Original articles
Ana L. Martínez-López, Carlos J. González-Navarro, Paula Aranaz, José L. Vizmanos, Juan M. Irache. In vivo testing of mucus-permeating nanoparticles for oral insulin delivery using Caenorhabditis elegans as a model under hyperglycemic conditions[J]. Acta Pharmaceutica Sinica B, 2021, 11(4): 989-1002

In vivo testing of mucus-permeating nanoparticles for oral insulin delivery using Caenorhabditis elegans as a model under hyperglycemic conditions
Ana L. Martínez-Lópeza, Carlos J. González-Navarrob, Paula Aranazb, José L. Vizmanosc,d, Juan M. Irachea
a NANO-VAC Research Group, Department of Chemistry and Pharmaceutical Technology, School of Pharmacy and Nutrition, University of Navarra, Pamplona 31080, Spain;
b Center for Nutrition Research, School of Pharmacy and Nutrition, University of Navarra, Pamplona 31080, Spain;
c Department of Biochemistry & Genetics, School of Science, University of Navarra, Pamplona 31080, Spain;
d Navarra Institute for Health Research(IdiSNA), Pamplona 31080, Spain
The aim was to evaluate the potential of mucus-permeating nanoparticles for the oral administration of insulin. These nanocarriers, based on the coating of zein nanoparticles with a polymer conjugate containing PEG, displayed a size of 260 nm with a negative surface charge and an insulin payload of 77 μg/mg. In intestinal pig mucus, the diffusivity of these nanoparticles (PPA-NPs) was found to be 20-fold higher than bare nanoparticles (NPs). These results were in line with the biodistribution study in rats, in which NPs remained trapped in the mucus, whereas PPA-NPs were able to cross this layer and reach the epithelium surface. The therapeutic efficacy was evaluated in Caenorhabditis elegans grown under high glucose conditions. In this model, worms treated with insulin-loaded in PPA-NPs displayed a longer lifespan than those treated with insulin free or nanoencapsulated in NPs. This finding was associated with a significant reduction in the formation of reactive oxygen species (ROS) as well as an important decrease in the glucose and fat content in worms. These effects would be related with the mucus-permeating ability of PPA-NPs that would facilitate the passage through the intestinal peritrophic-like dense layer of worms (similar to mucus) and, thus, the absorption of insulin.
Key words:    Nanoparticles    Oral delivery    Mucus-permeating    Biodistribution    Insulin    Caenorhabditis elegans    ROS    Lifespan    Zein    Epithelium   
Received: 2020-11-08     Revised: 2020-12-18
DOI: 10.1016/j.apsb.2021.02.020
Funds: The first author was supported by Postdoctoral Fellowship from the National Council for Science and Technology of Mexico (CONACyT, Grant No. 291231, Mexico).
Corresponding author: Juan M.Irache, jmirache@unav.es     Email:jmirache@unav.es
Author description:
PDF(KB) Free
Ana L. Martínez-López
Carlos J. González-Navarro
Paula Aranaz
José L. Vizmanos
Juan M. Irache

1. Muheem A, Shakeel F, Jahangir MA, Anwar M, Mallick N, Jain GK, et al. A review on the strategies for oral delivery of proteins and peptides and their clinical perspectives. Saudi Pharmaceut J 2016;24:413-28.
2. Cao SJ, Xu S, Wang HM, Ling Y, Dong J, Xia RD, et al. Nanoparticles:oral delivery for protein and peptide drugs. AAPS PharmSciTech 2019;20:1-11.
3. Han Y, Gao Z, Chen L, Kang L, Huang W, Jin M, et al. Multifunctional oral delivery systems for enhanced bioavailability of therapeutic peptides/proteins. Acta Pharm Sin B 2019;9:902-22.
4. Perry SL, McClements DJ. Recent advances in encapsulation, protection, and oral delivery of bioactive proteins and peptides using colloidal systems. Molecules 2020;25:1-26.
5. Al-Remawi M, Elsayed A, Maghrabi I, Hamaidi M, Jaber N. Chitosan/lecithin liposomal nanovesicles as an oral insulin delivery system. Pharmaceut Dev Technol 2017;22:390-8.
6. Zhang X, Qi J, Lu Y, He W, Li X, Wu W. Biotinylated liposomes as potential carriers for the oral delivery of insulin. Nanomedicine 2014; 10:167-76.
7. Lundquist P, Artursson P. Oral absorption of peptides and nanoparticles across the human intestine:opportunities, limitations and studies in human tissues. Adv Drug Deliv Rev 2016;106:256-76.
8. McClements DJ. Encapsulation, protection, and delivery of bioactive proteins and peptides using nanoparticle and microparticle systems:a review. Adv Colloid Interface Sci 2018;253:1-22.
9. Wong CY, Martinez J, Dass CR. Oral delivery of insulin for treatment of diabetes:status quo, challenges and opportunities. J Pharm Pharmacol 2016;68:1093-108.
10. Macedo A, Filipe P, Thomé NG, Vieira J, Oliveira C, Teodósio C, et al. A brief overview of the oral delivery of insulin as an alternative to the parenteral delivery. Curr Mol Med 2020;20:134-43.
11. Ensign LM, Cone R, Hanes J. Oral drug delivery with polymeric nanoparticles:the gastrointestinal mucus barriers. Adv Drug Deliv Rev 2012;64:557-70.
12. Pereira De Sousa I, Moser T, Steiner C, Fichtl B, BernkopSchnürch A. Insulin loaded mucus permeating nanoparticles:addressing the surface characteristics as feature to improve mucus permeation. Int J Pharm 2016;500:236-44.
13. Bourganis V, Karamanidou T, Samaridou E, Karidi K, Kammona O, Kiparissides C. On the synthesis of mucus permeating nanocarriers. Eur J Pharm Biopharm 2015;97:239-49.
14. Inchaurraga L, Martínez-López AL, Cattoz B, Gri PC, Wilcox M, Pearson P, et al. The effect of thiamine-coating nanoparticles on their biodistribution and fate following oral administration. Eur J Pharmaceut Sci 2019;128:81-90.
15. Gonzalez-Moragas L, Roig A, Laromaine A. C. elegans as a tool for in vivo nanoparticle assessment. Adv Colloid Interface Sci 2015;219:10-26.
16. Moraes BKS, Vieira SM, Salgueiro WG, Michels LR, Colomé LM, Avila DS, et al. Clozapine-loaded polysorbate-coated polymeric nanocapsules:physico-chemical characterization and toxicity evaluation in Caenorhabditis elegans model. J Nanosci Nanotechnol 2016; 16:1257-64.
17. Kaletta T, Hengartner MO. Finding function in novel targets:C. elegans as a model organism. Nat Rev Drug Discov 2006;5:387-99.
18. Kenyon C. The plasticity of aging:insights from long-lived mutants. Cell 2005;120:449-60.
19. Everman JL, Ziaie NR, Bechler J, Bermudez LE. Establishing Caenorhabditis elegans as a model for Mycobacterium avium subspecies hominissuis infection and intestinal colonization. Biol Open 2015;4:1330-5.
20. Dimov I, Maduro MF. The C. elegans intestine:organogenesis, digestion, and physiology. Cell Tissue Res 2019;377:383-96.
21. Laughlin ST, Bertozzi CR. In vivo imaging of Caenorhabditis elegans glycans. ACS Chem Biol 2009;4:1068-72.
22. Martínez-López AL, Pangua C, Reboredo C, Campión R, MoralesGracia J, Irache JM. Protein-based nanoparticles for drug delivery purposes. Int J Pharm 2020;581:119289.
23. Peñalva R, Esparza I, González-Navarro CJ, Quincoces G, Peñuelas I, Irache JM. Zein nanoparticles for oral folic acid delivery. J Drug Deliv Sci Technol 2015;30:450-7.
24. Inchaurraga L, Martínez-López AL, Martin-Arbella N, Irache JM. Zein-based nanoparticles for the oral delivery of insulin. Drug Deliv Transl Res 2020;10:1601-11.
25. Lucio D, Martínez-Ohárriz MC, Gu Z, He Y, Aranaz P, Vizmanos JL, et al. Cyclodextrin-grafted poly(anhydride) nanoparticles for oral glibenclamide administration. In vivo evaluation using C. elegans. Int J Pharm 2018;547:97-105.
26. Yoncheva K, Lizarraga E, Irache JM. Pegylated nanoparticles based on poly(methyl vinyl ether-co-maleic anhydride):preparation and evaluation of their bioadhesive properties. Eur J Pharmaceut Sci 2005;24:411-9.
27. Doktorovova S, Shegokar R, Martins-Lopes P, Silva AM, Lopes CM, Müller RH, et al. Modified Rose Bengal assay for surface hydrophobicity evaluation of cationic solid lipid nanoparticles (cSLN). Eur J Pharmaceut Sci 2012;45:606-12.
28. Abdulkarim M, Agulló N, Cattoz B, Griffiths P, Bernkop-Schnürch A, Borros SG, et al. Nanoparticle diffusion within intestinal mucus:threedimensional response analysis dissecting the impact of particle surface charge, size and heterogeneity across polyelectrolyte, pegylated and viral particles. Eur J Pharm Biopharm 2015;97:230-8.
29. Inchaurraga L, Martínez-López AL, Abdulkarim M, Gumbleton M, Quincoces G, Peñuelas I, et al. Modulation of the fate of zein nanoparticles by their coating with a Gantrez® AN-thiamine polymer conjugate. Int J Pharm X 2019;1:100006.
30. Schindelin J, Arganda-Carreras I, Frise E, Kaynig V, Longair M, Pietzsch T, et al. Fiji:an open-source platform for biological-image analysis. Nat Methods 2012;9:676-82.
31. Inchaurraga L, Martín-Arbella N, Zabaleta V, Quincoces G, Peñuelas I, Irache JM. In vivo study of the mucus-permeating properties of PEG-coated nanoparticles following oral administration. Eur J Pharm Biopharm 2015;97:280-9.
32. Brenner S. The genetics of Caenorabditis elegans. Genetics 1974;77:71-94.
33. Martorell P, Llopis S, González N, Montón F, Ortiz P, Genovés S, et al. Caenorhabditis elegans as a model to study the effectiveness and metabolic targets of dietary supplements used for obesity treatment:the specific case of a conjugated linoleic acid mixture (Tonalin). J Agric Food Chem 2012;60:11071-9.
34. Mendler M, Schlotterer A, Ibrahim Y, Kukudov G, Fleming T, Bierhaus A, et al. daf-16/FOXO and glod-4/glyoxalase-1 are required for the life-prolonging effect of human insulin under high glucose conditions in Caenorhabditis elegans. Diabetologia 2014;58:393-401.
35. Navarro-Herrera D, Aranaz P, Eder-Azanza L, Zabala M, Hurtado C, Romo-Hualde A, et al. Dihomo-gamma-linolenic acid induces fat loss in:C. elegans in an omega-3-independent manner by promoting peroxisomal fatty acid β-oxidation. Food Funct 2018;9:1621-37.
36. Wang Z, Ma X, Li J, Cui X. Peptides from sesame cake extend healthspan of Caenorhabditis elegans via upregulation of skn-1 and inhibition of intracellular ROS levels. Exp Gerontol 2016;82:139-49.
37. Schlotterer A, Kukudov G, Bozorgmehr F, Hutter H, Du X, Oikonomou D, et al. C. elegans as model for the study of high glucose-mediated life span reduction. Diabetes 2009;58:2450-6.
38. Aranaz P, Navarro-Herrera D, Romo-Hualde A, Zabala M, LópezYoldi M, González-Ferrero C, et al. Broccoli extract improves high fat diet-induced obesity, hepatic steatosis and glucose intolerance in Wistar rats. J Funct Foods 2019;59:319-28.
39. Wu H, Taki FA, Zhang Y, Dobbins DL, Pan X. Evaluation and identification of reliable reference genes for toxicological study in Caenorhabditis elegans. Mol Biol Rep 2014;41:3445-55.
40. Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2-ΔΔCT method. Methods 2001;25:402-8.
41. Lee SJ, Murphy CT, Kenyon C. Glucose Shortens the Life Span of C. elegans by downregulating DAF-16/FOXO activity and aquaporin gene expression. Cell Metabol 2009;10:379-91.
42. Tullet JMA, Hertweck M, An JH, Baker J, Hwang JY, Liu S, et al. Direct inhibition of the longevity-promoting factor SKN-1 by insulinlike signaling in C. elegans. Cell 2008;132:1025-38.
43. Schubert S, Delaney Jr JT, Schubert US. Nanoprecipitation and nanoformulation of polymers:from history to powerful possibilities beyond poly(lactic acid). Soft Matter 2011;7:1581-8.
44. Li YP, Pei YY, Zhang XY, Gu ZH, Zhou ZH, Yuan WF, et al. PEGylated PLGA nanoparticles as protein carriers:synthesis, preparation and biodistribution in rats. J Control Release 2001;71:203-11.
45. Mura S, Hillaireau H, Nicolas J, Kerdine-Römer S, Le Droumaguet B, Deloménie C, et al. Biodegradable nanoparticles meet the bronchial airway barrier:how surface properties affect their interaction with mucus and epithelial cells. Biomacromolecules 2011;12:4136-43.
46. Agarwal V, Nazzal S, Reddy IK, Khan MA. Transport studies of insulin across rat jejunum in the presence of chicken and duck ovomucoids. J Pharm Pharmacol 2001;53:1131-8.
47. Zhu G, Yin F, Wang L, Wei W, Jiang L, Qin J. Modeling type 2 diabetes-like hyperglycemia in C. elegans on a microdevice. Integr Biol (Cam) 2016;8:30-8.
48. Lu Z, Qiu Z. High glucose concentration restricts fat consumption in Caenorhabditis elegans. Int J Clin Exp Med 2017;10:10554-9.
49. Schulz TJ, Zarse K, Voigt A, Urban N, Birringer M, Ristow M. Glucose restriction extends Caenorhabditis elegans life span by inducing mitochondrial respiration and increasing oxidative stress. Cell Metabol 2007;6:280-93.
50. Kimura KD, Tissenbaum HA, Liu Y, Ruvkun G. daf-2, an insulin receptor-like gene that regulates longevity and diapause in Caenorhabditis elegans. Science 1997;277:942-6.
51. Sun X, Chen W-D, Wang Y-D. DAF-16/FOXO Transcription factor in aging and longevity. Front Pharmacol 2017;8:548.
52. Pierce SB, Costa M, Wisotzkey R, Devadhar S, Homburger SA, Buchman AR, et al. Regulation of DAF-2 receptor signaling by human insulin and ins-1, a member of the unusually large and diverse C. elegans insulin gene family. Genes Dev 2001;15:672-86.
53. Tullet JMA, Green JW, Au C, Benedetto A, Thompson MA, Clark E, et al. The SKN-1/Nrf 2 transcription factor can protect against oxidative stress and increase lifespan in C. elegans by distinct mechanisms. Aging Cell 2017;16:1191-4.
54. Zarse K, Schmeisser S, Groth M, Priebe S, Beuster G, Kuhlow D, et al. Impaired insulin/IGF1 signaling extends life span by promoting mitochondrial L-proline catabolism to induce a transient ROS signal. Cell Metabol 2012;15:451-65.
55. Pang S, Lynn DA, Lo JY, Paek J, Curran SP. SKN-1 and Nrf 2 couples proline catabolism with lipid metabolism during nutrient deprivation. Nat Commun 2014;5:5048.
Similar articles:
1.Yang Zhao, Xiangrong Yu, Jia Li.Manipulation of immune-vascular crosstalk: new strategies towards cancer treatment[J]. Acta Pharmaceutica Sinica B, 2020,10(11): 2018-2036
2.Yangyang Li, Qiaolin Wei, Fei Ma, Xin Li, Fengyong Liu, Min Zhou.Surface-enhanced Raman nanoparticles for tumor theranostics applications[J]. Acta Pharmaceutica Sinica B, 2018,8(3): 349-359
3.Li Tian, Linfeng Lu, James Feng, Marites P. Melancon.Radiopaque nano and polymeric materials for atherosclerosis imaging, embolization and other catheterization procedures[J]. Acta Pharmaceutica Sinica B, 2018,8(3): 360-370
Similar articles: