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.

Lipopolysaccharide removal affinity matrices based on novel cationic amphiphilic peptides.

Lipopolysaccharide (LPS) is one of the most challenging contaminants in biopharmaceutical industries. Cationic amphiphilic peptides (CAPs) -based affinity matrices can be potent tools for LPS removal in such situations. In this study, two newly designed CAPs derived from the LPS binding site of factor C of horseshoe crab S3E3 and S3E3A were immobilized chemo-selectively on diaminodipropylamine (DADPA) and iodoacetyl functionalized Sepharose beads. Both peptides were immobilized via their carboxyl or sulfhydryl (thiol) groups by amide or thioether bonds, respectively. The generated four affinity matrices were used to remove LPS from bovine serum albumin (BSA). The effects of different influential factors including pH, NaCl, Ethylenediaminetetraacetic acid (EDTA), and LPS concentrations on LPS removal efficiency and protein recovery were investigated by Plackett Burman (PB) method.Statistical analysis revealed that immobilized S3E3 removed LPS more effectively than immobilized S3E3A. Increasing pH and LPS concentration had negative effects on LPS removal efficiency and protein recovery. Increasing NaCl concentration improved protein recovery but reduced LPS removal efficiency. Other factors such as EDTA and type of buffer had no significant effect on the measured responses.

Enhanced antitumor activity of bovine lactoferrin through immobilization onto functionalized nano graphene oxide: an in vitro/in vivo study.

This study aims to improve the anticancer activity of bovine lactoferrin through enhancing its stability by immobilization onto graphene oxide. Bovine lactoferrin was conjugated onto graphene oxide and the conjugation process was confirmed by FT-IR, SDS-PAGE, and UV spectrophotometry. Physical characterization was performed by DLS analysis and atomic force microscopy. The cytotoxicity and cellular uptake of the final construct (CGO-PEG-bLF) was inspected on lung cancer TC-1 cells by MTT assay and flow cytometry/confocal microscopy. The anticancer mechanism of the CGO-PEG-bLF was studied by cell cycle analysis, apoptosis assay, and western blot technique. Finally, the anticancer activity of CGO-PEG-bLF was assessed in an animal model of lung cancer. Size and zeta potential of CGO-PEG-bLF was obtained in the optimum range. Compared with free bLF, more cytotoxic activity, cellular uptake and more survival time was obtained for CGO-PEG-bLF. CGO-PEG-bLF significantly inhibited tumor growth in the animal model. Cell cycle arrest and apoptosis were more induced by CGO-PEG-bLF. Moreover, exposure to CGO-PEG-bLF decreased the phospho-AKT and pro-Caspase 3 levels and increased the amount of cleaved caspase 3 in the treated cells. This study revealed the potential of CGO-PEG as a promising nanocarrier for enhancing the therapeutic efficacy of anticancer agents.

Niosomal delivery of simvastatin to MDA-MB-231 cancer cells.

OBJECTIVE: The objective of this study was to use nano-niosomal formulations to deliver simvastatin as a poor-water soluble drug into breast cancer cells. SIGNIFICANCE: Our study focused on the problem associated with poor water-soluble drugs which have significant biological activity in vivo. METHODS: Different niosomal formulations of simvastatin were prepared and characterized in terms of morphology, size, encapsulation efficiency (EE), and release kinetic. Antiproliferative activity and the mechanism were assessed by quantitative real-time PCR and flow cytometry. Moreover, confocal microscopy was employed to analyze the cell uptake of simvastatin loaded niosomes to the cancerous cells. RESULTS: Size, polydispersity index (PDI), and EE of the best formulation were obtained as 164.8 nm, 0.232, and 97%, respectively. The formulated simvastatin had a spherical shape and showed a slow release profile of the drug after 72 h. Stability data elucidated an increase in mean diameter and PDI which was lower for 4 °C than 25 °C. Confocal microscopy showed the localization of drug loaded niosomes in the cancer cells. The MTT assay revealed both free drug and drug loaded niosomes exhibited a dose-dependent cytotoxicity against breast cancer cells (MDA-MB-231 cells). Flow cytometry and qPCR analysis revealed drug loaded niosomes exert their cytotoxicity on cancerous cells via regulation of apoptotic and anti-apoptotic genes. CONCLUSION: The prepared niosomal simvastatin showed good physicochemical and biological properties than free drug. Our study suggests that niosomal delivery could be considered as a promising strategy for the delivery of poor water-soluble drugs to cancer cells.

Effect of glutamic acid elimination/substitution on the biological activities of S3 cationic amphiphilic peptides.

Cationic amphiphilic peptides (CAPs) are usually classified as bacterial membrane targeting molecules. Rational design and modification of cationic and amphiphilic properties of CAPs have made them to be used in new medical and biotechnological applications. However, CAPs modification and development strategies are challenging issues due to the risk of cytotoxicity or hemolytic activity. In this research, modified variants of S3 peptide were introduced. S3 is a linear 34 amino acid peptide derived from the lipopolysaccharide (LPS) binding site of factor C in horseshoe crab's hemolymph. Net positive charges of variants (S3E3 and S3E3A) increased by either eliminating negatively charged residues of the peptides or substituting them with alanine. Different biological activities of new variants including LPS binding affinity, antimicrobial activity, cytotoxicity against human breast tumor cell line, and hemolytic property were studied and compared to those of S3 peptide. S3E3 variant showed 68.5% higher LPS binding affinity, 40.4% stronger anti-microbial activity, conserved hemolytic property with the same anti-cancer activity compared to S3peptide. These results revealed that elimination/substitution of negatively charged residues will be a proper strategy for modification of S3 peptide.

Increment in protease activity of Lysobacter enzymogenes strain by ultra violet radiation.

BACKGROUND AND OBJECTIVES: Increasing the amount of protease from microbial sources is in the focus of attention. Random mutagenesis by physical methods like ultraviolet (UV) radiation is a cost effective and convenient procedure for strain improvement. Therefore, in the present study attempts were made to investigate the effect of UV radiation on Lysobacter enzymogenes in order to increase its protease activity. MATERIALS AND METHODS: UV mutagenesis was induced in L. enzymogenes fresh culture at the distance of 20 cm from light source for different exposure times of 70, 90, 150 and 200 seconds. The mutated isolates were randomly cultured from the nutrient agar medium to casein agar plate, as a selective medium. The primary screening was performed by observing hydrolysis of casein in the plate and the secondary screening was carried out on skim milk agar on the basis of zone of hydrolysis using bacterial supernatants. Quantification of protease activity was done by Anson's method using tyrosine as standard. RESULTS: UV radiation resulted in obtaining 12 mutants out of 100 examined L. enzymogenes strains with increased protease activity. The mutant M2, at 90s exposure time was selected as the best mutant bacterium which produced 1.96 fold more protease over the parent strain. CONCLUSION: Random mutation by UV radiation is a simple and convenient method to increase the protease activity of Lysobacter enzymogenes. Furthermore, it seems that the middle time of exposure to UV, 90 s, was the best time because it can induce mutagenesis but did not hamper the bacteria growth and viability.

A turn-on graphene quantum dot and graphene oxide based fluorometric aptasensor for the determination of telomerase activity.

A turn-on fluorometric assay is described for determination of the activity of enzyme telomerase. For this purpose, graphene quantum dots (GQDs) were first modified with the telomeric sequence (5'-amino-AATCCGTCGAGCAGAGTT-3') via a condensation reaction. Injection of graphene oxide causes instant quenching of the blue fluorescence of the GQDs. Addition of cell extract containing telomerase, triggers the extension of telomer via addition of specific sequence (TTAGGG)n to its 3' end. Fluorescence, best measured at excitation/emission wavelengths of 390/446 nm, is subsequently restored due to folding of the extended telomeric sequence into G-quadruplex structure. The method was applied to the determination of telomerase activity in crude cell extracts of as little as 10 HeLa cells. The linear dynamic range extends from 10 to 6500 cells. Graphical abstractIn this study, a new turn-on graphene quantum dotm and graphene oxide based fluorometric assay is developed for the determination of telomerase activity.

Enhancing bioactivity, physicochemical, and pharmacokinetic properties of a nano-sized, anti-VEGFR2 Adnectin, through PASylation technology.

The crucial role of VEGF receptor 2 (VEGFR2) signaling in the angiogenesis and metastasis of solid tumors has prompted the development of inhibitors with minimal bystander effects. Recently, Adnectin C has attracted attention for cancer treatment. To overcome the problematic properties of Adnectin, a novel form of Adnectin C has been designed by its fusion to a biodegradable polymeric peptide containing Pro/Ala/Ser (PAS) repetitive residues. E. coli-expressed recombinant fused and unfused proteins were compared in terms of bioactivity, physicochemical, and pharmacokinetic properties using standard methods. Dynamic light scattering (DLS) analysis of PASylated adnectin C revealed an approximate 2-fold increase in particle size with a slight change in the net charge. Additionally, fusion of the PAS sequence improved its stability against the growth of thermo-induced aggregated forms. The high receptor-binding and improved binding kinetic parameters of PASylated Adnectin C was confirmed by ELISA and surface plasmon resonance assays, respectively. Pharmacokinetic studies showed a noticeable increase in the terminal half-life of Adnectin C-PAS#1(200) by a factor of 4.57 after single dose by intravenous injection into female BALB/c mice. The results suggest that PASylation could offer a superior delivery strategy for developing Adnectin-derived drugs with improved patient compliance.

Delivery of vinblastine-containing niosomes results in potent in vitro/in vivo cytotoxicity on tumor cells.

Vinblastine (VB), as a chemotherapeutic agent, is widely used in treatment of different types of cancer. However, its clinical application is limited due to its low water solubility, side effects, and multidrug resistance. The aim of this study was to increase the therapeutic efficacy of VB using drug delivery systems. For this purpose, a PEGylated niosomal formulation of vinblastine (Pn-VB) was prepared by thin film hydration method and physicochemically characterized. Drug release pattern was performed by dialysis diffusion method. The cytotoxicity of Pn-VB was investigated against murine lung cancer TC-1 cells using MTT assay and its tumor inhibitory effect was evaluated in lung tumor-bearing C57BL/6 mice. Mean particle size, zeta potential, entrapment, and loading efficiency of niosomes were obtained to be about 234.3 ± 11.4 nm, -34.6 ± 4.2 mV, 99.92 ± 1.6%, and 2.673 ± 0.30%, respectively. While, the mean particle size and zeta potential for non-PEGylated niosomes were obtained about 212.4 nm and -31.4 mV, respectively. The in vitro release pattern of drug from niosomes showed a sustained release behavior. Pn-VB indicated a significant increase in toxicity against TC-l cells as compared to free VB. In animal model, Pn-VB exhibited stronger tumor inhibitory effect and longer life time in comparison to free VB. In conclusion, Pn-VB showed appropriate stability, high-entrapment efficacy, lower releasing rate, and stronger cytotoxic activity against lung cancer TC-1 cells as compared to free drug. Thus, the Pn-VB could be a promising formulation for delivery of vinblastine to tumor cells with enhanced drug bioavailability and therapeutic efficacy.

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.

Folic acid conjugated nanoliposomes as promising carriers for targeted delivery of bleomycin.

Targeted drug delivery has received considerable attention due to its key role in improving therapeutic efficacy and reducing the side effects of anticancer drugs. Bleomycin (BLM) is an anticancer antibiotic with short half-life, low therapeutic and high side effects that limit its clinical applications. This study aims to evaluate the anticancer potential of folate-targeted liposomal bleomycin (FL-BLM) and its free-folate form (L-BLM) on two different cancer cell lines including human cervix carcinoma HeLa, and human breast carcinoma MCF-7 cells. Furthermore, the effect of FL-BLM in induction of apoptosis and cell cycle arrest was studied by flow cytometry. FL-BLM was prepared by thin film hydration method and folic acid was conjugated to nanoliposomes by post insertion technique. Anticancer activity was evaluated by MTT assay. The cytotoxicity of FL-BLM against HeLa cells was significantly increased compared to L-BLM and conventional BLM. Flow cytometry and annexin-V analysis indicated that FL-BLM effectively induced apoptosis and cell-cycle arrest in HeLa cells especially at G2/M phase. In addition, the uptake of FL-BLM by Hela cells was significantly increased as compared to the MCF-7 cells. Overall, our findings indicated that FL-BLM may be promising formulation for targeted drug delivery to folate receptor-positive tumour cells.

Enhanced antitumor effect of targeted nanoliposomal bleomycin.

Folate receptor (FR)-mediated drug delivery is a promising approach for active targeting of drugs to the FR-positive tumor cells. Bleomycin (BLM) is an antitumor antibiotic with poor therapeutic activity as a result of its limited diffusion into tumor cells. The aim of this study was to investigate whether FR-targeted PEGylated nanoliposomes (FPNL) can effectively deliver BLM to tumor cells and enhance its in vitro and in vivo efficacy. FPNL and PNL (non-targeted) were prepared by thin film hydration method, and their physiochemical properties, cellular uptake, tissue distribution and tumor inhibitory effects were investigated. In Lewis lung cancer (LLC1) cells, FPNL containing BLM showed 2.38-fold and 3.26-fold higher cytotoxicity compared to PNL-BLM and free BLM, respectively. Moreover, the uptake of FPNL by these cells was increased as compared to the PNL. Furthermore, FPNL showed significantly higher tumor distribution of BLM in the LLC1 cells and more tumor inhibition efficacy compared to free BLM and PNL. Both formulations of nanoliposomes had longer plasma half-life than that of free BLM. Therefore, FPNL may be suitable carriers for targeted drug delivery to FR-positive tumor cells.

Preparation, characterization, and in vitro evaluation of bleomycin-containing nanoliposomes.

Bleomycin is an anticancer drug used against various types of cancers. The aim of this study was to prepare a new PEGylated and non-PEGylated nanoliposomal formulation of bleomycin (PEG-nLip-BLM and nLip-BLM) and evaluate their anticancer activity in different tumor cell lines. The liposomes were prepared by thin-film hydration method, and then, bleomycin (BLM) was loaded to the prepared vesicles. The size, zeta potential, entrapment efficiency, loading rate, release profile, and cytotoxicity of liposomal formulations in TC-1, LLC1, and HFLF-PI5 cell lines were investigated. Mean particle size and zeta potential of the PEG-nLip-BLM and nLip-BLM were found to be 99.4 ± 4.6 nm and -34.83 ± 4.7 mV; and 112.2 ± 7.2 nm and -27.5 ± 3.2 mV, respectively, which were stable for at least 2 months. Encapsulation and loading efficiency of BLM for PEG-nLip-BLM and nLip-BLM were obtained about 83.1 ± 4.2% and 14.3 ± 2.5%; and 78.3 ± 8.6% and 11.1 ± 3.3%, respectively. Drug release study showed a slow release pattern without considerable burst effect. The liposomal formulations indicated lower toxicity compared to free drug in case of TC-1 and HFLF-PI5 cells, but their cytotoxicity against LLC1 cells was significantly higher than free drug. The results of this study indicated that PEG-nLip-BLM can be a suitable candidate for drug delivery to solid tumors.

Immunoisolated transplantation of purified langerhans islet cells in testis cortex of male rats for treatment of streptozotocin induced diabetes mellitus.

The objective of this study is to induce experimental diabetes mellitus by streptozotocin in normal adult Wistar rats via comparison of changes in body weight, consumption of food, volume of water, urine and levels of glucose, insulin and C-peptide in serum, between normal and diabetic rats. Intra-venous injection of 60 mg/kg dose of streptozotocin in 250-300 g (75-90 days) adult Wistar rats makes pancreas swell and causes degeneration in Langerhans islet ß-cells and induces experimental diabetes mellitus in 2-4 days. For a microscopic study of degeneration of Langerhans islet ß-cells of diabetic rats, biopsy from pancreas tissue of diabetic and normal rats, staining and comparison between them, were done. In this process, after collagenase digestion of pancreas, islets were isolated, dissociated and identified by dithizone method and then with enzymatic procedure by DNase and trypsin, the islet cells changed into single cells and ß-cells were identified by immune fluorescence method and then assayed by flow-cytometer. Donor tissue in each step of work was prepared from 38 adult male Wistar rats weighted 250-300 g (75-90 days). Transplantation was performed in rats after 2-4 weeks of diabetes induction. In this study, the levels of insulin, C-peptide and glucose in diabetic rats reached to normal range as compared to un-diabetic rats in 20 days after transplantation of islet cells. Transplantation was performed under the cortex of testis as immunoisolated place for islet cells transplantation.

Magnetic resonance contrast media sensing in vivo molecular imaging agents: an overview.

Metabolic imaging is commonly performed by nuclear medicine facilities such as PET or SPECT, etc. The production and biomedical applications of bio-molecular sensing in vivo MRI metabolic contrast agents has recently become of great universal research interest, which follows its great success as a potential cost effective, less radioactive, nuclear medicine alternative. Temperature, redox potential, enzyme activity, free radial/metal ion responsive and/or pH sensitive molecular metabolic MR contrast agents are among the famous instances exemplified, which basically promote MR image contrast enhancement ability to distinguish molecular metabolic/gene expression features. Overall, these MRI contrast agents provide a framework to achieve a greater degree of accuracy from MRI as a low cost, more available facility, non radioactive radiation producing and highly sensitive biomedical tool to propound as a new suggesting opponent for PET nuclear medicine imaging. In the present review, the design, development, examination and future of the above agents will be discussed in detail.

Design, modeling, expression, and chemoselective PEGylation of a new nanosize cysteine analog of erythropoietin.

BACKGROUND: Recombinant human erythropoietin (rhEPO) is considered to be one of the most pivotal pharmaceutical drugs in the market because of its clinical application in the treatment of anemia-associated disorders worldwide. However, like other therapeutic proteins, it does not have suitable pharmacokinetic properties for it to be administrated at least two to three times per week. Chemoselective cysteine PEGylation, employing molecular dynamics and graphics in in silico studies, can be considered to overcome such a problem. METHODS: A special kind of EPO analog was elicited based on a literature review, homology modeling, molecular dynamic simulation, and factors affecting the PEGylation reaction. Then, cDNA of the selected analog was generated by site-directed mutagenesis and subsequently cloned into the expression vector. The construct was transfected to Chinese hamster ovary/dhfr(-) cells, and highly expressed clones were selected via methotrexate amplification. Ion-immobilized affinity and size exclusion (SE) chromatography techniques were used to purify the expressed analog. Thereafter, chemoselective PEGylation was performed and a nanosize PEGylated EPO was obtained through dialysis. The in vitro biologic assay and in vivo pharmacokinetic parameters were studied. Finally, E31C analog Fourier transform infrared, analytical SE-high-performance liquid chromatography, zeta potential, and size before and after PEGylation were characterized. RESULTS: The findings indicate that a novel nanosize EPO31-PEG has a five-fold longer terminal half-life in rats with similar biologic activity compared with unmodified rhEPO in proliferation cell assay. The results also show that EPO31-PEG size and charge versus unmodified protein was increased in a nanospectrum, and this may be one criterion of EPO biologic potency enhancement. DISCUSSION: This kind of novel engineered nanosize PEGylated EPO has remarkable advantages over rhEPO.