Generation of a fluorescent oncoprotein in soluble form and its delivery into mammalian cells.

BACKGROUND: Protein fusion technology was widely used to improve expression, purification and solubility of the recombinant proteins expressed in E. coli for in vitro/in vivo delivery. METHODS: We developed a method for successful expression of soluble +36GFP-A2-E7 protein in E. coli and its effective delivery into mammalian cells. At first, the plasmid harboring +36GFP-A2-E7 was transformed into E. coli Rosetta competent cells. Then, the recombinant protein fused to histidine tag was expressed and purified using affinity chromatography. Different conditions such as inducer dose, time and temperature of induction, pH and urea concentration were evaluated. Finally, the delivery of the recombinant protein was detected in HEK-293T cells using fluorescent microscopy and flow cytometry. RESULTS: Our data showed that the expressed protein formed inclusion bodies at 37 °C and 3 h post-induction. The soluble protein was generated using 0.5 mM IPTG and growth at 16 °C for 20 h, and purified by low concentrations of urea and 200 mM imidazole. The soluble fraction of +36GFP-A2-E7 protein could significantly represent higher fluorescent property and stronger delivery into mammalian cells compared to the insoluble form. CONCLUSION: Generally, soluble form of fusion protein retained its biological activity and could directly penetrate into the cells without the fusion tags (Tab. 1, Fig. 7, Ref. 23).

Evaluation of TOPAS MC tool performance in optical photon transport and radioluminescence-based dosimetry.

Radiation therapy plays a pivotal role in modern cancer treatment, demanding precise and accurate dose delivery to tumor sites while minimizing harm to surrounding healthy tissues. Monte Carlo simulations have emerged as indispensable tools for achieving this precision, offering detailed insights into radiation transport and interaction at the subatomic level. As the use of scintillation and luminescence dosimetry becomes increasingly prevalent in radiation therapy, there arises a need for validated Monte Carlo tools tailored to optical photon transport applications. In this paper, an evaluation process of the TOPAS (TOol for PArticle Simulation) Monte Carlo tool for Cerenkov light generation, optical photon transport and radioluminescence based dosimetry is presented. Three distinct sources of validation data are utilized: one from a published set of experimental results and two others from simulations performed with the Geant4 code. The methodology employed for evaluation includes the selection of benchmark experiments, making use of opt3 and opt4 Geant4 physics models and simulation setup, with observed slight discrepancies within the calculation uncertainties. Additionally, the complexities and challenges associated with modeling optical photons generation through luminescence or Cerenkov radiation and their transport are discussed. The results of our evaluation suggests that TOPAS can be used to reliably predict Cerenkov generation, luminescence phenomenon and the behavior of optical photons in common dosimetry scenarios.

Osteoimmunomodulatory GelMA/liposome coatings to promote bone regeneration of orthopedic implants.

Despite being the most widely used biomaterials in orthopedic surgery, metallic implants do not induce new bone growth because they are bioinert. Surface biofunctionalization of implants with immunomodulatory mediators is a recent approach to promote osteogenic factors that facilitate bone regeneration. Liposomes (Lip) can be used as a low-cost, efficient and simple immunomodulator to stimulate immune cells in favor of bone regeneration. Even though liposomal coating systems have been reported previously, their main disadvantage is their limited ability to preserve liposome integrity after drying. In order to address this issue, we developed a hybrid system in which liposomes could be embedded in a polymeric hydrogel namely gelatin methacryloyl (GelMA). Specifically, we have developed a novel versatile coating strategy using electrospray technology to coat implants with GelMA/Liposome without using adhesive intermediate layer. The two differently charged Lip (i.e., anionic and cationic) were blended with GelMA and coated via electrospray technology on the bone-implant surfaces. The results showed that the developed coating withstood mechanical stress during surgical replacement, and Lip inside GelMA coating stayed intact in different storage conditions for a minimum of 4 weeks. Surprisingly, bare Lip, either cationic or anionic, improved the osteogenesis of human Mesenchymal Stem Cells (MSCs) by inducing pro-inflammatory cytokines, even at a low dosage of Lip released from the GelMA coating. More importantly, we showed that the inflammatory response could be fine-tuned by selecting the Lip concentration, Lip/hydrogel ratio, and coating thickness to determine the timing of the release such that we can accommodate different clinical needs. These promising results pave the way to use these Lip coatings to load different types of therapeutic cargo for bone-implant applications.

Protective effects of subchronic caffeine administration on cisplatin induced urogenital toxicity in male mice.

In Cisplatin treated group, the degeneration intensity of the kidneys the diameter of seminiferous tubules as well as the apoptotic index in testes and kidney were increased. In Caffeine+Cisplatin treated groups, the total body weight, the weight of testes and kidneys and also the histopathological data did not show significant differences. The motility of sperm in cisplatin group reduced but in Caffeine+Cisplatin groups this parameter was increased. These data suggest that caffeine recovers toxicity induced by cisplatin in both kidneys and testes of mice.

Controlled temperature-mediated curcumin release from magneto-thermal nanocarriers to kill bone tumors.

Systemic chemotherapy has lost its position to treat cancer over the past years mainly due to drug resistance, side effects, and limited survival ratio. Among a plethora of local drug delivery systems to solve this issue, the combinatorial strategy of chemo-hyperthermia has recently received attention. Herein we developed a magneto-thermal nanocarrier consisted of superparamagnetic iron oxide nanoparticles (SPIONs) coated by a blend formulation of a three-block copolymer Pluronic F127 and F68 on the oleic acid (OA) in which Curcumin as a natural and chemical anti-cancer agent was loaded. The subsequent nanocarrier SPION@OA-F127/F68-Cur was designed with a controlled gelation temperature of the shell, which could consequently control the release of curcumin. The release was systematically studied as a function of temperature and pH, via response surface methodology (RSM). The bone tumor killing efficacy of the released curcumin from the carrier in combination with the hyperthermia was studied on MG-63 osteosarcoma cells through Alamar blue assay, live-dead staining and apoptosis caspase 3/7 activation kit. It was found that the shrinkage of the F127/F68 layer stimulated by elevated temperature in an alternative magnetic field caused the curcumin release. Although the maximum release concentration and cell death took place at 45 °C, treatment at 41 °C was chosen as the optimum condition due to considerable cell apoptosis and lower side effects of mild hyperthermia. The cell metabolic activity results confirmed the synergistic effects of curcumin and hyperthermia in killing MG-63 osteosarcoma cells.