Nucleic Acid Delivery Systems Research Articles

Efficient Transfection of Large Plasmids Encoding HIV-1 into Human Cells-A High Potential Transfection System Based on a Peptide Mimicking Cationic Lipid.

One major disadvantage of nucleic acid delivery systems is the low transfection or transduction efficiency of large-sized plasmids into cells. In this communication, we demonstrate the efficient transfection of a 15.5 kb green fluorescent protein (GFP)-fused HIV-1 molecular clone with a nucleic acid delivery system prepared from the highly potent peptide-mimicking cationic lipid OH4 in a mixture with the phospholipid DOPE (co-lipid). For the transfection, liposomes were loaded using a large-sized plasmid (15.5 kb), which encodes a replication-competent HIV type 1 molecular clone that carries a Gag-internal green fluorescent protein (HIV-1 JR-FL Gag-iGFP). The particle size and charge of the generated nanocarriers with 15.5 kb were compared to those of a standardized 4.7 kb plasmid formulation. Stable, small-sized lipoplexes could be generated independently of the length of the used DNA. The transfer of fluorescently labeled pDNA-HIV1-Gag-iGFP in HEK293T cells was monitored using confocal laser scanning microscopy (cLSM). After efficient plasmid delivery, virus particles were detectable as budding structures on the plasma membrane. Moreover, we observed a randomized distribution of fluorescently labeled lipids over the plasma membrane. Obviously, a significant exchange of lipids between the drug delivery system and the cellular membranes occurs, which hints toward a fusion process. The mechanism of membrane fusion for the internalization of lipid-based drug delivery systems into cells is still a frequently discussed topic.

Photocontrolled miR-148b nanoparticles cause apoptosis, inflammation and regression of Ras induced epidermal squamous cell carcinomas in mice

Despite evidence that microRNAs (miRNAs) are essential in modulating tumorigenesis, a major challenge in cancer treatment is to achieve tumor-specific selectivity and efficient yet safe delivery of miRNAs in vivo. In this study, we have developed a light-inducible silver nanoparticle nucleic acid delivery system that demonstrates precise spatiotemporal control, high cellular uptake, low cytotoxicity, escape from endosomes and release of functional miRNA into the cytosol. Using this approach, we delivered exogenous miR-148b to induce apoptosis in Ras-expressing keratinocytes and murine squamous cell carcinoma cells while avoiding cytotoxicity in untransformed keratinocytes. When administered to transgenic mice with HRasG12V-driven skin tumors, a single dose of silver nanoparticle conjugates followed by 415 nm LED irradiation at the tumor site caused a rapid and sustained reduction in tumor volume by 92.8%, recruited T cells to the tumor site, and acted as a potent immunomodulator by polarizing the cytokine balance toward Th1 both locally and systemically. In summary, our results demonstrate that spatiotemporal controlled miR-148b mimic delivery can promote tumor regression efficiently and safely.

Combination Therapy and Nanoparticulate Systems: Smart Approaches for the Effective Treatment of Breast Cancer.

Breast cancer has become one of the biggest concerns for oncologists in the past few decades because of its unpredictable etiopathology and nonavailability of personalized translational medicine. The number of women getting affected by breast cancer has increased dramatically, owing to lifestyle and environmental changes. Besides, the development of multidrug resistance has become a challenge in the therapeutic management of breast cancer. Studies reveal that the use of monotherapy is not effective in the management of breast cancer due to high toxicity and the development of resistance. Combination therapies, such as radiation therapy with adjuvant therapy, endocrine therapy with chemotherapy, and targeted therapy with immunotherapy, are found to be effective. Thus, multimodal and combination treatments, along with nanomedicine, have emerged as a promising strategy with minimum side effects and drug resistance. In this review, we emphasize the multimodal approaches and recent advancements in breast cancer treatment modalities, giving importance to the current data on clinical trials. The novel treatment approach by targeted therapy, according to type, such as luminal, HER2 positive, and triple-negative breast cancer, are discussed. Further, passive and active targeting technologies, including nanoparticles, bioconjugate systems, stimuli-responsive, and nucleic acid delivery systems, including siRNA and aptamer, are explained. The recent research exploring the role of nanomedicine in combination therapy and the possible use of artificial intelligence in breast cancer therapy is also discussed herein. The complexity and dynamism of disease changes require the constant upgrading of knowledge, and innovation is essential for future drug development for treating breast cancer.

The Cell Membrane of Sulfolobus spp.-Homeoviscous Adaption and Biotechnological Applications.

The microbial cell membrane is affected by physicochemical parameters, such as temperature and pH, but also by the specific growth rate of the host organism. Homeoviscous adaption describes the process of maintaining membrane fluidity and permeability throughout these environmental changes. Archaea, and thereby, Sulfolobus spp. exhibit a unique lipid composition of ether lipids, which are altered in regard to the ratio of diether to tetraether lipids, number of cyclopentane rings and type of head groups, as a coping mechanism against environmental changes. The main biotechnological application of the membrane lipids of Sulfolobus spp. are so called archaeosomes. Archaeosomes are liposomes which are fully or partly generated from archaeal lipids and harbor the potential to be used as drug delivery systems for vaccines, proteins, peptides and nucleic acids. This review summarizes the influence of environmental parameters on the cell membrane of Sulfolobus spp. and the biotechnological applications of their membrane lipids.

A Nanoprimer To Improve the Systemic Delivery of siRNA and mRNA.

Despite tremendous interest in gene therapies, the systemic delivery of nucleic acids still faces substantial challenges. To successfully administer nucleic acids, one approach is to encapsulate them in lipid nanoparticles (LNPs). However, LNPs administered intravenously substantially accumulate in the liver where they are taken up by the reticuloendothelial system (RES). Here, we administer prior to the LNPs a liposome designed to transiently occupy liver cells, the Nanoprimer. This study demonstrates that the pretreatment of mice with the Nanoprimer decreases the LNPs' uptake by the RES. By accumulating rapidly in the liver cells, the Nanoprimer improves the bioavailability of the LNPs encapsulating human erythropoietin (hEPO) mRNA or factor VII (FVII) siRNA, leading respectively to more hEPO production (by 32%) or FVII silencing (by 49%). The use of the Nanoprimer offers a new strategy to improve the systemic delivery of RNA-based therapeutics.

A Study of the Cellular Uptake of Magnetic Branched Amphiphilic Peptide Capsules.

Understanding cellular uptake mechanisms of nanoparticles with therapeutic potential has become critical in the field of drug delivery. Elucidation of cellular entry routes can aid in the dissection of the complex intracellular trafficking and potentially allow for the manipulation of nanoparticle fate after cellular delivery (i.e., avoid lysosomal degradation). Branched amphiphilic peptide capsules (BAPCs) are peptide nanoparticles that have been and are being explored as delivery systems for nucleic acids and other therapeutic molecules in vitro and in vivo. In the present study, we determined the cellular uptake routes of BAPCs with and without a magnetic nanobead core (BAPc-MNBs) in two cell lines: macrophages and intestinal epithelial cells. We also studied the influence of size and growth media composition in this cellular process. Substituting the water-filled core with magnetic nanobeads might provide the peptide bilayer nanocapsules with added functionalities, facilitating their use in bio/immunoassays, magnetic field guided drug delivery, and magnetofection among others. Results suggest that BAPc-MNBs are internalized into the cytosol using more than one endocytic pathway. Flow cytometry and analysis of reactive oxygen and nitrogen species (ROS/RNS) demonstrated that cell viability was minimally impacted by BAPc-MNBs. Cellular uptake pathways of peptide vesicles remain poorly understood, particularly with respect to endocytosis and intracellular trafficking. Outcomes from these studies provide a fundamental understanding of the cellular uptake of this peptide-based delivery system which will allow for strengthening of their delivery capabilities and expanding their applications both in vitro and in vivo.

Extracellular vesicle-mediated nucleic acid transfer and reprogramming in the tumor microenvironment

Extracellular vesicles (EVs) have garnered much attention as key mediators of intercellular communication within the tumor microenvironment (TME) as well as at distinct metastatic sites. Nucleic acid molecules are the important components of the EV cargo. Characterizing EVs and strategies for modulating the nucleic acid content to promote anti-tumoral functions has led to the emerging role of EVs as potential novel targets for cancer therapy. Recent approaches of engineering the EVs to reach targeted sites have bought this to the forefront for nucleic acid delivery. In this article, we discuss EV biology with recent methods to analyze their nucleic acid contents. We emphasize the role of EV-mediated nucleic acid transfer in the TME assisting in tumor progression and metastasis and further review the strategies for modulating the nucleic acid content in EV for suppressing tumor growth and immune activation. The article further discusses the recent developments in generating EV mimics as nucleic acid delivery systems.

Сationic liposomes as delivery systems for nucleic acids

Objectives. Gene therapy is based on the introduction of genetic material into cells, tissues, or organs for the treatment of hereditary or acquired diseases. A key factor in the success of gene therapy is the development of delivery systems that can efficiently transfer genetic material to the place of their therapeutic action without causing any associated side effects. Over the past 10 years, significant effort has been directed toward creating more efficient and biocompatible vectors capable of transferring nucleic acids (NAs) into cells without inducing an immune response. Cationic liposomes are among the most versatile tools for delivering NAs into cells; however, the use of liposomes for gene therapy is limited by their low specificity. This is due to the presence of various biological barriers to the complex of liposomes with NA, including instability in biological fluids, interaction with serum proteins, plasma and nuclear membranes, and endosomal degradation. This review summarizes the results of research in recent years on the development of cationic liposomes that are effective in vitro and in vivo. Particular attention is paid to the individual structural elements of cationic liposomes that determine the transfection efficiency and cytotoxicity. The purpose of this review was to provide a theoretical justification of the most promising choice of cationic liposomes for the delivery of NAs into eukaryotic cells and study the effect of the composition of cationic lipids (CLs) on the transfection efficiency in vitro.Results. As a result of the analysis of the related literature, it can be argued that one of the most promising delivery systems of NAs is CL based on cholesterol and spermine with the addition of a helper lipid DOPE. In addition, it was found that varying the composition of cationic liposomes, the ratio of CL to NA, or the size and zeta potential of liposomes has a significant effect on the transfection efficiency.Conclusions. Further studies in this direction should include optimization of the conditions for obtaining cationic liposomes, taking into account the physicochemical properties and established laws. It is necessary to identify mechanisms that increase the efficiency of NA delivery in vitro by searching for optimal structures of cationic liposomes, determining the ratio of lipoplex components, and studying the delivery efficiency and properties of multicomponent liposomes.

Multi-targeted Antisense Oligonucleotide Delivery by a Framework Nucleic Acid for Inhibiting Biofilm Formation and Virulence

HighlightsA framework nucleic acid delivery system was developed through self-assembly, which can deliver antisense oligonucleotides against multiple targets in bacterial cells.The ASOs-tFNAs (750 nM) was found to simultaneously inhibit the expression of gtfBCD, gbpB, and ftf, and significantly reduce the extracellular polysaccharide synthesis and biofilm thickness.

Degradable branched polycationic systems for nucleic acid delivery.

Nowadays, nucleic acid-based therapy has become a promising way for the treatment of various malignant diseases like cancers. However, the process of nucleic acid delivery is hampered by several extracellular and intracellular obstacles. To overcome these obstacles, many nucleic acid delivery carriers have been developed to achieve improved safety and enhanced gene transfection performance. Among these carriers, degradable branched polycations have attracted much attention due to their good responsive degradability and impressive transfection performances. In this review, we mainly summarized the redox- and pH-responsive degradable branched polycationic systems used for nucleic acid delivery. For responsive degradable branched polycationic systems, the preparation methods were introduced, where amino-epoxy ring-opening reaction as a novel step growth approach was mainly presented. The properties of redox- and pH-responsive degradable branched polycationic systems were subsequently introduced and highlighted. We hope this brief review will motivate the delicate design of responsive degradable branched polycationic systems for potential clinical applications. This article is categorized under: Biology-Inspired Nanomaterials > Nucleic Acid-Based Structures.

Deciphering the internalization mechanism of WRAP:siRNA nanoparticles

Gene silencing mediated by double-stranded small interfering RNA (siRNA) has been widely investigated as a potential therapeutic approach for a variety of diseases and, indeed, the first therapeutic siRNA was approved by the FDA in 2018. As an alternative to the traditional delivery systems for nucleic acids, peptide-based nanoparticles (PBNs) have been applied successfully for siRNA delivery. Recently, we have developed amphipathic cell-penetrating peptides (CPPs), called WRAP allowing a rapid and efficient siRNA delivery into several cell lines at low doses (20 to 50 nM).In this study, using a highly specific gene silencing system, we aimed to elucidate the cellular uptake mechanism of WRAP:siRNA nanoparticles by combining biophysical, biological, confocal and electron microscopy approaches. We demonstrated that WRAP:siRNA complexes remain fully active in the presence of chemical inhibitors of different endosomal pathways suggesting a direct cell membrane translocation mechanism. Leakage studies on lipid vesicles indicated membrane destabilization properties of the nanoparticles and this was supported by the measurement of WRAP:siRNA internalization in dynamin triple-KO cells. However, we also observed some evidences for an endocytosis-dependent cellular internalization. Indeed, nanoparticles co-localized with transferrin, siRNA silencing was inhibited by the scavenger receptor A inhibitor Poly I and nanoparticles encapsulated in vesicles were observed by electron microscopy in U87 cells.In conclusion, we demonstrate here that the efficiency of WRAP:siRNA nanoparticles is mainly based on the use of multiple internalization mechanisms including direct translocation as well as endocytosis-dependent pathways.

Molecular and Coarse-Grained Modeling to Characterize and Optimize Dendrimer-Based Nanocarriers for Short Interfering RNA Delivery.

Dendrimer nanocarriers are unique hyper-branched polymers with biomolecule-like properties, representing a promising prospect as a nucleic acid delivery system. The design of effective dendrimer-based gene carriers requires considering several parameters, such as carrier morphology, size, molecular weight, surface chemistry, and flexibility/rigidity. In detail, the rational design of the dendrimer surface chemistry has been ascertained to play a crucial role on the efficiency of interaction with nucleic acids. Within this framework, advances in the field of organic chemistry have allowed us to design dendrimers with even small difference in the chemical structure of their surface terminals. In this study, we have selected two different cationic phosphorus dendrimers of generation 3 functionalized, respectively, with pyrrolidinium (DP) and morpholinium (DM) surface groups, which have demonstrated promising potential for short interfering RNA (siRNA) delivery. Despite DP and DM differing only for one atom in their chemical structure, in vitro and in vivo experiments have highlighted several differences between them in terms of siRNA complexation properties. In this context, we have employed coarse-grained molecular dynamics simulation techniques to shed light on the supramolecular characteristics of dendrimer–siRNA complexation, the so-called dendriplex formations. Our data provide important information on self-assembly dynamics driven by surface chemistry and competition mechanisms.

Amphiphilic Polypeptides for VEGF siRNA Delivery into Retinal Epithelial Cells.

Polyethyleneimine, poly-L-lysine, chitosan and some others cationic polymers have been thoroughly studied as nucleic acid delivery systems in gene therapy. However, the drug release from these systems proceeds at a very low rate due to extremely high binding between a carrier and gene material. To reduce these interactions and to enhance drug release, we developed a set of amphiphilic polypeptides containing positively and negatively charged amino acids as well as a hydrophobic one. The copolymers obtained were characterized by size-exclusion chromatography, static light scattering, HPLC amino acid analysis and 1HNMR spectroscopy. All copolymers formed particles due to a self-assembly in aqueous media. Depending on polypeptide composition, the formation of particles with hydrodynamic diameters from 180 to 900 nm was observed. Stability of polymer particles, loading and release efficiency were carefully studied. Cellular uptake of the particles was efficient and their cytotoxicity was negligible. The application of polymer carriers, containing siRNA, to vascular endothelial growth factor (VEGF-A165) silencing of ARPE-19 cells was successful. The gene silencing was confirmed by suppression of both messenger RNA and protein expression.

Investigation of Nanoparticles for Nucleic Acid Delivery System

Investigation of Nanoparticles for Nucleic Acid Delivery System

Tunable Composition of Dynamic Non-Viral Vectors over the DNA Polyplex Formation and Nucleic Acid Transfection.

Polyethylene glycol (PEG) functionalization of non-viral vectors represents a powerful tool through the formation of an overall surface charge shielding ability, which is fundamental for efficient nucleic acid delivery systems. The degree of non-viral vector PEGylation and the molecular weight of utilized PEG is crucial since the excessive use of PEG units may lead to a considerable reduction of the DNA-binding capacity and, subsequently, in a reduction of in vitro transfection efficiency. Herein, we report a detailed study on a series of dynamic combinatorial frameworks (DCFs) containing PEGylated squalene, poly-(ethyleneglycol)-bis(3-aminopropyl) of different lengths, and branched low molecular weight polyethylenimine components, reversibly connected in hyperbranched structures, as efficient dynamic non-viral vectors. The obtained frameworks were capable of forming distinct supramolecular amphiphilic architectures, shown by transmission electron microscopy (TEM) and dynamic light scattering (DLS), with sizes and stability depending on the length of PEG units. The interaction of PEGylated DCFs with nucleic acids was investigated by agarose gel retardation assay and atomic force microscopy (AFM), while their transfection efficiency (using pCS2+MT-Luc DNA as a reporter gene) and cytotoxicity were evaluated in HeLa cells. In addition, the data on the influence of the poly-(ethyleneglycol)-bis(3-aminopropyl) length in composition of designed frameworks over transfection efficiency and tolerance in human cells were analyzed and compared.

Endocytic Profiling of Cancer Cell Models Reveals Critical Factors Influencing LNP-Mediated mRNA Delivery and Protein Expression.

Lipid nanoparticles have great potential for delivering nucleic-acid-based therapeutics, but low efficiency limits their broad clinical translation. Differences in transfection capacity between invitro models used for nanoparticle pre-clinical testing are poorly understood. To address this, using a clinically relevant lipid nanoparticle (LNP) delivering mRNA, we highlight specific endosomal characteristics in invitro tumor models that impact protein expression. A 30-cell line LNP-mRNA transfection screen identified three cell lines having low, medium, and high transfection that correlated with protein expression when they were analyzed in tumor models. Endocyticprofiling of these cell lines identified major differences in endolysosomal morphology, localization, endocytic uptake, trafficking, recycling, and endolysosomal pH, identified using a novel pH probe. High-transfecting cells showed rapid LNP uptake and trafficking through an organized endocytic pathway to lysosomes or rapid exocytosis. Low-transfecting cells demonstrated slower endosomal LNP trafficking to lysosomes and defective endocytic organization and acidification. Our data establish that efficient LNP-mRNA transfection relies on an early and narrow endosomal escape window prior to lysosomal sequestration and/or exocytosis. Endocytic profiling should form an important pre-clinical evaluation step for nucleic acid delivery systems to inform model selection and guide delivery-system design for improved clinical translation.

The Impact of Alkyl-Chain Purity on Lipid-Based Nucleic Acid Delivery Systems - Is the Utilization of Lipid Components with Technical Grade Justified?

The physicochemical properties and transfection efficacies of two samples of a cationic lipid have been investigated and compared in 2D (monolayers at the air/liquid interface) and 3D (aqueous bulk dispersions) model systems using different techniques. The samples differ only in their chain composition due to the purity of the oleylamine (chain precursor). Lipid 8 (using the oleylamine of technical grade for cost‐efficient synthesis) shows lateral phase separation in the Langmuir layers. However, the amount of attached DNA, determined by IRRAS, is for both samples the same. In 3D systems, lipid 8 p forms cubic phases, which disappear after addition of DNA. At physiological temperatures, both lipids (alone and in mixture with cholesterol) assemble to lamellar aggregates and exhibit comparable DNA delivery efficiency. This study demonstrates that non‐lamellar structures are not compulsory for high transfection rates. The results legitimate the utilization of oleyl chains of technical grade in the synthesis of cationic transfection lipids.

Reduction-Responsive Nucleic Acid Delivery Systems To Prevent In-Stent Restenosis in Rabbits.

Cardiovascular and cerebrovascular ischemic diseases seriously affect human health. Endovascular stent placement is an effective treatment but always leads to in-stent restenosis (ISR). Gene-eluting stent, which combines gene therapy with stent implantation, is a potential method to prevent ISR. In this study, an efficient gene-eluting stent was designed on the basis of one new nucleic acid delivery system to decrease the possibility of ISR. The reduction-responsive branched nucleic acid vector (SKP) with low cytotoxicity was first synthesized via ring-opening reaction. The impressive in vitro transfection performances of SKP were proved using luciferase reporter, enhanced green fluorescent protein plasmid, and vascular endothelial growth factor plasmid (pVEGF). Subsequently, SKP/pVEGF complexes were coated on the surfaces of pretreated clinical stents to construct gene-eluting stents (S-SKP/pVEGF). Antirestenosis performance of S-SKP/pVEGF was evaluated via implanting stents into rabbit aortas. S-SKP/pVEGF could lead to the localized upregulation of VEGF proteins, improve the progress of re-endothelialization, and inhibit the development of ISR in vivo. Such efficient pVEGF-eluting stent with responsive nucleic acid delivery systems is very promising to prevent in-stent restenosis of cerebrovascular diseases.

Nanocarrier-based systems for targeted and site specific therapeutic delivery.

Systemic drug delivery methods such as oral or parenteral administration of free drugs possess relatively low treatment efficiency and marked adverse side effects. The use of nanoparticles for drug delivery in most cases substantially enhances drug efficacy, improves pharmacokinetics and drug release and limits their side effects. However, further enhancement in drug efficacy and significant limitation of adverse side effects can be achieved by specific targeting of nanocarrier-based delivery systems especially in combination with local administration. The present review describes major advantages and limitations of organic and inorganic nanocarriers or living cell-based drug and nucleic acid delivery systems. Among these, different nanoparticles, supramolecular gels, therapeutic cells as living drug carriers etc. have emerged as a new frontier in modern medicine.

Efficient bioactive oligonucleotide-protein conjugation for cell-targeted cancer therapy.

Oligonucleotide‐protein conjugates have important applications in biomedicine. Simple and efficient methods are described for the preparation of these conjugates. Specifically, we describe a new method in which a bifunctional linker is attached to thiol‐oligonucleotide to generate a reactive intermediate that is used to link to the protein. Having similar conjugation efficacy compared with the classical method in which the bifunctional linker is attached first to the protein, this new approach produces significantly more active conjugates with higher batch to batch reproducibility. In a second approach, direct conjugation is proposed using oligonucleotides carrying carboxyl groups. These methodologies have been applied to prepare nanoconjugates of an engineered nanoparticle protein carrying a T22 peptide with affinity for the CXCR4 chemokine receptor and oligomers of the antiproliferative nucleotide 2’‐deoxy‐5‐fluorouridine in a very efficient way. The protocols have potential uses for the functionalization of proteins, amino‐containing polymers or amino‐lipids in order to produce complex therapeutic nucleic acid delivery systems.