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PurposePaclitaxel is a first-line drug for the therapy of lung cancer, however, drug resistance is a serious limiting factor, related to overexpression of anti-apoptotic proteins like survivin. To overcome this phenomenon, developing novel ultrasound responsive nanobubbles - nanosized drug delivery system- for the delivery of paclitaxel and siRNA in order to silence survivin expression in the presence of ultrasound was aimed.MethodsPaclitaxel-carrying nanobubble formulation was obtained by modifying the multistep method. Then, the complex formation of the nanobubbles - paclitaxel formulation with survivin-siRNA, was examined in terms of particle size, polydispersity index, zeta potential, and morphology. Furthermore, siRNA binding and protecting ability, cytotoxicity, cellular uptake, gene silencing, and induction of apoptosis studies were investigated in terms of lung cancer cells.ResultsDeveloped nanobubbles have particle sizes of 218.9–369.6 nm, zeta potentials of 27–34 mV, were able to protect siRNA from degradation and delivered siRNA into the lung cancer cells. Survivin expression was significantly lower compared with the control groups and enhanced apoptosis was induced by the co-delivery of survivin-siRNA and paclitaxel. Furthermore, significantly higher effects were obtained in the presence of ultrasound induction.ConclusionThe ultrasound responsive nanobubble system carrying paclitaxel and survivin-siRNA is a promising and effective approach against lung cancer cells.

Toxoplasma gondii is a zoonotic parasite that infects almost all warm-blooded animals and humans and results in serious health problems. There is no drug treatment for chronic toxoplasmosis. Thus, a safe and protective vaccine is required by the community to combat against the chronic infection caused by T. gondii in humans and animals. Rhoptry (ROP) proteins of T. gondii have important roles in host cell invasion, penetration, and biogenesis of the parasitophorous vacuole. In this study, we aimed to develop a novel vaccine with recombinant ROP6 protein (rROP6), which was shown to be highly immunogenic in protein microarray screening with blood samples collected from animal models infected with T. gondii oocysts or tissue cysts. Initially, a comprehensive in silico analyses was performed to design ROP6 protein to be used as vaccine antigen. Then, rROP6 protein was expressed in Saccharomyces cerevisiae INVSc1 cells and purified by affinity chromatography. Next, rROP6 protein adjuvanted with Freund’s (rROP6 + Freund) was administered to BALB/c mice two times at three-week intervals. Humoral and cellular immune responses were analyzed by Western blot, ELISA, flow cytometry, and cytokine ELISA. Protective efficacy was determined by orally infecting mice with T. gondii PRU strain tissue cysts. The level of protection was analyzed by investigating tissue cysts in brain homogenate of mice using microscopy and qPCR. According to the results, rROP6 + Freund induced a strong IgG response compared to only rROP6 ( P  < 0.01) and the rate of CD8 + T lymphocytes secreting IFN- γ significantly increased in mice administered with rROP6 + Freund compared to other mice groups ( P  < 0.05). According to challenge results, all the rROP6 + Freund vaccine administered mice survived and the number of tissue cysts and the amount of T. gondii DNA decreased significantly compared to controls ( P  < 0.01; P  < 0.0001). In conclusion, compared to control groups, rROP6 + Freund vaccine induced a high level of protective immunity and provided a significant level of protection as demonstrated by significant reduction in tissue cysts. We conclude that the recombinant ROP6 protein produced in S. cerevisiae is an immunogenic, protective, and promising vaccine candidate antigen that can be used in vaccine formulations against chronic toxoplasmosis.

Severe acute respiratory syndrome coronavirus 2 had devastating consequences for human health. Despite the introduction of several vaccines, COVID-19 continues to pose a serious health risk due to emerging variants of concern. DNA vaccines gained importance during the pandemic due to their advantages such as induction of both arms of immune response, rapid development, stability, and safety profiles. Here, we report the immunogenicity and protective efficacy of a DNA vaccine encoding spike protein with D614G mutation (named pcoSpikeD614G) and define a large-scale production process. According to the in vitro studies, pcoSpikeD614G expressed abundant spike protein in HEK293T cells. After the administration of pcoSpikeD614G to BALB/c mice through intramuscular (IM) route and intradermal route using an electroporation device (ID + EP), it induced high level of anti-S1 IgG and neutralizing antibodies ( P  < 0.0001), strong Th1-biased immune response as shown by IgG2a polarization ( P  < 0.01), increase in IFN-γ levels ( P  < 0.01), and increment in the ratio of IFN-γ secreting CD4 + (3.78–10.19%) and CD8 + (5.24–12.51%) T cells. Challenging K18-hACE2 transgenic mice showed that pcoSpikeD614G administered through IM and ID + EP routes conferred 90–100% protection and there was no sign of pneumonia. Subsequently, pcoSpikeD614G was evaluated as a promising DNA vaccine candidate and scale-up studies were performed. Accordingly, a large-scale production process was described, including a 36 h fermentation process of E. coli DH5α cells containing pcoSpikeD614G resulting in a wet cell weight of 242 g/L and a three-step chromatography for purification of the pcoSpikeD614G DNA vaccine.

Aim: A successful gene therapy requires a delivery system for overcoming various biological barriers. For this, we adapted the factorial design and response surface methodology to the cationic solid lipid nanoparticle production process. Methods: Screening and optimization of formulations were carried out with factorial design with 3 factors and 3 levels using Box-Behnken Design. Then, solid lipid nanoparticles were physicochemically characterized. Furthermore, optimal SLN formulation is examined in terms of complex formation with plasmid DNA, its protection potential against nucleases, cytotoxicity profile, and storage stability. Results: Response-surface analyses demonstrated that the selected quadratic model holds significance for particle size and zeta potential. The interaction of independent variables was statistically determined. Optimization and prediction were performed using obtained second-order polynomial equations. Optimal formulation and complexes were found to be nanosized, positively charged and their polydispersity-index values below 0.3 as an indicator of being monodispersed. Cytotoxicity of the optimal formulation is compatible for further studies and no significant increase was observed in particle size until day 21 and until day 60 for polydispersity-index. Conclusion: Optimal formulation provides a good basis as a gene delivery system was produced with developed systematic. Briefly, this methodology could be used to obtain SLNs with desired conditions.

Background This study is to compare the two different techniques used in the labeling of implant-based fixed prostheses, using the square code and using microchip labeling techniques, taking into account the properties of an ideal prosthetic labeling technique. Sixty implants fixed prostheses were produced, 30 for each group. Square codes were created on the lingual bands of implants fixed prostheses which are the samples of the group that were labelled by using square code, and microchips were placed in the implant abutments of the samples in the group that were labelled with the microchip. Results The thermal cycle test was used to compare the long-life cycle of the samples, and no deformation was found. A survey attended by 51 dentists was created to evaluate the techniques’ effects on their aesthetic appeal and their application. The data found was statistically evaluated, and the result was statistically insignificant ( p  < 0.05). Conclusions Microchips’ data storage capacity was found more successful; however, according to their resistance to heat and their costs, the square code, was more advantageous.

In recent years, non-viral delivery systems for plasmid DNA have become particularly important. They can overcome the disadvantages of viral systems such as insertional mutagenesis and unpredicted immunogenicity. Some additional advantages of non-viral gene delivery systems are; good stability, low cost, targetability, delivery of a high amount of genetic materials. The aim of the study was to develop novel non-viral nanosystems suitable for gene delivery. Two formulations were developed for this purpose: water-in-oil microemulsion (ME) and solid lipid nanoparticles (SLN). The microemulsion was composed of Peceol, Tween 80, Plurol oleique, ethanol and water. The SLN was consisting of Precirol, Esterquat-1 (EQ1), Tween 80, Lecithin, ethanol and water. Characterization studies were carried out by measuring particle size, zeta potential, viscosity and pH. TEM imaging was performed on SLN formulations. Protection against DNase I degradation was examined. Cytotoxicity and transfection efficacy of selected formulations were tested on L929 mouse fibroblast cells. Particle sizes of complexes were below 100 nm and with high positive zeta potential. TEM images revealed that SLNs are spherical. The SLN:DNA complexes have low toxicity and good transfection ability. All results showed that the developed SLN formulations can be considered as suitable non-viral gene delivery systems.

Gene therapy has emerged as a promising strategy for tissue regeneration, offering the potential to address the limitations of conventional treatments across musculoskeletal, cardiovascular, nervous, and epithelial tissues.Recent advances in molecular biology and genetics, such as clustered regularly interspaced short palindromic repeats (CRISPR) gene editing, have enhanced the precision and efficacy of gene therapy approaches for tissue engineering.Technological advances in 3D bioprinting and gene delivery systems have expanded the possibilities for creating customized scaffolds and improving targeted gene delivery for tissue engineering applications.Clinical trials demonstrate the potential of gene therapy in tissue engineering, though challenges remain in areas such as safety, long-term efficacy, and regulatory approval.Future directions include integrating artificial intelligence and machine learning to optimize gene therapy protocols, developing personalized approaches, and combining gene therapy with other advanced technologies for enhanced tissue regeneration outcomes. Gene therapy has emerged as a promising strategy for tissue regeneration, offering the potential to address the limitations of conventional treatments. In this review we present an overview of applications of gene therapy in tissue regeneration, emphasizing recent advancements and future directions. Our work addresses gaps in the current literature by examining developments in molecular biology and genetics, such as clustered regularly interspaced short palindromic repeats (CRISPR) gene editing, advances in 3D bioprinting, and progress in gene delivery for tissue engineering. We describe case studies and clinical trials that demonstrate the potential of gene therapy applications in tissue engineering. We conclude by highlighting challenges and future directions, including emerging technologies and personalized gene-based approaches for tissue engineering research. Gene therapy has emerged as a promising strategy for tissue regeneration, offering the potential to address the limitations of conventional treatments. In this review we present an overview of applications of gene therapy in tissue regeneration, emphasizing recent advancements and future directions. Our work addresses gaps in the current literature by examining developments in molecular biology and genetics, such as clustered regularly interspaced short palindromic repeats (CRISPR) gene editing, advances in 3D bioprinting, and progress in gene delivery for tissue engineering. We describe case studies and clinical trials that demonstrate the potential of gene therapy applications in tissue engineering. We conclude by highlighting challenges and future directions, including emerging technologies and personalized gene-based approaches for tissue engineering research.

Objectives: Gene therapy approaches have become increasingly attractive in the medical, pharmaceutical, and biotechnological industries due to their applicability in the treatment of diseases with no effective conventional therapy. Non-viral delivery using cationic solid lipid nanoparticles (cSLNs) can be useful to introduce large nucleic acids to target cells. A careful selection of components and their amounts is critical to obtain a successful delivery system. In this study, solid Witepsol nanoparticles were formulated, characterized, and evaluated in vitro for gene delivery purposes. Materials and Methods: Solid Witepsol nanoparticles were formulated through the microemulsion dilution technique using two grades of Witepsol and three surfactants, namely Cremephor RH40, Kolliphor HS15, and Peceol. Dimethyldioctadecylammonium bromide was incorporated into the system as a cationic lipid. Twelve combinations of these ingredients were formulated. The obtained nanoparticles were then evaluated for particle size, zeta potential, DNA binding and protection ability, cytotoxicity, and transfection ability. Results: Particle sizes of the prepared cationic cSLNs were between 13.43±0.06 and 68.80±0.78 nm. Their zeta potential, which is important for DNA binding efficiency, was determined at >+40 mV. Gel retardation assays revealed that the obtained cSLNs can form a compact complex with plasmid DNA (pDNA) encoding green fluorescent protein and that this complex can protect pDNA from DNase I-mediated degradation. Cytotoxicity evaluation of nanoparticles was performed on the L929 cell line. In vitro transfection data revealed that solid Witepsol nanoparticles could effectively transfect fibroblasts. Conclusion: Our findings indicate that solid Witepsol nanoparticles prepared using the microemulsion dilution technique are promising non-viral delivery systems for gene therapy.

Toxoplasma gondii is an obligate intracellular parasite that can infect a variety of mammals including humans and causes toxoplasmosis. Unfortunately, a protective and safe vaccine against toxoplasmosis hasn’t been developed yet. In this study, we developed a DNA vaccine encoding the SRS13 protein and immunized BALB/c mice thrice with pVAX1-SRS13 through the intramuscular route (IM) or intradermally using an electroporation device (ID + EP). The immunogenicity of pVAX1-SRS13 was analyzed by ELISA, Western blot, cytokine ELISA, and flow cytometry. The protective efficacy of the pVAX1-SRS13 was investigated by challenging mice orally with T. gondii PRU strain tissue cysts. The results revealed that pVAX1-SRS13 administered through IM or ID + EP routes induced high level of anti-SRS13 IgG antibody responses (P = 0.0037 and P < 0.0001). The IFN-γ level elicited by the pVAX1-SRS13 (ID + EP) was significantly higher compared to the control group (P = 0.00159). In mice administered with pVAX1-SRS13 (ID + EP), CD8+ cells secreting IFN-γ was significantly higher compared to pVAX1-SRS13 (IM) (P = 0.0035) and the control group (P = 0.0068). Mice vaccinated with the SRS13 DNA vaccine did not induce significant IL-4 level. Moreover, a significant reduction in the number of tissue cysts and the load of T. gondii DNA was detected in brains of mice administered with pVAX1-SRS13 through ID + EP and IM routes compared to controls. In conclusion, the SRS13 DNA vaccine was found to be highly immunogenic and confers strong protection against chronic toxoplasmosis.

No significant cytotoxicity was observed on COS-7 cells in the concentration range of 4-20 pL/well. [...]transfection studies revealed that the optimal system (GMS-MNP-1) showed significantly higher transfection efficacy comparing the naked plasmid or non-magnetic version of nanoparticle under a magnetic field (p>0.05). Since nanoparticles are small enough to easily enter almost all body areas, including the circulatory system and cells, they have become the basis of basic biomedical research, diagnostic science, and therapeutic applications of nanotechnology (Selmani et al., 2022). For MNP-based in vitro transfection, the particle-DNA complex is applied to the cell culture and a magnet or electromagnet is placed on the underside of this cell culture, which can generate a magnet-like electromagnetic field. [...]the sedimentation and transfection rate of the DNA-particle complex increases (Dowaidar et al., 2017). Characterization Magnetic properties of GMS-MNPs were confirmed by using Lakeshore Vibrating Sample Magnetometer (VSM). [...]the particle size and zeta potential of nanoparticles were evaluated with the dynamic light scattering (DLS) method.