Lavandula stoechas extract incorporated polylactic acid nanofibrous mats as an antibacterial and cytocompatible wound dressing.

In recent years, great efforts have been devoted to the design and production of bioactive wound dressings that promote skin regeneration and prevent infection. Many plant extracts and essential oils have been widely accepted in traditional medicine for a wide variety of medicinal purposes, especially wound healing. Over the past decade, many studies have focused on manufacturing and designing wound dressings containing plant compounds and extracts. In this study, Lavandula stoechas extract (LSE) (0.25 %, 0.5 %, and 1%wt) incorporated-polylactic acid (PLA) nanofibrous mats were successfully produced and characterized. Microstructural analysis by SEM revealed that the fiber diameter changed with the increase in the amount of LSE. Also, the nanofibrous mats were evaluated for their in vitro antibacterial, cytotoxicity, and wound healing properties for their use as a wound dressing material. According to the results of the disc diffusion test, PLA nanofibrous mats containing LSE %1 showed 9.65 ± 0.46 and 7.37 ± 0.03 inhibition zone (mm) against E. coli and S. aureus, respectively. According to the results of the in vitro wound healing assay, mats containing 0.5 % LSE showed better-wound closure activity compared to the control. Our results show that LSE-incorporated nanofibrous dressings can be an effective alternative with good antimicrobial activity.

Development of an Antiviral Ion-Activated In Situ Gel Containing 18ß-Glycyrrhetinic Acid: A Promising Alternative against Respiratory Syncytial Virus.

The human respiratory syncytial virus (hRSV) is a major cause of serious lower respiratory infections and poses a considerable risk to public health globally. Only a few treatments are currently used to treat RSV infections, and there is no RSV vaccination. Therefore, the need for clinically applicable, affordable, and safe RSV prevention and treatment solutions is urgent. In this study, an ion-activated in situ gelling formulation containing the broad-spectrum antiviral 18ß-glycyrrhetinic acid (GA) was developed for its antiviral effect on RSV. In this context, pH, mechanical characteristics, ex vivo mucoadhesive strength, in vitro drug release pattern, sprayability, drug content, and stability were all examined. Rheological characteristics were also tested using in vitro gelation capacity and rheological synergism tests. Finally, the cytotoxic and antiviral activities of the optimized in situ gelling formulation on RSV cultured in the human laryngeal epidermoid carcinoma (HEp-2) cell line were evaluated. In conclusion, the optimized formulation prepared with a combination of 0.5% w/w gellan gum and 0.5% w/w sodium carboxymethylcellulose demonstrated good gelation capacity and sprayability (weight deviation between the first day of the experiment (T0) and the last day of the experiment (T14) was 0.34%), desired rheological synergism (mucoadhesive force (Fb): 9.53 Pa), mechanical characteristics (adhesiveness: 0.300 ± 0.05 mJ), ex vivo bioadhesion force (19.67 ± 1.90 g), drug content uniformity (RSD%: 0.494), and sustained drug release over a period of 6 h (24.56% ± 0.49). The optimized formulation demonstrated strong anti-hRSV activity (simultaneous half maximal effective concentration (EC50) = 0.05 µg/mL; selectivity index (SI) = 306; pre-infection EC50 = 0.154 µg/mL; SI = 100), which was significantly higher than that of ribavirin (EC50 = 4.189 µg/mL; SI = 28) used as a positive control against hRSV, according to the results of the antiviral activity test. In conclusion, this study showed that nasal in situ gelling spray can prevent viral infection and replication by directly inhibiting viral entry or modulating viral replication.

The Effect of Smoking on Inactivated and mRNA Vaccine Responses Applied to Prevent COVID-19 in Multiple Sclerosis.

Introduction: Coronavirus disease 2019 (COVID-19) is the biggest health challenge of recent times. Studies so far reveal that vaccination is the only way to prevent this pandemic. There may be factors that decrease or increase vaccine effectiveness. In multiple sclerosis (MS), some of these factors may cause changes in the effectiveness of the vaccine, depending on the nature of the disease and disease-modifying treatments (DMT). In this study, we aimed to investigate the relationship between antibody titer and smoking in non-treated and DMT-treated MS patients who received inactivated vaccine (Sinovac) and messenger RNA BNT162b2 (BioNTech) mRNA vaccines. Method: Vaccine antibody responses were measured between 4-12 weeks after two doses of inactivated vaccine and mRNA vaccines. Patients were separated into 6 groups as: patients with MS without treatment PwMS w/o T, ocrelizumab, fingolimod, interferons (interferon beta-1a and interferon beta-1b), dimethyl fumarate, and teriflunomide. Antibody titers of smokers and non-smokers were compared for both vaccines and for each group. Results: The study included 798 patients. In the mRNA vaccine group, smokers (n=148; 2982±326 AU/mL) had lower antibody titers compared to the non-smokers (n=244; 5903±545 AU/mL) in total (p=0.020). In the inactivated vaccine group, no significant difference was detected between smokers (n=136; 383±51 AU/mL) and non-smokers (n=270; 388±49 AU/mL) in total (p=0.149). In both vaccine groups, patients receiving ocrelizumab and fingolimod had lower antibody titers than those receiving other DMTs or PwMS w/o T. In untreated MS patients, antibody levels in smokers were lower than in non-smokers in the mRNA vaccine group. No difference was found between antibody levels of smokers and non-smokers in any of the inactivated vaccine groups. Conclusion: Ocrelizumab and fingolimod have lower antibody levels than PwMS w/o T or other DMTs in both mRNA and inactivated vaccine groups. Smoking decreases antibody levels in the mRNA vaccine group, while it has no effect in the inactivated vaccine group.

Methylprednisolone 100 mg tablet formulation with pea protein: experimental approaches over intestinal permeability and cytotoxicity.

OBJECTIVE: This study was carried out to transform the hydrolyzed pea protein into a pharmaceutical tablet form by masking methylprednisolone. SIGNIFICANCE: This study provides some crucial contributions in showing how functional excipients such as pea protein, which are generally used in food industries, can be used in pharmaceutical product formulations and their effects. METHODS: Methylprednisolone was formulated using spray drying technology. Design Expert Software (Version 13) was used for the statistical analysis. The in vitro cytotoxic effects for NIH/3T3 mouse fibroblast cells were investigated by XTT cell viability assay. HPLC was used to analyze the Caco-2 permeability studies and dissolution tests. RESULTS: The optimum formulation was evaluated against the reference product by performing cytotoxicity and cell permeability studies. According to our test results, Papp (apparent permeability) values of Methylprednisolone were measured around 3 × 10-6 cm/s and Fa (fraction absorbed) values around 30%. These data indicate that Methylprednisolone HCl has 'moderate permeability' and our study confirmed that it could have belonged to BCS Class II-IV since both low solubility and moderate permeability. CONCLUSION: The findings offer valuable information to guide and inform the use of pea protein in pharmaceutical formulations. Significant effects on methylprednisolone tablet formulation designed with the philosophy of quality by design (QbD) of pea protein have been demonstrated by both in vitro and cell studies.

mRNA versus inactivated virus COVID-19 vaccines in multiple sclerosis: Humoral responses and protectivity-Does it matter?

BACKGROUND: COVID-19 vaccines are recommended for people with multiple sclerosis (pwMS). Adequate humoral responses are obtained in pwMS receiving disease-modifying therapies (DMTs) after vaccination, with the exception of those receiving B-cell-depleting therapies and non-selective S1P modulators. However, most of the reported studies on the immunity of COVID-19 vaccinations have included mRNA vaccines, and information on inactivated virus vaccine responses, long-term protectivity, and comparative studies with mRNA vaccines are very limited. Here, we aimed to investigate the association between humoral vaccine responses and COVID-19 infection outcomes following mRNA and inactivated virus vaccines in a large national cohort of pwMS receiving DMTs. METHODS: This is a cross-sectional and prospective multicenter study on COVID-19-vaccinated pwMS. Blood samples of pwMS with or without DMTs and healthy controls were collected after two doses of inactivated virus (Sinovac) or mRNA (Pfizer-BioNTech) vaccines. PwMS were sub-grouped according to the mode of action of the DMTs that they were receiving. SARS-CoV-2 IgG titers were evaluated by chemiluminescent microparticle immunoassay. A representative sample of this study cohort was followed up for a year. COVID-19 infection status and clinical outcomes were compared between the mRNA and inactivated virus groups as well as among pwMS subgroups. RESULTS: A total of 1484 pwMS (1387 treated, 97 untreated) and 185 healthy controls were included in the analyses (male/female: 544/1125). Of those, 852 (51.05%) received BioNTech, and 817 (48.95%) received Sinovac. mRNA and inactivated virus vaccines result in similar seropositivity; however, the BioNTech vaccination group had significantly higher antibody titers (7.175±10.074) compared with the Sinovac vaccination group (823±1.774) (p<0.001). PwMS under ocrelizumab, fingolimod, and cladribine treatments had lower humoral responses compared with the healthy controls in both vaccine types. After a mean of 327±16 days, 246/704 (34.9%) of pwMS who were contacted had COVID-19 infection, among whom 83% had asymptomatic or mild disease. There was no significant difference in infection rates of COVID-19 between participants vaccinated with BioNTech or Sinovac vaccines. Furthermore, regression analyses show that no association was found regarding age, sex, Expanded Disability Status Scale score (EDSS), the number of vaccination, DMT type, or humoral antibody responses with COVID-19 infection rate and disease severity, except BMI Body mass index (BMI). CONCLUSION: mRNA and inactivated virus vaccines had similar seropositivity; however, mRNA vaccines appeared to be more effective in producing SARS-CoV-2 IgG antibodies. B-cell-depleting therapies fingolimod and cladribine were associated with attenuated antibody titer. mRNA and inactive virus vaccines had equal long-term protectivity against COVID-19 infection regardless of the antibody status.

Synthesis characterisation and neuroprotectivity of Silybum marianum extract loaded chitosan nanoparticles.

AIM: Silybum marianum extract (SME) possesses neuroprotective potency through its high antioxidant content. We attempted to increase the effectiveness of SME by encapsulating them in chitosan. Neuroprotective potency of SME and SME-loaded chitosan nanoparticles (SME-CNPs) were shown in SH-SY5Y cell line against H2O2-induced oxidative stress. METHODS: We produced CNPs and SME-CNPs by ionic gelation method and properly determined their physical characteristics. Encapsulation efficiency, loading capacity, and in vitro release tests were performed for SME-CNPs. The neurotoxicity and neuroprotective efficiency in SH-SY5Y cell line against H2O2 was also investigated. RESULTS: The size of SME-CNPs was 168.2 ± 11.12 nm with zeta potential 10.6 ± 1.0 mV. The encapsulation efficiency and loading capacity were successfully achieved at 96.6% and 1.89% respectively. SME and SME-CNPs improved cell viability higher than 80%, and SME-CNPs exhibited significant neuroprotective effects against H2O2 damage. CONCLUSIONS: It was concluded that SME and SME-CNPs highly prevent damage caused by H2O2 and reduce cell damage in vitro by their neuroprotective effects.

Immunogenic evaluation of multi-epitope peptide-loaded PCPP microparticles as a vaccine candidate against Toxoplasma Gondii.

Toxoplasmosis is a major health problem and socioeconomic burden, affecting around 30-50% of the global population. Poly(dicarboxylatophenoxy)phosphazene (PCPP) polymer was chosen as adjuvant for the immunogenic peptide antigen. Peptide-loaded PCPP microparticles were synthesized via the coacervation method and the characterization studies of microparticles were conducted to determine their size, charge, morphology, encapsulation efficacy, and loading capacity. To evaluate in vivo efficacy of the vaccine candidate, Balb/c mice were immunized with the formulations. Brain and spleen tissues were isolated from animals to investigate cytokine levels, lymphocyte proliferation, and brain cyst formation. As a result, antibody and cytokine responses in groups immunized with peptide-loaded PCPP microparticles were found to be significantly higher when compared to the control group. In conclusion, our novel multi-epitope peptide-loaded PCPP microparticle-based vaccine formulation demonstrated considerable humoral and cellular immune responses against T. gondii and protected mice against T. gondii infection during Toxoplasmosis.

Safety and immunogenicity of an inactivated whole virion SARS-CoV-2 vaccine, TURKOVAC, in healthy adults: Interim results from randomised, double-blind, placebo-controlled phase 1 and 2 trials.

BACKGROUND: Development of safe and effective vaccine options is crucial to the success of fight against COVID-19 pandemic. Herein, we report interim safety and immunogenicity findings of the phase 1&2 trials of ERUCoV-VAC, an inactivated whole virion SARS-CoV-2 vaccine. METHODS: Double-blind, randomised, single centre, phase 1 and 2 trials included SARS-CoV-2 seronegative healthy adults aged 18-55 years (18-64 in phase 2). All participants, except the first 4 in phase 1 who received ERUCoV-VAC 3 µg or 6 µg unblinded and monitored for 7 days for safety purposes, were assigned to receive two intramuscular doses of ERUCoV-VAC 3 µg or 6 µg (an inactivated vaccine containing alhydrogel as adjuvant) or placebo 21 days apart (28 days in phase 2) according to computer-generated randomisation schemes. Both trials are registered at ClinicalTrials.gov (phase 1, NCT04691947 and phase 2, NCT04824391). RESULTS: Forty-four participants (3 µg [n:17], 6 µg [n:17], placebo [n:10]) in phase 1 and 250 (3 µg [n:100], 6 µg [n:100], placebo [n:50]) in phase 2 received ≥1 dose. In phase 1 trial, 25 adverse events AEs (80 % mild) occured in 15 participants (34.1 %) until day 43. There was no dose-response relationship noted in safety events in ERUCoV-VAC recipients (p = 0.4905). Pain at injection site was the most common AE (9/44;20.5 %). Both doses of ERUCoV-VAC 3 µg and 6 µg groups were comparable in inducing SARS-CoV-2 wild-type neutralising antibody (MNT50): GMTs (95 %CI) were 8.3 (6.4-10.3) vs. 8.6 (7.0-10.2) at day 43 (p = 0.7357) and 9.7 (6.0-13.4) vs. 10.8 (8.8-12.8) at day 60 (p = 0.8644), respectively. FRNT50 confirmed MNT50 results: SARS-CoV-2 wild-type neutralising antibody GMTs (95 %CI) were 8.4 (6.3-10.5) vs. 9.0 (7.2-10.8) at day 43 (p = 0.5393) and 11.0 (7.0-14.9) vs. 12.3 (10.3-14.5) at day 60 (p = 0.8578). Neutralising antibody seroconversion rates (95 %CI) were 86.7 % (59.5-98.3) vs 94.1 % (71.3-99.8) at day 43 (p = 0.8727) and 92.8 % (66.1-99.8) vs. 100 % (79.4-100.0) at day 60 (p = 0.8873), in ERUCoV-VAC 3 µg and 6 µg groups, respectively. In phase 2 trial, 268 AEs, (67.2 % moderate in severity) occured in 153 (61.2 %) participants. The most common local and systemic AEs were pain at injection site (23 events in 21 [8.4 %] subjects) and headache (56 events in 47 [18.8 %] subjects), respectively. Pain at injection site was the only AE with a significantly higher frequency in the ERUCoV-VAC groups than in the placebo arm in the phase 2 study (p = 0.0322). ERUCoV-VAC groups were comparable in frequency of AEs (p = 0.4587). ERUCoV-VAC 3 µg and 6 µg groups were comparable neutralising antibody (MNT50): GMTs (95 %CI) were 30.0 (37.9-22.0) vs. 34.9 (47.6-22.1) at day 43 (p = 0.0666) and 34.2 (23.8-44.5) and 39.6 (22.7-58.0) at day 60, (p = 0.2166), respectively. FRNT50 confirmed MNT50 results: SARS-CoV-2 wildtype neutralising antibody GMTs were 28.9 (20.0-37.7) and 30.1 (18.5-41.6) at day 43 (p = 0.3366) and 34.2 (23.8-44.5) and 39.6 (22.7-58.0) at day 60 (p = 0.8777). Neutralising antibody seroconversion rates (95 %CI) were 95.7 % (91.4-99.8) vs. 98.9 % (96.9-100.0) at day 43 (p = 0.8710) and 96.6 % (92.8-100.0) vs 98.9 % (96.7-100.0) at day 60 (p = 0.9129) in ERUCoV-VAC 3 µg and 6 µg groups, respectively. CONCLUSIONS: Two-dose regimens of ERUCoV-VAC 3 µg and 6 µg 28 days both had an acceptable safety and tolerability profile and elicited comparable neutralising antibody responses and seroconversion rates exceeding 95 % at day 43 and 60 after the first vaccination. Data availability Data will be made available on request.

Development of piperine nanoemulsions: an alternative topical application for hypopigmentation.

This study, it was aimed to develop a topical piperine nanoemulsion (P-NE) using an ultrasonic emulsification process to find an alternative treatment option for some hypopigmentation disorders such as vitiligo. Results showed that 150 mg piperine loaded NE with 1:2 oil phase to Smix ratio and manufactured with 20 min ultrasonication duration with the pre-emulsification step was the most durable formulation with a mean globule size of 216.00 ± 2.65, a PdI value of 0.094 ± 0.02 and a zeta potential value of -27.50 ± 2.48 mV. After three months of storage, the selected P-NE (coded as F3P2) remained kinetically stable without visual changes. This formulation displayed a sustained release pattern with a release of 81.92% ± 3.04% piperine after 72 h. According to our in vitro activity experiments, it was determined that the P-NE had no toxic effect including the dose of 5 mg/mL, and the highest P-NE formulation dose of 5 mg/mL increased tyrosinase activity by 32.77% ± 9.09% and melanogenesis activity by 34.90% ± 0.73%. In conclusion, it was demonstrated that the P-NE formulation may serve as a promising therapy for the efficient treatment of vitiligo. Moreover, P-NE formulation may also help in preventing irregular pigmentation and skin cancer, associated with the conventional treatment methods.

Preparation of chitosan nanoparticles as Ginkgo Biloba extract carrier: In vitro neuroprotective effect on oxidative stress-induced human neuroblastoma cells (SH-SY5Y).

Ginkgo biloba (Gb) is an ancient Chinese tree cultivated for its health-promoting properties. Moreover, Gb extract has a therapeutic effect, especially on neurodegenerative diseases. In this study, Gb extract-loaded chitosan nanoparticles (Gb-CsNPs) were synthesized by ionic gelation method. Size and zeta potential of the nanoparticles were analyzed and Scanning Electron Microscopy (SEM) and Fourier Transform Spectroscopy (FT-IR) were performed. Besides, encapsulation efficacy and loading capacity were calculated, and in vitro release, and cellular uptake studies were carried out. The biocompatibility of Gb-CsNPs was demonstrated and their neuroprotective activity was investigated on oxidative stress-induced SH-SY5Y cells. Apoptotic cells were monitored by DAPI, and cell migration was examined by in vitro scratch assay. Results showed that Gb-CsNPs had an average size of 104.4 nm, their zeta potential and polydispersity index (PDI) values were 29.3 mV, and 0.09 respectively. Encapsulation efficacy and loading capacity were found as 97.4% and 40%, respectively. It has been revealed that Gb-CsNPs were biocompatible and showed neuroprotective activity by increasing cell viability from 60% to 92.3%. Consequently, neuroprotective effect of the Gb extract was increased by chitosan encapsulation. This formulation is a candidate to be used as a food supplement after being supported by future in vivo studies.

Comparison of the anticancer effect of microalgal oils and microalgal oil-loaded electrosprayed nanoparticles against PC-3, SHSY-5Y and AGS cell lines.

Many of the bioactive substances used in pharmaceutical industry are easily affected by temperature, light and oxygen, and are easily degraded during storage and processing, and exhibit poor adsorption properties during digestion, which limits their direct use. Microalgae are rich in oils which have antimicrobial properties and antioxidants that attract attention in both food and pharmaceutical sectors in recent years. Studies to encapsulate bioactive compound-rich microalgae oils with nanotechnological approaches to improve the physical and chemical stability are relatively new, and it is promising to apply these approaches for pharmaceutical purposes. In this study, cytotoxic effects of oil extracts of Botryococcus braunii and Microcystis aeruginosa and their oil-loaded nanoparticles on L929 cell line, PC-3 prostate cell line, SHSY-5Y neuroblastoma cell line and AGS gastric adenocarcinoma cell line were investigated. The obtained extracts were found to have no cytotoxic effect on L929 cells. However, they showed cytotoxic effect on cancer cells. As for the nanoparticles; a gradual release was determined and the stability of the nanoparticle structure was shown. In the light of obtained findings, it was considered that nanoparticles produced with oil extracts of microalgae which have bioactive substances, have potential to be evaluated especially in pharmaceutical and cosmetic fields.

Conjugation and Characterization of Latex Particles with Toxoplasma gondii-specific Immunoglobulin Y Antibodies for Diagnostic Aim and Evaluation Efficiency in In Vitro Culture.

Toxoplasma gondii is a parasite that causes severe health problems in the world. Toxoplasmosis, an infection caused by T. gondii, leads to high risk of mortality in patients with immunodeficiency, transplantation, and cancer. Besides that, it causes miscarriages in pregnancy, various abnormalities such as hydrocephalus in infants and congenital diseases. Because the clinical indication of the disease is not specific, it is confused with many diseases, and this leads to the necessity of directly detecting the presence of the toxoplasmosis. Therefore, various diagnostic assays are needed for the diagnosis of the disease. Amongs them, latex agglutination assay is widely used for the detection of specific antibodies or antigens in samples. Latex particles are coated with immunogenic molecules (antigens) to detect antibodies in the blood or used to identify antigens when coated with specific antibodies. In both, aggregation of latex particles results in agglutination. Monoclonal antibodies are often used in latex agglutination assay as in other diagnostic methods. However, monoclonal antibodies can be produced in low quantities at a high cost. Besides, to produce monoclonal antibodies, an experienced staff, a well-equipped cell culture laboratory, a long period of time, and a burdened budget are needed. In recent years, as an alternative to monoclonal antibodies, immunoglobulin Y (IgY) antibodies, which are obtained from chicken eggs, and specifically produced against desired antigenic constructs, have become quite attractive in terms of both low cost and abundant production without requiring infrastructure. In contrast, the latex assay based on IgY antibodies for use in the diagnosis of T. gondii has not been developed. This study aimed to conjugate T. gondii-specific IgY antibodies to latex particles, characterize the particles by Fourier transform infrared spectroscopy, scanning electron microscopy, and spectroscopic methods, and finally demonstrate the interaction with T.gondii parasites in culture with scanning electron microscopy analysis.

Synthesis of biologically active copper oxide nanoparticles as promising novel antibacterial-antibiofilm agents.

In this study, we aimed to synthesize copper oxide nanoparticles (CuONPs) mediated by plant extract in an environmentally friendly way and to reveal their potential biological activities. Here we synthesized CuONPs by using different concentrations of aqueous leaf extract of Thymbra spicata at 80 °C to obtain Ts1CuONPs and Ts2CuONPs. Biosynthesized nanoparticles were characterized by using UV-Vis, AFM, FTIR, SEM-EDS, TEM, DLS and zeta potential analysis. The antibacterial activity of the nanoparticles was determined by calculation of the inhibition zone and minimum inhibitory concentration against selected bacterial strains. Moreover, the antioxidant activity of the as-synthesized nanoparticles was evaluated based on DPPH radical scavenging activity. The results indicate that the as-synthesized NPs have an average size of 26.8 and 21 nm for Ts1CuONPs and Ts2CuONPs, respectively. The formed CuONPs have more antibacterial action on gram-positive bacteria compared to gram-negative bacteria. In addition, CuONPs demonstrated good inhibition activity against biofilm formation of Staphylococcus aureus (S. aureus). Furthermore, the results showed that the smaller size of the CuONPs caused the higher cytotoxicity on L929 mouse fibroblast cells. The as-synthesized CuONPs exhibit antibacterial and antibiofilm potential against S. aureus, indicating that they may be attractive candidates to use in future therapeutic applications.

In Silico design of AVP (4-5) peptide and synthesis, characterization and in vitro activity of chitosan nanoparticles.

BACKGROUND: Arginine-vasopressin (AVP) is a neuropeptide and provides learning and memory modulation. The AVP (4-5) dipeptide corresponds to the N-terminal fragment of the major vasopressin metabolite AVP (4-9), has a neuroprotective effect and used in the treatment of Alzheimer's and Parkinson's disease. METHODS: The main objective of the present study is to evaluate the molecular mechanism of AVP (4-5) dipeptide and to develop and synthesize chitosan nanoparticle formulation using modified version of ionic gelation method, to increase drug effectiveness. For peptide loaded chitosan nanoparticles, the synthesized experiment medium was simulated for the first time by molecular dynamics method and used to determine the stability of the peptide, and the binding mechanism to protein (HSP70) was also investigated by molecular docking calculations. A potential pharmacologically features of the peptide was also characterized by ADME (Absorption, Distribution, Metabolism and Excretion) analysis. The characterization, in vitro release study, encapsulation efficiency and loading capacity of the peptide loaded chitosan nanoparticles (CS NPs) were performed by Dynamic Light Scattering (DLS), UV-vis absorption (UV), Scanning Electron Microscopy (SEM), Fourier transform infrared (FT-IR) spectroscopy techniques. Additionally, in vitro cytotoxicity of the peptide on human neuroblastoma cells (SH-SY5Y) was examined with XTT assay and the statistical analysis was evaluated. RESULTS: The results showed that; hydrodynamic size, zeta potential and polydispersity index (PdI) of the peptide-loaded CS NPs were 167.6 nm, +13.2 mV, and 0.211, respectively. In vitro release study of the peptide-loaded CS NPs showed that 17.23% of the AVP (4-5)-NH2 peptide was released in the first day, while 61.13% of AVP (4-5)-NH2 peptide was released in the end of the 10th day. The encapsulation efficiency and loading capacity were 99% and 10%, respectively. According to the obtained results from XTT assay, toxicity on SHSY-5Y cells in the concentration from 0.01 µg/µL to 30 µg/µL were evaluated and no toxicity was observed. Also, neuroprotective effect was showed against H2O2 treatment. CONCLUSION: The experimental medium of peptide-loaded chitosan nanoparticles was created for the first time with in silico system and the stability of the peptide in this medium was carried out by molecular dynamics studies. The binding sites of the peptide with the HSP70 protein were determined by molecular docking analysis. The size and morphology of the prepared NPs capable of crossing the blood-brain barrier (BBB) were monitored using DLS and SEM analyses, and the encapsulation efficiency and loading capacity were successfully performed with UV Analysis. In vitro release studies and in vitro cytotoxicity analysis on SHSY-5Y cell lines of the peptide were conducted for the first time. Grapical abstract.

In silico Analysis of Sulpiride, Synthesis, Characterization and In vitro Studies of its Nanoparticle for the Treatment of Schizophrenia.

BACKGROUND: Sulpiride, which has selective dopaminergic blocking activity, is a substituted benzamide antipsychotic drug playing a prominent role in the treatment of schizophrenia, which more selective and primarily blocks dopamine D2 and D3 receptor. OBJECTIVE: This study has two main objectives, firstly; the molecular modeling studies (MD and Docking, ADME) were conducted to define the molecular profile of sulpiride and sulpiridereceptor interactions, another to synthesize polymeric nanoparticles with chitosan, having the advantage of slow/controlled drug release, to improve drug solubility and stability, to enhance utility and reduce toxicity. METHODS: Molecular dynamic simulation was carried out to determine the conformational change and stability (in water) of the drug and the binding profile of D3 dopamine receptor was determined by molecular docking calculations. The pharmacological properties of the drug were revealed by ADME analysis. The ionic gelation method was used to prepare sulpiride loaded chitosan nanoparticles (CS NPs). The Dynamic Light Scattering (DLS), UV-vis absorption (UV), Scanning Electron Microscopy (SEM), Fourier transform infrared (FT-IR) spectroscopy techniques were carried out to characterize the nanoparticles. In vitro cell cytotoxicity experiments examined with MTT assay on mouse fibroblast (L929), human neuroblastoma (SH-SY5Y) and glioblastoma cells (U-87). The statistical evaluations were produced by ANOVA. RESULTS: The residues (ASP-119, PHE-417) of D3 receptor provided a stable docking with the drug, and the important pharmacological values (blood brain barrier, Caco-2 permeability and human oral absorption) were also determined. The average particle size, PdI and zeta potential value of sulpiride- loaded chitosan NPs having a spherical morphology were calculated as 96.93 nm, 0.202 and +7.91 mV. The NPs with 92.8% encapsulation and 28% loading efficiency were found as a slow release profile with 38.49% at the end of the 10th day. Due to the formation of encapsulation, the prominent shifted wave numbers for C-O, S-O, S-N stretching, S-N-H bending of Sulpiride were also identified. Mitochondrial activity of U87, SHSY-5Y and L929 cell line were assayed and evaluated using the SPSS program. CONCLUSION: To provide more efficient use of Sulpiride having a low bioavailability of the gastrointestinal tract, the nanoparticle formulation with high solubility and bioavailability was designed and synthesized for the first time in this study for the treatment of schizophrenia. In addition to all pharmacological properties of drug, the dopamine blocking activity was also revealed. The toxic effect on different cell lines have also been interpreted.

Application of an immunoglobulin Y-alkaline phosphatase bioconjugate as a diagnostic tool for influenza A virus.

The diagnosis of influenza A virus is essential since it can be confused with influenza A like illness and lead to inaccurate drug prescription. In this study, the M2e peptide, a strategic antigen that is conserved in all virus subtypes, was used as a diagnostic marker of influenza A. For the first time, M2e-specific IgY antibody was covalently conjugated to alkaline phosphatase (ALP) enzyme in the presence of glutaraldehyde. The antibody-enzyme bioconjugate was characterized by fluorescence and Fourier-transform infrared spectroscopy. Subsequently, the diagnostic value of this bioconjugate was evaluated by direct sandwich ELISA using nasopharyngeal swab samples positive/negative for H1N1 and H3N2, which were previously analyzed by rRT-PCR for influenza. In conclusion, the M2e-specific IgY-ALP bioconjugate demonstrated positive results for Influenza A in samples that were diagnosed as Influenza A via the RT-PCR method.

Production and characterization of a conserved M2e peptide-based specific IgY antibody: evaluation of the diagnostic potential via conjugation with latex nanoparticles.

Antibodies play an important role in combating and controlling viral diseases such as influenza. Immunoglobulin Y (IgY) antibodies have several advantages such as a less invasive manufacturing process, ease of isolation, higher affinity compared with IgG antibodies, and cost-effectiveness. To date, although specific IgY production has been performed for different strains of influenza A, to the best of our knowledge, an IgY against the M2e peptide has not been produced. In the current study, IgY antibodies are produced, purified, and characterized using the M2e peptide sequence for the first time with the intent to apply them for the diagnosis of influenza A virus. Anti-M2e IgY antibodies are obtained from eggs using a two-step purification method. The activity and characterization of the antibodies are determined using an enzyme-linked immunosorbent assay, a nano-spectrophotometer, an SDS-Page assay, and a Western Blot analysis. Finally, anti-M2e IgY antibodies are conjugated to the latex nanoparticles, and the reaction between the influenza A virus and the nanoparticles is demonstrated using light microscopy, transmission electron microscopy, and energy dispersive X-ray spectroscopy. In conclusion, this study shows that anti-M2e IgY antibodies can contribute to the diagnosis, treatment, and prevention of the influenza A virus.

Papain Loaded Poly(ε-Caprolactone) Nanoparticles: In-silico and In-Vitro Studies.

Papain is a protease enzyme with therapeutic properties that are very valuable for medical applications. Poly(ε-caprolactone) (PCL) is an ideal polymeric carrier for controlled drug delivery systems due to its low biodegradability and its high biocompatibility. In this study, the three-dimensional structure and action mechanism of papain were investigated by in vitro and in silico experiments using molecular dynamics (MD) and molecular docking methods to elucidate biological functions. The results showed that the size of papain-loaded PCL nanoparticles (NPs) and the polydispersity index (PDI) of the NPs were 242.9 nm and 0.074, respectively. The encapsulation efficiency and loading efficiency were 80.4 and 27.2%, respectively. Human embryonic kidney cells (HEK-293) were used for determining the cytotoxicity of papain-loaded PCL and PCL nanoparticles. The in vitro cell culture showed that nanoparticles are not toxic at low concentrations, while toxicity slightly increases at high concentrations. In silico studies, which were carried out with MD simulations and ADME analysis showed that the strong hydrogen bonds between the ligand and the papain provide stability and indicate the regions in which the interactions occur.

Molecular docking of immunogenic peptide of Toxoplasma gondii and encapsulation with polymer as vaccine candidate.

Toxoplasma gondii is one of the most widely spread parasitic organisms in the world. T. gondii causes primary, chronic infection and mortality. Major surface antigen 1 is the most abundant tachyzoite surface protein and highly conserved between species and causes strong humoural response. Some studies showed that the peptide sequence of surface antigen has immunity. Therefore, tachyzoite surface antigenic peptide sequence is one of the good candidates for vaccine development. However, conformational information and delivery systems are very important parameters for vaccine development. Computational chemistry which is used as an effective method to perform drug or vaccine design provides important information on structure-activity relationship, biological effects of functional groups, molecular geometry, design of enzyme inhibitors and antagonists. The interaction of immunological peptides with protein systems was carried out by means of computing the free energy of binding using the molecular docking technique. Due to the major histocompatibility complex (MHC), proteins play a substantial role for adaptive immunity, the crystal structure of a MHC class I, which plays a pivotal role in the adaptive branch of the immune system, was preferred for docking calculations. A delivery system based on poly(lactic-co-glycolic acid) (PLGA) nanoparticles and peptide loaded PLGA nanoparticles was prepared in this study to improve the bioavailability of tachyzoite surface antigenic peptide sequence. Double emulsion method (water-in-oil-in-water or w/o/w) was used for synthesis of PLGA and peptide loaded PLGA nanoparticles. The average particle size, polydispersity index and zeta potential values of PLGA and peptide loaded PLGA nanoparticles were measured with zeta-sizer by using dynamic light scattering (DLS) technique. The scanning electron microscope (SEM) (Zeiss Supra 50 V) was used for imagining the peptide loaded PLGA nanoparticles. Cell toxicity of nanoparticles was assayed on AGS (gastric adenocarcinoma) cell line. To evaluate mitochondrial activity of cells and toxicity studies, XTT methods were carried out. In this study, we aimed to obtain specific immunological peptide loaded PLGA nanoparticles and characterize the formation with FTIR, zeta sizer and SEM imaging, and evaluate cytotoxicity and carry out molecular docking calculations of peptide-MHC protein in order to enlight in vivo events as vaccine candidate against T. gondii.