bims-novged 2022-03-27 papers

on Non-viral vectors for gene delivery

Issue of 2022–03–27
nine papers selected by
the Merkel lab, Ludwig-Maximilians University and Benjamin Winkeljann, Ludwig-Maximilians University

PAMAM versus PEI complexation for siRNA delivery: interaction with model lipid membranes and cellular uptake.
Magnetic Nanoparticles as a Component of Peptide-Based DNA Delivery System for Suicide Gene Therapy of Uterine Leiomyoma.
Enzyme-sensitive nanoparticles, smart TAT and cetuximab conjugated immunoliposomes to overcome multidrug resistance in breast cancer cells.
Gene-Delivery Ability of New Hydrogenated and Partially Fluorinated Gemini bispyridinium Surfactants with Six Methylene Spacers.
mRNA Lipoplexes with Cationic and Ionizable α-Amino-lipophosphonates: Membrane Fusion, Transfection, mRNA Translation and Conformation.
Surface-Engineered Mucus Penetrating Nucleic Acid Delivery Systems with Cell Penetrating Peptides for the Lungs.
Physical and chemical advances of synthetic delivery vehicles to enhance mRNA vaccine efficacy.
3D Melanoma Cocultures as Improved Models for Nanoparticle-Mediated Delivery of RNA to Tumors.
Dually Active Polycation/miRNA Nanoparticles for the Treatment of Fibrosis in Alcohol-Associated Liver Disease.

Pharm Res. 2022 Mar 22.

PAMAM versus PEI complexation for siRNA delivery: interaction with model lipid membranes and cellular uptake.

Patrick K C Chang, Clive A Prestidge, Kristen E Bremmell.

   PURPOSE: Cationic polymers have many advantages as vectors for mediated cellular entry and delivery of siRNA. However, toxicity related to their cationic charge has compromised clinical use. It is hypothesized that the siRNA-vector complex composition and properties can be controlled to optimize therapeutic performance. Here we investigate siRNA complexes with branched polyethylenimine (bPEI) versus generation 4 polyamidoamine dendrimers (PAMAM) on interactions with immobilized lipid membranes, and cellular uptake and toxicity.
METHODS: A model siRNA was complexed with either PAMAM or bPEI, and their size and zeta-potential characterized. Interaction of the complexes and parent polymers with lipid bilayers was investigated using atomic force microscopy and correlated with the uptake and toxicity in HeLa cells.
RESULTS: PAMAM and its siRNA complexes formed circular shaped micron-sized holes in lipid bilayers, while bPEI formed nanoscale holes. Flow cytometry and fluorescence microscopy demonstrated PAMAM-siRNA complexes to have a higher cellular uptake than bPEI-siRNA complexes. bPEI-siRNA complexes did not impact on viability, however PAMAM-siRNA complexes demonstrated increasing cell toxicity as N/P ratio increased. PAMAM-siRNA complexes accumulated around the cell nucleus, while PEI-siRNA complexes were located closer to the cell wall.
CONCLUSION: Complexation of PAMAM dendrimer or bPEI with siRNA modified physicochemical properties of the parent polymer, however it did not impact on the mechanism of interaction with model lipid bilayers or how the polymer/siRNA complex interacted and was internalized by HeLa cells. Interaction of siRNA polymer complexes with cells is related to the action of the parent polymer.

Keywords:  AFM; PEI; dendrimer; lipid-bilayer; siRNA

DOI:  https://doi.org/10.1007/s11095-022-03229-7
Bioengineering (Basel). 2022 Mar 08. pii: 112. [Epub ahead of print]9(3):

Magnetic Nanoparticles as a Component of Peptide-Based DNA Delivery System for Suicide Gene Therapy of Uterine Leiomyoma.

Sofia Shtykalova, Anna Egorova, Marianna Maretina, Vladislav Baranov, Anton Kiselev.

  Suicidegene therapy is considered a promising approach for the treatment of uterine leiomyoma (UL), a benign tumor in women characterized by precise localization. In this study, we investigate the efficiency of αvβ3 integrin-targeted arginine-rich peptide carrier R6p-cRGD electrostatically bound to magnetic nanoparticles (MNPs) for targeted DNA delivery into the UL cells. The physico-chemical and cytotoxic properties, transfection efficiency, and specificity of R6p-cRGD/DNA/MNPs polyplexes were evaluated. The addition of MNPs resulted in a decrease in the time needed for successful transfection with simultaneous increase in efficiency. We revealed a therapeutic effect on primary UL cells after delivery of plasmid encoding the herpes simplex virus type 1 (HSV-1) thymidine kinase gene. Treatment with ganciclovir resulted in 20% efficiency of suicide gene therapy in UL cells transfected with the pPTK-1 plasmid. Based on these results, we conclude that the use of cationic peptide carriers with MNPs can be promising for the development of modular non-viral carriers for suicide gene delivery to UL cells.

Keywords:  DNA delivery; gene therapy; integrins; magnetic nanoparticles; peptide-based carriers; thymidine kinase; uterine leiomyoma

DOI:  https://doi.org/10.3390/bioengineering9030112
Toxicol Appl Pharmacol. 2022 Mar 18. pii: S0041-008X(22)00134-X. [Epub ahead of print]441 115989

Enzyme-sensitive nanoparticles, smart TAT and cetuximab conjugated immunoliposomes to overcome multidrug resistance in breast cancer cells.

Mohsen Safaei, Pegah Khosravian, Sedighe Kazemi Sheykhshabani, Gashtasb Mardani, Fatemeh Elahian, Seyed Abbas Mirzaei.

  Due to recent advances in the field of small molecule-based drugs, designing an efficient siRNA delivery system seems essential. Here, modified sets of lipids conjugated with cell-penetrating TAT peptide, MMP2 enzyme-sensitive moiety, and cetuximab antibodies against the EGF receptor were synthesized, purified and verified on HPLC, TLC, SEM, and DLS analyses. Different cellular and molecular experiments were designed to evaluate the transfection efficiency, targeting properties, and functions, including cytotoxicity assay, resensitization assessments, flow cytometry-based uptake assay, BCRP silencing efficiency, real-time PCR, and western blotting. The final targeted liposomes represented an average diameter of 160 nm; zeta-potential and siRNA encapsulation rates were respectively around -28.9 ± 3.16 mV and 88.3 ± 0.9 w/w. The siBCRP carried by the TAT+Cetuximab+ liposome led to an increase in the tumoricidal effect of mitoxantrone by a reduction in IC50 value by 4-fold (*** P < 0.001). Flow cytometry results showed that the cellular uptake rate of final immunoliposomes was significantly higher than the naked liposomes (*** P < 0.001). The Targeted siRNA encapsulating liposomes decreased BCRP transcript and protein levels in MCF7-MX cells by 0.24 and 0.2-fold after 48 h, respectively. Due to the silencing results of the BCRP by the encapsulated siRNA and the inhibitory effects of cetuximab on the EGFR, this formulation could widely be utilized as a carrier for tumor-directed gene delivery.

Keywords:  Breast cancer; EGFR; Gene therapy; Immunoliposome; Tumor targeting; siRNA

DOI:  https://doi.org/10.1016/j.taap.2022.115989
Int J Mol Sci. 2022 Mar 11. pii: 3062. [Epub ahead of print]23(6):

Gene-Delivery Ability of New Hydrogenated and Partially Fluorinated Gemini bispyridinium Surfactants with Six Methylene Spacers.

Michele Massa, Mirko Rivara, Gaetano Donofrio, Luigi Cristofolini, Erica Peracchia, Carlotta Compari, Franco Bacciottini, Davide Orsi, Valentina Franceschi, Emilia Fisicaro.

  The pandemic emergency determined by the spreading worldwide of the SARS-CoV-2 virus has focused the scientific and economic efforts of the pharmaceutical industry and governments on the possibility to fight the virus by genetic immunization. The genetic material must be delivered inside the cells by means of vectors. Due to the risk of adverse or immunogenic reaction or replication connected with the more efficient viral vectors, non-viral vectors are in many cases considered as a preferred strategy for gene delivery into eukaryotic cells. This paper is devoted to the evaluation of the gene delivery ability of new synthesized gemini bis-pyridinium surfactants with six methylene spacers, both hydrogenated and fluorinated, in comparison with compounds with spacers of different lengths, previously studied. Results from MTT proliferation assay, electrophoresis mobility shift assay (EMSA), transient transfection assay tests and atomic force microscopy (AFM) imaging confirm that pyridinium gemini surfactants could be a valuable tool for gene delivery purposes, but their performance is highly dependent on the spacer length and strictly related to their structure in solution. All the fluorinated compounds are unable to transfect RD-4 cells, if used alone, but they are all able to deliver a plasmid carrying an enhanced green fluorescent protein (EGFP) expression cassette, when co-formulated with 1,2-dioleyl-sn-glycero-3-phosphoethanolamine (DOPE) in a 1:2 ratio. The fluorinated compounds with spacers formed by six (FGP6) and eight carbon atoms (FGP8) give rise to a very interesting gene delivery activity, greater to that of the commercial reagent, when formulated with DOPE. The hydrogenated compound GP16_6 is unable to sufficiently compact the DNA, as shown by AFM images.

Keywords:  DNA-surfactant interaction; atomic force microscopy on DNA; gene delivery; gene therapy; heterocyclic gemini cationic surfactants; nonviral vectors; partially fluorinated gemini surfactants

DOI:  https://doi.org/10.3390/ijms23063062
Pharmaceutics. 2022 Mar 07. pii: 581. [Epub ahead of print]14(3):

mRNA Lipoplexes with Cationic and Ionizable α-Amino-lipophosphonates: Membrane Fusion, Transfection, mRNA Translation and Conformation.

Sohail Akhter, Mathieu Berchel, Paul-Alain Jaffrès, Patrick Midoux, Chantal Pichon.

  Cationic liposomes are attractive carriers for mRNA delivery. Here, mRNA lipoplexes (LX) were prepared with the cationic lipids α-aminolipophosphonate (3b) or imidazolium lipophosphoramidate (2) associated with various α-aminolipophosphonates co-lipids comprising protonable groups (imidazole or pyridine) and DOPE. Physicochemical parameters of liposomes and their membrane fusion activity were measured. LXs comprising either 3b- or 2- allowed transfection of ~25% and 40% of dendritic cells with low cytotoxicity, respectively; the efficiency increased up to 80% when 2 was combined with the imidazole-based co-lipid 1. The transfections were high with 3b/1, 3b/DOPE, 2/1 and 2/DOPE LXs. We observed that the transfection level was not well correlated with the acid-mediated membrane fusion activity of liposomes supposed to destabilize endosomes. The mRNA release from LXs and its translation capacity after release were studied for the most efficient LXs. The results showed that the more mRNA was condensed, the poorer the translation efficiency after release was. In contrast to DNA, circular dichroism performed on mRNA complexed with 2/DOPE revealed the presence of denatured mRNA in LXs explaining this lack of translation efficiency. This is an important parameter that should be stressed for the preparation of mRNA LXs with a conserved mRNA translation activity.

Keywords:  cationic liposomes; dendritic cells; imidazolium lipophosphoramidate; lipoplexes; mRNA transfection; α-aminolipophosphonate

DOI:  https://doi.org/10.3390/pharmaceutics14030581
Mol Pharm. 2022 Mar 25.

Surface-Engineered Mucus Penetrating Nucleic Acid Delivery Systems with Cell Penetrating Peptides for the Lungs.

Anupama Kumari, Simanti Pal, Betsy Reshma G, Franklin Pulikkottil Mohny, Nidhi Gupta, Chirag Miglani, Bijay Pattnaik, Asish Pal, Munia Ganguli.

  Nucleic acids, both DNA and small RNAs, have emerged as potential therapeutics for the treatment of various lung disorders. However, delivery of nucleic acids to the lungs is challenging due to the barrier property imposed by mucus, which is further reinforced in disease conditions such as chronic obstructive pulmonary disease and asthma. The presence of negatively charged mucins imparts the electrostatic barrier property, and the mesh network structure of mucus provides steric hindrance to the delivery system. To overcome this, the delivery system either needs to be muco-inert with a low positive charge such that the interactions with mucus are minimized or should have the ability to transiently dismantle the mucus structure for effective penetration. We have developed a mucus penetrating system for the delivery of both small RNA and plasmid DNA independently. The nucleic acid core consists of a nucleic acid (pDNA/siRNA) and a cationic/amphipathic cell penetrating peptide. The mucus penetrating coating consists of the hydrophilic biopolymer chondroitin sulfate A (CS-A) conjugated with a mucolytic agent, mannitol. We hypothesize that the hydrophilic coating of CS-A would reduce the surface charge and decrease the interaction with negatively charged mucins, while the conjugated mannitol residues would disrupt the mucin-mucin interaction or decrease the viscosity of mucus by increasing the influx of water into the mucus. Our results indicate that CS-A-mannitol-coated nanocomplexes possess reduced surface charge, reduced viscosity of artificial mucus, and increased diffusion in mucin suspension as well as increased penetration through the artificial mucus layer as compared to the non-coated ones. Further, the coated nanocomplexes showed low cytotoxicity as well as higher transfection in A-549 and BEAS-2B cells as compared to the non-coated ones.

Keywords:  cell penetrating peptide; chondroitin sulfate coating; lung delivery system; mannitol; mucus penetration; nucleic acid delivery

DOI:  https://doi.org/10.1021/acs.molpharmaceut.1c00770
J Control Release. 2022 Mar 18. pii: S0168-3659(22)00155-9. [Epub ahead of print]

Physical and chemical advances of synthetic delivery vehicles to enhance mRNA vaccine efficacy.

Hyun Jin Kim, Su Kyoung Seo, Ha Yeon Park.

  The successful translation of mRNA vaccines slows down the spread of viral infectious diseases, which may be accomplished by developing novel chemically modified nucleotides (or nucleosides) and highly efficient, safe mRNA delivery vehicles. Delivery vehicles protect vulnerable antigen mRNA and increase the uptake of mRNA into antigen-presenting cells in the peripheral tissue or lymph nodes. This review introduces essential characteristics of mRNA vaccines (e.g., particle sizes, colloidal stability, surface charges/endosomal escape ability, and ligand conjugation) that may be used to generate high immune responses against foreign antigens. The significance and mechanism of each characteristic are described based on the results obtained from in vitro and in vivo studies. We also discuss the development of next generation delivery vehicles for future mRNA vaccines.

Keywords:  Cationic polymer; Ionizable lipid; Nanomedicine; Nanoparticle; mRNA vaccine

DOI:  https://doi.org/10.1016/j.jconrel.2022.03.029
Cells. 2022 Mar 17. pii: 1026. [Epub ahead of print]11(6):

3D Melanoma Cocultures as Improved Models for Nanoparticle-Mediated Delivery of RNA to Tumors.

Maximilian E A Schäfer, Florian Keller, Jens Schumacher, Heinrich Haas, Fulvia Vascotto, Ugur Sahin, Mathias Hafner, Rüdiger Rudolf.

  Cancer therapy is an emergent application for mRNA therapeutics. While in tumor immunotherapy, mRNA encoding for tumor-associated antigens is delivered to antigen-presenting cells in spleen and lymph nodes, other therapeutic options benefit from immediate delivery of mRNA nanomedicines directly to the tumor. However, tumor targeting of mRNA therapeutics is still a challenge, since, in addition to delivery of the cargo to the tumor, specifics of the targeted cell type as well as its interplay with the tumor microenvironment are crucial for successful intervention. This study investigated lipoplex nanoparticle-mediated mRNA delivery to spheroid cell culture models of melanoma. Insights into cell-type specific targeting, non-cell-autonomous effects, and penetration capacity in tumor and stroma cells of the mRNA lipoplex nanoparticles were obtained. It was shown that both coculture of different cell types as well as three-dimensional cell growth characteristics can modulate distribution and transfection efficiency of mRNA lipoplex formulations. The results demonstrate that three-dimensional coculture spheroids can provide a valuable surplus of information in comparison to adherent cells. Thus, they may represent in vitro models with enhanced predictivity for the in vivo activity of cancer nanotherapeutics.

Keywords:  cancer; in vitro in vivo correlation (IVIVC); lipoplex; mRNA; nanoparticles; tumor models; tumor targeting

DOI:  https://doi.org/10.3390/cells11061026
Pharmaceutics. 2022 Mar 18. pii: 669. [Epub ahead of print]14(3):

Dually Active Polycation/miRNA Nanoparticles for the Treatment of Fibrosis in Alcohol-Associated Liver Disease.

Chuhan Zhang, Yu Hang, Weimin Tang, Diptesh Sil, Heather C Jensen-Smith, Robert G Bennett, Benita L McVicker, David Oupický.

  Alcohol-associated liver disease (AALD) is a major cause of liver disorders worldwide. Current treatment options are limited, especially for AALD-associated fibrosis. Promising approaches include RNA interference for miR-155 overexpression in Kupffer cells (KCs), as well as the use of CXCR4 antagonists that inhibit the activation of hepatic stellate cells (HSCs) through the CXCL12/CXCR4 axis. The development of dual-functioning nanoparticles for the effective delivery of antifibrotic RNA together with a CXCR4 inhibitor thus promises to improve the treatment of AALD fibrosis. In this study, cholesterol-modified polymeric CXCR4 inhibitor (Chol-PCX) was synthesized and used to encapsulate anti-miR-155 or non-coding (NC) miRNA in the form of Chol-PCX/miRNA nanoparticles. The results indicate that the nanoparticles induce a significant miR-155 silencing effect both in vitro and in vivo. Treatment with the Chol-PCX/anti-miR-155 particles in a model of moderate alcohol consumption with secondary liver insult resulted in a significant reduction in aminotransferase enzymes as well as collagen content in the liver parenchyma. Overall, our data support the use of Chol-PCX as a carrier for anti-miR-155 for the combined therapeutic inhibition of CXCR4 and miR-155 expression as a way to improve fibrotic damage in the liver.

Keywords:  CXCR4; alcohol-associated liver disease (AALD); miR-155; nanoparticles

DOI:  https://doi.org/10.3390/pharmaceutics14030669