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Non-viral vectors based on cationic niosomes and minicircle DNA technology enhance gene delivery efficiency for biomedical applications in retinal disorders
Abstract
Low transfection efficiency is a major challenge to overcome in non-viral approaches to reach clinical practice. Our aim was to explore new strategies to achieve more efficient non-viral gene therapies for clinical applications and in particular, for retinal diseases. Cationic niosomes and three GFP-encoding genetic materials consisting on minicircle (2.3 kb), its parental plasmid (3.5 kb) and a larger plasmid (5.5 kb) were combined to form nioplexes. Once fully physicochemically characterized, in vitro experiments in ARPE-19 retina epithelial cells showed that transfection efficiency of minicircle nioplexes doubled that of plasmids ones, maintaining good cell viability in all cases. Transfections in retinal primary cells and injections of nioplexes in rat retinas confirmed the higher capacity of cationic niosomes vectoring minicircle to deliver the genetic material into retina cells. Therefore, nioplexes based on cationic niosomes vectoring minicircle DNA represent a potential tool for the treatment of inherited retinal diseases.
... In doing this, the size of the construct can be reduced by 2-3 kb, depending on the backbone size in the parental plasmid. the retinal pigment epithelium, minicircles also outperformed plasmid delivery when assessing GFP reporter expression from equivalent transgenes [24]. Following the delivery of these constructs to the neural retina, GFP expression was observed but not quantitatively compared [24]. ...
... the retinal pigment epithelium, minicircles also outperformed plasmid delivery when assessing GFP reporter expression from equivalent transgenes [24]. Following the delivery of these constructs to the neural retina, GFP expression was observed but not quantitatively compared [24]. Minicircle delivery to the eye will be considered further later on in this review, but it is clear from the data described so far that minicircles offer therapeutic advantages by increased and sustained transgene expression compared to plasmid vectors. ...
... Investigations in mouse lungs yielded similar results, with minicircles achieving 6.5 times higher levels of luciferase activity compared to the equivalent plasmid-treated samples and, once more, expressions from minicircle-treated mice were sustained at a higher level and for longer than in plasmid-treated mice [23]. For ARPE19 cells, an in vitro model of the retinal pigment epithelium, minicircles also outperformed plasmid delivery when assessing GFP reporter expression from equivalent transgenes [24]. Following the delivery of these constructs to the neural retina, GFP expression was observed but not quantitatively compared [24]. ...
Non-viral gene therapy has the potential to overcome several shortcomings in viral vector-based therapeutics. Methods of in vivo plasmid delivery have developed over recent years to increase the efficiency of non-viral gene transfer, yet further improvements still need to be made to improve their translational capacity. Gene therapy advances for inherited retinal disease have been particularly prominent over the recent decade but overcoming physical and physiological barriers present in the eye remains a key obstacle in the field of non-viral ocular drug delivery. Minicircles are circular double-stranded DNA vectors that contain expression cassettes devoid of bacterial DNA, thereby limiting the risks of innate immune responses induced by such elements. To date, they have not been extensively used in pre-clinical studies yet remain a viable vector option for the treatment of inherited retinal disease. Here, we explore the potential of minicircle DNA delivery to the neural retina as a gene therapy approach. We consider the advantages of minicircles as gene therapy vectors as well as review the challenges involved in optimising their delivery to the neural retina.
... [ 10,15]. Various niosomes formulations based on the combination of the cationic lipid DOTMA with different non-ionic surfactants and helper lipids have demonstrated successful transfection of retina pigment epithelial cells [24,35] or primary neuronal cells [20]. However, up to date very little is known about the use of niosomes as delivery systems to genetically modify MSCs, which is a target cell population in different regenerative medicine approaches [3]. ...
... Storage of niosomes at 25 • C during several weeks may affect main physicochemical parameters influencing gene delivery process [35]. In agreement with previous works [35], nioplexes formed from long-term stored niosomes showed an increase in size. ...
... Storage of niosomes at 25 • C during several weeks may affect main physicochemical parameters influencing gene delivery process [35]. In agreement with previous works [35], nioplexes formed from long-term stored niosomes showed an increase in size. The surface charge resulted only modified in those nioplexes formed at cationic lipid/DNA ratio of 10/1. ...
Gene transfer to mesenchymal stem cells (MSCs) has arisen as a powerful approach to increase the therapeutic potential of this effective cell population. Over recent years, niosomes have emerged as self-assembled carriers with promising performance for gene delivery. The aim of our work was to develop effective niosomes-based DNA delivery platforms for targeting MSCs. Niosomes based on 1,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA; 0, 7 or 15%) as cationic lipid, cholesterol as helper lipid, and polysorbate 60 as non-ionic surfactant, were prepared using a reverse phase evaporation technique. Niosomes dispersions (filtered or not) and their corresponding nioplexes with a lacZ plasmid were characterized in terms of size, charge, protection, and complexation abilities. DOTMA concentration had a large influence on the physicochemical properties of resulting nioplexes. Transfection efficiency and cytotoxic profiles were assessed in two immortalized cell lines of MSCs. Niosomes formulated with 15% DOTMA provided the highest values of β-galactosidase activity, being similar to those achieved with Lipofectamine®, but showed less cytotoxicity. Filtration of niosomes dispersions before adding to the cells resulted in a loss of their biological activities. Storage of niosomes formulations (for 30 days at room temperature) caused minor modification of their physicochemical properties but also attenuated the transfection capability of the nioplexes. Differently, addition of the lysosomotropic agent sucrose into the culture medium during transfection or to the formulation itself improved the transfection performance of non-filtered niosomes. Indeed, steam heat-sterilized niosomes prepared in sucrose medium demonstrated transfection capability.
... However, the main drawbacks of plasmid administration include the immune response generated against the bacterial elements [7] and, in some cases, the big size of the plasmid that decreases transfection efficiency process [8]. To minimize such disadvantages, unmethylated cytosine-phosphate-guanine (CpG) dinucleotides from bacterial origin and other not relevant sequences related to the origin of replication and the resistance to antibiotics have been removed from conventional plasmids resulting in minicircle DNAs (mcDNAs), which reduce immunogenic response and enhance transfection efficiency, allowing a sustained expression of the therapeutic gene (transgene) [9]. Other different approach include the administration of exogenous genetic material in the form of small interfering RNA (siRNA), or aptamers to inhibit protein expression by different mechanisms at a post-transcriptional level, or the synthesis of antisense oligonucleotides (ASOs) that can regulate the expression of both precursor RNA (pre-RNA) or mature RNA in the nucleus or cytosol, respectively [10][11][12][13]. ...
... Due to the electrostatic interactions between the positively charged amine groups of the cationic lipids incorporated into the niosome vesicles and the negatively charged phosphate groups of the genetic material, nioplexes can be easily obtained at different cationic lipid/genetic material ratios [76]. In the case the obtained niosomes are not going to be used soon, they can be stored at 4 °C during several weeks, without affecting the main physicochemical parameters that influence the gene delivery process [9]. ...
... For that purpose, specific fluorescent endocytic markers such as dextrans, cholera toxin B, or transferrin can be used to stain the most representative endocytosis pathways (macropinocitosis, caveolae, and clathrin-mediated endocytosis, respectively). The colocalization of such dyes with fluorescent labelled niosomes, or preferably, fluorescent plasmids attached on the surface of niosomes can be qualitatively evaluated by confocal microscopy [91], or quantified by different overlay coefficients, such as Mander´s or Pearson´s colocalization coefficients [9,114] ( Figure 5). Additionally, intracellular trafficking studies can be completed with lysosome markers such as lysotrackers [90] or with different uptake inhibitors such as genistein, wortmannin, or chlorpromazine, to inhibit selectively caveolae, macropinocytosis, or clathrin-mediated endocytosis, respectively [115]. ...
Non-viral vectors have emerged as a promising alternative to viral gene delivery systems due to their safer profile. Among non-viral vectors, recently, niosomes have shown favorable properties for gene delivery, including low toxicity, high stability, and easy production. The three main components of niosome formulations include a cationic lipid that is responsible for the electrostatic interactions with the negatively charged genetic material, a non-ionic surfactant that enhances the long-term stability of the niosome, and a helper component that can be added to improve its physicochemical properties and biological performance. This review is aimed at providing recent information about niosome-based non-viral vectors for gene delivery purposes. Specially, we will discuss the composition, preparation methods, physicochemical properties, and biological evaluation of niosomes and corresponding nioplexes that result from the addition of the genetic material onto their cationic surface. Next, we will focus on the in situ application of such niosomes to deliver the genetic material into immune-privileged tissues such as the brain cortex and the retina. Finally, as future perspectives, non-invasive administration routes and different targeting strategies will be discussed.
... Non-viral vectors are usually based on different biocompatible nanoparticles, which represent a safer strategy than viruses and their elaboration process is easier and cheaper. Although in many occasions non-viral vectors are still unable to reach the high transfection levels of viral counterparts, continuous advances in the field bring us closer to this goal [4][5][6][7]. In fact, currently some lipid nanoparticles are already commercialized for drug and RNA delivery, such as Onivyde ® [8] and Onpattro™ [9], among others. ...
... Titration of niosome formulations into DNA showed that formulations 1 and 2 reached to a plateau or saturation point at DTPA/pEGFP mass ratios around 2/1-3/1, whereas saturation with formulation 3 required a mass ratio of 9/1, which suggests a less binding affinity for DNA molecules. Therefore, it can be concluded that the addition of chloroquine increased the DNA binding affinity of niosome formulations, which is in accordance with previous reports [4] and would also in part explain the higher transfection efficiency of these formulations compared to formulation 3. Additionally, ITC results suggested that variations reported in the structural and functional properties of nioplexes based in formulations 1 and 2 at mass ratios above 2/1 likely reflect a redistribution of DNA leading to nioplexes with a decreasing fraction of plasmid molecules bound as niosome concentration was further increased. Besides, the titration results were in accordance with the results obtained from nioplexes packing since the presence of chloroquine promoted greater order of niosome membrane packing. ...
Lipid nanocarriers, such as niosomes, are considered attractive candidates for non-viral gene delivery due to their suitable biocompatibility and high versatility. In this work, we studied the influence of incorporating chloroquine in niosomes biophysical performance, as well as the effect of non-ionic surfactant composition and protocol of incorporation in their biophysical performance. An exhaustive comparative evaluation of three niosome formulations differing in these parameters was performed, which included the analysis of their thermal stability, rheological behavior, mean particle size, dispersity, zeta potential, morphology, membrane packing capacity, affinity to bind DNA, ability to release and protect the genetic material, buffering capacity and ability to escape from artificially synthesized lysosomes. Finally, in vitro biological studies were, also, performed in order to determine the compatibility of the formulations with biological systems, their transfection efficiency and transgene expression. Results revealed that the incorporation of chloroquine in niosome formulations improved their biophysical properties and the transfection efficiency, while the substitution of one of the non-ionic surfactants and the phase of addition resulted in less biophysical variations. Of note, the present work provides several biophysical parameters and characterization strategies that could be used as gold standard for gene therapy nanosystems evaluation.
... Interestingly, small plasmidic cassettes as mcDNA have been recently developed to mitigate some disadvantages associated with the use of conventional plasmids [31]. Such mcDNAs contain a minimal expression cassette, where the bacterial backbone DNA has been eliminated, which reduces the unwanted immunogenic responses and enhances the transfection efficiency due to the reduced size of this CpG-free genetic material [8,44]. The cytotoxic effect of the non-viral vectors can be qualitatively evaluated by different techniques based on microscopy analyses [45]. ...
... In this case, due to the intrinsic characteristics of primary culture cells, transfection efficiency values normally decrease when compared to immortalized cell lines. Consequently, studies performed in primarily culture cells or in other difficult-totransfect cell lines such as neurons are normally used to report the kind of cells that have been transfected by immunohistochemistry techniques rather than the transfection efficiency in quantitative terms [23,44]. ...
Efficient delivery of genetic material into cells is a critical process to translate gene therapy into clinical practice. In this sense, the increased knowledge acquired during past years in the molecular biology and nanotechnology fields has contributed to the development of different kinds of non-viral vector systems as a promising alternative to virus-based gene delivery counterparts. Consequently, the development of non-viral vectors has gained attention, and nowadays, gene delivery mediated by these systems is considered as the cornerstone of modern gene therapy due to relevant advantages such as low toxicity, poor immunogenicity and high packing capacity. However, despite these relevant advantages, non-viral vectors have been poorly translated into clinical success. This review addresses some critical issues that need to be considered for clinical practice application of non-viral vectors in mainstream medicine, such as efficiency, biocompatibility, long-lasting effect, route of administration, design of experimental condition or commercialization process. In addition, potential strategies for overcoming main hurdles are also addressed. Overall, this review aims to raise awareness among the scientific community and help researchers gain knowledge in the design of safe and efficient non-viral gene delivery systems for clinical applications to progress in the gene therapy field.
... Although non-toxic and biodegradable, chitosan nanoparticles are recognized to accomplish prolonged gradual emancipation of the capacity, escalation bioavailability, and enhance therapeutic effectiveness (Safdar et al., 2019). To benefit from the affluence usefulness of manufacturing, great revenue at squat cost, and greater loading capability, the manufacturing procedure for nanoparticles such as chitosan NPs might be enhanced (Gallego et al., 2019;Kamel and El-Sayed, 2019). According to (Quester et al., 2022), chitosan-based nanoparticles comprising α-lipoic acid could pass the gastrointestinal barrier and emancipate their antioxidant consignment while remaining stable in stomach-like circumstances. ...
A significant public health issue worldwide is metabolic syndrome, a cluster of metabolic illnesses that comprises insulin resistance, obesity, dyslipidemia, hyperglycemia, and hypertension. The creation of natural treatments and preventions for metabolic syndrome is crucial. Chitosan, along with its nanoformulations, is an oligomer of chitin, the second-most prevalent polymer in nature, which is created via deacetylation. Due to its plentiful biological actions in recent years, chitosan and its nanoformulations have drawn much interest. Recently, the chitosan nanoparticle-based delivery of CRISPR-Cas9 has been applied in treating metabolic syndromes. The benefits of chitosan and its nanoformulations on insulin resistance, obesity, diabetes mellitus, dyslipidemia, hyperglycemia, and hypertension will be outlined in the present review, highlighting potential mechanisms for the avoidance and medication of the metabolic syndromes by chitosan and its nanoformulations.
... Niosomes are bilayer vesicles composed of three main components-cationic lipids, "helper" components, and non-ionic surfactants-and can bind to DNA (forming nioplexes), exhibit long-term stability and proper physicochemical properties, and have relatively low preparation costs [15][16][17][18][19]. Our group already has extensive experience using niosomes as vectors for genetic material in the retina [19][20][21][22][23] and the brain cortex [24][25][26], achieving encouraging results in both. This opens the possibility of developing a safer and non-toxic genetic therapy for the treatment of multiple diseases that affect neural tissues. ...
Citation: Celdrán, J.D.; Humphreys, L.; González, D.; Soto-Sánchez, C.; Martínez-Navarrete, G.; Maldonado, I.; Gallego, I.; Villate-Beitia, I.; Sainz-Ramos, M.; Puras, G.; et al. Assessment of Different Niosome Formulations for Optogenetic Applications: Morphological and Electrophysiological Effects. Abstract: Gene therapy and optogenetics are becoming promising tools for treating several nervous system pathologies. Currently, most of these approaches use viral vectors to transport the genetic material inside the cells, but viruses present some potential risks, such as marked immunogenicity, insertional mutagenesis, and limited insert gene size. In this framework, non-viral nanoparticles, such as niosomes, are emerging as possible alternative tools to deliver genetic material, avoiding the aforementioned problems. To determine their suitability as vectors for optogenetic therapies in this work, we tested three different niosome formulations combined with three optogenetic plasmids in rat cortical neurons in vitro. All niosomes tested successfully expressed optogenetic channels, which were dependent on the ratio of niosome to plasmid, with higher concentrations yielding higher expression rates. However, we found changes in the dendritic morphology and electrophysiological properties of transfected cells, especially when we used higher concentrations of niosomes. Our results highlight the potential use of niosomes for optogenetic applications and suggest that special care must be taken to achieve an optimal balance of niosomes and nucleic acids to achieve the therapeutic effects envisioned by these technologies.
... 81 Despite these difficulties, some studies reported retinal delivery of DNA plasmid or minicircle in mouse models of disease-associated genes like RPE65. 82,83 These vectors also offer an opportunity to deliver genemodifying tools in mRNA or proteins form, limiting their intracellular presence and potentially reducing the risk of off- 70,84,85 So far, CRISPR/Cas9 transfer into the retina in its ribonucleoprotein (RNP) conformation has been used to target the VEGFA gene, whose overexpression is involved in AMD. Recent studies delivered RNP complexes using LNPs and achieved significant editing in RPE cells. ...
Owing to their small size and safety profiles, adeno-associated viruses (AAVs) have become the vector of choice for gene therapy applications in the retina. In addition to the naturally occurring AAVs, several engineered variants with enhanced properties are being developed for experimental and therapeutic applications. Nonetheless, there are still some challenges impeding successful application of AAVs for a broader range of retinal gene therapies. The small size of AAV particles ensures efficient tissue transduction but also limits the packaging capacity to a few kilobases. Further, AAV’s ability to cross retinal barriers is still an obstacle to pan-retinal transduction of the outer retina with tolerable doses. Lastly, despite overall safety, there have been recent reports of immune responses to AAVs in the eye. Hence, evaluation and prediction of immune responses to AAVs has come to be considered an integral part of future clinical success. This review focuses on the use of AAV in clinical trials for retinal diseases, and discusses developments of variants and novel strategies to overcome immune responses to AAVs.
... Transfection results showed that DFSWCNT can be applied as a vector for EGFP plasmid at DFSWCNT/EGFP plasmid mass ratio 2:1 or 5:1 which had high live transfected cells as well as high cell viability percentage. So, DFSWCNT could act as a vehicle in gene delivery systems[91][92][93] such as cationic niosomes[94][95][96] . The results of qualitative analysis of transfection efficiency via examination of HEK-293 cells under fluorescence microscopy 48 h after transfection can be observed inFig. ...
In the present work, single wall carbon nanotubes (SWCNT) were successively functionalized with phospholipid DSPE-PEG carboxylic acid, and then, with ethylenediamine (EDA), to obtain double functionalized single wall carbon nanotube (DFSWCNT). Then, DFSWCNT was applied as a carrier for delivering amphotericin B (Amb) and EGFP plasmid. FSWCNT’s concentration obtained via UV–visible analysis was 0.99 mg/mL. The TGA analysis results provided the lost weights of DSPE-PEG-COOH, EDA, Amb and SWCNT impurities. XPS results showed that carbon atoms’ percentage decreased during the functionalization processes from 97.2% (SWCNT) to 76.4% (FSWCNT) and 69.9% (DFSWNCT). Additionally, the oxygen atoms’ percentage increased from 2.3% (SWCNT) to 21% and 22.5% for FSWCNT and DFSWCNT, respectively. New bonds such as C–N and N–C=O appeared in the synthesized nanocarrier. The I G /I D ratio in Raman analysis decreased from 7.15 (SWCNT) to 4.08 (FSWCNT). The amount of Amb released to phosphate buffer saline medium was about 33% at pH = 5.5 and 75% at pH = 7.4 after 48 h. CCK8 results confirmed that the toxicity of functionalized SWCNT had decreased. In a 2:1 ratio of DFSWCNT/EGFP plasmid, the cell viability (87%) and live transfected cells (56%) were at their maximum values. The results indicate that carbon nanotubes have the potential to be applied as drug/gene delivery systems with outstanding properties such as high loading capacity and easy penetration to cell membrane.
... The cationic liposomes with a positive charge binding to DNA by electrostatic interaction have larger packaging capacity than anionic liposomes. Currently, strategies to ameliorate liposomes are to permit targeting specific cell types because the liposomes adhere to cells non-specifically and further undergo endocytosis (Gallego et al., 2019). ...
Pulmonary arterial hypertension (PAH) is a rare but progressive and lethal vascular disease of diverse etiologies, mainly caused by proliferation of endothelial cells, smooth muscle cells in the pulmonary artery, and fibroblasts, which ultimately leads to right-heart hypertrophy and cardiac failure. Recent genetic studies of childhood-onset PAH report that there is a greater genetic burden in children than in adults. Since the first-identified pathogenic gene of PAH, BMPR2, which encodes bone morphogenetic protein receptor 2, a receptor in the transforming growth factor-β superfamily, was discovered, novel causal genes have been identified and substantially sharpened our insights into the molecular genetics of childhood-onset PAH. Currently, some newly identified deleterious genetic variants in additional genes implicated in childhood-onset PAH, such as potassium channels (KCNK3) and transcription factors (TBX4 and SOX17), have been reported and have greatly updated our understanding of the disease mechanism. In this review, we summarized and discussed the advances of genetic variants underlying childhood-onset PAH susceptibility and potential mechanism, and the most promising BMPR2 gene therapy and gene delivery approaches to treat childhood-onset PAH in the future.
... Retinal primary cell transfection and niopleXes injection in rat retinitis acknowledged the more excellent capability of cationic niosomes vectoring minicircle to transport the genetic substance into retina cells. (Gallego et al., 2019) other diseases (Table 5). ...
The buildup of nonionic surfactants in the aqueous environment produces niosomes. The usage of niosomes is becoming increasingly frequent due to their sustainability, low cost of components and assembly, large-scale manufacture, and, finally, easy maintenance of the niosomes to the other. Because of their nonionic characteristics, niosomes play a critical role in medication delivery systems. Controlled release and targeted distribution of niosomes to treat cancer, infectious illnesses, and other disorders are one of their most important properties. Niosomes can also be injected by ocular and transdermal routes, which are less common than oral and parenteral administration. Using niosomes to manufacture biotechnology goods and novel vaccines is one of the most exciting research fields today. The molecular structure of niosomes, the physicochemical characteristics of nonionic surfactants in their formulation, the influence of external stimuli on niosomes, the many methods of niosomes administration, and their diverse therapeutic qualities are all explored in this study.
... Primary CNS cells were extracted from the brain cortex and retinal tissue of E17−E18 rat embryos (Sprague Dawley) and processed as described elsewhere. 36,37 Lipofectamine 2000 (Invitrogen, California, USA) at 2/1 ratio was employed as a positive control. Each condition was performed in triplicate. ...
Nanodiamonds (NDs) are promising materials for gene delivery because of their unique physicochemical and biological features, along with their possibility of combination with other nonviral systems. Our aim was to evaluate the biophysical performance of NDs as helper components of niosomes, named nanodiasomes, to address a potential nonviral gene delivery nanoplatform for therapeutic applications in central nervous system (CNS) diseases. Nanodiasomes, niosomes, and their corresponding complexes, obtained after genetic material addition at different ratios (w/w), were evaluated in terms of physicochemical properties, cellular uptake, intracellular disposition, biocompatibility, and transfection efficiency in HEK-293 cells. Nanodiasomes, niosomes, and complexes fulfilled the physicochemical features for gene therapy applications. Biologically, the incorporation of NDs into niosomes enhanced 75% transfection efficiency (p < 0.001) and biocompatibility (p < 0.05) to values over 90%, accompanied by a higher cellular uptake (p < 0.05). Intracellular trafficking analysis showed higher endocytosis via clathrins (p < 0.05) in nanodiaplexes compared with nioplexes, followed by higher lysosomal colocalization (p < 0.05), that coexisted with endosomal escape properties, whereas endocytosis mediated by caveolae was the most efficient pathway in the case of nanodiaplexes. Moreover, studies in CNS primary cells revealed that nanodiaplexes successfully transfected neuronal and retinal cells. This proof-of-concept study points out that ND integration into niosomes represents an encouraging nonviral nanoplatform strategy for the treatment of CNS diseases by gene therapy.
... Chitosan nanoparticles are known to easily achieve a sustained slow release of the cargo as well as increase bioavailability and therapeutic efficiency, while being biodegradable and non-toxic [47]. The production process for nanoparticles such as chitosan NPs could be improved to gain the advantages of ease of production, high yield at low cost, and larger loading capacity [48][49][50][51][52]. ...
The addition of the antioxidant α-lipoic acid (ALA) to a balanced diet might be crucial for the prevention of comorbidities such as cardiovascular diseases, diabetes, and obesity. Due to its low half-life and instability under stomach-like conditions, α-lipoic acid was encapsulated into chitosan nanoparticles (Ch-NPs). The resulting chitosan nanoparticles containing 20% w/w ALA (Ch-ALA-NPs) with an average diameter of 44 nm demonstrated antioxidant activity and stability under stomach-like conditions for up to 3 h. Furthermore, fluorescent Ch-ALA-NPs were effectively internalized into 3T3-L1 fibroblasts and were able to cross the intestinal barrier, as evidenced by everted intestine in vitro experiments. Thus, chitosan-based nanoparticles seem to be an attractive administration method for antioxidants, or other sensible additives, in food.
... Disease associated with retinal degeneration constitutes an important health challenge that severely affects the quality of life of patients and has a significant socioeconomic impact [4]. Recently, hydrophilic cargos, like nucleic acids [5] or cGMP analog [6], has attracted major interest for the development of new retinal disease treatments. These hydrophilic cargos may require encapsulation in a nanocarrier to reach their intracellular gue, Czech Republic), deionized water (VWR, Prague, Czech Republic), human adult retinal pigment epithelial cells (ARPE-19 cell, ATCC), 661W cell (generously provided by Dr. Muayyad Al-Ubaidi, University of Oklahoma), Dulbecco's modified Eagle's medium and Ham's F12 nutrient mixture (DMEM/F12; Gibco, Rodano, Italy), low glucose (1 mg/mL) Dulbecco's modified Eagle's medium (DMEM; Gibco, Rodano, Italy), fetal bovine serum (FBS; Gibco, Rodano, Italy), glutamine (Sigma Aldrich, Milan, Italy), penicillin-streptomycin (Sigma Aldrich, Milan, Italy), Accutase ® solution (Sigma-Aldrich, Milan, Italy), paraformaldehyde (PFA; Sigma Aldrich, Milan, Italy), anti-Zonula occludens-1 (ZO-1) antibody (Invitrogen, Rodano, Italy), goat anti-rabbit secondary antibody (Life Technologies, Rodano, Italy), 4',6-diamidino-2-phenylindole, dihydrochloride (DAPI; Sigma Aldrich, Milan, Italy), colorimetric methyl-thiazolyl diphenyl-tetrazolium bromide (MTT; Sigma Aldrich, Milan, Italy). ...
In this study, we developed a novel solid lipid nanoparticle (SLN) formulation for drug delivery of small hydrophilic cargos to the retina. The new formulation, based on a gel core and composite shell, allowed up to two-fold increase in the encapsulation efficiency. The type of hydrophobic polyester used in the composite shell mixture affected the particle surface charge, colloidal stability, and cell internalization profile. We validated SLNs as a drug delivery system by performing the encapsulation of a hydrophilic neuroprotective cyclic guanosine monophosphate analog, previously demonstrated to hold retinoprotective properties, and the best formulation resulted in particles with a size of ±250 nm, anionic charge > −20 mV, and an encapsulation efficiency of ±60%, criteria that are suitable for retinal delivery. In vitro studies using the ARPE-19 and 661W retinal cell lines revealed the relatively low toxicity of SLNs, even when a high particle concentration was used. More importantly, SLN could be taken up by the cells and the release of the hydrophilic cargo in the cytoplasm was visually demonstrated. These findings suggest that the newly developed SLN with a gel core and composite polymer/lipid shell holds all the characteristics suitable for the drug delivery of small hydrophilic active molecules into retinal cells.
... Thus, the effectiveness of AAV-mediated gene therapy will be significantly improved. In addition to the viral gene delivery system, the non-viral vectors based on cationic niosomes (Gallego et al. 2019;Mashal et al. 2019) and lipid nanoparticles (Patel et al. 2019) have been utilized in the rodents, the non-viral vector gene delivery system shows great potentials in therapeutic modality, for its low immunogenicity and high packing DNA size. Applying the non-viral vector to the retina organoids, is also worth trying. ...
The evolution of pluripotent stem cell-derived retinal organoids (ROs) has brought remarkable opportunities for developmental studies while also presenting new therapeutic avenues for retinal diseases. With a clear understanding of how well these models mimic native retinas, such preclinical models may be crucial tools that are widely used for the more efficient translation of studies into novel treatment strategies for retinal diseases. Genetic modifications or patient-derived ROs can allow these models to simulate the physical microenvironments of the actual disease process. However, we are currently at the beginning of the three-dimensional (3D) RO era, and a general quantitative technology for analyzing ROs derived from numerous differentiation protocols is still missing. Continued efforts to improve the efficiency and stability of differentiation, as well as understanding the disparity between the artificial retina and the native retina and advancing the current treatment strategies, will be essential in ensuring that these scientific advances can benefit patients with retinal disease. Herein, we briefly discuss RO differentiation protocols, the current applications of RO as a disease model and the treatments for retinal diseases by using RO modeling, to have a clear view of the role of current ROs in retinal development and diseases.
... Despite their benefit, the translation of pDNA from benchtop-to-bedside remains highly compromised by their transient expression levels and unwanted backbone elements (bacterial origins of replication and antibiotic resistance). Therefore, minicircle DNAs (mcDNAs) were designed with a minimal backbone to meet the clinical requirements for safe and long-lasting therapeutic transgene expression under the control of mammalian promoters [27,28]. The decreased backbone size was shown to be directly correlated with the levels and extent of transgene expression in mammalian cells [29]. ...
The cell-based approach in gene therapy arises as a promising strategy to provide safe, targeted, and efficient gene delivery. Owing to their unique features, as homing and tumor-tropism, mesenchymal stem cells (MSCs) have recently been introduced as an encouraging vehicle in gene therapy. Nevertheless, non-viral transfer of nucleic acids into MSCs remains limited due to various factors related to the main stakeholders of the process (e.g., nucleic acids, carriers, or cells). In this review, we have summarized the main types of nucleic acids used to transfect MSCs, the pros and cons, and applications of each. Then, we have emphasized on the most efficient lipid-based carriers for nucleic acids to MSCs, their main features, and some of their applications. While a myriad of studies have demonstrated the therapeutic potential for engineered MSCs therapy in various illnesses , optimization for clinical use is an ongoing challenge. On the way of improvement, genetically modified MSCs have been combined with various novel techniques and tools (e.g., exosomes, spheroids, 3D-Bioprinting, etc.,) aiming for more efficient and safe applications in biomedicine.
... Small hydrophilic cargo, like DNA [13] or cGMP analogue [14], are of major interest for treatment development. However, encapsulation of small hydrophilic molecules is challenging. ...
In this study, we developed a novel solid lipid nanoparticle (SLN) formulation for drug delivery of small hydrophilic cargos to the retina. The new formulation, based on a gel core and composite shell, allowed up to two times increase in the encapsulation efficiency. The type of hydrophobic polyester used in the composite shell mixture affected the particle surface charge, colloidal stability, and cell internalization profile. We validated the SLN as a drug delivery system by performing encapsulation of a hydrophilic neuroprotective cyclic guanosine monophosphate analogue, previously demonstrated to hold retinoprotective properties, and the best formulation resulted in particles with size of ± 250 nm, anionic charge > -20 mV, and an encapsulation efficiency value of ±60%, criteria that are suitable for retinal delivery. In vitro studies using ARPE-19 and 661W retinal cell lines revealed a relatively low SLN toxicity, even when high particle concentration was used. More importantly, SLN could be uptaken by the cells and the release of the hydrophilic cargo in the cytoplasm was visually demonstrated. These findings suggest that the newly developed SLN with a gel core and composite polymer/lipid shell holds all characteristics suitable for drug delivery of small hydrophilic active molecules into retinal cell.
... Four different cationic niosomes, which presented the best results in previous studies carried out in the laboratory [39,40], were prepared using the reverse-phase evaporation. For the niosome named GPxT-CQ 2.5 mg (0.05% w/v), chloroquine diphosphate (Sigma-Aldrich, St. Louis, MO, USA) was dissolved in milliQ water in order to obtain the aqueous phase. ...
Background:
Mesenchymal stem cells (MSCs) are stem cells present in adult tissues. They can be cultured, have great growth capacity, and can differentiate into several cell types. The isolation of urine-derived mesenchymal stem cells (hUSCs) was recently described. hUSCs present additional benefits in the fact that they can be easily obtained noninvasively. Regarding gene delivery, nonviral vectors based on cationic niosomes have been used and are more stable and have lower immunogenicity than viral vectors. However, their transfection efficiency is low and in need of improvement.
Methods:
We isolated hUSCs from urine, and the cell culture was tested and characterized. Different cationic niosomes were elaborated using reverse-phase evaporation, and they were physicochemically characterized. Then, they were screened into hUSCs for transfection efficiency, and their internalization was evaluated.
Results:
GPxT-CQ at a lipid/DNA ratio of 5:1 (w/w) had the best transfection efficiency. Intracellular localization studies confirmed that nioplexes entered mainly via caveolae-mediated endocytosis.
Conclusions:
In conclusion, we established a protocol for hUSC isolation and their transfection with cationic niosomes, which could have relevant clinical applications such as in gene therapy. This methodology could also be used for creating cellular models for studying and validating pathogenic genetic variants, and even for performing functional studies. Our study increases knowledge about the internalization of tested cationic niosomes in these previously unexplored cells.
... Tweens belong to a family of PEG surfactants with branched PEG chains attaching to lipid tails with different lengths, and therefore, are ideal candidates to test our hypothesis [30]. Tween 80 and Tween 20 have a similar branched PEG structure linking to an unsaturated C17 and a saturated C11 tail, respectively [31][32][33]. Importantly, both Tween 80 and 20 are biocompatible, have been clinically used in injectable formulations, can stabilize lipid bilayers in serum, and enhance gene delivery efficiency [34][35][36]. Therefore, we employed Tween 80 and 20 to replace a widely used PEG attaching to two stearoyl tails (PEG-DSPE) and subsequently studied the size, charge, stability and DNA encapsulation efficiency of the resulting LNPs. ...
Targeted delivery of nucleic acids to lymph nodes is critical for the development of effective vaccines and immunotherapies. However, it remains challenging to achieve selective lymph node delivery. Current gene delivery systems target mainly to the liver and typically exhibit off-target transfection at various tissues. Here we report novel lipid nanoparticles (LNPs) that can deliver plasmid DNA (pDNA) to a draining lymph node, thereby significantly enhancing transfection at this target organ, and substantially reducing gene expression at the intramuscular injection site (muscle). In particular, we discovered that LNPs stabilized by 3% Tween 20, a surfactant with a branched poly(ethylene glycol) (PEG) chain linking to a short lipid tail, achieved highly specific transfection at the lymph node. This was in contrast to conventional LNPs stabilized with a linear PEG chain and two saturated lipid tails (PEG-DSPE) that predominately transfected at the injection site (muscle). Interestingly, replacing Tween 20 with Tween 80, which has a longer unsaturated lipid tail, led to a much lower transfection efficiency. Our work demonstrates the importance of PEGylation in selective organ targeting of nanoparticles, provides new insights into the structure-property relationship of LNPs, and offers a novel, simple, and practical PEGylation technology to prepare the next generation of safe and effective vaccines against viruses or tumours.
... Along with the design of proper non-viral vectors for gene therapy purposes, the size and composition of the genetic material to deliver have to be considered. In this sense, several efforts have been conducted in order to enhance the performance of conventional plasmids, including the removal of bacterial backbones, the optimization of the promoters or the elimination of CpG islands, which confer not only lower immunogenicity but also higher transfection efficiencies and sustained transgene expression (Gallego et al., 2019;Hyde et al., 2008). The therapeutic pGM169 plasmid, which encodes for a correct copy of the CFTR gene, is currently used in CF clinical trials as an optimized gene construct vectored by liposome based non-viral vectors (Alton et al., 2015;Hyde et al., 2008). ...
... In a recent study [116], niosomes made by cationic lipid 1,2-di-octadecenyl-3trimethylammonium propane, Squalene, and Tween 20 have been combined with GFP-encoding genetic materials consisting on minicircle (MC-GFP, 2.3 kb), its parental plasmid (pGFP, 3.5 kb), and a larger plasmid (pEGFP, 5.5 kb). Their efficacy in retinal disease treatment has been investigated. ...
We are witnessing an exponential increase in the use of different nanomaterials in a plethora of biomedical fields. We are all aware of how nanoparticles (NPs) have influenced and revolutionized the way we supply drugs or how to use them as therapeutic agents thanks to their tunable physico-chemical properties. However, there is still a niche of applications where NP have not yet been widely explored. This is the field of ocular delivery and NP-based therapy, which characterizes the topic of the current review. In particular, many efforts are being made to develop nanosystems capable of reaching deeper sections of the eye such as the retina. Particular attention will be given here to noble metal (gold and silver), and to polymeric nanoparticles, systems consisting of lipid bilayers such as liposomes or vesicles based on nonionic surfactant. We will report here the most relevant literature on the use of different types of NPs for an efficient delivery of drugs and bio-macromolecules to the eyes or as active therapeutic tools.
... Compared with non-viral vectors, the transfection rate and immunogenicity of the viral vector are both higher, but the vector is more toxic, the capacity of the target gene is smaller, the targeting specificity is worse, the preparation is more complicated, and the cost is higher. Compared with viral vectors, non-viral vectors feature an ease of production, high yield, and low cost, and they can be widely used for drug delivery [174][175][176][177][178]. Chitosan derivative nanoparticles, as non-viral vectors, have excellent solubility, biodegradability, biocompatibility, non-toxicity, and a higher transfection rate than chitosan nanoparticles [73,98,179]. ...
Chitosan is a product of the deacetylation of chitin, which is widely found in nature. Chitosan is insoluble in water and most organic solvents, which seriously limits both its application scope and applicable fields. However, chitosan contains active functional groups that are liable to chemical reactions; thus, chitosan derivatives can be obtained through the chemical modification of chitosan. The modification of chitosan has been an important aspect of chitosan research, showing a better solubility, pH-sensitive targeting, an increased number of delivery systems, etc. This review summarizes the modification of chitosan by acylation, carboxylation, alkylation, and quaternization in order to improve the water solubility, pH sensitivity, and the targeting of chitosan derivatives. The applications of chitosan derivatives in the antibacterial, sustained slowly release, targeting, and delivery system fields are also described. Chitosan derivatives will have a large impact and show potential in biomedicine for the development of drugs in future.
Ocular gene therapy targets eye diseases at the genetic level. Systemic transport of nucleic acids to ocular tissues poses a significant challenge, as the effectiveness of crossing the blood‐retina barrier limits nucleic acid penetration. Therefore, local administration, such as topical, periocular, or intraocular, can improve the outcome of in vivo gene therapy by bypassing the first‐pass effect and minimizing the systemic toxic effects. The eye is an immune‐privileged organ with limited local immune response, making it an ideal candidate for local gene therapy. Ocular gene therapy offers a promising solution for the treatment of a wide range of retinal diseases including age‐related macular degeneration, diabetic retinopathy, retinitis pigmentosa, and Leber congenital amaurosis. Gene therapy enables replacement of mutated genes essential for visual function, delivery of genes expressing neurotrophic factors and anti‐apoptosis factors for retinal degeneration, and delivery of genes expressing anti‐angiogenic proteins for ocular neovascularization. This perspective discusses the potential of nanoparticles for nucleic acid delivery to the retina, explores challenges, and evaluates different delivery methods, including non‐viral agents such as liposomes and polymers. These nonviral agents present advantages over traditional viral vectors, showing promise in overcoming limitations and offering a viable option for retinal gene therapy.
Clinically, with the treatment of cardiovascular disease in addition to surgery and interventional therapy, drug therapy is also a common means. With the development of medicine, new dosage forms and new technologies are playing a more and more important role. Designing new type of drug delivery systems (DDS) to achieve precise drug release and prolonging drug action time has become a hot spot; the application of DDS in clinic is also increasing gradually. A variety of biomedical materials are used in different types of DDS. These biomaterials include natural polymer, synthetic polymers, metals, and ceramics for the preparation of different drug delivery systems. DDS includes liposomes, micelles, polymer nanoparticles, metal nanoparticles, dendrimers, hydrogels, viral vectors, and so on. In this chapter, details of them in CVD will be discussed.
A significant public health issue worldwide is metabolic syndrome, a cluster of metabolic illnesses that comprises
insulin resistance, obesity, dyslipidemia, hyperglycemia, and hypertension. The creation of natural treatments and
preventions for metabolic syndrome is crucial. Chitosan, along with its nanoformulations, is an oligomer of chitin,
the second-most prevalent polymer in nature, which is created via deacetylation. Due to its plentiful biological
actions in recent years, chitosan and its nanoformulations have drawn much interest. Recently, the chitosan
nanoparticle-based delivery of CRISPR-Cas9 has been applied in treating metabolic syndromes. The benefits of
chitosan and its nanoformulations on insulin resistance, obesity, diabetes mellitus, dyslipidemia, hyperglycemia,
and hypertension will be outlined in the present review, highlighting potential mechanisms for the avoidance
and medication of the metabolic syndromes by chitosan and its nanoformulations
Polymeric drug delivery technology, which allows for medicinal ingredients to enter a cell more easily, has advanced considerably in recent decades. Innovative medication delivery strategies use biodegradable and bio-reducible polymers, and progress in the field has been accelerated by future possible research applications. Natural polymers utilized in polymeric drug delivery systems include arginine, chitosan, dextrin, polysaccharides, poly(glycolic acid), poly(lactic acid), and hyaluronic acid. Additionally, poly(2-hydroxyethyl methacrylate), poly(N-isopropyl acrylamide), poly(ethylenimine), dendritic polymers, biodegradable polymers, and bioabsorbable polymers as well as biomimetic and bio-related polymeric systems and drug-free macromolecular therapies have been employed in polymeric drug delivery. Different synthetic and natural biomaterials are in the clinical phase to mitigate different diseases. Drug delivery methods using natural and synthetic polymers are becoming increasingly common in the pharmaceutical industry, with biocompatible and bio-related copolymers and dendrimers having helped cure cancer as drug delivery systems. This review discusses all the above components and how, by combining synthetic and biological approaches, micro- and nano-drug delivery systems can result in revolutionary polymeric drug and gene delivery devices.
In recent years, various conventional formulations have been used for the treatment and/or management of ocular medical conditions. Diabetic retinopathy, a microvascular disease of the retina, remains the leading cause of visual disability in patients with diabetes. Currently, for treating diabetic retinopathy, only intraocular, intravitreal, periocular injections, and laser photocoagulation are widely used. Frequent administration of these drugs by injections may lead to serious complications, including retinal detachment and endophthalmitis. Although conventional ophthalmic formulations like eye drops, ointments, and suspensions are available globally, these formulations fail to achieve optimum drug therapeutic profile due to immediate nasolacrimal drainage, rapid tearing, and systemic tearing toxicity of the drugs. To achieve better therapeutic outcomes with prolonged release of the therapeutic agents, nano-drug delivery materials have been investigated. These nanocarriers include nanoparticles, solid lipid nanoparticles (SLN), nanostructured lipid carriers (NLC), dendrimers, nanofibers, in-situ gel, vesicular carriers, niosomes, and mucoadhesive systems, among others. The nanocarriers carry the potential benefits of site-specific delivery and controlled and sustained drug release profile. In the present article, various nanomaterials explored for treating diabetic retinopathy are reviewed.
Dermal drug delivery has emerged as a promising alternative to traditional methods of drug administration due to its non-invasive nature and ease of use. However, the stratum corneum, the outermost layer of the skin, presents a significant barrier to drug penetration. Niosomes, self-assembled vesicular structures composed of nonionic surfactants and cholesterol, have been extensively investigated as a means of overcoming this barrier and improving the efficacy of dermal drug delivery. This review summarizes the current state of research on the use of niosomes in dermal drug delivery in cosmetics, with a particular focus on their formulation, characterization, and application in the delivery of various drug classes. The review highlights the advantages of niosomes over conventional drug delivery methods, including improved solubility and stability of drugs, controlled release, and enhanced skin permeation. The review also discusses the challenges associated with niosome-based drug delivery, such as their complex formulation and optimization, and the need for further studies on their long-term safety and toxicity.
Senescence is a process characterized by a prolonged irreversible cell-cycle arrest. The accumulation of senescent cells in tissues is related to aging and to the development of age-related diseases. Recently, gene therapy has emerged as a powerful tool for treating age-associated diseases by the transference of specific genes into the target cell population. However, the high sensitivity of senescent cells significantly precludes their genetic modification via classical viral and non-viral systems. Niosomes are self-assembled non-viral nanocarriers that exhibit important advantages due to their elevated cytocompatibility, versatility, and cost-efficiency, arising as a new alternative for genetic modification of senescent cells. In this work, we explore for the first time the use of niosomes for genetic modification of senescent umbilical cord-derived mesenchymal stem cells. We report that niosome composition greatly affected transfection efficiency; those formulations prepared in medium with sucrose and containing cholesterol as helper lipid being the most suitable to transfect senescent cells. Moreover, resulting niosome formulations exhibited a superior transfection efficiency with a markedly less cytotoxicity than the commercial reagent Lipofectamine. These findings highlight the potentiality of niosomes as effective vectors for genetic modification of senescent cells, providing new tools for the prevention and/or treatment of age-related diseases.
Amongst different routes of drug delivery systems, ophthalmic drug delivery still requires a careful investigation and strict parameter measurements because the eyes are one of the most sensitive parts of the body and require special attention. The conventional systems for eyes lead to rapid elimination of formulation and hence very small contact time on the ocular epithelium. The current review article covers various types of polymers used in ocular drug delivery along with their applications/ limitations. Polymers are widely used by researchers in prodrug techniques and as a penetration enhancer in ocular delivery. This article covers the role and use of different polymeric systems which makes the final formulation a promising candidate for ophthalmic drug delivery.
The researchers are still facing multiple challenges in order to maintain the therapeutic concentration of the drug in the eyes because of its complex structure. There are several barriers that further restrict the intraocular entry of the drug. In order to remove/reduce such challenges, these days various types of polymers are used for ocular delivery in order to develop different drug carrier systems for better efficacy and stability. The polymers used are highly helpful in increasing residence time by increasing the viscosity at the ocular epithelium layer. Such preparations also get easily permeated in ocular cells. The combination of different polymeric properties makes the final formulation stable with prolonged retention, high viscosity, high permeability, and better bioavailability, making the final formulation a promising candidate for ocular drug delivery.
In the present work, single wall carbon nanotubes (SWCNT) were functionalized with phospholipid DSPE-PEG carboxylic acid, and then, with ethylenediamine (EDA), to obtain double functionalized single wall carbon nanotube (DFSWCNT). We applied DFSWCNT as a carrier for delivering amphotericin B (Amb) and EGFP plasmid. FSWCNT’s concentration obtained via UV-visible analysis was 0.99 mg/mL. The TGA analysis results provided the lost weights of DSPE-PEG-COOH, EDA, Amb and SWCNT impurities. XPS results showed that carbon atoms’ percentage decreased during the functionalization process from 97.2% (SWCNT) to 76.4% (FSWCNT) and 69.9% (DFSWNCT). Additionally, the oxygen atoms’ percentage increased from 2.3% (SWCNT) to 21% and 22.5% for FSWCNT and DFSWCNT, respectively. New bonds such as C-N and N-C = O appeared in the synthesized nanocarrier. The I G /I D ratio in Raman analysis decrease from 7.15 (SWCNT) to 4.08 (FSWCNT). The amount of Amb released to phosphate buffer saline medium was about 33% at pH = 5.5 and 75% at pH = 7.4 after 48 h. CCK8 results confirmed that the toxicity of functionalized SWCNT decreased. In a 2:1 ratio of DFSWCNT/EGFP plasmid, the cell viability (87%) and live transfected cells (56%) were at their maximum values. processes indicate that carbon nanotubes have the potential to be applied as both drug/gene delivery systems with outstanding properties such as high loading capacity and easy penetration to the cell membrane.
Lentiviral vectors (LVs) play an important role in gene therapy and have proven successful in clinical trials. LVs are capable of integrating specific genetic materials into the target cells and allow for long-term expression of the cDNA of interest. The use of non-integrating LVs (NILVs) reduces insertional mutagenesis and the risk of malignant cell transformation over integrating lentiviral vectors. NILVs enable transient expression or sustained episomal expression, especially in non-dividing cells. Important modifications have been made to the basic human immunodeficiency virus (HIV) structures to improve the safety and efficacy of LVs. NILV-aided transient expression has led to more pre-clinical studies on primary immunodeficiencies, cytotoxic cancer therapies, and hemoglobinopathies. Recently, the third generation of self-inactivating LVs was applied in clinical trials for recombinant protein production, vaccines, gene therapy, cell imaging, and induced pluripotent stem cell (iPSC) generation. This review discusses the basic lentiviral biology and the four systems used for generating NILV designs. Mutations or modifications in LVs and their safety are addressed with reference to pre-clinical studies. The detailed application of NILVs in promising pre-clinical studies is also discussed.
In designing a new drug, considering the preferred route of administration, various requirements must be fulfilled. Active molecules pharmacokinetics should be reliable with a valuable drug profile as well as well-tolerated. Over the past 20 years, nanotechnologies have provided alternative and complementary solutions to those of an exclusively pharmaceutical chemical nature since scientists and clinicians invested in the optimization of materials and methods capable of regulating effective drug delivery at the nanometer scale. Among the many drug delivery carriers, lipid nano vesicular ones successfully support clinical candidates approaching such problems as insolubility, biodegradation, and difficulty in overcoming the skin and biological barriers such as the blood-brain one. In this review, the authors discussed the structure, the biochemical composition, and the drug delivery applications of lipid nanovesicular
The aim was to evaluate relevant biophysic processes related to the physicochemical features and gene transfection mechanism when sphingolipids are incorporated into a cationic niosome formulation for non-viral gene delivery to central nervous system. For that, two formulations named niosphingosomes and niosomes devoid of sphingolipid extracts, as control, were developed by the oil-in water emulsion technique. Both formulations and the corresponding complexes, obtained upon the addition of the reporter EGFP plasmid, were physicochemically and biologically characterized and evaluated. Compared to niosomes, niosphingosomes, and the corresponding complexes decreased particle size and increased superficial charge. Although there were not significant differences in the cellular uptake, cell viability and transfection efficiency increased when human retinal pigment epithelial (ARPE-19) cells were exposed to niosphingoplexes. Endocytosis via caveolae decreased in the case of niosphingoplexes, which showed higher co-localization with lysosomal compartment, and endosomal escape properties. Moreover, niosphingoplexes transfected not only primary central nervous system cells, but also different cells in mouse retina, depending on the administration route, and brain cortex. These preliminary results suggest that niosphingosomes represent a promising non-viral vector formulation purposed for the treatment of both retinal and brain diseases by gene therapy approach.
Retinal diseases, including age-related macular degeneration (AMD), are a major cause of blindness. Efficient delivery of therapeutic genes to retinal cells to treat retinal disease is a formidable challenge. In this study, we developed a core-shell nanoplatform composed of a core and two external layers for targeted delivery of the gene to the retina. The inner core was composed of amino acid-functionalized dendrimers and a nuclear localization signal (NLS) for DNA complexation, nuclear transport and efficient transfection. The inner core was coated in a lipid bilayer that comprised pH-sensitive lipids as the inner shell layer. Hyaluronic acid (HA)-1,2-dioleoylphosphatidylethanolamine (DOPE) as the outermost shell layer was used for retinal cell targeting. This core-shell nanoplatform was developed so that the mobility in the vitreous body of these negatively charged carriers would not be affected by their surface charge, allowing diffusion into the retina, uptake into the retinal cells via CD44-mediated internalization, and finally transport into the nucleus by the NLS. The designed nanoparticles showed safety both in vitro and in vivo and inhibited the expression of VEGF under hypoxia-mimicking conditions. In vitro angiogenesis assays exhibited significant inhibitory effects on cell migration and tube formation. The in vivo assays indicated that this nanoplatform could be delivered to the retina. Taken together, this nanoplatform has the potential to transfer gene material into the retina for the treatment of retinal diseases, including AMD.
Statement of significance
It remains a challenge to develop an efficient nonviral vector for gene therapy, especially retinal gene therapy. Various barriers exist in gene delivery and the unique ocular environment, making gene delivery to the retina difficult. In this study, we designed a negatively charged core-shell nanoplatform (HD-NPPND) for the targeted delivery of gene to the retina. The developed nanoplatform possessed excellent transfection efficiency and safety both in vitro and in vivo. It efficiently delivered a gene to the retina. The results of this study suggested that this core-shell nanoplatform has the potential to deliver genes to the retina to treat retinal diseases, including age-related macular degeneration (AMD).
Novel gene therapy treatments for inherited retinal diseases have been at the forefront of translational medicine over the past couple of decades. Since the discovery of CRISPR mechanisms and their potential application for the treatment of inherited human conditions, it seemed inevitable that advances would soon be made using retinal models of disease. The development of CRISPR technology for gene therapy and its increasing potential to selectively target disease-causing nucleotide changes has been rapid. In this chapter, we discuss the currently available CRISPR toolkit and how it has been and can be applied in the future for the treatment of inherited retinal diseases. These blinding conditions have until now had limited opportunity for successful therapeutic intervention, but the discovery of CRISPR has created new hope of achieving such, as we discuss within this chapter.
The tragic deaths of three patients in a recent AAV-based X-linked myotubular myopathy clinical trial highlight once again the pressing need for safe and reliable gene delivery vectors. Non-viral minimized DNA vectors offer one possible way to meet this need. Recent pre-clinical results with minimized DNA vectors have yielded promising outcomes in cancer therapy, stem cell therapy, stem cell reprograming, and other uses. Broad clinical use of these vectors, however, remains to be realized. Further advances in vector design and production are ongoing. An intriguing and promising potential development results from manipulation of the specific shape of non-viral minimized DNA vectors. By improving cellular uptake and biodistribution specificity, this approach could impact gene therapy, DNA nanotechnology, and personalized medicine.
While bone has a capability to heal itself, there is a great difficulty in reconstituting large bone defects created by heavy trauma or the resection of malignant tumor. Also, osteonecrosis of the femoral head (ONFH), which is caused by obstruction of the blood supply to bone cells and occurs in the young, is not amenable to successful bone regeneration. We developed VEGF- and BMP2-transfected adipose stem cells (ASCs) using electroporation that can effectively treat bone defects by providing rapid angiogenesis and osteogenesis. The optimal ratio of BMP2- to VEGF- transfected ASCs to enhance both osteogenesis and angiogenesis was 9:1. BMP2-/VEGF-transfected ASCs administered in this ratio effectively healed critical-size calvarial defects and long-bone segmental defects in immunosuppressed rats. The implanted cells did not migrate out of the implantation site by the 56th day. TAZ, TEAD, and ANKRD1 were overexpressed in BMP2-/VEGF-transfected ASCs, possibly proposing the mechanism of enhanced bone regeneration and angiogenesis. Our results suggest the possibility of using gene-cell therapy that can induce rapid angiogenesis and osteogenesis in inhospitable avascular environments including large bone defects and ONFH.
Finding a safe and efficient gene delivery vector is a major international challenge facing the development of gene therapy. Tannic acid (TA) is a natural cross-linker owing to its hydroxyl and carboxyl groups that can interact with biopolymers for different biomaterial design. In this work, three polyethyleneimine-modified TA polymers were prepared, and the polymers were characterized by FTIR, UV-vis, elemental analysis and 1H NMR. The potential of PTAs as gene vector was studied in vitro, including DNA loading capacity, DNA protection ability and biocompatibility. In addition, the particle size, zeta potential, DNA encapsulation efficiency, cell uptake and transfection efficiency of the PTA-pDNA polyplexes were also studied. The results showed that PTA2k and PTA30k could completely condense DNA at N/P of 2, and PTA600 could only completely condense DNA at N/P of 50. The PTA/pDNA polyplexes could protect DNA from degrading by DNA enzymes and could be efficiently uptaked by cells. Biocompatibility assay showed that PTA had no significant cytotoxicity and effect on cell proliferation compared to PEI. At low N/P ratios of 1-4, PTA showed higher transfection efficiency than PEI, and the transfection efficiency increased with the increase of PEI molecular weight in PTA. At N/P of 3, PTA30k showed the highest transfection efficiency of 23.8%, while PEI30k showed only 6.7%. These results indicate that PTA is a promising candidate vector for safe and efficient gene delivery.
Gene-directed tissue repair offers the clinician, human or veterinary, the chance to enhance cartilage regeneration and repair at a molecular level. Non-viral plasmid vectors have key biosafety advantages over viral vector systems for regenerative therapies due to their episomal integration however, conventional non-viral vectors can suffer from low transfection efficiency. Our objective was to identify and validate in vitro a novel non-viral gene expression vector that could be utilized for ex vivo and in vivo delivery to stromal-derived mesenchymal stem cells (MSCs). Minicircle plasmid DNA vector containing green fluorescent protein (GFP) was generated and transfected into adipose-derived MSCs from three species: canine, equine and rodent and transfection efficiency was determined. Both canine and rat cells showed transfection efficiencies of approximately 40% using minicircle vectors with equine cells exhibiting lower transfection efficiency. A Sox9-expressing minicircle vector was generated and transfected into canine MSCs. Successful transfection of the minicircle-Sox9 vector was confirmed in canine cells by Sox9 immunostaining. This study demonstrate the application and efficacy of a novel non-viral expression vector in canine and equine MSCs. Minicircle vectors have potential use in gene-directed regenerative therapies in non-rodent animal models for treatment of cartilage injury and repair.
PIM1, a pro-survival gene encoding a serine/ threonine kinase, influences cell proliferation and survival. Modification of cardiac progenitor cells (CPCs) or cardiomyocytes with PIM1 using a lentivirus-based delivery method showed long-term improved cardiac function after myocardial infarction (MI). However, lentivirus based delivery methods have stringent FDA regulation with respect to clinical trials. To provide an alternative and low risk PIM1 delivery method, this study examined the use of a non-viral modified plasmid-minicircle (MC) as a vehicle to deliver PIM1 into mouse CPCs (mCPCs) in vitro and the myocardium in vivo. MC containing a turbo gfp reporter gene (gfp-MC) was used as a transfection and injection control. PIM1 was subcloned into gfp-MC (PIM1-MC) and then transfected into mCPCs at an efficiency of 29.4±3.7%. PIM1-MC engineered mCPCs (PIM1-mCPCs) exhibit significantly (P<0.05) better survival rate under oxidative treatment. PIM1-mCPCs also exhibit 1.9±0.1 and 2.2±0.2 fold higher cell proliferation at 3 and 5 days post plating, respectively, as compared to gfp-MC transfected mCPCs control. PIM1-MC was injected directly into ten-week old adult FVB female mice hearts in the border zone immediately after MI. Delivery of PIM1 into myocardium was confirmed by GFP⁺ cardiomyocytes. Mice with PIM1-MC injection showed increased protection compared to gfp-MC injection groups measured by ejection fraction at 3 and 7 days post injury (P = 0.0379 and P = 0.0262 by t-test, respectively). Success of PIM1 delivery and integration into mCPCs in vitro and cardiomyocytes in vivo by MC highlights the possibility of a non-cell based therapeutic approach for treatment of ischemic heart disease and MI.
Uses of viral vectors have thus far eclipsed uses of non-viral vectors for gene therapy delivery in the clinic. Viral vectors, however, have certain issues involving genome integration, the inability to be delivered repeatedly, and possible host rejection. Fortunately, development of non-viral DNA vectors has progressed steadily, especially in plasmid vector length reduction, now allowing these tools to fill in specifically where viral or other non-viral vectors may not be the best options. In this review, we examine the improvements made to non-viral DNA gene therapy vectors, highlight opportunities for their further development, address therapeutic needs for which their use is the logical choice, and discuss their future expansion into the clinic.
Background:
Niosomes are non-ionic surfactant vesicles used as drug carriers for encapsulating both hydrophobic and hydrophilic drugs. The aim of this study is to evaluate the effect of different surfactants on the physical properties and stability of carvedilol niosomes designed to improve oral bioavailability.
Materials and methods:
Different niosomal formulations were prepared using a film hydration method, with various mixtures of different non-ionic surfactants including Span 20, 40, and 60, and also Tween 20, 40, and 60, along with cholesterol. The physicochemical characteristics of the formulations were evaluated in vitro.
Results:
The drug encapsulation efficiency was reduced by using lauryl (C12) chain containing surfactants, that is, Span/Tween. Cholesterol content and drug entrapment were the main factors affecting the mean particle size of the niosomes. The drug release profiles from most of the formulations were fitted well with the Baker-Lonsdale model, indicating a diffusion-based drug release mechanism. Niosomes prepared from 50 and 40% of the cholesterol with 25 or 30% of Span/Tween 60 showed the highest stability due to their high transition temperature and solid state feature of these surfactants.
Conclusions:
From the results obtained, it may be concluded that nanoniosomes are promising stable carriers for the oral delivery of carvedilol.
In this work, we carried out a comparative study of four different niosome formulations based on the same cationic lipid and non-ionic tensoactive. The niosomes prepared by oil-in-water emulsion technique (o/w) only differed in the helper lipid composition: squalene, cholesterol, squalane or no helper lipid. Niosomes and nioplexes elaborated upon the addition of pCMS-EGFP reporter plasmid were characterized in terms of size, zeta potential and polydispersity index. The capacity of the niosomes to condense, release and protect the DNA against enzymatic degradation was evaluated by agarose gel electrophoresis. In vitro experiments were carried out to evaluate transfection efficiency and cell viability in retinal pigment epithelial cells. Moreover, uptake and intracellular trafficking studies were performed to further understand the role of the helper lipids in the transfection process. Interestingly, among all tested formulations, niosomes elaborated with squalene as helper lipid were the most efficient transfecting cells. Such transfection efficiency could be attributed to their higher cellular uptake and the particular entry pathways used, where macropinocytosis pathway and lysosomal release played an important role. Therefore, these results suggest that helper lipid composition is a crucial step to be considered in the design of niosome formulation for retinal gene delivery applications since clearly modulates the cellular uptake, internalization mechanism and consequently, the final transfection efficiency.
Advanced age-related macular degeneration (AMD) is the leading cause of blindness in the elderly, with limited therapeutic options. Here we report on a study of >12 million variants, including 163,714 directly genotyped, mostly rare, protein-altering variants. Analyzing 16,144 patients and 17,832 controls, we identify 52 independently associated common and rare variants (P < 5 [times] 10-8) distributed across 34 loci. Although wet and dry AMD subtypes exhibit predominantly shared genetics, we identify the first genetic association signal specific to wet AMD, near MMP9 (difference P value = 4.1 [times] 10-10). Very rare coding variants (frequency <0.1%) in CFH, CFI and TIMP3 suggest causal roles for these genes, as does a splice variant in SLC16A8. Our results support the hypothesis that rare coding variants can pinpoint causal genes within known genetic loci and illustrate that applying the approach systematically to detect new loci requires extremely large sample sizes.
The present study aimed to evaluate the incorporation of protamine into niosome/DNA vectors to analyze the potential application of this novel ternary formulation to deliver the pCMS-EGFP plasmid into the rat retina. Binary vectors based on niosome/DNA and ternary vectors based on protamine/DNA/niosomes were prepared and physicochemically characterized. In vitro experiments were performed in ARPE-19 cells. At 1/1/5 protamine/DNA/niosome mass ratio, the resulted ternary vectors had 150 nm size, positive charge, spherical morphology, and condensed, released and protected the DNA against enzymatic digestion. The presence of protamine in the ternary vectors improved transfection efficiency, cell viability and DNA condensation. After ocular administration, the EGFP expression was detected in different cell layers of the retina depending on the administration route without any sign of toxicity associated to the formulations. While subretinal administration transfected mainly photoreceptors and retinal pigment epithelial cells at the site of injection, intravitreal administration produced a more uniform distribution of the protein expression through the inner layers of the retina. The protein expression in the retina persisted for at least one month after both administrations. Our study highlights the flattering properties of protamine/DNA/niosome ternary vectors for efficient and safe gene delivery to the rat retina.
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”.
We designed niosomes based on three lipids that differed only in the polar-head group to analyze their influence on the transfection efficiency. These lipids were characterized by small-angle X-ray scattering before being incorporated into the niosomes which were characterized in terms of pKa, size, zeta potential, morphology and physical stability. Nioplexes were obtained upon the addition of a plasmid. Different ratios (w/w) were selected to analyze the influence of this parameter on size, charge and the ability to condense, release and protect the DNA. In vitro transfection experiments were performed in HEK-293, ARPE-19 and MSC-D1 cells. Our results show that the chemical composition of the cationic head-group clearly affects the physicochemical parameters of the niosomes and especially the transfection efficiency. Only niosomes based on cationic lipids with a dimethyl amino head group (lipid ) showed a transfection capacity when compared with their counterparts amino (lipid ) and tripeptide head-groups (lipid ). Regarding cell viability, we clearly observed that nioplexes based on the cationic lipid had a more deleterious effect than their counterparts, especially in ARPE-19 cells at 20/1 and 30/1 ratios. Similar studies could be extended to other series of cationic lipids in order to progress in the research on safe and efficient non-viral vectors for gene delivery purposes.
Objective(s): Polyethylenimine (PEI) is a potent non-viral gene delivery carrier. PEI surface charge plays a major role in its condensation ability which in turn is a necessary requirement for high transfection efficiency. As the PEI charge density is dependent on pH, the effect of pH changing PEI on PEI condensation ability, transfection efficiency, and cytotoxicity was investigated.
Materials and Methods: In order to compare the different values of pH, 25 kDa PEI solutions were prepared at the pH values of 5, 7 and 10, afterward complexed with two plasmids encoding reporter genes of luciferase and enhanced green fluorescence protein to evaluate the capability of polymers in plasmid delivery at two incubation time (4 versus 24 hr).
Results: The condensation ability of PEI in acidic, neutral and basic environments was similar. At low pH value, the polyplex had negative surface charge whereas at a higher pH value, the surface charge increased and became positive. The higher transfection efficiency and lower cytotoxicity were achieved by the polyplexes prepared at the pH value of 5 with longer incubation time of 24 hr.
Conclusion: The results showed the impact of pH value of PEI on biological and biophysical properties of polyplexes. Although the proton sponge effect can justify several properties of PEI, these results suggest that the proton sponge hypothesis may not be applicable for polymers with low buffering capacity at the pH values around 5.
Gene therapy products for the treatment of genetic diseases are currently in clinical trials, and one of these, an adeno-associated viral (AAV) product, has recently been licensed in Europe. AAV vectors have achieved positive results in a number of clinical and pre-clinical settings, including hematologic disorders such as the hemophilias, Gaucher's disease, hemochromatosis, and the porphyrias. Because AAV vectors are administered directly to the patient, the likelihood of a host immune response is high, as shown by human studies. Preexisting and/or recall responses to the wild-type virus from which the vector is engineered, or to the transgene product itself, can interfere with therapeutic efficacy if not identified and managed optimally. Small-scale clinical studies have enabled investigators to dissect the innate and adaptive immune responses to the AAV vector capsid and to the transgene product, facilitating the formulation of strategies to manage these responses to allow long-term expression of the therapeutic gene. However, a comprehensive understanding of the determinants of immunogenicity of AAV vectors, and of the potential associated toxicities, is still lacking. Careful immunosurveillance conducted as part of ongoing clinical studies will provide the basis for understanding the intricacies of the immune response in AAV-mediated gene transfer, facilitating safe and effective therapies for genetic disease.
During the past decade formulation of vesicles as a tool to improve drug delivery, has created a lot of interest amongst the scientist working in the area of drug delivery systems. Vesicular system such as liposomes, niosomes, transferosomes, pharmacosomes and ethosomes provide an alternative to improve the drug delivery. Niosomes play an important role owing to their nonionic properties, in such drug delivery system. Design and development of novel drug delivery system (NDDS) has two prerequisites. First, it should deliver the drug in accordance with a predetermined rate and second it should release therapeutically effective amount of drug at the site of action. Conventional dosage forms are unable to meet these requisites. Niosomes are essentially non-ionic surfactant based multilamellar or unilamellar vesicles in which an aqueous solution of solute is entirely enclosed by a membrane resulting from the organization of surfactant macromolecules as bilayer. Niosomes are formed on hydration of non-ionic surfactant film which eventually hydrates imbibing or encapsulating the hydrating aqueous solution. The main aim of development of niosomes is to control the release of drug in a sustained way, modification of distribution profile of drug and for targeting the drug to the specific body site. This paper deals with composition, characterization/evaluation, merits, demerits and applications of niosomes.
In vivo gene replacement for the treatment of inherited disease is one of the most compelling concepts in modern medicine. Adeno-associated virus (AAV) vectors have been extensively used for this purpose and have shown therapeutic efficacy in a range of animal models. Successful translation to the clinic was initially slow, but long-term expression of donated genes at therapeutic levels has now been achieved in patients with inherited retinal disorders and haemophilia B. Recent exciting results have raised hopes for the treatment of many other diseases. As we discuss here, the prospects and challenges for AAV gene therapy are to a large extent dependent on the target tissue and the specific disease.
The interaction of particles with cells is known to be strongly influenced by particle size, but little is known about the interdependent role that size, shape, and surface chemistry have on cellular internalization and intracellular trafficking. We report on the internalization of specially designed, monodisperse hydrogel particles into HeLa cells as a function of size, shape, and surface charge. We employ a top-down particle fabrication technique called PRINT that is able to generate uniform populations of organic micro- and nanoparticles with complete control of size, shape, and surface chemistry. Evidence of particle internalization was obtained by using conventional biological techniques and transmission electron microscopy. These findings suggest that HeLa cells readily internalize nonspherical particles with dimensions as large as 3 μm by using several different mechanisms of endocytosis. Moreover, it was found that rod-like particles enjoy an appreciable advantage when it comes to internalization rates, reminiscent of the advantage that many rod-like bacteria have for internalization in nonphagocytic cells.
• PRINT
• shape
• size
• surface charge
Clinical applications of gene therapy mainly depend on the development of efficient gene transfer vectors. Large DNA molecules
can only be transfected into cells by using synthetic vectors such as cationic lipids and polymers. The present investigation
was therefore designed to explore the physicochemical properties of cationic lipid-DNA particles, with plasmids ranging from
900 to 52 500 bp. The colloidal stability of the lipoplexes formed by complexing lipopolyamine micelles with plasmid DNA of
various lengths, depending on the charge ratio, resulted in the formation of three domains, respectively corresponding to
negatively, neutrally and positively charged lipoplexes. Lipoplex morphology and structure were determined by the physicochemical
characteristics of the DNA and of the cationic lipid. Thus, the lamellar spacing of the structure was determined by the cationic
lipid and its spherical morphology by the DNA. The main result of this study was that the morphological and structural features
of the lipopolyamine-DNA complexes did not depend on plasmid DNA length. On the other hand, their gene transfer capacity was
affected by the size of plasmid DNA molecules which were sandwiched between the lipid bilayers. The most effective lipopolyamine-DNA
complexes for gene transfer were those containing the shortest plasmid DNA.
The purpose of our study was to evaluate the biodistribution of rAAV vectors following subretinal or intravitreal injection. In rats, we performed subretinal or intravitreal injections of rAAV-2/2.CMV.gfp. In large animals, rAAV-2/4.CMV.gfp or rAAV-2/5.CMV.gfp was delivered into the subretinal space while rAAV-2/2.CMV.gfp was delivered either to the subretinal space or to the vitreous. In euthanized animals, we undertook a complete necropsy. In animals maintained alive, we collected blood and tissue samples from the submandibular lymph node, liver, and gonads. We analyzed total DNA, extracted from various tissue samples and peripheral blood mononuclear cells (PBMC), by PCR. Following subretinal or intravitreal injections in rats and in large animals, vector sequences were not detected in the liver or in the gonads but were occasionally found in PBMC. An unexpected result was the detection of rAAV sequences in the optic nerve following subretinal injection. The most striking finding was the detection of vector sequences in the brain, along the visual pathway, in rAAV-2/2 intravitreally injected dogs. These findings raise safety concerns regarding intraocular administration of rAAV vectors and will have an impact on the development of future gene therapy trials for retinal diseases.
The present study aimed to evaluate the incorporation of the natural lipid lycopene into niosome formulations based on cationic lipid DOTMA and polysorbate 60 non-ionic surfactant to analyze the potential application of this novel formulation to deliver genetic material into the rat retina. Both niosomes with and without lycopene were prepared by the reverse phase evaporation method and physicochemically characterized in terms of size, zeta potential, polydispersity index and capacity to condense, release and protect the DNA against enzymatic digestion. In vitro experiments were performed in ARPE-19 cells after complexion of niosomes with pCMS-EGFP plasmid at appropriate cationic lipid/DNA ratios. At 18/1 mass ratio, nioplexes containing lycopene had nanometric size, positive zeta potential, low polydispersity and were able to condense, release and protect DNA. Percentage of transfected cell was around 35% without compromising cell viability. The internalization pathways studies revealed a preference to caveolae mediated endocytosis and macropinocytosis, which could circumvent lysosomal degradation. Both subretinal and intravitreal administrations to the rat retina showed that nioplexes were able to transfect efficiently the outer segments of the retina, which offer reasonable hope for the treatment of many inherited retinal diseases by a safe non-viral vector formulation after the less invasive intravitreal administration.
Nanotechnology-based gene delivery is the division of nanomedicine concerned with the synthesis, characterization, and functionalization of nanomaterials to be used in targeted-gene delivery applications. Nanomaterial-based gene delivery systems hold great promise for curing fatal inherited and acquired diseases, including neurological disorders, cancer, cardiovascular diseases, and acquired immunodeficiency syndrome (AIDS). However, their use in clinical applications is still controversial. To date, the Food and Drug Administration (FDA) has not approved any gene delivery system because of the unknown long-term toxicity and the low gene transfection efficiency of nanomaterials in vivo. Compared to viral vectors, nonviral gene delivery vectors are characterized by a low preexisting immunogenicity, which is important for preventing a severe immune response. In addition, nonviral vectors provide higher loading capacity and ease of fabrication. For these reasons, this review article focuses on applications of nonviral gene delivery systems, including those based on lipids, polymers, graphene, and other inorganic nanoparticles, and discusses recent advances in nanomaterials for gene therapy. Methods of synthesizing these nanomaterials are briefly described from a materials science perspective. Also, challenges, critical issues, and concerns about the in vivo applications of nanomaterial-based gene delivery systems are discussed. It should be noted that this article is not a comprehensive review of the literature.
Genetically engineered neural stem cell (NSC) transplant populations offer key benefits in regenerative neurology, for release of therapeutic biomolecules in ex vivo gene therapy. NSCs are 'hard-to-transfect' but amenable to 'magnetofection'. Despite the high clinical potential of this approach, the low and transient transfection associated with the large size of therapeutic DNA constructs is a critical barrier to translation. We demonstrate for the first time that DNA minicircles (small DNA vectors encoding essential gene expression components but devoid of a bacterial backbone, thereby reducing construct size versus conventional plasmids) deployed with magnetofection achieve the highest, safe non-viral DNA transfection levels (up to 54%) reported so far for primary NSCs. Minicircle-functionalized magnetic nanoparticle (MNP)-mediated gene delivery also resulted in sustained gene expression for up to four weeks. All daughter cell types of engineered NSCs (neurons, astrocytes and oligodendrocytes) were transfected (in contrast to conventional plasmids which usually yield transfected astrocytes only), offering advantages for targeted cell engineering. In addition to enhancing MNP functionality as gene delivery vectors, minicircle technology provides key benefits from safety/scale up perspectives. Therefore, we consider the proof-of-concept of fusion of technologies used here offers high potential as a clinically translatable genetic modification strategy for cell therapy.
Objectives
Up to now, little research has been focussed on discovering how zeta potential independently affects polymeric nanoparticle (NP) cytotoxicity.Methods
Polymeric nanoparticles of gradient zeta potential ranging from −30 mv to +40 mv were fabricated using the same poly-3-hydroxybutyrate-co-3-hydroxyhexanoate (PHBHHx) biopolymer. Interaction forces between nanoparticles and cells were measured by atomic force microscopy (AFM). Cytotoxicity of the nanoparticles to cells was investigated by using MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide) assay.ResultsFour kinds of nanoparticle with similar sizes and gradient zeta potentials, were fabricated. Those with positive surface charges were found to be more toxic than those with negative surface charges. Positively charged nanoparticles or nanoparticles with higher ‘like’ charges, offered higher interaction force with cells.Conclusion
This work proposes a novel approach for investigating interaction between NPs and cells, and discloses the importance of controlling zeta potential in developing NPs-based formulations in the future.
INTRODUCTION:
Nucleic-acid-based biopharmaceuticals enclose a remarkable potential for treating debilitating or life-threatening diseases that currently remain incurable. This promising area of research envisages the creation of state-of-the-art DNA vaccines, pluripotent cells or gene-based therapies, which can be used to overcome current issues. To achieve this goal, DNA minicircles are emerging as ideal nonviral vectors due to their safety and persistent transgene expression in either quiescent or actively dividing cells.
AREAS COVERED:
This review focuses on the characteristics of minicircle DNA (mcDNA) technology and the current advances in their production. The possible modifications to further improve minicircle efficacy are also emphasized and discussed in light of recent advances. As a final point, the main therapeutic applications of mcDNA are summarized, with a special focus on pluripotent stem cells production and cancer therapy.
EXPERT OPINION:
Achieving in-target and persistent transgene expression is a challenging issue that is of critical importance for a successful therapeutic outcome. The use of miniaturized mcDNA cassettes with additional modifications that increase and prolong expression may contribute to an improved generation of biopharmaceuticals. The unique features of mcDNA render it an attractive alternative to overcome current technical issues and to bridge the significant gap that exists between basic research and clinical applications.
Introduction:
Nucleic-acid-based biopharmaceuticals enclose a remarkable potential for treating debilitating or life-threatening diseases that currently remain incurable. This promising area of research envisages the creation of state-of-the-art DNA vaccines, pluripotent cells or gene-based therapies, which can be used to overcome current issues. To achieve this goal, DNA minicircles are emerging as ideal nonviral vectors due to their safety and persistent transgene expression in either quiescent or actively dividing cells.
Areas covered:
This review focuses on the characteristics of minicircle DNA (mcDNA) technology and the current advances in their production. The possible modifications to further improve minicircle efficacy are also emphasized and discussed in light of recent advances. As a final point, the main therapeutic applications of mcDNA are summarized, with a special focus on pluripotent stem cells production and cancer therapy.
Expert opinion:
Achieving in-target and persistent transgene expression is a challenging issue that is of critical importance for a successful therapeutic outcome. The use of miniaturized mcDNA cassettes with additional modifications that increase and prolong expression may contribute to an improved generation of biopharmaceuticals. The unique features of mcDNA render it an attractive alternative to overcome current technical issues and to bridge the significant gap that exists between basic research and clinical applications.
Niosomes represent a recent promising approach for gene delivery purposes. We elaborated a novel niosome formulation based on the 2,3-di(tetradecyloxy)propan-1-amine cationic lipid, combined with squalene and polysorbate 80 to evaluate the transfection efficiency in rat retinas. Niosomes prepared by the solvent emulsification-evaporation technique were mixed with the pCMSEGFP plasmid to form lipoplexes which were characterized in terms of morphology, size, surface charge, and DNA condensation, protection and release. In vitro studies were conducted to evaluate transfection efficiency, viability and internalization mechanism in HEK-293 and ARPE-19 cells. The efficacy of the most promising formulation was evaluated in rat eyes by monitoring the expression of the EGFP after intravitreal and subretinal injections. Lipoplexes at 15/1 ratio were 200 nm in size, 25mV in zeta potential and exhibited spherical morphology. At this ratio, niosomes condensed and protected the DNA from enzymatic digestion. Lipoplexes successfully transfected HEK-293 and specially ARPE-19 cells, without affecting the viability. Whereas lipoplexes entered mainly retinal cells by clathrin-mediated endocytosis, HEK-293 cells showed a higher caveolae-dependent entry. After ocular administration, the expression of EGFP was detected in different cells of the retina depending on the administration route. This novel niosome formulation represents a promising approach to deliver genetic material into the retina to treat inherited retinal diseases.
Gene therapy strategies for the treatment of inherited retinal diseases have made major advances in recent years. This review focuses on adeno-associated viral (AAV) vector approaches to treat retinal degeneration and, thus, prevent or delay the onset of blindness. Data from human clinical trials of gene therapy for retinal disease show encouraging signs of safety and efficacy from AAV vectors. Recent progress in enhancing cell-specific targeting and transduction efficiency of the various retinal layers plus the use of AAV-delivered growth factors to augment the therapeutic effect and limit cell death suggest even greater success in future human trials is possible.
The interaction of DNA with monolayers of the cationic lipid dimethyldioctadecylammonium bromide, with/without 50 mol % of a neutral "helper" lipid, either dioleoylphosphatidylethanolamine or cholesterol, has been studied using specular neutron reflection, surface pressure-area isotherms, and Brewster angle microscopy. The amount of DNA bound to the lipid head groups has been comprehensively quantified in the range of 8-39 vol% of DNA with respect to the monolayer composition (monolayers composed of dimethyldioctadecylammonium bromide binding the most DNA and monolayers containing dioleoylphosphatidylethanolamine binding the least) and surface pressure (DNA binding being greatest at highest surface pressures). Surprisingly, regardless of these variables, the thickness of the DNA-containing layer remained approximately constant between 18 and 25 Å. This systematic study is the first direct quantification of the binding of DNA with two different helper-lipid-containing multicomponent monolayers, an important step toward understanding interaction parameters in more realistic models of gene delivery systems.
Glaucoma is a group of diseases characterized by progressive optic nerve degeneration that results in visual field loss and irreversible blindness. A crucial element in the pathophysiology of all forms of glaucoma is the death of retinal ganglion cells (RGCs), a population of CNS neurons with their soma in the inner retina and axons in the optic nerve. Strategies that delay or halt RGC loss have been recognized as potentially beneficial to preserve vision in glaucoma; however, the success of these approaches depends on an in-depth understanding of the mechanisms that lead to RGC dysfunction and death. In recent years, there has been an exponential increase in valuable information regarding the molecular basis of RGC death stemming from animal models of acute and chronic optic nerve injury as well as experimental glaucoma. The emerging landscape is complex and points at a variety of molecular signals - acting alone or in cooperation - to promote RGC death. These include: axonal transport failure, neurotrophic factor deprivation, toxic pro-neurotrophins, activation of intrinsic and extrinsic apoptotic signals, mitochondrial dysfunction, excitotoxic damage, oxidative stress, misbehaving reactive glia and loss of synaptic connectivity. Collectively, this body of work has considerably updated and expanded our view of how RGCs might die in glaucoma and has revealed novel, potential targets for neuroprotection.
Corneal gene therapy can potentially treat acquired and inherited corneal disorders that otherwise lead to blindness. In a previous study on the development of effective vectors for corneal gene delivery, we showed that a particular formulation of chitosan-DNA nanoparticles, based on ultrapure chitosan oligomers injected into rat corneas, led to transgene expression that was 5.4-fold higher than that obtained using polyethylenimine-DNA nanoparticles.
In the present study, we investigate the same formulation of chitosan-DNA nanoparticles as carriers of six different plasmids for corneal gene delivery. Size, zeta potential, the ability to condense plasmid DNA, and transfection efficiency in cell cultures and in rat corneas, were all investigated.
Size, zeta potential, the ability to condense plasmid DNA, and transfection efficiency in cell cultures did not substantially vary for nanoparticles based on different plasmids. One day post-injection of nanoparticles into rat corneas, we found that a CpG-free plasmid DNA, pCpG-Luc, which has an EF1α promoter, led to transgene expression that was 7.1-fold higher than that for gWiz-Luc, a commercially available plasmid DNA with a cytomegalovirus (CMV) promoter used in our previous study; 116.8-fold higher than that for pEPI-CMV, a commercially available plasmid that has a scaffold/matrix attachment region (S/MAR) sequence and a CMV promoter; and 76.8-fold higher than that for pEPI-UbC, an experimental plasmid that has an S/MAR sequence and a ubiquitin C promoter.
The present study reveals the potential of comparing various plasmids as an approach for enhancing transgene expression. The delivery system designed in the present study represents the next step in the development of effective vectors for corneal gene therapy.
Ocular gene therapy is becoming a well-established field. Viral gene therapies for the treatment of Leber's congentinal amaurosis (LCA) are in clinical trials, and many other gene therapy approaches are being rapidly developed for application to diverse ophthalmic pathologies. Of late, development of non-viral gene therapies has been an area of intense focus and one technology, polymer-compacted DNA nanoparticles, is especially promising. However, development of pharmaceutically and clinically viable therapeutics depends not only on having an effective and safe vector but also on a practical treatment strategy. Inherited retinal pathologies are caused by mutations in over 220 genes, some of which contain over 200 individual disease-causing mutations, which are individually very rare. This review will focus on both the progress and future of nanoparticles and also on what will be required to make them relevant ocular pharmaceutics.
Poorly soluble drugs are often a challenging problem in drug formulation, especially when the drug is not soluble in either aqueous media or organic solvents. Attempts to overcome the solubility problem are, e.g. solubilisation with mixed micelles or forming a complex using cyclodextrines, but these approaches are of limited success. Another problem with new high potential drug is that these drugs often show bioavailability problems. One tried to improve the in vivo performance of poorly soluble drugs by reducing the particles size of the drug thus leading to an increased surface area and an increased dissolution velocity (Müller et al., 1994, 1999). Some of these problems occurred with tarazepide and therefore it was tried to create a formulation with this drug as nanosuspension which is suitable for intravenous administration.
Recognition by innate immune cells of the pathogen associated molecular patterns (PAMP) lipopolysaccharide (LPS) from Gram-negative bacteria and bacterial CpG-DNA depends on Toll-like receptor4 (TLR4) and TLR9, respectively. To define differences in the response to these distinct PAMP we compared a key intracellular event, namely recruitment of myeloid differentiation marker 88 (MyD88) to the respective PAMP-initiated TLR signaling. Using MyD88-GFP fusion protein expressing macrophages we demonstrate that LPS and CpG-DNA trigger signaling from two different cellular locations: theformer at the cell membrane and the latter at the lysosomal compartment. While LPS does not require endocytosis to functionally associate with the membrane expressed TLR4/MD2 complex, internalization and endosomal maturation is conditional for CpG-DNA to activate TLR9. In support of these data TLR9 is not localized at the cell surface, but intracellularily. These data stress the need to characterize individual TLR at the very beginning of signal initiation in order to understand their diverse biological functions.
This review describes the use of nanoparticles based on solid lipids for the parenteral application of drugs. Firstly, different types of nanoparticles based on solid lipids such as "solid lipid nanoparticles" (SLN), "nanostructured lipid carriers" (NLC) and "lipid drug conjugate" (LDC) nanoparticles are introduced and structural differences are pointed out. Different production methods including the suitability for large scale production are described. Stability issues and drug incorporation mechanisms into the particles are discussed. In the second part, the biological activity of parenterally applied SLN and biopharmaceutical aspects such as pharmacokinetic profiles as well as toxicity aspects are reviewed.
Cationic liposome has been studied as one of the most promising non-viral gene delivery systems. However, it has major drawbacks such as the formation of large aggregates at higher concentrations and the instability in the serum due to cationic lipid. As an alternative gene delivery system, cationic emulsion was formulated and transfection efficiency was evaluated in vitro and in vivo, in comparison with cationic liposome. Cationic emulsion was prepared with varying compositions of 3 beta [N-(N',N'-dimethylaminoethane) carbamoyl] cholesterol (DC-Chol), dioleoylphosphatidyl ethanolamine (DOPE), caster oil and Tween 80. Cationic liposome was prepared with DC-Chol and DOPE. The particle size of all the DNA/lipid complexes varied from 150 to 230 nm. The in vitro transfection efficiency of plasmid DNA was assessed by the expression of green fluorescent protein as a reporter. Of various formulations, cationic emulsion E2 (DC-Chol/DOPE/Castor Oil/Tween 80 = 0.3:0.3:0.3:0.15) and cationic liposome L3 (DC-Chol/DOPE = 0.6:0.3) showed improved transfection. DNA/E2 complexes exhibited higher transfection efficiencies (17.39+/-0.58%) in comparison with DNA/L3 complexes (11.47+/-0.59%). DNA/E2 complexes also showed a better physical stability and a stronger serum resistance than DNA/L3 complexes. Moreover, the cytotoxicity of DNA/E2 complexes was comparable to that of DNA/L3 complexes. When DNA/lipid complexes were intravenously administered, DNA/E2 complexes showed a prolonged circulation in blood and mRNA expression in various tissues compared with DNA/L3 complexes. These results suggest that cationic emulsion E2 could be a potential gene delivery system in clinical approaches because of enhanced in vivo gene transfer with low toxicity.
In this study, we explored the use of electroporation or media that promote lipoplex formation for nonviral gene transfer in the eye. There was no detectable staining for LacZ after subretinal, intravitreous, or periocular injection of a plasmid containing a CMV promoter expression cassette for LacZ, but when plasmid injection in each of the three sites was combined with electroporation, there was efficient transduction. Specific staining for LacZ was seen in retinal pigmented epithelial (RPE) cells after subretinal injection of a plasmid containing a vitelliform macular dystrophy 2 (VMD2) promoter expression cassette, demonstrating that this approach can be used to evaluate purported tissue-specific promoters in vivo. Electroporation with 10 V/mm resulted in strong LacZ staining, but was damaging to photoreceptors; substantial transduction with no evidence of retinal damage was seen using 3.4 V/mm. Staining for LacZ was also seen after subretinal or periocular, but not intravitreous, injection of plasmid DNA in medium containing 40% Lipofectamine2000 (Lf). Injection of 40% Lf into the subretinal space caused damage to photoreceptors, but subretinal injection of plasmid DNA in medium containing 10% NeuroPorter resulted in transduction of RPE cells with no adverse effects on retinal morphology or function as assessed by electroretinograms (ERGs). After either electroporation or lipofection, LacZ staining was detectable for at least 14 days, and could be reinduced by a second procedure. These data suggest that electroporation or lipofection can be used as experimental tools for ocular gene transfer to evaluate tissue-specific promoter fragments or to evaluate the effects of transgene expression in the retina. Also, with additional optimization, nonviral gene transfer may prove to be a valuable approach for the treatment of retinal and choroidal diseases.
The clinical success of gene therapy is critically dependent on the development of efficient and safe gene delivery reagents, popularly known as "transfection vectors." The transfection vectors commonly used in gene therapy are mainly of two types: viral and non-viral. The efficiencies of viral transfection vectors are, in general, superior to their non-viral counterparts. However, the myriads of potentially adverse immunogenic aftermaths associated with the use of viral vectors are increasingly making the non-viral gene delivery reagents as the vectors of choice. Among the existing arsenal of non-viral gene delivery reagents, the distinct advantages associated with the use of cationic transfection lipids include their: (a) robust manufacture; (b) ease in handling and preparation techniques; (c) ability to inject large lipid:DNA complexes; and (d) low immunogenic response. The present review highlights the major achievements in the area of designing efficacious cationic transfection lipids, some of the more recent advances in the field of cationic liposomes-mediated gene transfer and targeted gene delivery, some unresolved issues and challenges in liposomal gene delivery, and future promises of cationic liposomes as gene-carriers in non-viral gene therapy.
Increasing attention has been paid to technology used for the delivery of genetic materials into cells for gene therapy and the generation of genetically engineered cells. So far, viral vectors have been mainly used because of their inherently high transfection efficiency of gene. However, there are some problems to be resolved for the clinical applications, such as the pathogenicity and immunogenicity of viral vectors themselves. Therefore, many research trials with non-viral vectors have been performed to enhance their efficiency to a level comparable to the viral vector. Two directions of these trials exist: Material improvement of non-viral vectors and their combination with various external physical stimuli. In this study gelatin was selected as a non-viral carrier for DNA. To give a positive charge to gelatin, different extents introduction of ethylenediamine (Ed), spermidine (Sd), and spermine (Sm) were reacted with gelatin in the presence of a water-soluble carbodiimide. When positively charged gelatin derivatives (Ed, Sd, and Sm) were mixed with negatively charged DNA, a self assembly of DNA nanoparticle (complex) was formed within few minutes through electrostatic interaction. Irrespective of the type of gelatin derivatives, the apparent molecular size of DNA was reduced by increasing the gelatin/DNA mixing ratio to attain a saturated value of about 150 nm. The condensed gelatin/DNA complexes showed the zeta potential of 10-15 mV. The amount of DNA internalized into the cells was significantly increased by the complexation with every gelatin derivative. The cells incubated with the gelatin/DNA complexes exhibited significantly stronger luciferase activities than naked plasmid DNA. This study clearly demonstrates and self-assembled DNA complexes has potential as a gene delivery vechile and are stable to transfer genetic materials to cells.
Minicircle DNA vectors achieve sustained expression reflected by active chromatin and transcriptional level
Minicircle DNA vectors achieve sustained expression reflected by active
chromatin and transcriptional level. Mol Ther 2013;21:131-8.