Isolation and structure elucidation of the compounds from Teucrium hyrcanicum L. and the investigation of cytotoxicity, antioxidant activity, and protective effect on hydrogen peroxide-induced oxidative stress.

BACKGROUND: Teucrium hyrcanicum L. (family Lamiaceae) is widely distributed in the North and Northwest of Iran. It has been used in the form of tea, tonic, and tincture for the treatment of various diseases such as cough, rheumatism, and fever. METHODS: In this study, the total phenolic and flavonoid contents, antioxidant and cytotoxic activities of methanol extract and different fractions of T. hyrcanicum were measured. Furthermore, the potential ability of T. hyrcanicum to protect against H2O2-induced oxidative stress was tested on the NIH3T3 cell line. Then, the isolation and structure elucidation of the compounds were performed on the most potent fractions. Finally, the quantification of isolated compounds in methanol extract (ME) was done by the HPLC method. Isolated phytochemicals were assessed for the cytotoxic and antioxidant activities. RESULTS: The results indicated that the methanol fraction (MF) had the highest amount of phenolic and flavonoid contents (69.36 mg GAE/g extract and 68.95 mg QE/g extract). The highest radical scavenging activities were observed from MF and ME (IC50 44.32 and 61.12 µg.ml-1, respectively). The best cytotoxicity was obtained by ethyl acetate fraction (EF) against A431 and MCF7 cell lines (IC50 values of 235.4and 326.6 µg.ml-1, respectively). The pretreatment with MF exerts the highest reduction in malondialdehyde (MDA) formation (IC50 2.51 µM, p < 0.001) compared to the H2O2 group (5.77 µM). Also, MF significantly inhibited H2O2-induced Glutathione (GSH) oxidation (p < 0.001). Furthermore, two phenolic compounds, acteoside and quercetin, were isolated and identified in MF and EF, respectively. The IC50 values of acteoside and quercetin in the DPPH assay were 7.19 and 5.56 µg.ml-1, respectively. Both quercetin and acteoside significantly reduced the MDA formation and inhibited GSH oxidation, which was comparable with BHA (as a standard antioxidant) (p < 0.05). Acteoside demonstrated significant cytotoxicity against all tested cell lines (IC50 = 32 to 145 µg.ml-1). The HPLC quantification of isolated compounds revealed that the quantity of acteoside and quercetin in ME were 93.31 and 16.87 µg.mg-1, respectively. CONCLUSION: The isolated compounds (quercetin and acteoside) had significant antioxidant activities and revealed a protective effect on H2O2-induced oxidative stress which was comparable with BHA.

Ion release, biocompatibility, and bioactivity of resin-modified calcium hydroxide cavity liners.

BACKGROUND: The placement of liners near the pulp area is essential for therapeutic effects and maintaining pulp health while stimulating the formation of tertiary dentin. This in vitro study aimed to evaluate the calcium release, pH, biocompatibility, solubility, and bioactivity of three resin-modified calcium hydroxide cavity liners. METHODS: The disc specimens of each cavity liner were prepared using polyethylene molds of 7 mm in diameter and 2 mm in height (n = 10). Three light-cure liners evaluated include Ultra-Blend Plus (UB), Base-it (BI), and Master Dent (MD). The samples were then immersed in flasks containing 10 mL of distilled water. Calcium ion release, pH, and solubility were evaluated in two weeks of incubation. The cytotoxicity of extracts adjacent to the specimens was evaluated by MTT assay using NIH/3T3 cells after 1, 3, and 7 days of incubation. The ability to induce the nucleation of calcium phosphates (CaPs) after 28-day immersion in a simulated body fluid was investigated by SEM-EDX analysis. Statistical analysis was performed using ANOVA, Kruskal-Wallis, and repeated measures tests at the significant level of 0.05. RESULTS: There was a significant difference in the release of calcium ions among the three liners investigated on days 1, 7, and 14 (p < 0.05). UB liners exhibited a significantly higher amount of calcium release than the other two liners, followed by BI, and MD. On day 1, there was no significant difference in the average pH among the three liners. However, after day 7, the MD liner showed a significant decrease in pH compared to the other two liners. BI liner demonstrated the highest level of biocompatibility, followed by the MD and UB liners. UB showed a high calcium release, solubility with no alkalizing activity, and the formation of more mature Ca-rich apatite deposits than the other two liners. CONCLUSION: Based on the results of this study, the cavity liner material's performance is material dependent. It can impact ion release, biocompatibility, and bioactivity which are important factors to consider in clinical practice. Further studies are needed to investigate the long-term effects of different liner materials on oral tissues.

Virtual spaced-learning method, during COVID-19 for Pharm D students.

BACKGROUND: The coronavirus (COVID-19) outbreak basically changed teaching methods across the world, and learning was almost replaced by virtual learning during the pandemic. Also, the spacing effect is one of the most well-established phenomena in the science of learning. Using temporal intervals for re-exposing learners to information over time (spaced learning) leads to more effective retention of knowledge compared to having information presented at a single time (massed learning). Hence, we designed a virtual spaced learning method to reap the benefits of virtual learning and spaced learning concomitantly. METHODS/APPROACH: An interventional semi- experimental survey among 66 Pharm D students was designed and implemented. Students were divided into two groups (spaced vs mass learning) in the national integrated virtual education platform (NAVID) as the matrix for teaching as well as evaluation. Classes were conducted in the following sequence: 1- answering the pre-test, 2- watching and listening to the educational content (separately for each group), 3- answering the post-test (n = 1). The pre/post-test consisted of 10 four-choice questions based on the Kirkpatrick Model extracted from the educational content. RESULTS/OUTCOMES: Findings revealed that the average score was not significantly different between the post-tests of the spaced learning and mass learning (7.26 ± 2.26 vs 6.5 ± 2.5) methods utilizing the independent t- test (p ≥ 0.05). CONCLUSIONS: Since no statistically significant improvement was observed in the virtual spaced learning group compared to the control group, it seems that clarifying the significant influence of the spaced learning strategy in pharmacy education requires longer period of study, or study on less complex or skill-based topics for further evaluation.

Histocompatibility of Light-Curing Composites Used in Pediatric Dentistry in Human Oral Fibroblast Cells.

Statement of the Problem: Tooth-colored composites are used to repair caries lesions and other dental defects, particularly in anterior regions in children. Although a wide range of composites is using, little attention has been paid to the important indicators such as biological profiles or products released from these materials. Purpose: The current study aimed to compare the histocompatibility and cytotoxicity of light-curing resin used to repair children's teeth with different brands (3M, DenFil, and Opallis) in curing times of 20 and 40 seconds in human oral fibroblast cells (HGF1). Materials and Method: In this in vitro study, Three types of flow composites (3M, Opallis, and DenFil), all at A2 shade, were used. The composites were at 4×2mm with separate exposure times of 20 and 40 seconds. MTT test was used to determine the cytotoxicity of composites on oral fibroblast cells. This test is based on the conversion of tetrazolium bromide to a purple compound known as formazan that its color intensity can be evaluated using the ELISA. The higher intensity of the color reveals the higher survival rate of the mitochondria, which indicates less toxicity. One-way variance analysis and unpaired t-test were used to compare the cytotoxicity of each brand in two conditions of 20 and 40 seconds of curing. Statistical significance was considered when p< 0.05. Results: 3M and Opallis composites were significantly reduced vitality of cells compared to control group in both 20s and 40s curing status. While DenFil brand did not show marked cytotoxicity. In each brand, there are no significant deference between 20s and 40s curing times. Conclusion: Histocompatibility depends on the type of composite resin. In the current study, DenFil brand showed the highest histocompatibility, followed by 3M and Opallis.

Formulation of Cinnamon (Cinnamomum verum) oil loaded solid lipid nanoparticles and evaluation of its antibacterial activity against Multi-drug Resistant Escherichia coli.

Today, the increment in microbial resistance has guided the researches focus into new antimicrobial compounds or transmission systems. Escherichia coli (E. coli) is an opportunistic pathogen, producing a biofilm responsible for a wide range of nosocomial infections which are often difficult to eradicate with available antibiotics. On the other hand, Cinnamomum verum (cinnamon oil) (CO) is widely used as a natural antibacterial agent and Solid lipid nanoparticles (SLNs) are promising carriers for antibacterial compounds due to their lipophilic nature and ease of transmission through the bacterial cell wall. In this study, nanoparticles containing cinnamon oil (CO-SLN) were prepared by dual emulsion method and evaluated in terms of particle size, shape, entrapment efficiency (EE), transmission electron microscopy (TEM), oil release kinetics, and cell compatibility. The antibacterial activity of CO-SLN and CO against 10 drug-resistant E. coli strains was investigated. The anti-biofilm activity of CO-SLN on the selected pathogen was also investigated. Nanoparticles with an average size of 337.6 nm, and zeta potential of -26.6 mV were fabricated and their round shape was confirmed by TEM images. The antibacterial effects of CO-SLN and CO were reported with MIC Value of 60-75 µg/mL and 155-165 µg/mL and MBC value of 220-235 µg/ml and 540-560 µg/ml, respectively. On the other hand, CO-SLN with 1/2 MIC concentration had the greatest inhibition of biofilm formation in 24 h of incubation (55.25%). The data presented indicate that the MIC of CO-SLN has significantly reduced and it seems that SLN has facilitated and promoted CO transmission through the cell membrane.

Assessment of Protective Effects of Carvacrol on Haloperidol-Induced Oxidative Stress and Genotoxicity in Human Peripheral Blood Lymphocytes.

Haloperidol is a first-generation antipsychotic drug that has several indications in a wide range of mental conditions. The extensive prescription of haloperidol is correlated with some less-known adverse effects such as genotoxicity. Carvacrol is a monoterpenoid mainly found in oregano and thyme. It has the potential to scavenge free radicals in addition to increasing antioxidant defense enzyme activities and glutathione levels. In this study, we attempted to explore the possible potential of haloperidol in inducing genotoxicity in human peripheral lymphocytes as well as the protective role of carvacrol against this effect. The lymphocytes were divided into separate groups as follows: control group (cosolvent and NS); carvacrol group (5 µM); haloperidol group (25, 50, and 100 ng/ml); haloperidol (25, 50, and 100 ng/ml) + carvacrol (5 µM); positive control (0.8 µg/ml Cisplatin). After 24 hours of treatment, we conducted a cytokinesis-Block micronucleus test and an alkaline comet assay in order to determine genetic damage. Additionally, we measured glutathione and MDA levels as the biomarkers associated with oxidative stress. Significant increases in the levels of genotoxicity biomarkers (micronucleus frequency, DNA percentage in tail and tail moment) were observed in haloperidol-treated cells. The result of our oxidative stress tests also demonstrated that haloperidol had the potential to induce oxidative stress via reducing the levels of glutathione and increasing lipid peroxidation. Treatment with carvacrol significantly decreased the genotoxic events. It can be presumed that the induction of oxidative stress by haloperidol is the critical event associated with haloperidol-mediated genotoxicity. Therefore, using carvacrol as a natural antioxidant protected human lymphocytes against haloperidol genetic damage.

CRISPR/Cas9-medaited knockout of endogenous T-cell receptor in Jurkat cells and generation of NY-ESO-1-specific T cells: An in vitro study.

Adoptive transfer of T-cell receptor (TCR)-engineered T cells has been successful in mediating favorable clinical outcomes. TCR-engineered T cells can be applied for targeting cancers whose associated antigens are intracellular and presented through major histocompatibility complexes (MHC). The mispairing of the exogenous TCR chains with the endogenous TCR chains leads to functionally impaired TCR-engineered T cells. The CRISPR/Cas9 genome-editing system can be utilized for the knockout of the endogenous TCR in T cells before introducing the exogenous TCR chains. In this study, we used the lentiviral delivery of CRISPR/Cas9 for disrupting the expression of the endogenous TCR in the Jurkat cell line. Next, an exogenous TCR targeting human leukocyte antigen (HLA)-A*0201-restricted New York esophageal squamous cell carcinoma 1 (NY-ESO-1) peptide was transduced into the TCR-knockout (KO) Jurkat cells. Further, we assessed lentiviral transduction efficacy using tetramer assay and evaluated the functionality of the NY-ESO-1-specific TCR-engineered T cells by quantifying the cell surface expression of CD69 upon co-cultivation with peptide-pulsed T2 cells. We successfully knocked out the endogenous TCR in âˆ¼40% of the Jurkat cells. TCR-KO cells were selected and subjected to express NY-ESO-1-specific TCRs using lentiviral vectors. Flow cytometry analysis confirmed that up to 55% of the cells expressed the transgenic TCR on their surface. The functionality assay demonstrated that >90% of the engineered cells expressed CD69 when co-cultured with peptide-pulsed T2 cells. Conclusively, we developed a pipeline to engineer Jurkat cells using the state-of-the-art technique CRISPR/Cas9 and generated TCR-engineered cells that can become activated by a tumor-specific antigen.

Phytochemical analysis, antioxidant, cytotoxic, and antimicrobial activities of golden chamomile (Matricaria aurea (Loefl.) Schultz Bip).

Matricaria aurea (Loefl.) Schultz Bip. (Asteraceae), known as golden chamomile, has been traditionally used for the treatment of various diseases. In this study, total phenolic, flavonoid, and tannin contents of total extract and different fractions of this plant were determined. The antioxidant, cytotoxic, and antimicrobial activities were also evaluated. Moreover, the phenolic profiles of selected fractions were determined by HPLC and LC-MS/MS analysis. Results demonstrated total phenolic contents of 37.8-57.2 mg GAE/g and total flavonoid contents of 3.0-111.2 mg QE/g. The ethyl acetate and methanol fractions (EF and MF) had the highest concentrations of phenolic, tannin, and flavonoid compounds. In both DPPH radical scavenging assay and phosphomolybdenum reduction assay, EF showed the best antioxidant activity, followed by MF. EF and MF indicated also the best antibacterial activities against Bacillus subtilis (MIC 1.56 and 12.5 mg ml-1) and Staphylococcus aureus (MIC 0.78 and 12.5 mg ml-1). Hexane fraction (HF) had no antibacterial effect. None of the samples had antifungal effect. MTT (3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay revealed for EF and HF the highest antiproliferative activities (IC50 values ranged from 111.8 to 294.6 µg ml-1). The presence of chlorogenic acid, ferulic acid, and luteolin-7-O-glucoside in MF, and p-coumaric acid in EF was confirmed and quantified.

Anti-inflammatory effect of pregabalin on acetic acid-induced colitis in the rats.

BACKGROUND AND PURPOSE: Inflammatory bowel disease (IBD) is a chronic gastrointestinal disease characterized by the inflammation of the intestine. The available medicinal treatments for IBD are not efficacious enough since they exert various adverse effects. Therefore, the search for new therapeutic agents should be continued. The present study aimed to assess the anti-inflammatory effects of pregabalin on acetic acid-induced colitis in rats. EXPERIMENTAL APPROACH: Using 2 mL of 3% acetic acid solution, colitis was intra-rectally induced in rats. Animals were randomly divided into 6 groups including the normal group, colitis control group, pregabalin treatment groups (30, 50, and 100 mg/kg; i.p., respectively), and dexamethasone treatment group (1 mg/kg; i.p.). Macroscopic, microscopic, and biochemical (myeloperoxidase, tumor necrosis factor-alpha, interleukin-6, and interleukin-1 beta) examinations were used to evaluate the efficacy of pregabalin in the inflamed colon. FINDINGS/RESULTS: All the applied doses of pregabalin significantly decreased the severity of macroscopic and microscopic colonic damages including ulcer severity, ulcer area, percentage of necrosis, and total colitis index compared to the colitis control group. These results were confirmed by the reduced colonic concentration of tumor necrosis factor-alpha, interleukin-6, interleukin-1 beta, and myeloperoxidase activity. CONCLUSION AND IMPLICATIONS: Results of this study indicated that pregabalin administration has beneficial effects upon the treatment of experimental colitis, which might be partly due to its anti-inflammatory properties.

Apoptosis Effects of Oxalis corniculata L. Extract on Human MCF-7 Breast Cancer Cell Line:.

Background: Recently, the non-toxic properties of natural plant products have gained more focus as anticancer agents. Therefore, this study aimed to assess the apoptosis effects of the ethanolic extract of Oxalis corniculata on the MCF-7 breast cancer cell line.Materials and Methods: In this experimental study, aerial parts of O. corniculata were collected in Lahijan city (Iran), and after confirmation, they were dried and extracted with ethanol for 24 h. Then, the total phenolic and flavonoid contents of the extract were measured. The 2,2-diphenyl-1-picrylhydrazyl radical scavenging assay was used to measure the antioxidant properties of the extract. Selected cell lines (MCF-7 and human dermal fibroblast) were cultured in 6-wells dishes (1×106 cells/well). After 72 h of treating the extract, cytotoxicity was assessed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay. The expression of apoptotic genes (such as p53, bcl-2, bax, and CD95) was studied by real-time polymerase chain reaction (PCR). Results: The extract's total phenolic content was 31.30±02 µg of gallic acid equivalents/mg of dry extract, and the total flavonoid content was 49.61±04 µg of quercetin as equivalents/mg of extract. The antioxidant activity ofO. corniculata was measured at the dose of 619.2 µg/µl, indicating that it decreases cancer cell viability and enhances apoptosis. Within the half maximal inhibitory concentrations, real-time PCR revealed substantial increases in p53 (P<0.001), CD95 (P<0.05), and bcl-2 expression (P<0.05) in MCF-7 cells treated with O. corniculata. Conclusion: This study suggests that O. corniculata may cause apoptosis by oxidative stress in cancer cells.[GMJ.2022;11:e2484].

Toward a universal influenza virus vaccine: Some cytokines may fulfill the request.

The influenza virus annually causes widespread damages to the health and economy of the global community. Vaccination is currently the most crucial strategy in reducing the number of patients. Genetic variations, the high diversity of pandemic viruses, and zoonoses make it challenging to select suitable strains for annual vaccine production. If new pandemic viruses emerge, it will take a long time to produce a vaccine according to the new strains. In the present study, intending to develop a universal influenza vaccine, new bicistronic DNA vaccines were developed that expressed NP or NPm antigen with one of modified IL-18/ IL-17A/ IL-22 cytokine adjuvants. NPm is a mutant form of the antigen that has the ability for cytoplasmic accumulation. In order to investigate and differentiate the role of each of the components of Th1, Th2, Th17, and Treg cellular immune systems in the performance of vaccines, Treg competent and Treg suppressed mouse groups were used. Mice were vaccinated with Foxp3-FC immunogen to produce Treg suppressed mouse groups. The potential of the vaccines to stimulate the immune system was assessed by IFN-γ/IL-17A Dual FluoroSpot. The vaccine's ability to induce humoral immune response was determined by measuring IgG1, IgG2a, and IgA-specific antibodies against the antigen. Kinetics of Th1, Th2, and Th17 cellular immune responses after vaccination, were assessed by evaluating the expression changes of IL-17A, IFN-γ, IL-18, IL-22, IL-4, and IL-2 cytokines by semi-quantitative real-time RT-PCR. To assess the vaccines' ability to induce heterosubtypic immunity, challenge tests with homologous and heterologous viruses were performed and then the virus titer was measured in the lungs of animals. Evaluation of the data obtained from this study showed that the DNA-vaccines coding NPm have more ability to induces a potent cross-cellular immune response and protective immunity than DNA-vaccines coding NP. Although the use of IL-18/ IL-17A/ IL-22 genetic adjuvants enhanced immune responses and protective immunity, Administration of NPm in combination with modified IL-18 (Igk-mIL18-IgFC) induced the most effective immunity in Treg competent mice group.

Generating the Engineered Form of a Nanobody Against Placenta Growth Factor with High Antiangiogenesis Potential.

Antibody engineering is a dynamic field in antibody industry. Over 30% of novel monoclonal antibodies (mAbs) in R&D and clinical trials are engineered forms. Affinity enhancement contributes to the development of new binders that are not only effective in low dose and cost but also improve some drawbacks of antibody production. After previous successful work on in silico affinity maturation of nanobody against placenta growth factor and finding the best engineered nanobodies (Mut2:S31D and Mut4:R45E), according to bioinformatic parameters and molecular dynamics (MD) simulation results, in this study we focused on experimental confirmation of affinity enhancement of a mutant form of nanobody. So, we cloned and expressed two of four mutant forms in pHEN6c vector. Affinity binding was assayed by enzyme-linked immunosorbent assay on purified mutants, with results showing that 10-time enhancement in affinity compared with the native form associated with MD simulation results. We checked the effectiveness of these mutant nanobodies in angiogenesis inhibition by human umbilical vein endothelial cell proliferation and 3D capillary tube formation. EC50 of mut2, mut4, and native in proliferation assay was 110, 140, and 190 ng/mL, respectively, and that in 3D capillary tube formation was 80, 83, and 100 ng/mL. The results of functional studies revealed strong effectiveness of Mut2 followed by Mut4 compared with the native form. Our study confirmed that in silico approach could facilitate development of novel versions of mAb with better characteristics, which could save cost and time.

Aptasensors as a new sensing technology developed for the detection of MUC1 mucin: A review.

Mucin 1 protein (MUC1) is a membrane-associated glycoprotein overexpressed in the majority of human malignancies and considered as a predominant protein biomarker in cancers. Owing to the crucial role of MUC1 in cancer dissemination and metastasis, detection and quantification of this biomarker is of great importance in clinical diagnostics. Today, there exist a wide variety of strategies for the determination of various types of disease biomarkers, especially MUC1. In this regard, aptamers, as artificial single-stranded DNA or RNA oligonucleotides with catalytic and receptor properties, have drawn lots of attention for the development of biosensing platforms. So far, various sensitivity-enhancement techniques in combination with a broad range of smart nanomaterials have integrated into the design of novel aptamer-based biosensors (aptasensors) to improve detection limit and sensitivity of analyte determination. This review article provides a brief classification and description of the research progresses of aptamer-based biosensors and nanobiosensors for the detection and quantitative determination of MUC1 based on optical and electrochemical platforms.

Purification and Characterization of Recombinant Darbepoetin Alfa from Leishmania tarentolae.

Darbepoetin alfa is a biopharmaceutical glycoprotein that stimulates erythropoiesis and is used to treat anemia, which associated with renal failure and cancer chemotherapy. We herein describe the structural characterization of recombinant darbepoetin alfa produced by Leishmania tarentolae T7-TR host. The DNA expression cassette was integrated into the L. tarentolae genome through homologous recombination. Transformed clones were selected by antibiotic resistance, diagnostic PCRs, and protein expression analysis. The structure of recombinant darbepoetin alfa was analyzed by isoelectric focusing, ultraviolet-visible spectrum, and circular dichroism (CD) spectroscopy. Expression analysis showed the presence of a protein band at 40 kDa, and its expression level was 51.2 mg/ml of culture medium. Darbepoetin alfa have 5 isoforms with varying degree of sialylation. The UV absorption and CD spectra were analogous to original drug (Aranesp), which confirmed that the produced protein was darbepoetin alfa. Potency test results revealed that the purified protein was biologically active. In brief, the structural and biological characteristics of expressed darbepoetin alfa were very similar to Aranesp which has been normally expressed in CHO. Our data also suggest that produced protein has potential to be developed for clinical use.

Generation and characterization of nanobodies targeting PSMA for molecular imaging of prostate cancer.

Nanobodies show attractive characteristics for tumor targeting in cancer diagnosis and therapy. A radiolabeled nanobody binding the prostate-specific membrane antigen (PSMA) could offer a noninvasive strategy to select prostate cancer patients eligible for PSMA-targeted therapies. We here describe the generation, production and in vivo evaluation of anti-PSMA nanobodies. Nanobodies were derived from heavy-chain-only antibodies, raised in immunized dromedaries. Binding characteristics were evaluated through ELISA and flow cytometry. Selected nanobodies were radiolabeled with (99m) Tc at their hexahistidine tail, after which cell binding capacity and internalization were evaluated on PSMA(pos) LNCaP and PSMA(neg) PC3 cell lines. In vivo tumor targeting was analyzed in both LNCaP and PC3 xenografted mice through SPECT/microCT and tissue sampling. A panel of 72 generated clones scored positive on ELISA, all contributing to three nanobody groups, of which group 3 dominated with 70 clones. ELISA and FACS analysis led to the selection of two dominant nanobodies. (99m) Tc-labeled PSMA6 and PSMA30 both showed specific binding on LNCAP cells, but not on PC3 cells. (99m) Tc-PSMA30 internalized significantly more in LNCaP cells compared to (99m) Tc-PSMA6. Higher absolute tumor uptake and tumor-to-normal organ ratios were observed for (99m) Tc-PSMA30 compared with (99m) Tc-PSMA6 and a (99m) Tc-control nanobody in LNCaP but not in PC3 tumor-bearing mice. PSMA30 nanobody has improved targeting characteristics both in vitro as well as in vivo compared with PSMA6 and the control nanobody, and was therefore selected as our in-house-developed lead compound for PSMA targeting.

Camel heavy chain antibodies against prostate-specific membrane antigen.

Prostate-specific membrane antigen (PSMA), a type II integral membrane glycoprotein, is highly overexpressed in all forms of prostate cancer tissues. It has also been demonstrated in a wide range of neovasculature of non-prostatic solid tumors, including bladder, pancreas, lung, kidney, colorectal, and gastric cancers. Given the unique expression of PSMA, it is considered an alluring target for antibody-based imaging and therapy of cancer. In the present study, the production and characterization of camel heavy chain antibodies (HCAbs) specific for the external domain of the PSMA are reported. Due to the absence of the CH1 domain, HCAbs are smaller than their counterparts in conventional antibodies. In this study, camel antibodies were generated through immunization of Camelus dromedarius with a synthetic 28 amino acid peptide corresponding to the external surface domain of antigen and PSMA-expressing cell lines. Different binding properties to protein A and protein G affinity columns were deployed to separate three subclasses of camel IgG. The affinity purified HCAbs bound selectively to the synthetic peptide in enzyme linked immunosorbent assay (ELISA) and reacted specifically with PSMA-expressing cell line through immunocytochemistry study. Currently, we are attempting to develop recombinant variable domain of these heavy chain antibodies (VHH or nanobody) for tumor imaging and cancer therapy.