bims-novged Biomed News
on Non-viral vectors for gene delivery
Issue of 2024‒04‒14
eleven papers selected by
the Merkel lab, Ludwig-Maximilians University
  1. Ionizable Lipids from Click Reactions for Lipid Nanoparticle Assembling and mRNA Delivery.
  2. Identification of film-based formulations that move mRNA lipid nanoparticles out of the freezer.
  3. Co-delivery of Cas9 mRNA and guide RNAs for editing of LGMN gene represses breast cancer cell metastasis.
  4. Simple Scattering: Lipid nanoparticle structural data repository.
  5. Inflammatory-Targeted Lipid Carrier as a New Nanomaterial to Formulate an Inhaled Drug Delivery System.
  6. Escape sites from the endo-lysosomal trafficking route manipulate exocytosis of nanoparticles in polar epithelium.
  7. High Charge Density in Peptide Dendrimers is Required to Destabilize Membranes: Insights into Endosome Evasion.
  8. Regulatory Insights into Nanomedicine and Gene Vaccine Innovation: Safety Assessment, Challenges, and Regulatory Perspectives.
  9. Molecular modeling of the carbohydrate corona formation on a polyvinyl chloride nanoparticle and its impact on the adhesion to lipid bilayers.
  10. Anti-EGFR ScFv functionalized exosomes delivering LPCAT1 specific siRNAs for inhibition of lung cancer brain metastases.
  11. A Scalable Microfluidic Platform for Nanoparticle Formulation: For Exploratory- and Industrial-Level Scales.
  1. J Phys Chem B. 2024 Apr 08.
    Ionizable Lipids from Click Reactions for Lipid Nanoparticle Assembling and mRNA Delivery.
    Feiyang Xu, Xiao Si, Yixiang Wang, Cheng Sun, Minglun Liu, Yan Zhang, Xiaoyu Xu, Tian Tian.
      Ionizable lipid-containing lipid nanoparticles (LNPs) are regarded as promising nonviral vectors for gene therapy delivery systems. Rationale design of the ionizable lipid structure based on initial screening of ionizable lipid molecule libraries combined with systematic comparison and analysis on the physical chemical parameters related to delivery efficiency greatly accelerated the discovery of novel LNP candidates for delivering various nucleic acid therapeutics like mRNAs (mRNAs). Based on the copper-catalyzed azide-alkyne click reaction, which is highly efficient and biocompatible, we were able to obtain the lipid molecule library containing a common triazole moiety between different lipid tails and various substituents as hydrophilic head groups. Herein, we systematically investigated the change of pKa values of different ionizable lipid molecules with different substituents as head groups in the click-based lipid library, mapping the pKa value change to different steps in the process of the LNP assembly and mRNA delivery. Systematic analyses on the data including the pKa value of the ionized lipids and the encapsulation and delivery efficiency of mRNA in LNPs with these ionized lipids provided the possibility of rational design on the head and tail structure for the triazole containing ionized lipids to realize highly efficient delivery of different mRNAs.
    DOI:  https://doi.org/10.1021/acs.jpcb.3c07600
  2. Mol Ther Nucleic Acids. 2024 Jun 11. 35(2): 102179
    Identification of film-based formulations that move mRNA lipid nanoparticles out of the freezer.
    Trang Nguyen Kieu Doan, Madison M Davis, Maria A Croyle.
      COVID-19 vaccines consisting of mRNA lipid nanoparticles (LNPs) encoding the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein antigen protected millions of people from severe disease; however, they must be stored frozen prior to use. The objective of this study was to evaluate the compatibility and stability of mRNA LNPs within a polymer-based film matrix. An optimized formulation of polymer base, glycerol, surfactants, and PEGylated lipid that prevents damage to the LNP due to physical changes during the film-forming process (osmotic stress, surface tension, spatial stress, and water loss) was identified. Surfactants added to LNP stock prior to mixing with other film components contributed to this effect. Formulations prepared at pH ≥ 8.5 extended transfection efficiency beyond 4 weeks at 4°C when combined with known nucleic acid stabilizers. mRNA LNPs were most stable in films when manufactured in an environment of ∼50% relative humidity. The optimized formulation offers 16-week stability at 4°C.
    Keywords:  LNP; MT: Delivery Strategies; film; humidity; lipid nanoparticle; mRNA; storage; temperature; thermostability; vaccines
    DOI:  https://doi.org/10.1016/j.omtn.2024.102179
  3. Sci Rep. 2024 04 06. 14(1): 8095
    Co-delivery of Cas9 mRNA and guide RNAs for editing of LGMN gene represses breast cancer cell metastasis.
    Yue Wang, Yatu Peng, Guanghui Zi, Jin Chen, Baowei Peng.
      Legumain (or asparagine endopeptidase/AEP) is a lysosomal cysteine endopeptidase associated with increased invasive and migratory behavior in a variety of cancers. In this study, co-delivery of Cas9 mRNA and guide RNA (gRNA) by lipid nanoparticles (LNP) for editing of LGMN gene was performed. For in-vitro transcription (IVT) of gRNA, two templates were designed: linearized pUC57-T7-gRNA and T7-gRNA oligos, and the effectiveness of gRNA was verified in multiple ways. Cas9 plasmid was modified and optimized for IVT of Cas9 mRNA. The effects of LGMN gene editing on lysosomal/autophagic function and cancer cell metastasis were investigated. Co-delivery of Cas9 mRNA and gRNA resulted in impaired lysosomal/autophagic degradation, clone formation, migration, and invasion capacity of cancer cells in-vitro. Experimental lung metastasis experiment indicates co-delivery of Cas9 mRNA and gRNA by LNP reduced the migration and invasion capacity of cancer cells in-vivo. These results indicate that co-delivery of Cas9 mRNA and gRNA can enhance the efficiency of CRISPR/Cas9-mediated gene editing in-vitro and in-vivo, and suggest that Cas9 mRNA and gRNA gene editing of LGMN may be a potential treatment for breast tumor metastasis.
    DOI:  https://doi.org/10.1038/s41598-024-58765-6
  4. Front Mol Biosci. 2024 ;11 1321364
    Simple Scattering: Lipid nanoparticle structural data repository.
    Lee Joon Kim, David Shin, Wellington C Leite, Hugh O'Neill, Oliver Ruebel, Andrew Tritt, Greg L Hura.
      Lipid nanoparticles (LNPs) are being intensively researched and developed to leverage their ability to safely and effectively deliver therapeutics. To achieve optimal therapeutic delivery, a comprehensive understanding of the relationship between formulation, structure, and efficacy is critical. However, the vast chemical space involved in the production of LNPs and the resulting structural complexity make the structure to function relationship challenging to assess and predict. New components and formulation procedures, which provide new opportunities for the use of LNPs, would be best identified and optimized using high-throughput characterization methods. Recently, a high-throughput workflow, consisting of automated mixing, small-angle X-ray scattering (SAXS), and cellular assays, demonstrated a link between formulation, internal structure, and efficacy for a library of LNPs. As SAXS data can be rapidly collected, the stage is set for the collection of thousands of SAXS profiles from a myriad of LNP formulations. In addition, correlated LNP small-angle neutron scattering (SANS) datasets, where components are systematically deuterated for additional contrast inside, provide complementary structural information. The centralization of SAXS and SANS datasets from LNPs, with appropriate, standardized metadata describing formulation parameters, into a data repository will provide valuable guidance for the formulation of LNPs with desired properties. To this end, we introduce Simple Scattering, an easy-to-use, open data repository for storing and sharing groups of correlated scattering profiles obtained from LNP screening experiments. Here, we discuss the current state of the repository, including limitations and upcoming changes, and our vision towards future usage in developing our collective knowledge base of LNPs.
    Keywords:  SAS; database; lipid nanoparticles; small-angle scattering; structure-activity relationship; vaccines; web application
    DOI:  https://doi.org/10.3389/fmolb.2024.1321364
  5. Molecules. 2024 Apr 03. pii: 1616. [Epub ahead of print]29(7):
    Inflammatory-Targeted Lipid Carrier as a New Nanomaterial to Formulate an Inhaled Drug Delivery System.
    Eleonora Maretti, Federica Gioia, Cecilia Rustichelli, Susanna Molinari, Eliana Leo.
      There is a pressing need for efficacious therapies in the field of respiratory diseases and infections. Lipid nanocarriers, administered through aerosols, represent a promising tool for maximizing therapeutic concentration in targeted cells and minimizing systemic exposure. However, this approach requires the application of efficient and safe nanomaterials. Palmitoylethanolamide (PEA), an endocannabinoid-like endogenous lipid, plays a crucial role in providing protective mechanisms during inflammation, making it an interesting material for preparing inhalable lipid nanoparticles (LNPs). This report aims to preliminarily explore the in vitro behavior of LNPs prepared with PEA (PEA-LNPs), a new inhalable inflammatory-targeted nanoparticulate drug carrier. PEA-LNPs exhibited a size of about 250 nm, a rounded shape, and an marked improvement in PEA solubility in comparison to naked PEA, indicative of easily disassembled nanoparticles. A twin glass impinger instrument was used to screen the aerosol performance of PEA-LNP powders, obtained via freeze-drying in the presence of two quantities of mannose as a cryoprotectant. Results indicated that a higher amount of mannose improved the emitted dose (ED), and in particular, the fine particle fraction (FPF). A cytotoxicity assay was performed and indicated that PEA-LNPs are not toxic towards the MH-S alveolar macrophage cell line up to concentrations of 0.64 mg/mL, and using coumarin-6 labelled particles, a rapid internalization into the macrophage was confirmed. This study demonstrates that PEA could represent a suitable material for preparing inhalable lipid nanocarrier-based dry powders, which signify a promising tool for the transport of drugs employed to treat respiratory diseases and infections.
    Keywords:  DPI; PEA; Palmitoylethanolamide; aerosol; dry powder; lung disease; nanocarrier; nanoparticles; pulmonary administration; respirability
    DOI:  https://doi.org/10.3390/molecules29071616
  6. Biomater Sci. 2024 Apr 09.
    Escape sites from the endo-lysosomal trafficking route manipulate exocytosis of nanoparticles in polar epithelium.
    Lingling Wang, Yuting Li, Xi Liu, Liyun Xing, Ruinan Wu, Yuan Huang.
      The endo-lysosomal pathway is a major barrier for the trans-epithelial transport of nanoparticles (NPs), but escape strategies could facilitate trans-epithelial delivery. Based on the polarization properties of the epithelium, different escape compartments may result in different exocytosis fates of NPs and further affect the delivery efficiency. Therefore, optimizing the escape sites is critical for trans-epithelial delivery. Here, commonly used PEG-coated-poly(lactic-co-glycolic acid) (PLGA)-based nanoparticles were fabricated as model nanoparticles (MNPs) and the intestinal epithelium was chosen as the polarized epithelium. The MNPs were incubated with different endosomolytic agents for early endosomal escape, late endosomal escape and lysosomal escape, respectively. According to in vitro and in vivo studies, MNPs escaping from early endosomes and late endosomes exhibited stronger capacity for trans-epithelial transport than those escaping from lysosomes. By further probing into the mechanism, we surprisingly found that although MNPs escaping from early endosomes quickly egressed from the apical side of epithelia, they were subsequently followed by "reuptake" via caveolae and trafficked through the endoplasmic reticulum-Golgi apparatus (ER/GA) secretory pathway, achieving efficient trans-epithelial transport; MNPs escaping from late endosomes, which were located near the nucleus, were prone to enter the ER/GA for efficient basolateral exocytosis. However, MNPs escaping from lysosomes were detained within cells by autophagosomes. Collectively, our research suggested that early endosomes and late endosomes were ideal escape sites for trans-epithelial delivery.
    DOI:  https://doi.org/10.1039/d4bm00174e
  7. J Chem Inf Model. 2024 Apr 08.
    High Charge Density in Peptide Dendrimers is Required to Destabilize Membranes: Insights into Endosome Evasion.
    Filipe E P Rodrigues, Tamis Darbre, Miguel Machuqueiro.
      Peptide dendrimers are a type of branched, symmetric, and topologically well-defined molecule that have already been used as delivery systems for nucleic acid transfection. Several of the most promising sequences showed high efficiency in many key steps of transfection, namely, binding siRNA, entering cells, and evading the endosome. However, small changes to the peptide dendrimers, such as in the hydrophobic core, the amino acid chirality, or the total available charges, led to significantly different experimental results with unclear mechanistic insights. In this work, we built a computational model of several of those peptide dendrimers (MH18, MH13, and MH47) and some of their variants to study the molecular details of the structure and function of these molecules. We performed CpHMD simulations in the aqueous phase and in interaction with a lipid bilayer to assess how conformation and protonation are affected by pH in different environments. We found that while the different peptide dendrimer sequences lead to no substantial structural differences in the aqueous phase, the total charge and, more importantly, the total charge density are key for the capacity of the dendrimer to interact and destabilize the membrane. These dendrimers become highly charged when the pH changes from 7.5 to 4.5, and the presence of a high charge density, which is decreased for MH47 that has four fewer titratable lysines, is essential to trigger membrane destabilization. These findings are in excellent agreement with the experimental data and help us to understand the high efficiency of some dendrimers and why the dendrimer MH47 is unable to complete the transfection process. This evidence provides further understanding of the mode of action of these peptide dendrimers and will be pivotal for the future design of new sequences with improved transfection capabilities.
    DOI:  https://doi.org/10.1021/acs.jcim.4c00018
  8. Acta Biomater. 2024 Apr 09. pii: S1742-7061(24)00183-1. [Epub ahead of print]
    Regulatory Insights into Nanomedicine and Gene Vaccine Innovation: Safety Assessment, Challenges, and Regulatory Perspectives.
    Eliana B Souto, Cristina Blanco-Llamero, Karolline Krambeck, Neelakanta Sarvashiva Kiran, Chandrashekar Yashaswini, Humzah Postwala, Patricia Severino, Ronny Priefer, Bhupendra Gopalbhai Prajapati, Rahul Maheshwari.
      This analysis explores the principal regulatory concerns linked to nanomedicines and gene vaccines, including the complexities involved and the perspectives on how to navigate them. In the realm of nanomedicines, ensuring the safety of nanomaterials is paramount due to their unique characteristics and potential interactions with biological systems. Regulatory bodies are actively formulating guidelines and standards to assess the safety and risks associated with nanomedicine products, emphasizing the need for standardized characterization techniques to accurately gauge their safety and effectiveness. Regarding gene vaccines, regulatory frameworks must be tailored to address the distinct challenges posed by genetic interventions, necessitating special considerations in safety and efficacy evaluations, particularly concerning vector design, target specificity, and long-term patient monitoring. Ethical concerns such as patient autonomy, informed consent, and privacy also demand careful attention, alongside the intricate matter of intellectual property rights, which must be balanced against the imperative of ensuring widespread access to these life-saving treatments. Collaborative efforts among regulatory bodies, researchers, patent offices, and the private sector are essential to tackle these challenges effectively, with international cooperation being especially crucial given the global scope of nanomedicine and genetic vaccine development. Striking the right balance between safeguarding intellectual properties and promoting public health is vital for fostering innovation and ensuring equitable access to these ground-breaking technologies, underscoring the significance of addressing these regulatory hurdles to fully harness the potential benefits of nanomedicine and gene vaccines for enhancing healthcare outcomes on a global scale. STATEMENT OF SIGNIFICANCE: Several biomaterials are being proposed for the development of nanovaccines, from polymeric micelles, PLGA-/PEI-/PLL-nanoparticles, solid lipid nananoparticles, cationic lipoplexes, liposomes, hybrid materials, dendrimers, carbon nanotubes, hydrogels, to quantum dots. Lipid nanoparticles (LNPs) have gained tremendous attention since the US Food and Drug Administration (FDA) approval of Pfizer and Moderna's COVID-19 vaccines, raising public awareness to the regulatory challenges associated with nanomedicines and genetic vaccines. This review provides insights into the current perspectives and potential strategies for addressing these issues, including clinical trials. By navigating these regulatory landscapes effectively, we can unlock the full potential of nanomedicine and genetic vaccines using a range of promising biomaterials towards improving healthcare outcomes worldwide.
    Keywords:  Ethical considerations; Gene vaccines; Intellectual property rights; Nanomedicines; Regulatory issues; Safety assessment
    DOI:  https://doi.org/10.1016/j.actbio.2024.04.010
  9. J Chem Phys. 2024 Apr 14. pii: 144901. [Epub ahead of print]160(14):
    Molecular modeling of the carbohydrate corona formation on a polyvinyl chloride nanoparticle and its impact on the adhesion to lipid bilayers.
    Daniel G Angelescu.
      The pervasive presence of nanoplastics (NPs) in the environment has gained increasing attention due to their accumulation in living organisms. These emerging contaminants inevitably interact with extracellular polymeric substances along respiratory or gastrointestinal tracts, and diverse organic coating on the surface of NPs, known as bio- or eco-corona, is formed. Although its impact on altering the NP properties and potential cell internalization has been extensively examined, studies on its role in NP partitioning in the cell membrane are elusive yet. In this work, molecular dynamics is used to investigate the formation of chitosan (CT) corona centered on a polyvinyl chloride (PVC) nanoparticle and the uptake of the resulting complex onto lipid membranes. Coarse-grained models compatible with the newly developed Martini 3.0 force field are implemented for the two polymers employing the atomistic properties as targets in the parameterization. The reliability of the coarse-grained polymer models is demonstrated by reproducing the structural properties of the PVC melt and of solvated CT strands, as well as by determining the conformation adopted by the latter at the NP surface. Results show that the spontaneous binding of CT chains of high and intermediate protonation degrees led to the formation of soft and hard corona that modulates the interaction of PVC core with model membranes. The structural changes of the corona adsorbed at the lipid-water interface enable a subsequent transfer of the NP to the center of the saturated lipid membranes and a complete or partial transition to a snorkel conformation depending on the hydrophilic/hydrophobic balance in the CT-PVC complex. Overall, the computational investigation of the coarse-grained model system provides implications for understanding how the eco-corona development influences the uptake and implicit toxicology of NPs.
    DOI:  https://doi.org/10.1063/5.0198254
  10. J Nanobiotechnology. 2024 Apr 08. 22(1): 159
    Anti-EGFR ScFv functionalized exosomes delivering LPCAT1 specific siRNAs for inhibition of lung cancer brain metastases.
    Jun Jiang, Yuan Lu, Jie Chu, Xiao Zhang, Chao Xu, Shaojie Liu, Zhuo Wan, Jiawei Wang, Lu Zhang, Kui Liu, Zhenhua Liu, Angang Yang, Xinling Ren, Rui Zhang.
      Brain metastasis (BM) is one of the leading causes of cancer-related deaths in patients with advanced non-small cell lung cancer (NSCLC). However, limited treatments are available due to the presence of the blood-brain barrier (BBB). Upregulation of lysophosphatidylcholine acyltransferase 1 (LPCAT1) in NSCLC has been found to promote BM. Conversely, downregulating LPCAT1 significantly suppresses the proliferation and metastasis of lung cancer cells. In this study, we firstly confirmed significant upregulation of LPCAT1 in BM sites compared to primary lung cancer by analyzing scRNA dataset. We then designed a delivery system based on a single-chain variable fragment (scFv) targeting the epidermal growth factor receptor (EGFR) and exosomes derived from HEK293T cells to enhance cell-targeting capabilities and increase permeability. Next, we loaded LPCAT1 siRNA (siLPCAT1) into these engineered exosomes (exoscFv). This novel scFv-mounted exosome successfully crossed the BBB in an animal model and delivered siLPCAT1 to the BM site. Silencing LPCAT1 efficiently arrested tumor growth and inhibited malignant progression of BM in vivo without detectable toxicity. Overall, we provided a potential platform based on exosomes for RNA interference (RNAi) therapy in lung cancer BM.
    Keywords:  Blood-brain barrier; Brain metastasis; Drug delivery; Engineered exosomes; Lung cancer
    DOI:  https://doi.org/10.1186/s12951-024-02414-7
  11. Nano Lett. 2024 Apr 08.
    A Scalable Microfluidic Platform for Nanoparticle Formulation: For Exploratory- and Industrial-Level Scales.
    Islam Seder, Tao Zheng, Jing Zhang, César Cruz Rojas, Seyed Hossein Helalat, Rodrigo Coronel Téllez, Yi Sun.
      Nanoparticle synthesis on microfluidic platforms provides excellent reproducibility and control over bulk synthesis. While there have been plenty of platforms for producing nanoparticles (NPs) with controlled physicochemical properties, such platforms often operate in a narrow range of predefined flow rates. The flow rate limitation restricts either up-scalability for industrial production or down-scalability for exploratory research use. Here, we present a universal flow rate platform that operates over a wide range of flow rates (0.1-75 mL/min) for small-scale exploratory research and industrial-level synthesis of NPs without compromising the mixing capabilities. The wide range of flow rate is obtained by using a coaxial flow with a triangular microstructure to create a vortex regardless of the flow regime (Reynolds number). The chip synthesizes several types of NPs for gene and protein delivery, including polyplex, lipid NPs, and solid polymer NPs via self-assembly and precipitation, and successfully expresses GFP plasmid DNA in human T cells.
    Keywords:  lipid nanoparticle; nanoparticle synthesis; polyplex; scalable microfluidic chip
    DOI:  https://doi.org/10.1021/acs.nanolett.3c05057
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