Search Results (37)

The synthesis of novel biodegradable polymers as non-viral vectors remains one of the challenging tasks in the field of gene delivery. In this study, the synthesis of the polysaccharide-g-polypeptide copolymers, namely, hyaluronic acid-g-polylysine (HA-g-PLys), using a copper-free strain-promoted azide-alkyne cycloaddition reaction was proposed. For this purpose, hyaluronic acid was modified with dibenzocyclooctyne moieties, and poly-L-lysine with a terminal azido group was obtained using ring-opening polymerization of N-carboxyanhydride of the corresponding protected amino acid, initiated with the amino group azido-PEG₃-amine. Two HA-g-PLys samples with different degrees of grafting were synthesized, and the structures of all modified and synthesized polymers were confirmed using ¹H NMR and FTIR spectroscopy. The HA-g-PLys samples obtained were able to form nanoparticles in aqueous media due to self-assembly driven by electrostatic interactions. The binding of DNA and model siRNA by copolymers to form polyplexes was analyzed using ethidium bromide, agarose gel electrophoresis, and SybrGreen I assays. The hydrodynamic diameter of polyplexes was ˂300 nm (polydispersity index, PDI ˂ 0.3). The release of a model fluorescently-labeled oligonucleotide in the complex biological medium was significantly higher in the case of HA-g-PLys as compared to that in the case of PLys-based polyplexes. In addition, the cytotoxicity in normal and cancer cells, as well as the ability of HA-g-PLys to facilitate intracellular delivery of anti-GFP siRNA to NIH-3T3/GFP+ cells, were evaluated. Full article

Background: Genome editing at specific loci is an innovative therapeutic approach; however, it faces many challenges, so optimizing delivery vectors is essential to enhance the safety and efficacy of the CRISPR/Cas9 system. This study investigated whether the hydrodynamic administration of liposomal CRISPR/Cas9 complexes (LCs) in newborn mice induces off-target events or tumors. Methods: Liposomes were obtained through microfluidization. The CRISPR/Cas9 plasmid and a donor plasmid containing the Idua cDNA (alpha-L-iduronidase enzyme) were incorporated by adsorption, and complexes (LCs) were characterized regarding physicochemical properties. C57BL/6 newborn mice were divided in two groups, one received the complexes through hydrodynamic intravenous injection (n = 15) and the other was used as control (n = 15). After 21 months, mice were euthanized and organs were analyzed regarding histological characteristics. Lungs and liver were analyzed by qPCR searching for potential off-target sites in chromosomes 2, 5, 11, and 17 and on-target site in chromosome 6, identified by COSMID. Sequences were analyzed using an ICE tool for indels detection. Results: LCs exhibited 136 nm mean vesicle diameter with PDI below 0.15 and a zeta potential around +43 mV. Immediate biodistribution was predominant in the lungs and liver. There was no significant increase in tumor induction (20% in LCs vs. 33% in control). Molecular analyses indicated 0% off-target effects and around 3% on-target events. Conclusions: We conclude that this set of experiments demonstrates the potential of the chosen gRNA sequence to perform safe gene editing at the murine ROSA26 locus, corroborating the safety of the CRISPR/Cas9 gene editing platform. Full article

Hepatocellular carcinoma (HCC), the predominant primary liver tumor, remains one of the most lethal cancers worldwide, despite the advances in therapy in recent years. In addition to the traditional chemically and dietary-induced HCC models, a broad spectrum of novel preclinical tools have been generated following the advent of transgenic, transposon, organoid, and in silico technologies to overcome this gloomy scenario. These models have become rapidly robust preclinical instruments to unravel the molecular pathogenesis of liver cancer and establish new therapeutic approaches against this deadly disease. The present review article aims to summarize and discuss the commonly used preclinical models for HCC, evaluating their strengths and weaknesses. Full article

Intracellular delivery, the process of transporting substances into cells, is crucial for various applications, such as drug delivery, gene therapy, cell imaging, and regenerative medicine. Among the different approaches of intracellular delivery, mechanoporation stands out by utilizing mechanical forces to create temporary pores on cell membranes, enabling the entry of substances into cells. This method is promising due to its minimal contamination and is especially vital for stem cells intended for clinical therapy. In this review, we explore various mechanoporation technologies, including microinjection, micro–nano needle arrays, cell squeezing through physical confinement, and cell squeezing using hydrodynamic forces. Additionally, we highlight recent research efforts utilizing mechanoporation for stem cell studies. Furthermore, we discuss the integration of mechanoporation techniques into microfluidic platforms for high-throughput intracellular delivery with enhanced transfection efficiency. This advancement holds potential in addressing the challenge of low transfection efficiency, benefiting both basic research and clinical applications of stem cells. Ultimately, the combination of microfluidics and mechanoporation presents new opportunities for creating comprehensive systems for stem cell processing. Full article

The formulation of novel delivery protocols for the targeted delivery of genes into hepatocytes by receptor mediation is important for the treatment of liver-specific disorders, including cancer. Non-viral delivery methods have been extensively studied for gene therapy. Gold nanoparticles (AuNPs) have gained attention in nanomedicine due to their biocompatibility. In this study, AuNPs were synthesized and coated with polymers: chitosan (CS), and polyethylene glycol (PEG). The targeting moiety, lactobionic acid (LA), was added for hepatocyte-specific delivery. Physicochemical characterization revealed that all nano-formulations were spherical and monodispersed, with hydrodynamic sizes between 70 and 250 nm. Nanocomplexes with pCMV-Luc DNA (pDNA) confirmed that the NPs could bind, compact, and protect the pDNA from nuclease degradation. Cytotoxicity studies revealed that the AuNPs were well tolerated (cell viabilities > 70%) in human hepatocellular carcinoma (HepG2), embryonic kidney (HEK293), and colorectal adenocarcinoma (Caco-2) cells, with enhanced transgene activity in all cells. The inclusion of LA in the NP formulation was notable in the HepG2 cells, which overexpress the asialoglycoprotein receptor on their cell surface. A five-fold increase in luciferase gene expression was evident for the LA-targeted AuNPs compared to the non-targeted AuNPs. These AuNPs have shown potential as safe and suitable targeted delivery vehicles for liver-directed gene therapy. Full article

Understanding the regulation of transgene expression is critical for the success of plasmid-based gene therapy and vaccine development. In this study, we used two sets of plasmid vectors containing secreted embryonic alkaline phosphatase or the mouse IL-10 gene as a reporter and investigated the role of promoter elements in regulating transgene expression in vivo. We demonstrated in mice that hydrodynamic transfer of plasmids with the CMV promoter resulted in a high level of reporter gene expression that declined rapidly over time. In contrast, when plasmids with albumin promoters were used, a lower but sustained gene expression pattern was observed. We also found that plasmids containing a shorter CMV promoter sequence with fewer transcription factor binding sites showed a decrease in the peak level of gene expression without changing the overall pattern of reporter gene expression. The replacement of regulatory elements in the CMV promoter with a single regulatory element of the albumin promoter changed the pattern of transient gene expression seen in the CMV promoter to a pattern of sustained gene expression identical to that of a full albumin promoter. ChIP analyses demonstrated an elevated binding of acetylated histones and TATA box-binding protein to the promoter carrying regulatory elements of the albumin promoter. These results suggest that the strength of a promoter is determined by the number of appropriate transcription factor binding sites, while gene expression persistence is determined by the presence of regulatory elements capable of recruiting epigenetic modifying complexes that make the promoter accessible for transcription. This study provides important insights into the mechanisms underlying gene expression regulation in vivo, which can be used to improve plasmid-based gene therapy and vaccine development. Full article

The liver plays a crucial role in glucose metabolism. Obesity and a diet rich in fats (HFD) contribute to the accumulation of intracellular lipids. The aim of the study was to explore the involvement of acyl-CoA synthetase 1 (ACSL1) in bioactive lipid accumulation and the induction of liver insulin resistance (InsR) in animals fed an HFD. The experiments were performed on male C57BL/6 mice divided into the following experimental groups: 1. Animals fed a control diet; 2. animals fed HFD; and 3. HFD-fed animals with the hepatic ACSL1 gene silenced through a hydrodynamic gene delivery technique. Long-chain acyl-CoAs, sphingolipids, and diacylglycerols were measured by LC/MS/MS. Glycogen was measured by means of a commercially available kit. The protein expression and phosphorylation state of the insulin pathway was estimated by Western blot. HFD-fed mice developed InsR, manifested as an increase in fasting blood glucose levels (202.5 mg/dL vs. 130.5 mg/dL in the control group) and inhibition of the insulin pathway, which resulted in an increase in the rate of gluconeogenesis (0.420 vs. 0.208 in the control group) and a decrease in the hepatic glycogen content (1.17 μg/mg vs. 2.32 μg/mg in the control group). Hepatic ACSL1 silencing resulted in decreased lipid content and improved insulin sensitivity, accounting for the decreased rate of gluconeogenesis (0.348 vs. 0.420 in HFD_(+/+)) and the increased glycogen content (4.3 μg/mg vs. 1.17 μg/mg in HFD_(+/+)). The elevation of gluconeogenesis and the decrease in glycogenesis in the hepatic tissue of HFD-fed mice resulted from cellular lipid accumulation. Inhibition of lipid synthesis through silencing ACSL1 alleviated HFD-induced hepatic InsR. Full article

Geraniol (GE) is a monoterpene alcohol with excellent antifungal activity. However, its low solubility and high volatility impair its use. Nanoemulsions (NE) are excellent delivery systems for poorly soluble and volatile drugs, achieving controlled release of the active ingredient. The aim of this study was to improve the delivery of geraniol (GE) incorporated in NE against Candida albicans in order to evaluate the antibiofilm effect and cytotoxicity. Nanoemulsion containing 10% oil phase (cholesterol) (w/w), 10% surfactant (mixture of soy phosphatidylcholine and Brij 58; 1:2) (w/w), and 80% aqueous phase (phosphate buffer) (w/w) was synthesized. Incorporation of GE was carried out by sonication and the final compounds were characterized by hydrodynamic diameter, polydispersity index (PDI), and zeta potential (ZP), in addition to evaluation of physicochemical stability after 6 months and 1 year. The GE-NE effect was evaluated on Candida albicans biofilms and cytotoxic effect was evaluated on immortalized normal oral cell line NOK-Si. The diameter of GE-NE was 232.3 ± 2.7 nm and PDI 0.155 with exhibited homogeneity and stability in solution. GE-NE showed antibiofilm activity at a concentration of 75 μg/mL with reduction of >6.0 log₁₀, and no cytotoxicity against NOK-Si cells at concentrations below 150 μg/mL was observed. GE-NE proved to be a promising candidate for prevention and treatment of fungal diseases. Full article

The estrogen metabolite 2-methoxyestradiol (2ME) is a promissory anticancer drug mainly because of its pro-apoptotic properties in cancer cells. However, the therapeutic use of 2ME has been hampered due to its low solubility and bioavailability. Thus, it is necessary to find new ways of administration for 2ME. Zeolites are inorganic aluminosilicates with a porous structure and are considered good adsorbents and sieves in the pharmaceutical field. Here, mordenite-type zeolite nanoparticles were loaded with 2ME to assess its efficiency as a delivery system for prostate cancer treatment. The 2ME-loaded zeolite nanoparticles showed an irregular morphology with a mean hydrodynamic diameter of 250.9 ± 11.4 nm, polydispersity index of 0.36 ± 0.04, and a net negative surface charge of −34 ± 1.73 meV. Spectroscopy with UV-vis and Attenuated Total Reflectance Infrared Fourier-Transform was used to elucidate the interaction between the 2ME molecules and the zeolite framework showing the formation of a 2ME-zeolite conjugate in the nanocomposite. The studies of adsorption and liberation determined that zeolite nanoparticles incorporated 40% of 2ME while the liberation of 2ME reached 90% at pH 7.4 after 7 days. The 2ME-loaded zeolite nanoparticles also decreased the viability and increased the mRNA of the 2ME-target gene F-spondin, encoded by SPON1, in the human prostate cancer cell line LNCaP. Finally, the 2ME-loaded nanoparticles also decreased the viability of primary cultures from mouse prostate cancer. These results show the development of 2ME-loaded zeolite nanoparticles with physicochemical and biological properties compatible with anticancer activity on the human prostate and highlight that zeolite nanoparticles can be a good carrier system for 2ME. Full article

In this paper, Au nanocages (AuNCs) loaded with the MRI contrast agent gadolinium (Gd) and capped with the tumor-targeting gene survivin (Sur–AuNC•Gd–Cy7 nanoprobes) were designed and applied as a targeted imaging agent for pancreatic cancer. With its capacity to transport fluorescent dyes and MR imaging agents, the gold cage is an outstanding platform. Furthermore, it has the potential to transport different drugs in the future, making it a unique carrier platform. The utilization of Sur–AuNC•Gd–Cy7 nanoprobes has proven to be an effective means of targeting and localizing survivin-positive BxPC-3 cells within their cytoplasm. By targeting survivin, an antiapoptotic gene, the Sur–AuNC•Gd–Cy7 nanoprobe was able to induce pro-apoptotic effects in BxPC-3 pancreatic cancer cells. The biocompatibility of AuNCs•Gd, AuNCs•Gd–Cy7 nanoparticles, and Sur–AuNC•Gd–Cy7 nanoprobes is evaluated through the hemolysis rate assay. The stability of AuNCs•Gd, AuNCs•Gd–Cy7 nanoparticles, and Sur–AuNC•Gd–Cy7 nanoprobes was evaluated by determining their hydrodynamic dimensions following storage in different pH solutions for a corresponding duration. Excellent biocompatibility and stability of the Sur–AuNC•Gd–Cy7 nanoprobes will facilitate their further utilization in vivo and in vitro. The surface-bound survivin plays a role in facilitating the Sur–AuNC•Gd–Cy7 nanoprobes’ ability to locate the BxPC-3 tumor. The probe was modified to incorporate Gd and Cy7, thereby enabling the simultaneous utilization of magnetic resonance imaging (MRI) and fluorescence imaging (FI) techniques. In vivo, the Sur–AuNC•Gd–Cy7 nanoprobes were found to effectively target and localize survivin-positive BxPC-3 tumors through the use of MRI and FI. After being injected via the caudal vein, the Sur–AuNC•Gd–Cy7 nanoprobes were found to accumulate effectively in an in situ pancreatic cancer model within 24 h. Furthermore, these nanoprobes were observed to be eliminated from the body through the kidneys within 72 h after a single injection. This characteristic is crucial for a diagnostic agent. Based on the aforementioned outcomes, the Sur–AuNC•Gd–Cy7 nanoprobes have significant potential advantages for the theranostic treatment of pancreatic cancer. This nanoprobe possesses distinctive characteristics, such as advanced imaging abilities and specific drug delivery, which offer the possibility of enhancing the precision of diagnosis and efficacy of treatment for this destructive illness. Full article

Chitosan is extensively studied as a carrier for gene delivery and is an attractive non-viral gene vector owing to its polycationic, biodegradable, and biocompatible nature. Thus, it is essential to understand the chemistry of self-assembled chitosan–DNA complexation and their structural and functional properties, enabling the formation of an effective non-viral gene delivery system. In this study, two parent chitosans (samples NAS-032 and NAS-075; Mw range ~118–164 kDa) and their depolymerised derivatives (deploy nas-032 and deploy nas-075; Mw range 6–14 kDa) with degrees of acetylation 43.4 and 4.7%, respectively, were used to form polyelectrolyte complexes (PECs) with DNA at varying [–NH₃⁺]/[–PO₄⁻] (N/P) molar charge ratios. We investigated the formation of the PECs using ζ-potential, asymmetric flow field-flow fractionation (AF4) coupled with multiangle light scattering (MALS), refractive index (RI), ultraviolet (UV) and dynamic light scattering (DLS) detectors, and TEM imaging. PEC formation was confirmed by ζ-potential measurements that shifted from negative to positive values at N/P ratio ~2. The radius of gyration (R_g) was determined for the eluting fractions by AF4-MALS-RI-UV, while the corresponding hydrodynamic radius (R_h), by the DLS data. We studied the influence of different cross-flow rates on AF4 elution patterns for PECs obtained at N/P ratios 5, 10, and 20. The determined rho shape factor (ρ = R_g/R_h) values for the various PECs corresponded with a sphere morphology (ρ ~0.77–0.85), which was consistent with TEM images. The results of this study represent a further step towards the characterisation of chitosan–DNA PECs by the use of multi-detection AF4 as an important tool to fractionate and infer aspects of their morphology. Full article

The development of effective anti-cancer therapeutics remains one of the current pharmaceutical challenges. The joint delivery of chemotherapeutic agents and biopharmaceuticals is a cutting-edge approach to creating therapeutic agents of enhanced efficacy. In this study, amphiphilic polypeptide delivery systems capable of loading both hydrophobic drug and small interfering RNA (siRNA) were developed. The synthesis of amphiphilic polypeptides included two steps: (i) synthesis of poly-αl-lysine by ring-opening polymerization and (ii) its post-polymerization modification with hydrophobic l-amino acid and l-arginine/l-histidine. The obtained polymers were used for the preparation of single and dual delivery systems of PTX and short double-stranded nucleic acid. The obtained double component systems were quite compact and had a hydrodynamic diameter in the range of 90–200 nm depending on the polypeptide. The release of PTX from the formulations was studied, and the release profiles were approximated using a number of mathematical dissolution models to establish the most probable release mechanism. A determination of the cytotoxicity in normal (HEK 293T) and cancer (HeLa and A549) cells revealed the higher toxicity of the polypeptide particles to cancer cells. The separate evaluation of the biological activity of PTX and anti-GFP siRNA formulations testified the inhibitory efficiency of PTX formulations based on all polypeptides (IC₅₀ 4.5–6.2 ng/mL), while gene silencing was effective only for the Tyr-Arg-containing polypeptide (56–70% GFP knockdown). Full article

The principle of hydrodynamic delivery was initially used to develop a method for the delivery of plasmids into mouse hepatocytes through tail vein injection and has been expanded for use in the delivery of various biologically active materials to cells in various organs in a variety of animal species through systemic or local injection, resulting in significant advances in new applications and technological development. The development of regional hydrodynamic delivery directly supports successful gene delivery in large animals, including humans. This review summarizes the fundamentals of hydrodynamic delivery and the progress that has been made in its application. Recent progress in this field offers tantalizing prospects for the development of a new generation of technologies for broader application of hydrodynamic delivery. Full article

Hydrodynamic transfection (HT) or hydrodynamic tail vein injection (HTVi) is among the leading technique that is used to deliver plasmid genes mainly into the liver of live mice or rats. The DNA constructs are composed of coupled plasmids, while one contains the gene of interest that stably integrate into the hepatocyte genome with help of the other consisting sleeping beauty transposase system. The rapid injection of a large volume of DNA-solution through the tail vein induces an acute cardiac congestion that refluxed into the liver, mainly in acinus zone 3, also found through our EM study. Although, HT mediated hydrodynamic force can permeabilizes the fenestrated sinusoidal endothelium of liver, but the mechanism of plasmid incorporation into the hepatocytes remains unclear. Therefore, in the present study, we have hydrodynamically injected 2 mL volume of empty plasmid (transposon vector) or saline solution (control) into the tail vein of anesthetized C57BL/6J/129Sv mice. Liver tissue was resected at different time points from two animal group conditions, i.e., one time point per animal (1, 5, 10–20, 60 min or 24 and 48 hrs after HT) or multiple time points per animal (0, 1, 2, 5, 10, 20 min) and quickly fixed with buffered 4% osmium tetroxide. The tissues fed with only saline solution was also resected and fixed in the similar way. EM evaluation from the liver ultrathin sections reveals that swiftly after 1 min, the hepatocytes near to the central venule in the acinus zone 3 shows cytoplasmic membrane-bound vesicles. Such vesicles increased in both numbers and size to vacuoles and precisely often found in the proximity to the nucleus. Further, EM affirm these vacuoles are also optically empty and do not contain any electron dense material. Although, some of the other hepatocytes reveals sign of cell damage including swollen mitochondria, dilated endoplasmic reticulum, Golgi apparatus and disrupted plasma membrane, but most of the hepatocytes appeared normal. The ultrastructural findings in the mice injected with empty vector or saline injected control mice were similar. Therefore, we have interpreted the vacuole formation as nonspecific endocytosis without specific interactions at the plasma membrane. Full article

The study of gold nanoparticles (AuNPs) in the blood flow has emerged as an area of interest for numerous researchers, due to its many biomedical applications, such as cancer radiotherapy, DNA and antigens, drug and gene delivery, in vitro evaluation, optical bioimaging, radio sensitization and laser phototherapy of cancer cells and tumors. Gold nanoparticles can be amalgamated in various shapes and sizes. Due to this reason, gold nanoparticles can be diffused efficiently, target the diseased cells and destroy them. The current work studies the effect of gold nanoparticles of different shapes on the electro-magneto-hydrodynamic (EMHD) peristaltic propulsion of blood in a micro-channel under various effects, such as activation energy, bioconvection, radiation and gyrotactic microorganisms. Four kinds of nanoparticle shapes, namely bricks, cylinders and platelets, are considered. The governing equations are simplified under the approximations of low Reynolds number (LRN), long wavelength (LWL) and Debye–Hückel linearization (DHL). The numerical solutions for the non-dimensional equations are solved using the computational software MATLAB with the help of the bvp4c function. The influences of different physical parameters on the flow and thermal characteristics are computed through pictorial interpretations. Full article