Delineating effect of headgroup and preparation method on transfection versus toxicity of DNA-loaded lipid nanocarriers
Abstract
Aim: To perform a parallel comparison of key parameters affecting the safety and efficiency of lipid-based nanovectors (i.e., complexing headgroups, composition and preparation method). Materials & methods: Various cationic and ionizable headgroups were screened for formulating lipoplexes with GFP–plasmid DNA. Ethanol injection and microfluidics were used to prepare nanoparticles with GFP–plasmid DNA complexed on the surface or within the interior of lipid bilayers. Results: Lipoplexes composed of sphingomyelin 102 exhibited the highest transfection efficiency given their higher cellular uptake in BRAF inhibitor-resistant melanoma cells. Lipid nanoparticles demonstrated acceptable transfection efficiency and high spheroid penetration while protecting plasmid DNA under simulated physiological conditions. Conclusion: Selecting the right complexing lipid and preparation method is critical for developing lipid nanocarriers to treat intractable diseases.
Plain language summary
Certain genetic diseases or cancers can be treated with gene therapy. This involves the delivery of working genes to cells with a faulty copy. These genes are often contained in a circular piece of DNA called a plasmid. Plasmids can be delivered by a variety of structures called vectors. These vectors include altered viruses as well as lipid-based nanovectors, which are nanoscale spheres of phospholipids, a type of fat. Plasmids can either be internalized inside these spheres in lipid nanoparticles (LNPs) or attached to the surface in nanocomplexes. A section of the phospholipid called the headgroup faces outward in lipid-based nanovectors. The chemical makeup of these headgroups determines the function of the lipid-based nanovector. This study aimed to determine an optimal lipid-based nanovector in gene delivery by testing a variety and identifying which was most effective at delivering a gene that makes cells fluoresce green when successfully delivered. The more intense the fluorescence, the greater the degree of successful gene delivery. This study found that LNPs were more effective at delivering plasmid DNA than nanocomplexes and were safer and protected plasmid DNA better. The best performing LNP contained the lipid sphingomyelin 102, which is biodegradable. Therefore, the optimized LNP formulation employing sphingomyelin 102 as a biodegradable lipid could be an efficient nonviral gene-delivery system and will be further investigated to target drug-resistant melanoma, a type of skin cancer.
Tweetable abstract
Optimized ionizable ‘state-of-the-art’ LNPs displayed high transfection efficiency, 3D tumor penetration and systemic safety of entrapped pDNA compared with lipoplexes and will be explored to target drug-resistant melanoma. #LNPs #Microfluidics #Lipoplexes #PlasmidDNADelivery #NonViralGeneTherapy
Graphical abstract
Papers of special note have been highlighted as: • of interest; •• of considerable interest
References
- 1. . A review of the tortuous path of nonviral gene delivery and recent progress. Int. J. Biol. Macromol. 183, 2055–2073 (2021).
- 2. Microfluidic formulation of DNA-loaded multicomponent lipid nanoparticles for gene delivery. Pharmaceutics 13(8), 1292 (2021).
- 3. . Clinical progress of nanomedicine-based RNA therapies. Bioact. Mater. 12, 203–213 (2022).
- 4. . Lipid-based nanoparticles in the clinic and clinical trials: from cancer nanomedicine to COVID-19 vaccines. Vaccines (Basel) 9(4), 359 (2021).
- 5. CRISPR/Cas9 genome editing for tissue-specific in vivo targeting: nanomaterials and translational perspective. Adv. Sci. (Weinh.) 10(19), 2207512 (2023).
- 6. . Structure and function of cationic and ionizable lipids for nucleic acid delivery. Pharm. Res. 40(1), 27–46 (2023). •• Demonstrates the importance of type of cationic or ionizable lipid for efficient complexation of nucleic acids for enhanced cellular uptake, endosomal escape and successful delivery in target cells.
- 7. . Deciphering the functional composition of fusogenic liposomes. Int. J. Mol. Sci. 19(2), 346 (2018).
- 8. . Arginoplexes: an arginine-anchored nanoliposomal carrier for gene delivery. J. Nanopart. Res. 16, 1–10 (2014).
- 9. . DNA pre-condensation with an amino acid-based cationic amphiphile. A viable approach for liposome-based gene delivery. Mol. Membr. Biol. 25(1), 23–34 (2008).
- 10. An ionizable lipid toolbox for RNA delivery. Nat. Commun. 12(1), 7233 (2021). • Provides critical attributes of various novel generations of ionizable lipids with improved transfection efficiency, endosomal escape capability and biodegradability of resulting lipid nanoparticles (LNPs).
- 11. . The role of lipid components in lipid nanoparticles for vaccines and gene therapy. Adv. Drug Deliv. Rev. 188, 114416 (2022).
- 12. . Recent advancements in lipid–mRNA nanoparticles as a treatment option for cancer immunotherapy. J. Pharm. Investig. 52(4), 415–426 (2022).
- 13. PTEN loss confers BRAF inhibitor resistance to melanoma cells through the suppression of BIM expression. Cancer Res. 71(7), 2750–2760 (2011).
- 14. PTEN loss-of-function alterations are associated with intrinsic resistance to BRAF inhibitors in metastatic melanoma. JCO Precis. Oncol. 1, 1–15 (2017).
- 15. . BRD4 PROTAC as a novel therapeutic approach for the treatment of vemurafenib resistant melanoma: preformulation studies, formulation development and in vitro evaluation. Eur. J. Pharm. Sci. 138, 105039 (2019).
- 16. Development of dual ARV-825 and nintedanib-loaded PEGylated nano-liposomes for synergistic efficacy in vemurafnib-resistant melanoma. Pharmaceutics 13(7), 1005 (2021).
- 17. . A novel gene therapy for neurodegenerative Lafora disease via EPM2A-loaded DLinDMA lipoplexes. Nanomedicine (Lond.) 16(13), 1081–1095 (2021).
- 18. . Galactose-decorated liver tumor-specific nanoliposomes incorporating selective BRD4-targeted PROTAC for hepatocellular carcinoma therapy. Heliyon 8(1), e08702 (2022).
- 19. . Development and optimization of stealth liposomal system for enhanced in vitro cytotoxic effect of quercetin. J. Drug Deliv. Sci. Technol. 55, 101477 (2020).
- 20. . Aerosolized nanoliposomal carrier of remdesivir: an effective alternative for COVID-19 treatment in vitro. Nanomedicine (Lond.) 16(14), 1187–1202 (2021).
- 21. . Nanoformulation of proteolysis targeting chimera targeting ‘undruggable’ c-Myc for the treatment of pancreatic cancer. Nanomedicine (Lond.) 15(18), 1761–1777 (2020).
- 22. . Susceptibility of lung carcinoma cells to nanostructured lipid carrier of ARV-825, a BRD4 degrading proteolysis targeting chimera. Pharm. Res. 39(11), 2745–2759 (2022).
- 23. Ionization and structural properties of mRNA lipid nanoparticles influence expression in intramuscular and intravascular administration. Commun. Biol. 4(1), 956 (2021).
- 24. . Non-viral delivery of nucleic acids: insight into mechanisms of overcoming intracellular barriers. Front. Pharmacol. 9, 971 (2018).
- 25. . The importance of apparent pKa in the development of nanoparticles encapsulating siRNA and mRNA. Trends Pharmacol. Sci. 42(6), 448–460 (2021). •• Proves that the pH sensitivity of ionizable lipids is dependent on the acid dissociation constant (pKa) of the headgroup and plays a vital role in facilitating the bilayer-destabilizing capacity and relative endosomal escape potential of lipid nanocarriers encapsulating nucleic acids.
- 26. . Toxicity of cationic lipids and cationic polymers in gene delivery. J. Control. Rel. 114(1), 100–109 (2006).
- 27. . Protective effect of edaravone against cationic lipid-mediated oxidative stress and apoptosis. Biol. Pharm. Bull. 44(1), 144–149 (2021).
- 28. . Biodegradable cationic and ionizable cationic lipids: a roadmap for safer pharmaceutical excipients. Small 19(17), e2206968 (2023). • Shows that the presence of ester bonds in the hydrophobic chains of biodegradable lipids like SM-102 could result in safer formulations for nucleic acid delivery given the rapid elimination due to enzymatic cleavage of ester bonds in vivo.
- 29. . Evaluation of cellular uptake and intracellular trafficking as determining factors of gene expression for amino acid-substituted gemini surfactant-based DNA nanoparticles. J. Nanobiotechnol. 10, 7 (2012). •• Reveals the essential role of cellular uptake in regulating the number of particles internalized in target cells for enhanced endosomal escape and efficient gene expression.
- 30. . Inside out: optimization of lipid nanoparticle formulations for exterior complexation and in vivo delivery of saRNA. Gene Ther. 26(9), 363–372 (2019).
- 31. Comparison of physicochemical properties of lipoparticles as mRNA carrier prepared by automated microfluidic system and bulk method. Pharmaceutics 14(6), 1297 (2022).
- 32. . Optimization of lipid nanoparticles for saRNA expression and cellular activation using a design-of-experiment approach. Mol. Pharm. 19(6), 1892–1905 (2022).
- 33. . Role of cellular retention and intracellular state in controlling gene delivery efficiency of multiple nonviral carriers. ACS Omega 4(24), 20547–20557 (2019).
- 34. Hyper-branched cationic cyclodextrin polymers for improving plasmid transfection in 2D and 3D spheroid cells. Pharmaceutics 14(12), 2690 (2022).
- 35. Head-to-head comparison of the penetration efficiency of lipid-based nanoparticles into tumor spheroids. ACS Omega 5(33), 21162–21171 (2020). • Characterizes the potential of multicellular 3D spheroids for mimicking the penetration barriers seen in vivo to study the uptake and transfection efficiency of nonviral gene-delivery carriers.
- 36. Penetration and uptake of nanoparticles in 3D tumor spheroids. Bioconjug. Chem. 30(5), 1371–1384 (2019).
- 37. Lipid composition dictates serum stability of reconstituted high-density lipoproteins: implications for in vivo applications. Nanoscale 10(16), 7420–7430 (2018). •• Indicates that the stability of nucleic acid-loaded nanoparticles in a physiologically relevant milieu has direct relevance to their in vivo applications.
- 38. Effect of the nanoformulation of siRNA–lipid assemblies on their cellular uptake and immune stimulation. Int. J. Nanomed. 12, 5121 (2017). • Proves that the siRNA uptake efficiency and systemic safety of LNPs are considerably higher than those of lipoplexes.
