Self-assembled peptide nanoparticles for efficient delivery of methotrexate into cancer cells.

The low cellular uptake of Methotrexate (MTX), a commonly used anticancer drug, is a big challenge for efficient cancer therapy. Self-assembled peptide nanoparticles (SAPNs) are one of the major classes of peptide vectors that have gained much attention toward novel drug delivery systems. In the present study, different sequences of cell-penetrating peptides including R2W4R2 and W3R4W3 and their SAPNs (R2W4R2-E12 and W3R4W3-E12) were designed for efficient delivery of MTX into MCF7 breast cancer cells. Based on electron microscopy results, the obtained SAPNs were in nano scale with spherical shape. There was a positive relationship between the free energy of water to octanol transferring and cellular penetration of designed nanostructures. The R2W4R2 possessed proper free energy and ability to form a spherical structure and hydrophobic-hydrophobic interactions, therefore, exhibited more cellular penetration than W3R4W3. The cellular uptake of obtained nanoparticles was examined by flow cytometry and fluorescence microscopy, in which, R2W4R2 and R2W4R2-E12 showed more appropriate penetration into MCF7 cells than W3R4W3 and W3R4W3-E12. The cytotoxicity of MTX-loaded peptides and SAPNs was examined by MTT assay. As a result, at higher concentrations, the R2W4R2 and R2W4R2-E12 showed higher cytotoxic behavior than their counterparts. Despite their enhanced cellular internalization, the cytotoxic behavior of MTX-loaded SAPNs at lower concentrations was relatively less than free MTX, which could be ascribed to the gradual nature of drug detachment from these conjugates. Therefore, R2W4R2 could be considered as an efficient choice to enhance the therapeutic efficiency of MTX in cancer treatments.

Method for assembling and expressing multiple genes in the nucleus of microalgae.

The green alga, Chlamydomonas reinhardtii, is a model organism used in the study of photosynthesis and biotechnological research. Despite its importance, a complete set of genetic tools has yet to be developed. Here, we report the development of a new method for constructing a multi-gene pathway in Saccharomyces cerevisiae and integrating the assembled pathway into the nuclear genome of C. reinhardtii. To demonstrate the use of this method, we assembled and functionally expressed up to three reporter proteins (Ble, AphVIII, and GFP) simultaneously in the nucleus of C. reinhardtii. This new molecular tool should aid efforts to engineer microalgae for biofuel and biopharmaceutical production.

Estimating Hidden Population Size of COVID-19 using Respondent-Driven Sampling Method - A Systematic Review.

INTRODUCTION: Currently, the ongoing COVID-19 pandemic is posing a challenge to health systems worldwide. Unfortunately, the true number of infections is underestimated due to the existence of a vast number of asymptomatic infected individual's proportion. Detecting the actual number of COVID-19-affected patients is critical in order to treat and prevent it. Sampling of such populations, so-called hidden or hard-to-reach populations, is not possible using conventional sampling methods. The objective of this research is to estimate the hidden population size of COVID-19 by using respondent-driven sampling (RDS) methods. METHODS: This study is a systematic review. We have searched online databases of PubMed, Web of Science, Scopus, Embase, and Cochrane to identify English articles published from the beginning of December 2019 to December 2022 using purpose-related keywords. The complete texts of the final chosen articles were thoroughly reviewed, and the significant findings are condensed and presented in the table. RESULTS: Of the 7 included articles, all were conducted to estimate the actual extent of COVID-19 prevalence in their region and provide a mathematical model to estimate the asymptomatic and undetected cases of COVID-19 amid the pandemic. Two studies stated that the prevalence of COVID-19 in their sample population was 2.6% and 2.4% in Sierra Leone and Austria, respectively. In addition, four studies stated that the actual numbers of infected cases in their sample population were significantly higher, ranging from two to 50 times higher than the recorded reports. CONCLUSIONS: In general, our study illustrates the efficacy of RDS in the estimation of undetected asymptomatic cases with high cost-effectiveness due to its relatively trouble-free and low-cost methods of sampling the population. This method would be valuable in probable future epidemics.

Method to assemble and integrate biochemical pathways into the chloroplast genome of Chlamydomonas reinhardtii.

Recombinant protein expression in the chloroplasts of green algae has recently become more routine; however, the heterologous expression of multiple proteins or complete biosynthetic pathways remains a significant challenge. Here, we show that a modified DNA Assembler approach can be used to rapidly assemble multiple-gene biosynthetic pathways in yeast and then integrate these assembled pathways at a site-specific location in the chloroplast genome of the microalgal species Chlamydomonas reinhardtii. As a proof of concept, this method was used to successfully integrate and functionally express up to three reporter proteins (AphA6, AadA, and GFP) in the chloroplast of C. reinhardtii. An analysis of the relative gene expression of the engineered strains showed significant differences in the mRNA expression levels of the reporter genes and thus highlights the importance of proper promoter/untranslated region selection when constructing a target pathway. This new method represents a useful genetic tool in the construction and integration of complex biochemical pathways into the chloroplast genome of microalgae and should aid current efforts to engineer algae for biofuels production and other desirable natural products.

Expression of SCUBE2 and BCL2 Predicts Favorable Response in ERα Positive Breast Cancer.

BACKGROUND: The study aimed at evaluating steroid biomarker genes (ERα, PGR, ERß) and determining the expression level of estrogen-regulated genes (SCUB2 and BCL2) and growth factors receptors (HER2 and IGFR1) in cancer tissue samples obtained from Iranian patients with breast cancer. Moreover, relationships with clinicopathologic aspects of tumor and response to treatment were studied. METHODS: The current study was conducted on 246 breast tissue samples. The expression levels of these genes and their relationships with clinicopathologic aspects and treatment response were evaluated. RESULTS: Based on immunohistochemistry (IHC) results, 12% of the ER negative patients expressed ERα. Comparing the effects of ERα and coexpression of BCL2 and SCUBE2 on the survival of the patients demonstrated remarkably poorer survival in ERα positive, SCUBE2, and BCL2 negative groups in comparison with other patients, which was statistically significant in the log-rank analysis (P = 0.01). Evaluation of the effects of coexpression of HER2 and IGFR1 on patients' survival demonstrated a worse survival rate in patients with positive expression of both receptors, which was insignificant. CONCLUSION: Many studies suggest that PGR alone is not enough for the functional evaluation of ERα. Evaluation of the progesterone receptor expression as well as other genes such as BLC2, SCUBE2, and IGFR1, seems necessary to evaluate functionality.

The impact of Helicobacter pylori infection on gut microbiota-endocrine system axis; modulation of metabolic hormone levels and energy homeostasis.

The gut microbiota is a complex ecosystem that is involved in the development and preservation of the immune system, energy homeostasis and nutritional status of the host. The crosstalk between gut microbiota and the host cells modulates host physiology and metabolism through different mechanisms. Helicobacter pylori (H. pylori) is known to reside in the gastric mucosa, induce inflammation, and alter both gastric and intestinal microbiota resulting in a broad spectrum of diseases, in particular metabolic syndrome-related disorders. Infection with H. pylori have been shown to affect production level and physiological regulation of the gut metabolic hormones such as ghrelin and leptin which are involved in food intake, energy expenditure and body mass. In this study, we reviewed and discussed data from the literature and follow-up investigations that links H. pylori infection to alterations of the gut microbiota and metabolic hormone levels, which can exert broad influences on host metabolism, energy homeostasis, behavior, appetite, growth, reproduction and immunity. Also, we discussed the strong potential of fecal microbiota transplantation (FMT) as an innovative and promising investigational treatment option for homeostasis of metabolic hormone levels to overcome H. pylori-associated metabolic syndrome-related disorders.

Nanoparticulate delivery of irinotecan active metabolite (SN38) in murine colorectal carcinoma through conjugation to poly (2-ethyl 2-oxazoline)-b-poly (L-glutamic acid) double hydrophilic copolymer.

SN-38 is the active metabolite of irinotecan, an FDA-approved chemotherapeutic agent indicated for colorectal carcinoma, which would not be clinically applicable due to its very poorly soluble and hydrolytic degradation properties. To overcome these limitations, it was proposed to conjugate SN38 to residing carboxylic acid residues in poly (2-ethyl 2-oxazoline) block poly (L-glutamic acid), inducing nano-assembly in aqueous medium. Following a series of reactions including poly (2-ethyl oxazoline) macro-initiated ring opening polymerization of N-carboxyanhydride, deprotection of benzyl group and chemical conjugation of SN38 via biodegradable ester linkage, the as-synthesized product was characterized by dynamic light scattering, ζ potential and transmission electron microscopy. The resulting particles presented about 90% loading efficiency with a mean size of 90 nm. Upon incubation with colorectal carcinoma CT26 cell line, higher association of SN-38 fluorescence and significantly more specific cytotoxicity was noticed for the SN38 conjugated particles than free drug. Therapeutic applicability of the as-synthesized product was evaluated in CT26 allograft tumor model in BALB/c mice, showing superior efficiency of the SN38 conjugated particles particularly in tumors with sizes larger than 200 mm3 than parent irinotecan and reduced mortality rate by 2.5 times. Conclusively, the poly (2-ethyl 2-oxazoline) decorated nano-conjugates of poly (L-glutamic acid) and SN38 can be regarded as a novel and potentially efficient drug delivery system for advanced colorectal carcinoma.

Sonochemical synthesis and characterization of a new nano Ce(III) coordination supramolecular compound; highly sensitive direct fluorescent sensor for Cu2.

Micro and nano-structures of a new Ce(III) Coordination supramolecular compound, [Ce (1,5-NDS)1.5(H2O)5]n,1, (1,5-Naphthalenedisulfonic acid), were prepared using hydrothermal and sonochemical approaches, respectively. These new micro and nano structures were characterized by elemental analysis, IR spectra, thermal gravimetric analysis (TGA), scanning electron microscopy (SEM), and powder X-ray diffraction. The X-ray single crystal structure determination of 1 shows that it features a neutral 2D framework based on the [Ce2H20O16S2] clusters as a secondary building unit (SBU) which shows a sql/Shubnikov tetragonal plane net. Moreover by considering the H-bonds, the final structure can be considered as 3D supramolecular network. The influence of ultrasound irradiation time on the morphology and size of the nanostructure 1 was investigated. The results indicated that by increasing the time of ultrasonic radiation, smaller nanostructures form and morphological changes occur. Fluorescent properties of the nanoparticles of 1 were also investigated. Coordination polymer 1 shows high fluorescence intensity and good tendency to copper ion that can be used as an optical sensor for selective and sensitive determination of Cu2+ in aqueous media with detection limit of 3.0µM.

Effectiveness of Solution-Focused Brief Therapy (SFBT) on Depression and Perceived Stress in Patients with Breast Cancer.

BACKGROUND: Attending to psychological status in patients with breast cancer, because of expanded damage and mortality in these patients, is important. The present study investigated the effectiveness of Solution-Focused Brief Therapy (SFBT) on depression and Perceived Stress in Patients with breast cancer. MATERIALS AND METHODS: This research was a semi-experimental with pretest, post-test and follow-up (1 month), which was conducted from November to February, 2016. In this study, 30 patients with breast cancer who attended Imam Hossein Hospital in Tehran city were selected by convenience sampling method and randomly were assigned in 2 experimental (n=15) and control groups (n=15) and Cohen's Perceived Stress Scale and Center Epidemiological Studies Depression Scale were administrated as pretest. Experimental group received 8 sessions of Solution-Focused Brief Therapy SFBT and control group received no intervention. At the end, post-test was administrated on two groups and, repeated measure multi-variable method was used for data analysis by SPSS-21 software. RESULTS: The results of the present study indicated that there were significant differences between the experimental and control groups after administrating SFBT. Thus, the mean of depression and perceived stress of experimental group decreased (P<0.001). CONCLUSION: The result of study that showed SFBT is effective in decreasing depression and perceived stress in patients with breast cancer. Therefore, in order to improve the positive psychological state in these patients psychological screening must be performed and if needed clinical trials and appropriate intervention be considered.

Synthesis and cellular characterization of various nano-assemblies of cell penetrating peptide-epirubicin-polyglutamate conjugates for the enhancement of antitumor activity.

A new class of cell penetrating peptides (CPPs) named peptide amphiphile was designed to improve the intracellular uptake and the antitumor activity of epirubicin (EPR). Various amphiphilic CPPs were synthesized by solid phase peptide synthesis method and were chemically conjugated to EPR. Their corresponding nanoparticles (CPPs-E4 and CPPs-E8) were prepared via non-covalent binding of the peptides and polyanions. Cytotoxicity and anti-proliferative activity were evaluated by MTT assay. Cellular uptake was examined by flow cytometry and fluorescence microscopy. The CPPs exhibited slight cytotoxicity. Binding of polyglutamate to CPPs (CPPs-E4 and CPPs-E8 nanoparticles) decreased their cytotoxicity. CPPs-E8 nanoparticles showed lower cytotoxicity than CPPs-E4 nanoparticles. Cellular uptake of K3W4K3-E8, K2W4K2-E8 and W3K4W3-E8 reached 100% with no difference between each of the mentioned CPPs and its nanoparticles at 50 µM. The anti-proliferative activity of EPR was enhanced following conjugation to peptides and nanoparticles at 25 µM. CPPs-EPR-E4 and CPPs-E8-EPR nanoparticles displayed higher anti-proliferative activity than CPPs-EPR at 25 µM. CPPs-E8-EPR nanoparticles showed higher anti-proliferative activity than CPPs-E4-EPR. K3W4K3-E8-EPR nanoparticles exhibited the highest anti-proliferative activity at 25 µM. The synthesized peptide nanoparticles are proposed as suitable carriers for improving the intracellular delivery of EPR into tumor cells with low cytotoxicity and high antitumor activity.

miR-21, An Oncogenic Target miRNA for Cancer Therapy: Molecular Mechanisms and Recent Advancements in Chemo and Radio-resistance.

In the past decade, miRNAs have been extensively attracted the scientist's attentions as tumor suppressors or oncogenes that have been implicated in tumor progression, metastasis and intrinsic resistance to various cancer therapies. microRNA-21 (miR-21) demonstrates a potential oncogenic function and targets tumor inhibitor proteins in almost all types of cancer. miR-21 overexpression has been studied in terms of cell proliferation, migration, invasion, metastasis, and apoptosis regulation. Inhibition of miRNA expression using antisense technology by various nanovectors of different sizes, shapes and compositions has been evolved progressively to overcome the barriers confronted by miRNA delivery. Application of miR-21 antisense oligonucleotides for treating cancerous cells has become a promising achievement for cancer therapy. Moreover, miR-21 can mediate resistance to radiation and chemotherapy. The expanding role of miR-21 functions in human cancers with an emphasis on its regulatory targets and mechanisms, miR-21 related achievements against cancer promotion have been discussed.

Cellular uptake and anti-tumor activity of gemcitabine conjugated with new amphiphilic cell penetrating peptides.

Gemcitabine (Gem) is used as a single agent or in combination with other anticancer agents to treat many types of solid tumors. However, it has many limitations such as a short plasma half-life, dose-limiting toxicities and drug resistance. Cell-penetrating peptides (CPPs) are short peptides which may deliver a large variety of cargo molecules into the cancerous cells. The current study was designed to evaluate the antiproliferative activity of gemcitabine chemically conjugated to CPPs. The peptides were synthesized using solid phase synthesis procedure. The uptake efficiency of CPPs into cells was examined by flow cytometry and fluorescent microscopy. The synthesized peptides were chemically conjugated to Gem and the in vitro cytotoxicity of conjugates was tested by MTT assay on A594 cell line. According to the obtained results, cellular uptake was increased with increasing the concentration of CPPs. On the other hand the coupling of Gem with peptides containing block sequence of arginine (R5W3R4) and some alternating sequences (i.e. [RW]6 and [RW]3) exhibited improved antitumor activity of the drug. The findings in this study support the advantages of using cell-penetrating peptides for improving intracellular delivery of Gem into tumor as well as its activity.

Chemically crosslinked nanogels of PEGylated poly ethyleneimine (l-histidine substituted) synthesized via metal ion coordinated self-assembly for delivery of methotrexate: Cytocompatibility, cellular delivery and antitumor activity in resistant cells.

Self-assembled nanogels were engineered by forming Zn(2+)-coordinated micellar templates of PEGylated poly ethyleneimine (PEG-g-PEI), chemical crosslinking and subsequent removal of the metal ion. Creation of stable micellar templates is a crucial step for preparing the nanogels. To this aim, imidazole moieties were introduced to the polymer by Fmoc-l-histidine using carbodiimide chemistry. It was hypothesized the nanogels loaded with methotrexate (MTX), a chemotherapeutic agent, circumvent impaired carrier activity in HepG2 cells (MTX-resistant hepatocellular carcinoma). So, the nanogels were post-loaded with MTX and characterized by (1)H-NMR, FTIR, dynamic light scattering-zeta potential, atomic force microscopy, and drug release experiments. Cellular uptake and the antitumor activity of MTX-loaded nanogels were investigated by flow cytometry and MTT assay. Discrete, spherical and uniform nanogels, with sizes about 77-83 nm and a relatively high drug loading (54 ± 4% w/w), showed a low polydispersity and neutral surface charges. The MTX-loaded nanogels, unlike empty nanogels, lowered viability of HepG2 cells; the nanogels demonstrated cell-cycle arrest and apoptosis comparably higher than MTX as free drug that was shown to be through i) cellular uptake of the nanogels by clathrin-mediated transport and ii) endosomolytic activity of the nanogels in HepG2 cells. These findings indicate the potential antitumor application of this preparation, which has to be investigated in-vivo.

Effect of poly-glutamate on uptake efficiency and cytotoxicity of cell penetrating peptides.

Cell penetrating peptides (CPPs) were developed as vehicles for efficient delivery of various molecules. An ideal CPP-peptide should not display any toxicity against cancer cells as well as healthy cells and efficiently enter into the cell. Because of the cationic nature and the intrinsic vector capabilities, these peptides can cause cytotoxicity. One of the possible reasons for toxicity of CPPs is direct translocation and consequently, pore formation on the plasma membrane. In this study it was demonstrated that interaction of poly-glutamate with CPP considerably reduced their cytotoxicity in A549 cell. This strategy could be useful for efficient drug delivery mediated by CPP.

The Relation Between Thermodynamic and Structural Properties and Cellular Uptake of Peptides Containing Tryptophan and Arginine.

PURPOSE: Cell-penetrating peptides (CPPs) are used for delivering drugs and other macromolecular cargo into living cells. In this paper, we investigated the relationship between the structural/physicochemical properties of four new synthetic peptides containing arginine-tryptophan in terms of their cell membrane penetration efficiency. METHODS: The peptides were prepared using solid phase synthesis procedure using FMOC protected amino acids. Fluorescence-activated cell sorting and fluorescence imaging were used to evaluate uptake efficiency. Prediction of the peptide secondary structure and estimation of physicochemical properties was performed using the GOR V method and MPEx 3.2 software (Wimley-White scale, helical wheel projection and total hydrophobic moment). RESULTS: Our data showed that the uptake efficiency of peptides with two tryptophans at the C- and N-terminus were significantly higher (about 4-fold) than that of peptides containing three tryptophans at both ends. The distribution of arginine at both ends also increased the uptake efficiency 2.52- and 7.18-fold, compared with arginine distribution at the middle of peptides. CONCLUSION: According to the obtained results the value of transfer free energies of peptides from the aqueous phase to membrane bilayer could be a good predictor for the cellular uptake efficiency of CPPs.

Synthesis and in vitro evaluation of amphiphilic peptides and their nanostructured conjugates.

PURPOSE: Breast cancer is the second leading cancer type among people of advanced countries. Various methods have been used for cancer treatment such as chemotherapy and radiotherapy. In the present study we have designed and synthesized a new group of drug delivery systems (DDS) containing a new class of Cell Penetrating Peptides (CPPs) named Peptide Amphiphiles (PAs). METHODS: Two PAs and anionic peptides were synthesized using solid phase peptide synthesis (SPPS), namely [KW]4, [KW]5, E4 and E8. Then nano-peptides were synthesized by non-covalent binding between PAs and poly anions as [KW]4-E4, [KW]4-E8, [KW]5-E4 and [KW]5-E8. RESULTS: Flow cytometry studies showed that increased chain length of PAs with a higher ratio between hydrophobicity and net charge results in increased intracellular uptake by MCF7 cells after 2h incubation. Moreover, nano-peptides showed greater intracellular uptake compared to PAs. Anti-proliferative assay revealed that by increasing chain length of PAs, the toxicity effect on MCF7 cells is reduced, however nano-peptides did not show significant toxicity on MCF7 cells even at high concentration levels. CONCLUSION: These data suggest that due to the lack of toxicity effect at high concentration levels and also high cellular uptake, nano-peptides are more suitable carrier compared to PAs for drug delivery.

A multifunctional peptide based on the neutrophil immune defense molecule, CAP37, has antibacterial and wound-healing properties.

CAP37, a protein constitutively expressed in human neutrophils and induced in response to infection in corneal epithelial cells, plays a significant role in host defense against infection. Initially identified through its potent bactericidal activity for Gram-negative bacteria, it is now known that CAP37 regulates numerous host cell functions, including corneal epithelial cell chemotaxis. Our long-term goal is to delineate the domains of CAP37 that define these functions and synthesize bioactive peptides for therapeutic use. We report the novel finding of a multifunctional domain between aa 120 and 146. Peptide analogs 120-146 QR, 120-146 QH, 120-146 WR, and 120-146 WH were synthesized and screened for induction of corneal epithelial cell migration by use of the modified Boyden chamber assay, antibacterial activity, and LPS-binding activity. In vivo activity was demonstrated by use of mouse models of sterile and infected corneal wounds. The identity of the amino acid at position 132 (H vs. R) was important for cell migration and in vivo corneal wound healing. All analogs demonstrated antimicrobial activity. However, analogs containing a W at position 131 showed significantly greater antibacterial activity against the Gram-negative pathogen Pseudomonas aeruginosa. All analogs bound P. aeruginosa LPS. Topical administration of analog 120-146 WH, in addition to accelerating corneal wound healing, effectively cleared a corneal infection as a result of P. aeruginosa. In conclusion, we have identified a multifunctional bioactive peptide, based on CAP37, that induces cell migration, possesses antibacterial and LPS-binding activity, and is effective at healing infected and noninfected corneal wounds in vivo.

Production of xylitol by recombinant microalgae.

Microalgae have received significant attention recently as a potential low-cost host for the production of next-generation biofuels and natural products. Here we show that the chloroplast genome of the eukaryotic green microalga Chlamydomonas reinhardtii can be genetically engineered to produce xylitol through the introduction of a gene encoding a xylose reductase (XR) from the fungi Neurospora crassa. Increased levels of heterologous protein accumulation and xylitol production were achieved by synthesizing the XR gene in the chloroplast codon bias and by driving expression of the codon-optimized XR gene using a 16S/atpA promoter/5'-UTR fusion. These results demonstrate the feasibility of engineering microalgae to produce xylitol, and show the importance of codon optimizing the XR gene and using the 16S/atpA promoter/5'-UTR fusion to express XR in the chloroplast of C. reinhardtii.