bims-novged 2022-02-27 papers

bims-novged

Biomed News

on Non-viral vectors for gene delivery

Issue of 2022‒02‒27
twelve papers selected by
Benjamin Winkeljann
Ludwig-Maximilians University

Effect of Cationic Lipid Nanoparticle Loaded siRNA with Stearylamine against Chikungunya Virus.
Determination of Optimum Ratio of Cationic Polymers and Small Interfering RNA with Agarose Gel Retardation Assay.
Chitosan-Crosslinked Low Molecular Weight PEI-Conjugated Iron Oxide Nanoparticle for Safe and Effective DNA Delivery to Breast Cancer Cells.
Augmented lipid-nanoparticle-mediated in vivo genome editing in the lungs and spleen by disrupting Cas9 activity in the liver.
Microfluidic technologies and devices for lipid nanoparticle-based RNA delivery.
Anionic Lipid Nanoparticles Preferentially Deliver mRNA to the Hepatic Reticuloendothelial System.
Endothelial Cell-Targeting, ROS-Ultrasensitive Drug/siRNA Co-Delivery Nanocomplexes Mitigate Early-Stage Neutrophil Recruitment For the Anti-Inflammatory Treatment of Myocardial Ischemia Reperfusion Injury.
Synthesis and Characterization of Fatty Acid Grafted Chitosan Polymeric Micelles for Improved Gene Delivery of VGF to the Brain through Intranasal Route.
Dual-RNA controlled delivery system inhibited tumor growth by apoptosis induction and TME activation.
In vivo mRNA delivery to virus-specific T cells by light-induced ligand exchange of MHC class I antigen-presenting nanoparticles.
Lipid nanoparticle-encapsulated mRNA antibody provides long-term protection against SARS-CoV-2 in mice and hamsters.
Complex pBAE nanoparticles cell trafficking: tracking both position and composition using super resolution microscopy.

Molecules. 2022 Feb 09. pii: 1170. [Epub ahead of print]27(4):

Effect of Cationic Lipid Nanoparticle Loaded siRNA with Stearylamine against Chikungunya Virus.

Manish Kumar Jeengar, Mallesh Kurakula, Poonam Patil, Ashwini More, Ramakrishna Sistla, Deepti Parashar.

  Chikungunya is an infectious disease caused by mosquito-transmitted chikungunya virus (CHIKV). It was reported that NS1 and E2 siRNAs administration demonstrated CHIKV inhibition in in vitro as well as in vivo systems. Cationic lipids are promising for designing safe non-viral vectors and are beneficial in treating chikungunya. In this study, nanodelivery systems (hybrid polymeric/solid lipid nanoparticles) using cationic lipids (stearylamine, C9 lipid, and dioctadecylamine) and polymers (branched PEI-g-PEG -PEG) were prepared, characterized, and complexed with siRNA. The four developed delivery systems (F1, F2, F3, and F4) were assessed for stability and potential toxicities against CHIKV. In comparison to the other nanodelivery systems, F4 containing stearylamine (Octadecylamine; ODA), with an induced optimum cationic charge of 45.7 mV in the range of 152.1 nm, allowed maximum siRNA complexation, better stability, and higher transfection, with strong inhibition against the E2 and NS1 genes of CHIKV. The study concludes that cationic lipid-like ODA with ease of synthesis and characterization showed maximum complexation by structural condensation of siRNA owing to high transfection alone. Synergistic inhibition of CHIKV along with siRNA was demonstrated in both in vitro and in vivo models. Therefore, ODA-based cationic lipid nanoparticles can be explored as safe, potent, and efficient nonviral vectors overcoming siRNA in vivo complexities against chikungunya.

Keywords:  cationic lipids; chikungunya; nanodelivery systems; non-viral vectors; siRNA; stearylamine

DOI:  https://doi.org/10.3390/molecules27041170
Methods Mol Biol. 2022 ;2434 117-128

Determination of Optimum Ratio of Cationic Polymers and Small Interfering RNA with Agarose Gel Retardation Assay.

Omer Aydin, Dilek Kanarya, Ummugulsum Yilmaz, Cansu Ümran Tunç.

  Nanomaterials have aroused attention in the recent years for their high potential for gene delivery applications. Most of the nanoformulations used in gene delivery are positively charged to carry negatively charged oligonucleotides. However, excessive positively charged carriers are cytotoxic. Therefore, the complexed oligonucleotide/nanoparticles should be well-examined before the application. In that manner, agarose gel electrophoresis, which is a basic method utilized for separation, identification, and purification of nucleic acid molecules because of its poriferous nature, is one of the strategies to determine the most efficient complexation rate. When the electric field is applied, RNA fragments can migrate through anode due to the negatively charged phosphate backbone. Because RNA has a uniform mass/charge ratio, RNA molecules run in agarose gel proportional according to their size and molecular weight. In this chapter, the determination of complexation efficiency between cationic polymer carriers and small interfering RNA (siRNA) cargos by using agarose gel electrophoresis is described. siRNA/cationic polymer carrier complexes are placed in an electric field and the charged molecules move through the counter-charged electrodes due to the phenomenon of electrostatic attraction. Nucleic acid cargos are loaded to cationic carriers via the electrostatic interaction between positively charged amine groups (N) of the carrier and negatively charged phosphate groups (P) of RNA. The N/P ratio determines the loading efficiency of the cationic polymer carrier. In here, the determination of N/P ratio, where the most efficient complexation occurs, by exposure to the electric field with a gel retardation assay is explained.

Keywords:  Agarose gel retardation assay; Gene delivery; N/P ratio; Nanoparticles; Small interfering ribonucleic acid (siRNA); siRNA/cationic polymer carrier complex

DOI:  https://doi.org/10.1007/978-1-0716-2010-6_7
Nanomaterials (Basel). 2022 Feb 09. pii: 584. [Epub ahead of print]12(4):

Chitosan-Crosslinked Low Molecular Weight PEI-Conjugated Iron Oxide Nanoparticle for Safe and Effective DNA Delivery to Breast Cancer Cells.

Guanyou Lin, Jianxi Huang, Mengyuan Zhang, Shanshan Chen, Miqin Zhang.

  Breast cancer has attracted tremendous research interest in treatment development as one of the major threats to public health. The use of non-viral carriers for therapeutic DNA delivery has shown promise in treating various cancer types, including breast cancer, due to their high DNA loading capacity, high cell transfection efficiency, and design versatility. However, cytotoxicity and large sizes of non-viral DNA carriers often raise safety concerns and hinder their applications in the clinic. Here we report the development of a novel nanoparticle formulation (termed NP-Chi-xPEI) that can safely and effectively deliver DNA into breast cancer cells for successful transfection. The nanoparticle is composed of an iron oxide core coated with low molecular weight (800 Da) polyethyleneimine crosslinked with chitosan via biodegradable disulfide bonds. The NP-Chi-xPEI can condense DNA into a small nanoparticle with the overall size of less than 100 nm and offer full DNA protection. Its biodegradable coating of small-molecular weight xPEI and mildly positive surface charge confer extra biocompatibility. NP-Chi-xPEI-mediated DNA delivery was shown to achieve high transfection efficiency across multiple breast cancer cell lines with significantly lower cytotoxicity as compared to the commercial transfection agent Lipofectamine 3000. With demonstrated favorable physicochemical properties and functionality, NP-Chi-xPEI may serve as a reliable vehicle to deliver DNA to breast cancer cells.

Keywords:  breast cancer; gene therapy; iron oxide nanoparticles

DOI:  https://doi.org/10.3390/nano12040584
Nat Biomed Eng. 2022 Feb;6(2): 157-167

Augmented lipid-nanoparticle-mediated in vivo genome editing in the lungs and spleen by disrupting Cas9 activity in the liver.

Cory D Sago, Melissa P Lokugamage, David Loughrey, Kevin E Lindsay, Robert Hincapie, Brandon R Krupczak, Sujay Kalathoor, Manaka Sato, Elisa Schrader Echeverri, Jordan P Fitzgerald, Zubao Gan, Lena Gamboa, Kalina Paunovska, Carlos A Sanhueza, Marine Z C Hatit, M G Finn, Philip J Santangelo, James E Dahlman.

Systemically delivered lipid nanoparticles are preferentially taken up by hepatocytes. This hinders the development of effective, non-viral means of editing genes in tissues other than the liver. Here we show that lipid-nanoparticle-mediated gene editing in the lung and spleen of adult mice can be enhanced by reducing Cas9-mediated insertions and deletions in hepatocytes via oligonucleotides disrupting the secondary structure of single-guide RNAs (sgRNAs) and also via their combination with short interfering RNA (siRNA) targeting Cas9 messenger RNA (mRNA). In SpCas9 mice with acute lung inflammation, the systemic delivery of an oligonucleotide inhibiting an sgRNA targeting the intercellular adhesion molecule 2 (ICAM-2), followed by the delivery of the sgRNA, reduced the fraction of ICAM-2 indels in hepatocytes and increased that in lung endothelial cells. In wild-type mice, the lipid-nanoparticle-mediated delivery of an inhibitory oligonucleotide, followed by the delivery of Cas9-degrading siRNA and then by Cas9 mRNA and sgRNA, reduced the fraction of ICAM-2 indels in hepatocytes but not in splenic endothelial cells. Inhibitory oligonucleotides and siRNAs could be used to modulate the cell-type specificity of Cas9 therapies.

DOI: https://doi.org/10.1038/s41551-022-00847-9
J Control Release. 2022 Feb 17. pii: S0168-3659(22)00091-8. [Epub ahead of print]

Microfluidic technologies and devices for lipid nanoparticle-based RNA delivery.

Masatoshi Maeki, Shuya Uno, Ayuka Niwa, Yuto Okada, Manabu Tokeshi.

  In 2021, mRNA vaccines against COVID-19 were approved by the Food and Drug Administration. mRNA vaccines are important for preventing severe COVID-19 and returning to normal life. The development of RNA-delivery technology, including mRNA vaccines, has been investigated worldwide for ~30 years. Lipid nanoparticles (LNPs) are a breakthrough technology that stably delivers RNA to target organs, and RNA-loaded LNP-based nanomedicines have been studied for the development of vaccines and nanomedicines for RNA-, gene-, and cell-based therapies. Recently, microfluidic devices and technologies have attracted attention for the production of LNPs, particularly RNA-loaded LNPs. Microfluidics provides many advantages for RNA-loaded LNP production, including precise LNP size controllability, high reproducibility, high-throughput optimization of LNP formulation, and continuous LNP-production processes. In this review, we summarize microfluidic-based RNA-loaded LNP production and its applications in RNA-based therapy and genome editing.

Keywords:  Lipid nanoparticles; Microfluidic device; RNA delivery; mRNA vaccine

DOI:  https://doi.org/10.1016/j.jconrel.2022.02.017
Adv Mater. 2022 Feb 25. e2201095

Anionic Lipid Nanoparticles Preferentially Deliver mRNA to the Hepatic Reticuloendothelial System.

Roy Pattipeiluhu, Gabriela Arias-Alpizar, Genc Basha, Karen Y T Chan, Jeroen Bussmann, Thomas H Sharp, Mohammad-Amin Moradi, Nico Sommerdijk, Edward N Harris, Pieter R Cullis, Alexander Kros, Dominik Witzigmann, Frederick Campbell.

  Lipid nanoparticles (LNPs) are the leading non-viral technology for the delivery of exogenous RNA to target cells in vivo. As systemic delivery platforms, these technologies are exemplified by Onpattro® , an approved LNP-based RNA interference (RNAi) therapy, administered intravenously and targeted to parenchymal liver cells. The discovery of systemically administered LNP technologies capable of preferential RNA delivery beyond hepatocytes has, however, proven more challenging. Here, preceded by comprehensive mechanistic understanding of in vivo nanoparticle biodistribution and bodily clearance, we rationally design an LNP-based mRNA delivery platform to preferentially target the hepatic reticuloendothelial system (RES). Evaluated in embryonic zebrafish, validated in mice and directly compared to LNP-mRNA systems based on the lipid composition of Onpattro® , RES-targeted LNPs significantly enhance mRNA expression both globally within the liver and specifically within hepatic RES cell types. Hepatic RES targeting requires just a single lipid change within the formulation of Onpattro® to switch LNP surface charge from neutral to anionic. This technology not only provides new opportunities to treat liver-specific and systemic diseases in which RES cell types play a key role but, more importantly, exemplifies that rational design of advanced RNA therapies must be preceded by a robust understanding of the dominant nano-bio interactions involved. This article is protected by copyright. All rights reserved.

Keywords:  Stabilin-2; embryonic zebrafish; lipid nanoparticles; mRNA delivery; reticuloendothelial system

DOI:  https://doi.org/10.1002/adma.202201095
Acta Biomater. 2022 Feb 18. pii: S1742-7061(22)00095-2. [Epub ahead of print]

Endothelial Cell-Targeting, ROS-Ultrasensitive Drug/siRNA Co-Delivery Nanocomplexes Mitigate Early-Stage Neutrophil Recruitment For the Anti-Inflammatory Treatment of Myocardial Ischemia Reperfusion Injury.

Mengying Hou, Xuejie Wu, Ziyin Zhao, Qiurong Deng, Yongbing Chen, Lichen Yin.

  Neutrophils serve as a key contributor to the pathophysiology of myocardial ischemia reperfusion injury (MIRI), because the unregulated activation and infiltration of neutrophils lead to overwhelming inflammation in the myocardium to cause tissue damage. Herein, endothelial cell-targeting and ROS-ultrasensitive nanocomplexes (NCs) were developed to mediate efficient co-delivery of VCAM-1 siRNA (siVCAM-1) and dexamethasone (DXM), which cooperatively inhibited neutrophil recruitment by impeding neutrophil migration and adhesion. RPPT was first synthesized via crosslinking of PEI 600 with ditellurium followed by modification with PEG and the endothelial cell-targeting peptide cRGD. RPPT was allowed to envelope the DXM-loaded PLGA nanoparticles and condense the siVCAM-1. After systemic administration in rats experiencing MIRI, the cRGD-modified NCs efficiently targeted and entered the inflamed endothelial cells, wherein RPPT was sensitively degraded by over-produced reactive oxygen species (ROS) to trigger intracellular siVCAM-1 release and potentiate the VCAM-1 silencing efficiency. As a consequence of the complementary function of DXM and siVCAM-1, the NCs notably mitigated neutrophil infiltration into ischemic myocardium, provoking potent anti-inflammatory efficacy to reduce MIRI and recover cardiac function. The present study offers an effective approach for the controlled co-delivery of siRNA and drug cargoes, and it also highlights the importance of multi-dimensional manipulation of neutrophils in anti-inflammatory treatment. STATEMENT OF SIGNIFICANCE: The unregulated activation and infiltration of neutrophils lead to overwhelming inflammation in the myocardium after myocardial ischemia reperfusion injury (MIRI). Here, endothelial cell-targeting and ROS-ultrasensitive nanocomplexes (NCs), comprised of PLGA NPs decorated with cRGD-poly(ethylene glycol) (PEG)-modified, ditellurium-crosslinked PEI (RPPT), were developed to mediate efficient co-delivery of VCAM-1 siRNA (siVCAM-1) and dexamethasone (DXM). DXM and siVCAM-1 with complementary functions inhibited both the migration and adhesion of neutrophils, efficiently interventing the neutrophil recruitment and interrupting the self-amplified inflammation cascade in the injured myocardium. The molecular design of RPPT renders an effective example for constructing polymeric materials with high ROS sensitivity, and it resolves the critical dilemma related to polycation-mediated siRNA delivery, such as siRNA encapsulation versus release, and transfection efficiency versus toxicity.

Keywords:  Anti-inflammation; Ditellurium-crosslinked polyethylenimine; Drug/siRNA co-delivery; Myocardial ischemia reperfusion injury (MIRI); Neutrophil recruitment; ROS responsiveness

DOI:  https://doi.org/10.1016/j.actbio.2022.02.018
Biomedicines. 2022 Feb 19. pii: 493. [Epub ahead of print]10(2):

Synthesis and Characterization of Fatty Acid Grafted Chitosan Polymeric Micelles for Improved Gene Delivery of VGF to the Brain through Intranasal Route.

Richard Nii Lante Lamptey, Avinash Gothwal, Riddhi Trivedi, Sanjay Arora, Jagdish Singh.

  Multifunctional fatty acid grafted polymeric micelles are an effective and promising approach for drug and gene delivery to the brain. An alternative approach to bypass the blood-brain barrier is administration through intranasal route. Multifunctional fatty acid grafted polymeric micelles were prepared and characterized for pVGF delivery to the brain. In vitro pVGF expression was analyzed in bEnd.3 cells, primary astrocytes, and neurons. Comparative in-vivo pVGF expression was analyzed to evaluate the effective route of administration between intranasal and intravenous. Biocompatible, multifunctional polymeric micelles were prepared, having an average size of 200 nm, and cationic zeta potential. Modified polymers were found to be hemo- and cyto-compatible. When transfected with the different modified chitosan formulations, significantly (p < 0.05) higher VGF expression was observed in primary astrocytes and neurons using the mannose, Tat peptide, and oleic acid grafted chitosan polymer. Compared to intravenous administration, intranasal administration of pVGF in polyplex formulation led to significantly (p < 0.05) higher pVGF expression. Developed multifunctional polymeric micelles were an effective pVGF delivery platform to the brain. Mannose and Tat ligand tagging improved the pVGF delivery to the brain.

Keywords:  blood–brain barrier; chitosan grafted micelles; intranasal; polymer; polyplex; synthesis; targeted gene delivery; transfection

DOI:  https://doi.org/10.3390/biomedicines10020493
J Control Release. 2022 Feb 18. pii: S0168-3659(22)00096-7. [Epub ahead of print]

Dual-RNA controlled delivery system inhibited tumor growth by apoptosis induction and TME activation.

Sibei Lei, Yan Gao, Jingmei Li, Xiaohua Chen, Weilin Zhou, Jieping Wu, Pingchuan Ma, Ke Men, Xingmei Duan.

  Drug-controlled release is recognized as effective for improving compliance with treatment and obtaining better therapeutic efficacy with less toxicity in cancer treatment. However, few reports in this area are involved in nucleic acids delivery, especially in RNA therapeutics delivery. In this study, an injectable hydrogel Methacrylated gelatin (GM) scaffold was introduced into a dual-RNA hybrid delivery complex hybrid lipid particle (HLP) to form a G-HLP/RNAs system. This system can control the release of both siRNA and mRNA and was found to be efficient for protecting these RNAs from biodegradation and retaining their therapeutic effect over 7 days. Further, a tumor environment (TME)-activation function after peritumoral injection of mocked GM scaffold was observed. Then, matured DC cells and activated T-cells were detected by the addition of HLP/RNAs complex, thus verifying the immunoactivation function of GM scaffold and its ability to reserve immune cells and antigens. Finally, two doses of G-HLP/RNAs treatment efficiently suppressed C26 tumor growth in mice with a tumor weight inhibition rate of 71.9%. Owing to its ability to achieve RNA drug-controlled release, alter TME, and induce tumor apoptosis, the G-HLP/RNAs system may become a valuable tool for cancer gene therapy.

Keywords:  Controlled release; Hydrogel; Tumor microenvironment; mRNA gene therapy; siRNA gene therapy

DOI:  https://doi.org/10.1016/j.jconrel.2022.02.022
Sci Adv. 2022 Feb 25. 8(8): eabm7950

In vivo mRNA delivery to virus-specific T cells by light-induced ligand exchange of MHC class I antigen-presenting nanoparticles.

Fang-Yi Su, Qingyang Henry Zhao, Shreyas N Dahotre, Lena Gamboa, Swapnil Subhash Bawage, Aaron D Silva Trenkle, Ali Zamat, Hathaichanok Phuengkham, Rafi Ahmed, Philip J Santangelo, Gabriel A Kwong.

Simultaneous delivery of mRNA to multiple populations of antigen (Ag)-specific CD8+ T cells is challenging given the diversity of peptide epitopes and polymorphism of class I major histocompatibility complexes (MHCI). We developed Ag-presenting nanoparticles (APNs) for mRNA delivery using pMHCI molecules that were refolded with photocleavable peptides to allow rapid ligand exchange by UV light and site-specifically conjugated with a lipid tail for postinsertion into preformed mRNA lipid nanoparticles. Across different TCR transgenic mouse models (P14, OT-1, and Pmel), UV-exchanged APNs bound and transfected their cognate Ag-specific CD8+ T cells equivalent to APNs produced using conventionally refolded pMHCI molecules. In mice infected with PR8 influenza, multiplexed delivery of UV-exchanged APNs against three immunodominant epitopes led to ~50% transfection of a VHH mRNA reporter in cognate Ag-specific CD8+ T cells. Our data show that UV-mediated peptide exchange can be used to rapidly produce APNs for mRNA delivery to multiple populations of Ag-specific T cells in vivo.

DOI: https://doi.org/10.1126/sciadv.abm7950
Cell Res. 2022 Feb 24.

Lipid nanoparticle-encapsulated mRNA antibody provides long-term protection against SARS-CoV-2 in mice and hamsters.

Yong-Qiang Deng, Na-Na Zhang, Yi-Fei Zhang, Xia Zhong, Sue Xu, Hong-Ying Qiu, Tie-Cheng Wang, Hui Zhao, Chao Zhou, Shu-Long Zu, Qi Chen, Tian-Shu Cao, Qing Ye, Hang Chi, Xiang-Hui Duan, Dan-Dan Lin, Xiao-Jing Zhang, Liang-Zhi Xie, Yu-Wei Gao, Bo Ying, Cheng-Feng Qin.

Monoclonal antibodies represent important weapons in our arsenal to against the COVID-19 pandemic. However, this potential is severely limited by the time-consuming process of developing effective antibodies and the relative high cost of manufacturing. Herein, we present a rapid and cost-effective lipid nanoparticle (LNP) encapsulated-mRNA platform for in vivo delivery of SARS-CoV-2 neutralization antibodies. Two mRNAs encoding the light and heavy chains of a potent SARS-CoV-2 neutralizing antibody HB27, which is currently being evaluated in clinical trials, were encapsulated into clinical grade LNP formulations (named as mRNA-HB27-LNP). In vivo characterization demonstrated that intravenous administration of mRNA-HB27-LNP in mice resulted in a longer circulating half-life compared with the original HB27 antibody in protein format. More importantly, a single prophylactic administration of mRNA-HB27-LNP provided protection against SARS-CoV-2 challenge in mice at 1, 7 and even 63 days post administration. In a close contact transmission model, prophylactic administration of mRNA-HB27-LNP prevented SARS-CoV-2 infection between hamsters in a dose-dependent manner. Overall, our results demonstrate a superior long-term protection against SARS-CoV-2 conferred by a single administration of this unique mRNA antibody, highlighting the potential of this universal platform for antibody-based disease prevention and therapy against COVID-19 as well as a variety of other infectious diseases.

DOI: https://doi.org/10.1038/s41422-022-00630-0
ChemMedChem. 2022 Feb 25.

Complex pBAE nanoparticles cell trafficking: tracking both position and composition using super resolution microscopy.

Roger Riera, Jana Tauler, Natàlia Feiner-Gracia, Salvador Borrós, Cristina Fornaguera, Lorenzo Albertazzi.

  Nanomedicine emerged some decades ago with the hope to be the solution for most unmet medical needs. Understanding materials that are made of multiple components, like gene carriers, is key for their arrival to clinics. However, tracking materials at nanoscale is challenging to their reduced size, below the resolution limit of most conventional techniques. In this context, we propose the use of direct stochastic optical reconstruction microscopy (dSTORM) to study time stability and cell trafficking after transfection of oligopeptide end-modified poly(beta aminoester) (OM-pBAE) nanoparticles. We selected different combinations of cationic end oligopeptides (arginine - R; histidine - H; and lysine - K) among polymer libraries, since the oligopeptide combination demonstrated to be useful for different applications, such as vaccination and gene silencing. After fluorescently labelling polymers and/or the encapsulated plasmid, we demonstrate that their time evolution as well as their cell uptake and trafficking are dependent on the oligopeptide. Therefore, their biological behavior as a function of oligopeptide combination can be explained at a molecular level thanks to dSTORM. This study opens the pave to broad mechanistic studies at nanoscale that could enable a rational selection of specific pBAE nanoparticles composition after determining their stability and cell trafficking.

Keywords:  cell trafficking; direct stochastic optical reconstruction microscopy (dSTORM); nanoparticles stability; poly (beta aminoester) nanoparticles

DOI:  https://doi.org/10.1002/cmdc.202100633