Immunogenicity and protection efficacy of a COVID-19 DNA vaccine encoding spike protein with D614G mutation and optimization of large-scale DNA vaccine production.

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.

A novel DNA vaccine encoding the SRS13 protein administered by electroporation confers protection against chronic toxoplasmosis.

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.

Design of Liposome Formulations for CRISPR/Cas9 Enzyme Immobilization: Evaluation of 5-Alpha-Reductase Enzyme Knockout for Androgenic Disorders.

The enzyme steroid type II 5-alpha-reductase (SRD5α2) is responsible for the conversion of testosterone to dihydrotestosterone (DHT), which is involved in prostate cancer, benign prostatic hyperplasia, and androgenic alopecia. Inhibition of SRD5α2 activity has been explored and presented as a potential treatment for these conditions, but current drugs have side effects and alternative treatment approaches are needed. The CRISPR/Cas9 system, an innovative gene-editing tool, shows potential for targeting the SRD5α2 gene knockout as a therapeutic approach. Liposomes have been used for the immobilization and delivery of different proteins, and studies have shown that liposomes can enhance the stability and activity of enzymes. In this study, we provided the immobilization of Cas9 protein by encapsulating it in a novel cationic liposome formulation that carries sgRNA on its outer surface for gene delivery approaches. This novel delivery system has shown promising results in terms of physicochemical properties, stability, cytotoxicity, in vitro cellular uptake, and gene knockout efficiency, together with providing flexibility in sgRNA selection. The optimized final formulations showed an average diameter of 229.1 ± 3.66 nm, a polydispersity index of 0.089 ± 0.013, and a zeta potential value of 25.7 ± 0.87 mV. The encapsulation efficiency of the developed formulations has been revealed as 80.60%. The cellular uptake efficiency was evaluated and measured as 45.6% for the final formulation. Furthermore, the Lipo/Cas9:sgRNA (1.5:1) formulation decreased the relative SRD5α2 mRNA expression by 29.7% compared to the control group. The results of this study reveal that the liposomal formulation based on enzyme immobilization of Cas9 protein using CRISPR technology, an innovative gene-editing tool for SRD5α2 suppression, might be an alternative treatment option for prostate cancer or BPH treatment without current drug side effects.

Interactions of Tissue-Resident T Cells.

Resident memory T cells (TRM) are non-circulating cells that play a critical role in protection from local infections and cancers. Flow cytometric and transcriptional analyses of these cells have defined their distinct phenotypes; imaging allows study of their morphology, localization, and interactions within tissues. Here, we describe commonly used methods to generate cutaneous CD8+ TRM and to prepare skin samples for analysis, including staining of cryostat sections, epidermal sheets, and tissue whole mounts.

Design, synthesis, in vitro - In vivo biological evaluation of novel thiazolopyrimidine compounds as antileishmanial agent with PTR1 inhibition.

The leishmaniasis are a group of vector-borne diseases caused by a protozoan parasite from the genus Leishmania. In this study, a series of thiazolopyrimidine derivatives were designed and synthesized as novel antileishmanial agents with LmPTR1 inhibitory activity. The final compounds were evaluated for their in vitro antipromastigote activity, LmPTR1 and hDHFR enzyme inhibitory activities, and cytotoxicity on RAW264.7 and L929 cell lines. Based on the bioactivity results, three compounds, namely L24f, L24h and L25c, were selected for evaluation of their in vivo efficacy on CL and VL models in BALB/c mice. Among them, two promising compounds, L24h and L25c, showed in vitro antipromastigote activity against L. tropica with the IC50 values of 0.04 µg/ml and 6.68 µg/ml; against L. infantum with the IC50 values of 0.042 µg/ml and 6.77 µg/ml, respectively. Moreover, the title compounds were found to have low in vitro cytotoxicity on L929 and RAW264.7 cell lines with the IC50 14.08 µg/ml and 21.03 µg/ml, and IC50 15.02 µg/ml and 8.75 µg/ml, respectively. LmPTR1 enzyme inhibitory activity of these compounds was determined as 257.40 µg/ml and 59.12 µg/ml and their selectivity index (SI) over hDHFR was reported as 42.62 and 7.02, respectively. In vivo studies presented that L24h and L25c have a significant antileishmanial activity against footpad lesion development of CL and at weight measurement of VL group in comparison to the reference compound, Glucantime®. Also, docking studies were carried out with selected compounds and other potential Leishmania targets to detect the putative targets of the title compounds. Taken together, all these findings provide an important novel lead structure for the antileishmanial drug development.

Design and evaluation of erucic acid-phytosphingosine structured cationic nanoemulsions as a plasmid DNA delivery system against breast cancer cells.

This study is focused on the preparation and characterization of erucic acid (EA) and phytosphingosine (PS) containing cationic nanoemulsions (NEs) for plasmid DNA (pDNA) delivery. Repurposing of cationic agents guided us to PS, previously used for enhanced interaction with negatively charged surfaces. It was reported that EA might act anti-tumoral on C6 glioma, melanoma, neuroblastoma, and glioblastoma. However, there is only one study about mixed oleic acid-EA liposomes. This gap attracted our interest in the possible synergistic effects of PS and EA on MDA-MB-231 and MCF-7 breast cancer cells. Three cationic NEs (NE 1, NE 2, and NE 3) were prepared and characterized in terms of droplet size (DS), polydispersity index (PDI), and zeta potential (ZP) before and after complexation with pDNA, long-term stability, SDS release, cytotoxicity, and transfection studies. The cationic NEs had DSs of <200 nm, PDIs <0.3, and ZPs > +30 mV. Long-term stability studies revealed that NE 2 and NE 3 were stable. NE 1-pDNA had appropriate particle properties. NE 2 reduced the viability of MDA-MB-231 cells to 11% and of MCF-7 cells to 13% and resulted in the highest number of transfected cells. To sum up, NE 2 containing PS and EA is appropriate for delivering pDNA.

Atezolizumab-Conjugated Poly(lactic acid)/Poly(vinyl alcohol) Nanoparticles as Pharmaceutical Part Candidates for Radiopharmaceuticals.

The necessity of new drugs for lung cancer therapy and imaging is increasing each day. The development of new drugs that are capable of reaching the tumor with specificity and selectivity is required. In this direction, the design of nanoparticles for tumor therapy represents an important alternative. The aim of this study was to develop, characterize, and evaluate target-specific atezolizumab-conjugated poly(lactic acid)/poly(vinyl alcohol) (PLA/PVA) nanoparticles as pharmaceutical fragment candidates for new radiopharmaceuticals. For this purpose, PLA/PVA nanoparticle formulations were prepared by the double emulsification/solvent evaporation method with a high-speed homogenizer. A special focus was oriented to the selection of a suitable method for modification of the nanoparticle surface with a monoclonal antibody. For this purpose, atezolizumab was bound to the nanoparticles during the preparation by solvent evaporation or either by adsorption or covalent binding. PLA/PVA/atezolizumab nanoparticles are characterized by dynamic light scattering, Raman spectroscopy, scanning electron microscopy, and atomic force microscopy. An in vitro assay was performed to evaluate the antibody binding efficiency, stability, and cytotoxicity [A549 (lung cancer cell) and L929 (healthy fibroblast cell)]. The results showed that a spherical nanoparticle with a size of 230.6 ± 1.768 nm and a ζ potential of -2.23 ± 0.55 mV was produced. Raman spectroscopy demonstrated that the monoclonal antibody was entrapped in the nanoparticle. The high antibody binding efficiency (80.58%) demonstrated the efficacy of the nanosystem. The cytotoxic assay demonstrated the safety of the nanoparticle in L929 and the effect on A549. In conclusion, PLA/PVA/atezolizumab nanoparticles can be used as drug delivery systems for lung cancer diagnosis and therapy.

Investigation of the potential therapeutic effect of cationic lipoplex mediated fibroblast growth factor-2 encoding plasmid DNA delivery on wound healing.

BACKGROUND: Developing an alternative and efficient therapy for wound healing has been an important research topic for pharmaceutical sciences. A straightforward but effective system for delivering fibroblast growth factor-2 (FGF-2) encoding plasmid DNA (pFGF-2) for wound healing therapy was aimed to develop in this study. METHODS: In order to provide the delivery of pFGF-2, a delivery vector, namely, cationic lipid nanoparticle (cLN) was developed by the melt-emulsification process, complexed with pFGF-2 to form a lipoplex system and further characterized. The pFGF-2 binding and protecting ability of lipoplexes were evaluated. The cytotoxicity and transfection efficiency of the lipoplexes, FGF-2 expression levels, and in vitro wound healing ability have been investigated on the L929 fibroblast cell line. RESULTS: The obtained lipoplex system has a particle size of 88.53 nm with a low PDI (0.185), and zeta potential values of 27.8 mV with a spherical shape. The ability of cLNs to bind pFGF-2 and protect against nucleases was demonstrated by gel retardation assay. Furthermore, the developed FGF-2 carrying lipoplexes system showed significant transfection and FGF-2 expression ability comparing naked plasmid. Finally, scratch assay revealed that the developed system is able to promote in vitro cell proliferation/migration in 48 h. CONCLUSION: Promising results have been achieved with the use of lipoplexes carrying pFGF-2, and this approach could be considered as a potentially applicable concept for the future gene-based wound healing therapies.

Development and Evaluation of Solid Witepsol Nanoparticles for Gene Delivery

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.

Special Focus Issue Part II: Recruitment of solid lipid nanoparticles for the delivery of CRISPR/Cas9: primary evaluation of anticancer gene editing.

Aim: The CRISPR/Cas9 system is a promising gene-editing tool for various anticancer therapies; however, development of a biocompatible, nonviral and efficient delivery of CRISPR/Cas9 expression systems remains a challenge. Materials & methods: Solid lipid nanoparticles (SLNs) were produced based on pseudo and 3D ternary plots. Obtained SLNs and their complexes with PX458 plasmid DNA were characterized and evaluated in terms of cytotoxicity and transfection efficiency. Results: SLNs were found to be nanosized, monodispersed, stable and nontoxic. Furthermore, they revealed similar transfection efficiency as the positive control. Conclusion: Overall, we have achieved a good SLN basis for CRISPR/Cas9 delivery and have the potential to produce SLNs with targeted anticancer properties by modifying production parameters and components to facilitate translating CRISPR/Cas9 into preclinical studies.

CD49a Regulates Cutaneous Resident Memory CD8+ T Cell Persistence and Response.

CD8+ tissue-resident memory T cells (TRM) persist at sites of previous infection, where they provide rapid local protection against pathogen challenge. CD8+ TRM expressing the α1 chain (CD49a) of integrin VLA-1 have been identified within sites of resolved skin infection and in vitiligo lesions. We demonstrate that CD49a is expressed early following T cell activation in vivo, and TGF-ß and IL-12 induce CD49a expression by CD8+ T cells in vitro. Despite this rapid expression, CD49a is not required for the generation of a primary CD8+ T cell response to cutaneous herpes simplex virus (HSV) infection, migration of CD8+ T cells across the epidermal basement membrane, or positioning of TRM within basal epidermis. Rather, CD49a supports CD8+ TRM persistence within skin, regulates epidermal CD8+ TRM dendritic extensions, and increases the frequency of IFN-γ+ CD8+ TRM following local antigen challenge. Our results suggest that CD49a promotes optimal cutaneous CD8+ TRM-mediated immunity.

Enhanced Cellular Uptake and Gene Silencing Activity of Survivin-siRNA via Ultrasound-Mediated Nanobubbles in Lung Cancer Cells.

PURPOSE: Paclitaxel 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. METHODS: Paclitaxel-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. RESULTS: Developed 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. CONCLUSION: The ultrasound responsive nanobubble system carrying paclitaxel and survivin-siRNA is a promising and effective approach against lung cancer cells.

Design, synthesis, and in vitro biological evaluation of novel thiazolopyrimidine derivatives as antileishmanial compounds.

A series of thiazolopyrimidine derivatives was designed and synthesized as a Leishmania major pteridine reductase 1 (LmPTR1) enzyme inhibitor. Their LmPTR1 inhibitor activities were evaluated using the enzyme produced by Escherichia coli in a recombinant way. The antileishmanial activity of the selected compounds was tested in vitro against Leishmania sp. Additionally, the compounds were evaluated for cytotoxic activity against the murine macrophage cell line RAW 264.7. According to the results, four compounds displayed not only a potent in vitro antileishmanial activity against promastigote forms but also low cytotoxicity. Among them, compound L16 exhibited an antileishmanial activity for both the promastigote and amastigote forms of L. tropica, with IC50 values of 7.5 and 2.69 µM, respectively. In addition, molecular docking studies and molecular dynamics simulations were also carried out in this study. In light of these findings, the compounds provide a new potential scaffold for antileishmanial drug discovery.

Development and characterization of nanobubbles containing paclitaxel and survivin inhibitor YM155 against lung cancer.

Lung cancer remains 23% of cancer-related death worldwide, ranking on first place for men and second place for women. Almost each cancer type has a great deal in common, overexpression of the apoptosis inhibitor survivin. Chemotherapy with anticancer drugs is leading to side effects. Drug targeting by the use of nanobubbles is a useful strategy to reduce side effects. Nanobubbles in cancer are one of the most investigated carriers in the last years. The size of nanobubbles (1-500 nm) is bigger than the pore size of healthy tissues, but smaller than the pores of cancer tissues. Thus, it is not possible for the drug to leave the blood stream and enter the tissue, but it can enter the cancer tissue through the pores, where it can accumulate. Therefore, the probability of undesired side effects decreases. For that reason, the development of nanobubbles containing paclitaxel and survivin inhibitor sepantronium bromide (YM155) were carried out. Characterization studies in terms of particle size, size distribution, zeta potential and morphology, and investigation of their effects on lung cancer cells were performed. To the best of our knowledge, there is no information in the literature about combining paclitaxel and YM155 loaded nanobubbles with ultrasound exposure.

Radiation-Induced Targeted Nanoparticle-Based Gene Delivery for Brain Tumor Therapy.

Targeted therapy against the programmed cell death ligand-1 (PD-L1) blockade holds considerable promise for the treatment of different tumor types; however, little effect has been observed against gliomas thus far. Effective glioma therapy requires a delivery vehicle that can reach tumor cells in the central nervous system, with limited systemic side effect. In this study, we developed a cyclic peptide iRGD (CCRGDKGPDC)-conjugated solid lipid nanoparticle (SLN) to deliver small interfering RNAs (siRNAs) against both epidermal growth factor receptor (EGFR) and PD-L1 for combined targeted and immunotherapy against glioblastoma, the most aggressive type of brain tumors. Building on recent studies showing that radiation therapy alters tumors for enhanced nanotherapeutic delivery in tumor-associated macrophage-dependent fashion, we showed that low-dose radiation primes targeted SLN uptake into the brain tumor region, leading to enhanced downregulation of PD-L1 and EGFR. Bioluminescence imaging revealed that radiation therapy followed by systemic administration of targeted SLN leads to a significant decrease in glioblastoma growth and prolonged mouse survival. This study combines radiation therapy to prime the tumor for nanoparticle uptake along with the targeting effect of iRGD-conjugated nanoparticles to yield a straightforward but effective approach for combined EGFR inhibition and immunotherapy against glioblastomas, which can be extended to other aggressive tumor types.

Preparation and characterization of non-viral gene delivery systems with pEGFP-C1 Plasmid DNA

ABSTRACT 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.

Improved Method for Solid Lipid Nanoparticle Preparation Based on Hot Microemulsions: Preparation, Characterization, Cytotoxicity, and Hemocompatibility Evaluation.

The ease of application and no requirement of extra energy input make the microemulsion method favorable for solid lipid nanoparticles (SLNs) production. Very limited data are available to date on preparation of SLNs from pre-screened microemulsion phase diagrams. The purpose of this study was to investigate the microemulsion formation area with solid lipids using hot ternary phase diagrams at elevated temperatures and to use selected microemulsions for SLN production. Also, we aimed to characterize obtained SLNs in terms of physicochemical properties, in vitro cell toxicity, and hemolysis. Phase diagrams of solid lipids were screened at elevated temperatures and oil-in-water microemulsion regions were determined. Microemulsions were selected, and SLNs were produced by modification of the microemulsion dilution method and characterized in terms of visual appearance, turbidity, particle size, and zeta potential. Cytotoxicity of nanoparticles was tested on L929 mouse skin fibroblast cells. Hemolytic potential was assessed in vitro using freshly isolated erythrocytes. The phase diagram screening and the modified hot microemulsion dilution method enabled production of SLNs with particle size below 100 nm. We found evidence that the solid lipids in the SLNs produced by the new method remain in supercooled liquid state. Nanoparticles prepared by the new method exhibit lower toxicity on L929 cells and have lower hemolytic potential than the formulations prepared by direct mixing of the components. The method can be used to prepare SLNs with controllable composition and small particle size below 100 nm. These SLNs are low toxic and can be used for drug delivery purposes.