A novel nanobody-based immunocytokine of a mutant interleukin-2 as a potential cancer therapeutic.

The immunotherapeutic application of interleukin-2 (IL-2) in cancer treatment is limited by its off-target effects on different cell populations and insufficient activation of anti-tumor effector cells at the site of the tumor upon tolerated doses. Targeting IL-2 to the tumor microenvironment by generating antibody-cytokine fusion proteins (immunocytokine) would be a promising approach to increase efficacy without associated toxicity. In this study, a novel nanobody-based immunocytokine is developed by the fusion of a mutant (m) IL-2 with a decreased affinity toward CD25 to an anti-vascular endothelial growth factor receptor-2 (VEGFR2) specific nanobody, denoted as VGRmIL2-IC. The antigen binding, cell proliferation, IFN-γ-secretion, and cytotoxicity of this new immunocytokine are evaluated and compared to mIL-2 alone. Furthermore, the pharmacokinetic properties are analyzed. Flow cytometry analysis shows that the VGRmIL2-IC molecule can selectively target VEGFR2-positive cells. The results reveal that the immunocytokine is comparable to mIL-2 alone in the stimulation of Primary Peripheral Blood Mononuclear Cells (PBMCs) and cytotoxicity in in vitro conditions. In vivo studies demonstrate improved pharmacokinetic properties of VGRmIL2-IC in comparison to the wild or mutant IL-2 proteins. The results presented here suggest VGRmIL2-IC could be considered a candidate for the treatment of VEGFR2-positive tumors.

Development of a bivalent protein-based vaccine candidate against invasive pneumococcal diseases based on novel pneumococcal surface protein A in combination with pneumococcal histidine triad protein D.

Extensive efforts have been made toward improving effective strategies for pneumococcal vaccination, focusing on evaluating the potential of multivalent protein-based vaccines and overcoming the limitations of pneumococcal polysaccharide-based vaccines. In this study, we investigated the protective potential of mice co-immunization with the pneumococcal PhtD and novel rPspA proteins against pneumococcal sepsis infection. The formulations of each antigen alone or in combination were administered intraperitoneally with alum adjuvant into BALB/c mice three times at 14-day intervals. The production of antigen-specific IgG, IgG1 and IgG2a subclasses, and IL-4 and IFN-γ cytokines, were analyzed. Two in vitro complement- and opsonophagocytic-mediated killing activities of raised antibodies on day 42 were also assessed. Finally, the protection against an intraperitoneal challenge with 106 CFU/mouse of multi-drug resistance of Streptococcus pneumoniae ATCC49619 was investigated. Our findings showed a significant increase in the anti-PhtD and anti-rPspA sera IgG levels in the immunized group with the PhtD+rPspA formulation compared to each alone. Moreover, the results demonstrated a synergistic effect with a 6.7- and 1.3- fold increase in anti-PhtD and anti-rPspA IgG1, as well as a 5.59- and 1.08- fold increase in anti-PhtD and anti-rPspA IgG2a, respectively. Co-administration of rPspA+PhtD elicited a mixture of Th-2 and Th-1 immune responses, more towards Th-2. In addition, the highest complement-mediated killing activity was observed in the sera of the immunized group with PhtD+rPspA at 1/16 dilution, and the opsonophagocytic activity was increased from 74% to 86.3%. Finally, the survival rates showed that mice receiving the rPspA+PhtD formulation survived significantly longer (100%) than those receiving protein alone or PBS and exhibited the strongest clearance with a 2 log10 decrease in bacterial load in the blood 24h after challenge compared to the control group. In conclusion, the rPspA+PhtD formulation can be considered a promising bivalent serotype-independent vaccine candidate for protection against invasive pneumococcal infection in the future.

Synthesis and characterization of Gd3+-loaded hyaluronic acid-polydopamine nanoparticles as a dual contrast agent for CT and MRI scans.

Magnetic resonance imaging and computed tomography (CT) suffer from low contrast sensitivity and potential toxicity of contrast agents. To overcome these limitations, we developed and tested a new class of dual contrast agents based on polydopamine nanoparticles (PDA-NPs) that are functionalized and targeted with hyaluronic acid (HA). These nanoparticles (NPs) are chelated with Gd3+ to provide suitable contrast. The targeted NPs were characterized through ultraviolet-visible spectroscopy (UV-vis), scanning electron microscopy (SEM), infrared Fourier transform (FTIR), and dynamic light scattering (DLS). The cytotoxicity was investigated on HEK293 cells using an MTT assay. The contrast property of synthesized Gd3+/PDA/HA was compared with Barium sulfate and Dotarem, as commercial contrast agents (CAs) for CT and MRI, respectively. The results illustrated that synthesized PDA-NPs have a spherical morphology and an average diameter of 72 nm. A distinct absorption peak around 280 nm in the UV-vis spectrum reported the self-polymerization of PDA-NPs. The HA coating on PDA-NPs was revealed through a shift in the FTIR peak of C=O from 1618 cm-1 to 1635 cm-1. The Gd3+ adsorption on PDA/HA-NPs was confirmed using an adsorption isotherm assay. The developed CA showed low in vitro toxicity (up to 158.98 µM), and created a similar contrast in MRI and CT when compared to the commercial agents. The r1 value for PDA/HA/Gd3+ (6.5 (mg/ml)-1 s-1) was more than Dotarem (5.6 (mg/ml)-1 s-1) and the results of the hemolysis test showed that at concentrations of 2, 4, 6, and 10 mg/ml, the hemolysis rate of red blood cells is very low. Additionally, the results demonstrated that PDA/HA/Gd3+ could better target the CD44+-expressing cancer cells than PDA/Gd3+. Thus, it can be concluded that lower doses of developed CA are needed to achieve similar contrast of Dotarem, and the developed CA has no safety concerns in terms of hemolysis. The stability of PDA/HA/Gd3+ has also been evaluated by ICP-OES, zeta potential, and DLS during 3 days, and the results suggested that Gd-HA NPs were stable.

Discovery of potential FGFR3 inhibitors via QSAR, pharmacophore modeling, virtual screening and molecular docking studies against bladder cancer.

BACKGROUND: Fibroblast growth factor receptor 3 is known as a favorable aim in vast range of cancers, particularly in bladder cancer treatment. Pharmacophore and QSAR modeling approaches are broadly utilized for developing novel compounds for the determination of inhibitory activity versus the biological target. In this study, these methods employed to identify FGFR3 potential inhibitors. METHODS: To find the potential compounds for bladder cancer targeting, ZINC and NCI databases were screened. Pharmacophore and QSAR modeling of FGFR3 inhibitors were utilized for dataset screening. Then, with regard to several factors such as Absorption, Distribution, Metabolism, Excretion and Toxicity (ADMET) properties and Lipinski's Rule of Five, the recognized compounds were filtered. In further step, utilizing the flexible docking technique, the obtained compounds interactions with FGFR3 were analyzed. RESULTS: The best five compounds, namely ZINC09045651, ZINC08433190, ZINC00702764, ZINC00710252 and ZINC00668789 were selected for Molecular Dynamics (MD) studies. Off-targeting of screened compounds was also investigated through CDD search and molecular docking. MD outcomes confirmed docking investigations and revealed that five selected compounds could make steady interactions with the FGFR3 and might have effective inhibitory potencies on FGFR3. CONCLUSION: These compounds can be considered as candidates for bladder cancer therapy with improved therapeutic properties and less adverse effects.

A Novel Immunotoxin Targeting Epithelial Cell Adhesion Molecule Using Single Domain Antibody Fused to Diphtheria Toxin.

Epithelial Cell Adhesion Molecule (EpCAM) is overexpressed in a variety of cancers such as colon, stomach, pancreas, and prostate adenocarcinomas. Inhibition of EpCAM is considered as a potential target for cancer therapy. In current study, anti-EpCAM immunotoxin (α-EpCAM IT) was developed using genetic fusion of α-EpCAM single domain antibody (nanobody) (α-EpCAM Nb) to truncated form of diphtheria toxin. The expression of recombinant α-EpCAM IT was induced by Isopropyl ß-d-1-thiogalactopyranoside (IPTG) and confirmed by SDS-PAGE and western blot. Recombinant α-EpCAM IT was purified from the inclusion bodies and refolded using urea gradient procedure. The cytotoxicity and apoptosis activity of α-EpCAM IT on EpCAM over-expressing (MCF7), low-expressing (HEK293), and no-expressing (HUVEC) cells were evaluated by 3-4,5-Dimethylthiazol-2-yl (MTT) assay and annexin V-FITC-PI assay as well. In addition, anti-tumor activity of α-EpCAM IT was evaluated on nude mice bearing MCF7 tumor cells. Results showed success expression and purification of α-EpCAM IT. The α-EpCAM IT showed time and dose-dependent anti-proliferative activity on MCF-7 cells. However, α-EpCAM IT did not show any anti-proliferative activity on HEK293 and HUVEC cells as well. In addition, the annexin V-FITC-PI assay results showed that α-EpCAM IT significantly increased apoptotic rate in MCF-7 cells with no effect on HEK293 and HUVEC as well. Moreover, α-EpCAM IT significantly reduced tumor size in vivo study. The achieved results indicate the potential of designing α-EpCAM IT as a novel therapeutic for cancer therapy.

A quartz crystal microbalance biosensor based on polyethylenimine-modified gold electrode to detect hepatitis B biomarker.

Biomarkers-based QCM-biosensors are suitable tools for the label-free detection of infectious diseases. In the current study, a QCM-biosensor was developed for the detection of HBsAg. Briefly, anti-HBsAg antibodies were covalently bound to the primary amines after PEI and thiolated-PEI surface modifications of gold-electrode. After RSM optimization, the statistical analysis revealed no significant difference between the immobilization yields of modified layers. Therefore, the PEI-modified QCM-biosensor was selected for further analysis. The PEI-surface was evaluated by FESEM, AFM, ATR-FTIR, and CA measurement. The surface hydrophilicity and its roughness were increased after PEI-coating. Also, FTIR confirmed the PEI-layering on the gold-surface. RSM optimization increased the antibody immobilization yield up to 80%. The QCM-biosensor showed noteworthy results with a wide dynamic range of 1-1 × 103 ng/mL, LOD of 3.14 ng/mL, LOQ of 9.52 ng/mL, and detection capability in human-sera, which were comparable with the ELISA. The mean accuracy of the QCM-biosensor was obtained at 91% when measured by the spike recovery test using human-sera. The biosensor was completely regenerated using 50 mM NaOH and 1% SDS. The benefits provided by the developed biosensor such as broad dynamic range, sensitivity, selectivity, stability, regenerate ability, and low cost suggest its potential application for the non-invasive and timely monitoring of HBV-biomarker.

Development of Streptococcus equisimilis Group G Mutant Strains with Ability to Produce Low Polydisperse and Low-Molecular-Weight Hyaluronic Acid

Background: Background: Hyaluronic acid (HA), a natural polymer with wide applications in biomedicine and cosmetics, is mainly produced by Streptococcal fermentation at industrial scale. In the present study, chemical random mutagenesis was used for development of Streptococcus equisimilis group G mutant strains with high HA productivity. Methods: Methods: The optimum of the pH of culture condition and cultivation time for HA production by wild strain group G were assessed. At first, two rounds of mutation at different concentrations of NTG was used for mutagenesis. Then, the nonhemolytic and hyaluronidase-negative mutants were screened on the blood and HA agar. HA productivity and molecular weight were determined by carbazole assay, agarose gel electrophoresis and specific staining. Moreover, stability of the high producer mutants was evaluated within 10 generations. Results: Results: The results showed that the wild-type strain produced 1241 ± 2.1 µg/ml of HA at pH 5.5 and 4 hours of cultivation, while the screened mutants showed a 16.1-45.5% increase in HA production. Two mutant strains, named Gm2-120-21-3 (2470 ± 8.1 µg/ml) and Gm2-120-21-4 (2856 ± 4.2 µg/ml), indicated the highest titer and a consistent production. The molecular weight (Mw) of HA for the mutants was less than 160 kDa, considering as a low Mw HA. Conclusion: Conclusion: The mutant strains producing a low polydisperse, as well as low Mw of HA with high titer might be regarded as potential industrial strains for HA production after further safety investigations.

A comprehensive review on the applications of carbon-based nanostructures in wound healing: from antibacterial aspects to cell growth stimulation.

A wound is defined as damage to the integrity of biological tissue, including skin, mucous membranes, and organ tissues. The treatment of these injuries is an important challenge for medical researchers. Various materials have been used for wound healing and dressing applications among which carbon nanomaterials have attracted significant attention due to their remarkable properties. In the present review, the latest studies on the application of carbon nanomaterials including graphene oxide (GO), reduced graphene oxide (rGO), carbon dots (CDs), carbon quantum dots (CQDs), carbon nanotubes (CNTs), carbon nanofibers (CNFs), and nanodiamonds (NDs) in wound dressing applications are evaluated. Also, a variety of carbon-based nanocomposites with advantages such as biocompatibility, hemocompatibility, reduced wound healing time, antibacterial properties, cell-adhesion, enhanced mechanical properties, and enhanced permeability to oxygen has been reported for the treatment of various wounds.

Fabrication of a magnetic alginate-silk fibroin hydrogel, containing halloysite nanotubes as a novel nanocomposite for biological and hyperthermia applications.

In this study, the main focus was on designing and synthesizing a novel magnetic nanobiocomposite and its application in hyperthermia cancer treatment. Regarding this aim, sodium alginate (SA) hydrogel with CaCl2 cross-linker formed and modified by silk fibroin (SF) natural polymer and halloysite nanotubes (HNTs), followed by in situ Fe3O4 magnetic nanoparticles preparation. No important differences were detected in red blood cells (RBCs) hemolysis, confirming the high blood compatibility of the treated erythrocytes with this nanobiocomposite. Moreover, the synthesized SA hydrogel/SF/HNTs/Fe3O4 nanobiocomposite does not demonstrate toxicity toward HEK293T normal cell line after 48 and 72 h. The anticancer property of SA hydrogel/SF/HNTs/Fe3O4 nanobiocomposites against breast cancer cell lines was corroborated. The magnetic saturation of the mentioned magnetic nanobiocomposite was 15.96 emu g-1. The specific absorption rate (SAR) was measured to be 22.3 W g-1 by applying an alternating magnetic field (AMF). This novel nanobiocomposite could perform efficiently in the magnetic fluid hyperthermia process, according to the obtained results.

pH-Responsive PEGylated Niosomal Nanoparticles as an Active-Targeting Cyclophosphamide Delivery System for Gastric Cancer Therapy.

A PEGylated niosomal formulation of cyclophosphamide (Nio-Cyclo-PEG) was prepared using a central composite design and characterized in terms of drug loading, size distribution, and average size. The stability of formulations was also studied at different conditions. In vitro cytotoxicity of drug delivery formulations was assessed on gastric cancer cells using MTT assay. The mechanism of cytotoxicity was studied at the transcriptional level by real-time PCR on Caspase3, Caspase9, CyclinD, CyclinE, MMP-2, and MMP-9 genes, while apoptosis was investigated with flow cytometry. The anti-metastatic property was evaluated using the scratch method. Propidium iodide staining was used to study the cell cycle. The results indicated that the as-designed nanocarrier exhibited a controlled drug release pattern with improved nanoparticle stability. It was found that the living cancer cells treated with Nio-Cyclo-PEG showed a significant decrease in number when compared with the niosomal carrier without PEG (Nio-Cyclo) and free drug (Cyclo). Moreover, the drug-loaded nanocarrier induced planned death (apoptosis) in the cancer cells through the regulation of Caspase3, Caspase9, CyclinD, CyclinE, MMP-9, and MMP-2 gene expression, indicating that the Nio-Cyclo-PEG formulation could significantly inhibit the cell cycle at the sub G1 phase as well as prevent the migration of cancer cells. In conclusion, Nio-Cyclo-PEG as developed in this study could serve as an active-targeting drug delivery nanocarriers for gastric cancer therapy with high efficacy and minimal side effects on healthy tissues/cells.