{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,16]],"date-time":"2026-10-16T18:34:24Z","timestamp":1792175664033,"version":"4.4.0"},"reference-count":32,"publisher":"MDPI AG","issue":"5","license":[{"start":{"date-parts":[[2026,2,27]],"date-time":"2026-02-27T00:00:00Z","timestamp":1772150400000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"Program for Talents and Team of Science and Technology in University of Yunnan Province","award":["202105AE160010"],"award-info":[{"award-number":["202105AE160010"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["IJMS"],"abstract":"<jats:p>Lipid nanoparticles (LNPs) have been widely utilized as carriers for nucleic acid drug delivery; however, their inherent heterogeneity impedes the accurate characterization of physicochemical and biological properties. Conventional analytical methods are inherently limited in resolving such heterogeneity. This study employed sucrose density gradient centrifugation (S-DGC) to separate LNP subpopulations of varying densities. It investigated the effects of lipid formulation parameters\u2014including nitrogen-to-phosphorus (N\/P) ratio, lipid composition, polyethylene glycol (PEG) concentration\u2014and microfluidic preparation conditions (flow rate) on LNP heterogeneity and biological functionality. Formulation stability was assessed via freeze\u2013thaw testing. The results demonstrated that S-DGC could effectively separate LNP subpopulations with divergent densities and physicochemical characteristics. Changes in the N\/P ratio and lipid composition significantly modulate subphase distribution and properties. When cholesterol (Chol) and distearoylphosphatidylcholine (DSPC) are absent from the formulation, LNPs aggregate in the low-density layer (0\u201310% sucrose density layer). The concentration of PEGylated lipids serves as a critical regulatory factor. When the concentration increased from 0.5% to 2.5%, the LNP particle size decreased from approximately 202 nm to 118.7 nm. Furthermore, the S-DGC profile indicates that LNP transitions from an aggregated low-density distribution to a uniformly dense subpopulation concentrated within the 0\u201320% sucrose layer, where transfection efficiency is optimal. In freeze\u2013thaw stability assessment, unprotected LNP exhibited a drastic decline in encapsulation efficiency to 5.3% after three freeze\u2013thaw cycles at \u221280 \u00b0C. The S-DGC diagram revealed aggregation in the 20\u201330% high-density region. However, adding 5% sucrose maintained encapsulation efficiency above 96%. This study confirms that the S-DGC analytical platform serves as a potent tool for resolving LNP heterogeneity and correlating formulation structure with function. Based on these findings, this study contends that during the early prescription development of LNP-based nucleic acid therapeutics, formulation screening should not be confined to meeting overall particle size and encapsulation rate targets. Instead, S-DGC can be employed to proactively identify and minimize ineffective subpopulations (such as particles distributed in extremely high or low density zones), thereby enhancing product quality uniformity and predictability from the outset of R&amp;D.<\/jats:p>","DOI":"10.3390\/ijms27052239","type":"journal-article","created":{"date-parts":[[2026,2,27]],"date-time":"2026-02-27T15:52:38Z","timestamp":1772207558000},"page":"2239","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":3,"title":["Investigation of Heterogeneity of Lipid Nanoparticles for Nucleic Acid Drug Delivery via Sucrose Gradient Density Centrifugation"],"prefix":"10.3390","volume":"27","author":[{"given":"Yujia","family":"Hong","sequence":"first","affiliation":[{"name":"College of Pharmacy, Dali University, No. 2 Hongsheng Road, Dali 671003, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Guanghui","family":"Zi","sequence":"additional","affiliation":[{"name":"College of Pharmacy, Dali University, No. 2 Hongsheng Road, Dali 671003, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0004-7844-2391","authenticated-orcid":false,"given":"Baowei","family":"Peng","sequence":"additional","affiliation":[{"name":"College of Pharmacy, Dali University, No. 2 Hongsheng Road, Dali 671003, China"},{"name":"Yunnan Provincial Key Laboratory of Entomological Biopharmaceutical R&D, College of Pharmacy, Dali University, Xueren Road, Dali 671003, China"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2026,2,27]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"1078","DOI":"10.1038\/s41578-021-00358-0","article-title":"Lipid nanoparticles for mRNA delivery","volume":"6","author":"Hou","year":"2021","journal-title":"Nat. Rev. Mater."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"577","DOI":"10.1007\/s11262-024-02102-6","article-title":"Advances in the study of LNPs for mRNA delivery and clinical applications","volume":"60","author":"Wang","year":"2024","journal-title":"Virus Genes"},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"e202400938","DOI":"10.1002\/cmdc.202400938","article-title":"Advances in Lipid Nanoparticle-Based Disease Treatment","volume":"20","author":"Zhou","year":"2025","journal-title":"ChemMedChem"},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"739","DOI":"10.1038\/s41571-023-00811-9","article-title":"Targeting cancer with mRNA\u2013lipid nanoparticles: Key considerations and future prospects","volume":"20","author":"Kon","year":"2023","journal-title":"Nat. Rev. Clin. Oncol."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"115196","DOI":"10.1016\/j.addr.2024.115196","article-title":"Intranasal drug delivery: The interaction between nanoparticles and the nose-to-brain pathway","volume":"207","author":"Chen","year":"2024","journal-title":"Adv. Drug Deliv. Rev."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"117465","DOI":"10.1016\/j.biopha.2024.117465","article-title":"Innovative lipid nanoparticles: A cutting-edge approach for potential renal cell carcinoma therapeutics","volume":"180","author":"Shahzad","year":"2024","journal-title":"Biomed. Pharmacother."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"114990","DOI":"10.1016\/j.addr.2023.114990","article-title":"Lipid nanoparticles (LNPs) for in vivo RNA delivery and their breakthrough technology for future applications","volume":"200","author":"Jeong","year":"2023","journal-title":"Adv. Drug Deliv. Rev."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"e2303261","DOI":"10.1002\/adma.202303261","article-title":"Lipid Nanoparticle (LNP) Enables mRNA Delivery for Cancer Therapy","volume":"35","author":"Zong","year":"2023","journal-title":"Adv. Mater."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"114416","DOI":"10.1016\/j.addr.2022.114416","article-title":"The role of lipid components in lipid nanoparticles for vaccines and gene therapy","volume":"188","author":"Albertsen","year":"2022","journal-title":"Adv. Drug Deliv. Rev."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"37","DOI":"10.1007\/s11095-018-2566-3","article-title":"Application of a Quality-By-Design Approach to Optimise Lipid-Polymer Hybrid Nanoparticles Loaded with a Splice-Correction Antisense Oligonucleotide: Maximising Loading and Intracellular Delivery","volume":"36","author":"Thanki","year":"2019","journal-title":"Pharm. Res."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"103505","DOI":"10.1016\/j.drudis.2023.103505","article-title":"Design of lipid-based nanoparticles for delivery of therapeutic nucleic acids","volume":"28","author":"Mendonca","year":"2023","journal-title":"Drug Discov. Today"},{"key":"ref_12","doi-asserted-by":"crossref","unstructured":"Catenacci, L., Rossi, R., Sechi, F., Buonocore, D., Sorrenti, M., Perteghella, S., Peviani, M., and Bonferoni, M.C. (2024). Effect of Lipid Nanoparticle Physico-Chemical Properties and Composition on Their Interaction with the Immune System. Pharmaceutics, 16.","DOI":"10.3390\/pharmaceutics16121521"},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"397","DOI":"10.1021\/mp3005337","article-title":"Assessing the Heterogeneity Level in Lipid Nanoparticles for siRNA Delivery: Size-Based Separation, Compositional Heterogeneity, and Impact on Bioperformance","volume":"10","author":"Zhang","year":"2012","journal-title":"Mol. Pharm."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"15729","DOI":"10.1021\/acsnano.4c02341","article-title":"Single-Particle Spectroscopic Chromatography Reveals Heterogeneous RNA Loading and Size Correlations in Lipid Nanoparticles","volume":"18","author":"Li","year":"2024","journal-title":"ACS Nano"},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"4725","DOI":"10.1021\/la903616p","article-title":"Prolonging Density Gradient Stability","volume":"26","author":"Jerri","year":"2009","journal-title":"Langmuir"},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"5570","DOI":"10.1021\/acs.analchem.4c00031","article-title":"Analytical Characterization of Heterogeneities in mRNA-Lipid Nanoparticles Using Sucrose Density Gradient Ultracentrifugation","volume":"96","author":"Vaidya","year":"2024","journal-title":"Anal. Chem."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"3097","DOI":"10.1021\/acsami.4c20077","article-title":"Transfection Potency of Lipid Nanoparticles Containing mRNA Depends on Relative Loading Levels","volume":"17","author":"Liao","year":"2024","journal-title":"ACS Appl. Mater. Interfaces"},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"114819","DOI":"10.1016\/j.ejpb.2025.114819","article-title":"The effect of mRNA-lipid nanoparticle composition on stability during microneedle patch manufacturing","volume":"215","author":"Sakers","year":"2025","journal-title":"Eur. J. Pharm. Biopharm."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"2300006","DOI":"10.1002\/anbr.202300006","article-title":"Ionizable Lipid Nanoparticles for mRNA Delivery","volume":"3","author":"Tang","year":"2023","journal-title":"Adv. NanoBiomed Res."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"114726","DOI":"10.1016\/j.ejpb.2025.114726","article-title":"From in vitro to in vivo: The Dominant role of PEG-Lipids in LNP performance","volume":"212","author":"Borah","year":"2025","journal-title":"Eur. J. Pharm. Biopharm."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"1059","DOI":"10.1002\/mabi.201300046","article-title":"The Effect of N\/P Ratio on the In Vitro and In Vivo Interaction Properties of PEGylated Poly [2-(dimethylamino)ethyl methacrylate]-Based siRNA Complexes","volume":"13","author":"Gary","year":"2013","journal-title":"Macromol. Biosci."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"21733","DOI":"10.1039\/C9NR09347H","article-title":"On the role of helper lipids in lipid nanoparticle formulations of siRNA","volume":"11","author":"Kulkarni","year":"2019","journal-title":"Nanoscale"},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"80","DOI":"10.1016\/j.jconrel.2022.02.017","article-title":"Microfluidic technologies and devices for lipid nanoparticle-based RNA delivery","volume":"344","author":"Maeki","year":"2022","journal-title":"J. Control. Release"},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"119792","DOI":"10.1016\/j.ijpharm.2020.119792","article-title":"PEG shedding-rate-dependent blood clearance of PEGylated lipid nanoparticles in mice: Faster PEG shedding attenuates anti-PEG IgM production","volume":"588","author":"Suzuki","year":"2020","journal-title":"Int. J. Pharm."},{"key":"ref_25","doi-asserted-by":"crossref","unstructured":"Liu, W., Zhang, M., Lv, H., and Yang, C. (2025). Formulation-Driven Optimization of PEG-Lipid Content in Lipid Nanoparticles for Enhanced mRNA Delivery In Vitro and In Vivo. Pharmaceutics, 17.","DOI":"10.3390\/pharmaceutics17080950"},{"key":"ref_26","doi-asserted-by":"crossref","unstructured":"Uddin, M.N., and Roni, M.A. (2021). Challenges of Storage and Stability of mRNA-Based COVID-19 Vaccines. Vaccines, 9.","DOI":"10.3390\/vaccines9091033"},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"120586","DOI":"10.1016\/j.ijpharm.2021.120586","article-title":"mRNA-lipid nanoparticle COVID-19 vaccines: Structure and stability","volume":"601","author":"Schoenmaker","year":"2021","journal-title":"Int. J. Pharm."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"172240","DOI":"10.1016\/j.scitotenv.2024.172240","article-title":"Impact of physicochemical properties on biological effects of lipid nanoparticles: Are they completely safe","volume":"927","author":"Yuan","year":"2024","journal-title":"Sci. Total. Environ."},{"key":"ref_29","doi-asserted-by":"crossref","unstructured":"Kim, Y., Park, J., Choi, J., Kim, M., Seo, G., Kim, J., Park, J.A., Lim, K.S., Ha, S.J., and Kim, H.O. (2025). Physiological Barriers to Nucleic Acid Therapeutics and Engineering Strategies for Lipid Na-noparticle Design, Optimization, and Clinical Translation. Pharmaceutics, 17.","DOI":"10.3390\/pharmaceutics17101309"},{"key":"ref_30","doi-asserted-by":"crossref","unstructured":"Aldosari, B.N., Alfagih, I.M., and Almurshedi, A.S. (2021). Lipid Nanoparticles as Delivery Systems for RNA-Based Vaccines. Pharmaceutics, 13.","DOI":"10.3390\/pharmaceutics13020206"},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"11329","DOI":"10.1039\/D5NR00433K","article-title":"PEGylated lipid screening, composition optimization, and structure\u2013activity relationship determination for lipid nanoparticle-mediated mRNA delivery","volume":"17","author":"Liu","year":"2025","journal-title":"Nanoscale"},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"195","DOI":"10.1089\/nat.2019.0784","article-title":"Formulation of Biocompatible Targeted ECO\/siRNA Nanoparticles with Long-Term Stability for Clinical Translation of RNAi","volume":"29","author":"Ayat","year":"2019","journal-title":"Nucleic Acid Ther."}],"container-title":["International Journal of Molecular Sciences"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1422-0067\/27\/5\/2239\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,2,27]],"date-time":"2026-02-27T15:53:04Z","timestamp":1772207584000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1422-0067\/27\/5\/2239"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,2,27]]},"references-count":32,"journal-issue":{"issue":"5","published-online":{"date-parts":[[2026,3]]}},"alternative-id":["ijms27052239"],"URL":"https:\/\/doi.org\/10.3390\/ijms27052239","relation":{},"ISSN":["1422-0067"],"issn-type":[{"value":"1422-0067","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,2,27]]}}}