bims-novged Biomed News
on Non-viral vectors for gene delivery
Issue of 2024–05–19
ten papers selected by
the Merkel lab, Ludwig-Maximilians University
  1. Electrophoretic Microfluidic Characterization of mRNA- and pDNA-Loaded Lipid Nanoparticles.
  2. Accelerating ionizable lipid discovery for mRNA delivery using machine learning and combinatorial chemistry.
  3. Polymer design via SHAP and Bayesian machine learning optimizes pDNA and CRISPR ribonucleoprotein delivery.
  4. Impact of Nebulization on the Physicochemical Properties of Polymer-Lipid Hybrid Nanoparticles for Pulmonary Drug Delivery.
  5. Targeted gene delivery systems for T-cell engineering.
  6. Effective delivery of anti-PD-L1 siRNA with human heavy chain ferritin (HFn) in acute myeloid leukemia cell lines.
  7. Stimuli-sensitive Chitosan-based Nanosystems-immobilized Nucleic Acids for Gene Therapy in Breast Cancer and Hepatocellular Carcinoma.
  8. Advances in Microfluidic-based Core@Shell Nanoparticles Fabrication for Cancer Applications.
  9. Gemcitabine-Phospholipid Complex Loaded Lipid Nanoparticles for Improving Drug Loading, Stability, and Efficacy against Pancreatic Cancer.
  10. Inhalable spray-dried porous microparticles containing dehydroandrographolide succinate phospholipid complex capable of improving and prolonging pulmonary anti-inflammatory efficacy in mice.
  1. ACS Appl Mater Interfaces. 2024 May 16.
    Electrophoretic Microfluidic Characterization of mRNA- and pDNA-Loaded Lipid Nanoparticles.
    Adriana Coll De Peña, Daniel Zimmer, Everett Gutterman-Johns, Nicole M Chen, Anubhav Tripathi, Christina M Bailey-Hytholt.
      Lipid nanoparticles (LNPs) are clinically advanced nonviral gene delivery vehicles with a demonstrated ability to address viral, oncological, and genetic diseases. However, the further development of LNP therapies requires rapid analytical techniques to support their development and manufacturing. The method developed and described in this paper presents an approach to rapidly and accurately analyze LNPs for optimized therapeutic loading by utilizing an electrophoresis microfluidic platform to analyze the composition of LNPs with different clinical lipid compositions (Onpattro, Comirnaty, and Spikevax) and nucleic acid (plasmid DNA (pDNA) and messenger RNA (mRNA)) formulations. This method enables the high-throughput screening of LNPs using a 96- or 384-well plate with approximate times of 2-4 min per sample using a total volume of 11 μL. The lipid analysis requires concentrations approximately between 109 and 1010 particles/mL and has an average precision error of 10.4% and a prediction error of 19.1% when compared to using a NanoSight, while the nucleic acid analysis requires low concentrations of 1.17 ng/μL for pDNA and 0.17 ng/μL for mRNA and has an average precision error of 4.8% and a prediction error of 9.4% when compared to using a PicoGreen and RiboGreen assay. In addition, our method quantifies the relative concentration of nucleic acid per LNP. Utilizing this approach, we observed an average of 263 ± 62.2 mRNA per LNP and 126.3 ± 21.2 pDNA per LNP for the LNP formulations used in this study, where the accuracy of these estimations is dependent on reference standards. We foresee the utility of this technique in the high-throughput characterization of LNPs during manufacturing and formulation research and development.
    Keywords:  electrophoresis; lipid nanoparticles; messenger RNA; microfluidics; nucleic acid encapsulation; plasmid DNA
    DOI:  https://doi.org/10.1021/acsami.4c00208
  2. Nat Mater. 2024 May 13.
    Accelerating ionizable lipid discovery for mRNA delivery using machine learning and combinatorial chemistry.
    Bowen Li, Idris O Raji, Akiva G R Gordon, Lizhuang Sun, Theresa M Raimondo, Favour A Oladimeji, Allen Y Jiang, Andrew Varley, Robert S Langer, Daniel G Anderson.
      To unlock the full promise of messenger (mRNA) therapies, expanding the toolkit of lipid nanoparticles is paramount. However, a pivotal component of lipid nanoparticle development that remains a bottleneck is identifying new ionizable lipids. Here we describe an accelerated approach to discovering effective ionizable lipids for mRNA delivery that combines machine learning with advanced combinatorial chemistry tools. Starting from a simple four-component reaction platform, we create a chemically diverse library of 584 ionizable lipids. We screen the mRNA transfection potencies of lipid nanoparticles containing those lipids and use the data as a foundational dataset for training various machine learning models. We choose the best-performing model to probe an expansive virtual library of 40,000 lipids, synthesizing and experimentally evaluating the top 16 lipids flagged. We identify lipid 119-23, which outperforms established benchmark lipids in transfecting muscle and immune cells in several tissues. This approach facilitates the creation and evaluation of versatile ionizable lipid libraries, advancing the formulation of lipid nanoparticles for precise mRNA delivery.
    DOI:  https://doi.org/10.1038/s41563-024-01867-3
  3. Chem Sci. 2024 May 15. 15(19): 7219-7228
    Polymer design via SHAP and Bayesian machine learning optimizes pDNA and CRISPR ribonucleoprotein delivery.
    Rishad J Dalal, Felipe Oviedo, Michael C Leyden, Theresa M Reineke.
      We present the facile synthesis of a clickable polymer library with systematic variations in length, binary composition, pKa, and hydrophobicity (clog P) to optimize intracellular pDNA and CRISPR-Cas9 ribonucleoprotein (RNP) performance. We couple physicochemical characterization and machine learning to interpret quantitative structure-property relationships within the combinatorial design space. For the first time, we reveal unexpected disparate design parameters for nucleic acid carriers; via explainable machine learning on 432 formulations, we discover that lower polymer pKa and higher percentages of benzimidazole ethanethiol enhance pDNA delivery, yet polymer length and captamine cation identity improve RNP delivery. Closed-loop Bayesian optimization of 552 formulation ratios further enhances in vitro performance. The top three polymers yield a higher signal and stable transgene expression over 20 days in vivo, and a 1.7-fold enhancement over controls. Our facile coupling of synthesis, characterization, and machine analysis provides powerful tools to quantitate performance parameters accelerating next-generation vehicles for nucleic acid medicines.
    DOI:  https://doi.org/10.1039/d3sc06920f
  4. Int J Mol Sci. 2024 May 05. pii: 5028. [Epub ahead of print]25(9):
    Impact of Nebulization on the Physicochemical Properties of Polymer-Lipid Hybrid Nanoparticles for Pulmonary Drug Delivery.
    Andrea Gonsalves, Jyothi U Menon.
      Nanoparticles (NPs) have shown significant potential for pulmonary administration of therapeutics for the treatment of chronic lung diseases in a localized and sustained manner. Nebulization is a suitable method of NP delivery, particularly in patients whose ability to breathe is impaired due to lung diseases. However, there are limited studies evaluating the physicochemical properties of NPs after they are passed through a nebulizer. High shear stress generated during nebulization could potentially affect the surface properties of NPs, resulting in the loss of encapsulated drugs and alteration in the release kinetics. Herein, we thoroughly examined the physicochemical properties as well as the therapeutic effectiveness of Infasurf lung surfactant (IFS)-coated PLGA NPs previously developed by us after passing through a commercial Aeroneb® vibrating-mesh nebulizer. Nebulization did not alter the size, surface charge, IFS coating and bi-phasic release pattern exhibited by the NPs. However, there was a temporary reduction in the initial release of encapsulated therapeutics in the nebulized compared to non-nebulized NPs. This underscores the importance of evaluating the drug release kinetics of NPs using the inhalation method of choice to ensure suitability for the intended medical application. The cellular uptake studies demonstrated that both nebulized and non-nebulized NPs were less readily taken up by alveolar macrophages compared to lung cancer cells, confirming the IFS coating retention. Overall, nebulization did not significantly compromise the physicochemical properties as well as therapeutic efficacy of the prepared nanotherapeutics.
    Keywords:  Aeroneb; aerosol; biomimetic; cancer; lung surfactant; macrophage; nebulizer; pulmonary drug delivery; vibrating-mesh nebulizer
    DOI:  https://doi.org/10.3390/ijms25095028
  5. Cell Oncol (Dordr). 2024 May 16.
    Targeted gene delivery systems for T-cell engineering.
    Fengling Wang, Yong Huang, JiaQian Li, Weilin Zhou, Wei Wang.
      T lymphocytes are indispensable for the host systems of defense against pathogens, tumors, and environmental threats. The therapeutic potential of harnessing the cytotoxic properties of T lymphocytes for antigen-specific cell elimination is both evident and efficacious. Genetically engineered T-cells, such as those employed in CAR-T and TCR-T cell therapies, have demonstrated significant clinical benefits in treating cancer and autoimmune disorders. However, the current landscape of T-cell genetic engineering is dominated by strategies that necessitate in vitro T-cell isolation and modification, which introduce complexity and prolong the development timeline of T-cell based immunotherapies. This review explores the complexities of gene delivery systems designed for T cells, covering both viral and nonviral vectors. Viral vectors are known for their high transduction efficiency, yet they face significant limitations, such as potential immunogenicity and the complexities involved in large-scale production. Nonviral vectors, conversely, offer a safer profile and the potential for scalable manufacturing, yet they often struggle with lower transduction efficiency. The pursuit of gene delivery systems that can achieve targeted gene transfer to T cell without the need for isolation represents a significant advancement in the field. This review assesses the design principles and current research progress of such systems, highlighting the potential for in vivo gene modification therapies that could revolutionize T-cell based treatments. By providing a comprehensive analysis of these systems, we aim to contribute valuable insights into the future development of T-cell immunotherapy.
    Keywords:  Nonviral vector; T-cell based immunotherapy; Targeted gene delivery system; Viral vector
    DOI:  https://doi.org/10.1007/s13402-024-00954-6
  6. Med Oncol. 2024 May 13. 41(6): 149
    Effective delivery of anti-PD-L1 siRNA with human heavy chain ferritin (HFn) in acute myeloid leukemia cell lines.
    Misagh Rajabinejad, Reza Valadan, Mohsen Tehrani, Ahmad Najafi, Reza Negarandeh, Majid Saeedi, Hossein Asgarian-Omran.
      Because of the high biocompatibility, self-assembly capability, and CD71-mediated endocytosis, using human heavy chain ferritin (HFn) as a nanocarrier would greatly increase therapeutic effectiveness and reduce possible adverse events. Anti-PD-L1 siRNA can downregulate the level of PD-L1 on tumor cells, resulting in the activation of effector T cells against leukemia. Therefore, this study aimed to produce the tumor-targeting siPD-L1/HFn nanocarrier. Briefly, the HFn coding sequence was cloned into a pET-28a, and the constructed expression plasmid was subsequently transformed into E. coli BL21. After induction of Isopropyl β-D-1-thiogalactopyranoside (IPTG), HFn was purified with Ni-affinity chromatography and dialyzed against PBS. The protein characteristics were analyzed using SDS-PAGE, Western Blot, and Dynamic light scattering (DLS). The final concentration was assessed using the Bicinchoninic acid (BCA) assay. The encapsulation was performed using the standard pH system. The treatment effects of siPD-L1/HFn were carried out on HL-60 and K-562 cancer cell lines. The RT-PCR was used to determine the mRNA expression of PD-L1. The biocompatibility and excretion of siPD-L1/HFn have also been evaluated. The expression and purity of HFn were well verified through SDS-PAGE, WB, and DLS. RT-PCR analyses also showed significant siRNA-mediated PD-L1 silencing in both HL-60 and K-562 cells. Our study suggested a promising approach for siRNA delivery. This efficient delivery system can pave the way for the co-delivery of siRNAs and multiple chemotherapies to address the emerging needs of cancer combination therapy.
    Keywords:  Human heavy chain ferritin; Nanoparticle; PD-L1; siRNA
    DOI:  https://doi.org/10.1007/s12032-024-02393-7
  7. Curr Top Med Chem. 2024 May 14.
    Stimuli-sensitive Chitosan-based Nanosystems-immobilized Nucleic Acids for Gene Therapy in Breast Cancer and Hepatocellular Carcinoma.
    Seyed Morteza Naghib, Bahar Ahmadi, M R Mozafari.
      Chitosan-based nanoparticles have emerged as a promising tool in the realm of cancer therapy, particularly for gene delivery. With cancer being a prevalent and devastating disease, finding effective treatment options is of utmost importance. These nanoparticles provide a unique solution by encapsulating specific genes and delivering them directly to cancer cells, offering immense potential for targeted therapy. The biocompatibility and biodegradability of chitosan, a naturally derived polymer, make it an ideal candidate for this purpose. The nanoparticles protect the genetic material during transportation and enhance its cellular uptake, ensuring effective delivery to the site of action. Furthermore, the unique properties of chitosan-based nanoparticles allow for the controlled release of genes, maximizing their therapeutic effect while minimizing adverse effects. By advancing the field of gene therapy through the use of chitosan-based nanoparticles, scientists are making significant strides toward more humane and personalized treatments for cancer patients.
    Keywords:  Breast cancer; Chitosan; DNA; Gene delivery; Hepatocellular carcinoma.; Nanoparticle; RNA
    DOI:  https://doi.org/10.2174/0115680266293173240506054439
  8. Adv Healthc Mater. 2024 May 12. e2400946
    Advances in Microfluidic-based Core@Shell Nanoparticles Fabrication for Cancer Applications.
    Duarte R S Almeida, João Ferreira Gil, Antonio José Guillot, Jiachen Li, Ricardo J B Pinto, Hélder A Santos, Gil Gonçalves.
      Current research in cancer therapy focuses on personalized therapies, through nanotechnology-based targeted drug delivery systems. Particularly, controlled drug release with core-shell nanoparticles can be designed to safely transport various active agents, optimizing delivery to specific organs and tumors, minimizing side effects. The use of microfluidics in this field has stood out against conventional methods by allowing precise control over parameters like size, structure, composition, and mechanical/biological properties of nanoscale carriers. This review compiles applications of microfluidics in the production of core-shell nanoparticles for cancer therapy, discussing the versatility inherent in various microchannel and/or micromixer setups and showcasing how these setups can be utilized individually or in combination, as well as how this technology allowed the development of new advances in more efficient and controlled fabrication of core-shell nanoformulations. Recent biological studies have achieved an effective, safe and controlled delivery of otherwise unreliable encapsulants such as siRNA, pDNA, and cisplatin as a result of precisely tuned fabrication of nanocarriers, showing that this technology is paving the way for innovative strategies in cancer therapy nanofabrication, characterized by continuous production and high reproducibility. Finally, this review analyses the technical, biological, and technological limitations that currently prevent this technology from becoming the standard. This article is protected by copyright. All rights reserved.
    Keywords:  multiphasic nanoparticles; nanomedicine; theragnostic microfluidics; tumor targeting
    DOI:  https://doi.org/10.1002/adhm.202400946
  9. Mol Pharm. 2024 May 15.
    Gemcitabine-Phospholipid Complex Loaded Lipid Nanoparticles for Improving Drug Loading, Stability, and Efficacy against Pancreatic Cancer.
    Chander Parkash Dora, Varun Kushwah, Vivek Yadav, Kaushik Kuche, Sanyog Jain.
      This study aims to encapsulate gemcitabine (GEM) using a phospholipid complex (PLC) in lipid nanoparticles (NPs) to achieve several desirable outcomes, including high drug loading, uniform particle size, improved therapeutic efficacy, and reduced toxicities. The successful preparation of GEM-loaded lipid NPs (GEM-NPs) was accomplished using the emulsification-solidification method, following optimization through Box-Behnken design. The size of the GEM-NP was 138.5 ± 6.7 nm, with a low polydispersity index of 0.282 ± 0.078, as measured by a zetasizer and confirmed by transmission electron and atomic force microscopy. GEM-NPs demonstrated sustained release behavior, surpassing the performance of the free GEM and phospholipid complex. Moreover, GEM-NPs exhibited enhanced cytotoxicity, apoptosis, and cell uptake in Panc-2 and Mia PaCa cells compared to the free GEM. The in vivo pharmacokinetics revealed approximately 4-fold higher bioavailability of GEM-NPs in comparison with free GEM. Additionally, the pharmacodynamic evaluation conducted in a DMBA-induced pancreatic cancer model, involving histological examination, serum IL-6 level estimation, and expression of cleaved caspase-3, showed the potential of GEM-NPs in the management of pancreatic cancer. Consequently, the lipid NP-based approach developed in our investigation demonstrates high stability and uniformity and holds promise for enhancing the therapeutic outcomes of GEM.
    Keywords:  TPGS; gemcitabine; lipid nanoparticles; pancreatic cancer; phospholipid complex
    DOI:  https://doi.org/10.1021/acs.molpharmaceut.3c00983
  10. Drug Deliv Transl Res. 2024 May 17.
    Inhalable spray-dried porous microparticles containing dehydroandrographolide succinate phospholipid complex capable of improving and prolonging pulmonary anti-inflammatory efficacy in mice.
    Wei-Ya Chen, Jia-Xing Wei, Chen-Yang Yu, Chun-Yu Liu, Yong-Hong Liao.
      Due to the unique physiological barriers within the lungs, there are considerable challenges in developing drug delivery systems enabling prolonged drug exposure to respiratory epithelial cells. Here, we report a PulmoSphere-based dry powder technology that incorporates a drug-phospholipid complex to promote intracellular retention of dehydroandrographolide succinate (DAS) in respiratory epithelial cells following pulmonary delivery. The DAS-phospholipid complex has the ability to self-assemble into nanoparticles. After spray-drying to produce PulmoSphere microparticles loaded with the drug-phospholipid complex, the rehydrated microparticles discharge the phospholipid complex without altering its physicochemical properties. The microparticles containing the DAS-phospholipid complex exhibit remarkable aerodynamic properties with a fine particle fraction of ∼ 60% and a mass median aerodynamic diameter of ∼ 2.3 μm. These properties facilitate deposition in the alveolar region. In vitro cell culture and lung tissue explants experiments reveal that the drug-phospholipid complex prolongs intracellular residence time and lung tissue retention due to the slow intracellular disassociation of drug from the complex. Once deposited in the lungs, the DAS-phospholipid complex loaded microparticles increase and extend drug exposure to the lung tissues and the immune cells compared to the free DAS counterpart. The improved drug exposure to airway epithelial cells, but not immune cells, is related to a prolonged duration of pulmonary anti-inflammation at decreased doses in a mouse model of acute lung injury induced by lipopolysaccharide. Overall, the phospholipid complex loaded microparticles present a promising approach for improved treatment of respiratory diseases, e.g. pneumonia and acute respiratory distress syndrome.
    Keywords:  Acute lung injury; Lung retention; Phospholipid complex; PulmoSphere microparticles; Pulmonary delivery; Sustained release
    DOI:  https://doi.org/10.1007/s13346-024-01626-6
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