bims-novged 2023-11-26 papers

bims-novged

Biomed News

on Non-viral vectors for gene delivery

Issue of 2023–11–26
eleven papers selected by
the Merkel lab, Ludwig-Maximilians University

Clickable Polyethyleneimine Incorporated into Triblock Copolymeric Micelles as an Efficient Platform in the Delivery of siRNA to NSCLC Cells.
Poly(N-methyl-N-vinylacetamide): A Strong Alternative to Peg for Lipid-Based Nanocarriers Delivering Sirna.
PEI functionalized cell membrane for tumor targeted and glutathione responsive gene delivery.
Fusogenic Coiled-Coil Peptides Enhance Lipid Nanoparticle-Mediated mRNA Delivery upon Intramyocardial Administration.
Second-Generation Polyamidoamine Dendrimer Conjugated with Oligopeptides Can Enhance Plasmid DNA Delivery In Vitro.
Combinatorial development of nebulized mRNA delivery formulations for the lungs.
A preparation method for mRNA-LNPs with improved properties.
Delivery of Mrna Vaccine with 1, 2-diesters-derived Lipids Elicits Fast Liver Clearance for Safe and Effective Cancer Immunotherapy.
Effective delivery of miR-511-3p with mannose-decorated exosomes with RNA nanoparticles confers protection against asthma.
Phase-Separated Lipid-Based Nanoparticles: Selective Behavior At The Nano-Bio Interface.
Cell interactions with lipid nanoparticles possessing different internal nanostructures: Liposomes, bicontinuous cubosomes, hexosomes, and discontinuous micellar cubosomes.

Int J Pharm. 2023 Nov 22. pii: S0378-5173(23)01054-2. [Epub ahead of print] 123632

Clickable Polyethyleneimine Incorporated into Triblock Copolymeric Micelles as an Efficient Platform in the Delivery of siRNA to NSCLC Cells.

Franck Marquet, Harry Hagen, Mattia Stanchieri, Véronique Serre Beinier, Gianvito Grasso, Andrea Danani, Viorica Patrulea, Gerrit Borchard.

  The efficacy of transfection vectors to cross the endosomal membrane into the cytosol is a central aspect in the development of nucleic acid-based therapeutics. The challenge remains the same: Delivery, Delivery, Delivery. Despite a rational and appropriate construct of triblock polymeric micelles, which could serve as an ideal platform for the co-delivery of siRNAs and hydrophobic anticancer drugs, we show here its inability to properly convey oligonucleotides to their final destination. In order to overcome biological barriers, a linear PEI comprising two orthogonal groups was synthesized, holding an appropriate balance between safety and efficacy. Micellar carriers were then formulated with this polymer to enhance endosomal siRNA release. This chemical technology also addresses the two major challenges to consider when developing novel micellar products for siRNA delivery, namely cytotoxicity of polycations and endosomal escape. Herein, we demonstrate successful release of siRNA using a polymer tailoring strategy combined with a relevant in vitro approach, considering STAT3 as a promising target in the treatment of non-small cell lung cancer (NSCLC).

Keywords:  Endosomal escape; Lung cancer; Polyethylenimine; siRNA delivery

DOI:  https://doi.org/10.1016/j.ijpharm.2023.123632
Adv Healthc Mater. 2023 Nov 23. e2302712

Poly(N-methyl-N-vinylacetamide): A Strong Alternative to Peg for Lipid-Based Nanocarriers Delivering Sirna.

Manon Berger, François Toussaint, Sanaa Ben Djemaa, Erik Maquoi, Hélène Pendeville, Brigitte Evrard, Christine Jerôme, Jeanne Leblond Chain, Anna Lechanteur, Denis Mottet, Antoine Debuigne, Géraldine Piel.

  Lipid-based nanocarriers such as liposomes or lipid nanoparticles (LNPs) have demonstrated high interest in delivering genetic material. This has been recently emphasized by the approval of Onpattro® and COVID-19 vaccines. PEGylation is known to provide lipid-based nanocarriers with stealth properties, but it also leads to reduced cellular uptake and endosomal escape, and to the production of anti-PEG antibodies causing accelerated blood clearance (ABC) and hypersensitivity reactions (HSR). This work highlights the great potential of amphiphilic poly(N-methyl-N-vinylacetamide) (PNMVA) derivatives as alternatives to lipid-PEG for post-insertion into lipoplexes and pre-insertion into LNPs designed for siRNA delivery. PNMVA compounds with different degrees of polymerization and hydrophobic segments, such as octadecyl and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), were synthesized. Among them, DSPE-PNMVA efficiently integrated into lipoplex and LNP membranes and prevented protein corona formation around these lipid carriers, exhibiting stealth properties comparable to DSPE-PEG. However, unlike DSPE-PEG, DSPE-PNMVA24 showed no adverse impact on lipoplexes cell uptake and endosomal escape. In in vivo study with mice, DSPE-PNMVA24 lipoplexes demonstrated no liver accumulation, indicating good stealth properties, extended circulation time after a second dose, reduced immunological reaction, and no systemic pro-inflammatory response. Safety of DSPE-PNMVA24 was confirmed at the cellular level and in animal models of zebrafish and mice. Overall, DSPE-PNMVA is an advantageous substitute to DSPE-PEG for siRNA delivery, offering comparable stealth and toxicity properties while improving efficacy of the lipid-based carriers by minimizing the dilemma effect and reducing immunological reactions, meaning no ABC or HSR effects. This article is protected by copyright. All rights reserved.

Keywords:  PEG alternative; lipid nanoparticles; lipoplexes; poly(N-methyl-N-vinylacetamide); siRNA delivery

DOI:  https://doi.org/10.1002/adhm.202302712
Int J Biol Macromol. 2023 Nov 21. pii: S0141-8130(23)05253-4. [Epub ahead of print] 128354

PEI functionalized cell membrane for tumor targeted and glutathione responsive gene delivery.

Mengying Wang, Haoxiang Huang, Yanlin Sun, Mingjie Wang, Zhaojun Yang, Yong Shi, Liang Liu.

  Polyethylenimine (PEI) is a broadly exploited cationic polymer due to its remarkable gene-loading capacity. However, the high cytotoxicity caused by its high surface charge density has been reported in many cell lines, limiting its application significantly. In this study, two different molecular weights of PEI (PEI10k and PEI25k) were crosslinked with red blood cell membranes (RBCm) via disulfide bonds to form PEI derivatives (RMPs) with lower charge density. Furthermore, the targeting molecule folic acid (FA) molecules were further grafted onto the polymers to obtain FA-modified PEI-RBCm copolymers (FA-RMP25k) with tumor cell targeting and glutathione response. In vitro experiments showed that the FA-RMP25k/DNA complex had satisfactory uptake efficiency in both HeLa and 293T cells, and did not cause significant cytotoxicity. Furthermore, the uptake and transfection efficiency of the FA-RMP25k/DNA complex was significantly higher than that of the PEI25k/DNA complex, indicating that FA grafting can increase transfection efficiency by 15 %. These results suggest that FA-RMP25k may be a promising non-viral gene vector with potential applications in gene therapy.

Keywords:  Cell membrane; Gene vector; Glutathione response; Polyethylenimine; Tumor targeting

DOI:  https://doi.org/10.1016/j.ijbiomac.2023.128354
ACS Nano. 2023 Nov 20.

Fusogenic Coiled-Coil Peptides Enhance Lipid Nanoparticle-Mediated mRNA Delivery upon Intramyocardial Administration.

Ye Zeng, Mariona Estapé Senti, M Clara I Labonia, Panagiota Papadopoulou, Maike A D Brans, Inge Dokter, Marcel H Fens, Alain van Mil, Joost P G Sluijter, Raymond M Schiffelers, Pieter Vader, Alexander Kros.

  Heart failure is a serious condition that results from the extensive loss of specialized cardiac muscle cells called cardiomyocytes (CMs), typically caused by myocardial infarction (MI). Messenger RNA (mRNA) therapeutics are emerging as a very promising gene medicine for regenerative cardiac therapy. To date, lipid nanoparticles (LNPs) represent the most clinically advanced mRNA delivery platform. Yet, their delivery efficiency has been limited by their endosomal entrapment after endocytosis. Previously, we demonstrated that a pair of complementary coiled-coil peptides (CPE4/CPK4) triggered efficient fusion between liposomes and cells, bypassing endosomal entrapment and resulting in efficient drug delivery. Here, we modified mRNA-LNPs with the fusogenic coiled-coil peptides and demonstrated efficient mRNA delivery to difficult-to-transfect induced pluripotent stem-cell-derived cardiomyocytes (iPSC-CMs). As proof of in vivo applicability of these fusogenic LNPs, local administration via intramyocardial injection led to significantly enhanced mRNA delivery and concomitant protein expression. This represents the successful application of the fusogenic coiled-coil peptides to improve mRNA-LNPs transfection in the heart and provides the potential for the advanced development of effective regenerative therapies for heart failure.

Keywords:  fusogenic coiled-coil; iPSC-CM; intramyocardial delivery; lipid nanoparticles; mRNA delivery

DOI:  https://doi.org/10.1021/acsnano.3c05341
Molecules. 2023 Nov 17. pii: 7644. [Epub ahead of print]28(22):

Second-Generation Polyamidoamine Dendrimer Conjugated with Oligopeptides Can Enhance Plasmid DNA Delivery In Vitro.

Seongyeon Kim, Le Thi Thuy, Jeil Lee, Joon Sig Choi.

  Poly(amidoamine) (PAMAM) dendrimers have attracted considerable attention in the field of gene therapy due to their flexibility in introducing different functional moieties and reduced toxicity at low generations. However, their transfection efficiency remains a limitation. Therefore, an essential approach for improving their transfection efficiency as gene carriers involves modifying the structure of PAMAM by conjugating functional groups around their surface. In this study, we successfully conjugated an RRHRH oligopeptide to the surface of PAMAM generation 2 (PAMAM G2) to create RRHRH-PAMAM G2. This construction aims to condense plasmid DNA (pDNA) and facilitate its penetration into cell membranes, leading to its promising potential for gene therapy. RRHRH-PAMAM G2/pDNA complexes were smaller than 100 nm and positively charged. Nano-polyplexes can enter the cell and show a high transfection efficiency after 24 h of transfection. The RRHRH-PAMAM G2 was non-toxic to HeLa, NIH3T3, A549, and MDA-MB-231 cell lines. These results strongly suggest that RRHRH-PAMAM G2 holds promise as a gene carrier for gene therapy owing to its biocompatibility and ability to deliver genes to the cell.

Keywords:  PAMAM; RRHRH; gene carrier; oligopeptide; transfection

DOI:  https://doi.org/10.3390/molecules28227644
Nat Nanotechnol. 2023 Nov 20.

Combinatorial development of nebulized mRNA delivery formulations for the lungs.

Allen Y Jiang, Jacob Witten, Idris O Raji, Feyisayo Eweje, Corina MacIsaac, Sabrina Meng, Favour A Oladimeji, Yizong Hu, Rajith S Manan, Robert Langer, Daniel G Anderson.

Inhaled delivery of mRNA has the potential to treat a wide variety of diseases. However, nebulized mRNA lipid nanoparticles (LNPs) face several unique challenges including stability during nebulization and penetration through both cellular and extracellular barriers. Here we develop a combinatorial approach addressing these barriers. First, we observe that LNP formulations can be stabilized to resist nebulization-induced aggregation by altering the nebulization buffer to increase the LNP charge during nebulization, and by the addition of a branched polymeric excipient. Next, we synthesize a combinatorial library of ionizable, degradable lipids using reductive amination, and evaluate their delivery potential using fully differentiated air-liquid interface cultured primary lung epithelial cells. The final combination of ionizable lipid, charge-stabilized formulation and stability-enhancing excipient yields a significant improvement in lung mRNA delivery over current state-of-the-art LNPs and polymeric nanoparticles.

DOI: https://doi.org/10.1038/s41565-023-01548-3
J Control Release. 2023 Nov 16. pii: S0168-3659(23)00734-4. [Epub ahead of print]364 632-643

A preparation method for mRNA-LNPs with improved properties.

Cong Geng, Kefan Zhou, Ying Yan, Chan Li, Beibei Ni, Jiangman Liu, Yeming Wang, Xiaoyan Zhang, Dazhuang Wang, Lu Lv, Yongchuan Zhou, Anhua Feng, Yajuan Wang, Chunlei Li.

  The properties of mRNA lipid nanoparticles (mRNA-LNPs), including size, empty particles, morphology, storage stability, and transfection potency, are critically dependent on the preparation methods. Here, a Two-step tangential-flow filtration (TFF) method was successfully employed to improve the properties of mRNA-LNPs during the preparation process. This method involves an additional ethanol removal step prior to the particle fusion process. Notably, this innovative approach has yielded mRNA-LNPs with larger particles, a reduced proportion of empty LNPs, optimized storage stability (at least 6 months at 2-8 °C), improved in vitro transfection efficiency, and minimized distribution in the heart and blood in vivo. In summary, this study represents the implementation of the innovative Two-step TFF method in the preparation of mRNA-LNPs. Our findings indicate substantial enhancements in the properties of our mRNA-LNPs, specifically with regard to the percentage of empty LNPs, stability, transfection efficiency, and in vivo distribution. These improvements have the potential to optimize their industrial applicability and expand their clinical use.

Keywords:  Empty lipid nanoparticles; Ethanol removal; Lipid nanoparticles; Particle fusion; Stability

DOI:  https://doi.org/10.1016/j.jconrel.2023.11.017
Adv Healthc Mater. 2023 Nov 21. e2302691

Delivery of Mrna Vaccine with 1, 2-diesters-derived Lipids Elicits Fast Liver Clearance for Safe and Effective Cancer Immunotherapy.

Yuandong Xu, Yuexiao Hu, Heng Xia, Shiqiang Zhang, Hanqi Lei, Binyuan Yan, Ze Xiu Xiao, Jinjin Chen, Jun Pang, Gao-Feng Zha.

  messenger RNA (mRNA) vaccine has been explored as a promising strategy for cancer immunotherapy, but the side effects especially the liver-related damage caused by LNP raise concerns about its safety. In this study, we designed a novel library of 248 ionizable lipids comprising 1,2-diesters via a two-step process involving the epoxide ring-opening reaction with carboxyl group-containing alkyl chains followed by an esterification reaction with the tertiary amines. Owing to the special chemical structure of 1,2-diesters, our top-performing lipids and formulations exhibited a faster clearance rate in the liver, contributing to increased stability and higher safety compared with DLin-MC3-DMA. Moreover, our LNP showed superior intramuscular mRNA delivery and elicited robust antigen-specific immune activation. The vaccinations delivered by our LNP system suppressed tumor growth and prolonged survival in both model human papillomavirus E7 and ovalbumin antigen-expressing tumor models. Finally, we further optimized the structure of lipids which enhanced the protein expression in the spleen and draining lymph nodes compared with ALC-0315 lipid in Comirnaty. In conclusion, the 1, 2-diester-derived lipids exhibited rapid liver clearance and effective anti-cancer efficiency to different types of antigens-expressing tumor models, which might be a safe and universal platform for mRNA vaccines. This article is protected by copyright. All rights reserved.

Keywords:  Immunotherapy; Lipid nanoparticle; mRNA delivery

DOI:  https://doi.org/10.1002/adhm.202302691
J Control Release. 2023 Nov 21. pii: S0168-3659(23)00751-4. [Epub ahead of print]

Effective delivery of miR-511-3p with mannose-decorated exosomes with RNA nanoparticles confers protection against asthma.

Wei Tu, Xinyue Hu, Rongjun Wan, Xiaojun Xiao, Yingchun Shen, Prakhyath Srikaram, Sai Nithin Avvaru, Fuhan Yang, Fengmei Pi, Yufeng Zhou, Mei Wan, Peisong Gao.

  Our previous studies have shown that miR-511-3p treatment has a beneficial effect in alleviating allergic airway inflammation. Here, we sought to explore its therapeutic potential in animal models and gain a deeper understanding of its therapeutic value for asthma. miR-511-3p knockout mice (miR-511-3p-/-) were generated by CRISPR/Cas and showed exacerbated airway hyper-responsiveness and Th2-associated allergic airway inflammation compared with wild-type (WT) mice after exposed to cockroach allergen. RNA nanoparticles with mannose decorated EV-miR-511-3p were also created by loading miR-511-3p mimics into the mannose decorated EVs with engineered RNA nanoparticle PRNA-3WJ (Man-EV-miR-511-3p). Intra-tracheal inhalation of Man-EV-miR-511-3p, which could effectively penetrate the airway mucus barrier and deliver functional miR-511-3p to lung macrophages, successfully reversed the increased airway inflammation observed in miR-511-3p-/- mice. Through microarray analysis, complement C3 (C3) was identified as one of the major targets of miR-511-3p. C3 was increased in LPS-treated macrophages but decreased after miR-511-3p treatment. Consistent with these findings, C3 expression was elevated in the lung macrophages of an asthma mouse model but decreased in mice treated with miR-511-3p. Further experiments, including miRNA-mRNA pulldown and luciferase reporter assays, confirmed that miR-511-3p directly binds to C3 and activates the C3 gene. Thus, miR-511-3p represents a promising therapeutic target for asthma, and RNA nanotechnology reprogrammed EVs are efficient carriers for miRNA delivery for disease treatment.

Keywords:  Asthma; Complement C3; Inflammation; Macrophage; miRNA

DOI:  https://doi.org/10.1016/j.jconrel.2023.11.034
Adv Mater. 2023 Nov 21. e2310872

Phase-Separated Lipid-Based Nanoparticles: Selective Behavior At The Nano-Bio Interface.

Panagiota Papadopoulou, Rianne van der Pol, Niek van Hilten, Winant L van Os, Roy Pattipeiluhu, Gabriela Arias-Alpizar, Renzo A Knol, Willem Noteborn, Mohammad-Amin Moradi, Maria J Ferraz, Johannes M F G Aerts, Nico Sommerdijk, Frederick Campbell, H Jelger Risselada, G J Agur Sevink, Alexander Kros.

  The membrane-protein interface on lipid-based nanoparticles influences their in vivo behavior. Better understanding may evolve current drug delivery methods towards effective targeted nanomedicine. Previously, we demonstrated the cell-specific accumulation of a liposome formulation in vivo, through the selective recognition of lipid phase separation by triglyceride lipases. This exemplified how liposome morphology and composition can determine nanoparticle-protein interactions. Here, we investigate the lipase-induced compositional and morphological changes of phase-separated liposomes - which bear a lipid droplet in their bilayer - and unravel how lipases recognize and bind to the particles. We observe the selective lipolytic degradation of the phase-separated lipid droplet, while nanoparticle integrity remains intact. Next, we identify the Tryptophan-rich loop of the lipase - a region responsible for endogenous lipoprotein binding - as the region with which the enzymes bind to the particles. This preferential binding is due to lipid packing defects induced on the liposome surface by phase separation. In parallel, we build upon the existing knowledge that phase separation leads to in vivo selectivity, to generate phase-separated mRNA-LNPs that target cell subsets in zebrafish embryos, with subsequent mRNA delivery and protein expression. Together, these findings can expand our current knowledge on selective nanoparticle-protein communications and in vivo behavior, aspects that will assist to gain control of lipid-based nanoparticles. This article is protected by copyright. All rights reserved.

Keywords:  cell targeting; lipases; lipid-based nanoparticles; mRNA-delivery; nano-bio interface; phase-separated

DOI:  https://doi.org/10.1002/adma.202310872
J Colloid Interface Sci. 2023 Nov 13. pii: S0021-9797(23)02177-X. [Epub ahead of print]656 409-423

Cell interactions with lipid nanoparticles possessing different internal nanostructures: Liposomes, bicontinuous cubosomes, hexosomes, and discontinuous micellar cubosomes.

Sue Lyn Yap, Haitao Yu, Shiyao Li, Calum J Drummond, Charlotte E Conn, Nhiem Tran.

   HYPOTHESIS: Lyotropic liquid crystalline nanoparticles (LLCNPs) with complex internal nanostructures hold promise for drug delivery. Cubosomes, in particular, have garnered interest for their ability to fuse with cell membranes, potentially bypassing endosomal escape challenges and improving cellular uptake. The mesostructure of nanoparticles plays a crucial role in cellular interactions and uptake. Therefore, we hypothesise that the specific internal mesophase of the LLCNPs will affect their cellular interactions and uptake efficiencies, with cubosomes exhibiting superior cellular uptake compared to other LLCNPs.
EXPERIMENTS: LLCNPs with various mesophases, including liposomes, cubosomes, hexosomes, and micellar cubosomes, were formulated and characterised. Their physicochemical properties and cytotoxicity were assessed. Chinese Hamster Ovarian (CHO) cells were treated with fluorescently labelled LLCNPs, and their interactions were monitored and quantified through confocal microscopy and flow cytometry.
FINDINGS: The non-lamellar LLCNPs showed significantly higher cellular interactions compared to liposomes, with cubosomes exhibiting the highest level. However, there was no significant difference in relative cell uptake between cubosomes, hexosomes, and micellar cubosomes. Cell uptake experiments at 4 °C revealed the presence of an energy-independent uptake mechanism. This study provides the first comparative analysis of cellular interactions and uptake efficiencies among LLCNPs with varying mesophases, while maintaining similar size, composition, and surface charge.

Keywords:  Cellular uptake; Cubosomes; Hexosomes; Lipid nanoparticles; Liposomes; Lyotropic liquid crystalline nanoparticles (LLCNPs); Membrane fusion; Micellar cubosomes

DOI:  https://doi.org/10.1016/j.jcis.2023.11.059