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Pharmaceutics
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Lipid-Based Nanocarriers for Non-Viral Gene Delivery
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Lipid-Based Nanocarriers for Non-Viral Gene Delivery

A special issue of Pharmaceutics (ISSN 1999-4923). This special issue belongs to the section "Gene and Cell Therapy".

Deadline for manuscript submissions: closed (20 October 2021) | Viewed by 23361

Special Issue Editors

Prof. María Luisa Moyá

E-Mail Website
Guest Editor
Departament of Physical Chemistry, University of Seville, Sevilla, Spain
Interests: surfactants; calixarenes; polymers; nanomedicine; non-viral gene delivery systems
Dr. Manuel López-López

E-Mail Website
Guest Editor
Department of Chemical Engineering, Physical Chemistry and Materials Science, University of Huelva, Huelva, Spain
Interests: nanomaterials; carbon nanotubes; surfactants; polymers; micelles; liposomes; nanoparticles; DNA; drug delivery nanocarriers; gene transfection; polymers; Physical Chemistry; Kinetics; Thermodynamics.
Special Issues, Collections and Topics in MDPI journals

Special Issue Information

Dear Colleagues,

Gene therapy shows enormous potential for the treatment of cancer and monogenic disorders, the latter being caused by single mutations in a specific (set of) gene(s). Most of the initiated clinical studies used viral delivery systems (vectors), but a low success rate was found due to limitations with respect to immunogenicity and general toxicity. For this reason, in recent decades, tremendous efforts have been made to advance the design and preparation of non-viral gene delivery systems, including lipid-based nanocarriers. They can deliver an encoding gene sequence specifically to the target tissue, which makes the expression of therapeutic proteins in diseased cells possible. Besides, they show reduced immunogenicity, large payloads, safety, and ease manufacturing. However, the wide-spread use of lipid-based vectors in clinical trials is still restricted by low transfection efficiency in certain tissues and unreliable targeting. Therefore, research on efficient non-viral lipid- based gene delivery nanocarriers is mandatory.

This Special Issue invites international researchers in the area of the design and preparation of new lipid-based nanocarriers for gene delivery. Studies focused on elucidating how factors such as structure-activity relationships, additional lipid components present in the nanocarriers, gene delivery systems-to-nucleic acid ratio, formulation buffers and nanocarriers preparation, among others, can increase gene transfection and achieve clinical utility.

Prof. María Luisa Moyá
Dr. Manuel López-López
Guest Editors

Manuscript Submission Information

Manuscripts should be submitted online at www.mdpi.com by registering and logging in to this website. Once you are registered, click here to go to the submission form. Manuscripts can be submitted until the deadline. All submissions that pass pre-check are peer-reviewed. Accepted papers will be published continuously in the journal (as soon as accepted) and will be listed together on the special issue website. Research articles, review articles as well as short communications are invited. For planned papers, a title and short abstract (about 100 words) can be sent to the Editorial Office for announcement on this website.

Submitted manuscripts should not have been published previously, nor be under consideration for publication elsewhere (except conference proceedings papers). All manuscripts are thoroughly refereed through a single-blind peer-review process. A guide for authors and other relevant information for submission of manuscripts is available on the Instructions for Authors page. Pharmaceutics is an international peer-reviewed open access monthly journal published by MDPI.

Please visit the Instructions for Authors page before submitting a manuscript. The Article Processing Charge (APC) for publication in this open access journal is 2900 CHF (Swiss Francs). Submitted papers should be well formatted and use good English. Authors may use MDPI's English editing service prior to publication or during author revisions.

Keywords

  • lipid-based nanocarriers
  • nucleic acids compacting agents
  • lipoplexes
  • non-viral delivery systems
  • nanomedicine
  • nanotechnology
  • DNA delivery
  • RNA delivery
  • lipofection
  • gene therapy

Published Papers (7 papers)

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Research

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16 pages, 2554 KiB  
Article
In Vitro CRISPR/Cas9 Transfection and Gene-Editing Mediated by Multivalent Cationic Liposome–DNA Complexes
by Diana A. Sousa, Ricardo Gaspar, Celso J. O. Ferreira, Fátima Baltazar, Ligia R. Rodrigues and Bruno F. B. Silva
Pharmaceutics 2022, 14(5), 1087; https://doi.org/10.3390/pharmaceutics14051087 - 19 May 2022
Cited by 8 | Viewed by 3347
Abstract
Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated nuclease 9 (Cas9) gene-editing offers exciting new therapeutic possibilities for disease treatment with a genetic etiology such as cancer, cardiovascular, neuronal, and immune disorders. However, its clinical translation is being hampered by the lack [...] Read more.
Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated nuclease 9 (Cas9) gene-editing offers exciting new therapeutic possibilities for disease treatment with a genetic etiology such as cancer, cardiovascular, neuronal, and immune disorders. However, its clinical translation is being hampered by the lack of safe, versatile, and effective nonviral delivery systems. Herein we report on the preparation and application of two cationic liposome–DNA systems (i.e., lipoplexes) for CRISPR/Cas9 gene delivery. For that purpose, two types of cationic lipids are used (DOTAP, monovalent, and MVL5, multivalent with +5e nominal charge), along with three types of helper lipids (DOPC, DOPE, and monoolein (GMO)). We demonstrated that plasmids encoding Cas9 and single-guide RNA (sgRNA), which are typically hard to transfect due to their large size (>9 kb), can be successfully transfected into HEK 293T cells via MVL5-based lipoplexes. In contrast, DOTAP-based lipoplexes resulted in very low transfection rates. MVL5-based lipoplexes presented the ability to escape from lysosomes, which may explain the superior transfection efficiency. Regarding gene editing, MVL5-based lipoplexes achieved promising GFP knockout levels, reaching rates of knockout superior to 35% for charge ratios (+/−) of 10. Despite the knockout efficiency being comparable to that of Lipofectamine 3000® commercial reagent, the non-specific gene knockout is more pronounced in MVL5-based formulations, probably resulting from the considerable cytotoxicity of these formulations. Altogether, these results show that multivalent lipid-based lipoplexes are promising CRISPR/Cas9 plasmid delivery vehicles, which by further optimization and functionalization may become suitable in vivo delivery systems. Full article
(This article belongs to the Special Issue Lipid-Based Nanocarriers for Non-Viral Gene Delivery)
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12 pages, 1605 KiB  
Article
Microfluidic Formulation of DNA-Loaded Multicomponent Lipid Nanoparticles for Gene Delivery
by Erica Quagliarini, Serena Renzi, Luca Digiacomo, Francesca Giulimondi, Barbara Sartori, Heinz Amenitsch, Valentina Tassinari, Laura Masuelli, Roberto Bei, Lishan Cui, Junbiao Wang, Augusto Amici, Cristina Marchini, Daniela Pozzi and Giulio Caracciolo
Pharmaceutics 2021, 13(8), 1292; https://doi.org/10.3390/pharmaceutics13081292 - 19 Aug 2021
Cited by 24 | Viewed by 6462
Abstract
In recent years, lipid nanoparticles (LNPs) have gained considerable attention in numerous research fields ranging from gene therapy to cancer immunotherapy and DNA vaccination. While some RNA-encapsulating LNP formulations passed clinical trials, DNA-loaded LNPs have been only marginally explored so far. To fulfil [...] Read more.
In recent years, lipid nanoparticles (LNPs) have gained considerable attention in numerous research fields ranging from gene therapy to cancer immunotherapy and DNA vaccination. While some RNA-encapsulating LNP formulations passed clinical trials, DNA-loaded LNPs have been only marginally explored so far. To fulfil this gap, herein we investigated the effect of several factors influencing the microfluidic formulation and transfection behavior of DNA-loaded LNPs such as PEGylation, total flow rate (TFR), concentration and particle density at the cell surface. We show that PEGylation and post-synthesis sample concentration facilitated formulation of homogeneous and small size LNPs with high transfection efficiency and minor, if any, cytotoxicity on human Embryonic Kidney293 (HEK-293), spontaneously immortalized human keratinocytes (HaCaT), immortalized keratinocytes (N/TERT) generated from the transduction of human primary keratinocytes, and epidermoid cervical cancer (CaSki) cell lines. On the other side, increasing TFR had a detrimental effect both on the physicochemical properties and transfection properties of LNPs. Lastly, the effect of particle concentration at the cell surface on the transfection efficiency (TE) and cell viability was largely dependent on the cell line, suggesting that its case-by-case optimization would be necessary. Overall, we demonstrate that fine tuning formulation and microfluidic parameters is a vital step for the generation of highly efficient DNA-loaded LNPs. Full article
(This article belongs to the Special Issue Lipid-Based Nanocarriers for Non-Viral Gene Delivery)
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20 pages, 3391 KiB  
Article
Folate-Equipped Cationic Liposomes Deliver Anti-MDR1-siRNA to the Tumor and Increase the Efficiency of Chemotherapy
by Daniil V. Gladkikh, Aleksandra V. Sen′kova, Ivan V. Chernikov, Tatyana O. Kabilova, Nelly A. Popova, Valery P. Nikolin, Elena V. Shmendel, Mikhail A. Maslov, Valentin V. Vlassov, Marina A. Zenkova and Elena L. Chernolovskaya
Pharmaceutics 2021, 13(8), 1252; https://doi.org/10.3390/pharmaceutics13081252 - 13 Aug 2021
Cited by 11 | Viewed by 2287
Abstract
In this study, we examined the in vivo toxicity of the liposomes F consisting of 1,26-bis(cholest-5-en-3-yloxycarbonylamino)-7,11,16,20-tetraazahexacosan tetrahydrochloride, lipid-helper 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine and folate lipoconjugate (O-{2-[rac-2,3-di(tetradecyloxy)prop-1-yloxycarbonyl]aminoethyl}-O’-[2-(pteroyl-L-glutam-5-yl)aminoethyl]octadecaethyleneglycol) and investigated the antitumor effect of combined antitumor therapy consisting of MDR1-targeted siMDR/F complexes [...] Read more.
In this study, we examined the in vivo toxicity of the liposomes F consisting of 1,26-bis(cholest-5-en-3-yloxycarbonylamino)-7,11,16,20-tetraazahexacosan tetrahydrochloride, lipid-helper 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine and folate lipoconjugate (O-{2-[rac-2,3-di(tetradecyloxy)prop-1-yloxycarbonyl]aminoethyl}-O’-[2-(pteroyl-L-glutam-5-yl)aminoethyl]octadecaethyleneglycol) and investigated the antitumor effect of combined antitumor therapy consisting of MDR1-targeted siMDR/F complexes and conventional polychemotherapy using tumor xenograft initiated in immunodeficient mice. Detailed analysis of acute and chronic toxicity of this liposomal formulation in healthy C57BL/6J mice demonstrated that formulation F and parent formulation L (without folate lipoconjugate) have no acute and chronic toxicity in mice. The study of the biodistribution of siMDR/F lipoplexes in SCID mice with xenograft tumors formed by tumor cells differing in the expression level of folate receptors showed that the accumulation in various types of tumors strongly depends on the abandons of folate receptors in tumor cells and effective accumulation occurs only in tumors formed by cells with the highest FR levels. Investigating the effects of combined therapy including anti-MDR1 siRNA/F complexes and polychemotherapy on a multidrug-resistant KB-8-5 tumor xenograft in SCID mice demonstrated that siMDR/F increases the efficiency of polychemotherapy: the treatment leads to pronounced inhibition of tumor growth, reduced necrosis and inflammation, and stimulates apoptosis in KB-8-5 tumor tissue. At the same time, it does not induce liver toxicity in tumor-bearing mice. These data confirm that folate-containing liposome F mediated the extremely efficient delivery of siRNA in FR-expressing tumors in vivo and ensured the safety and effectiveness of its action. Full article
(This article belongs to the Special Issue Lipid-Based Nanocarriers for Non-Viral Gene Delivery)
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19 pages, 1779 KiB  
Article
Gemini Cationic Lipid-Type Nanovectors Suitable for the Transfection of Therapeutic Plasmid DNA Encoding for Pro-Inflammatory Cytokine Interleukin-12
by Natalia Sánchez-Arribas, María Martínez-Negro, Clara Aicart-Ramos, Conchita Tros de Ilarduya, Emilio Aicart, Andrés Guerrero-Martínez and Elena Junquera
Pharmaceutics 2021, 13(5), 729; https://doi.org/10.3390/pharmaceutics13050729 - 15 May 2021
Cited by 2 | Viewed by 2211
Abstract
Ample evidence exists on the role of interleukin-12 (IL-12) in the response against many pathogens, as well as on its remarkable antitumor properties. However, the unexpected toxicity and disappointing results in some clinical trials are prompting the design of new strategies and/or vectors [...] Read more.
Ample evidence exists on the role of interleukin-12 (IL-12) in the response against many pathogens, as well as on its remarkable antitumor properties. However, the unexpected toxicity and disappointing results in some clinical trials are prompting the design of new strategies and/or vectors for IL-12 delivery. This study was conceived to further endorse the use of gemini cationic lipids (GCLs) in combination with zwitterionic helper lipid DOPE (1,2-dioleoyl-sn-glycero-3-phosphatidyl ethanol amine) as nanovectors for the insertion of plasmid DNA encoding for IL-12 (pCMV-IL12) into cells. Optimal GCL formulations previously reported by us were selected for IL-12-based biophysical experiments. In vitro studies demonstrated efficient pCMV-IL12 transfection by GCLs with comparable or superior cytokine levels than those obtained with commercial control Lipofectamine2000*. Furthermore, the nanovectors did not present significant toxicity, showing high cell viability values. The proteins adsorbed on the nanovector surface were found to be mostly lipoproteins and serum albumin, which are both beneficial to increase the blood circulation time. These outstanding results are accompanied by an initial physicochemical characterization to confirm DNA compaction and protection by the lipid mixture. Although further studies would be necessary, the present GCLs exhibit promising characteristics as candidates for pCMV-IL12 transfection in future in vivo applications. Full article
(This article belongs to the Special Issue Lipid-Based Nanocarriers for Non-Viral Gene Delivery)
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23 pages, 3821 KiB  
Article
Cationic Single-Chained Surfactants with a Functional Group at the End of the Hydrophobic Tail DNA Compacting Efficiency
by José Antonio Lebrón, Pilar López-Cornejo, Elena García-Dionisio, Pablo Huertas, Margarita García-Calderón, María Luisa Moyá, Francisco José Ostos and Manuel López-López
Pharmaceutics 2021, 13(4), 589; https://doi.org/10.3390/pharmaceutics13040589 - 20 Apr 2021
Cited by 8 | Viewed by 2418
Abstract
The interaction between calf-thymus DNA, ctDNA, and various single-chained surfactants with different functional groups at the end of hydrophobic tail was studied with the goal of investigating the influence of the functional group nature on surfactant DNA compacting efficiency. The surfactants investigated were [...] Read more.
The interaction between calf-thymus DNA, ctDNA, and various single-chained surfactants with different functional groups at the end of hydrophobic tail was studied with the goal of investigating the influence of the functional group nature on surfactant DNA compacting efficiency. The surfactants investigated were dodecyltriethylammonium bromide (DTEABr), triethyl(1-phenoxydodecyl)ammonium bromide (12PhBr), triethyl(2-naphthoxydodecyl)ammonium bromide (12NBr) and 11-(isonicotinoyloxy)-N,N,N-triethyl-1-undecanaminium bromide (11PyBr). Results made evident that the surfactants’ tendencies to self-aggregation is the key factor determining their efficiency to compact the nucleic acid. Subsequently, DOPE/12NBr/pEGFP-C1 lipoplexes, with different cationic surfactant molar fractions (α) and mass ratios (L/D), were prepared and characterized. DOPE is a zwitterionic phospholipid 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine, and the plasmid pEGFP-C1 carries a GFP coding sequence with the necessary regulatory elements for constitutive expression of the gene in human cells. 12NBr was chosen because it was the most efficient DNA compacting agent among the surfactants investigated. Finally, the cytotoxicity and transfection efficiency (TE) of DOPE/12NBr/pDNA lipoplexes, with different compositions, were investigated. Full article
(This article belongs to the Special Issue Lipid-Based Nanocarriers for Non-Viral Gene Delivery)
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14 pages, 4113 KiB  
Article
Viscous Core Liposomes Increase siRNA Encapsulation and Provides Gene Inhibition When Slightly Positively Charged
by Shayan Ahmed, Hugo Salmon, Nicholas Distasio, Hai Doan Do, Daniel Scherman, Khair Alhareth, Maryam Tabrizian and Nathalie Mignet
Pharmaceutics 2021, 13(4), 479; https://doi.org/10.3390/pharmaceutics13040479 - 01 Apr 2021
Cited by 8 | Viewed by 2941
Abstract
Since its discovery, evidence that siRNA was able to act as an RNA interference effector, led to its acceptation as a novel medicine. The siRNA approach is very effective, due to its catalytic mechanism, but still the limitations of its cellular delivery should [...] Read more.
Since its discovery, evidence that siRNA was able to act as an RNA interference effector, led to its acceptation as a novel medicine. The siRNA approach is very effective, due to its catalytic mechanism, but still the limitations of its cellular delivery should be addressed. One promising form of non-viral gene delivery system is liposomes. The variable and versatile nature of the lipids keeps the possibility to upgrade the liposomal structure, which makes them suitable for encapsulation and delivery of drugs. However, to avoid the limitation of fast release for the hydrophilic drug, we previously designed viscous core liposomes. We aimed in this work to evaluate if these viscous core liposomes (NvcLs) could be of interest for siRNA encapsulation. Then, we sought to add a limited amount of positive charges to provide cell interaction and transfection. Cationic lipid dimyristoylaminopropylaminopropyl or the polymer poly(ethylenimine) were incorporated in NvcL to produce positively charged viscous core liposomes (PvcL) by a customized microfluidic device. We found that NvcLs increased the encapsulation efficiency and loading content with regards to the neutral liposome. Both PvcLPEI and PvcLDMAPAP exhibited transfection and GFP knock-down (≈40%) in both 2D and 3D cell cultures. Finally, the addition of slight positive charges did not induce cell toxicity. Full article
(This article belongs to the Special Issue Lipid-Based Nanocarriers for Non-Viral Gene Delivery)
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Review

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27 pages, 5817 KiB  
Review
Lipophilic Polyamines as Promising Components of Liposomal Gene Delivery Systems
by Pavel A. Puchkov and Michael A. Maslov
Pharmaceutics 2021, 13(6), 920; https://doi.org/10.3390/pharmaceutics13060920 - 21 Jun 2021
Cited by 9 | Viewed by 2676
Abstract
Gene therapy requires an effective and safe delivery vehicle for nucleic acids. In the case of non-viral vehicles, including cationic liposomes, the structure of compounds composing them determines the efficiency a lot. Currently, cationic amphiphiles are the most frequently used compounds in liposomal [...] Read more.
Gene therapy requires an effective and safe delivery vehicle for nucleic acids. In the case of non-viral vehicles, including cationic liposomes, the structure of compounds composing them determines the efficiency a lot. Currently, cationic amphiphiles are the most frequently used compounds in liposomal formulations. In their structure, which is a combination of hydrophobic and cationic domains and includes spacer groups, each component contributes to the resulting delivery efficiency. This review focuses on polycationic and disulfide amphiphiles as prospective cationic amphiphiles for gene therapy and includes a discussion of the mutual influence of structural components. Full article
(This article belongs to the Special Issue Lipid-Based Nanocarriers for Non-Viral Gene Delivery)
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