Reaction-Induced Self-Assembly of Polymyxin Mitigates Cytotoxicity and Reverses Drug Resistance.

Polymyxins have been regarded as an efficient therapeutic against many life-threatening, multidrug resistant Gram-negative bacterial infections; however, the cytotoxicity and emergence of drug resistance associated with polymyxins have greatly hindered their clinical potential. Herein, the reaction-induced self-assembly (RISA) of polymyxins and natural aldehydes in aqueous solution is presented. The resulting assemblies effectively mask the positively charged nature of polymyxins, reducing their cytotoxicity. Moreover, the representative PMBA4 (composed of polymyxin B (PMB) and (E)-2-heptenal (A4)) assemblies demonstrate enhanced binding to Gram-negative bacterial outer membranes and exhibit multiple antimicrobial mechanisms, including increased membrane permeability, elevated bacterial metabolism, suppression of quorum sensing, reduced ATP synthesis, and potential reduction of bacterial drug resistance. Remarkably, PMBA4 assemblies reverse drug resistance in clinically isolated drug-resistant strains of Gram-negative bacteria, demonstrating exceptional efficacy in preventing and eradicating bacterial biofilms. PMBA4 assemblies efficiently eradicate Gram-negative bacterial biofilm infections in vivo and alleviate inflammatory response. This RISA strategy offers a practical and clinically applicable approach to minimize side effects, reverse drug resistance, and prevent the emergence of resistance associated with free polymyxins.

Nucleic acid-responsive smart systems for controlled cargo delivery.

Stimulus-responsive delivery systems allow controlled, highly regulated, and efficient delivery of various cargos while minimizing side effects. Owing to the unique properties of nucleic acids, including the ability to adopt complex structures by base pairing, their easy synthesis, high specificity, shape memory, and configurability, they have been employed in autonomous molecular motors, logic circuits, reconfigurable nanoplatforms, and catalytic amplifiers. Moreover, the development of nucleic acid (NA)-responsive intelligent delivery vehicles is a rapidly growing field. These vehicles have attracted much attention in recent years due to their programmable, controllable, and reversible properties. In this work, we review several types of NA-responsive controlled delivery vehicles based on locks and keys, including DNA/RNA-responsive, aptamer-responsive, and CRISPR-responsive, and summarize their advantages and limitations.

The effect of aqueous extract of Iranian oak (Quercus brantii) on antioxidant capacity and oxidative stress in beta-thalassemia patients: Randomized controlled trial.

BACKGROUND AND AIM: Frequent administration of blood in ß-thalassemia patients can lead to over-loaded iron, a reduction in the levels of antioxidant activities in the body, and oxidative stress. This study was done to evaluate the antioxidant and protective effect of aqueous oak (Quercus brantii) extract supplementation on these patients. METHODS: This clinical trial was performed on 60 major ß thalassemia patients dividing them into intervention and control groups. In addition to taking desferrioxamine (DFO), the control and intervention groups received respectively placebo capsule supplementation and aqueous Quercus extract capsules (300 mg/day) for 3 months. Serum lipid profiles (LDL-c, HDL-c, triglyceride), Total Antioxidant Capacity (TAC), Glucose, Uric acid, urea nitrogen (BUN), Creatinine, LFT (Liver Function Tests) such as SGOT, SGPT, ALP, Total bilirubin, Direct bilirubin, ferritin, MDA and carbonyl protein (CO) levels were measured before and after the period. In addition, the activity of catalase (CAT), and superoxide dismutase (SOD) was measured in the red blood cell. Furthermore, antioxidant activity and total phenolic content of aqueous Quercus were recorded to standardize capsule formulation. RESULTS: Mean serum MDA, and protein CO, significantly decreased in the intervention group with ß-TM after 3 months of treatment with Quercus extract. In addition, the superoxide dismutase (SOD) enzyme and Total antioxidant capacity (TAC) significantly increased in comparison with the control group. Changes in serum creatinine, BUN, and alanine transferase were not significant. In the study, Quercus extract capsules contain 48/56 mg gallic acid/g (dry extract) total phenol, 58/6 mg/g (dry extract), and flavonoids of 63/8 µg/ml antioxidant power which by GC/MS analysis has been measured. At the end of the study, serum MDA decreased from 48.65 ± 8.74 to 43.94 ± 10.39 µ mol/l after administration of oak extract and protein CO dropped from 2.44 ± 0.38 to 1.2 ± 0.31 nmol DNPH/mg protein after administration of the oak extract. At the end of the study serum, TAC increased in patients interventional group from 907 ± 319 to 977 ± 327 µmol FeSO4/l compared to the control group 916 ± 275 to 905.233 ± 233 µmol FeSO4/l with placebo, and SOD increased from 1577 ± 325 to 2079 ± 554 U/l (compared to 1687 ± 323 U/l with placebo). The treatment effect of Quercus was measured using a mixed-effects model of variance analysis for changes in MDA, protein CO, TAC, and SOD, with significant effects being demonstrated for each laboratory parameter (P = 0.15, P = 0.001, P = 0.02, and P < 0.003, respectively). CONCLUSIONS: Aqueous Quercus extract, due to its high antioxidant potential, reduced MDA, serum carbonyl protein, and increased superoxide dismutase activity effectively decreased serum OS and enhanced serum antioxidant capacity in patients with ß-thalassemia major. oak given as an adjuvant therapy to standard iron chelators may provide an improvement in the OS measurements obtained in these patients. REGISTRATION INFORMATION: This study was submitted, evaluated, and approved by the Iranian Registry of Clinical Trials (IRCT: http://www.irct.ir; IRCT2015101411819N4), which was established for national medical schools in Iran.

Grape seed extract supplementation in non-alcoholic fatty liver disease.

Background: Despite rising non-alcoholic fatty liver disease (NAFLD) prevalence and its impact on liver health, there's a lack of studies on grape seed extract's (GSE) effect on oxidative stress and quality of life (QoL) in NAFLD patients. This study aims to fill this gap by the potential benefits of GSE in reducing oxidative stress and improving QoL. Methods: In this randomized clinical trial study, fifty patients with NAFLD were randomly assigned to receive either 2 tablets of GSE containing 250 mg of proanthocyanidins or placebo (25 participants in each group) for two months. QoL was evaluated using the SF-36 questionnaire, and oxidative stress variables (TAC, MDA, SOD, GPx, CAT, and IL-6) were measured at the beginning and end of the study. Results: Compared with the control group, the group supplemented with GSE experienced greater reductions in IL-6 and MDA (3.14±1.43 pg/ml vs. 2.80±0.31 pg/ml; 4.16±2.09 µM vs. 4.59±1.19 µM, p for all <0.05), as well as greater increases in TAC, SOD, and GPx levels (0.18±0.08 mM vs. -0.03±0.09 mM; 10.5±6.69 U/ml vs. 8.93±1.63 U/ml; 14.7±13.4 U/ml vs. 8.24±3.03 U/ml, p for all <0.05). Furthermore, the QoL questionnaire showed that physical limitations, general health, and total physical health were significantly improved in the GSE group compared with the placebo (17.0±42.0 vs. -12.0±37.5; 3.80±14.8 vs. -3.92±9.55; 5.08 5.26 vs. -7.01±13.7, p for all <0.05). Conclusions: GSE can be effective in improving oxidative stress and QoL in patients with NAFLD. More studies are needed to confirm the results of this study.

Applications of Innovation Technologies for Personalized Cancer Medicine: Stem Cells and Gene-Editing Tools.

Personalized medicine is a new approach toward safer and even cheaper treatments with minimal side effects and toxicity. Planning a therapy based on individual properties causes an effective result in a patient's treatment, especially in a complex disease such as cancer. The benefits of personalized medicine include not only early diagnosis with high accuracy but also a more appropriate and effective therapeutic approach based on the unique clinical, genetic, and epigenetic features and biomarker profiles of a specific patient's disease. In order to achieve personalized cancer therapy, understanding cancer biology plays an important role. One of the crucial applications of personalized medicine that has gained consideration more recently due to its capability in developing disease therapy is related to the field of stem cells. We review various applications of pluripotent, somatic, and cancer stem cells in personalized medicine, including targeted cancer therapy, cancer modeling, diagnostics, and drug screening. CRISPR-Cas gene-editing technology is then discussed as a state-of-the-art biotechnological advance with substantial impacts on medical and therapeutic applications. As part of this section, the role of CRISPR-Cas genome editing in recent cancer studies is reviewed as a further example of personalized medicine application.

Effects of virtual interventions based on the theory of planned behavior to improve obesity-preventive lifestyle among girls, during COVID-19 pandemic.

BACKGROUND: Adolescence is a critical period for the spread of obesity and overweight. This research was conducted with the aim of determining the effect of an educational intervention based on the theory of planned behavior on promoting obesity-related behaviors in overweight female students in Gachsaran. METHODS: this quasi-experimental study was conducted on 90 female students of the first secondary school in the form of two intervention and control groups. Information related to nutritional status and the structures of the theory of planned behavior were collected using a researcher-made questionnaire whose validity and reliability have been confirmed. The educational intervention was carried out during five virtual training sessions. The data obtained three months after the intervention were analyzed using SPSS statistical software, version 20, using independent t-tests, paired t-tests, and equivalent non-parametric tests. RESULTS: The present study showed that the scores of the constructs of awareness, perceived behavior control, subjective norms, intention, and nutritional behaviors were significantly improved after the intervention (p < 0.001). The results of the Mann-Whitney test showed that the two intervention and control groups did not have a significant difference in terms of the average overall physical activity score after the intervention (p = 0.078). CONCLUSION: The results of the present study showed that training based on the theory of planned behavior in the conditions of COVID-19 disease and in a virtual form had an effect on nutritional behavior but could not increase physical activity behavior in adolescents with weight loss.

Quantitative measurement of CA 15-3 cancer biomarker using an electrochemical aptasensor based on the electrodeposition of Au thin film on cauliflower-like rGO-MoS2 nanocomposite.

The present research was conducted to design and construct an electrochemical aptasensor for evaluating carbohydrate antigen 15-3 (CA15-3) as a biomarker for breast cancer. The aptasensor has been fabricated by a gold thin film (AuTF) electrodeposited on a cauliflower-like reduced graphene oxide-molybdenum sulfide nanocomposite (rGO-MoS2). The modified electrode's surface was used to immobilize the thiolated aptamer, which was subsequently treated with CA 15-3 antigen. The aptasensor fabrication process was assessed using electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV). This research also applied EIS to the quantitative measurement of CA 15-3 antigen by the proposed aptasensor. The interfacial charge transfer resistance (Rct) alteration before and after incubation of CA 15-3 by the immobilized aptamer was considered a signal for the quantitative measurement of CA 15-3. A linear concentration ranging from 5.0 to 200.0 U mL-1 with a detection limit of 3.0 × 10-1 U mL-1 was obtained for CA 15-3 using the EIS method. This designed aptasensor indicates satisfactory repeatability and stability, good selectivity, and high sensitivity. Moreover, clinical samples were assayed by the prepared aptasensor and compared with the ELISA method, yielding acceptable results. The recovery and relative standard deviation (RSD) of CA 15-3 in human serum samples were in the range 95.0 to 107.0% and 3.5 to 7.5%, respectively.

Mechanism and antibacterial synergies of poly(Dabco-BBAC) nanoparticles against multi-drug resistant Pseudomonas aeruginosa isolates from human burns.

Multi-drug resistant bacteria are a major problem in the treatment of infectious diseases, such as pneumonia, meningitis, or even coronavirus disease 2019 (COVID-19). Cationic nanopolymers are a new type of antimicrobial agent with high efficiency. We synthesized and characterized cationic polymer based on 1,4-diazabicyclo [2.2.2] octane (DABCO) and Bis (bromoacetyl)cystamine (BBAC), named poly (DABCO-BBAC) nanoparticles(NPs), and produced 150 nm diameter NPs. The antibacterial activity of poly (DABCO-BBAC) against eight multi drug resistant (MDR) Pseudomonas aeruginosa isolates from human burns, its possible synergistic effect with gentamicin, and the mechanism of action were examined. Poly(DABCO-BBAC) could effectively inhibit and kill bacterial strains at a very low concentration calculated by minimum inhibitory concentration (MIC) assay. Nevertheless, its synergism index with gentamicin showed an indifferent effect. Moreover, transmission electron microscopy and lipid peroxidation assays showed that poly (DABCO-BBAC) distorted and damaged the bacterial cell wall. These results suggest that the poly (DABCO-BBAC) could be an effective antibacterial agent for MDR clinical pathogens.

Two-Tailed Dynamic Covalent Amphiphile Combats Bacterial Biofilms.

Drug combination provides an efficient pathway to combat drug resistance in bacteria and bacterial biofilms. However, the facile methodology to construct the drug combinations and their applications in nanocomposites is still lacking. Here the two-tailed antimicrobial amphiphiles (T2 A2 ) composed of nitric oxide (NO)-donor (diethylenetriamine NONOate, DN) and various natural aldehydes are reported. T2 A2 self-assemble into nanoparticles due to their amphiphilic nature, with remarkably low critical aggregation concentration. The representative cinnamaldehyde (Cin)-derived T2 A2 (Cin-T2 A2 ) assemblies demonstrate excellent bactericidal efficacy, notably higher than free Cin and free DN. Cin-T2 A2 assemblies kill multidrug-resistant staphylococci and eradicate their biofilms via multiple mechanisms, as proved by mechanism studies, molecular dynamics simulations, proteomics, and metabolomics. Furthermore, Cin-T2 A2 assemblies rapidly eradicate bacteria and alleviate inflammation in the subsequent murine infection models. Together, the Cin-T2 A2 assemblies may provide an efficient, non-antibiotic alternative in combating the ever-increasing threat of drug-resistant bacteria and their biofilms.

Co-delivery of curcumin and Bcl-2 siRNA to enhance therapeutic effect against breast cancer cells using PEI-functionalized PLGA nanoparticles.

PURPOSE: Breast cancer is the second major cause of death worldwide among women. Co-delivery of anticancer drugs and nucleic acids targeting the apoptosis pathway could be a promising new approach. METHODS: In the present study, we synthesized a novel nanostructure for the co-delivery of curcumin and siRNA to breast cancer cells. Curcumin-loaded polylactic-co-glycolic acid (PLGA) was synthesized using an O/W emulsion-solvent diffusion method. It was coated with polyethylenimine (PEI) and subsequently complexed with Bcl-2 siRNA. Also, nanoparticles were characterized such as zeta potential, size distribution and drug encapsulation. Finally, the cytotoxicity of NP and Bcl-2 expression was evaluated. RESULTS: The curcumin-loaded PLGA nanoparticles were 70 nm in size, and increased to 84 nm after incorporation of PEI plus Bcl-2 siRNA. The encapsulation ratio of the drug in our nanoparticle was 78%. Cellular internalization of PLGA-CUR-PEI/Bcl-2 siRNA NPs was confirmed by fluorescence microscopy with the broadcasting of the fluorescence in the cytoplasm and into the nucleus. The results of the cell viability assay revealed that curcumin-loaded PLGA coated with PEI and Bcl-2 siRNA exhibited the highest cytotoxicity against the T47D cell line, while the siRNA decreased the Bcl-2 expression by 90.7%. CONCLUSION: The co-delivery of curcumin plus Bcl-2 siRNA with the PLGA-PEI nanosystem could be a synergistic drug carrier against breast cancer cells.

Saponin and fluorine-modified polycation as a versatile gene delivery system.

Despite the development of many novel carriers for the delivery of various types of genetic material, the lack of a delivery system with high efficiency and low cytotoxicity is a major bottleneck. Herein, low molecular weight polyethylenimine (PEI1.8k) was functionalized with saponin residues using phenylboronic acid (PBA) as an ATP-responsive cross-linker, and a fluorinated side chain to construct PEI-PBA-SAP-F polycation as a highly efficient delivery vector. This vehicle could transfect small plasmid DNA (∼3 kb) with outstanding efficiency into various cells, including HEK 293T, NIH3T3, A549, PC12, MCF7 and HT-29, as well as robust transfection of a large plasmid (∼9 kb) into HEK 293T cells. The carrier indicated good transfection efficacy even at high concentration of serum and low doses of plasmid. The use of green fluorescent protein (GFP) knock-out analysis demonstrated transfection of different types of CRISPR/Cas9 complexes (Cas9/sgRNA ribonucleoproteins RNP, plasmid encoding Cas9 plus sgRNA targeting GFP, Cas9 expression plasmid plusin vitro-prepared sgRNA). In summary, we report an effective PEI-PBA-SAP-F gene carrier with the appropriate lipophilic/cationic balance for biomedical applications.

Poly-L-lysine/hyaluronan nanocarriers as a novel nanosystem for gene delivery.

The present research comes up with a novel DNA-loaded poly-L-lysine (PLL)/hyaluronan (HA) nanocarrier (DNA-loaded PLL/HA NCs) for gene delivery applications, as a promising candidate for gene delivery into diverse cells. A straightforward approach was employed to prepare such a nanosystem through masking DNA-loaded PLL molecules by HA. Fourier-transform infrared (FTIR) spectroscopy, dynamic light scattering (DLS), field emission-scanning electron microscopy (FE-SEM) and transmission electron microscopy (TEM) were used to analyse the interaction of the molecules as well as the physicochemical properties of the NCs. The NCs showed a negative charge of -24 ± 3 mV, with an average size of 138 ± 6 nm, in an ellipsoid-shape with smooth surfaces. The DNA loading efficiency (LE) measured by DNA absorbance was around 95 %. The MTT assay showed that the developed NCs are non-toxic to the cells. Furthermore, the uptake of the DNA-loaded PLL/HA NCs by the human embryonic kidney (HEK)-293T cells was evaluated by a flow cytometry method, and demonstrated high potential cellular uptake over 90% for transferring the gene to HEK-293T cells at the optimised conditions. Therefore, the DNA-loaded PLL/HA NCs are the potent strategy for developing nanosystems for gene delivery applications.

In vitro elimination of EL4 cancer cells from spermatogonia stem cells by miRNA-143- and 206-loaded folic acid-conjugated PLGA nanoparticles.

Aim: MiRNA's-143 and -206 are powerful apoptotic regulators in cancer cells. This study aimed to use miRNA-143- and 206-loaded poly(lactic-co-glycolic) acid (PLGA) nanoparticles conjugated with folic acid to induce apoptosis in the EL4 cancer cells. Materials & methods: The therapy was conducted in six groups: treatment with both miRNAs simultaneously (mixed miRNAs), miRNA-206 treatment, miRNA-143 treatment, blank PLGA, blank polyethylenimine (PEI) and complex PEI-miRNAs. Results: In terms of viability, in mixed miRNAs no synergistic effect was observed on EL4 cell elimination. However, in the single miRNA-206 group, a stronger apoptotic effect was observed than the mixed miRNAs group and single miRNA-143 group alone. Conclusion: MiRNAs' apoptotic induction effects in cancer cells were found to be remarkable.

Histidine-enhanced gene delivery systems: The state of the art.

Gene therapy has emerged as a promising tool for treating different intractable diseases, particularly cancer or even viral diseases such as COVID-19 (coronavirus disease 2019). In this context, various non-viral gene carriers are being explored to transfer DNA or RNA sequences into target cells. Here, we review the applications of the naturally occurring amino acid histidine in the delivery of nucleic acids into cells. The biocompatibility of histidine-enhanced gene delivery systems has encouraged their wider use in gene therapy. Histidine-based gene carriers can involve the modification of peptides, dendrimers, lipids or nanocomposites. Several linear polymers, such as polyethylenimine, poly-l-lysine (synthetic) or dextran and chitosan (natural), have been conjugated with histidine residues to form complexes with nucleic acids for intracellular delivery. The challenges, opportunities and future research trends of histidine-based gene deliveries are investigated.

Smart Strategies for Precise Delivery of CRISPR/Cas9 in Genome Editing.

The emergence of CRISPR/Cas technology has enabled scientists to precisely edit genomic DNA sequences. This approach can be used to modulate gene expression for the treatment of genetic disorders and incurable diseases such as cancer. This potent genome-editing tool is based on a single guide RNA (sgRNA) strand that recognizes the targeted DNA, plus a Cas nuclease protein for binding and processing the target. CRISPR/Cas has great potential for editing many genes in different types of cells and organisms both in vitro and in vivo. Despite these remarkable advances, the risk of off-target effects has hindered the translation of CRISPR/Cas technology into clinical applications. To overcome this hurdle, researchers have devised gene regulatory systems that can be controlled in a spatiotemporal manner, by designing special sgRNA, Cas, and CRISPR/Cas delivery vehicles that are responsive to different stimuli, such as temperature, light, magnetic fields, ultrasound (US), pH, redox, and enzymatic activity. These systems can even respond to dual or multiple stimuli simultaneously, thereby providing superior spatial and temporal control over CRISPR/Cas gene editing. Herein, we summarize the latest advances on smart sgRNA, Cas, and CRISPR/Cas nanocarriers, categorized according to their stimulus type (physical, chemical, or biological).

Could artificial intelligence revolutionize the development of nanovectors for gene therapy and mRNA vaccines?

Gene therapy enables the introduction of nucleic acids like DNA and RNA into host cells, and is expected to revolutionize the treatment of a wide range of diseases. This growth has been further accelerated by the discovery of CRISPR/Cas technology, which allows accurate genomic editing in a broad range of cells and organisms in vitro and in vivo. Despite many advances in gene delivery and the development of various viral and non-viral gene delivery vectors, the lack of highly efficient non-viral systems with low cellular toxicity remains a challenge. The application of cutting-edge technologies such as artificial intelligence (AI) has great potential to find new paradigms to solve this issue. Herein, we review AI and its major subfields including machine learning (ML), neural networks (NNs), expert systems, deep learning (DL), computer vision and robotics. We discuss the potential of AI-based models and algorithms in the design of targeted gene delivery vehicles capable of crossing extracellular and intracellular barriers by viral mimicry strategies. We finally discuss the role of AI in improving the function of CRISPR/Cas systems, developing novel nanobots, and mRNA vaccine carriers.

Immunohistochemical Expression Pattern of MLH1, MSH2, MSH6, and PMS2 in Tumor Specimen of Iranian Gastric Carcinoma Patients.

BACKGROUND: Gastric cancer is the third leading cause of cancer-related death. Determining molecular and histopathologic tumor features, which may contribute to the development or progression of gastric cancer, can improve the prognosis. Expression patterns of DNA repair proteins such as MLH1, MSH2, MSH6, and PMS2 that are associated with microsatellite instability (MSI) are some of the markers that are useful in predicting the prognosis of gastric cancer. PURPOSE: The purpose was to determine the immunohistochemical expression pattern of MLH1, MSH2, MSH6, and PMS2 in tumor specimens of Iranian gastric carcinoma patients. METHODS: In this prospective cohort, 186 consecutive patients with gastric cancer, attending Taleghani Hospital, were enrolled. The immunohistochemical expression patterns of MLH1, MSH2, MSH6, and PMS2 in tumor specimens among them were determined. RESULTS: The results of this study demonstrated that 91.4% of our gastric cancer patients were negative for MSI, and 8.6% of them were MSI positive. The positive MSI was seen in 5.9% and 15.7% of male and female subjects, respectively, with a significant difference (P = 0.043). The other variables were not related to MSI results (P > 0.05). CONCLUSION: According to the obtained results, the expression of MLH1, MSH2, MSH6, and PMS2 in tumor specimens is positive in 8.6% of the total Iranian gastric cancer sample size, which is mainly positive in female subjects. However, it is not related to the location and stage of the tumor.

Antibacterial, antibiofilm, anti-inflammatory, and wound healing effects of nanoscale multifunctional cationic alternating copolymers.

Infectious diseases caused by new or unknown bacteria and viruses, such as anthrax, cholera, tuberculosis and even COVID-19, are a major threat to humanity. Thus, the development of new synthetic compounds with efficient antimicrobial activity is a necessity. Herein, rationally designed novel multifunctional cationic alternating copolymers were directly synthesized through a step-growth polymerization reaction using a bivalent electrophilic cross-linker containing disulfide bonds and a diamine heterocyclic ring. To optimize the activity of these alternating copolymers, several different diamines and cross-linkers were explored to find the highest antibacterial effects. The synthesized nanopolymers not only displayed good to excellent antibacterial activity as judged by minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) against Staphylococcus aureus, Enterococcus faecalis, Pseudomonas aeruginosa, and Escherichia coli, but also reduced the number of biofilm cells even at low concentrations, without killing mammalian cells. Furthermore, in vivo experiments using infected burn wounds in mice demonstrated good antibacterial activity and stimulated wound healing, without causing systemic inflammation. These findings suggest that the multifunctional cationic nanopolymers have potential as a novel antibacterial agent for eradication of multidrug resistant bacterial infections.

Smart arginine-equipped polycationic nanoparticles for p/CRISPR delivery into cells.

An efficient and safe delivery system for the transfection of CRISPR plasmid (p/CRISPR) into target cells can open new avenues for the treatment of various diseases. Herein, we design a novel nonvehicle by integrating an arginine-disulfide linker with low-molecular-weight PEI (PEI1.8k) for the delivery of p/CRISPR. These PEI1.8k-Arg nanoparticles facilitate the plasmid release and improve both membrane permeability and nuclear localization, thereby exhibiting higher transfection efficiency compared to native PEI1.8kin the delivery of nanocomplexes composed of PEI1.8k-Arg and p/CRISPR into conventional cells (HEK 293T). This nanovehicle is also able to transfect p/CRISPR in a wide variety of cells, including hard-to-transfect primary cells (HUVECs), cancer cells (HeLa), and neuronal cells (PC-12) with nearly 5-10 times higher efficiency compared to the polymeric gold standard transfection agent. Furthermore, the PEI1.8k-Arg nanoparticles can edit the GFP gene in the HEK 293T-GFP reporter cell line by delivering all possible forms of CRISPR/Cas9 system (e.g. plasmid encoding Cas9 and sgRNA targeting GFP, and Cas9/sgRNA ribonucleoproteins (RNPs) as well as Cas9 expression plasmid andin vitro-prepared sgRNA) into HEK 293T-GFP cells. The successful delivery of p/CRISPR into local brain tissue is also another remarkable capability of these nanoparticles. In view of all the exceptional benefits of this safe nanocarrier, it is expected to break new ground in the field of gene editing, particularly for therapeutic purposes.

The Role of MicroRNA 143 and MicroRNA 206 in The Regulation of Apoptosis in Mouse Lukemia Cancer Cells and Spermatogonial Cells.

OBJECTIVE: In cancer treatments, smart gene delivery via nanoparticles (NPs) can be targeted for cancer cells, while concurrently minimizing damage to healthy cells. This study assessed the efficiency of poly lactic-co-glycolic acid (PLGA)-miR 143/206 transfection on apoptosis in mouse leukemia cancer cells (El4) and spermatogonial stem cells (SSCs). MATERIALS AND METHODS: In this experimental study, neonatal mouse spermatogonia cells and EL4 cancer cell lines were used. MicroRNA-PLGA NPs were prepared, characterized, and targeted with folate. Several doses were evaluated to obtain a suitable miR dose that can induce appropriate apoptosis in EL4 cells, while not harming SSCs. Cells were treated separately at 3 doses of each miR (for miR 143, doses of 25, 50 and 75 nmol and for miR 206, doses of 50, 100 and 150 nmol). The experiments were performed at 24, 48 and 72 hours. Viability and apoptosis were investigated by MTT and Annexin Kits. RESULTS: Based on MTT assay results, the optimal dose of miR 143 was 75 nmol (59.87 ± 2.85 % SSC and 35.3 ± 0.78 % EL4) (P≤0.05), and for miR 206, the optimal dose was 150 nmol (54.82 ± 6.7 % SSC and 33.92 ± 3.01% EL4) (P≤0.05). The optimal time was 48 hours. At these doses, the survival rate of the EL4 cells was below the half maximal inhibitory concentration (IC50) and SSC survival was above 50%. Annexin V staining also confirmed the selected doses (for miR 143 total apoptosis was 6.62% ± 1.8 SSC and 37.4% ± 4.2 EL4 (P≤0.05), and miR 206 was (10.98% ± 1.5 SSC and 36.4% ± 3.7 EL4, P≤0.05). CONCLUSION: Using intelligent transfection by NPs, we were able to induce apoptosis on EL4 cells and maintain acceptable SSC survival rates.