bims-novged 2023-01-08 papers

bims-novged

Biomed News

on Non-viral vectors for gene delivery

Issue of 2023‒01‒08
eleven papers selected by
the Merkel lab
Ludwig-Maximilians University

Lipid Nanoparticle Composition Drives mRNA Delivery to the Placenta.
Structure and Function of Cationic and Ionizable Lipids for Nucleic Acid Delivery.
Layer-by-Layer siRNA Particle Assemblies for Localized Delivery of siRNA to Epithelial Cells through Surface-Mediated Particle Uptake.
Why mRNA-ionizable LNPs formulations are so short-lived: causes and way-out.
The Delivery of ABE mRNA to the Adult Murine Liver by Lipid Nanoparticles (LNPs).
Efficient delivery of VEGF-A mRNA for promoting diabetic wound healing via ionizable lipid nanoparticles.
Multivalent mannose-conjugated siRNA causes robust gene silencing in pancreatic macrophages in vivo.
Atomistic Insights into Organization of RNA-Loaded Lipid Nanoparticles.
High-Performance Delivery of CRISPRi System via Lipid-Polymer Hybrid Nanoparticles combined with Ultrasound-Mediated Microbubble Destruction for Tumor-Specific Gene Repression.
pH-responsive nanoparticles based on POEOMA-b-PDPA block copolymers for RNA encapsulation, protection and cell delivery.
Supramolecular Peptide Nanofiber/PLGA Nanocomposites for Enhancing Pulmonary Drug Delivery.

bioRxiv. 2022 Dec 22. pii: 2022.12.22.521490. [Epub ahead of print]

Lipid Nanoparticle Composition Drives mRNA Delivery to the Placenta.

Rachel E Young, Katherine M Nelson, Samuel I Hofbauer, Tara Vijayakumar, Mohamad-Gabriel Alameh, Drew Weissman, Charalampos Papachristou, Jason P Gleghorn, Rachel S Riley.

Ionizable lipid nanoparticles (LNPs) have gained attention as mRNA delivery platforms for vaccination against COVID-19 and for protein replacement therapies. LNPs enhance mRNA stability, circulation time, cellular uptake, and preferential delivery to specific tissues compared to mRNA with no carrier platform. However, LNPs have yet to be developed for safe and effective mRNA delivery to the placenta as a method to treat placental dysfunction. Here, we develop LNPs that enable high levels of mRNA delivery to trophoblasts in vitro and to the placenta in vivo with no toxicity. We conducted a Design of Experiments to explore how LNP composition, including the type and molar ratio of each lipid component, drives trophoblast and placental delivery. Our data revealed that a specific combination of ionizable lipid and phospholipid in the LNP design yields high transfection efficiency in vitro . Further, we present one LNP platform that exhibits highest delivery of placental growth factor mRNA to the placenta in pregnant mice, which demonstrates induced protein synthesis and secretion of a therapeutic protein. Lastly, our high-performing LNPs have no toxicity to both the pregnant mice and fetuses. Our results demonstrate the feasibility of LNPs as a platform for mRNA delivery to the placenta. Our top LNPs may provide a therapeutic platform to treat diseases that originate from placental dysfunction during pregnancy.

DOI: https://doi.org/10.1101/2022.12.22.521490
Pharm Res. 2023 Jan 04.

Structure and Function of Cationic and Ionizable Lipids for Nucleic Acid Delivery.

Da Sun, Zheng-Rong Lu.

  Hereditary genetic diseases, cancer, and infectious diseases are affecting global health and become major health issues, but the treatment development remains challenging. Gene therapies using DNA plasmid, RNAi, miRNA, mRNA, and gene editing hold great promise. Lipid nanoparticle (LNP) delivery technology has been a revolutionary development, which has been granted for clinical applications, including mRNA vaccines against SARS-CoV-2 infections. Due to the success of LNP systems, understanding the structure, formulation, and function relationship of the lipid components in LNP systems is crucial for design more effective LNP. Here, we highlight the key considerations for developing an LNP system. The evolution of structure and function of lipids as well as their LNP formulation from the early-stage simple formulations to multi-components LNP and multifunctional ionizable lipids have been discussed. The flexibility and platform nature of LNP enable efficient intracellular delivery of a variety of therapeutic nucleic acids and provide many novel treatment options for the diseases that are previously untreatable.

Keywords:  cationic lipid; ionizable lipid; lipid nucleic acid nanoparticles; nucleic acid delivery; structural effect

DOI:  https://doi.org/10.1007/s11095-022-03460-2
ACS Appl Bio Mater. 2023 Jan 04.

Layer-by-Layer siRNA Particle Assemblies for Localized Delivery of siRNA to Epithelial Cells through Surface-Mediated Particle Uptake.

Aaron Lee, Natalia Gosnell, Daela Milinkovic, Patricia Taladriz-Blanco, Barbara Rothen-Rutishauser, Alke Petri-Fink.

  Localized delivery of small interfering RNA (siRNA) is a promising approach for spatial control of cell responses at biomaterial interfaces. Layer-by-layer (LbL) assembly of siRNA with cationic polyelectrolytes has been used in film and nanoparticle vectors for transfection. Herein, we combine the ability of particles to efficiently deliver siRNA with the ability of film polyelectrolyte multilayers to act locally. LbL particles were prepared with alternating layers of poly(l-arginine) and siRNA and capped with hyaluronic acid. Negatively charged LbL particles were subsequently assembled on the poly(l-lysine)-functionalized substrate to form a LbL particle-decorated surface. Cells grown in contact with the particle-decorated surface were able to survive, internalize particles, and undergo gene silencing. This work shows that particle-decorated surfaces can be engineered by using electrostatic interactions and used to deliver therapeutic payloads for cell-instructive biointerfaces.

Keywords:  drug delivery; layer-by-layer; particle surface; particle uptake; siRNA; transfection

DOI:  https://doi.org/10.1021/acsabm.2c00668
Expert Opin Drug Deliv. 2023 Jan 01. 1-13

Why mRNA-ionizable LNPs formulations are so short-lived: causes and way-out.

Anindita De, Young Tag Ko.

  INTRODUCTION: Messenger ribonucleic acid (mRNA) and small interfering RNA (siRNA) are biological molecules that can be heated, frozen, lyophilized, precipitated, or re-suspended without degradation. Currently, ionizable lipid nanoparticles (LNPs) are a promising approach for mRNA therapy. However, the long-term shelf-life stability of mRNA-ionizable LNPs is one of the open questions about their use and safety. At an acidic pH, ionizable lipids shield anionic mRNA. However, the stability of mRNA under storage conditions remains a mystery. Moreover, ionizable LNPs excipients also cause instability during long-term storage.AREA COVERED: This paper aims to illustrate why mRNA-ionizable LNPs have such a limited storage half-life. For the first time, we compile the tentative reasons for the short half-life and ultra-cold storage of mRNA-LNPs in the context of formulation excipients. The article also provided possible ways of prolonging the lifespan of mRNA-ionizable LNPs during long storage.
EXPERT OPINION: mRNA-ionizable LNPs are the future of genetic medicine. Current limitations of the formulation can be overcome by an advanced drying process or a whole new hybrid formulation strategy to extend the shelf life of mRNA-ionizable LNPs. A breakthrough technology may open up new research directions for producing thermostable and safe mRNA-ionizable LNPs at room temperature.

Keywords:  Ionizable LNPs; hybrid formulation; internal structure; mRNA hydrolytic degradation; shelf life stability

DOI:  https://doi.org/10.1080/17425247.2023.2162876
Methods Mol Biol. 2023 ;2606 159-170

The Delivery of ABE mRNA to the Adult Murine Liver by Lipid Nanoparticles (LNPs).

Dong Yang, Chun-Qing Song.

  A genetic disorder is a disease caused by an abnormal DNA sequence, and almost half of the known pathogenic monogenetic mutations are caused by G-to-A mutation (Landrum et al., Nucleic Acids Res 44:D862-868, 2016). Adenine base editors (ABE), developed from the CRISPR, hold the great promise to mediate the A-to-G transition in genomic DNA while not inducing DNA cleavage (Gaudelli et al., Nature 551:464-471, 2017). Additionally, lipid nanoparticles (LNPs), as a non-viral delivery, are able to deliver the ABE mRNAs and gRNA to the target tissues (Newby and Liu, Mol Ther 29:3107-3124, 2021). This chapter mainly introduces the production and LNP delivery of ABE mRNA and gRNA.

Keywords:  ABE mRNA; Delivery; LNPs; Murine liver

DOI:  https://doi.org/10.1007/978-1-0716-2879-9_12
Int J Pharm. 2022 Dec 28. pii: S0378-5173(22)01120-6. [Epub ahead of print]632 122565

Efficient delivery of VEGF-A mRNA for promoting diabetic wound healing via ionizable lipid nanoparticles.

Wenhui Zha, Ji Wang, Zongke Guo, Yanhao Zhang, Yang Wang, Shuo Dong, Chao Liu, Hanlei Xing, Xinsong Li.

  Diabetes is often accompanied by chronic non-healing wounds, and vascularendothelial growth factor A (VEGF-A) is crucial in the treatment of chronic diabetic wounds. However, the application of VEGF-A protein in clinic is limited due to poor absorption and short half-life of protein macromolecule. Herein, we employed an emerging protein replacement therapy by delivering VEGF-A mRNA into the body to express the desired protein to accelerate diabetic wound healing. Primarily, VEGF-A mRNA was synthesized by an in vitro transcription (IVT) method and encapsulated with an ionizable lipid-mediated nanoparticles (LNP) delivery system via a microfluidic method. The resultant LNP/VEGF-A mRNA were characterized by using dynamic light scattering (DLS) and transmission electron microscope(TEM). The nanoparticles have regular spherical morphology with an average particle size of 101.17 nm, a narrow polydispersity (PDI) of 0.17 and negative Zeta potential of -3.05 mV. The bioactivities of the nanoparticles formulation were evaluated against HUVEC cells through cell proliferation, migration and tube formation assays. It was found that the LNP/VEGF-A mRNA nanoparticles could promote endothelial cell proliferation. In addition, they exhibited successful mRNA delivery and high VEGF-A protein expression in vitro and in vivo by means of Western Blot assay and in vivo imaging system (IVIS). Finally, C57BL/6 diabetic mice model was established and intradermally treated with the LNP/VEGF-A mRNA nanoparticles. It was found that the LNP/VEGF-A mRNA treated wounds were almost healed after 14 days with an average wound area of 2.4 %, compared with the PBS group of 21.4 %. Apparently, the nanoparticles formulation was able to significantly expedite diabetic wound healing. The histological analysis containing H&E, Masson's trichrome staining and CD31 further confirmed the healing efficacy and low toxicity of the formulation. Taken together, the LNP/VEGF-A mRNA nanoparticles can be taken up by cells to express protein effectively and improve diabetic wound healing, which might have potential application in the treatment of chronic diabetic wounds as a protein replacement therapy.

Keywords:  Diabetic wound; Ionizable lipid nanoparticle; Protein replacement; VEGF-A mRNA

DOI:  https://doi.org/10.1016/j.ijpharm.2022.122565
Eur J Pharm Biopharm. 2023 Jan 02. pii: S0939-6411(22)00322-8. [Epub ahead of print]

Multivalent mannose-conjugated siRNA causes robust gene silencing in pancreatic macrophages in vivo.

Kazuto Yamazaki, Kenji Kubara, Yuta Suzuki, Taro Hihara, Daisuke Kurotaki, Tomohiko Tamura, Masashi Ito, Kappei Tsukahara.

  Nucleic acid therapeutics have been utilized for gene regulation, and their recent advancement has led to approval of novel drugs for liver-related disorders. However, systemic extrahepatic delivery remains challenging. Here, we report newly designed mannose-conjugated oligonucleotides for delivering oligonucleotides to macrophages by leveraging the mannose receptor, C-type 1 (MRC1, CD206), which is abundantly expressed in macrophages. We investigated the relationship between cellular uptake and multivalency (mono to tetra) of mannose ligands or linker length and selected a trivalent-mannose ligand. Trivalent-mannose (Man3)-conjugated siRNA induced concentration-dependent gene silencing in both human CD206-overexpressing cells and human macrophages in vitro. After subcutaneous injection into mice, we observed a high distribution of Man3-conjugated oligonucleotides in the liver and pancreata as well as cellular uptake into Kupffer cells and pancreatic macrophages. A single subcutaneous injection of Man3-conjugated siRNA (10 mg/kg) targeting β2-microglobulin (B2M) silenced B2m mRNA expression by ∼ 50% and decreased its protein levels in mouse pancreatic macrophages compared to those in saline-treated mice. Of note, multiple subcutaneous injections decreased B2m gene expression and B2M protein levels by ∼ 80% and ∼ 85%, respectively. These results show that mannose-conjugation with oligonucleotides is expected to help deliver oligonucleotides to macrophages and regulate gene expression in vivo, particularly in the pancreas.

Keywords:  CD206; Macrophages; Mannose ligands; Nucleic acid therapeutics; Pancreas

DOI:  https://doi.org/10.1016/j.ejpb.2022.12.017
J Phys Chem B. 2023 Jan 05.

Atomistic Insights into Organization of RNA-Loaded Lipid Nanoparticles.

Markéta Paloncýová, Martin Šrejber, Petra Čechová, Petra Kührová, Filip Zaoral, Michal Otyepka.

RNA-based therapies have shown promise in a wide range of applications, from cancer therapy, treatment of inherited diseases to vaccination. Encapsulation of RNA into ionizable lipid (IL) containing lipid nanoparticles (LNPs) has enabled its safe and targeted delivery. We present here the simulations of the self-assembly process of pH-sensitive RNA-carrying LNPs and their internal morphology. At low pH, the simulations confirm a lipid core encapsulating RNA in the hexagonal phase. Our all-atom and coarse-grained simulations show that an RNA molecule inside an LNP is protected from interactions with ions by being enveloped in the charged ILs. At neutral pH, representing the environment after LNP administration into human tissues, LNPs expelled most of the encapsulated RNA and water and formed separate bulk IL-rich and ordered the helper-lipid-rich phase. Helper lipids arranged themselves to be in contact with RNA or water. The presented models provide atomistic understanding of the LNP structure and open a way to investigate them in silico, varying the LNP composition or interacting with other biostructures aiming at increasing the efficiency of RNA-based medicine.

DOI: https://doi.org/10.1021/acs.jpcb.2c07671
Adv Healthc Mater. 2023 Jan 02. e2203082

High-Performance Delivery of CRISPRi System via Lipid-Polymer Hybrid Nanoparticles combined with Ultrasound-Mediated Microbubble Destruction for Tumor-Specific Gene Repression.

Yan Li, Pengying Wu, Mingting Zhu, Meiling Liang, Lei Zhang, Yujin Zong, Mingxi Wan.

  The dCas9-based CRISPR interference (CRISPRi) system efficiently silences genes without causing detectable off-target activity, thus showing great potential for the treatment of cancer at the transcriptional level. However, due to the large size of the commonly used CRISPRi system, effective delivery of the system has been a challenge that hinders its application in the clinic. Herein, a combination of pH-responsive lipid-polymer hybrid nanoparticles (PLPNs) and ultrasound-mediated microbubble destruction (UMMD) is used for the delivery of the CRISPRi system. The core-shell structure of PLPNs can effectively be loaded with the CRISPRi plasmid, increases the time spent in the circulating in vivo, and "actively target" cancer cells. Moreover, the combination of PLPNs with UMMD achieves a higher cellular uptake of the CRISPRi plasmid in vitro and retention in vivo. Furthermore, when PLPNs loaded with a CRISPRi plasmid that targets microRNA-10b (miR-10b) are used in combination with UMMD, it results in the effective repression of miR-10b in breast cancer, simultaneous disturbance of multiple cell migration and invasion-related signaling pathways, and a significant inhibition of lung metastasis. Thus, the established system presents a versatile, highly efficient, and safe strategy for delivery of the CRISPRi system both in vitro and in vivo. This article is protected by copyright. All rights reserved.

Keywords:  CRISPRi system; breast cancer; lipid-polymer hybrid nanoparticles; miR-10b; ultrasound-mediated microbubble destruction

DOI:  https://doi.org/10.1002/adhm.202203082
Biomater Adv. 2022 Dec 23. pii: S2772-9508(22)00544-1. [Epub ahead of print]145 213267

pH-responsive nanoparticles based on POEOMA-b-PDPA block copolymers for RNA encapsulation, protection and cell delivery.

Bruno Baptista, Andreia S R Oliveira, Patrícia Mendonça, Arménio C Serra, Jorge F J Coelho, Fani Sousa.

  The use of gene-based products, such as DNA or RNA, is increasingly being explored for various innovative therapies. However, the success of these strategies is highly dependent on the effective delivery of these biomolecules to target cells. Therefore, the development of pH-responsive nanoparticles comprises the creation of intelligent delivery systems with high therapeutic efficiency. In this work, the pH-responsiveness of the poly(2-(diisopropylamino)ethyl methacrylate)) (PDPA) block was investigated for the encapsulation and delivery of small RNAs (sRNA) to cancer cells. The pH responsiveness was dependent on the protonation profile of the tertiary amines of PDPA, which directly affected the electrostatic interactions established with RNA. Thus, block copolymers based on poly(oligo(ethylene oxide) methyl ether methacrylate) (POEOMA) and PDPA, POEOMA-b-PDPA, were synthesized by supplemental activator and reducing agent atom transfer radical polymerization (SARA ATRP). The structure of the block copolymers was characterized by size exclusion chromatography and 1H NMR spectroscopy. The copolymers allowed effective complexation of model sRNAs and a pre-miRNA with efficiencies of about 89 % and 91 %, respectively. The characterization by dynamic light scattering revealed that these systems had sizes between 76 and 1375 nm. It was also found that the morphology of the polyplexes depended on the pH, since the preparation at a pH lower than the pKa of the copolymers resulted in spherical but polydisperse particles, while higher pH values resulted in nanoparticles with more homogeneous size, but altered morphology. Moreover, due to pH-responsiveness, it was achieved the release of RNA at pH higher than the pKa of the copolymers, while maintaining its integrity. The polyplexes also showed a high potential to protect RNA from RNases. The transfection of a lung cancer model (A549) and fibroblast cell lines showed that these polyplexes did not cause cell toxicity. In addition, the polyplexes enabled the effective transfection of the A549 cell line with pre-miRNA-29b and miRNA-29b, resulting in a decrease of expression levels of the target DNMT3B gene by approximately 51 % and 47 %, respectively. Overall, the POEOMA-b-PDPA copolymers proved to be a promising strategy for developing responsive delivery systems, that can play a critical role in some diseases, such as cancer, where pH varies between the intra and extracellular environments.

Keywords:  POEOMA-b-PDPA; Pre-miRNA-29b; cancer; pH-responsive nanocarriers; sRNA

DOI:  https://doi.org/10.1016/j.bioadv.2022.213267
ACS Appl Mater Interfaces. 2022 Dec 28. 14(51): 56498-56509

Supramolecular Peptide Nanofiber/PLGA Nanocomposites for Enhancing Pulmonary Drug Delivery.

Uday Chintapula, Su Yang, Trinh Nguyen, Yang Li, Justyn Jaworski, He Dong, Kytai T Nguyen.

  Effective drug delivery to pulmonary sites will benefit from the design and synthesis of novel drug delivery systems that can overcome various tissue and cellular barriers. Cell penetrating peptides (CPPs) have shown promise for intracellular delivery of various imaging probes and therapeutics. Although CPPs improve delivery efficacy to a certain extent, they still lack the scope of engineering to improve the payload capacity and protect the payload from the physiological environment in drug delivery applications. Inspired by recent advances of CPPs and CPP-functionalized nanoparticles, in this work, we demonstrate a novel nanocomposite consisting of fiber-forming supramolecular CPPs that are coated onto polylactic-glycolic acid (PLGA) nanoparticles to enhance pulmonary drug delivery. These nanocomposites show a threefold higher intracellular delivery of nanoparticles in various cells including primary lung epithelial cells, macrophages, and a 10-fold increase in endothelial cells compared to naked PLGA nanoparticles or a twofold increase compared to nanoparticles modified with traditional monomeric CPPs. Cell uptake studies suggest that nanocomposites likely enter cells through mixed macropinocytosis and passive energy-independent mechanisms, which is followed by endosomal escape within 24 h. Nanocomposites also showed potent mucus permeation. More importantly, freeze-drying and nebulizing formulated nanocomposite powder did not affect their physiochemical and biological activity, which further highlights the translative potential for use as a stable drug carrier for pulmonary drug delivery. We expect nanocomposites based on peptide nanofibers, and PLGA nanoparticles can be custom designed to encapsulate and deliver a wide range of therapeutics including nucleic acids, proteins, and small-molecule drugs when employed in inhalable systems to treat various pulmonary diseases.

Keywords:  PLGA nanoparticles; endosomal escape; nanocomposites; nanofiber; peptide self-assembly; pulmonary delivery

DOI:  https://doi.org/10.1021/acsami.2c15204