Formulation of protein-loaded nanoparticles via freeze-drying.

Publisher: Springer Country of Publication: United States NLM ID: 101540061 Publication Model: Print-Electronic Cited Medium: Internet ISSN: 2190-3948 (Electronic) Linking ISSN: 2190393X NLM ISO Abbreviation: Drug Deliv Transl Res Subsets: MEDLINE

Original Publication: New York : Springer

Freeze Drying* ; Nanoparticles*/chemistry ; Nanoparticles*/administration & dosage ; Insulin*/chemistry ; Insulin*/administration & dosage ; Sucrose*/chemistry ; Sucrose*/analogs & derivatives; Animals ; Zinc/chemistry ; Zinc/administration & dosage ; Administration, Oral ; Mannitol/chemistry ; Particle Size ; Humans ; Drug Stability ; Male ; Drug Compounding/methods

Several nanotechnology-based formulation strategies have been reported for the oral administration of biological drugs. However, a prerequisite often overlooked in developing these formulations is their adaptation to a solid dosage form. This study aimed to incorporate a freeze-drying step, using either mannitol or sucrose laurate (SLAE), into the formulation of new insulin-zinc nanocomplexes to render them resistant to intestinal fluids while maintaining a high protein loading. The resulting freeze-dried insulin-zinc nanocomplexes exhibited physicochemical properties consistent with the target product profile, including a particle size of ∼ 100 nm, a zeta potential close to neutrality (∼ -15 mV) and a high association efficiency (> 90%). Importantly, integrating the freeze-drying step in the formulation significantly improved the colloidal stability of the system and preserved the stability of the insulin molecules. Results from in vitro and in vivo studies indicated that the insulin activity remained fully retained throughout the entire formulation and freeze-drying processes. In brief, we present a novel protein formulation strategy that incorporates a critical freeze-drying step, resulting in a dry powder enabling efficient protein complexation with zinc and optimized for oral administration.
(© 2024. Controlled Release Society.)

Zelikin AN, Ehrhardt C, Healy AM. Materials and methods for delivery of biological drugs. Nat Chem [Internet]. 2016;8:997–1007.  https://doi.org/10.1038/nchem.2629 .
Byrne J, Huang HW, McRae JC, Babaee S, Soltani A, Becker SL, et al. Devices for drug delivery in the gastrointestinal tract: a review of systems physically interacting with the mucosa for enhanced delivery. Adv Drug Deliv Rev [Internet]. 2021;177: 113926. https://doi.org/10.1016/j.addr.2021.113926 . (PMID: 10.1016/j.addr.2021.11392634403749)
Brown TD, Whitehead KA, Mitragotri S. Materials for oral delivery of proteins and peptides. Nat Rev Mater [Internet]. 2020;5:127–48.  https://doi.org/10.1038/s41578-019-0156-6 .
Wu J, Zheng Y, Liu M, Shan W, Zhang Z, Huang Y. Biomimetic viruslike and charge reversible nanoparticles to sequentially overcome mucus and epithelial barriers for oral insulin delivery. ACS Appl Mater Interfaces [Internet]. 2018;10:9916–28.  https://doi.org/10.1021/acsami.7b16524 .
Ji N, Hong Y, Gu Z, Cheng L, Li Z, Li C. Chitosan coating of zein-carboxymethylated short-chain amylose nanocomposites improves oral bioavailability of insulin in vitro and in vivo. JCR [Internet]. 2019;313:1–13.  https://doi.org/10.1016/j.jconrel.2019.10.006 .
Wang A, Yang T, Fan W, Yang Y, Zhu Q, Guo S, et al. Protein corona liposomes achieve efficient oral insulin delivery by overcoming mucus and epithelial barriers. Adv Healthc Mater [Internet]. 2019;8:1–11.
Xu Y, De Keersmaecker H, Braeckmans K, De Smedt S, Cani PD, Préat V, et al. Targeted nanoparticles towards increased L cell stimulation as a strategy to improve oral peptide delivery in incretin-based diabetes treatment. Biomaterials [Internet]. 2020;255: 120209. https://doi.org/10.1016/j.biomaterials.2020.120209 . (PMID: 10.1016/j.biomaterials.2020.120209325800987116363)
Samaridou E, Kalamidas N, Santalices I, Crecente-Campo J, Alonso MJ. Tuning the PEG surface density of the PEG-PGA enveloped Octaarginine-peptide nanocomplexes. Drug Deliv Transl Res [Internet]. 2020;10:241–58.  https://doi.org/10.1007/s13346-019-00678-3 .
Niu Z, Samaridou E, Jaumain E, Coëne J, Ullio G, Shrestha N et al. PEG-PGA enveloped octaarginine-peptide nanocomplexes: an oral peptide delivery strategy. JCR [Internet]. 2018;276:125–39.  https://doi.org/10.1016/j.jconrel.2018.03.004 .
Han X, Lu Y, Xie J, Zhang E, Zhu H, Du H, et al. Zwitterionic micelles efficiently deliver oral insulin without opening tight junctions. Nat Nanotechnol [Internet]. 2020;15:605–14. https://doi.org/10.1038/s41565-020-0693-6 . (PMID: 10.1038/s41565-020-0693-6324833197534179)
Zhou Y, Chen Z, Zhao D, Li D, He C, Chen X. A pH-triggered self-unpacking capsule containing zwitterionic hydrogel-coated MOF nanoparticles for efficient oral Exendin-4 delivery. Adv Mater [Internet]. 2021;33:1–10.
Thwala LN, Delgado DP, Leone K, Marigo I, Benetti F, Chenlo M, et al. Protamine nanocapsules as carriers for oral peptide delivery. JCR [Internet]. 2018;291:157–68.
Niu Z, Tedesco E, Benetti F, Mabondzo A, Montagner IMIM, Marigo I, et al. Rational design of polyarginine nanocapsules intended to help peptides overcoming intestinal barriers. JCR [Internet]. 2017;263:4–17.
Lakkireddy HR, Urmann M, Besenius M, Werner U, Haack T, Brun P, et al. Oral delivery of diabetes peptides — comparing standard formulations incorporating functional excipients and nanotechnologies in the translational context. Adv Drug Deliv Rev [Internet]. 2016;106:196–222. https://doi.org/10.1016/j.addr.2016.02.011 . (PMID: 10.1016/j.addr.2016.02.01126964477)
Trenkenschuh E, Friess W. Freeze-drying of nanoparticles: how to overcome colloidal instability by formulation and process optimization. Eur J Pharm Biopharm [Internet]. 2021;165:345–60. https://doi.org/10.1016/j.ejpb.2021.05.024 . (PMID: 10.1016/j.ejpb.2021.05.02434052428)
Mohammady M, mohammadi Y, Yousefi G. Freeze-drying of pharmaceutical and nutraceutical nanoparticles: the effects of formulation and technique parameters on nanoparticles characteristics. J Pharm Sci [Internet]. 2020;109:3235–47.  https://doi.org/10.1016/j.xphs.2020.07.015 .
Fonte P, Reis S, Sarmento B. Facts and evidences on the lyophilization of polymeric nanoparticles for drug delivery. JCR [Internet]. 2016;225:75–86.  https://doi.org/10.1016/j.jconrel.2016.01.034 .
Abdelwahed W, Degobert G, Stainmesse S, Fessi H. Freeze-drying of nanoparticles: Formulation, process and storage considerations. Adv Drug Deliv Rev [Internet]. 2006;58:1688–713. https://linkinghub.elsevier.com/retrieve/pii/S0169409X06001840 .
De Jaeghere F, Allémann E, Feijen J, Kissel T, Doelker E, Gurny R. Freeze-drying and lyopreservation of diblock and triblock Poly(Lactic Acid)–Poly(Ethylene Oxide) (PLA–PEO) copolymer nanoparticles. Pharm Dev Technol [Internet]. 2000;5:473–83. Available from: http://www.tandfonline.com/doi/full/10.1081/PDT-100102031 .
Kesenci K, Motta A, Fambri L, Migliaresi C. Poly(ε-caprolactone-co-D, L-lactide)/silk fibroin composite materials: preparation and characterization. J Biomater Sci Polym Ed [Internet]. 2001;12:337–51.  https://doi.org/10.1163/156856201750180852 .
Jeong Y-I, Shim Y-H, Kim C, Lim G-T, Choi K-C, Yoon C. Effect of cryoprotectants on the reconstitution of surfactant-free nanoparticles of poly(DL-lactide-co-glycolide). J Microencapsul [Internet]. 2005;22:593–601.  https://doi.org/10.1080/02652040500162659 .
Fonte P, Andrade F, Azevedo C, Pinto J, Seabra V, van de Weert M, et al. Effect of the freezing step in the stability and bioactivity of protein-loaded PLGA nanoparticles upon lyophilization. Pharm Res [Internet]. 2016;33:2777–93. https://doi.org/10.1007/s11095-016-2004-3 . (PMID: 10.1007/s11095-016-2004-327444681)
Meulewaeter S, Nuytten G, Cheng MHY, De Smedt SC, Cullis PR, De Beer T, et al. Continuous freeze-drying of messenger RNA lipid nanoparticles enables storage at higher temperatures. JCR [Internet]. 2023;357:149–60.  https://doi.org/10.1016/j.jconrel.2023.03.039 .
Santalices I, Gonella A, Torres D, Alonso MJ. Advances on the formulation of proteins using nanotechnologies. J Drug Deliv Sci Technol [Internet]. 2017;42:155–80.  https://doi.org/10.1016/j.jddst.2017.06.018 .
Brayden DJ, Alonso M-J. Oral delivery of peptides: opportunities and issues for translation. Adv Drug Deliv Rev [Internet]. 2016;106:193–5.  https://doi.org/10.1016/j.addr.2016.10.005 .
Cázares-Delgadillo J, Naik A, Kalia YN, Quintanar-Guerrero D, Ganem-Quintanar A. Skin permeation enhancement by sucrose esters: a pH-dependent phenomenon. Int J Pharm [Internet]. 2005;297:204–12. https://doi.org/10.1016/j.ijpharm.2005.03.020 .
Li Y, Li J, Zhang X, Ding J, Mao S. Non-ionic surfactants as novel intranasal absorption enhancers: in vitro and in vivo characterization. Drug Deliv [Internet]. 2016;23:2272–9.  https://doi.org/10.3109/10717544.2014.971196 .
Guo Y, Baldelli A, Singh A, Fathordoobady F, Kitts D, Pratap-Singh A. Production of high loading insulin nanoparticles suitable for oral delivery by spray drying and freeze drying techniques. Sci Rep [Internet]. 2022;12:9949.  https://doi.org/10.1038/s41598-022-13092-6 .
Yamagata YIO. Sustained-release preparation containing a metal salt of a peptide. Patent. 2000. U.S. Patent No. 6,045,834.
Hallas-Møller K, Petersen K, Schlichtkrull J. Crystalline and amorphous insulin-zinc compounds with prolonged action. Science (1979) [Internet]. 1952;116:394–8.  https://doi.org/10.1126/science.116.3015.394 .
Thwala LN, Beloqui A, Csaba NS, González-Touceda D, Tovar S, Dieguez C, et al. The interaction of protamine nanocapsules with the intestinal epithelium: A mechanistic approach. JCR [Internet]. 2016;243:109–20. Available from: http://www.sciencedirect.com/science/article/pii/S0168365916309026#f0015 .
Santalices I, Vázquez-Vázquez C, Santander-Ortega MJ, Lozano V, Araújo F, Sarmento B, et al. A nanoemulsion/micelles mixed nanosystem for the oral administration of hydrophobically modified insulin. Drug Deliv Transl Res [Internet]. 2021;11:524–45. https://doi.org/10.1007/s13346-021-00920-x . (PMID: 10.1007/s13346-021-00920-x33575972)
McConnell EL, Basit AW, Murdan S. Measurements of rat and mouse gastrointestinal pH, fluid and lymphoid tissue, and implications for in-vivo experiments. JPP [Internet]. 2010;60:63–70.  https://doi.org/10.1211/jpp.60.1.0008 .
Harris IR, Höppner H, Siefken W, Wittern K-P, Farrell AM. Regulation of HMG-CoA synthase and HMG-CoA reductase by insulin and epidermal growth factor in HaCaT keratinocytes. JID [Internet]. 2000;114:83–7.  https://linkinghub.elsevier.com/retrieve/pii/S0022202X15407353 . (PMID: 10.1046/j.1523-1747.2000.00822.x)
Garcia-Rendueles AR, Rodrigues JS, Garcia-Rendueles MER, Suarez-Fariña M, Perez-Romero S, Barreiro F et al. Rewiring of the apoptotic TGF-β-SMAD/NFκB pathway through an oncogenic function of p27 in human papillary thyroid cancer. Oncogene [Internet]. 2017;36:652–66. https://doi.org/10.1038/onc.2016.233 .
Alonso MJ, Losa C, Calvo P, Vila-Jato, JL. Approaches to improve the association of amikacin sulphate to poly(alkylcyanoacrylate) nanoparticles. Int J Pharm [Internet]. 1991;68:69–76. https://doi.org/10.1016/0378-5173(91)90128-B .
de Waard H, Grasmeijer N, Hinrichs WLJ, Eissens AC, Pfaffenbach PPF, Frijlink HW. Preparation of drug nanocrystals by controlled crystallization: application of a 3-way nozzle to prevent premature crystallization for large scale production. Eur J Pharm Sci [Internet]. 2009;38:224–9. https://doi.org/10.1016/j.ejps.2009.07.005 .
de Waard H, Hinrichs WLJ, Frijlink HW. A novel bottom–up process to produce drug nanocrystals: Controlled crystallization during freeze-drying. JCR [Internet]. 2008;128:179–83. https://doi.org/10.1016/j.jconrel.2008.03.002 .
de Waard H, De Beer T, Hinrichs WLJ, Vervaet C, Remon J-P, Frijlink HW. Controlled crystallization of the lipophilic drug fenofibrate during freeze-drying: elucidation of the mechanism by in-line raman spectroscopy. AAPS J [Internet]. 2010;12:569–75.  http://link.springer.com/10.1208/s12248-010-9215-z . (PMID: 10.1208/s12248-010-9215-z206258652976986)
Oh KS, Lee KE, Han SS, Cho SH, Kim D, Yuk SH. Formation of core/shell nanoparticles with a lipid core and their application as a drug delivery system. Biomacromolecules [Internet]. 2005;6:1062–7.  https://doi.org/10.1021/bm049234r .
Yoshizane C, Mizote A, Yamada M, Arai N, Arai S, Maruta K, et al. Glycemic, insulinemic and incretin responses after oral trehalose ingestion in healthy subjects. Nutr J [Internet]. 2017;16:9. https://doi.org/10.1186/s12937-017-0233-x .
Wyatt PJ. Measurement of special nanoparticle structures by light scattering. Anal Chem [Internet]. 2014;86:7171–83. (PMID: 10.1021/ac500185w25047231)
McCartney F, Perinelli DR, Tiboni M, Cavanagh R, Lucarini S, Filippo Palmieri G, et al. Permeability-enhancing effects of three laurate-disaccharide monoesters across isolated rat intestinal mucosae. Int J Pharm [Internet]. 2021;601: 120593. https://doi.org/10.1016/j.ijpharm.2021.120593 . (PMID: 10.1016/j.ijpharm.2021.12059333857587)
Zhao Y, Liu A, Du Y, Cao Y, Zhang E, Zhou Q, et al. Effects of sucrose ester structures on liposome-mediated gene delivery. Acta Biomater [Internet]. 2018;72:278–86. https://doi.org/10.1016/j.actbio.2018.03.031 . (PMID: 10.1016/j.actbio.2018.03.03129609051)
Castañeda Ruiz AJ, Shetab Boushehri MA, Phan T, Carle S, Garidel P, Buske J, et al. Alternative excipients for protein stabilization in protein therapeutics: overcoming the limitations of polysorbates. Pharmaceutics [Internet]. 2022;14:2575.  https://doi.org/10.1016/j.actbio.2018.03.031 .
Sanchez SA, Gratton E, Zanocco AL, Lemp E, Gunther G. Sucrose monoester micelles size determined by fluorescence correlation spectroscopy (FCS). PLoS One [Internet]. 2011;6: e29278. https://doi.org/10.1371/journal.pone.0029278 . (PMID: 10.1371/journal.pone.002927822216230)
Abdelwahed W, Degobert G, Fessi H. Investigation of nanocapsules stabilization by amorphous excipients during freeze-drying and storage. Eur J Pharm Biopharm [Internet]. 2006;63:87–94. https://doi.org/10.1016/j.ejpb.2006.01.015 . (PMID: 10.1016/j.ejpb.2006.01.01516621490)
Allison SD, Dong A, Carpenter JF. Counteracting effects of thiocyanate and sucrose on chymotrypsinogen secondary structure and aggregation during freezing, drying, and rehydration. Biophys J [Internet]. 1996;71:2022–32. Available from: https://linkinghub.elsevier.com/retrieve/pii/S0006349596794006 .
Trenkenschuh E, Friess W. Freeze-drying of nanoparticles: how to overcome colloidal instability by formulation and process optimization. Eur J Pharm Biopharm [Internet]. 2021;165:345–60. https://doi.org/10.1016/s0006-3495(96)79400-6 .
Presas E, McCartney F, Sultan E, Hunger C, Nellen S, Alvarez CV, et al. Physicochemical, pharmacokinetic and pharmacodynamic analyses of amphiphilic cyclodextrin-based nanoparticles designed to enhance intestinal delivery of insulin. JCR [Internet]. 2018;286:402–14. https://doi.org/10.1016/j.jconrel.2018.07.045 .
O’Halloran TV, Kebede M, Philips SJ, Attie AD. Zinc, insulin, and the liver: a ménage à trois. J Clin Invest [Internet]. 2013;123:4136–9. (PMID: 10.1172/JCI72325240513733784553)
Brader ML, Sukumar M, Pekar AH, McClellan DS, Chance RE, Flora DB, et al. Hybrid insulin cocrystals for controlled release delivery. Nat Biotechnol [Internet]. 2002;20:800–4. https://doi.org/10.1172/JCI72325 .
Viehof A, Javot L, Béduneau A, Pellequer Y, Lamprecht A. Oral insulin delivery in rats by nanoparticles prepared with non-toxic solvents. Int J Pharm [Internet]. 2013;443:169–74.  https://doi.org/10.1038/nbt722 .
Makhlof A, Tozuka Y, Takeuchi H. Design and evaluation of novel pH-sensitive chitosan nanoparticles for oral insulin delivery. Eur J Pharm Sci [Internet]. 2011;42:445–51.  https://doi.org/10.1016/j.ijpharm.2013.01.017 .

281035 FP7 Ideas: European Research Council

Keywords: Freeze-drying; Intestinal absorption; Nanocomplexes; Nanostructures; Oral administration; Peptide/protein delivery

0 (Insulin)
57-50-1 (Sucrose)
J41CSQ7QDS (Zinc)
3OWL53L36A (Mannitol)

Date Created: 20240928 Date Completed: 20241023 Latest Revision: 20241113

20241114

10.1007/s13346-024-01712-9

39342023