mRNA-Based Nanomedicinal Products to Address Corneal Inflammation by Interleukin-10 Supplementation

The anti-inflammatory cytokine Interleukin-10 (IL-10) is considered an efficient treatment for corneal inflammation, in spite of its short half-life and poor eye bioavailability. In the present work, mRNA-based nanomedicinal products based on solid lipid nanoparticles (SLNs) were developed in order to produce IL-10 to treat corneal inflammation. mRNA encoding green fluorescent protein (GFP) or human IL-10 was complexed with different SLNs and ligands. After, physicochemical characterization, transfection efficacy, intracellular disposition, cellular uptake and IL-10 expression of the nanosystems were evaluated in vitro in human corneal epithelial (HCE-2) cells. Energy-dependent mechanisms favoured HCE-2 transfection, whereas protein production was influenced by energy-independent uptake mechanisms. Nanovectors with a mean particle size between 94 and 348 nm and a positive superficial charge were formulated as eye drops containing 1% (w/v) of polyvinyl alcohol (PVA) with 7.1–7.5 pH. After three days of topical administration to mice, all formulations produced GFP in the corneal epithelium of mice. SLNs allowed the obtaining of a higher transfection efficiency than naked mRNA. All formulations produce IL-10, and the interleukin was even observed in the deeper layers of the epithelium of mice depending on the formulation. This work shows the potential application of mRNA-SLN-based nanosystems to address corneal inflammation by gene augmentation therapy.

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Topical Administration of SLN-Based Gene Therapy for the Treatment of Corneal Inflammation by De Novo IL-10 Production

Pharmaceutics ◽

10.3390/pharmaceutics12060584 ◽

2020 ◽

Vol 12 (6) ◽

pp. 584 ◽

Cited By ~ 2

Author(s):

Mónica Vicente-Pascual ◽

Itziar Gómez-Aguado ◽

Julen Rodríguez-Castejón ◽

Alicia Rodríguez-Gascón ◽

Elisabetta Muntoni ◽

...

Keyword(s):

Gene Therapy ◽

Viral Vectors ◽

De Novo ◽

Topical Administration ◽

Interleukin 10 ◽

Viral Gene ◽

Clinical Approach ◽

Eye Drops ◽

Knock Out ◽

Corneal Inflammation

One of the main challenges in gene therapy is the issue of delivery, and it is especially relevant for the success of gene therapy in the cornea. In the present work, eye drops containing biocompatible non-viral vectors based on solid lipid nanoparticles (SLNs) as gene delivery systems to induce the expression of interleukin 10 (IL-10) were designed to address the treatment of corneal inflammation. Two kinds of SLNs combined with different ligands (protamine, dextran, or hyaluronic acid (HA)) and formulated with polyvinyl alcohol (PVA) were prepared. SLN-based vectors were characterized in terms of size, adhesiveness, viscosity, and pH, before topical administration to wild type and IL-10 knock out (KO) mice. The formulations showed a homogenous particle size below 400 nm and a positive surface charge to favor bioadhesion; the incorporation of PVA improved the corneal penetration. After three days of treatment by topical instillation, SLN-based vectors mainly transfected corneal epithelial cells, HA-formulations being the most effective ones. IL-10 was capable of reaching even the endothelial layer. Corneal sections showed no histological change and formulations seemed to be well tolerated after repeated topical administration. These promising results highlight the possible contribution of non-viral gene augmentation therapy to the future clinical approach of corneal gene therapy.

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Cationic vesicles of a carnitine-derived single-tailed surfactant: Physicochemical characterization and evaluation of in vitro gene transfection efficiency

Journal of Colloid and Interface Science ◽

10.1016/j.jcis.2014.08.049 ◽

2014 ◽

Vol 436 ◽

pp. 138-145 ◽

Cited By ~ 15

Author(s):

Trilochan Patra ◽

Subhajit Ghosh ◽

Joykrishna Dey

Keyword(s):

Transfection Efficiency ◽

Gene Transfection ◽

Physicochemical Characterization

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Nanoparticles Composed by Oligochitosan and Polyethylenimine for Gene Delivery

Applied Mechanics and Materials ◽

10.4028/www.scientific.net/amm.284-287.418 ◽

2013 ◽

Vol 284-287 ◽

pp. 418-422

Author(s):

Chi Hsien Liu ◽

Mei Shan Cheng

Keyword(s):

Gene Delivery ◽

Cell Lines ◽

Fluorescent Protein ◽

Synergistic Effects ◽

Image Analyzer ◽

Transfection Efficacy ◽

Fluorescence Protein ◽

Green Fluorescent ◽

3T3 Cell Lines

Cationic carriers including polyethylenimine, liposomes, and chitosan have been used to transfer plasmid DNA in vitro by condensing anionic DNA. Here, oligochitosan (OC) was found to have capacity for in-vitro gene delivery in four cell lines. Plasmid containing green fluorescent protein (GFP) gene was used as a reporter gene. The transfection efficacy and cell viability of the transfection vehicles were analyzed by using a high-throughput image analyzer. We found that DNA polyplexes formed by high dosage of OC could be efficiently delivered into the cells. The combination of OC and polyethylenimine (PEI) were found to significantly enhance the fluorescence protein expression. The introduction of oligochitosan in PEI-mediated transfection could increase the transfection efficacy and could reduce the toxicity of PEI. Additionally, the synergistic effects of PEI and OC were confirmed in CHO, Caco2, Hep-SK, and 3T3 cell lines. The detailed mechanism of PEI and oligochitosan on transfection was investigated by using gel retardation and DNase degradation experiments. A facile and inexpensive construction of gene delivering vehicles was developed herein by using oligochitosan and PEI.

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Effects of lyoprotectants on long-term stability and transfection efficacy of lyophilized poly(lactide-co-glycolide)-graft-polyethylenimine/plasmid DNA polyplexes

Nanomedicine ◽

10.2217/nnm-2021-0065 ◽

2021 ◽

Author(s):

Joshua Woo ◽

Jeoung Soo Lee

Keyword(s):

Plasmid Dna ◽

Fluorescent Protein ◽

Transfection Efficiency ◽

Dna Encoding ◽

Rat Glioma ◽

Term Stability ◽

Long Term Stability ◽

Glioma C6 ◽

Transfection Efficacy

Aim: We investigated the effect of lyoprotectants on the long-term stability and transfection efficiency of lyophilized (Lyo.) polyplexes prepared from poly(lactide-co-glycolide)-graft-polyethylenimine (PgP) and plasmid DNA encoding green fluorescent protein (pGFP). Materials & methods: Lyo. PgP/pGFP polyplexes prepared with/without lyoprotectants were stored at -20°C over 6 months. Polyplex stability was analyzed by gel electrophoresis and heparin competition assay. Transfection efficiency and cytotoxicity were evaluated in rat glioma (C6) cells in medium containing 10% serum. Results: Lyo. PgP/pGFP polyplexes prepared with 5% sucrose as a lyoprotectant remained stable up to 6 months and retained transfection efficiency up to 4 months. Conclusion: Lyo. PgP-based polyplexes retain bioactivity during extended storage, potentially enabling transport to remote regions and less stable settings, increasing access to life-changing gene therapy.

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CLONING OF HUMAN INTERLEUKIN-10 GENE AND TRANSGENE EXPRESSION IN RABBIT SYNOVIAL CELLS IN VITRO

Journal of Musculoskeletal Research ◽

10.1142/s0218957708001900 ◽

2008 ◽

Vol 11 (01) ◽

pp. 1-7 ◽

Cited By ~ 1

Author(s):

Xiangping Liu ◽

Haibo Wang ◽

Kun Yang ◽

Aihua Sui ◽

Yanming Wang ◽

...

Keyword(s):

Transgene Expression ◽

Fluorescent Protein ◽

Interleukin 10 ◽

Eukaryotic Expression ◽

Cell Culture Supernatant ◽

Enzyme Linked Immunosorbent Assay ◽

Synovial Cells ◽

Reading Frame ◽

Green Fluorescent

In this work, the full-length open reading frame of the human interleukin-10 (hIL-10) gene was amplified through reverse transcription–polymerase chain reaction (RT-PCR), and then PCR products were inserted into pcDNA4/HisMax to construct an eukaryotic expression vector. After optimization by green fluorescent protein (GFP), recombinant hIL-10 genes were transfected and expressed in rabbit synovial cells compounded with liposome in vitro. In cell culture supernatant, rhIL-10 was detected using enzyme-linked immunosorbent assay (ELISA) at time intervals of 12 hours, 24 hours, 48 hours, 72 hours, 7 days, and 14 days. After 12 hours of transfection, ELISA showed that transgene expression of hIL-10 in rabbit synovial cells was elevated; at 72 hours, hIL-10 expression reached its peak value; and then it declined gradually until 7 days, compared with the control. After 14 days, transgene expression ceased. Gene cloning of hIL-10 and its transgene expression in synovial cells therefore gives a basis for the gene therapy of rheumatoid arthritis.

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Gene Therapy: A Lipofection Approach for Gene Transfer into Primary Endothelial Cells

Cell Transplantation ◽

10.3727/000000002783985495 ◽

2002 ◽

Vol 11 (6) ◽

pp. 573-582 ◽

Cited By ~ 9

Author(s):

A. T. L. Young ◽

J. R. T. Lakey ◽

A. G. Murray ◽

R. B. Moore

Keyword(s):

Gene Therapy ◽

Endothelial Cells ◽

Gene Delivery ◽

Fluorescent Protein ◽

Transfection Efficiency ◽

Umbilical Vein ◽

Vector System ◽

Microscopic Evaluation

Despite the great potential of gene therapy to become a new treatment modality in future medicine, there are still many limitations to overcome before this gene approach can pass to the stage of human trial. The foremost obstacle is the development of a safe, efficient, and efficacious vector system for in vivo gene application. This study evaluated the efficacy of lipofection as a gene delivery vehicle into primary endothelial cells. Transfection efficiency of several lipid-based reagents (Effectene, Fugene 6, DOTAP) was examined at experimental temperatures of 37°C, 24°C, and 6°C. Human umbilical vein endothelial cells (HUVECs) were transfected with the enhanced green fluorescent protein (EGFP) using precise amounts of DNA (Effectene, 0.2 μg; Fugene 6, 0.5 μg; DOTAP, 2.5 μg) and lipids (Effectene, 10 μl; Fugene 6, 6 μl; DOTAP, 15 μl) optimized in our laboratory. Duration of incubation in the DNA/lipid transfection mixture varied for each lipid transfectant as follows: 5 h for both Fugene 6 and DOTAP and 3 h for Effectene. Efficiency of transfection was quantified by microscopic evaluation of EFGP expression in a minimum of 100 cells per group. Transfection efficiencies achieved with these lipofection agents were 34 ± 1.3% (mean ± SEM), 33 ± 1.4%, and 18 ± 1.5% for Effectene, Fugene 6, and DOTAP, respectively, at 37°C. Transfection results were lower at 24°C with mean efficiencies of 26 ± 2.4% for Effectene, 14 ± 2.9% for Fugene 6, and 15 ± 3.2% for DOTAP. Furthermore, mean efficiencies at 6°C were 6 ± 0.5%, 8 ± 1.5%, and 6 ± 0.0% for Effectene, Fugene 6, and DOTAP, respectively. Efficiency of transfection appeared to be temperature dependent (ANOVA; p < 0.0001). In spite of a significant decrease (37°C vs. 24°C: p < 0.0001; 37°C vs. 6°C: p < 0.0001; 24°C vs. 6°C: p < 0.0115) in transfection efficiency at low temperatures, the successful in vitro gene manipulation renders lipofection a potential gene delivery strategy for in vivo gene therapy.

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Strategies for simultaneous and successive delivery of RNA

Journal of Molecular Medicine ◽

10.1007/s00109-020-01956-1 ◽

2020 ◽

Vol 98 (12) ◽

pp. 1767-1779

Author(s):

Hanieh Moradian ◽

Andreas Lendlein ◽

Manfred Gossen

Keyword(s):

Messenger Rna ◽

Fluorescent Protein ◽

Transfection Efficiency ◽

Cell Types ◽

Viral Gene ◽

Cell Level ◽

Integrated Method ◽

Viral Gene Delivery ◽

Selection Of

AbstractAdvanced non-viral gene delivery experiments often require co-delivery of multiple nucleic acids. Therefore, the availability of reliable and robust co-transfection methods and defined selection criteria for their use in, e.g., expression of multimeric proteins or mixed RNA/DNA delivery is of utmost importance. Here, we investigated different co- and successive transfection approaches, with particular focus on in vitro transcribed messenger RNA (IVT-mRNA). Expression levels and patterns of two fluorescent protein reporters were determined, using different IVT-mRNA doses, carriers, and cell types. Quantitative parameters determining the efficiency of co-delivery were analyzed for IVT-mRNAs premixed before nanocarrier formation (integrated co-transfection) and when simultaneously transfecting cells with separately formed nanocarriers (parallel co-transfection), which resulted in a much higher level of expression heterogeneity for the two reporters. Successive delivery of mRNA revealed a lower transfection efficiency in the second transfection round. All these differences proved to be more pronounced for low mRNA doses. Concurrent delivery of siRNA with mRNA also indicated the highest co-transfection efficiency for integrated method. However, the maximum efficacy was shown for successive delivery, due to the kinetically different peak output for the two discretely operating entities. Our findings provide guidance for selection of the co-delivery method best suited to accommodate experimental requirements, highlighting in particular the nucleic acid dose-response dependence on co-delivery on the single-cell level.

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Retina-Targeted Delivery of 17β-Estradiol by the Topically Applied DHED Prodrug

Pharmaceutics ◽

10.3390/pharmaceutics12050456 ◽

2020 ◽

Vol 12 (5) ◽

pp. 456

Author(s):

Katalin Prokai-Tatrai ◽

Vien Nguyen ◽

Daniel L. De La Cruz ◽

Rebecca Guerra ◽

Khadiza Zaman ◽

...

Keyword(s):

Targeted Delivery ◽

Ex Vivo ◽

Estrogen Therapy ◽

Topical Administration ◽

The Body ◽

Female Rats ◽

Full Potential ◽

Eye Drops ◽

17Β Estradiol

The purpose of this study was to explore retina-targeted delivery of 17β-estradiol (E2), a powerful neuroprotectant, by its bioprecursor prodrug 10β,17β-dihydroxyestra-1,4-dien-3-one (DHED) administered as eye drops in animal models. Compared to the parent hormone, DHED displayed increased transcorneal flux ex vivo both with and without the presence of 2-hydroxypropyl-β-cyclodextrin used as a penetration-enhancing excipient in rat, rabbit, and pig. In vitro, the prodrug also showed facile bioactivation to E2 in the retina but not in the cornea. After topical administration to rats and rabbits, peak DHED-derived E2 concentrations reached 13 ± 5 ng/g and 18 ± 7 ng/g in the retina of female rats and rabbits, respectively. However, the prodrug remained inert in the rest of the body and, therefore, did not cause increase in circulating hormone concentration, as well as wet uterine and anterior pituitary weights as typical markers of E2′s endocrine impact. Altogether, our studies presented here have demonstrated the premise of topical retina-selective estrogen therapy by the DHED prodrug approach for the first time and provide compelling support for further investigation into the full potential of DHED for an efficacious and safe ocular neurotherapy.

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Investigation of Combined Cyclodextrin and Hydrogel Formulation for Ocular Delivery of Dexamethasone Acetate by Means of Experimental Designs

Pharmaceutics ◽

10.3390/pharmaceutics10040249 ◽

2018 ◽

Vol 10 (4) ◽

pp. 249 ◽

Cited By ~ 6

Author(s):

Roseline Mazet ◽

Luc Choisnard ◽

Delphine Levilly ◽

Denis Wouessidjewe ◽

Annabelle Gèze

Keyword(s):

Ex Vivo ◽

Aqueous Solubility ◽

Eye Drops ◽

Ocular Delivery ◽

Drug Bioavailability ◽

Corneal Inflammation ◽

Dexamethasone Acetate ◽

Great Propensity

Dexamethasone acetate (DXMa) has proven its efficiency to treat corneal inflammation, without a great propensity to increase intraocular pressure. Unfortunately, its poor aqueous solubility, associated with a rapid precorneal elimination, results in a low drug bioavailability and a low penetration after topical ocular administration. The main objective of this study was to improve the apparent aqueous solubility of DXMa using cyclodextrins. First, hydroxypropyl-β-CD (HPβCD) and hydroxypropyl-γ-CD (HPγCD) were used to enhance DXMa concentration in aqueous solution. The β and γ HPCD derivatives allowed the increase of the DXMa amount in solution at 25 °C by a factor of 500 and 1500, respectively. Second, with the aim of improving the persistence of the complex solution after instillation in the eye, the formulations of DXMa-based CD solutions with marketed ophthalmic gels (CELLUVISC®, GEL-LARMES®, and VISMED®) were investigated and optimized by means of special cubic mixture designs, allowing the defining of mixed gels loaded with 0.7% (HPβCD) and 2% (HPγCD) DXMa with osmolality within acceptable physiological range. Finally, in vitro drug release assays from the mixed gels were performed and compared with reference eye drops. Similarly to MAXIDEX® and DEXAFREE®, in the case of mixed gel containing HPβCD, more than 90% of the drug was released within 2 h, while in mixed gel containing HPγCD, the release of DXMa was partial, reaching ≈60% in 2 h. This difference will have to be further addressed with ex vivo and in vivo ocular delivery experiments.

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