Novel N-α-amino acid spacer-conjugated phthalimide-triazine derivatives: synthesis, antimicrobial and molecular docking studies.

To design and develop novel antimicrobial agents, a series of phthalimide-triazine-based derivatives (6a-6e) were synthesized, characterized and evaluated for their potential antibacterial activities. The compounds were prepared through reaction of 6-phenyl-1,3,5-triazine-2,4-diamine with phthalimide moiety containing aliphatic amino acid. Structural analysis of the synthesized compounds was carried out by various characterization techniques such as FT-IR, 1H and 13C-NMR and mass spectroscopy. After the confirmation of the structure, the antibacterial screening of the synthesized compounds was performed against two strains of Gram-positive (Staphylococcus aureus, and Bacillus subtilis) and two strains of Gram-negative (Escherichia coli and Salmonella enteritidis) bacteria. The results of antimicrobial activity showed that compound 6d was the most active against all the tested strains of microorganisms with the MIC value 1.25 µg/µl. The synthesized compounds were docked into the binding sites of E. coli-DNA gyrase B and S. aureus-DNA gyrase complex to explore their theoretically binding mode and possible interactions of these ligands with these two targets. Docking study showed the importance of both hydrogen bonding and hydrophobic interactions as a key interaction with the targets. Based on the obtained results, the hybrid derivatives of triazine and phthalimide could be regarded as efficient candidates for further molecular developments of antimicrobial agents.

Generation of HBsAg DNA aptamer using modified cell-based SELEX strategy.

Aptamers as potential alternatives for antibodies could be employed against hepatitis B surface antigen (HBsAg), the great hallmark and first serological marker in HBV, for further theragnostic applications. Therefore, isolation HBsAg specific aptamer was performed in this study with a modified Cell-SELEX method. HEK293T overexpressing HBsAg and HEK293T as target and control cells respectively, were incubated with single-stranded rounds of DNA library during six SELEX and Counter SELEX rounds. Here, we introduced the new modified Cell-SELEX using deoxyribonuclease I digestion to separate single stranded DNA aptamers against the HBsAg. Characterization and evaluation of selected sequences were performed using flow cytometry analysis. The results led to isolation of 15 different ssDNA clones in six rounds of selection which were categorized to four clusters based on common structural motifs. The evaluation of SELEX progress showed growth in aptamer affinity with increasing in the cycle number. Taken together, the application of modified cell-SELEX demonstrated the isolation of HBsAg-specific ssDNA aptamers with proper affinity. Modified cell-SELEX as an efficient method can shorten the selection procedure and increase the success rate while the benefits of cell-based SELEX will be retained. Selected aptamers could be applied in purification columns, diagnostic kits, and drug delivery system against HBV-related liver cancer.

SA/G hydrogel containing NRF2-engineered HEK-293-derived CM improves wound healing efficacy of WJ-MSCs in a rat model of excision injury.

AIM OF THE STUDY: Skin wounds are a major public health issue due to the lack of real effective remedies. Mesenchymal stem cells (MSCs) are considered as a promising therapeutic strategy for wound injuries; however, low survival rate following transplantation limited their application. In an attempt to introduce a novel potential wound dressing and improve wound healing properties, the current study was conducted. MATERIAL AND METHODS: we prepared conditioned medium (CM) harvested from HEK-293 cells overexpressing nuclear factor erythroid 2-related factor 2 (NRF2), a master regulator of antioxidant genes expression. Then, the CM was loaded in a biodegradable hydrogel. Next, in an animal model of full-thickness excision wound, wharton's jelly derived-mesenchymal stem cells (WJ-MSCs) were transplanted at the margins of the wound followed by application of the hydrogel on injury site. Finally, wound healing characteristics were evaluated by proper methods. RESULTS: Our findings revealed that, the NRF2-CM protected the WJ-MSCs against H2O2-induced toxicity in vitro. Furthermore, in vivo results showed that, SA/G hydrogel containing NRF2-CM significantly (P < 0.01) promoted WJ-MSCs survival, increased angiogenesis, accelerated wound contraction, and promoted wound healing compared to other groups. CONCLUSION: Though further preclinical and clinical studies regarding mechanisms behind the protection and also safety of the strategy are needed, our findings strongly suggest that the prepared wound dressing enhanced the efficacy of therapeutic potential of WJ-MSCs by providing an enriched/antioxidant niche support.

Expression and one step intein-mediated purification of biologically active human G-CSF in Escherichia coli.

Recombinant form of granulocyte colony stimulating factor (G-CSF) was first approved by FDA in 1998 for chemotherapy induced neutropenia. However, despite production of its biosimilars, less expensive production of G-CSF could reduce the overall therapeutic cost. The aim of this study was to evaluate the possibility of producing biologically active recombinant G-CSF via a single step purification procedure mediated by a self-cleavable intein. G-CSF was expressed by E. coli BL21 (DE3) through IPTG induction, followed by its purification using pH optimization on a chitin column. Western blotting, ELISA, size exclusion chromatography, circular diachorism, peptide mapping, and in vitro assays were performed to compare the structural similarity and biological activity of the purified G-CSF with Neupogen™. Protein purification was confirmed by revealing a band of approximately 18.8 kDa on SDS-PAGE. Bioactivity and physicochemical assays based on the US pharmacopeia showed almost identical or acceptable ranges of similarities between recombinant G-CSF and Neopogen™. this study, biologically active soluble recombinant G-CSF was successfully produced with high purity without using chaotropic solvents through a one-step procedure. This shorter and more efficient purification procedure can reduce the cost and time of G-CSF production which makes its industrial production more cost-effective and might be also applicable for production of other biopharmaceuticals.

Implication and role of neutrophil gelatinase-associated lipocalin in cancer: lipocalin-2 as a potential novel emerging comprehensive therapeutic target for a variety of cancer types.

Cancer is a leading cause of mortalities worldwide. Over the past few decades, exploration of molecular mechanisms behind cancer initiation and progression has been of great interest in the viewpoint of both basic and clinical scientists. It is generally believed that identification of key molecules implicated in cancer pathology not only improves our understanding of the disease, but also could result in introduction of novel therapeutic strategies. Neutrophil gelatinase-associated lipocalin (NGAL)/lipocalin-2 (LCN2) is a member of lipocalin superfamily with a variety of functions. Although the main function of LCN2 is still unknown, many studies confirmed its significant role in the initiation, progression, and metastasis of various types of cancer. Furthermore, aberrant expression of LCN2 is also concerned with the chemo- and radio-resistant phenotypes of tumors. Here, we will review the contribution of known functions of LCN2 to the pathophysiology of cancer. We also highlight how the deregulated expression of LCN2 is associated with a variety of fatal types of cancer for which there are no effective therapeutic modalities. The unique and multiple functions of LCN2 and its widespread expression in different types of cancer prompted us to suggest LCN2 could be considered either as a valuable diagnostic and prognostic biomarker or as a potential novel therapeutic target.

Transfer of healthy fibroblast-derived mitochondria to HeLa ρ0 and SAS ρ0 cells recovers the proliferation capabilities of these cancer cells under conventional culture medium, but increase their sensitivity to cisplatin-induced apoptotic death.

Mitochondrial dysfunction is known to contribute to cancer initiation, progression, and chemo-and radio-resistance. However, the precise role of mitochondria in cancer is controversial. Hence, here we tried to further clarify the role of mitochondria in cancer by transferring healthy mitochondria to cancer cells, and also to cells with depleted mitochondrial DNA (ρ0). Healthy mitochondria were isolated from WI-38 cells and were transferred to HeLa, SAS, HeLa ρ0, and SAS ρ0 cells. Then, cell proliferation was verified. In addition, the cells were treated by different concentrations of cisplatin and assessed for apoptosis induction and quantifying the mRNA expression of apoptosis-related genes. Results revealed that incubation of the HeLa, SAS and HeLa ρ0 cells with 5 µg/ml of the isolated mitochondria for 24 h significantly (p < 0.001) increased cell proliferation compared to non-treated controls. Interestingly, the mitochondria transfer rescued the ρ0 cells and made them capable of growing under conventional culture medium. However, the number of apoptotic cells was significantly higher in the HeLa ρ0 cells that received the mitochondria (HeLa-Fibro-Mit) compared to the HeLa ρ0. Furthermore, the expression level of BCL-2 anti-apoptotic gene was down-regulated in both HeLa-Fibro-Mit and SAS-Fibro-Mit cell lines while the expression levels of the BAX, caspase8, caspase9, and AIF pro-apoptotic genes were upregulated. Our findings indicated that although the response of cancer cells to the mitochondria transfer is cancer-type dependent, but the introduction of normal exogenous mitochondria to some cancer cells might be considered as a potential novel therapeutic strategy.

RGD peptide grafted polybutylene adipate-co-terephthalate/gelatin electrospun nanofibers loaded with a matrix metalloproteinase inhibitor drug for alleviating of wounds: an in vitro/in vivo study.

The objective of the present study was the fabrication of a wound dressing membrane based on RGD modified polybutylene adipate-co-terephthalate (PBAT)/gelatin nanofibrous structures loaded with doxycycline (DOX). This type of nanofiber for wound healing has not been reported so far and is quite novel. PBAT and gelatin nanofibers were separately electrospun using double needles electrospinning setup. Electrospinning variables were optimized to obtain bead-free thin nanofibers. The amount of drug loaded and release were measured in different concentrations of DOX and PBAT. MMPs inhibition was studied by polyacrylamide gel-zymography. Then, surface of the nanofibers was modified with RGD peptide, and their antimicrobial effect was investigated on Staphylococcus aureus and Pseudomonas aeruginosa. Effect of developed nanofibrous membranes on L929 fibroblast cells proliferation, adhesion and closure of excised wounds in rat were also studied. PBAT/gelatin nanofibrous structures with average fiber diameter of 75-529 nm were developed successfully. Drug release study revealed that about 65% of DOX was released from the optimized formulation (P17D1.6) after 20 h. The developed DOX loaded membrane inhibited the MMPs activity and showed no cytotoxicity. RGD surface-modified PBAT/gelatin nanofibers significantly improved the wound closure and histopathological results (re-epithelialization, collagen deposition, and angiogenesis) in rats compared to the control groups. Overall, RGD immobilized PBAT/gelatin nanofibrous membrane may have a potential application for wound healing.

Improvement of soluble expression of GM-CSF in the cytoplasm of Escherichia coli using chemical and molecular chaperones.

The most common approaches to improve soluble expression of heterologous proteins are applications of molecular chaperones such as DnaK, DnaJ, GrpE, GroEL and GroES. The aim of present study was to enhance soluble expression of granulocyte-macrophage colony-stimulating factor (GM-CSF) in Escherichia coli by different approaches including modification of cultivation and induction conditions, and thermally, genetically and chemically enhancement of expression of cellular chaperones. To genetically enhance amount of molecular chaperones, co-expression of pET28-GM-CSF and pKJE7 plasmids was performed. The soluble expressed protein was affinity purified and subjected to endotoxin removal. Co-expression with molecular chaperones significantly increased soluble expression of GM-CSF. Addition of chemical chaperones and osmolytes like NaCl (0.5 M), sucrose (0.5 M), sorbitol (0.5 M) and MgCl2 (1 mM) to growing media could improve solubility of GM-CSF. Biological activity of purified GM-CSF was confirmed based on its proliferative effect on HL-60 cell lines. The approach developed in the present study can be applied to improve soluble expression of other recombinant protein proteins.

Enhanced solubility, oral bioavailability and anti-osteoporotic effects of raloxifene HCl in ovariectomized rats by Igepal CO-890 nanomicelles.

The purpose of the present study was to enhance the bioavailability and anti-osteoporotic effects of raloxifene HCl (RH) by increasing its solubility and inhibition of the p-glycoprotein pump using surfactant micelles of Igepal CO-890. The micelles were prepared by the direct method and their critical micellar concentration, drug dissolution rate, saturated solubility, drug loading and surface morphology were defined. The cytotoxicity of Igepal CO-890 and its ability to inhibit the p-glycoprotein pump were studied on Caco-2 cells. The pharmacokinetic parameters were analyzed by oral administration of a single dose of 15 mg/kg in Wistar rats. Anti-osteoporotic effects were studied by measuring the calcium, phosphorous, and uterus weight of rats after one month of oral administration of 6 mg/kg/day of RH in ovariectomized rats. Igepal CO-890 micelles enhanced the RH solubility by about two-fold. The FT-IR and DSC studies indicated no interaction between the drug and the surfactant. XRD spectrum showed an amorphous state of RH in the micelles. The p-glycoprotein pump was inhibited by Igepal CO-890 in Caco-2 cells comparable to verapamil. Micelles increased the uterine weight and decreased the serum calcium and phosphorus significantly compared to the untreated drug. Oral bioavailability of RH increased about four-fold by nanomicelles.

Preparation and Characterization of Spray-Dried Inhalable Powders Containing Polymeric Micelles for Pulmonary Delivery of Paclitaxel in Lung Cancer.

PURPOSE: Local delivery of chemotherapeutic drugs to the lungs offers many advantages for lung cancer treatment compared to conventional systemic chemotherapy. In the present study, novel mixed polymeric micelles based on tocopheryl succinate-polyethylene glycol 1000 and 5000 Da (TPGS1K and TPGS5K) were synthesized and loaded with paclitaxel (PTX). Then, the optimized micelles were incorporated as colloidal drug delivery system into lactose carrier particles using a spray drying technique. METHODS: The mixed micelles of TPGS5K and TPGS1K in different molar ratios (10:0, 7:3, 5:5, 3:7, 0:10) were prepared and physicochemical properties including: particle size, zeta potential, critical micelle concentration (CMC), drug loading, drug release rate, and in vitro cytotoxicitywere investigated in details. The optimized nanoparticles were co-spray dried with lactose carriers to produce the spherical particle morphology of the inhalable particles. RESULTS: Particle sizes and zeta potentials of the different formulations varied in the range of 102 to 196 nm and -9.4 to -13.8 mV, respectively. The lowest CMC values were calculated for 5:5 and 7:3 combinations (16.33 and 17.89 µM, respectively). The drug release rate from different formulations were very slow and only 30% of the drug was released during 72 h. Cytotoxicity assay demonstrated increased cytotoxic activity of PTX-loaded mixed micelles compared to the free drug. The in vitro deposition data indicated that spray drying of PTX-loaded micelles with lactose resulted in the production of inhalable powders with the high fine particle fraction (60%). CONCLUSION: These results demonstrate that this novel PTX-loaded micelles embedded in dry powder inhalation aerosol platform has a great potential to be used in lung cancer treatment.

A novel mixed polymeric micelle for co-delivery of paclitaxel and retinoic acid and overcoming multidrug resistance: synthesis, characterization, cytotoxicity, and pharmacokinetic evaluation.

In the current study, retinoic acid (RA) was conjugated to Pluronic F127 (PF127) through an esterification process. Mixed micelles were formed with tocopheryl polyethylene glycol 1000 (TPGS) for co-delivery of paclitaxel (PTX) and RA to the cancer cells. Mixed micelles of RA-PF127 and TPGS in different weight ratios (10:0, 7:3, 5:5, 3:7, 0:10 w/w) were prepared and physicochemical properties including, particle size, zeta potential, critical micelle concentration (CMC), drug loading content, entrapment efficiency, drug release, cellular uptake and in vitro cytotoxicity, were investigated in details. Furthermore, the pharmacokinetics of PTX-loaded optimized mixed micelles were evaluated in Sprague-Dawley rats and compared with Stragen® (PTX in Cremophor EL®). Particle sizes and zeta potentials of the drug-loaded micelles were in the range of 102.6-223.5 nm and -5.3 to -9.6 mV, respectively. The 7:3 and 5:5 micellar combinations had lower CMC values (0.034-0.042 mg/mL) than 0:10 (0.124 mg/mL). The entrapment efficiencies of 10:0, 7:3, and 5:5 were 53.4 ± 9.3%, 61.3 ± 0.5%, and 78.7 ± 1.66%, respectively. The release rates of PTX from 7:3 and 5:5 mixed micelles were significantly slower than other formulations. Cytotoxicity assay demonstrated increased cytotoxic activity of PTX-loaded mixed micelles compared to free PTX. The Vd and t1/2ß of PTX-loaded RA-PF127/TPGS (7:3) were increased by 2.61- and 1.27-fold, respectively, while the plasma area under the curve (AUC) of the micelles was 2.03-fold lower than those of Stragen®. Therefore, these novel mixed micelles could be effectively used for delivery of PTX and RA to the cancer cells. Moreover, TPGS as part of micelle composition could enhance the therapeutic effect of PTX and reduce side effects.

Poly (butylene adipate-co-butylene terephthalate) nanoparticles prepared by electrospraying technique for docetaxel delivery in ovarian cancer induced mice.

OBJECTIVE: Ovarian cancer is still a major cause of morbidity and mortality. Docetaxel (DTX) is one of the most notable cytotoxic agents for treatment of ovarian cancer. However, its side effects proposed considerable problems to the patients. SIGNIFICANCE: Polymeric nanoparticles (NPs) of poly (butylene adipate-co-butylene terephthalate) (Ecoflex®), a biodegradable and biocompatible polymer, were prepared for the first time by the upgradeable electrospraying technique. METHODS: The formulation and procedure variables were optimized using Design Expert software, and effect of each variable on particle size, particle size distribution, drug entrapment efficiency, and drug release of the NPs were evaluated. Then, in vitro cytotoxicity, cellular uptake, X-ray diffraction pattern, and morphological characteristics of the optimized NPs were evaluated. Finally, in vivo efficacy of the DTX-loaded NPs was evaluated on tumor bearing nude mice. RESULTS: The optimum condition for production of NPs included voltage of 20 kV, 12 cm distance between electrodes, feeding rate of 1 mL/hr, polymer to drug ratio of 3:1, 1 w/v% of Pluronic-F127 and dichloromethane to dimethyl formamide ratio of 2.7:1. Fluorescent microscopy test showed the NPs were successfully up-taken by ovarian cancer cells. In vitro cytotoxicity test confirmed no cytotoxic effect caused by blank NPs, while cell viability of the DTX loaded NPs was significantly lower than the free DTX (p < .05). The NPs significantly enhanced anti-tumor efficacy of the drug in nude mice (p < .05). CONCLUSION: The Ecoflex® NPs could potentially provide a suitable alternative for currently available formulations of DTX.

Biologically Active Heterocyclic Hybrids Based on Quinazolinone, Benzofuran and Imidazolium Moieties: Synthesis, Characterization, Cytotoxic and Antibacterial Evaluation.

Cytotoxic and antimicrobial agents structurally based on quinazolinone, benzofuran and imidazole pharmacophores, have been designed and synthesized. Spectral (IR, 1 H-NMR) and elemental analysis data established the structures of these novel 3-[1-(1-benzofuran-2-yl)-2-(4-oxoquinazolin-3(4H)-yl)ethyl]-1-methyl-1H-imidazol-3-ium chloride hybrid derivatives. All the synthesized compounds were evaluated for in vitro cytotoxicity and antimicrobial activities. Cytotoxic evaluation using MTT assay revealed that compounds 12c, 12g and 12i exhibited significant cytotoxicity with IC50 values 1, 1, and 0.57 µm on this cell line, respectively. Biological activity of the synthesized compounds as antibacterial agent were also evaluated against three Gram-negative (Escherichia coli, Pseudomonas aeruginosa and Salmonella typhi), three Gram-positive (Staphylococcus aureus, Bacillus subtilis and Listeria monocitogenes) and one yeast-like fungi (Candida albicans) strains. All compounds 12a - 12i showed slightly higher activity against Gram-positive bacteria than the Gram-negative one. Among the nine new compounds screened, 3-[1-(5-bromo-1-benzofuran-2-yl)-2-(6-chloro-4-oxoquinazolin-3(4H)-yl)ethyl]-1-methyl-1H-imidazol-3-ium chloride (12e) has pronounced higher antimicrobial activity against all tested strains. These results demonstrated potential importance of molecular hybridization in the development of new lead molecules with major cytotoxicity and antimicrobial activity.

Expression of HCV Alternative Reading Frame Protein (Core+1/F) in Baculovirus Expression System and its Evaluation for Assessment of Specific Anti-core+1 Antibody in Iranian HCV Infected Patients.

BACKGROUND: Hepatitis C virus (HCV) genome contains an overlapping reading frame which results in alternative core protein (ARFP). Baculovirus expression system was used as a powerful eukaryotic vector system to express core+1/F protein for the first time. This recombinant core+1/F protein was used to assess the anti-core+1 antibody in anti-HCV drug resistant and sustained virologic response (SVR) patients. METHODS: The core+1 coding sequence from HCV genotype 1 was designed and synthesized in pUC57 vector. It was subcloned into baculovirus donor plasmid pFastBacTM HTA and transposed into baculovirus shuttle vector (bacmid) to transfect Sf9 cells. Recombinant core+1 protein was purified using Ni-NTA agarose under native condition and verified using SDS-PAGE electrophoresis and Western blotting. An enzyme-linked immunosorbent assay (ELISA) was developed using this purified protein to assess anti-core+1 antibody in 28 anti-HCV drug resistant patients and in 34 patients with sustained virologic response (SVR) in comparison with 31 healthy volunteers used as the negative control. RESULTS: Expression of HCV core+1 protein in Sf9 cells was confirmed by using SDS-PAGE and Western blotting. Antibody titer against core+1 protein in anti-HCV drug resistant patients was significantly higher than that in both the healthy volunteers and SVR patients (p < 0.0001). CONCLUSIONS: HCV core+1 protein was expressed successfully in a baculovirus expression system in high yield in order to develop an ELISA to assess the anti-core+1 antibody. Further studies are needed to reveal the potential application of core+1 protein in anti-HCV treatment prognosis.

Co-culture of bone marrow-derived mesenchymal stem cells overexpressing lipocalin 2 with HK-2 and HEK293 cells protects the kidney cells against cisplatin-induced injury.

Conditioned medium of mesenchymal stem cells (MSCs) is now being used for its cytoprotective effects, especially when the cells are equipped with cytoprotective factors to strengthen them against unfavorable microenvironments. Overexpression of Lcn2 in MSCs mimics in vivo kidney injury. Hence, unraveling how Lcn2-engineered MSCs affect kidney cells has been investigated. Cisplatin treated HK-2 or HEK293 kidney cells were co-cultivated with Lcn2 overexpressing MSCs in upper and lower chambers of transwell plates. Proliferation, apoptosis, and expression of growth factors and cytokines were assessed in the kidney cells. Co-cultivation with the MSCs-Lcn2 not only inhibited cisplatin-induced cytotoxicity in the HK-2 and HEK293 cells, but increased proliferation rate, prevented cisplatin-induced apoptosis, and increased expression of growth factors and the amount of antioxidants in the kidney cells. Thus Lcn2-engineered MSCs can ameliorate and repair injured kidney cells in vitro, which strongly suggests there are beneficial effects of the MSCs-Lcn2 in cell therapy of kidney injury.

Recombinant Production of TP4-LYC1, A New Chimeric Peptide with Targeted Cytotoxicity to HeLa Cells.

Background: Tilapia Piscidin 4 (TP4) showed potential anti-tumor effects against various cancer cells. Lycosine-1 (LYC1), is another Antimicrobial Peptides (AMP) from spider venom with targeted penetration to cancer cells without any adverse effects on normal cells. The aim of this study was to produce a soluble recombinant fusion peptide in order to diminish the cytotoxicity of TP4 against normal cells. Methods: In order to express of TP4-LYC-1, TP4, and LYC1 in fusion to the inteins1/2 of pTWIN-1 vector, induction condition was optimized to earn soluble peptides. Auto-cleavage induction of inteins1/2 was performed based on IMPACT® manual and their effect on cell viability of HeLa and HUVEC cells was surveyed by MTT assay. Results: The best condition for accessing the most soluble peptide in fusion to the inteins was approximately similar for all three peptides (0.1 mM of IPTG, at 22°C). After the induction of self-cleavage of inteins, a band in 3, 3, and 6 kDa was observed on tricine-SDS-PAGE. The IC50 values of TP4-LYC1 and TP4 against HeLa cells were calculated as 0.83, and 2.75 µM, respectively. Conclusion: In the present study, a novel chimeric peptide, TP4-LYC1, was successfully produced. This fusion protein can act as a safe bio-molecule with potent cytotoxic effects against cancer cells, but the penetration ability and determination of cell death mechanism must be performed in order to have more precise view on the apoptosis induction of this recombinant peptide.

Platelet-cloaked alginate-poly (ß-amino ester) a novel platform bioinspired polyelectrolyte nanoparticle for targeted delivery of carboplatin in breast cancer: An in vitro/in vivo study.

Triple-negative breast cancer (TNBC) has poor prognosis. Carboplatin (Crb) is a widely used chemotherapeutic agent, in TNBC but with serious systemic toxicity and poor tumor targeting. Bioinspired drug-loaded platelets (Plt) and Plt-coated nanocarriers evade macrophage phagocytosis by membrane proteins like CD47. The goal of this study was preparation of a novel alginate-poly (ß-amino ester) (PßAE) nanoparticles (NPs) for targeted delivery of Crb to TNBC cells by developing and comparison of two bioinspired carriers of Plt membrane (PltM) coated Crb-loaded alginate-poly (ß-amino ester) nanoparticles (PltM@Crb-PßAE-ALG NPs) and Plt loaded Crb (Plt@Crb). The NPs were prepared by ionic gelation and subsequently were coated by platelet membrane using ultra-sonication method. The loading efficiency, release profile, and in vitro cytotoxicity of both formulations were evaluated on HUVEC and 4 T1 cells. Additionally, the in vivo tumor targeting, therapeutic efficacy, and organ toxicity of the two formulations were assessed in a murine tumor model. Results showed both Plt@Crb and (PltM@Crb-PßAE-ALG NPs) exhibited high drug loading efficiency, sustained release, enhanced cytotoxicity against 4 T1 cells, and decreased cytotoxicity in normal cells (HUVEC) in vitro. In vivo studies revealed that although both formulations considerably improved tumor inhibition compared to free Crb, but the PltM@Crb-PßAE-ALG NPs demonstrated superior cytotoxicity and therapeutic efficacy, thanks to improved Crb's internalization efficiency, enhanced stability, and controlled release properties.

Lipocalin-2 Upregulation in Nasopharyngeal Carcinoma: A Novel Potential Diagnostic Biomarker.

Background: Lipocalin-2 (LCN2) deregulation has been reported in several types of cancer and is implicated in the proliferation, migration, angiogenesis, and progression of tumors. However, its aberrant expression has been rarely studied in nasopharyngeal carcinoma (NPC). In the present study, we investigated the expression of LCN2 in NPC patients. Methods: In this descriptive cross-sectional study, 29 NPC and 20 non-cancerous control paraffin pathology blocks were obtained from the seven-year (2011 to 2018) archive of Razi Laboratory in Rasht, Iran. LCN2 mRNA expression was evaluated through quantitative real-time PCR. In addition, immunohistochemistry was performed to evaluate LCN2 expression at the protein level. The fold change value and total immunostaining score (TIS) were applied for quantitative evaluation. The nonparametric Mann-Whitney U test and Fisher's exact test were used through GraphPad Prism 8.3.0 software. P<0.05 was considered statistically significant. Results: Our results revealed that LCN2 mRNA and protein levels in NPC tissues were significantly higher than control tissues (P=0.028 and P=0.002, respectively). At the protein level, 65.51% (19/29) of NPC patients were categorized as having high LCN2 expression (TIS>3) and 34.47% (10/29) as low expression (TIS≤3). While in the control group, 25% (5/20) of subjects represented a high expression of LCN2 (TIS>3), and 75% (15/20) showed no or weak expression (TIS≤3). No significant correlation was found between the overexpression of LCN2 at the protein level and the demographic features of the patients. Conclusion: Our findings suggest that LCN2 might be considered a potential new diagnostic marker for NPC. However, this warrants further studies.

Human umbilical cord-derived mesenchymal stem cells-harvested mitochondrial transplantation improved motor function in TBI models through rescuing neuronal cells from apoptosis and alleviating astrogliosis and microglia activation.

Each year, traumatic brain injury (TBI) causes a high rate of mortality throughout the world and those who survive have lasting disabilities. Given that the brain is a particularly dynamic organ with a high energy consumption rate, the inefficiency of current TBI treatment options highlights the necessity of repairing damaged brain tissue at the cellular and molecular levels, which according to research is aggravated due to ATP deficiency and reactive oxygen species surplus. Taking into account that mitochondria contribute to generating energy and controlling cellular stress, mitochondrial transplantation as a new treatment approach has lately reduced complications in a number of diseases by supplying healthy and functional mitochondria to the damaged tissue. For this reason, in this study, we used this technique to transplant human umbilical cord-derived mesenchymal stem cells (hUC-MSCs)-derived mitochondria as a suitable source for mitochondrial isolation into rat models of TBI to examine its therapeutic benefit and the results showed that the successful mitochondrial internalisation in the neuronal cells significantly reduced the number of brain cells undergoing apoptosis, alleviated astrogliosis and microglia activation, retained normal brain morphology and cytoarchitecture, and improved sensorimotor functions in a rat model of TBI. These data indicate that human umbilical cord-derived mesenchymal stem cells-isolated mitochondrial transplantation improves motor function in a rat model of TBI via rescuing neuronal cells from apoptosis and alleviating astrogliosis and microglia activation, maybe as a result of restoring the lost mitochondrial content.

Attempts to express the A1-GMCSF immunotoxin in the baculovirus expression vector system.

Immunotoxins are fusion proteins consisting of two elements, a targeting and a toxin moiety, and are designed for specific elimination of tumor cells. Previously we expressed a recombinant fusion protein consisting of the toxic fragment of Shiga toxin (A1) and GMCSF (A1-GMCSF) in Escherichia coli, and evaluated its cytotoxic properties in acute myeloid leukemia and colon carcinoma cell lines. In view of the specific cytotoxic effects of this immunotoxin, further detailed in-vitro and preclinical studies were undertaken. Large amounts of the recombinant protein of high purity and free of unwanted side products, such as lipopolysaccharides (LPS), were required. Since GMCSF is of mammalian origin and it requires proper disulfide bond formation, we intended to use the baculovirus expression vector system (BEVS) for the expression of the recombinant fusion protein. However, despite previous reports on the expression of several other immunotoxins by this system, the A1 derived fusion proteins revealed an inhibitory effect on baculoviral particle formation and even caused cell death in insect cells. This observation was further pursued and confirmed by the use of other baculoviral specific promoters. The salient features of this finding are described below.