Modulating mycobacterial envelope integrity for antibiotic synergy with benzothiazoles.

Developing effective tuberculosis drugs is hindered by mycobacteria's intrinsic antibiotic resistance because of their impermeable cell envelope. Using benzothiazole compounds, we aimed to increase mycobacterial cell envelope permeability and weaken the defenses of Mycobacterium marinum, serving as a model for Mycobacterium tuberculosis Initial hit, BT-08, significantly boosted ethidium bromide uptake, indicating enhanced membrane permeability. It also demonstrated efficacy in the M. marinum-zebrafish embryo infection model and M. tuberculosis-infected macrophages. Notably, BT-08 synergized with established antibiotics, including vancomycin and rifampicin. Subsequent medicinal chemistry optimization led to BT-37, a non-toxic and more potent derivative, also enhancing ethidium bromide uptake and maintaining synergy with rifampicin in infected zebrafish embryos. Mutants of M. marinum resistant to BT-37 revealed that MMAR_0407 (Rv0164) is the molecular target and that this target plays a role in the observed synergy and permeability. This study introduces novel compounds targeting a new mycobacterial vulnerability and highlights their cooperative and synergistic interactions with existing antibiotics.

Establishment of a Pilot Newborn Screening Program for Spinal Muscular Atrophy in Saint Petersburg.

Spinal muscular atrophy 5q (SMA) is one of the most common neuromuscular inherited diseases and is the most common genetic cause of infant mortality. SMA is associated with homozygous deletion of exon 7 in the SMN1 gene. Recently developed drugs can improve the motor functions of infants with SMA when they are treated in the pre-symptomatic stage. With aim of providing an early diagnosis, newborn screening (NBS) for SMA using a real-time PCR assay with dried blood spots (DBS) was performed from January 2022 through November 2022 in Saint Petersburg, which is a representative Russian megapolis. Here, 36,140 newborns were screened by the GenomeX real-time PCR-based screening test, and three genotypes were identified: homozygous deletion carriers (4 newborns), heterozygous carriers (772 newborns), and wild-type individuals (35,364 newborns). The disease status of all four newborns that screened positive for the homozygous SMN1 deletion was confirmed by alternate methods. Two of the newborns had two copies of SMN2, and two of the newborns had three copies. We determined the incidence of spinal muscular atrophy in Saint Petersburg to be 1 in 9035 and the SMA carrier frequency to be 1 in 47. In conclusion, providing timely information regarding SMN1, confirmation of disease status, and SMN2 copy number as part of the SMA newborn-screening algorithm can significantly improve clinical follow-up, testing of family members, and treatment of patients with SMA.

Assessment of Nuclear Gem Quantity for Evaluating the Efficacy of Antisense Oligonucleotides in Spinal Muscular Atrophy Cells.

Spinal muscular atrophy is a neuromuscular disorder caused by mutations in both copies of the survival motor neuron gene 1 (SMN1), which lead to reduction in the production of the SMN protein. Currently, there are several therapies that have been approved for SMA, with many more undergoing active research. While various biomarkers have been proposed for assessing the effectiveness of SMA treatment, a universally accepted one still has not been identified. This study aimed to describe a fast and reliable method using the number of gems in cell nuclei as a potential tool for assessment of splicing correction of oligonucleotide efficacy in SMA cells. To gain insight into whether the number of gems in cell nuclei varies based on their SMN genotype and whether the increase in gem number is associated with therapeutic response, we utilized fibroblast cell cultures obtained from a patient with SMA type II and from a healthy individual. We discovered a remarkable difference in the number of gems found in the nuclei of these cells, specifically when counting gems per 100 nuclei. The SMA fibroblasts treated with antisense oligonucleotide showed beneficial effects in correcting the abnormal splicing of SMN2 exon 7. It was observed that there was a significant increase in the number of gems in the treated cells compared to the intact SMA cells. The results obtained significantly correlate with an increase of full-length SMN transcript sharing. Based on our findings, we propose using the quantity of gems as a reliable biomarker for SMA drug development.

Antidepressant-like Effect of the Eburnamine-Type Molecule Vindeburnol in Rat and Mouse Models of Ultrasound-Induced Depression.

Vindeburnol (VIND, RU24722, BC19), a synthetic molecule derived from the eburnamine-vincamine alkaloid group, has many neuropsychopharmacological effects, but its antidepressant-like effects are poorly understood and have only been described in a few patents. To reliably estimate this effect, vindeburnol was studied in a model of long-term variable-frequency ultrasound (US) exposure at 20-45 kHz in male Wistar rats and BALB/c mice. Vindeburnol was administered chronically for 21 days against a background of simultaneous ultrasound exposure at a dose of 20 mg/kg intraperitoneally (IP). Using four behavioral tests, the sucrose preference test (SPT), the social interaction test (SIT), the open field test (OFT), and the forced swimming test (FST), we found that the treatment with the compound diminished depression-like symptoms in mice and rats. The compound restored the ultrasound-related reduced sucrose consumption to control levels and increased social interaction time in mice and rats compared with those in ultrasound-exposed animals. Vindeburnol showed contraversive results of horizontal and vertical activity in both species and generally did not increase locomotor activity. At the same time, the compound showed a specific effect in the FST, significantly reducing the immobility time. Moreover, we found an increase in norepinephrine, dopamine, and its metabolite levels in the brainstem, as well as an increase in dopamine, 3-methoxytyramine, and 3,4-dihydroxyphenylacetic acid levels in the striatum. We also observed a statistically significant increase in tyrosine hydroxylase (TH) levels in the region containing the locus coeruleus (LC). We suggest that using its distinct chemical structure and pharmacological activity as a starting point could boost antidepressant drug discovery.

Molecular mechanism of rhinovirus escape from the Pyrazolo[3,4-d]pyrimidine capsid-binding inhibitor OBR-5-340 via mutations distant from the binding pocket: Derivatives that brake resistance.

Rhinoviruses (RVs) cause the common cold. Attempts at discovering small molecule inhibitors have mainly concentrated on compounds supplanting the medium chain fatty acids residing in the sixty icosahedral symmetry-related hydrophobic pockets of the viral capsid of the Rhinovirus-A and -B species. High-affinity binding to these pockets stabilizes the capsid against structural changes necessary for the release of the ss(+) RNA genome into the cytosol of the host cell. However, single-point mutations may abolish this binding. RV-B5 is one of several RVs that are naturally resistant against the well-established antiviral agent pleconaril. However, RV-B5 is strongly inhibited by the pyrazolopyrimidine OBR-5-340. Here, we report on isolation and characterization of RV-B5 mutants escaping OBR-5-340 inhibition and show that substitution of amino acid residues not only within the binding pocket but also remote from the binding pocket hamper inhibition. Molecular dynamics network analysis revealed that strong inhibition occurs when an ensemble of several sequence stretches of the capsid proteins enveloping OBR-5-340 move together with OBR-5-340. Mutations abrogating this dynamic, regardless of whether being localized within the binding pocket or distant from it result in escape from inhibition. Pyrazolo [3,4-d]pyrimidine derivatives overcoming OBR-5-340 escape of various RV-B5 mutants were identified. Our work contributes to the understanding of the properties of capsid-binding inhibitors necessary for potent and broad-spectrum inhibition of RVs.

Comparative Study of Plastomes in Solanum tuberosum with Different Cytoplasm Types.

The potato is one of the most important food crops in the world. Improving the efficiency of potato breeding is of great importance for solving the global food problem. Today, researchers distinguish between six potato cytoplasm types: A, M, P, T, W, D. In the current study, the complete chloroplast genomes of Solanum tuberosum accessions with five out of the six major cytoplasmic genome types were sequenced (T-, W-, D-, A-, and P-genomes). A comparative analysis of the plastomes in potato accessions with different cytoplasm types was carried out for the first time. The time of origin of the different cytoplasm types was estimated. The presence of two main groups of chloroplast genomes among cultivated potato was confirmed. Based on the phylogenetic analysis of the complete plastome sequences, five main evolutionary branches of chloroplast genomes can be distinguished within the Petota section. Samples with A- and P- cytoplasm formed isolated and distant groups within a large and polymorphic group of samples with M-type cytoplasm, suggesting that A and P genomes arose independently. The findings suggest that the diversity of the T-genome in S. tuberosum Group Tuberosum could be initially low due to a bottle neck already existing at the origin of the Chilean clade. Differences in the rbcL gene sequence may be one of the factors causing differences in economically important traits in species with A and T-type cytoplasm. The data obtained will contribute to the development of methods for molecular marking of cytoplasm types and increase knowledge about the evolution and diversity of potato.

Unusual Oligomeric Laccase-like Oxidases from Ascomycete Curvularia geniculata VKM F-3561 Polymerizing Phenylpropanoids and Phenolic Compounds under Neutral Environmental Conditions.

The unique oligomeric alkaliphilic laccase-like oxidases of the ascomycete C. geniculata VKM F-3561 (with molecular masses about 1035 and 870 kDa) were purified and characterized for the first time. The ability of the enzymes to oxidize phenylpropanoids and phenolic compounds under neutral environmental conditions with the formation of previously unknown di-, tri-, and tetrameric products of transformation was shown. The possibility to obtain industrially valuable compounds (dihydroxybenzyl alcohol and hydroxytyrosol) from caffeic acid using laccase-like oxidases of C. geniculata VKM F-3561 has been shown. Complete nucleotide sequence of the laccase gene, which is expressed at the peak of alkaliphilic laccase activity of the fungus, and its promoter region were determined. Based on the phylogenetic analysis of the nucleotide sequence, the nearest relationship of the isolated laccase gene with similar genes of fungi of the genera Alternaria, Bipolaris, and Cochliobolus was shown. Homologous model of the laccase structure was predicted and a proton channel was found, which was presumably responsible for the accumulation and transport of protons to T2/T3-copper center in the alkaliphilic laccase molecule and providing the functional activity of the enzyme in the neutral alkaline environment conditions.

Development of 2'-O-Methyl and LNA Antisense Oligonucleotides for SMN2 Splicing Correction in SMA Cells.

Spinal muscular atrophy (SMA) is a devastating neurodegenerative disease caused by mutations in the SMN1 gene. Existing therapies demonstrate positive results on SMA patients but still might be ameliorated in efficacy and price. In the presented study we designed antisense oligonucleotides (AONs), targeting intronic splicing silencer sites, some were modified with 2'-O-methyl, others with LNA. The AONs have been extensively tested in different concentrations, both individually and combined, in order to effectively target the ISS-N1 and A+100G splicing silencer regions in intron 7 of the SMN2 gene. By treating SMA-cultured fibroblasts with certain AONs, we discovered a remarkable increase in the levels of full-length SMN transcripts and the number of nuclear gems. This increase was observed to be dose-dependent and reached levels comparable to those found in healthy cells. When added to cells together, most of the tested molecules showed a remarkable synergistic effect in correcting splicing. Through our research, we have discovered that the impact of oligonucleotides is greatly influenced by their length, sequence, and pattern of modification.

Biosynthesis of Functional Silver Nanoparticles Using Callus and Hairy Root Cultures of Aristolochia manshuriensis.

This study delves into the novel utilization of Aristolochia manshuriensis cultured cells for extracellular silver nanoparticles (AgNPs) synthesis without the need for additional substances. The presence of elemental silver has been verified using energy-dispersive X-ray spectroscopy, while distinct surface plasmon resonance peaks were revealed by UV-Vis spectra. Transmission and scanning electron microscopy indicated that the AgNPs, ranging in size from 10 to 40 nm, exhibited a spherical morphology. Fourier-transform infrared analysis validated the abilty of A. manshuriensis extract components to serve as both reducing and capping agents for metal ions. In the context of cytotoxicity on embryonic fibroblast (NIH 3T3) and mouse neuroblastoma (N2A) cells, AgNPs demonstrated varying effects. Specifically, nanoparticles derived from callus cultures exhibited an IC50 of 2.8 µg/mL, effectively inhibiting N2A growth, whereas AgNPs sourced from hairy roots only achieved this only at concentrations of 50 µg/mL and above. Notably, all studied AgNPs' treatment-induced cytotoxicity in fibroblast cells, yielding IC50 values ranging from 7.2 to 36.3 µg/mL. Furthermore, the findings unveiled the efficacy of the synthesized AgNPs against pathogenic microorganisms impacting both plants and animals, including Agrobacterium rhizogenes, A. tumefaciens, Bacillus subtilis, and Escherichia coli. These findings underscore the effectiveness of biotechnological methodologies in offering advanced and enhanced green nanotechnology alternatives for generating nanoparticles with applications in combating cancer and infectious disorders.

iRGD-Targeted Peptide Nanoparticles for Anti-Angiogenic RNAi-Based Therapy of Endometriosis.

Anti-angiogenic RNAi-based therapy can be considered as a possible strategy for the treatment of endometriosis (EM), which is the most common gynecological disease. Targeted delivery of siRNA therapeutics is a prerequisite for successful treatment without adverse effects. Here we evaluated the RGD1-R6 peptide carrier as a non-viral vehicle for targeted siRNA delivery to endothelial cells in vitro and endometrial implants in vivo. The physicochemical properties of the siRNA complexes, cellular toxicity, and GFP and VEGFA gene silencing efficiency were studied, and anti-angiogenic effects were proved in cellular and animal models. The modification of siRNA complexes with iRGD ligand resulted in a two-fold increase in gene knockdown efficiency and three-fold decrease in endothelial cells' migration in vitro. Modeling of EM in rats with the autotransplantation of endometrial tissue subcutaneously was carried out. Efficiency of anti-angiogenic EM therapy in vivo by anti-VEGF siRNA/RGD1-R6 complexes was evaluated by the implants' volume measurement, CD34 immunohistochemical staining, and VEGFA gene expression analysis. We observed a two-fold decrease in endometriotic implants growth and a two-fold decrease in VEGFA gene expression in comparison with saline-treated implants. RNAi-mediated therapeutic effects were comparable with Dienogest treatment efficiency in a rat EM model. Taken together, these findings demonstrate the advantages of RGD1-R6 peptide carrier as a delivery system for RNAi-based therapy of EM.

Whole-Genome Sequencing Revealed the Fusion Plasmids Capable of Transmission and Acquisition of Both Antimicrobial Resistance and Hypervirulence Determinants in Multidrug-Resistant Klebsiella pneumoniae Isolates.

Klebsiella pneumoniae, a member of the Enterobacteriaceae family, has become a dangerous pathogen accountable for a large fraction of the various infectious diseases in both clinical and community settings. In general, the K. pneumoniae population has been divided into the so-called classical (cKp) and hypervirulent (hvKp) lineages. The former, usually developing in hospitals, can rapidly acquire resistance to a wide spectrum of antimicrobial drugs, while the latter is associated with more aggressive but less resistant infections, mostly in healthy humans. However, a growing number of reports in the last decade have confirmed the convergence of these two distinct lineages into superpathogen clones possessing the properties of both, and thus imposing a significant threat to public health worldwide. This process is associated with horizontal gene transfer, in which plasmid conjugation plays a very important role. Therefore, the investigation of plasmid structures and the ways plasmids spread within and between bacterial species will provide benefits in developing prevention measures against these powerful pathogens. In this work, we investigated clinical multidrug-resistant K. pneumoniae isolates using long- and short-read whole-genome sequencing, which allowed us to reveal fusion IncHI1B/IncFIB plasmids in ST512 isolates capable of simultaneously carrying hypervirulence (iucABCD, iutA, prmpA, peg-344) and resistance determinants (armA, blaNDM-1 and others), and to obtain insights into their formation and transmission mechanisms. Comprehensive phenotypic, genotypic and phylogenetic analysis of the isolates, as well as of their plasmid repertoire, was performed. The data obtained will facilitate epidemiological surveillance of high-risk K. pneumoniae clones and the development of prevention strategies against them.

Efficacy of an isoxazole-3-carboxamide analog of pleconaril in mouse models of Enterovirus-D68 and Coxsackie B5.

Enteroviruses (EV) cause a number of life-threatening infectious diseases. EV-D68 is known to cause respiratory illness in children that can lead to acute flaccid myelitis. Coxsackievirus B5 (CVB5) is commonly associated with hand-foot-mouth disease. There is no antiviral treatment available for either. We have developed an isoxazole-3-carboxamide analog of pleconaril (11526092) which displayed potent inhibition of EV-D68 (IC50 58 nM) as well as other enteroviruses including the pleconaril-resistant Coxsackievirus B3-Woodruff (IC50 6-20 nM) and CVB5 (EC50 1 nM). Cryo-electron microscopy structures of EV-D68 in complex with 11526092 and pleconaril demonstrate destabilization of the EV-D68 MO strain VP1 loop, and a strain-dependent effect. A mouse respiratory model of EV-D68 infection, showed 3-log decreased viremia, favorable cytokine response, as well as statistically significant 1-log reduction in lung titer reduction at day 5 after treatment with 11526092. An acute flaccid myelitis neurological infection model did not show efficacy. 11526092 was tested in a mouse model of CVB5 infection and showed a 4-log TCID50 reduction in the pancreas. In summary, 11526092 represents a potent in vitro inhibitor of EV with in vivo efficacy in EV-D68 and CVB5 animal models suggesting it is worthy of further evaluation as a potential broad-spectrum antiviral therapeutic against EV.

Pyrrolo[2,3-e]indazole as a novel chemotype for both influenza A virus and pneumococcal neuraminidase inhibitors.

Influenza infections are often exacerbated by secondary bacterial infections, primarily caused by Streptococcus pneumoniae. Both respiratory pathogens have neuraminidases that support infection. Therefore, we hypothesized that dual inhibitors of viral and bacterial neuraminidases might be an advantageous strategy for treating seasonal and pandemic influenza pneumonia complicated by bacterial infections. By screening our in-house chemical library, we discovered a new chemotype that may be of interest for a further campaign to find small molecules against influenza. Our exploration of the pyrrolo[2,3-e]indazole space led to the identification of two hit compounds, 6h and 12. These molecules were well-tolerated by MDCK cells and inhibited the replication of H3N2 and H1N1 influenza A virus strains. Moreover, both compounds suppress viral and pneumococcal neuraminidases indicating their dual activity. Given its antiviral activity, pyrrolo[2,3-e]indazole has been identified as a promising scaffold for the development of novel neuraminidase inhibitors that are active against influenza A virus and S. pneumoniae.

Kidney-Related Function of Mitochondrial Protein Mitoregulin.

A small protein, Mitoregulin (Mtln), localizes in mitochondria and contributes to oxidative phosphorylation and fatty acid metabolism. Mtln knockout mice develop obesity on a high-fat diet, demonstrating elevated cardiolipin damage and suboptimal creatine kinase oligomerization in muscle tissue. Kidneys heavily depend on the oxidative phosphorylation in mitochondria. Here we report kidney-related phenotypes in aged Mtln knockout mice. Similar to Mtln knockout mice muscle mitochondria, those of the kidney demonstrate a decreased respiratory complex I activity and excessive cardiolipin damage. Aged male mice carrying Mtln knockout demonstrated an increased frequency of renal proximal tubules' degeneration. At the same time, a decreased glomerular filtration rate has been more frequently detected in aged female mice devoid of Mtln. An amount of Mtln partner protein, Cyb5r3, is drastically decreased in the kidneys of Mtln knockout mice.

Mitoregulin Contributes to Creatine Shuttling and Cardiolipin Protection in Mice Muscle.

Small peptides compose a large share of the mitochondrial proteome. Mitoregulin (Mtln) is a mitochondrial peptide known to contribute to the respiratory complex I functioning and other processes in mitochondria. In our previous studies, we demonstrated that Mtln knockout mice develop obesity and accumulate triglycerides and other oxidation substrates in serum, concomitant with an exhaustion of tricarboxylic acids cycle intermediates. Here we examined the functional role of Mtln in skeletal muscles, one of the major energy consuming tissues. We observed reduced muscle strength for Mtln knockout mice. Decrease of the mitochondrial cardiolipin and concomitant increase in monolysocardiolipin concentration upon Mtln inactivation is likely to be a consequence of imbalance between oxidative damage and remodeling of cardiolipin. It is accompanied by the mitochondrial creatine kinase octamer dissociation and suboptimal respiratory chain performance in Mtln knockout mice.

Non-Viral Carriers for Nucleic Acids Delivery: Fundamentals and Current Applications.

Over the past decades, non-viral DNA and RNA delivery systems have been intensively studied as an alternative to viral vectors. Despite the most significant advantage over viruses, such as the lack of immunogenicity and cytotoxicity, the widespread use of non-viral carriers in clinical practice is still limited due to the insufficient efficacy associated with the difficulties of overcoming extracellular and intracellular barriers. Overcoming barriers by non-viral carriers is facilitated by their chemical structure, surface charge, as well as developed modifications. Currently, there are many different forms of non-viral carriers for various applications. This review aimed to summarize recent developments based on the essential requirements for non-viral carriers for gene therapy.

Plasmid Composition, Antimicrobial Resistance and Virulence Genes Profiles of Ciprofloxacin- and Third-Generation Cephalosporin-Resistant Foodborne Salmonella enterica Isolates from Russia.

Salmonella enterica is an important foodborne pathogen worldwide. Ciprofloxacin and extended-spectrum cephalosporins are the common first-line antimicrobial drugs for the treatment of salmonellosis, antimicrobial resistance genes for which are mostly transferred via plasmids. The goal of this work was to perform genomic analysis of plasmids from foodborne S. enterica isolates obtained in Russia based on whole-genome sequencing. In the current study, 11 multidrug-resistant samples isolated in 2021 from 8 regions of Russia were selected based on their resistance to ciprofloxacin and third-generation cephalosporins (CIP-3rd). Whole-genome short-read sequencing (WGS) was performed for all isolates; the samples belonged to five different sequence types (ST32, ST469, ST11, ST142, and ST548) which had different profiles of antimicrobial resistance (AMR) and virulence genes. We have performed additional long-read sequencing of four representative S. enterica isolates, which showed that they carried pESI-like megaplasmids of 202-280 kb length harboring extended-spectrum ß-lactamase genes, fluoroquinolone, tetracycline, and aminoglycosides resistance genes, as well as several virulence determinants. We believe that the WGS data obtained will greatly facilitate further studies of foodborne S. enterica isolates epidemiology in terms of their self-transmissible plasmid composition that mediated antimicrobial resistance and virulence determinants conferring selective advantages of this important bacterial pathogen.

Polycondensed Peptide-Based Polymers for Targeted Delivery of Anti-Angiogenic siRNA to Treat Endometriosis.

Endometriosis (EM) is a prevalent gynecological disease characterized by the abnormal growth of tissue similar to the endometrium outside of the uterus. This condition is accompanied by the development of new blood vessels in endometriotic lesions. While surgical intervention is effective in removing endometriotic lesions, some patients require multiple surgeries. Therefore, finding non-surgical treatments for EM is of great interest. One of the promising approaches is anti-angiogenic therapy using siRNA-therapeutics to target the expression of the VEGFA gene. Peptide-based polymers have shown promise as siRNA delivery systems due to their biocompatibility and ease of modification. We conducted a study to evaluate the effectiveness of the R6p-cRGD peptide carrier as a non-viral vehicle for delivering siRNA to endothelial cells in vitro and endometrial implants in vivo. We investigated the physicochemical properties of the siRNA-complexes, assessed cellular toxicity, and examined the efficiency of GFP and VEGFA genes silencing. Furthermore, we tested the anti-angiogenic effects of these complexes in cellular and animal models. The transfection with siRNA complexes led to a significant increase in VEGFA gene knockdown efficiency and a decrease in the migration of endothelial cells. For the animal model, we induced endometriosis in rats by transplanting endometrial tissue subcutaneously. We evaluated the efficiency of anti-angiogenic therapy for EM in vivo using anti-VEGF siRNA/R6p-RGD complexes. During this assessment, we measured the volume of the implants, analyzed VEGFA gene expression, and conducted CD34 immunohistochemical staining. The results showed a significant decrease in the growth of endometriotic implants and in VEGFA gene expression. Overall, our findings demonstrate the potential of the R6p-cRGD peptide carrier as a delivery system for anti-angiogenic therapy of EM.

Serum-Resistant Ternary DNA Polyplexes for Suicide Gene Therapy of Uterine Leiomyoma.

Uterine leiomyoma (UL) is a prevalent benign tumor in women that frequently gives rise to a multitude of reproductive complications. The use of suicide gene therapy has been proposed as a highly promising method for treating UL. To achieve successful gene therapy, it is essential to develop carriers that can efficiently transport nucleic acids into targeted cells and tissues. The instability of polyplexes in blood and other biological fluids is a crucial factor to consider when using non-viral carriers. In this study, we present serum-resistant and cRGD-modified DNA complexes for targeted delivery genes to UL cells. Ternary polyplexes were formed by incorporating cystine-cross-linked polyglutamic acid modified with histidine residues. We employed two techniques in the production of cross-linked polyanionic coating: matrix polymerization and oxidative polycondensation. In this study, we investigated the physicochemical properties of ternary DNA complexes, including the size and zeta-potential of the nanoparticles. Additionally, we evaluated cellular uptake, toxicity levels, transfection efficiency and specificity in vitro. The study involved introducing the HSV-TK gene into primary UL cells as a form of suicide gene therapy modeling. We have effectively employed ternary peptide-based complexes for gene delivery into the UL organtypic model. By implementing in situ suicide gene therapy, the increase in apoptosis genes expression was detected, providing conclusive evidence of apoptosis occurring in the transfected UL tissues. The results of the study strongly suggest that the developed ternary polyplexes show potential as a valuable tool in the implementation of suicide gene therapy for UL.

Peptide-Based Nanoparticles for αvß3 Integrin-Targeted DNA Delivery to Cancer and Uterine Leiomyoma Cells.

Uterine leiomyoma is the most common benign tumor of the reproductive system. Current therapeutic options do not simultaneously meet the requirements of long-term efficiency and fertility preservation. Suicide gene delivery can be proposed as a novel approach to uterine leiomyoma therapy. Non-viral vehicles are an attractive approach to DNA delivery for gene therapy of both malignant and benign tumors. Peptide-based vectors are among the most promising candidates for the development of artificial viruses, being able to efficiently cross barriers of DNA transport to cells. Here we described nanoparticles composed of cysteine-crosslinked polymer and histidine-arginine-rich peptide modified with iRGD moiety and characterized them as vehicles for plasmid DNA delivery to pancreatic cancer PANC-1 cells and the uterine leiomyoma cell model. Several variants of nanoparticles were formulated with different targeting ligand content. The physicochemical properties that were studied included DNA binding and protection, interaction with polyanions and reducing agents, size, structure and zeta-potential of the peptide-based nanoparticles. Cytotoxicity, cell uptake and gene transfection efficiency were assessed in PANC-1 cells with GFP and LacZ-encoding plasmids. The specificity of gene transfection via αvß3 integrin binding was proved in competitive transfection. The therapeutic potential was evaluated in a uterine leiomyoma cell model using the suicide gene therapy approach. The optimal formulation was found to be at the polyplex with the highest iRGD moiety content being able to transfect cells more efficiently than control PEI. Suicide gene therapy using the best formulation resulted in a significant decrease of uterine leiomyoma cells after ganciclovir treatment. It can be concluded that the application of iRGD-modified peptide-based nanoparticles has a high potential for cellular delivery of DNA therapeutics in favor of uterine leiomyoma gene therapy.