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4 Polycationic polymers for nanoparticle production. (a) PEI (branched and linear), (b) PAMAM dendrimers and (c) PDMAEMA are representative polycationic polymers that have been thoroughly studied for polyplex formation and delivery of nucleic acids.
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There are significant engineering challenges in translating the remarkable medical implications of gene and nucleic acid delivery from cell and animal models into the clinic. Off-target effects and inefficient delivery to the proper intracellular compartment of the targeted cells are major obstacles to success. Systemic delivery of any viral or non...
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Nanoparticle (NP) therapeutics can improve the therapeutic index of chemoradiotherapy (CRT). However, the effect of NP physical properties, such particle size, on CRT is unknown. To address this, we examined the effects of NP size on biodistribution, efficacy and toxicity in CRT. PEG-PLGA NPs (50, 100, 150 nm mean diameters) encapsulating wotrmanni...
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... Tissue engineering (TE) aims to create new tissue or organ by combining a large number of cells together with biocompatible materials and cell transplantation fields for the treatment of damaged tissue or organ (Duvall et al., 2013a(Duvall et al., , 2013bEnderle & Bronzino, 2011). The fields of biomaterials, three-dimensional (3D) printing technologies, nanotechnology, induced pluripotent stem cells (iPSCs), and gene editing technologies (such as clustered regularly interspaced short palindromic repeats, CRISPR) are technologies that TE benefits from and are important for modeling disease and treatment modalities. ...
Glioblastoma is a highly malignant brain tumor classified as grade IV with a poor prognosis and approximately a year of survival rate. The molecular changes that trigger primary glioblastoma are usually epidermal growth factor receptor mutations and amplifications, Mouse Double Minute and TP53 mutations, p16 deletion, phosphatase and tensin homolog and telomerase promoter mutations. In the vast majority of glioblastomas, altered signaling pathways were identified as receptor tyrosine kinase/Ras/PI3K, p53. Isocitrate dehydrogenase 1/2 mutations have also been associated with poor prognosis in glioblastoma The treatment options are very limited and complicated because of the diverse composition and heterogeneity of the tumors and unresponsiveness to the treatments with the existence of barriers reaching the brain tissue. Despite new trials, drug candidates that appeared effective in cell culture or mouse models failed in the clinic. Recently, new sophisticated experimental systems, including the those that mimic the tumor microenvironment, have started being used by several research groups, which will allow accurate prediction of drug efficacy. Tissue engineering strategies are also being combined with innovative cancer models, including spheroids, tumorspheres, organotypic slices, explants, tumoroids, and organoids. Such 3D systems provide powerful tools for studying glioblastoma biology by representing the dynamic evolution of the disease from the early to the metastatic stages and enabling interaction with the microenvironment. In this review, we both enlighten the molecular mechanisms that lead to glioblastoma development and detailed information on the tissue engineering approaches that have been used to model glioblastoma and the tumor microenvironment with the advantages and disadvantages. We anticipate that these novel approaches could improve the reliability of preclinical data by reducing the need for animal models.
... Many attempts such as modification of electric field, buffers and electrodes have been made and the optimization of electric fields setting seems to be very important [30]. Many types of electrodes have been developed, and different materials have been used for electrodes production [67]. Using this technique, gene replacement can be designed in cases of retinal inheritance or corneal disease. ...
The aim of this document is to present an overview of gene electrotransfer in ophthalmological disorders. In order to ensure an adequate variety of the assessed studies, several electronic databases were considered and studies published between January 1998 and December 2021 were analysed. Three investigators carried out data extraction and analysis, focusing on both technical (i.e., electrical protocol, type of electrode, plasmid) and medical (i.e., type of study, threated disease) aspects and highlighting the main differences in terms of results obtained. Moreover, the IGEA experience in the project “Transposon-based, targeted ex vivo gene therapy to treat age-related macular degeneration” (TargetAMD) was reported in the results section. No clinical trial was found on international literature and on clinicaltrial.gov. Twelve preclinical studies were found including in vivo and ex-vivo applications. The studied showed that electrotransfer could be very efficient for plasmid DNA transfection. Many attempts such as modification of the electric field, buffers and electrodes have been made and the optimization of electric field setting seems to be very important. Using this technique, gene replacement can be designed in cases of retinal inheritance or corneal disease and a wide range of human eye diseases could, in the future, benefitfrom these gene therapy technologies.
... When it comes to genetic disorders, pDNA vectors are typically utilized to re-express an endogenous or non-functional gene. In an approach to treating autoimmune skin disorders, an attempt was made to deliver pDNA encoding IL-12 by Heller's team to treat melanoma [75,76]. However, the delivery of pDNA is restricted by DNA degrading nucleases present in tissues or blood, minimal distribution at the delivery site, deprived cell targeting, incapability to cross barrier membranes, less cellular uptake, and intracellular nucleocytoplasmic trafficking. ...
Various skin problems are affected significantly by a combination of factors that include genetics, environmental factors, particular infections, and hitherto unexplained variables. The structure and function of key cellular networks may be altered by a single mutation, leading to significant phenotypic changes and the development of abnormal skin disorders in affected individuals. In recent years, there has been a great deal of discussion about the huge medicinal potential of nucleic acids. Although nucleic acids may be delivered to the desired location, there are a number of difficulties that must be solved before they can be used in clinical trials. Providing a comprehensive overview of numerous facts that highlight a range of skin-related autoimmune illnesses, as well as the use of dermal nanomedicine as a powerful tool for personalized therapy in the context of nuclear acid delivery, is our primary purpose in this paper. The most recent emerging trends and up-to-date metadata on various perspectives of dermal nanomedicines in different autoimmune diseases and related skin conditions as potential candidates for nucleic acid delivery have been gathered from a variety of databases that have been searched for potentially existing research to obtain the most recent emerging trends and up-to-date metadata. Existing research indicates that nanomedicines integrating nucleic acid, including liposomes, dendrimers, polymeric micelles, and other forms of nucleic acid delivery, have significant promise in the pharmaceutical industry. The current investigation focuses on the delivery of nucleic acid medications through the dermal route, as well as the therapeutic applications of these agents. Furthermore, the current state of nanomedicines has been discussed, as well as their potential and use.
... As a rule of thumb, gene delivery techniques and technologies allow the delivery of NAs, including plasmid DNA (pDNA) and messenger RNA (mRNA), as well as short regulatory RNAs, such as small interfering RNA (siRNA), micro RNA (miRNA) and short hairpin RNA (shRNA), and antisense oligonucleotides (ASOs) (for additional information please refer to (Duvall et al., 2013;Giacca, 2010;Ginn et al., 2018)). Despite the delivery of naked NAs represents the safest way to transfect cells, such procedure is rather ineffective because they are inherently anionic at physiological pH such that they cannot passively enter cells. ...
Lipid-based carriers represent the most widely used alternative to viral vectors for gene expression and gene silencing purposes. This class of non-viral vectors is particularly attractive for their ease of synthesis and chemical modifications to endow them with desirable properties. Despite combinatorial approaches have led to the generation of a large number of cationic lipids displaying different supramolecular structures and improved behavior, additional effort is needed towards the development of more and more effective cationic lipids for transfection purposes.
With this review, we seek to highlight the great progress made in the design of each and every constituent domain of cationic lipids, that is, the chemical structure of the headgroup, linker and hydrophobic moieties, and on the specific effect on the assembly with nucleic acids. Since the complexity of such systems is known to affect their performances, the role of formulation, stability and phase behavior on the transfection efficiency of such assemblies will be thoroughly discussed. Our objective is to provide a conceptual framework for the development of ever more performing lipid gene delivery vectors.
... Transfection is a technique that incorporates foreign nucleic acids into host cells to produce either stably or transiently genetically modified cells [4]. Chemical-based transfection methods make use of synthetic or natural carriers to deliver genes into cells such as polymers, liposomes, dendrimers, and cationic lipid systems [5]. Despite of having various options of commercially available transfection reagents, current products are quite expensive and high cost has become an obstacle to conduct experimentation involving a lot of transfection procedure, especially in a large scale [6]. ...
Aims: This study aims to develop an efficient, and inexpensive transfection procedure using low cost polyethylenimine (PEI) as transfection reagent for overexpression of CYP2C9 in WRL 68 host cells. Introduction: Overexpression of CYP450 isozymes such as CYP2C9 has been a reliable approach in developing a high specificity in vitro tool for inhibition study of this liver enzyme. Transient or stable transfection are used to insert plasmid DNA into a mammalian host cell in order to produce proteins. There are many efficient transfection reagents available in the market for this purpose, however, it can be expensive, and a low-cost alternative is favorable. Methodology: Cytotoxicity of PEI was screened on four cell lines namely WRL 68, HepG2, MCF-7 and A549 using MTS assay. WRL 68 was transiently transfected with CYP2C9 plasmid using PEI and DNA concentration as well as transfection time was optimized for the best efficiency and expression. Expression of CYP2C9 protein was measured using qPCR and further evaluated with western blot analysis. Results: IC50 values of PEI are 0.327 ± 0.013 mg/mL (WRL-68), 0.395 ± 0.037 mg/mL (HepG2), 1.159 ± 0.032 mg/mL (A549) and 1.281 ± 0.000 mg/mL (MCF-7). Combination of 3ug plasmid DNA with 2.8 μM of PEI and 10.5 mM NaCl resulted in the highest transfection efficiency and expression after 48 hours. Conclusion: A low-cost and efficient PEI transient transfection procedure was optimized for CYP2C9 overexpression and useful for the purpose of further development of cell-based enzyme inhibition model.
... The primary rate limiting step in achieving better efficiency (transfection) is contributed to the anatomical barriers (epithelial, endothelial cell linings and extracellular matrix around the cells). Physical methods like electroporation, ultrasound assisted by micro bubbles, and magnetofection shows some promising outcome in overcoming this hurdle still it's problematic to some extent [32]. ...
Non-viral vectors are simple in theory but complex in practice. Apart from intra cellular and extracellular barriers, number of other
challenges also needs to be overcome in order to increase the effectiveness of non-viral gene transfer. These barriers are categorized
as production, formulation and storage. No one-size-fits-all solution to gene delivery, which is why in spite of various developments in
liposome, polymer formulation and optimization, new compounds are constantly being proposed and investigated. In this review, we will
see in detail about various types of non-viral vectors highlighting promising development and recent advances that had improved the
non-viral gene transfer efficiency of translating from “Bench to bedside”.
In recent years, cationic polymer vectors have been viewed as a promising method for delivering nucleic acids. With the advancement of synthetic polymer chemistry, we can control chemical structures and properties to enhance the efficacy of gene delivery. Herein, a facile, cost‐effective, and scalable method was developed to synthesize PEGylated PDMAEMA polymers (PEO‐PDMAEMA‐PEO), where PEGylation could enable prolonged polyplexes circulation time in the blood stream. Two polymers of different molecular weights were synthesized, and polymer/eGFP polyplexes were prepared and characterized. The correlation between polymers' molecular weight and physicochemical properties (size and zeta potential) of polyplexes was investigated. Lipofectamine 2000, a commercial non‐viral transfection reagent, was used as a standard control. PEO‐PDMAEMA‐PEO with higher molecular weight exhibited slightly better transfection efficiency than Lipofectamine 2000, and the cytotoxicity study proved that it could function as a safe gene vector. We believe that PEO‐PDMAEMA‐PEO could serve as a model to investigate more potential in the gene delivery area.
The use of gene-based products, such as DNA or RNA, is increasingly being explored for various innovative therapies. However, the success of these strategies is highly dependent on the effective delivery of these biomolecules to target cells. Therefore, the development of pH-responsive nanoparticles comprises the creation of intelligent delivery systems with high therapeutic efficiency. In this work, the pH-responsiveness of the poly(2-(diisopropylamino)ethyl methacrylate)) (PDPA) block was investigated for the encapsulation and delivery of small RNAs (sRNA) to cancer cells. The pH responsiveness was dependent on the protonation profile of the tertiary amines of PDPA, which directly affected the electrostatic interactions established with RNA. Thus, block copolymers based on poly(oligo(ethylene oxide) methyl ether methacrylate) (POEOMA) and PDPA, POEOMA-b-PDPA, were synthesized by supplemental activator and reducing agent atom transfer radical polymerization (SARA ATRP). The structure of the block copolymers was characterized by size exclusion chromatography and ¹H NMR spectroscopy. The copolymers allowed effective complexation of model sRNAs and a pre-miRNA with efficiencies of about 89 % and 91 %, respectively. The characterization by dynamic light scattering revealed that these systems had sizes between 76 and 1375 nm. It was also found that the morphology of the polyplexes depended on the pH, since the preparation at a pH lower than the pKa of the copolymers resulted in spherical but polydisperse particles, while higher pH values resulted in nanoparticles with more homogeneous size, but altered morphology. Moreover, due to pH-responsiveness, it was achieved the release of RNA at pH higher than the pKa of the copolymers, while maintaining its integrity. The polyplexes also showed a high potential to protect RNA from RNases. The transfection of a lung cancer model (A549) and fibroblast cell lines showed that these polyplexes did not cause cell toxicity. In addition, the polyplexes enabled the effective transfection of the A549 cell line with pre-miRNA-29b and miRNA-29b, resulting in a decrease of expression levels of the target DNMT3B gene by approximately 51 % and 47 %, respectively. Overall, the POEOMA-b-PDPA copolymers proved to be a promising strategy for developing responsive delivery systems, that can play a critical role in some diseases, such as cancer, where pH varies between the intra and extracellular environments.
The knowledge about non-coding RNAs (ncRNAs) is rapidly increasing with new data continuously emerging, regarding their diverse types, applications, and roles. Particular attention has been given to ncRNA with regulatory functions, which may have a critical role both in biological and pathological conditions. As a result of the diversity of ncRNAs and their ubiquitous involvement in several biologic processes, ncRNA started to be considered in the biomedical field, with immense potential to be exploited either as biomarkers or as therapeutic agents in certain pathologies. Indeed, ncRNA-based therapeutics have been proposed in many disorders and some even reached clinical trials. However, to prepare an RNA product suitable for pharmacological applications, certain criteria must be fulfilled, and it has to be guaranteed RNA purity, stability, and bioactivity. So, in this review, the different types of ncRNAs are identified and characterized, by describing their biogenesis, functions, and applications. A perspective on the main challenges and innovative approaches for the future and broad therapeutic application of RNA is also presented.
