Enzyme variants in biosynthesis and biological assessment of different molecular weight hyaluronan.

In the present study, low- and high-molecular-weight hyaluronic acids (LMW-HA and HMW-HA) were synthesized in vitro by truncated Streptococcus equisimilis hyaluronan synthases (SeHAS). The enzyme kinetic parameters were determined for each enzyme variant. The MW, structure, dispersity, and biological activity of polymers were determined by electrophoresis, FTIR spectroscopy, carbazole, cell proliferation, and cell migration assay, respectively. The specific activities were calculated as 7.5, 6.8, 4.9, and 2.8 µgHA µgenzyme-1 min-1 for SeHAS, HAS123, HAS23, and HASIntra, respectively. The results revealed SeHAS produced a polydisperse HMW-HA (268 kDa), while HAS123 and HAS23 produced a polydisperse LMW-HA (< 30 kDa). Interestingly, HASIntra produced a low-disperse LMW-HA. Kinetics studies revealed the truncated variants displayed increased Km values for two substrates when compared to the wild-type enzyme. Biological assessments indicated all LMW-HAs showed a dose-dependent proliferation activity on endothelial cells (ECs), whereas HMW-HAs exhibited an inhibitory effect. Also, LMW-HAs had the highest cell migration effect at 10 µg/mL, while at 200 µg/mL, both LMW- and HMW-HAs postponed the healing recovery rate. The study elucidated that the transmembrane domains (TMDs) of SeHAS affect the enzyme kinetics, HA-titer, HA-size, and HA-dispersity. These findings open new insight into the rational engineering of SeHAS to produce size-defined HA.

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.

The correlation of long non-coding RNAs IFNG-AS1 and ZEB2-AS1 with IFN-γ and ZEB-2 expression in PBMCs and clinical features of patients with coronary artery disease.

BACKGROUND: Aberrant expression of long non-coding RNAs (lncRNAs) can contribute to the pathogenesis of coronary artery disease (CAD). In this study, we aimed to evaluate the expression of lncRNA interferon γ-antisense 1 (IFNG-AS1), zinc finger E-box binding homeobox 2 antisense RNA 1 (ZEB2-AS1), and their direct target genes (IFN-γ and ZEB2, respectively) in peripheral blood mononuclear cell (PBMC) from CAD and healthy individuals. METHODS AND RESULTS: We recruited 40 CAD patients and 40 healthy individuals. After doing some bioinformatics analyses, the expressions of IFNG-AS1/ ZEB2-AS1 lncRNAs and IFN-γ/ ZEB2 in PBMCs were measured using quantitative real-time PCR. The possible correlation between the putative lncRNAs and disease severity was also assessed. Receiver operating characteristic (ROC) curve analysis was used to evaluate the predictive role of lncRNAs as diagnostic biomarkers in CAD patients. The expressions of IFNG-AS1 lncRNA as well as IFN-γ and ZEB2 genes were significantly reduced in CAD patients compared to healthy subjects. In contrast, the expression of ZEB2-AS1 was up-regulated in these patients. Linear regression analysis unveiled that there is a positive correlation between the expression of IFNG-AS1 and IFN-γ, also similarly, ZEB2-AS1 and ZEB2 in PBMCs of subjects. Moreover, the expression of IFNG-AS1 and ZEB2-AS1 correlated with the Gensini score. The area under the ROC curves ranged from 0.633-0.742 for ZEB2-AS1/ZEB2 and IFNG-AS1/IFN-γ, respectively. CONCLUSIONS: Our results indicated that the dysregulation of IFNG-AS1/IFN-γ and ZEB2-AS1/ZEB2 in PBMCs of CAD patients may be involved in CAD pathogenesis.

Angiogenic biomolecules specific nanobodies application in cancer imaging and therapy; review and updates.

Solid cancers are affiliate on angiogenesis for preservation. The FDA-approved monoclonal antibodies like Bevacizumab are currently being administered effectively as inhibitors of angiogenesis against various types of tumors. Despite the clinical achievements in this regard, monoclonal antibodies suffer from considerable disadvantages, including the potential to develop therapeutic resistance, a high production cost, and the restricted tumor penetration, which consequently limit their therapeutic applications. In the past decades, some significant methods such as miniaturization of the antibodies, containing those inhibiting tumor angiogenesis, have been proposed to enhance cancer therapeutics efficiency. Since their discovery, small single-domain antigen binding antibody fragments, known as nanobodies, have been broadly utilized in the fields of cancer research, diagnosis, and treatment. Due to their desired functional attributes and a unique structure, nanobodies are becoming promising therapeutic and diagnostic biomolecules in oncology field. Moreover, they displayed a substantial translational potential in preclinical and clinical studies. This review was performed with the aim of highlighting the potential of nanobodies in blocking the angiogenic process by targeting of angiogenic biomolecules for cancer therapy and the application of labeled nanobodies in non-invasive in vivo tumor imaging.

Anti-angiogenic peptides application in cancer therapy; a review.

Cancer is a disease advanced via surplus angiogenesis. The development of new anti-angiogenic therapeutic agents with more efficacy and fewer side effects is still quite necessary. Conventional therapies saving the life of many cancer patients but due to drug resistance and lack of specificity utilizing these methods is faced with limits. Recently, new therapeutic agents have been developed and used to treat cancers such as scaffold proteins, monoclonal antibodies, tyrosine kinase inhibitors, and peptides. In antiangiogenic drug development, anti-angiogenic peptides design is a significant aim. Peptides have developed as substantial therapeutics that are being carefully investigated in angiogenesis-dependent diseases because of their high penetrating rate into the cancer cells, high specificity, and low toxicity. In this review, we focus on anti-angiogenic peptides in the field of cancer therapy that are designed, screened, or derived from nanobodies, mimotopes, phage displays, and natural resources.

Isolation and characterization of halophilic bacteria with the ability of heavy metal bioremediation and nanoparticle synthesis from Khara salt lake in Iran.

Increasing environmental pollutants such as heavy metals have become one of the most severe health dangers because of rapid industrialization. Exposure to lead and nickel heavy toxic metals can lead to hazardous diseases affecting most of the organs in humans. Bioremediation is a process that uses the ability of microorganisms or plants to detoxify environmental contaminants at lower costs than physicochemical techniques. This study isolated halophilic bacteria from Khara salt lake in Iran and screened their ability to resist lead and nickel. After screening stages, three selected strains including Bacillus sp. A21, Oceanobacillus sp. A22 and Salinicoccus A43 were identified by16S rDNA sequencing and the related sequences were submitted to GeneBank with accession IDs MN588312, MN588313, and MN 588,314, respectively. These strains resist 7.2 mM, 4.1 mM, and 6.7 mM lead and 3.6 mM, 3.7 mM, and 4.1 mM nickel, respectively. Investigation of growth pattern and evaluation of bioremediation ability by atomic absorption spectroscopy revealed that Bacillus sp. A21 could decrease lead and nickel in culture medium up to 97.5% and 76%, respectively. Oceanobacillus sp. A22 showed higher lead bioremediation rate (98.8%) and lower nickel-bioremediation rate (73.5%). Salinicoccus sp. A43 showed the least bioremediation ability (92% lead and 71.7% nickel). The ability of selected strains to synthesize lead and nickel nanoparticles was evaluated using UV/Vis spectrophotometry and Energy-Dispersive X-ray Spectroscopy (EDX). Particle dimensions were measured using Scanning Electron Microscopy (SEM). Bacillus sp. A21 and Oceanobacillus sp. A22 strains were able to synthesize lead nanoparticles; however, Salinicoccus sp. A43 could synthesize both lead and nickel nanoparticles.

Designing and Development of a Tandem Bivalent Nanobody against VEGF165.

BACKGROUND: Inhibition of angiogenesis using monoclonal antibodies is an effective strategy in cancer therapy. However, they could not penetrate sufficiently into solid tumors. Antibody fragments have solved this issue. However, they suffer from short in vivo half-life. In the current study, a tandem bivalent strategy was used to enhance the pharmacokinetic parameters of an anti-VEGF165 nanobody. METHODS: Homology modeling and MD simulation were used to check the stability of protein. The cDNA was cloned into pHEN6C vector and the expression was investigated in WK6 Escherichia coli (E. coli) cells by SDS-PAGE and western blot. After purification, the size distribution of tandem bivalent nanobody was investigated by dynamic light scattering. Moreover, in vitro antiproliferative activity and pharmacokinetic study were studied in HUVECs and Balb/c mice, respectively. RESULTS: RMSD analysis revealed the tandem bivalent nanobody had good structural stability after 50 ns of simulation. A hinge region of llama IgG2 was used to fuse the domains. The expression was induced by 1 mM IPTG at 25°C for overnight. A 30 kDa band in 12% polyacrylamide gel and nitrocellulose paper has confirmed the expression. The protein was successfully purified using metal affinity chromatography. MTT assay revealed there is no significant difference between the antiproliferative activity of tandem bivalent nanobody and the native protein. The hydrodynamic radius and terminal half-life of tandem bivalent nanobody increased approximately 2-fold by multivalency compared to the native protein. CONCLUSION: Our data revealed that the physicochemical as well as in vivo pharmacokinetic parameters of tandem bivalent nanobody was significantly improved.

Crosstalk between MUC1 and VEGF in angiogenesis and metastasis: a review highlighting roles of the MUC1 with an emphasis on metastatic and angiogenic signaling.

VEGF and its receptor family (VEGFR) members have unique signaling transduction system that play significant roles in most pathological processes, such as angiogenesis in tumor growth and metastasis. VEGF-VEGFR complex is a highly specific mitogen for endothelial cells and any de-regulation of the angiogenic balance implicates directly in endothelial cell proliferation and migration. Moreover, it has been shown that overexpressing Mucin 1 (MUC1) on the surface of many tumor cells resulting in upregulation of numerous signaling transduction cascades, such as growth and survival signaling pathways related to RTKs, loss of cell-cell and cell-matrix adhesion, and EMT. It promotes gene transcription of pro-angiogenic proteins such as HIF-1α during periods of oxygen scarcity (hypoxia) to enhance tumor growth and angiogenesis stimulation. In contrast, the cytoplasmic domain of MUC1 (MUC1-C) inhibits apoptosis, which in turn, impresses upon cell fate. Besides, it has been established that reduction in VEGF expression level correlated with silencing MUC1-C level indicating the anti-angiogenic effect of MUC1 downregulation. This review enumerates the role of MUC1-C oncoprotein and VEGF in angiogenesis and metastasis and describes several signaling pathways by which MUC1-C would mediate the pro-angiogenic activities of cancer cells.

Challenges and advancements in the pharmacokinetic enhancement of therapeutic proteins.

Nowadays, proteins are frequently administered as therapeutic agents in human diseases. However, the main challenge regarding the clinical application of therapeutic proteins is short circulating plasma half-life that leads to more frequent injections for maintaining therapeutic plasma levels, increased therapy costs, immunogenic reactions, and low patient compliance. So, the development of novel strategies to enhance the pharmacokinetic profile of therapeutic proteins has attracted great attention in pharmaceuticals. So far, several techniques, each with their pros and cons, have been developed including chemical bonding to polymers, hyper glycosylation, Fc fusion, human serum albumin fusion, and recombinant PEG mimetics. These techniques mainly classify into three strategies; (i) the endosomal recycling of neonatal Fc receptor which is observed for immunoglobulins and albumin, (ii) decrease in receptor-mediated clearance, and (iii) increase in hydrodynamic radius through chemical and genetic modifications. Recently, novel PEG mimetic peptides like proline/alanine/serine repeat sequences are designed to overcome pitfalls associated with the previous technologies. Biodegradability, lack of or low immunogenicity, product homogeneity, and a simple production process, currently make these polypeptides as the preferred technology for plasma half-life extension of therapeutic proteins. In this review, challenges and pitfalls in the pharmacokinetic enhancement of therapeutic proteins using PEG-mimetic peptides will be discussed in detail.

New Proline, Alanine, Serine Repeat Sequence for Pharmacokinetic Enhancement of Anti-VEGF Single-Domain Antibody.

Therapeutic fragmented antibodies show a poor pharmacokinetic profile that leads to frequent high-dose administration. In the current study, for the first time, a novel proline, alanine, serine (PAS) repeat sequence called PAS#208 was designed to extend the plasma half-life of a nanosized anti-vascular endothelial growth factor-A single-domain antibody. Polyacrylamide gel electrophoresis, circular dichroism, dynamic light scattering, and electrophoretic light scattering were used to assess the physicochemical properties of the newly designed PAS sequence. The effect of PAS#208 on the biologic activity of a single-domain antibody was studied using an in vitro proliferation assay. The pharmacokinetic parameters, including terminal half-life, the volume of distribution, elimination rate constant, and clearance, were determined in mice model and compared with the native protein and PAS#1(200) sequence. The novel PAS repeat sequence showed comparable physicochemical, biologic, and pharmacokinetic features to the previously reported PAS#1(200) sequence. The PAS#208 increased the hydrodynamic radius and decreased significantly the electrophoretic mobility of the native protein without any change in zeta potential. Surprisingly, the fusion of PAS#208 to the single-domain antibody increased the binding potency. In addition, it did not alter the biologic activity and did not show any cytotoxicity on the normal cells. The PAS#208 sequence improved the terminal half-life (14-fold) as well as other pharmacokinetic parameters significantly. The simplicity as well as superior effects on half-life extension make PAS#208 sequence a novel sequence for in vivo pharmacokinetic enhancement of therapeutic fragmented antibodies. SIGNIFICANCE STATEMENT: In the current study, a new proline, alanine, serine (PAS) sequence was developed that showed comparable physicochemical, biological, and pharmacokinetic features to the previously reported PAS#1(200) sequence. The simplicity as well as superior effects on half-life extension make PAS#208 sequence a novel sequence for in vivo pharmacokinetic enhancement of recombinant small proteins.

Liposomal delivery of vascular endothelial growth factor/receptors and their inhibitors.

The development of nanocarrier delivery systems opened new opportunities in the nano-technology field. Liposomes, possessing useful characteristics including their biocompatibility, stability in colloidal solutions and biodegradability are appropriate candidates for drug delivery applications. Vascular endothelial growth factors (VEGF) and their receptors (VEGFR) play a key role in the development of angiogenesis in physiological and pathological conditions such as wound healing and cancer, respectively. To overcome the delivery problems (e.g. drug half-life, cellular uptake, rate of release and efficacy) of VEGF/VEGFRs and their inhibitors, many studies focussed on the use of liposomes as nanocarrier drug delivery system. In this review we aim to describe the state-of-the-art targeting of VEGF/VEGFR and delivery of its inhibitors with liposomal nanocarriers. The lessons from this technology of liposomal nanocarriers should be instructive for scientists developing nano-encapsulation and delivery systems and those focussing on tissue engineering and innovative cancer therapy applications.

Molecular dynamics simulation for rational protein engineering: Present and future prospectus.

Recently protein engineering has been used as a pivotal tool for designing proteins with improved characteristics. While the experimental methods might be laborious and time-consuming, in silico protein design is a time and cost-effective approach. Moreover, in some cases, protein modeling might be the only way to obtain structural information where the experimental techniques are inapplicable. Molecular dynamics (MD) simulation is a method that allows the motion of protein to be simulated in defined conditions on the basis of classical molecular dynamics. MD simulation could widely be used when protein design needs accurate modeling of the target protein dynamics and also descriptions of the relation between conformational changes and function of protein at the atomic level. In this review, the effectiveness and the power of MD simulation in designing proteins with improved characteristics will be discussed.

Development of a novel nano-sized anti-VEGFA nanobody with enhanced physicochemical and pharmacokinetic properties.

Since physiological and pathological processes occur at nano-environments, nanotechnology has considered as an efficient tool for designing of next generation specific biomolecules with enhanced pharmacodynamic and pharmacodynamic properties. In the current investigation, by control of the size and hydrodynamic volume at the nanoscale, for the first time, physicochemical and pharmacokinetic properties of an anti-VEGFA nanobody was remarkably improved by attachment of a Proline-Alanine-Serine (PAS) rich sequence. The results elucidated unexpected impressive effects of PAS sequence on physicochemical properties especially on size, hydrodynamics radius, and even solubility of nanobody. CD analysis revealed an increment in random coil structure of the PASylated protein in comparison to native one without any change in charge state or binding kinetic parameters of nanobody assessed by isoelectric focusing and surface plasmon resonance measurements, respectively. In vitro biological activities of nanobody were not affected by coupling of the PAS sequence. In contrast, the terminal half-life was significantly increased by a factor of 14 for the nanobody-PAS after single dose IV injection to the mice. Our study demonstrated that the control of size in the design of small therapeutic proteins has a promising effect on the stability and solubility, in addition to their physiochemical and pharmacokinetic properties. The designed new anti-VEGFA nanobody could promise a better therapeutic agent with a long administration intervals and lower dose, which in turn leads to a better patient compliance. Size adjustment of an anti-VEGF nanobody at the nanoscale by the attachment of a natural PAS polymer remarkably improves physicochemical properties, as well as a pharmacokinetic profile without any change in biological activity of the miniaturized antibody.

Single-Domain Antibodies or Nanobodies: A Class of Next-Generation Antibodies.

Nanobodies for the first time were identified in the sera of Camelidae. Single-domain antibodies or nanobodies are a class of next-generation antibodies that have specific features: small size (in nanoscale), high penetration in various tissues, high stability in hard situations and ease production process in microbial systems. In fact single-domain antibodies are the smallest fragment of the antibody with binding ability. Unique characteristics and features of nanobodies make them an appropriate candidate for further evaluation as the development of novel antibody-based therapeutics. In this regard single-domain antibodies are in the interest of many researchers as well as biopharmaceutical companies for diagnostic and therapeutic applications. Nowadays several single domain antibodies have been developed and evaluated in different clinical trials. Because of many advantages of single-domain antibodies over other formats of antibodies, they could be good replacement for other formats of antibodies in near future. Here, we review the biology, engineering platforms and application of nanobodies.