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Nonviral Approach for Targeted Nucleic Acid Delivery
Abstract and Figures
Despite their relatively lower efficiency, nonviral approaches are emerging as safer alternatives in gene therapy to viral vectors. Delivery of nucleic acids to the target site is an important factor for effective gene expression (plasmid DNA) or knockdown (siRNA) with minimal side effects. Direct deposition at the target site by physical methods, including ultrasound, electroporation and gene gun, is one approach for local delivery. For less accessible sites, the development of carriers that can home into the target tissue is required. Cationic peptides, lipoplexes, polyplexes and nanoplexes have been used as carriers for delivery of nucleic acids. Targeting ligands, such as cell targeting peptides, have also been applied to decorate delivery vehicles in order to enhance their efficacy. This review focuses on delivery strategies and recent progress in non-viral carriers and their modifications to improve their performance in targeting and transfection.
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... Because non-viral carriers show reduced pathogenicity, they are attractive candidates to be developed based on nanosized lipid or polymeric carriers [20]. Lipid-based nanocarriers, often the preferred choice, naturally interact with the cell membrane, mainly composed by lipids [21]. ...
Introduction:
During past years, lipid nanoparticles (LNPs) have emerged as promising carriers for RNA delivery, with several clinical trials focusing on both infectious diseases and cancer. More recently, the success of messenger RNA (mRNA) vaccines for the treatment of severe diseases such as acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is partially justified by the development of LNPs encapsulating mRNA for efficient cytosolic delivery.
Areas covered:
This review examines the production and formulation of LNPs by using microfluidic devices, the status of mRNA-loaded LNPs therapeutics and explores spray drying process, as a promising dehydration process to enhance LNP stability and provide alternative administration routes.
Expert opinion:
Microfluidic techniques for preparation of LNPs based on organic solvent injection method promotes the generation of stable, uniform, and monodispersed nanoparticles enabling higher encapsulation efficiency. In particular, the application of microfluidics for the fabrication of mRNA-loaded LNPs is based on rapid mixing of small volumes of ethanol solution containing lipids and aqueous solution containing mRNA. Control of operating parameters and formulation has enabled the optimization of nanoparticle physicochemical characteristics and encapsulation efficiency.
... The use of naked plasmid DNA (pDNA) encoding a therapeutic gene in combination either with electric fields, ultrasound, gene gun or hydrodynamic injection can provide significant transfection efficiency [1][2][3][4][5][6][7][8][9]. Cationic lipids and cationic polymers forming electrostatic complexes with pDNA encoding a therapeutic gene are attractive carriers to transfect cells in a nonviral manner [10][11][12][13][14][15][16]. Nevertheless, they still experience inferior transfection efficiency compared to viruses. ...
In nonviral gene therapy approaches, the linkage of signal molecules to plasmid DNA (pDNA) is of interest for guiding its delivery to the nucleus. Here, we report its linkage to a peptide (P79–98) mediating migration on microtubules by using a triplex-forming oligonucleotide (TFO). pDNA of 5 kbp and 21 kbp containing 6 and 36 oligopurine • oligopyrimidine sites (TH), respectively, inserted outside the luciferase gene sequence were used. TFO with a dibenzocyclooctyl (DBCO) group in 3’ end comprising some Bridged Nucleic Acid bases was conjugated by click chemistry with the peptide carrying an azide function in the C-terminal end. We found the formation of 6 and 18 triplex with pDNA of 5 kbp and 21 kbp, respectively. A twofold increase of the transfection efficiency was observed in the hind-limbs upon Hydrodynamic Limb Vein (HLV) injection in mice of naked P79–98 –pDNA of 21 kbp. This work paves the way for the selective equipping of pDNA with intracellular targeting molecules while preserving the full expression of the encoded gene.
... reported TAT peptide mediated nanofibers formation and its application to encapsulate the hydrophobic drug paclitaxel (PTX) with a high loading capacity (Zhang et al., 2013). Numerous groups have shown TAT peptide as drug and nucleic acid delivery systems (Jafari et al., 2012). Based on this rationale, TAT peptide has been used as efficient positive control to evaluate the efficiency of the peptide in this study. ...
Vascular endothelial growth factor (VEGF) is considered as one of the vital growth factors for angiogenesis, which is primarily responsible for the progress and maintenance of new vascular network in tumor. Numerous studies report that inhibition of VEGF-induced angiogenesis is a potent technique for cancer suppression. Recently, RNA interference, especially small interfering RNA (siRNA) signified a promising approach to suppress the gene expression. However, the clinical implementation of biological macromolecules such as siRNA is significantly limited because of stability and bioavailability issues. Herein, self-assembled peptide nanospheres have been generated from L,L-cyclic peptides using hydrophobic (Trp), positively charged (Arg) and cysteine (Cys) amino acid residues and demonstrated as vehicles for intracellular delivery of VEGF siRNA and VEGF antisense oligonucleotide. Formation of peptide nanostructures is confirmed by HR-TEM, AFM, SEM and DLS analysis. Possible mechanism of self-assembly of the cyclic peptides and their binding with macromolecules are demonstrated by in-silico analysis. Gel electrophoresis reveals that the newly generated peptide based organic materials exhibit strong binding affinity toward siRNAs / antisense oligonucleotides (ASOs) at optimum concentration. Flow cytometry and confocal microscopy results confirm the efficiency of the new biomaterials toward the intracellular delivery of fluorescent labeled siRNA / ASOs. Furthermore, VEGF expression evaluated by western blot and RT-PCR upon the delivery of functional VEGF siRNA/ASOs suggests that very low concentrations of VEGF siRNA/ASOs cause significant gene knockdown at protein and mRNA levels, respectively.
... Their positive charge, besides, permits collaborations with the contrarily charged cell layer and therefore entrance into the cell is permitted. (26) There are various reports on the utilization of different cationic lipids as nonviral gene conveyance vectors. (27) Cationic liposomes are utilized as of late is siRNA conveyance. ...
... The search for effective delivery systems for NA-based drugs is an urgent task. Viral vectors, liposomes, cationic polymers of various nature, transport peptides, and other transport systems are used for cell transfection [14]. Despite the efforts made, the task cannot be considered complete. ...
Newly emerged highly pathogenic A/H7N9 viruses with pandemic potential are effectively transmitted from birds to humans and require the development of novel antiviral drugs. For the first time, we studied the in vitro and in vivo antiviral activity against A/H7N9 of oligodeoxyribonucleotides (ODNs), which were delivered into the cells in the proposed TiO2-based nanocomposites (TiO2∼ODN). The highest inhibition of A/H7N9 in vitro (∼400-fold) and efficient, sequence-specific, and dose-dependent protection (up to 100%) of A/H7N9-infected mice was revealed when ODN was targeted to the conserved terminal 3'-noncoding region of viral (-)RNA. After the treatment with ODN, the virus titer values in the lungs of mice decreased by several orders of magnitude. The TiO2∼ODN nanocomposite did not show toxicity in mice under the treatment conditions. The proposed approach for effective inhibition of the A/H7N9 can be tested against other viruses, for example, new emerging influenza viruses and coronaviruses with pandemic potential.
... Due to the presence of surface charges (SCs), polyplexes offer effective complexation with genetic materials viz. siRNA for gene therapy, DNA for cancer therapy, etc. Polyplexes are polymeric complex of active to increase the solubility, delay the degradation step and offer site-specific action [5,6]. The adaptable structure enables the active to form complex linkage with polymers like polyethyleneimine and poly-l-lysine for controlled release pattern [7]. ...
Despite recent advances in the treatment of human colon cancer, the chemotherapeutic efficacy against colon cancer is still unsatisfactory. The complexity in colorectal cancer treatment leads to new research in combination therapy to overcome multidrug resistance in cancer and increase apoptosis. The objective of the present research work was to develop polyplexes for co-delivery of plasmid DNA with retinoic acid against colorectal cancer cell line (HCT-15). Plain polyplexes were prepared using chitosan and hyaluronic acid solution (0.1% w/v), whereas retinoic acid polyplexes were prepared using ethanol: water (1:9 v/v) system. The particle size was observed in the order of chitosan solution > blank polyplex > retinoic acid-loaded polyplex. Encapsulation efficiency of retinoic acid was found to be 81.51 ± 4.33% for retinoic acid-loaded polyplex formulation. The drug release was observed to be in a controlled pattern with 72.23 ± 1.32% release of retenoic acid from polyplex formulation. Cell line studies of the formulation displayed better cell inhibition and low cytotoxicity for the retinoic acid-loaded polyplexes in comparison to pure retinoic acid, thus demonstrating better potential action against colorectal cancer cell line HCT-15. Retinoic acid-loaded polyplexes indicated higher potential for the delivery of the active whereas the cell line studies displayed the efficacy of the formulation against colorectal cancer cell line HCT-15.
Duchenne Muscular Dystrophy and Cystic Fibrosis are two major monogenetic diseases which could be treated by non-viral gene therapy. For this purpose, plasmid DNA (pDNA) coding for the functional genes requires its equipment with signal molecules favouring its intracellular trafficking and delivery in the nucleus of the target cells. Here, two novel constructions of large pDNAs encoding the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) and full-length dystrophin (DYS) genes are reported. The expression of CFTR and DYS genes are driven respectively by the hCEF1 airway epithelial cells and spc5-12 muscle cells specific promoter. Those pDNAs encode also the luciferase reporter gene driven by the CMV promoter to evaluate gene delivery in animals by bioluminescence. In addition, oligopurine • oligopyrimidine sequences are inserted to enable equipment of pDNAs with peptides conjugated with a triple helix forming oligonucleotide (TFO). Furthermore, specific κB sequences are also inserted to promote their NFκB-mediated nuclear import. pDNA constructions are reported; transfection efficiency, tissue specific expression of CFTR and dystrophin in target cells, and triple helix formation are demonstrated. These plasmids are tools of interest to develop non-viral gene therapy of Cystic Fibrosis and Duchenne Muscular Dystrophy.
Herein, we developed a novel labelling precursor Fe-DFO-5 for plasmid DNA (pDNA) utilizing 89Zr as a radioisotope for PET imaging. 89Zr-labelled pDNA showed comparable gene expression to non-labelled pDNA. The biodistribution of 89Zr-labelled pDNA after local or systemic administration in mice was evaluated. Furthermore, this labelling method was also applied to mRNA.
When the brain or spinal cord are impaired by diseases or injury, it presents a unique challenge for both imaging and treatment of the impaired parts, due to high degree of protection the central nervous system has, which is aimed in maintaining brain homeostasis. One of these forms of protection is the blood‐brain barrier, which prevents harmful agents from entering the brain, however, it also prevents therapeutic agents from reaching their target inside the brain parenchyma in sufficient amounts which needed to provide a therapeutic effect. In that regard, nano system may provide a future solution, as they may contain multiple moieties which can simultaneously aid in crossing the blood brain barrier, target the diseased cells inside the brain parenchyma and carry a sufficient amount of therapeutic agents to treat the impairment. In may also contain an imaging core or moiety which then provides a dual modality system, which can treat and image at the same time. In the present chapter, such nano systems for potential treatment and imaging of central nervous system related diseases and injuries will be reviewed.
The impact of a localized application of ultrasound on gene transfer to primary tumors following systemic administration of cationic lipid based transfection complexes was investigated. We have previously shown that systemic administration of DOTMA (N-[(1-(2-3-dioleyloxy) propyl)]-N-N-N-trimethylammonium chloride):cholesterol-based transfection complexes to tumor-bearing mice resulted in expression in the tumor and other tissues, primarily the lungs. Application of ultrasound to the tumor before or after the injection resulted in a significant increase in gene transfer to the tumor with no increase observed in other tissues. The magnitude of increased expression ranged from three- to 270-fold depending upon the DNA dose. The following parameters were optimized for maximal increase: duration of ultrasound application, the time interval between plasmid injection and sonoporation, and plasmid dose. A combination of plasmid quantitation and fluorescence microscopy showed that ultrasound increased tumor uptake of the plasmid and that uptake was limited to the tumor vasculature. Using an IL- 12 expression plasmid, the combination of a single plasmid dose (10 microg) and ultrasound treatment produced significantly higher levels of IL-12 in tumor. This increased expression was sufficient to inhibit tumor growth compared with the control conditions. These data demonstrate the potential application of sonoporation as an effective method for enhancing the expression of systemically administered genes in tumor endothelium for cancer gene therapy.
At the time of the lightbulb's invention, not even the most imaginative thinkers could have predicted the prevention and treatment of disease through genes delivered by electric fields. In Electroporation Protocols: Preclinical and Clinical Gene Medicine, top researchers provide hands-on protocols describing the process of non-viral delivery of genetic materials such as DNA, oligo-DNA, and RNA into targeted immune cells, tissues, and model animals in order to better treat disease. Following the successful Methods in Molecular Biology™ series format, each method and protocol offers step-by-step, readily reproducible laboratory instructions, with the benefit of several introductory reviews to aid investigators new to this exciting technology.
Ideal for beginners and essential for the more knowledgable, Electroporation Protocols: Preclinical and Clinical Gene Medicine collects a comprehensive set of the newest discoveries and applications in a revolutionary field that offers so much to our fight against disease.
Background — Although viral vector systems are efficient to transfect foreign genes into blood vessels, safety issues remain in relation to human gene therapy. In this study, we examined the feasibility of a novel nonviral vector system by using high-frequency, low-intensity ultrasound irradiation for transfection into blood vessels.
Methods and Results — Luciferase plasmid mixed with or without echo contrast microbubble (Optison) was transfected into cultured human vascular smooth muscle cells (VSMC) and endothelial cells (EC) with the use of ultrasound. Interestingly, luciferase activity was markedly increased in both cell types treated with Optison. We then transfected luciferase plasmid mixed with Optison by means of therapeutic ultrasound into rat artery. Two days after transfection, luciferase activity was significantly higher in carotid artery transfected with luciferase gene with Optison and ultrasound than with plasmid alone. In addition, we transfected an anti-oncogene (p53) plasmid into carotid artery after balloon injury as a model of gene therapy for restenosis. Two weeks after transfection, the intimal-to-medial area ratio in rats transfected with wild-type p53 plasmid complexed with Optison by means of ultrasound was significantly decreased as compared with control, accompanied by a significant increase in p53 protein. No apparent toxicity such as inflammation could be detected in blood vessels transfected with plasmid DNA with ultrasound and Optison.
Conclusions — Overall, we demonstrated that an ultrasound transfection method with Optison enhanced transfection efficiency of naked plasmid DNA into blood vessels without any apparent toxicity. Transfection of p53 plasmid with the use of this method should be useful for safe clinical gene therapy without a viral vector system.
Background— Local gene therapy has enormous potential for the treatment of vascular disease. We determined whether diagnostic ultrasound-mediated destruction of plasmid-loaded albumin microbubbles is a feasible and efficient technique for local vascular gene delivery. For gene transfer, we used a phosphomimetic, active endothelial nitric oxide synthase (eNOS) construct in which Ser1177 was replaced by aspartic acid (S1177D) and exhibits a 2-fold higher basal activity than the wild-type enzyme.
Methods and Results— Gas-filled microbubbles (3.0±1.2 μm) were created by sonication of 5% human albumin in the presence of plasmid DNA encoding for LacZ or eNOS S1177D. Porcine coronary arteries were perfused with DNA-loaded albumin microbubbles in vitro, exposed to diagnostic ultrasound (5 seconds), and incubated for a further 24 hours. Detection of the β-galactosidase in LacZ-transfected vessels revealed a predominant staining of endothelial cells without any functional impairment of vasoreactivity. Western blotting demonstrated the expression of the eNOS S1177D construct in extracts from the transfected segments. Vascular responsiveness was tested with prostaglandin F2α and the NOS inhibitor Nωnitro-l-arginine. Compared with segments treated with the expression plasmid alone, the contractile response to prostaglandin F2α was impaired in segments transfected with eNOS S1177D, whereas the contractile response to the administration of Nωnitro-l-arginine was markedly enhanced.
Conclusions— Ultrasound-mediated destruction of eNOS S1177D DNA-loaded albumin microbubbles is a feasible and efficient method for vascular gene transfection. Transfection resulted in significant protein expression and enhanced NO-mediated relaxation of bradykinin-stimulated porcine coronary arteries.
This chapter describes a new strategy for the delivery of full-length proteins and peptides into mammalian cells based on a short amphipathic peptide carrier, Pep-1. This peptide carrier allows the delivery of several distinct proteins and peptides into different cell lines in a fully biologically active form, without the need for prior chemical covalent coupling or denaturation steps. Pep-1/macromolecule particle is favored as soon as it has crossed the cell membrane. This peptide-based protein delivery strategy presents several advantages, including rapid delivery of proteins into cells with very high efficiency, stability in physiological buffers, lack of toxicity, and lack of sensitivity to serum. Detection of fluorescently labeled antibodies in cells requires a large amount of antibody to be delivered. Depending on the sensitivity required to detect antibodies, it may be necessary to increase the amount of Pep-1 and antibody used. The advantages of the Pep-1 technology are directly associated with the mechanism through which this carrier promotes the delivery of proteins and peptides into cells.
Integrins, the major class of αβ heterodimeric transmembrane glycoprotein receptors, play crucial roles in mediating tumor angiogenesis. Genetic ablation experiments combined with use of antibodies/peptide ligands for blocking either α5 or β1 integrins have convincingly demonstrated α5β1 integrin to be unquestionably proangiogenic among the 24 known integrin receptors. Herein, we report on a novel RGDK-lipopeptide 1 that targets selectively α5β1 integrin and is capable of targeting genes to mouse tumor vasculatures.
Tissue engineering scaffolds with complex geometries can provide an architecture that directs tissue formation. Drug delivery from these scaffolds to promote regeneration is often challenging due to the complex fabrication processes. Surface-mediated DNA delivery from multiple channel bridges was applied to deliver lipoplexes in vivo to the injured spinal cord. The surface properties of the polymer, DNA deposition with or without drying, and the presence of ECM components were investigated. In vitro studies revealed that fibronectin produced greater expression levels and immobilization efficiencies compared with collagen, laminin, and no coating. In addition, lipoplex incubation on ECM-coated PLG increased expression relative to either of the drying methods. Additionally, the incubation method had more homogeneously distributed lipoplexes and a higher number of transfected cells relative to the dried conditions. Translation to three-dimensional bridges led to high levels of transgene expression in vitro. In vivo, lipoplexes immobilized to the bridge produced transgene expression levels in a rat spinal cord hemisection model that were 2-fold greater than naked plasmid. Additionally, expression with lipoplexes persisted for at least three weeks. Surface-mediated delivery can be applied to scaffolds with complex geometries to promote transgene expression in vivo.
In the absence of any ideal gene delivery carrier despite the recent explosion of novel carrier systems, the current trend is to explore the complementary synergy promised by a combination of delivery systems such as liposomes, which are the most widely researched versatile non-viral carriers, and tissue-engineered scaffolds with macrostructures of defined architecture comprised of natural or synthetic macromolecules. Here, we discuss the recent advances in liposomal gene delivery and the possible benefits of a combined liposome–scaffold approach, such as long-term expression, enhanced stability, reduction in toxicity and ability to produce spatio-temporal expression patterns. This approach is generating significant impact in the field as a result of its potential for extended localised gene delivery for applications in a variety of clinical conditions.
RNA interference (RNAi) is a sequence-specific gene-silencing mechanism triggered by synthetic small interfering RNA (siRNA), and is utilized in a wide range of fields including cancer gene therapy by down-regulating a specific target protein. In this study, for tumor-targeted siRNA delivery, we developed a folate-linked nanoparticle (NP-F), and evaluated the potential of NP-F-mediated tumor gene therapy in human nasopharyngeal KB cells, which overexpressed folate receptor (FR). NP-F was composed of cholesteryl-3beta-carboxyamidoethylene-N-hydroxyethylamine (OH-Chol), Tween 80 and folate-poly(ethylene glycol)-distearoylphosphatidylethanolamine conjugate (f-PEG(2000)-DSPE), and NP-P was substituted f-PEG(2000)-DSPE in NP-F PEG(2000)-DSPE for a non-targeting nanoparticle. The NP-F and siRNA complex (nanoplex) formed at a charge ratio (+/-) of 2/1 in the presence of 5mM NaCl was injectable size and increased transfection efficiency in the cells. NP-F showed a significantly higher intracellular amount of siRNA and stronger localization of siRNA in the cytoplasm than NP-P. When Her-2 siRNA was transfected into cells by NP-F and NP-P, NP-F significantly inhibited tumor growth, and selectively suppressed Her-2 protein expression more than NP-P. In in vivo gene therapy, a NP-F nanoplex of Her-2 siRNA by intratumoral injection significantly inhibited tumor growth of KB xenografts compared with control siRNA, but a NP-P nanoplex did not. These results of the experiments have provided optimal conditions to form folate-linked nanoparticle complexes with siRNA for folate-targeted gene therapy.