bims-novged 2024-04-07 papers

bims-novged

Biomed News

on Non-viral vectors for gene delivery

Issue of 2024–04–07
nine papers selected by
the Merkel lab, Ludwig-Maximilians University

Efficient in vitro and in vivo transfection of self-amplifying mRNA with linear poly(propylenimine) and poly(ethylenimine-propylenimine) random copolymers as non-viral carriers.
Exploring Mechanisms of Lipid Nanoparticle-Mucus Interactions in Healthy and Cystic Fibrosis Conditions.
Silicon Ether Ionizable Lipids Enable Potent mRNA Lipid Nanoparticles with Rapid Tissue Clearance.
Design, synthesis, and in vitro gene transfer efficacy of novel ionizable cholesterol derivatives.
Lipid Nanoparticle-Based Inhibitors for SARS-CoV-2 Host Cell Infection.
A blood-brain barrier- and blood-brain tumor barrier-penetrating siRNA delivery system targeting gliomas for brain tumor immunotherapy.
Folic Acid-Conjugated Brush Polymers Show Enhanced Blood-Brain Barrier Crossing in Static and Dynamic In Vitro Models Toward Brain Cancer Targeting Therapy.
Immunometabolic Reprogramming of Macrophages with Inhalable CRISPR/Cas9 Nanotherapeutics for Acute Lung Injury Intervention.
Revealing the Origin of Cooperative Adsorption of Chains on Nanoparticle Surfaces through Coarse-Grained Simulations.

J Mater Chem B. 2024 Apr 02.

Efficient in vitro and in vivo transfection of self-amplifying mRNA with linear poly(propylenimine) and poly(ethylenimine-propylenimine) random copolymers as non-viral carriers.

Lisa Opsomer, Somdeb Jana, Ine Mertens, Xiaole Cui, Richard Hoogenboom, Niek N Sanders.

Messenger RNA (mRNA) based vaccines have been introduced worldwide to combat the Covid-19 pandemic. These vaccines consist of non-amplifying mRNA formulated in lipid nanoparticles (LNPs). Consequently, LNPs are considered benchmark non-viral carriers for nucleic acid delivery. However, the formulation and manufacturing of these mRNA-LNP nanoparticles are expensive and time-consuming. Therefore, we used self-amplifying mRNA (saRNA) and synthesized novel polymers as alternative non-viral carrier platform to LNPs, which enable a simple, rapid, one-pot formulation of saRNA-polyplexes. Our novel polymer-based carrier platform consists of randomly concatenated ethylenimine and propylenimine comonomers, resulting in linear, poly(ethylenimine-ran-propylenimine) (L-PEIx-ran-PPIy) copolymers with controllable degrees of polymerization. Here we demonstrate in multiple cell lines, that our saRNA-polyplexes show comparable to higher in vitro saRNA transfection efficiencies and higher cell viabilities compared to formulations with Lipofectamine MessengerMAX™ (LFMM), a commercial, lipid-based carrier considered to be the in vitro gold standard carrier. This is especially true for our in vitro best performing saRNA-polyplexes with N/P 5, which are characterised with a size below 100 nm, a positive zeta potential, a near 100% encapsulation efficiency, a high retention capacity and the ability to protect the saRNA from degradation mediated by RNase A. Furthermore, an ex vivo hemolysis assay with pig red blood cells demonstrated that the saRNA-polyplexes exhibit negligible hemolytic activity. Finally, a bioluminescence-based in vivo study was performed over a 35-day period, and showed that the polymers result in a higher and prolonged bioluminescent signal compared to naked saRNA and L-PEI based polyplexes. Moreover, the polymers show different expression profiles compared to those of LNPs, with one of our new polymers (L-PPI250) demonstrating a higher sustained expression for at least 35 days after injection.

DOI: https://doi.org/10.1039/d3tb03003b
Adv Healthc Mater. 2024 Apr 02. e2304525

Exploring Mechanisms of Lipid Nanoparticle-Mucus Interactions in Healthy and Cystic Fibrosis Conditions.

Belal Tafech, Mohammad-Reza Rokhforouz, Jerry Leung, Molly Mh Sung, Paulo Jc Lin, Don D Sin, Daniel Lauster, Stephan Block, Bradley S Quon, Ying Tam, Pieter Cullis, James J Feng, Sarah Hedtrich.

  Mucus forms the first defense line of human lungs, and as such hampers the efficient delivery of therapeutics to the underlying epithelium. This holds particularly true for genetic cargo such as CRISPR-based gene editing tools which cannot readily surmount the mucosal barrier. While lipid nanoparticles (LNPs) emerged as versatile non-viral gene delivery systems that could help overcome the delivery challenge, many knowledge gaps remain, especially for diseased states such as cystic fibrosis (CF). This study provides fundamental insights into Cas9 mRNA or ribonucleoprotein-loaded LNP-mucus interactions in healthy and diseased states by assessing the impact of the genetic cargo, mucin sialylation, mucin concentration, ionic strength, pH, and polyethylene glycol (PEG) concentration and nature on LNP diffusivity leveraging experimental approaches and Brownian dynamics simulations. Taken together, this study identifies key mucus and LNP characteristics that are critical to enabling a rational LNP design for transmucosal delivery. This article is protected by copyright. All rights reserved.

Keywords:  brownian dynamics simulation; cystic fibrosis; lipid nanoparticles; particle diffusivity; transmucosal delivery

DOI:  https://doi.org/10.1002/adhm.202304525
ACS Nano. 2024 Apr 03.

Silicon Ether Ionizable Lipids Enable Potent mRNA Lipid Nanoparticles with Rapid Tissue Clearance.

Richard Holland, Kieu Lam, Sunny Jeng, Kevin McClintock, Lorne Palmer, Petra Schreiner, Mark Wood, Wenchen Zhao, James Heyes.

  The advent of mRNA for nucleic acid (NA) therapeutics has unlocked many diverse areas of research and clinical investigation. However, the shorter intracellular half-life of mRNA compared with other NAs may necessitate more frequent dosing regimens. Because lipid nanoparticles (LNPs) are the principal delivery system used for mRNA, this could lead to tolerability challenges associated with an accumulated lipid burden. This can be addressed by introducing enzymatically cleaved carboxylic esters into the hydrophobic domains of lipid components, notably, the ionizable lipid. However, enzymatic activity can vary significantly with age, disease state, and species, potentially limiting the application in humans. Here we report an alternative approach to ionizable lipid degradability that relies on nonenzymatic hydrolysis, leading to a controlled and highly efficient lipid clearance profile. We identify highly potent examples and demonstrate their exceptional tolerability in multiple preclinical species, including multidosing in nonhuman primates (NHP).

Keywords:  biodegradability; ionizable lipid; lipid nanoparticle; mRNA therapeutics; nanotechnology; nucleic acid

DOI:  https://doi.org/10.1021/acsnano.3c09028
J Liposome Res. 2024 Apr 02. 1-13

Design, synthesis, and in vitro gene transfer efficacy of novel ionizable cholesterol derivatives.

Yajing Wang, Jiahui Jiang, Ziwei Ding, Tao Zhang, Yingying Shi, Xianfeng Huang, Xiaozhong Shen.

  ABSTACTThe medicinal properties of genetic drugs are highly dependent on the design of delivery systems. Ionizable cationic lipids are considered core materials in delivery systems. However, there has not yet been a widespread consensus on the relationship between the wide diversity of lipid structure design and gene delivery efficiency. The aims of the research work were to synthesize ionizable cholesterol derivatives (iChol-lipids) and to evaluate their potential applications as gene delivery vector. A series of iChol-lipids with different head groups were synthesized with carbamate bond spacer. The chemical structures were characterized by 1H NMR, MS, melting range, and pKa. The interactions between iChol-lipids and MALAT1-siRNA were studied by molecular dynamics simulations and compared with market available DC-Chol, which revealed that hydrogen bonds, salt-bridge, and electrostatic interaction were probably involved. The self-assemble behaviors of these lipids were intensively investigated and evaluated by dynamic laser scattering in the presence of different helper lipids and PEGylated lipids. Their plasmid binding ability, transfection efficiency, hemolytic toxicity, and cytotoxicity were fully studied. IZ-Chol-LNPs was proved to be highly potential to effectively complex with DNA, and endosome escape mechanisms mediated by proton sponge effect was verified by pH-sensitive fluorescence probe BCFL.

Keywords:  Cationic cholesterol derivatives; MD simulation; cell transfection; endosome escape; nucleic acid drug delivery

DOI:  https://doi.org/10.1080/08982104.2024.2333755
Int J Nanomedicine. 2024 ;19 3087-3108

Lipid Nanoparticle-Based Inhibitors for SARS-CoV-2 Host Cell Infection.

Vinith Yathindranath, Nura Safa, Mateusz Marek Tomczyk, Vernon Dolinsky, Donald W Miller.

   Purpose: The global pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and the lingering threat to public health has fueled the search for effective therapeutics to treat SARS-CoV-2. This study aimed to develop lipid nanoparticle (LNP) inhibitors of SARS-CoV-2 entry to reduce viral infection in the nose and upper airway.
Methods: Two types of LNP formulations were prepared following a microfluidic mixing method. The LNP-Trap consisted of DOPC, DSPC, cholesterol, and DSPE-PEG-COOH modified with various spike protein binding ligands, including ACE2 peptide, recombinant human ACE2 (rhACE2) or monoclonal antibody to spike protein (mAb). The LNP-Trim consisted of ionizing cationic DLin-MC3-DMA, DSPC, cholesterol, and DMG-PEG lipids encapsulating siACE2 or siTMPRSS2. Both formulations were assayed for biocompatibility and cell uptake in airway epithelial cells (Calu-3). Functional assessment of activity was performed using SARS-CoV-2 spike protein binding assays (LNP-Trap), host receptor knockdown (LNP-Trim), and SARS-CoV-2 pseudovirus neutralization assay (LNP-Trap and LNP-Trim). Localization and tissue distribution of fluorescently labeled LNP formulations were assessed in mice following intranasal administration.
Results: Both LNP formulations were biocompatible based on cell impedance and MTT cytotoxicity studies in Calu-3 cells at concentrations as high as 1 mg/mL. LNP-Trap formulations were able to bind spike protein and inhibit pseudovirus infection by 90% in Calu-3 cells. LNP-Trim formulations reduced ACE2 and TMPRSS2 at the mRNA (70% reduction) and protein level (50% reduction). The suppression of host targets in Calu-3 cells treated with LNP-Trim resulted in over 90% inhibition of pseudovirus infection. In vivo studies demonstrated substantial retention of LNP-Trap and LNP-Trim in the nasal cavity following nasal administration with minimal systemic exposure.
Conclusion: Both LNP-Trap and LNP-Trim formulations were able to safely and effectively inhibit SARS-CoV-2 pseudoviral infection in airway epithelial cells. These studies provide proof-of-principle for a localized treatment approach for SARS-CoV-2 in the upper airway.

Keywords:  SARS-CoV-2; antiviral; gene knockdown; intranasal drug delivery; lipid nanoparticles

DOI:  https://doi.org/10.2147/IJN.S448005
J Control Release. 2024 Apr 02. pii: S0168-3659(24)00227-X. [Epub ahead of print]

A blood-brain barrier- and blood-brain tumor barrier-penetrating siRNA delivery system targeting gliomas for brain tumor immunotherapy.

Lin Tang, Rui Zhang, Yusi Wang, Mohan Liu, Die Hu, Yefeng Wang, Li Yang.

  Glioma is recognized as the most infiltrative and lethal form of central nervous system tumors and is known for its limited response to standard therapeutic interventions, high recurrence rate, and unfavorable prognosis. Recent progress in gene and immunotherapy presents a renewed sense of optimism in the treatment of glioblastoma. However, the barriers to overcome include the blood-brain barrier (BBB) and the blood-brain tumor barrier (BBTB), as well as the suppressive immune microenvironment. Overcoming these barriers remains a significant challenge. Here, we developed a lipid nanoparticle platform incorporating a dual-functional peptide (cholesterol-DP7-ACP-T7-modified DOTAP or DAT-LNP) capable of targeting glioma across the BBB and BBTB for brain tumor immunotherapy. This system was designed to achieve two key functions. First, the system could effectively penetrate the BBB during accumulation within brain tissue following intravenous administration. Second, this system enhances the maturation of dendritic cells, the polarization of M1 macrophages, and the activation of cytotoxic CD8+ T cells. This multifaceted approach effectively mitigates the immunosuppressive tumor microenvironment of glioma and promotes robust antitumor immune responses. Overall, the intravenous administration of the delivery system designed in this study demonstrates significant therapeutic potential for glioma and holds promising applications in the field of cancer immunotherapy.

Keywords:  Blood–brain barrier; Cholesterol-DP7-ACP-T7; Glioma; Tumor microenvironment

DOI:  https://doi.org/10.1016/j.jconrel.2024.04.006
ACS Biomater Sci Eng. 2024 Mar 31.

Folic Acid-Conjugated Brush Polymers Show Enhanced Blood-Brain Barrier Crossing in Static and Dynamic In Vitro Models Toward Brain Cancer Targeting Therapy.

Zihnil A I Mazrad, Ahmed Refaat, Joshua P Morrow, Nicolas H Voelcker, Joseph A Nicolazzo, Meike N Leiske, Kristian Kempe.

  Over the past decades, evidence has consistently shown that treatment of central nervous system (CNS)-related disorders, including Alzheimer's disease, Parkinson's disease, stroke, multiple sclerosis, and brain cancer, is limited due to the presence of the blood-brain barrier (BBB). To assist with the development of new therapeutics, it is crucial to engineer a drug delivery system that can cross the BBB efficiently and reach target cells within the brain. In this study, we present a potentially efficient strategy for targeted brain delivery through utilization of folic acid (FA)-conjugated brush polymers, that specifically target the reduced folate carrier (RFC, SLC19A1) expressed on brain endothelial cells. Here, azide (N3)-decorated brush polymers were prepared in a straightforward manner coupling a heterotelechelic α-NH2, ω-N3-poly(2-ethyl-2-oxazoline) (NH2-PEtOx-N3) to N-acylated poly(amino ester) (NPAE)-based brushes. Strain-promoted azide-alkyne cycloaddition (SPAAC) 'click chemistry' with DBCO-folic acid (FA) yielded FA-brush polymers. Interestingly, while azide functionalization of the brush polymers dramatically reduced their association to brain microvascular endothelial cells (hCMEC/D3), the introduction of FA to azide led to a substantial accumulation of the brush polymers in hCMEC/D3 cells. The ability of the polymeric brush polymers to traverse the BBB was quantitatively assessed using different in vitro BBB models including static Transwell and microfluidic platforms. FA-brush polymers showed efficient transport across hCMEC/D3 cells in a manner dependent on FA composition, whereas nonfunctionalized brush polymers exhibited limited trafficking under the same conditions. Further, cellular uptake inhibition studies suggested that the interaction and transport pathway of FA-brush polymers across BBB relies on the RFC-mediated pathways. The potential application of the developed FA-brush polymers in brain cancer delivery was also investigated in a microfluidic model of BBB-glioblastoma. Brush polymers with more FA units successfully presented an enhanced accumulation into U-87 MG glioma cells following its BBB crossing, compared to controls. These results demonstrate that FA-modified brush polymers hold a great potential for more efficient delivery of future brain therapeutics.

Keywords:  blood−brain barrier; brain targeting; brush polymers; click chemistry; folic acid conjugates; glioblastoma; microfluidic BBB-on-a-chip

DOI:  https://doi.org/10.1021/acsbiomaterials.3c01650
Acta Biomater. 2024 Apr 01. pii: S1742-7061(24)00168-5. [Epub ahead of print]

Immunometabolic Reprogramming of Macrophages with Inhalable CRISPR/Cas9 Nanotherapeutics for Acute Lung Injury Intervention.

Wanling Huang, Gaohong Fu, Yangeng Wang, Cheng Chen, Yilan Luo, Qiaoqiao Yan, Yang Liu, Chengqiong Mao.

  Acute lung injury (ALI) represents a critical respiratory condition typified by rapid-onset lung inflammation, contributing to elevated morbidity and mortality rates. Central to ALI pathogenesis lies macrophage dysfunction, characterized by an overabundance of pro-inflammatory cytokines and a shift in metabolic activity towards glycolysis. This study emphasizes the crucial function of glucose metabolism in immune cell function under inflammatory conditions and identifies hexokinase 2 (HK2) as a key regulator of macrophage metabolism and inflammation. Given the limitations of HK2 inhibitors, we propose the CRISPR/Cas9 system for precise HK2 downregulation. We developed an aerosolized core-shell liposomal nanoplatform (CSNs) complexed with CaP for efficient drug loading, targeting lung macrophages. Various CSNs were synthesized to encapsulate an mRNA based CRISPR/Cas9 system (mCas9/gHK2), and their gene editing efficiency and HK2 knockout were examined at both gene and protein levels in vitro and in vivo. The CSN-mCas9/gHK2 treatment demonstrated a significant reduction in glycolysis and inflammation in macrophages. In an LPS-induced ALI mouse model, inhaled CSN-mCas9/gHK2 mitigated the proinflammatory tumor microenvironment and reprogrammed glucose metabolism in the lung, suggesting a promising strategy for ALI prevention and treatment. This study highlights the potential of combining CRISPR/Cas9 gene editing with inhalation delivery systems for effective, localized pulmonary disease treatment, underscoring the importance of targeted gene modulation and metabolic reprogramming in managing ALI. STATEMENT OF SIGNIFICANCE: This study investigates an inhalable CRISPR/Cas9 gene editing system targeting pulmonary macrophages, with the aim of modulating glucose metabolism to alleviate Acute Lung Injury (ALI). The research highlights the role of immune cell metabolism in inflammation, as evidenced by changes in macrophage glucose metabolism and a notable reduction in pulmonary edema and inflammation. Additionally, observed alterations in macrophage polarization and cytokine levels in bronchoalveolar lavage fluid suggest potential therapeutic implications. These findings not only offer insights into possible ALI treatments but also contribute to the understanding of immune cell metabolism in inflammatory diseases, which could be relevant for various inflammatory and metabolic disorders.

Keywords:  Acute lung injury; HK2; aerosolized nanoparticles; gene editing; immune metabolism; inhalation

DOI:  https://doi.org/10.1016/j.actbio.2024.03.031
Langmuir. 2024 Apr 05.

Revealing the Origin of Cooperative Adsorption of Chains on Nanoparticle Surfaces through Coarse-Grained Simulations.

Mohammed Suliman Alshammasi, Peng Chen, Fernando A Escobedo.

This work aims to deepen our understanding of the molecular origin of the recently observed phenomenon of polymer cooperative adsorption onto faceted nanoparticle (NP) surfaces. By exploring a large parameter space for polymer/NP interactions through coarse-grained (CG) molecular dynamics (MD) simulations, it is found that consistent with experiments the presence or absence of cooperativity is related to solvent quality and relative interaction strengths between the polymer and the adsorbent. Specifically, positive cooperativity is associated with stronger polymer-polymer interaction than polymer-surface interactions and vice versa for negative cooperativity. This contrast in interaction energies manifests in positive cooperativity (i.e., increased affinity) and negative cooperativity (i.e., decreased affinity) as concentration increases. It is also found that increasing chain length strengthens cooperativity effects and that the nanoscale confinement of polymer chains to the adsorbing facet (due to weaker affinity to corners and edges) enhances positive cooperativity but weakens negative cooperativity. Moreover, adsorption onto a spherical NP shows stronger positive cooperativity but weaker negative cooperativity compared with adsorption onto a cubic NP of equal surface area. It was further found that as polymer bulk concentration increases, the free energy of adsorption decreases in positive cooperativity, increases in negative cooperativity, and is independent of concentration in noncooperative systems consistent with the phenomenological explanation of cooperativity. We further found that positive cooperativity is associated with growing fluctuations in the adsorption density at critical bulk polymer concentrations. This behavior can be attributed to the competition between enthalpic gains and entropic losses upon adsorption. Overall, our results shed light on the microscopic origin of cooperative adsorption and the role of solvent quality, which can be leveraged in, for example, controlling NP growth into target shapes and designing NP catalysts with improved performance.

DOI: https://doi.org/10.1021/acs.langmuir.3c03951