Surface functionalization of virus-like particles via bioorthogonal click reactions for enhanced cell-specific targeting.

Surface functionalization of nano drug carriers allows for precise delivery of therapeutic molecules to the target site. This technique involves attaching targeting molecules to the nanoparticle surface, facilitating selective interaction. In this study, we engineered virus-like particles (VLPs) to enhance their targeting capabilities. Azide groups incorporated on the lipid membranes of VLPs enabled bioorthogonal click reactions for conjugation with cycloalkyne-bearing molecules, providing efficient conjugation with high specificity. HIV-1 Gag VLPs were chosen due to their envelope, which allows host membrane component incorporation, and the Gag protein, which serves as a recognition motif for human T cells. This combination, along with antibody-mediated targeting, addresses the limitations of intracellular delivery to T cells, which typically exhibit low uptake of exogenous materials. The selective uptake of azide VLPs by CD3-positive T cells was evaluated in a co-culture system. Even without antibody conjugation, VLP uptake was enhanced in T cells, indicating their intrinsic targeting potential. Antibody conjugation further amplified this effect, demonstrating the synergistic benefits of the combined targeting approach. Our study shows that recombinant production of azide functionalized VLPs results in engineered nanoparticles that can be easily modified using bioorthogonal click reactions, providing high specificity and versatility for conjugation with various molecules, making it applicable to a wide range of biological products.

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.

Osteogenic induction of asiatic acid derivatives in human periodontal ligament stem cells.

Asiatic acid (AA) and asiaticoside, pentacyclic triterpenoid compounds derived from Centella asiatica, are known for their biological effects in promoting type I collagen synthesis and inducing osteogenesis of stem cells. However, their applications in regenerative medicine are limited due to their low potency and poor aqueous solubility. This work aimed to evaluate the osteogenic induction activity of AA derivatives in human periodontal ligament stem cells (hPDLSCs) in vitro. Four compounds were synthesised, namely 501, 502, 503, and 506. AA was used as the control. The 502 exhibited low water solubility, while the 506 compound showed the highest. The cytotoxicity analysis demonstrated that 503 caused significant deterioration in cell viability, while other derivatives showed no harmful effect on hPDLSCs. The dimethyl aminopropyl amine derivative of AA, compound 506, demonstrated a relatively high potency in inducing osteogenic differentiation. An elevated mRNA expression of osteogenic-related genes, BMP2, WNT3A, ALP, OSX and IBSP was observed with 506. Additionally, the expression of BMP-2 protein was enhanced with increasing dose of 506, and the effect was pronounced when the Erk signalling molecule was inhibited. The 506 derivative was proposed for the promotion of osteogenic differentiation in hPDLSCs by upregulating BMP2 via the Erk signalling pathway. The 506 molecule showed promise in bone tissue regeneration.

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.

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.

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.

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.

Comparative Study of Silk Fibroin-Based Hydrogels and Their Potential as Material for 3-Dimensional (3D) Printing.

Three-dimensional (3D) printing is regarded as a critical technology in material engineering for biomedical applications. From a previous report, silk fibroin (SF) has been used as a biomaterial for tissue engineering due to its biocompatibility, biodegradability, non-toxicity and robust mechanical properties which provide a potential as material for 3D-printing. In this study, SF-based hydrogels with different formulations and SF concentrations (1-3%wt) were prepared by natural gelation (SF/self-gelled), sodium tetradecyl sulfate-induced (SF/STS) and dimyristoyl glycerophosphorylglycerol-induced (SF/DMPG). From the results, 2%wt SF-based (2SF) hydrogels showed suitable properties for extrusion, such as storage modulus, shear-thinning behavior and degree of structure recovery. The 4-layer box structure of all 2SF-based hydrogel formulations could be printed without structural collapse. In addition, the mechanical stability of printed structures after three-step post-treatment was investigated. The printed structure of 2SF/STS and 2SF/DMPG hydrogels exhibited high stability with high degree of structure recovery as 70.4% and 53.7%, respectively, compared to 2SF/self-gelled construct as 38.9%. The 2SF/STS and 2SF/DMPG hydrogels showed a great potential to use as material for 3D-printing due to its rheological properties, printability and structure stability.

Crosslinked Silk Fibroin/Gelatin/Hyaluronan Blends as Scaffolds for Cell-Based Tissue Engineering.

3D porous scaffolds fabricated from binary and ternary blends of silk fibroin (SF), gelatin (G), and hyaluronan (HA) and crosslinked by the carbodiimide coupling reaction were developed. Water-stable scaffolds can be obtained after crosslinking, and the SFG and SFGHA samples were stable in cell culture medium up to 10 days. The presence of HA in the scaffolds with appropriate crosslinking conditions greatly enhanced the swellability. The microarchitecture of the freeze-dried scaffolds showed high porosity and interconnectivity. In particular, the pore size was significantly larger with an addition of HA. Biological activities of NIH/3T3 fibroblasts seeded on SFG and SFGHA scaffolds revealed that both scaffolds were able to support cell adhesion and proliferation of a 7-day culture. Furthermore, cell penetration into the scaffolds can be observed due to the interconnected porous structure of the scaffolds and the presence of bioactive materials which could attract the cells and support cell functions. The higher cell number was noticed in the SFGHA samples, possibly due to the HA component and the larger pore size which could improve the microenvironment for fibroblast adhesion, proliferation, and motility. The developed scaffolds from ternary blends showed potential in their application as 3D cell culture substrates in fibroblast-based tissue engineering.

Dual-functional liposomes for curcumin delivery and accelerating silk fibroin hydrogel formation.

The administration of a drug-loaded implantable hydrogel at the tumor site after surgical resection is a viable approach to prevent the local recurrence or metastasis. Dimyristoyl glycerophosphorylglycerol (DMPG)-based liposomes were developed for inducing the rapid gelation of silk fibroin (SF) and delivering an anticancer drug, curcumin. Curcumin was loaded in the liposomes and the stability of curcumin was enhanced. The gelation time of liposome-induced SF hydrogels ranged from 3 min to more than 6 h. The biological activity of liposome-SF hydrogels was evaluated in vitro using L929 fibroblasts and MDA-MB-231 breast cancer cells. The release of curcumin can inhibit the growth of cancer cells. Both cells cultured on the surface of the hydrogels loaded with curcumin displayed low cell survival due to the combination of low cell attachment and cytotoxicity of curcumin. Liposome-SF hydrogels show potential as a sealant administered at the tumor site to eliminate residual cancer cells after tumor removal.

Exploring the Gelation Mechanisms and Cytocompatibility of Gold (III)-Mediated Regenerated and Thiolated Silk Fibroin Hydrogels.

Accelerating the gelation of silk fibroin (SF) solution from several days or weeks to minutes or few hours is critical for several applications (e.g., cell encapsulation, bio-ink for 3D printing, and injectable controlled release). In this study, the rapid gelation of SF induced by a gold salt (Au3+) as well as the cytocompatibility of Au3+-mediated SF hydrogels are reported. The gelation behaviors and mechanisms of regenerated SF and thiolated SF (tSF) were compared. Hydrogels can be obtained immediately after mixing or within three days depending on the types of silk proteins used and amount of Au3+. Au3+-mediated SF and tSF hydrogels showed different color appearances. The color of Au-SF hydrogels was purple-red, whereas the Au-tSF hydrogels maintained their initial solution color, indicating different gelation mechanisms. The reduction of Au3+ by amino groups and further reduction to Au by tyrosine present in SF, resulting in a dityrosine bonding and Au nanoparticles (NPs) production, are proposed as underlying mechanisms of Au-SF gel formation. Thiol groups of the tSF reduced Au3+ to Au+ and formed a disulfide bond, before a formation of Au+-S bonds. Protons generated during the reactions between Au3+ and SF or tSF led to a decrease of the local pH, which affected the chain aggregation of the SF, and induced the conformational transition of SF protein to beta sheet. The cytocompatibility of the Au-SF and tSF hydrogels was demonstrated by culturing with a L929 cell line, indicating that the developed hydrogels can be promising 3D matrices for different biomedical applications.

Phospholipid-induced silk fibroin hydrogels and their potential as cell carriers for tissue regeneration.

Silk fibroin (SF) hydrogels can be obtained via self-assembly, but this process takes several days or weeks, being unfeasible to produce cell carrier hydrogels. In this work, a phospholipid, namely, 1,2-dimyristoyl-sn-glycero-3-phospho-(1'-rac-glycerol) sodium salt (DMPG), was used to induce and accelerate the gelation process of SF solutions. Due to the amphipathic nature and negative charge of DMPG, electrostatic and hydrophobic interactions between the phospholipids and SF chains will occur, inducing the structural transition of SF chains to the beta sheet and consequently a rapid gel formation is observed (less than 50 min). Moreover, the gelation time can be controlled by varying the lipid concentration. To assess the potential of the hydrogels as cell carriers, several mammalian cell lines, including L929, NIH/3T3, SaOS-2, and CaSki, were encapsulated into the hydrogel. The silk-based hydrogels supported the normal growth of fibroblasts, corroborating their cytocompatibility. Interestingly, an inhibition in the growth of cancer-derived cell lines was observed. Therefore, DMPG-induced SF hydrogels can be successfully used as a 3D platform for in situ cell encapsulation, opening promising opportunities in biomedical applications, such as in cell therapies and tissue regeneration.