No abstract

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.

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.

Rubus hyrcanus Juz. (Rosaceae), known as Caspian blackberry, is wildly distributed around the Caspian Sea. This study focused on antioxidant, cytotoxic, and antibacterial activities of total extracts and different fractions from the roots and leaves of this species. The total phenolics and flavonoid contents were also evaluated. Finally, the phenolic profiles of selected fractions were determined using HPLC–DAD and LC–MS/MS. The results indicated that the total phenolics content (TPC) of root total extract (RTE) was 3.5 times that of leaves (340.4 and 102.7 mg GAE/g, respectively). The TPC of three root fractions ranged from 226.6 to 392.9 mg GAE/g, while in leaves fractions, it ranged between 68.3 and 101.8 mg GAE/g. The total extract of leaves had higher contents of total flavonoids than roots (70.5 and 8.9 mg QE/g, respectively). The methanol fractions of both parts had the highest amounts of flavonoids. The root methanol fraction (RMF) had the best antioxidant effect in both DPPH radical scavenging assay (IC50: 9.16 μg ml−1) and total antioxidant capacity test (1010.5 mg ɑTE/g). The RMF and RTE had potent antibacterial activities against Bacillus subtilis and Staphylococcus aureus (MIC 1.5 mg ml−1). In the MTT assay, ethyl acetate fractions of roots and leaves exhibited the best cytotoxicity (IC50 247 and 227 μg ml−1, respectively) and the highest selectivity indexes (4.73 and 5.31, respectively). Phytochemical analysis revealed the presence of gallic acid, p-coumaric acid, and chlorogenic acid in leaves ethyl acetate fraction, chlorogenic acid in leaves methanol fraction, and gallic acid in the root ethyl acetate fraction.

No abstract

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.

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.

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.