bims-novged 2024-03-17 papers

bims-novged

Biomed News

on Non-viral vectors for gene delivery

Issue of 2024–03–17
ten papers selected by
the Merkel lab, Ludwig-Maximilians University

Which Lipid Nanoparticle (LNP) Designs Work? A Simple Kinetic Model Linking LNP Chemical Structure to In Vivo Delivery Performance.
Mechanistic insights into interactions between ionizable lipid nanodroplets and biomembranes.
Optimization of lipid assisted polymeric nanoparticles for siRNA delivery and cancer immunotherapy.
Selective Replacement of Cholesterol with Cationic Amphiphilic Drugs Enables the Design of Lipid Nanoparticles with Improved RNA Delivery.
Influence of ionizable lipid tail length on lipid nanoparticle delivery of mRNA of varying length.
Polymer-lipid hybrid nanomedicines to deliver siRNA in and against glioblastoma cells.
Inhalable porous particles as dual micro-nano carriers demonstrating efficient lung drug delivery for treatment of tuberculosis.
mRNAid, an open-source platform for therapeutic mRNA design and optimization strategies.
Effect of in vitro transcription conditions on yield of high quality messenger and self-amplifying RNA.
Host-Directed Virus-Mimicking Particles Interacting with the ACE2 Receptor Competitively Block Coronavirus SARS-CoV-2 Entry.

ACS Appl Mater Interfaces. 2024 Mar 11.

Which Lipid Nanoparticle (LNP) Designs Work? A Simple Kinetic Model Linking LNP Chemical Structure to In Vivo Delivery Performance.

Esther H Roh, Millicent O Sullivan, Thomas H Epps.

  Although lipid nanoparticles (LNPs) are the predominant nanocarriers for short-interfering RNA (siRNA) delivery, most therapies use nearly identical formulations that have taken 30 years to design but lack the diverse property ranges necessary for versatile application. This dearth in variety and the extended timeline for implementation are attributed to a limited understanding of how LNP properties facilitate overcoming biological barriers. Herein, a simple kinetic model was developed by using major rate-limiting steps for siRNA delivery, and this model enabled the identification of a critical parameter to predict LNP efficacy without extensive experimental testing. A volume-averaged log D, the "solubility" of charged molecules as a function of pH weighted by component volume fractions, resulted in a good correlation between LNP composition and siRNA delivery. Both the effects of modifying the structures of ionizable lipids and LNP composition on gene silencing were easily captured in the model predictions. Thus, this approach provides a robust LNP structure-activity relationship to dramatically accelerate the realization of effective LNP formulations.

Keywords:  endosomal escape; in vitro-in vivo translation; kinetic modeling; lipid nanoparticles; siRNA delivery

DOI:  https://doi.org/10.1021/acsami.3c15424
J Biomol Struct Dyn. 2024 Mar 15. 1-11

Mechanistic insights into interactions between ionizable lipid nanodroplets and biomembranes.

Petra Čechová, Markéta Paloncýová, Martin Šrejber, Michal Otyepka.

  Delivery of RNA into cells using lipid nanoparticles (LNPs) has been a significant breakthrough in RNA-based medicine, with clinical applicability expanded through the use of ionizable lipids (ILs). These unique lipids can alter their charge state in response to pH changes, which is crucial for pH-triggered endosomal escape and effective lipid-mediated RNA delivery. In this study, we conducted a comprehensive set of molecular dynamics (MD) simulations to investigate interactions between IL-containing lipid nanodroplets (LNDs) and cell membrane models. Using an atomistic resolution model, we investigated the merging process of LNDs with cell membrane models under neutral conditions relevant to an intercellular environment and acidic pH conditions found in late endosomes. Our observations revealed that at neutral pH, LNDs merged with lipid membranes while preserving the bilayer structure. Under acidic conditions, the LNDs remained attached to the bilayer without fusing into the membranes. Importantly, the presence of ILs did not disrupt the original biomembrane structure during the simulation period. The MD simulations provided valuable atomistic insights into the mechanism of interaction between IL-containing nanodroplets and biomembranes, which could aid the rational design of ILs to develop more efficient LNPs for RNA therapies.Communicated by Ramaswamy H. Sarma.

Keywords:  Ionizable lipids; ionizable lipid nanoparticles; lipid mixing; lipid nanoparticles; membrane simulations; molecular dynamics

DOI:  https://doi.org/10.1080/07391102.2024.2329307
Biomater Sci. 2024 Mar 12.

Optimization of lipid assisted polymeric nanoparticles for siRNA delivery and cancer immunotherapy.

Song Lin, Houjin Jing, Xiaojiao Du, Xianzhu Yang, Jun Wang.

To date, five siRNA-based medications have received clinical approval and have demonstrated remarkable therapeutic efficacy in treating various diseases. However, their application has been predominantly limited to liver-specific diseases due to constraints in siRNA delivery capabilities. In this study, we have developed a siRNA delivery system utilizing clinically approved mPEG-b-PLGA, a cationic lipid, and an ionizable lipid. We optimized this system by carefully adjusting their mass ratios, resulting in highly efficient gene silencing. Furthermore, the optimized nanoparticle formulation, which encapsulates siRNA targeting CD47, induces a robust immune response. This response effectively suppresses the progression of melanoma tumors by blocking this critical immune checkpoint.

DOI: https://doi.org/10.1039/d3bm02071a
Nano Lett. 2024 Mar 13. 24(10): 2961-2971

Selective Replacement of Cholesterol with Cationic Amphiphilic Drugs Enables the Design of Lipid Nanoparticles with Improved RNA Delivery.

Bram Bogaert, Aliona Debisschop, Thomas Ehouarne, Hannelore P Van Eeckhoutte, Joyceline De Volder, An Jacobs, Eline Pottie, Riet De Rycke, Aurélie Crabbé, Pieter Mestdagh, Ine Lentacker, Guy G Brusselle, Christophe Stove, Sandra Verstraelen, Tania Maes, Ken R Bracke, Stefaan C De Smedt, Koen Raemdonck.

  The delivery of RNA across biological barriers can be achieved by encapsulation in lipid nanoparticles (LNPs). Cationic amphiphilic drugs (CADs) are pharmacologically diverse compounds with ionizable lipid-like features. In this work, we applied CADs as a fifth component of state-of-the-art LNPs via microfluidic mixing. Improved cytosolic delivery of both siRNA and mRNA was achieved by partly replacing the cholesterol fraction of LNPs with CADs. The LNPs could cross the mucus layer in a mucus-producing air-liquid interface model of human primary bronchial epithelial cells following nebulization. Moreover, CAD-LNPs demonstrated improved epithelial and endothelial targeting following intranasal administration in mice, without a marked pro-inflammatory signature. Importantly, quantification of the CAD-LNP molar composition, as demonstrated for nortriptyline, revealed a gradual leakage of the CAD from the formulation during LNP dialysis. Altogether, these data suggest that the addition of a CAD prior to the rapid mixing process might have an impact on the composition, structure, and performance of LNPs.

Keywords:  cationic amphiphilic drugs; combination therapy; drug repurposing; inhalation therapy; lipid nanoparticles; mRNA; nebulization; siRNA

DOI:  https://doi.org/10.1021/acs.nanolett.3c03345
J Biomed Mater Res A. 2024 Mar 15.

Influence of ionizable lipid tail length on lipid nanoparticle delivery of mRNA of varying length.

Kaitlin Mrksich, Marshall S Padilla, Ryann A Joseph, Emily L Han, Dongyoon Kim, Rohan Palanki, Junchao Xu, Michael J Mitchell.

  RNA-based therapeutics have gained traction for the prevention and treatment of a variety of diseases. However, their fragility and immunogenicity necessitate a drug carrier. Lipid nanoparticles (LNPs) have emerged as the predominant delivery vehicle for RNA therapeutics. An important component of LNPs is the ionizable lipid (IL), which is protonated in the acidic environment of the endosome, prompting cargo release into the cytosol. Currently, there is growing evidence that the structure of IL lipid tails significantly impacts the efficacy of LNP-mediated mRNA translation. Here, we optimized IL tail length for LNP-mediated delivery of three different mRNA cargos. Using C12-200, a gold standard IL, as a model, we designed a library of ILs with varying tail lengths and evaluated their potency in vivo. We demonstrated that small changes in lipophilicity can drastically increase or decrease mRNA translation. We identified that LNPs formulated with firefly luciferase mRNA (1929 base pairs) and C10-200, an IL with shorter tail lengths than C12-200, enhance liver transfection by over 10-fold. Furthermore, different IL tail lengths were found to be ideal for transfection of LNPs encapsulating mRNA cargos of varying sizes. LNPs formulated with erythropoietin (EPO), responsible for stimulating red blood cell production, mRNA (858 base pairs), and the C13-200 IL led to EPO translation at levels similar to the C12-200 LNP. The LNPs formulated with Cas9 mRNA (4521 base pairs) and the C9-200 IL induced over three times the quantity of indels compared with the C12-200 LNP. Our findings suggest that shorter IL tails may lead to higher transfection of LNPs encapsulating larger mRNAs, and that longer IL tails may be more efficacious for delivering smaller mRNA cargos. We envision that the results of this project can be utilized as future design criteria for the next generation of LNP delivery systems for RNA therapeutics.

Keywords:  Ionizable lipid; lipid nanoparticle; mRNA

DOI:  https://doi.org/10.1002/jbm.a.37705
Int J Pharm. 2024 Mar 12. pii: S0378-5173(24)00228-X. [Epub ahead of print] 123994

Polymer-lipid hybrid nanomedicines to deliver siRNA in and against glioblastoma cells.

Arianna Rinaldi, Florence Dumas, Jason Thomas Duskey, Carol Imbriano, Silvia Belluti, Charlotte Roy, Ilaria Ottonelli, Maria Angela Vandelli, Barbara Ruozi, Emmanuel Garcion, Giovanni Tosi, Frank Boury.

  Small interfering RNA (siRNA) holds great potential to treat many difficult-to-treat diseases, but its delivery remains the central challenge. This study aimed at investigating the suitability of polymer-lipid hybrid nanomedicines (HNMed) as novel siRNA delivery platforms for locoregional therapy of glioblastoma. Two HNMed formulations were developed from poly(lactic-co-glycolic acid) polymer and a cationic lipid: 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) or 3ß-[N-(N',N'-dimethylaminoethane)-carbamoyl]cholesterol (DC-Chol). After characterization of the HNMeds, a model siRNA was complexed onto their surface to form HNMed/siRNA complexes. The physicochemical properties and siRNA binding ability of complexes were assessed over a range of nitrogen-to-phosphate (N/P) ratios to optimize the formulations. At the optimal N/P ratio of 10, complexes effectively bound siRNA and improved its protection from enzymatic degradation. Using the NIH3T3 mouse fibroblast cell line, DOTAP-based HNMeds were shown to possess higher cytocompatibility in vitro over the DC-Chol-based ones. As proof-of-concept, uptake and bioefficacy of formulations were also assessed in vitro on U87MG human glioblastoma cell line expressing luciferase gene. Complexes were able to deliver anti-luciferase siRNA and induce a remarkable suppression of gene expression. Noteworthy, the effect of DOTAP-based formulation was not only about three-times higher than DC-Chol-based ones, but also comparable to lipofectamine model transfection reagent. These findings set the basis to exploit this nanosystem for silencing relevant GB-related genes in further in vitro and in vivo studies.

Keywords:  DC-Chol; DOTAP; Glioblastoma; Nanomedicine; Polymer-lipid hybrid nanomedicines; Small interfering RNA

DOI:  https://doi.org/10.1016/j.ijpharm.2024.123994
J Control Release. 2024 Mar 11. pii: S0168-3659(24)00165-2. [Epub ahead of print]

Inhalable porous particles as dual micro-nano carriers demonstrating efficient lung drug delivery for treatment of tuberculosis.

Jesús E Campos Pacheco, Tetiana Yalovenko, Azra Riaz, Nikolay Kotov, Camilla Davids, Alva Persson, Peter Falkman, Adam Feiler, Gabriela Godaly, C Magnus Johnson, Mikael Ekström, Georgia A Pilkington, Sabrina Valetti.

  Inhalation therapy treating severe infectious disease is among the more complex and emerging topics in controlled drug release. Micron-sized carriers are needed to deposit drugs into the lower airways, while nano-sized carriers are of preference for cell targeting. Here, we present a novel and versatile strategy using micron-sized spherical particles with an excellent aerodynamic profile that dissolve in the lung fluid to ultimately generate nanoparticles enabling to enhance both extra- and intra-cellular drug delivery (i.e., dual micro-nano inhalation strategy). The spherical particles are synthesised through the condensation of nano-size amorphous silicon dioxide resulting in high surface area, disordered mesoporous silica particles (MSPs) with monodispersed size of 2.43 μm. Clofazimine (CLZ), a drug shown to be effective against multidrug-resistant tuberculosis, was encapsulated in the MSPs obtaining a dry powder formulation with high respirable fraction (F.P.F. <5 μm of 50%) without the need of additional excipients. DSC, XRPD, and Nitrogen adsorption/desorption indicate that the drug was fully amorphous when confined in the nano-sized pores (9-10 nm) of the MSPs (shelf-life of 20 months at 4 °C). Once deposited in the lung, the CLZ-MSPs exhibited a dual action. Firstly, the nanoconfinement within the MSPs enabled a drastic dissolution enhancement of CLZ in simulated lung fluid (i.e., 16-fold higher than the free drug), increasing mycobacterial killing than CLZ alone (p = 0.0262) and reaching concentrations above the minimum bactericidal concentration (MBC) against biofilms of M. tuberculosis (i.e., targeting extracellular bacteria). The released CLZ permeated but was highly retained in a Calu-3 respiratory epithelium model, suggesting a high local drug concentration within the lung tissue minimizing risk for systemic side effects. Secondly, the micron-sized drug carriers spontaneously dissolve in simulated lung fluid into nano-sized drug carriers (shown by Nano-FTIR), delivering high CLZ cargo inside macrophages and drastically decreasing the mycobacterial burden inside macrophages (i.e., targeting intracellular bacteria). Safety studies showed neither measurable toxicity on macrophages or Calu-3 cells, nor impaired epithelial integrity. The dissolved MSPs also did not show haemolytic effect on human erythrocytes. In a nutshell, this study presents a low-cost, stable and non-invasive dried powder formulation based on a dual micro-nano carrier to efficiently deliver drug to the lungs overcoming technological and practical challenges for global healthcare.

Keywords:  Clofazimine; Disordered mesoporous silica particles; Dissolution enhancement; Dried powder formulation; Dual micro-nano carrier; Lung drug delivery; Soluble carrier

DOI:  https://doi.org/10.1016/j.jconrel.2024.03.013
NAR Genom Bioinform. 2024 Mar;6(1): lqae028

mRNAid, an open-source platform for therapeutic mRNA design and optimization strategies.

Nikita Vostrosablin, Shuhui Lim, Pooja Gopal, Kveta Brazdilova, Sushmita Parajuli, Xiaona Wei, Anna Gromek, David Prihoda, Martin Spale, Anja Muzdalo, Jamie Greig, Constance Yeo, Joanna Wardyn, Petr Mejzlik, Brian Henry, Anthony W Partridge, Danny A Bitton.

Recent COVID-19 vaccines unleashed the potential of mRNA-based therapeutics. A common bottleneck across mRNA-based therapeutic approaches is the rapid design of mRNA sequences that are translationally efficient, long-lived and non-immunogenic. Currently, an accessible software tool to aid in the design of such high-quality mRNA is lacking. Here, we present mRNAid, an open-source platform for therapeutic mRNA optimization, design and visualization that offers a variety of optimization strategies for sequence and structural features, allowing one to customize desired properties into their mRNA sequence. We experimentally demonstrate that transcripts optimized by mRNAid have characteristics comparable with commercially available sequences. To encompass additional aspects of mRNA design, we experimentally show that incorporation of certain uridine analogs and untranslated regions can further enhance stability, boost protein output and mitigate undesired immunogenicity effects. Finally, this study provides a roadmap for rational design of therapeutic mRNA transcripts.

DOI: https://doi.org/10.1093/nargab/lqae028
Eur J Pharm Biopharm. 2024 Mar 08. pii: S0939-6411(24)00073-0. [Epub ahead of print] 114247

Effect of in vitro transcription conditions on yield of high quality messenger and self-amplifying RNA.

Petya G Popova, Melissa A Lagace, George Tang, Anna K Blakney.

  Messenger RNA (mRNA) and self-amplifying RNA (saRNA) vaccines against SARS-CoV-2 produced using in vitro transcription (IVT) were clinically approved in 2020 and 2022, respectively. While the industrial production of mRNA using IVT has been extensively optimized, the optimal conditions for saRNA have been explored to a lesser extent. Most T7 polymerase IVT protocols have been specifically optimized for mRNA which is ∼5-10-fold smaller than saRNA and may have profound effects on both the quality and yield of longer transcripts. Here, we optimized IVT conditions for simultaneously increasing the yield of full-length transcripts and reducing dsRNA formation through Design of Experiments. Using a definitive screening approach, we found that the key parameters are temperature and magnesium in the outcome of RNA quality (% full length transcript) and yield in small scale synthesis. The most important parameter for reducing dsRNA formation for both mRNA and saRNA was Mg2+ concentration (10 mM). We observed that a lower temperature was vital for production of high quality saRNA transcripts. mRNA quality was optimal at higher Mg2+ concentration (>40 mM). High quality transcripts correspond to significantly reduced product yield for saRNA, but not for mRNA. The differences between mRNA and saRNA requirements for high quality product and the relationship between high quality large saRNA molecules and low temperature synthesis have not been reported previously. These findings are key for informing future IVT parameters design and optimization for smaller and larger RNA transcripts.

Keywords:  Design of experiments; Double-stranded RNA; In vitro transcription; Messenger RNA; RNA quality; Self-amplifying RNA

DOI:  https://doi.org/10.1016/j.ejpb.2024.114247
Nano Lett. 2024 Mar 11.

Host-Directed Virus-Mimicking Particles Interacting with the ACE2 Receptor Competitively Block Coronavirus SARS-CoV-2 Entry.

Pei Zhang, Erik Niemelä, Sandra López Cerdá, Pasi Sorvisto, Jani Virtanen, Hélder A Santos.

  Herein, we fabricate host-directed virus-mimicking particles (VMPs) to block the entry of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) into host cells through competitive inhibition enabled by their interactions with the angiotensin-converting enzyme 2 (ACE2) receptor. A microfluidic platform is developed to fabricate a lipid core of the VMPs with a narrow size distribution and a low level of batch-to-batch variation. The resultant solid lipid nanoparticles are decorated with an average of 231 or 444 Spike S1 RBD protrusions mimicking either the original SARS-CoV-2 or its delta variant, respectively. Compared with that of the nonfunctionalized core, the cell uptake of the functionalized VMPs is enhanced with ACE2-expressing cells due to their strong interactions with the ACE2 receptor. The fabricated VMPs efficiently block the entry of SARS-CoV-2 pseudovirions into host cells and suppress viral infection. Overall, this study provides potential strategies for preventing the spread of SARS-CoV-2 or other coronaviruses employing the ACE2 receptor to enter into host cells.

Keywords:  SARS-CoV-2; Spike S1 receptor binding domain; angiotensin-converting enzyme 2; microfluidics; virus-mimicking particles

DOI:  https://doi.org/10.1021/acs.nanolett.3c04430