding bone regeneration of a calvarial defect in both normal and osteoporotic ovariectomized rat models. Our results, based on histological analysis in both animal models, suggested that both wild-type TMV and TMV-RGD1 functionalised hydrogels could accelerate bone regeneration, without systemic toxicity, evaluated by blood counts. New bone formation was intensified by the incorporation of the RGD-mutant viral particles. This finding increased the potential for use of the rod-shaped plant virus as a platform for the addition of powerful biofunctionality for tissue engineering applications. This study was approved by the Ethics Committee on Animal Use of the Zhenjiang Affiliated First People's Hospital affiliated to Jiangsu University.