bims-novged Biomed News
on Non-viral vectors for gene delivery
Issue of 2023–07–30
ten papers selected by
the Merkel lab, Ludwig-Maximilians University
  1. Development and Characterization of an In Vitro Cell-Based Assay to Predict Potency of mRNA-LNP-Based Vaccines.
  2. Delivery and Expression of mRNA in the Secondary Lymphoid Organs Drive Immune Responses to Lipid Nanoparticle-mRNA Vaccines after Intramuscular Injection.
  3. Protein Corona Investigations of Polyplexes with Varying Hydrophobicity - from Method Development to in vitro Studies.
  4. Development of an Alcohol Dilution-Lyophilization Method for the Preparation of mRNA-LNPs with Improved Storage Stability.
  5. Understanding structure activity relationships of Good HEPES lipids for lipid nanoparticle mRNA vaccine applications.
  6. Poly(vinyl pyrrolidone) derivatives as PEG alternatives for stealth, non-toxic and less immunogenic siRNA-containing lipoplex delivery.
  7. 3D Macrocyclic Structure Boosted Gene Delivery: Multi-Cyclic Poly(β-Amino Ester)s from Step Growth Polymerization.
  8. Enhanced anticancer activity of siRNA and drug codelivered by anionic biopolymer: overcoming electrostatic repulsion.
  9. Investigating the mechanism of action of DNA-loaded PEGylated lipid nanoparticles.
  10. Dual Targeted Nanoparticles for the Codelivery of Doxorubicin and siRNA Cocktails to Overcome Ovarian Cancer Stem Cells.
  1. Vaccines (Basel). 2023 Jul 10. pii: 1224. [Epub ahead of print]11(7):
    Development and Characterization of an In Vitro Cell-Based Assay to Predict Potency of mRNA-LNP-Based Vaccines.
    Nisarg Patel, Zach Davis, Carl Hofmann, Josef Vlasak, John W Loughney, Pete DePhillips, Malini Mukherjee.
      Messenger RNA (mRNA) vaccines have emerged as a flexible platform for vaccine development. The evolution of lipid nanoparticles as effective delivery vehicles for modified mRNA encoding vaccine antigens was demonstrated by the response to the COVID-19 pandemic. The ability to rapidly develop effective SARS-CoV-2 vaccines from the spike protein genome, and to then manufacture multibillions of doses per year was an extraordinary achievement and a vaccine milestone. Further development and application of this platform for additional pathogens is clearly of interest. This comes with the associated need for new analytical tools that can accurately predict the performance of these mRNA vaccine candidates and tie them to an immune response expected in humans. Described here is the development and characterization of an imaging based in vitro assay able to quantitate transgene protein expression efficiency, with utility to measure lipid nanoparticles (LNP)-encapsulated mRNA vaccine potency, efficacy, and stability. Multiple biologically relevant adherent cell lines were screened to identify a suitable cell substrate capable of providing a wide dose-response curve and dynamic range. Biologically relevant assay attributes were examined and optimized, including cell monolayer morphology, antigen expression kinetics, and assay sensitivity to LNP properties, such as polyethylene glycol-lipid (or PEG-lipid) composition, mRNA mass, and LNP size. Collectively, this study presents a strategy to quickly optimize and develop a robust cell-based potency assay for the development of future mRNA-based vaccines.
    Keywords:  LNP; bioassays; mRNA; potency; potency assays; vaccines
    DOI:  https://doi.org/10.3390/vaccines11071224
  2. Mol Pharm. 2023 Jul 26.
    Delivery and Expression of mRNA in the Secondary Lymphoid Organs Drive Immune Responses to Lipid Nanoparticle-mRNA Vaccines after Intramuscular Injection.
    Asuka Takanashi, Colin W Pouton, Hareth Al-Wassiti.
      Lipid nanoparticles (LNPs) are the prime delivery vehicle for mRNA vaccines. Previous hypotheses suggested that LNPs contribute to innate reactogenicity and lead to the establishment of a vaccine adaptive response. It has not been clear whether LNP adjuvancy in the muscle is the prime driver of adaptive immune responses or whether delivery to secondary lymphatic organs is necessary to induce strong adaptive responses. To address this, we formulated reporter gene (NLuc) or OVA mRNA into LNP or coadministered the mRNA with empty LNP. After IM injection, we correlated the delivery with adaptive immune responses. Additionally, we investigated humoral responses to modified mRNA encoding the SARS-CoV-2 spike protein. Compared to unformulated mRNA encoding nanoluciferase, with or without co-administered empty LNPs, LNP-formulated mRNA resulted in high levels of nanoluciferase in the secondary lymphoid organs. Similarly, LNP-mRNA encoding ovalbumin led to a cellular immune response against OVA while free mRNA, with or without empty adjuvanted LNPs, caused little or no immune response. Finally, only mice injected with LNP-formulated mRNA encoding SARS-CoV-2 spike protein elicited robust cellular and humoral immune responses. Our results suggest that the mRNA delivery and transfection of secondary lymphatic organs, not LNP adjuvancy or RNA expression in muscle, are the main drivers for adaptive immune response in mice. This work informs the design of next-generation mRNA delivery systems where better delivery to secondary lymphatic organs should lead to a better vaccine response.
    Keywords:  COVID19; delivery strategy; lipid nanoparticles; mRNA; nonviral delivery; vaccine
    DOI:  https://doi.org/10.1021/acs.molpharmaceut.2c01024
  3. Int J Pharm. 2023 Jul 21. pii: S0378-5173(23)00677-4. [Epub ahead of print] 123257
    Protein Corona Investigations of Polyplexes with Varying Hydrophobicity - from Method Development to in vitro Studies.
    Natascha Hartl, David C Jürgens, Simone Carneiro, Ann-Christine König, Ximian Xiao, Runhui Liu, Stefanie M Hauck, Olivia M Merkel.
      In the field of non-viral drug delivery, polyplexes (PXs) represent an advanced investigated and highly promising tool for the delivery of nucleic acids. Upon encountering physiological fluids, they adsorb biological molecules to form a protein corona (PC), that influence PXs biodistribution, transfection efficiencies and targeting abilities. In an effort to understand protein - PX interactions and the effect of PX material on corona composition, we utilized cationic branched 10kDa polyethyleneimine (b-PEI) and a hydrophobically modified nylon-3 polymer (NM0.2/CP0.8) within this study to develop appropriate methods for PC investigations. A centrifugation procedure for isolating hard corona - PX complexes (PCPXs) from soft corona proteins after incubating the PXs in fetal bovine serum (FBS) for PC formation was successfully optimized and the identification of proteins by a liquid chromatography-tandem mass spectrometry (LC-MS-MS) method clearly demonstrated that the PC composition is affected by the underlying PXs material. With regard to especially interesting functional proteins, which might be able to induce active targeting effects, several candidates could be detected on b-PEI and NM0.2/CP0.8 PXs. These results are of high interest to better understand how the design of PXs impacts the PC composition and subsequently PCPXs-cell interactions to enable precise adjustement of PXs for targeted drug delivery.
    Keywords:  Polyplexes; cell targeting; hydrophobically modified cationic polymers; protein corona; siRNA delivery
    DOI:  https://doi.org/10.1016/j.ijpharm.2023.123257
  4. Pharmaceutics. 2023 Jun 26. pii: 1819. [Epub ahead of print]15(7):
    Development of an Alcohol Dilution-Lyophilization Method for the Preparation of mRNA-LNPs with Improved Storage Stability.
    Daiki Shirane, Hiroki Tanaka, Yu Sakurai, Sakura Taneichi, Yuta Nakai, Kota Tange, Itsuko Ishii, Hidetaka Akita.
      The lipid nanoparticle (LNP) is one of the promising nanotechnologies for the delivery of RNA molecules, such as small interfering RNA (siRNA) and messenger RNA (mRNA). A series of LNPs that contain an mRNA encoding the antigen protein of SARS-CoV-2 were already approved as RNA vaccines against this infectious disease. Since LNP formulations are generally metastable, their physicochemical properties are expected to shift toward a more stable state during the long-time storage of suspensions. The current mRNA vaccines are supplied in the form of frozen formulations with a cryoprotectant for preventing deterioration. They must be stored in a freezer at temperatures from -80 °C to -15 °C. It is thought that therapeutic applications of this mRNA-LNP technology could be accelerated if a new formulation that permits mRNA-LNPs to be stored under milder conditions were available. We previously reported on a one-pot method for producing siRNA-encapsulated LNPs by combining freeze-drying technology with the conventional alcohol dilution method (referred to herein as the "alcohol dilution-lyophilization method"). In this study, this method was applied to the preparation of mRNA-LNPs to provide a freeze-dried formulation of mRNA LNPs. The resulting formulation can be stored at 4 °C for at least 4 months.
    Keywords:  freeze drying; lipid nanoparticle; mRNA delivery; storage stability
    DOI:  https://doi.org/10.3390/pharmaceutics15071819
  5. Biomaterials. 2023 Jul 08. pii: S0142-9612(23)00251-X. [Epub ahead of print]301 122243
    Understanding structure activity relationships of Good HEPES lipids for lipid nanoparticle mRNA vaccine applications.
    Rebecca L Goldman, Namratha Turuvekere Vittala Murthy, Trent P Northen, Anuranjani Balakrishnan, Sudha Chivukula, Hillary Danz, Timothy Tibbitts, Anusha Dias, Jorel Vargas, Dustin Cooper, Hardip Gopani, Angela Beaulieu, Kirill V Kalnin, Timothy Plitnik, Saswata Karmakar, Ramesh Dasari, Ryan Landis, Shrirang Karve, Frank DeRosa.
      Lipid nanoparticles (LNPs) have shown great promise as delivery vehicles to transport messenger ribonucleic acid (mRNA) into cells and act as vaccines for infectious diseases including COVID-19 and influenza. The ionizable lipid incorporated within the LNP is known to be one of the main driving factors for potency and tolerability. Herein, we describe a novel family of ionizable lipids synthesized with a piperazine core derived from the HEPES Good buffer. These ionizable lipids have unique asymmetric tails and two dissimilar degradable moieties incorporated within the structure. Lipids tails of varying lengths, degrees of unsaturation, branching, and the inclusion of additional ester moieties were evaluated for protein expression. We observed several key lipid structure activity relationships that correlated with improved protein production in vivo, including lipid tails of 12 carbons on the ester side and the effect of carbon spacing on the disulfide arm of the lipids. Differences in LNP physical characteristics were observed for lipids containing an extra ester moiety. The LNP structure and lipid bilayer packing, visualized through Cryo-TEM, affected the amount of protein produced in vivo. In non-human primates, the Good HEPES LNPs formulated with an mRNA encoding an influenza hemagglutinin (HA) antigen successfully generated functional HA inhibition (HAI) antibody titers comparable to the industry standards MC3 and SM-102 LNPs, demonstrating their promise as a potential vaccine.
    Keywords:  Cationic lipid; Drug delivery; Ionizable lipid; Lipid nanoparticle; Liposomes; Structure activity; mRNA vaccines
    DOI:  https://doi.org/10.1016/j.biomaterials.2023.122243
  6. J Control Release. 2023 Jul 21. pii: S0168-3659(23)00455-8. [Epub ahead of print]
    Poly(vinyl pyrrolidone) derivatives as PEG alternatives for stealth, non-toxic and less immunogenic siRNA-containing lipoplex delivery.
    Manon Berger, François Toussaint, Sanaa Ben Djemaa, Julie Laloy, Hélène Pendeville, Brigitte Evrard, Christine Jerôme, Anna Lechanteur, Denis Mottet, Antoine Debuigne, Geraldine Piel.
      The recent approval of Onpattro® and COVID-19 vaccines has highlighted the value of lipid nanoparticles (LNPs) for the delivery of genetic material. If it is known that PEGylation is crucial to confer stealth properties to LNPs, it is also known that PEGylation is responsible for the decrease of the cellular uptake and endosomal escape and for the production of anti-PEG antibodies inducing accelerated blood clearance (ABC) and hypersensitivity reactions. Today, the development of PEG alternatives is crucial. Poly(N-vinyl pyrrolidone) (PNVP) has shown promising results for liposome decoration but has never been tested for the delivery of nucleic acids. Our aim is to develop a series of amphiphilic PNVP compounds to replace lipids-PEG for the post-insertion of lipoplexes dedicated to siRNA delivery. PNVP compounds with different degrees of polymerization and hydrophobic segments, such as octadecyl and dioctadecyl and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), were generated. Based on the physicochemical properties and the efficiency to reduce protein corona formation, we showed that the DSPE segment is essential for the integration into the lipoplexes. Lipoplexes post-grafted with 15% DSPE-PNVP30 resulted in gene silencing efficiency close to that of lipoplexes grafted with 15% DSPE-PEG. Finally, an in vivo study in mice confirmed the stealth properties of DSPE-PNVP30 lipoplexes as well as a lower immune response ABC effect compared to DSPE-PEG lipoplexes. Furthermore, we showed a lower immune response after the second injection with DSPE-PNVP30 lipoplexes compared to DSPE-PEG lipoplexes. All these observations suggest that DSPE-PNVP30 appears to be a promising alternative to PEG, with no toxicity, good stealth properties and lower immunological response.
    Keywords:  Immune reaction; Lipoplexes; PEG derivatives; Poly(vinyl pyrrolidone); Protein corona; Stealth properties
    DOI:  https://doi.org/10.1016/j.jconrel.2023.07.031
  7. J Am Chem Soc. 2023 Jul 25.
    3D Macrocyclic Structure Boosted Gene Delivery: Multi-Cyclic Poly(β-Amino Ester)s from Step Growth Polymerization.
    Yinghao Li, Xianqing Wang, Zhonglei He, Melissa Johnson, Sigen A, Irene Lara-Sáez, Jing Lyu, Wenxin Wang.
      The topological structures of polymers play a critical role in determining their gene delivery efficiency. Exploring novel polymeric structures as gene delivery vectors is thus of great interest. In this work, a new generation of multi-cyclic poly(β-amino ester)s (CPAEs) with unique topology structure was synthesized for the first time via step growth polymerization. Through controlling the occurrence stage of cyclization, three types of CPAEs with rings of different sizes and topologies were obtained. In vitro experiments demonstrated that the CPAEs with macro rings (MCPAEs) significantly boosted the transgene expression comparing to their branched counterparts. Moreover, the MCPAE vector with optimized terminal group efficiently delivered the CRISPR plasmid coding both Staphylococcus aureus Cas9 nuclease and dual guide sgRNAs for gene editing therapy.
    DOI:  https://doi.org/10.1021/jacs.3c04191
  8. Nanomedicine (Lond). 2023 Jul 28.
    Enhanced anticancer activity of siRNA and drug codelivered by anionic biopolymer: overcoming electrostatic repulsion.
    Lipika Ray, Sutapa Ray.
      Aim: To codeliver an anticancer drug (doxorubicin) and siRNA in the form of nanoparticles into CD44-overexpressing colon cancer cells (HT-29) using an anionic, amphiphilic biopolymer comprising modified hyaluronic acid (6-O-[3-hexadecyloxy-2-hydroxypropyl]-hyaluronic acid). Materials & methods: Characterization of nanoparticles was performed using dynamic light scattering, scanning electron microscopy, transmission electron microscopy, molecular docking, in vitro drug release and gel mobility assays. Detailed in vitro experiments, including a gene silencing study and western blot, were also performed. Results: A 69% knockdown of the target gene was observed, and western blot showed 5.7-fold downregulation of the target protein. The repulsive forces between siRNA and 6-O-(3-hexadecyloxy-2-hydroxypropyl)-hyaluronic acid were overcome by hydrogen bonding and hydrophobic interactions. Conclusion: We successfully codelivered a drug and siRNA by anionic vector.
    Keywords:  anionic biopolymer; codelivery; doxorubicin; hyaluronic acid; nanoparticles; siRNA
    DOI:  https://doi.org/10.2217/nnm-2022-0225
  9. Nanomedicine. 2023 Jul 26. pii: S1549-9634(23)00048-5. [Epub ahead of print] 102697
    Investigating the mechanism of action of DNA-loaded PEGylated lipid nanoparticles.
    Luca Digiacomo, Serena Renzi, Erica Quagliarini, Daniela Pozzi, Heinz Amenitsch, Gianmarco Ferri, Luca Pesce, Valentina De Lorenzi, Giulia Matteoli, Francesco Cardarelli, Giulio Caracciolo.
      PEGylated lipid nanoparticles (LNPs) are commonly used to deliver bioactive molecules, but the role of PEGylation in DNA-loaded LNP interactions at the cellular and subcellular levels remains poorly understood. In this study, we investigated the mechanism of action of DNA-loaded PEGylated LNPs using gene reporter technologies, dynamic light scattering (DLS), synchrotron small angle X-ray scattering (SAXS), and fluorescence confocal microscopy (FCS). We found that PEG has no significant impact on the size or nanostructure of DNA LNPs but reduces their zeta potential and interaction with anionic cell membranes. PEGylation increases the structural stability of LNPs and results in lower DNA unloading. FCS experiments revealed that PEGylated LNPs are internalized intact inside cells and largely shuttled to lysosomes, while unPEGylated LNPs undergo massive destabilization on the plasma membrane. These findings can inform the design, optimization, and validation of DNA-loaded LNPs for gene delivery and vaccine development.
    Keywords:  DNA delivery; Nanoparticle-membrane interactions; PEGylation, lipid nanoparticles
    DOI:  https://doi.org/10.1016/j.nano.2023.102697
  10. Int J Mol Sci. 2023 Jul 18. pii: 11575. [Epub ahead of print]24(14):
    Dual Targeted Nanoparticles for the Codelivery of Doxorubicin and siRNA Cocktails to Overcome Ovarian Cancer Stem Cells.
    Li Chen, Jinlan Luo, Jingyuan Zhang, Siyuan Wang, Yang Sun, Qinying Liu, Cui Cheng.
      Most anticancer treatments only induce the death of ordinary cancer cells, while cancer stem cells (CSCs) in the quiescent phase of cell division are difficult to kill, which eventually leads to cancer drug resistance, metastasis, and relapse. Therefore, CSCs are also important in targeted cancer therapy. Herein, we developed dual-targeted and glutathione (GSH)-responsive novel nanoparticles (SSBPEI-DOX@siRNAs/iRGD-PEG-HA) to efficiently and specifically deliver both doxorubicin and small interfering RNA cocktails (siRNAs) (survivin siRNA, Bcl-2 siRNA and ABCG2 siRNA) to ovarian CSCs. They are fabricated via electrostatic assembly of anionic siRNAs and cationic disulfide bond crosslinking-branched polyethyleneimine-doxorubicin (SSBPEI-DOX) as a core. Interestingly, the SSBPEI-DOX could be degraded into low-cytotoxic polyethyleneimine (PEI). Because of the enrichment of glutathione reductase in the tumor microenvironment, the disulfide bond (-SS-) in SSBPEI-DOX can be specifically reduced to promote the controlled release of siRNA and doxorubicin (DOX) in the CSCs. siRNA cocktails could specifically silence three key genes in CSCs, which, in combination with the traditional chemotherapy drug DOX, induces apoptosis or necrosis of CSCs. iRGD peptides and "sheddable" hyaluronic acid (HA) wrapped around the core could mediate CSC targeting by binding with neuropilin-1 (NRP1) and CD44 to enhance delivery. In summary, the multifunctional delivery system SSBPEI-DOX@siRNAs/iRGD-PEG-HA nanoparticles displays excellent biocompatibility, accurate CSC-targeting ability, and powerful anti-CSC ability, which demonstrates its potential value in future treatments to overcome ovarian cancer metastasis and relapse. To support this work, as exhaustive search was conducted for the literature on nanoparticle drug delivery research conducted in the last 17 years (2007-2023) using PubMed, Web of Science, and Google Scholar.
    Keywords:  cancer stem cells; iRGD peptides; reduction sensitive; siRNAs; targeted delivery
    DOI:  https://doi.org/10.3390/ijms241411575
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