ACE mRNA (Additional Chimeric Element incorporated IVT mRNA) for Enhancing Protein Expression by Modulating Immunogenicity.

The development of in vitro transcribed mRNA (IVT mRNA)-based therapeutics/vaccines depends on the management of IVT mRNA immunogenicity. IVT mRNA, which is used for intracellular protein translation, often triggers unwanted immune responses, interfering with protein expression and leading to reduced therapeutic efficacy. Currently, the predominant approach for mitigating immune responses involves the incorporation of costly chemically modified nucleotides like pseudouridine (Ψ) or N1-methylpseudouridine (m1Ψ) into IVT mRNA, raising concerns about expense and the potential misincorporation of amino acids into chemically modified codon sequences. Here, an Additional Chimeric Element incorporated mRNA (ACE mRNA), a novel approach incorporating two segments within a single IVT mRNA structure, is introduced. The first segment retains conventional IVT mRNA components prepared with unmodified nucleotides, while the second, comprised of RNA/DNA chimeric elements, aims to modulate immunogenicity. Notably, ACE mRNA demonstrates a noteworthy reduction in immunogenicity of unmodified IVT mRNA, concurrently demonstrating enhanced protein expression efficiency. The reduced immune responses are based on the ability of RNA/DNA chimeric elements to restrict retinoic acid-inducible gene I (RIG-I) and stimulator of interferon genes (STING)-mediated immune activation. The developed ACE mRNA shows great potential in modulating the immunogenicity of IVT mRNA without the need for chemically modified nucleotides, thereby advancing the safety and efficacy of mRNA-based therapeutics/vaccines.

Development of mRNA Vaccines/Therapeutics and Their Delivery System.

The rapid development of mRNA vaccines has contributed to the management of the current coronavirus disease 2019 (COVID-19) pandemic, suggesting that this technology may be used to manage future outbreaks of infectious diseases. Because the antigens targeted by mRNA vaccines can be easily altered by simply changing the sequence present in the coding region of mRNA structures, it is more appropriate to develop vaccines, especially during rapidly developing outbreaks of infectious diseases. In addition to allowing rapid development, mRNA vaccines have great potential in inducing successful antigen-specific immunity by expressing target antigens in cells and simultaneously triggering immune responses. Indeed, the two COVID-19 mRNA vaccines approved by the U.S. Food and Drug Administration have shown significant efficacy in preventing infections. The ability of mRNAs to produce target proteins that are defective in specific diseases has enabled the development of options to treat intractable diseases. Clinical applications of mRNA vaccines/therapeutics require strategies to safely deliver the RNA molecules into targeted cells. The present review summarizes current knowledge about mRNA vaccines/ therapeutics, their clinical applications, and their delivery strategies.

Platelet-rich plasma encapsulation in hyaluronic acid/gelatin-BCP hydrogel for growth factor delivery in BCP sponge scaffold for bone regeneration.

Microporous calcium phosphate based synthetic bone substitutes are used for bone defect healing. Different growth factor loading has been investigated for enhanced bone regeneration. The platelet is a cellular component of blood which naturally contains a pool of necessary growth factors that mediate initiation, continuation, and completion of cellular mechanism of healing. In this work, we have investigated the encapsulation and immobilization of platelet-rich plasma (PRP) with natural polymers like hyaluronic acid (HA) and gelatin (Gel) and loading them in a biphasic calcium phosphate (BCP) scaffold, for a synthetic-allologous hybrid scaffold. Effect of PRP addition in small doses was evaluated for osteogenic potential in vitro and in vivo. BCP (10%) mixed HA-Gel hydrogel with or without PRP, was loaded into a BCP sponge scaffold. We investigated the hydrogel-induced improvement in mechanical property and PRP-mediated enhancement in biocompatibility. In vitro studies for cytotoxicity, cell attachment, and proliferation were carried out using MC3T3-E1 pre-osteoblast cells. In in vitro studies, the cell count, cell proliferation, and cell survival were higher in the scaffold with PRP loading than without PRP. However, in the in vivo studies using a rat model, the PRP scaffold was not superior to the scaffold without PRP. This discrepancy was investigated in terms of the interaction of PRP in the in vivo environment.

The effects of dimethyl 3,3'-dithiobispropionimidate di-hydrochloride cross-linking of collagen and gelatin coating on porous spherical biphasic calcium phosphate granules.

Collagen- and gelatin-coated porous spherical granule was prepared by slurry dripping process using biphasic calcium phosphate powder. The coating was stabilized by cross-linking with dimethyl 3,3'-dithiobispropionimidate di-hydrogenchloride (DTBP). Afer DTBP cross-linking, the nanostructure of collagen- and gelatin-coated surfaces was changed from smooth to fibrous and net-like structure. Excellent cross-linking of the coating was seen as indicated by the differential scanning calorimetry thermogram and the Fourier transform infrared spectroscopy spectra. After cross-linking the relative intensities of the Fourier transform infrared spectroscopy peaks were decreased and amide bands were shifted to the left. The interaction of gelatin with DTBP cross-linking agent was stronger than that with collagen according to differential scanning calorimetry and Fourier transform infrared spectroscopy results. The compressive strength of the granular bone substitutes increased significantly after the coating process and gelatin coated biphasic calcium phosphate granules showed highest value at 3.68 MPa after cross-linking. Porosity was greater than 63% and did not change significantly with coating. Biocompatibility investigation by in vitro and in vivo showed that the coating improved the cell proliferation marginally. However, the cross-linking process did not jeopardize the excellent biocompatibility of collagen and gelatin. The in vivo study confirms better bone formation behavior of the cross-linked gelatin and collagen coated samples investigated for 8 weeks in vivo.

Evaluation of the potential anti-adhesion effect of the PVA/Gelatin membrane.

A common and prevailing complication for patients with abdominal surgery is the peritoneal adhesion that follows during the post-operative recovery period. Biodegradable polymers have been suggested as a barrier to prevent the peritoneal adhesion. In this work, as a preventive method, PVA/Gelatin hydrogel-based membrane was investigated with various combinations of PVA and gelatin (50/50, 30/70/, and 10/90). Membranes were made by casting method using hot PVA-gelatin solution and the gelatin was cross-linked by exposing UV irradiation for 5 days to render stability of the produced sheathed form in the physiological environment. Physical crosslinking was chosen to avoid the problems of potential cytotoxic effect of chemical crosslinking. Their materials characterization and mechanical properties were evaluated by SEM surface characterization, hydrophilicity, biodegradation rate, and so forth. Cytocompatibility was observed by in vitro experiments with cell proliferation using confocal laser scanning microscopy and the MTT assay by L-929 mouse fibroblast cells. The fabricated PVA/Gel membranes were implanted between artificially defected cecum and peritoneal wall in rats and were sacrificed after 1 and 2 weeks post-operative to compare their tissue adhesion extents with that of control group where the defected surface was not separated by PVA/Gel membrane. The PVA/Gel membrane (10/90) significantly reduced the adhesion extent and showed to be a potential candidate for the anti-adhesion application.

Electrospun PLGA/gelatin fibrous tubes for the application of biodegradable intestinal stent in rat model.

A biodegradable fibrous tube was fabricated by electrospinning method using a combination of Poly(lactic-co-glycolic acid) (PLGA) and gelatin dissolved in trifluoroethanol (TFE). Different ratios of the two polymers (PLGA/Gelatin: 1/9, 3/7, 5/5) were used for electrospinning to determine the optimum condition appropriate for intestinal stent application. Fiber morphology was visualized and analyzed using a scanning electron microscope (SEM). Characterizations of physical properties were done according to its tensile strength, surface hydrophilicity, swelling ability, and biodegradability. Biocompatibility of the scaffolds was investigated in vitro using IEC-18 (Rat intestinal epithelial cell). Cell proliferation was quantified using MTT assay and cell adhesion behavior was visualized by SEM and confocal laser scanning microscope. PLGA/Gelatin (5/5) was determined to have adequate material properties and sufficient in vitro biocompatibility. This was then implanted in a male Sprague-Dawley rat for 14 days to determine in vivo behavior of the sample. Histological examination on the intestinal tissue surrounding the graft showed normal morphology comparable to non-implanted intestine.

Preparation and characterization of PLGA microspheres by the electrospraying method for delivering simvastatin for bone regeneration.

Microparticles formulated from poly (D,L-lactic-co-glycolide) (PLGA), a biodegradable polymer, have been investigated extensively as a drug delivery system. In this study, solid tiny PLGA microspheres were fabricated using the electrospraying method. PLGA polymer was dissolved in dichloromethane (DCM), and the solution was electrosprayed. The electrospraying conditions were adjusted so that the stream ejected from the needle was divided into spheres instead of continuous fibers or irregular-shaped particles. Several experiments were carried out using the PLGA-DCM source solution with different amounts of simvastatin (SIM), a drug that enhances bone regeneration, to understand this drug delivery system. The surface morphology and microstructure of the microspheres formed were characterized by scanning electron microscopy, X-ray diffraction, Fourier-transform infrared spectroscopy, and differential scanning calorimetry. The in vitro experiments on drug loading and drug release behavior of the microspheres suggested a drug encapsulation efficacy >90%. The drug was continuously released from the microspheres for >3 weeks. Other experiments, such as MTT, cell attachment and proliferation and reverse transcription-polymerase chain reaction showed good biocompatibility of the electrosprayed PLGA microspheres, which increased in the presence of SIM. Thus, electrosprayed PLGA microspheres have potential as a drug delivery system and application in bone tissue engineering.

In vitro and in vivo evaluation of electrospun PCL/PMMA fibrous scaffolds for bone regeneration.

Scaffolds were fabricated by electrospinning using polycaprolactone (PCL) blended with poly(methyl methacrylate) (PMMA) in ratios of 10/0, 7/3, 5/5 and 3/7. The PCL/PMMA ratio affected the fiber diameter, contact angle, tensile strength and biological in vitro and in vivo properties of the scaffolds, and the 7/3 ratio resulted in a higher mechanical strength than 5/5 and 3/7. In vitro cytotoxicity and proliferation of MG-63 osteoblast cells on these blended scaffolds were examined by MTT assay, and it was found that PCL/PMMA blends are suitable for osteoblast cell proliferation. Confocal images and expression of proliferating cell nuclear antigen confirmed the good proliferation and expression of cells on the 7/3 PCL/PMMA fibrous scaffolds. In vivo bone formation was examined using rat models, and bone formation was observed on the 7/3 PCL/PMMA scaffold within 2 months. In vitro and in vivo results suggest that 7/3 PCL/PMMA scaffolds can be used for bone tissue regeneration.