bims-novged 2023-08-13 papers

bims-novged

Biomed News

on Non-viral vectors for gene delivery

Issue of 2023–08–13
ten papers selected by
the Merkel lab, Ludwig-Maximilians University

Exploration of mRNA nanoparticles based on DOTAP through optimization of the helper lipids.
Modification of Branched poly(Ethylene Imine) with d-Fructose for Selective Delivery of siRNA into Human Breast Cancer Cells.
Throughput-scalable manufacturing of SARS-CoV-2 mRNA lipid nanoparticle vaccines.
Retracted: Design and In Vitro Evaluation of Novel Cationic Lipids for siRNA Delivery in Breast Cancer Cell Lines.
Physicochemical Targeting of Lipid Nanoparticles to the Lungs Induces Clotting: Mechanisms and Solutions.
Machine learning instructed microfluidic synthesis of curcumin-loaded liposomes.
Application of Design of Experiment in the Optimization of Apixaban-Loaded Solid Lipid Nanoparticles: In Vitro and In Vivo Evaluation.
Effect of Nanoparticle Weight on the Cellular Uptake and Drug Delivery Potential of PLGA Nanoparticles.
In vitro transcription of self-assembling DNA nanoparticles.
Lipid Nanoparticles Optimized for Targeting and Release of Nucleic Acid.

Biotechnol J. 2023 Aug 07. e2300123

Exploration of mRNA nanoparticles based on DOTAP through optimization of the helper lipids.

Xueni Ma, Fanqi Wu, Caihong Peng, Huiling Chen, Dekui Zhang, Tiyun Han.

  Lipid nanoparticles (LNPs) are one of the most efficient carriers for RNA packaging and delivery, and vaccines based on mRNA-LNPs have received substantial attention since the outbreak of the COVID-19 pandemic. LNPs based on 1,2-dioleoyl-3-trimethylammonium propane (DOTAP) have been widely used in preclinical and clinical settings. A novel non-viral gene delivery system called LNP3 was previously developed, which was composed of DOTAP, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), and cholesterol. One of the helper lipids in this carrier was DOPE, which belongs to phospholipids. Given that substituting DOPE with non-phospholipids as helper lipids can increase the delivery efficiency of some LNPs, this study aimed to examine whether non-phospholipids can be formulated with DOTAP as helper lipids. It was found that monoglycerides with C14:0, C16:0, C18:0, C18:1, and C18:2 mediated mRNA transfection, and the transfection efficiency varied between C18:0, C18:1, and C18:2. Furthermore, substituting of the glycerol with other moieties such as the cholesterol or the ethanolamine similarly mediated mRNA transfection. The introduction of cholesterol can further improve the transfection capacity of some DOTAP-based LNPs. One of the best-performing formulations, LNP3-MO, was used to mediate luciferase-mRNA expression in vivo, and the luminescence signal was found to be mainly enriched in the lung and spleen. In addition, the level of SARS-CoV-2 spike antibody in the serum increased after three doses of LNP3-MO mediated SARS-CoV-2 spike mRNA. Altogether, this study demonstrates that non-phospholipids are promising helper lipids that can be formulated with DOTAP to facilitate efficient delivery of mRNAs in vitro and in vivo with organ-specific targeting.

Keywords:  DOTAP; SARS-CoV-2 spike antibody; helper lipids; lipid nanoparticles; mRNA delivery; non-phospholipids

DOI:  https://doi.org/10.1002/biot.202300123
Macromol Biosci. 2023 Aug 10. e2300135

Modification of Branched poly(Ethylene Imine) with d-Fructose for Selective Delivery of siRNA into Human Breast Cancer Cells.

Jan Matthias Peschel, Liên Sabrina Reichel, Tim Hoffmann, Christoph Enzensperger, Ulrich Sigmar Schubert, Anja Traeger, Michael Gottschaldt.

  Branched poly(ethylene imine) (bPEI) is frequently used in RNA interference (RNAi) experiments as a cationic polymer for the delivery of small interfering RNA (siRNA) because of its ability to form stable polyplexes that facilitate siRNA uptake. However, the use of bPEI in gene delivery is limited by its cytotoxicity and a need for target specificity. In this work, bPEI is modified with d-fructose to improve biocompatibility and target breast cancer cells through the overexpressed GLUT5 transporter. Fructose-substituted bPEI (Fru-bPEI) is accessible in three steps starting from commercially available protected fructopyranosides and bPEI. Several polymers with varying molecular weight, degree of substitution, and linker positions on d-fructose (C1 and C3) are synthesized and characterized with NMR spectroscopy, size exclusion chromatography, and elemental analysis. In vitro biological screenings show significantly reduced cytotoxicity of 10 kDa bPEI after fructose functionalization, specific uptake of siRNA polyplexes, and targeted GFP knockdown in triple-negative breast cancer cells (MDA-MB-231) compared to non-cancer cells (HEK-293T). This article is protected by copyright. All rights reserved.

Keywords:  GLUT5; bPEI; breast cancer; fructose; gene delivery; siRNA

DOI:  https://doi.org/10.1002/mabi.202300135
Proc Natl Acad Sci U S A. 2023 08 15. 120(33): e2303567120

Throughput-scalable manufacturing of SARS-CoV-2 mRNA lipid nanoparticle vaccines.

Sarah J Shepherd, Xuexiang Han, Alvin J Mukalel, Rakan El-Mayta, Ajay S Thatte, Jingyu Wu, Marshall S Padilla, Mohamad-Gabriel Alameh, Neha Srikumar, Daeyeon Lee, Drew Weissman, David Issadore, Michael J Mitchell.

  Lipid nanoparticles (LNPs) are a potent delivery technology that have made it possible for the recent clinical breakthroughs in mRNA therapeutics and vaccines. A key challenge to the broader implementation of mRNA therapeutics and vaccines is the development of technology to produce precisely defined LNP formulations, with throughput that can scale from discovery to commercial manufacturing and meet the stringent manufacturing standards of the pharmaceutical industry. To address these challenges, we have developed a microfluidic chip that incorporates 1×, 10×, or 256× LNP-generating units that achieve scalable production rates of up to 17 L/h of precisely defined LNPs. Using these chips, we demonstrate that LNP physical properties and potency in vivo are unchanged as throughput is scaled. Our chips are fabricated out of silicon and glass substrates, which have excellent solvent compatibility, compatibility with pharmaceutical manufacturing, and can be fully reset and reused. SARS-CoV-2 mRNA-LNP vaccines formulated by our chips triggered potent antibody responses in a preclinical study. These results demonstrate the feasibility of directly translating microfluidic-generated LNPs to the scale necessary for commercial production.

Keywords:  drug delivery; mRNA; nanomedicine; nanoparticle; vaccines

DOI:  https://doi.org/10.1073/pnas.2303567120
Evid Based Complement Alternat Med. 2023 ;2023 9892315

Retracted: Design and In Vitro Evaluation of Novel Cationic Lipids for siRNA Delivery in Breast Cancer Cell Lines.

Evidence-Based Complementary And Alternative Medicine.

[This retracts the article DOI: 10.1155/2022/9231641.].

DOI: https://doi.org/10.1155/2023/9892315
bioRxiv. 2023 Jul 25. pii: 2023.07.21.550080. [Epub ahead of print]

Physicochemical Targeting of Lipid Nanoparticles to the Lungs Induces Clotting: Mechanisms and Solutions.

Serena Omo-Lamai, Marco E Zamora, Manthan N Patel, Jichuan Wu, Jia Nong, Zhicheng Wang, Alina Peshkova, Liam S Chase, Eno-Obong Essien, Vladimir Muzykantov, Oscar Marcos-Contreras, Jacob W Myerson, Jacob S Brenner.

Lipid nanoparticles (LNPs) have become the dominant drug delivery technology in industry, holding the promise to deliver RNA to up- or down-regulate any protein of interest. LNPs have been targeted to specific cell types or organs by physicochemical targeting, in which LNP's lipid compositions are adjusted to find mixtures with the desired tropism. In a popular approach, physicochemical targeting is accomplished by formulating with charged lipids. Negatively charged lipids localize LNPs to the spleen, and positively charged lipids to the lungs. Here we found that lung-tropic LNPs employing cationic lipids induce massive thrombosis. We demonstrate that thrombosis is induced in the lungs and other organs, and greatly exacerbated by pre-existing inflammation. This clotting is induced by a variety of formulations with cationic lipids, including LNPs and non-LNP nanoparticles. The mechanism depends on the LNPs binding to fibrinogen and inducing platelet and thrombin activation. Based on these mechanisms, we engineered multiple solutions which enable positively charged LNPs to target the lungs while not inducing thrombosis. Our findings implicate thrombosis as a major barrier that blood erects against LNPs with cationic components and illustrate how physicochemical targeting approaches must be investigated early for risks and re-engineered with a careful understanding of biological mechanisms.

DOI: https://doi.org/10.1101/2023.07.21.550080
Biomed Microdevices. 2023 Aug 05. 25(3): 29

Machine learning instructed microfluidic synthesis of curcumin-loaded liposomes.

Valentina Di Francesco, Daniela P Boso, Thomas L Moore, Bernhard A Schrefler, Paolo Decuzzi.

  The association of machine learning (ML) tools with the synthesis of nanoparticles has the potential to streamline the development of more efficient and effective nanomedicines. The continuous-flow synthesis of nanoparticles via microfluidics represents an ideal playground for ML tools, where multiple engineering parameters - flow rates and mixing configurations, type and concentrations of the reagents - contribute in a non-trivial fashion to determine the resultant morphological and pharmacological attributes of nanomedicines. Here we present the application of ML models towards the microfluidic-based synthesis of liposomes loaded with a model hydrophobic therapeutic agent, curcumin. After generating over 200 different liposome configurations by systematically modulating flow rates, lipid concentrations, organic:water mixing volume ratios, support-vector machine models and feed-forward artificial neural networks were trained to predict, respectively, the liposome dispersity/stability and size. This work presents an initial step towards the application and cultivation of ML models to instruct the microfluidic formulation of nanoparticles.

Keywords:  Artificial Intelligence; Artificial neural network; Drug delivery; Microfluidics; Nanomedicine

DOI:  https://doi.org/10.1007/s10544-023-00671-1
AAPS PharmSciTech. 2023 Aug 08. 24(6): 167

Application of Design of Experiment in the Optimization of Apixaban-Loaded Solid Lipid Nanoparticles: In Vitro and In Vivo Evaluation.

Safaa E Ramadan, Sanaa A El-Gizawy, Mohamed A Osman, Mona F Arafa.

  Solid lipid nanoparticles (SLnPs) are usually utilized as lipid-based formulations for enhancing oral bioavailability of BCS class IV drugs. Accordingly, the objective of this work was to investigate the effect of formulation and processing variables on the properties of the developed SLnPs for oral delivery of apixaban. Randomized full factorial design (24) was employed for optimization of SLnPs. With two levels for each independent variable, four factors comprising both formulations and processing factors were chosen: the GMS content (A), the Tween 80 content (B), the homogenization time (C), and the content of poloxamer 188 used (D). The modified hot homogenization and sonication method was employed in the formulation of solid lipid nanoparticles loaded with apixaban (APX-SLnPs). The size of APX-SLnPs formulations was measured to lie between 116.7 and 1866 nm, polydispersity index ranged from 0.385 to 1, and zeta potential was discovered to be in the range of - 12.6 to - 38.6 mV. The entrapping efficiency of APX-SLnPs formulations was found to be in the range of 22.8 to 96.7%. The optimized formulation was evaluated in vivo after oral administration to rats. Oral administration of APX-SLnPs resulted in significant prolongation in bleeding time compared with both positive and negative control. This indicates the ability of this system to enhance drug therapeutic effect either by increasing intestinal absorption or trans-lymphatic transport. So, this study highlighted the capability of SLnPs to boost the pharmacological effect of apixaban.

Keywords:  apixaban; bleeding time; factorial design; poloxamer; solid lipid nanoparticles

DOI:  https://doi.org/10.1208/s12249-023-02628-2
ACS Omega. 2023 Aug 01. 8(30): 27146-27155

Effect of Nanoparticle Weight on the Cellular Uptake and Drug Delivery Potential of PLGA Nanoparticles.

Prabhat Kattel, Shoukath Sulthana, Jiří Trousil, Dinesh Shrestha, David Pearson, Santosh Aryal.

Biodegradable and biocompatible polymeric nanoparticles (NPs) stand out as a key tool for improving drug bioavailability, reducing the inherent toxicity, and targeting the intended site. Most importantly, the ease of polymer synthesis and its derivatization to add functional properties makes them potentially ideal to fulfill the requirements for intended therapeutic applications. Among many polymers, US FDA-approved poly(l-lactic-co-glycolic) acid (PLGA) is a widely used biocompatible and biodegradable co-polymer in drug delivery and in implantable biomaterials. While many studies have been conducted using PLGA NPs as a drug delivery system, less attention has been given to understanding the effect of NP weight on cellular behaviors such as uptake. Here we discuss the synthesis of PLGA NPs with varying NP weights and their colloidal and biological properties. Following nanoprecipitation, we have synthesized PLGA NP sizes ranging from 60 to 100 nm by varying the initial PLGA feed in the system. These NPs were found to be stable for a prolonged period in colloidal conditions. We further studied cellular uptake and found that these NPs are cytocompatible; however, they are differentially uptaken by cancer and immune cells, which are greatly influenced by NPs' weight. The drug delivery potential of these nanoparticles (NPs) was assessed using doxorubicin (DOX) as a model drug, loaded into the NP core at a concentration of 7.0 ± 0.5 wt % to study its therapeutic effects. The results showed that both concentration and treatment time are crucial factors for exhibiting therapeutic effects, as observed with DOX-NPs exhibiting a higher potency at lower concentrations. The observations revealed that DOX-NPs exhibited a higher cellular uptake of DOX compared to the free-DOX treatment group. This will allow us to reduce the recommended dose to achieve the desired effect, which otherwise required a large dose when treated with free DOX. Considering the significance of PLGA-based nanoparticle drug delivery systems, we anticipate that this study will contribute to the establishment of design considerations and guidelines for the therapeutic applications of nanoparticles.

DOI: https://doi.org/10.1021/acsomega.3c02273
Sci Rep. 2023 Aug 10. 13(1): 12961

In vitro transcription of self-assembling DNA nanoparticles.

Chang Yong Oh, Eric R Henderson.

Nucleic acid nanoparticles are playing an increasingly important role in biomolecular diagnostics and therapeutics as well as a variety of other areas. The unique attributes of self-assembling DNA nanoparticles provide a potentially valuable addition or alternative to the lipid-based nanoparticles that are currently used to ferry nucleic acids in living systems. To explore this possibility, we have assessed the ability of self-assembling DNA nanoparticles to be constructed from complete gene cassettes that are capable of gene expression in vitro. In the current report, we describe the somewhat counter-intuitive result that despite extensive crossovers (the stereochemical analogs of Holliday junctions) and variations in architecture, these DNA nanoparticles are amenable to gene expression as evidenced by T7 RNA polymerase-driven transcription of a reporter gene in vitro. These findings, coupled with the vastly malleable architecture and chemistry of self-assembling DNA nanoparticles, warrant further investigation of their utility in biomedical genetics.

DOI: https://doi.org/10.1038/s41598-023-39777-0
Adv Mater. 2023 Aug 07. e2305300

Lipid Nanoparticles Optimized for Targeting and Release of Nucleic Acid.

Yaru Jia, Xiuguang Wang, Luwei Li, Fangzhou Li, Jinchao Zhang, Xing-Jie Liang.

  Lipid nanoparticles (LNPs) are currently the most promising clinical nucleic acids drug delivery vehicles. LNPs prevent the degradation of cargo nucleic acids during blood circulation. Upon entry into the cell, specific components of the lipid nanoparticles can promote the endosomal escape of nucleic acids. These are the basic properties of lipid nanoparticles as nucleic acid carriers. As LNPs exhibit hepatic aggregation characteristics, enhancing targeting out of the liver is a crucial way to improve LNPs administrated in vivo. Meanwhile, endosomal escape of nucleic acids loaded in LNPs is often considered inadequate, and therefore, much effort is devoted to enhancing the intracellular release efficiency of nucleic acids. Here, we concluded different strategies to efficiently deliver nucleic acid delivery from LNPs and investigated their mechanisms. In addition, based on the information on LNPs that are in clinical trials or have completed clinical trials, we discussed the issues that are necessary to be approached in the clinical translation of LNPs, which we hope will shed light on the development of LNP nucleic acid drugs. This article is protected by copyright. All rights reserved.

Keywords:  endosomal escape; lipid nanoparticles; nucleic acid delivery; organ targeting

DOI:  https://doi.org/10.1002/adma.202305300