AAPS PharmSciTech

, Volume 19, Issue 7, pp 3152–3164 | Cite as

Amphotericin B Loaded Chitosan Nanoparticles: Implication of Bile Salt Stabilization on Gastrointestinal Stability, Permeability and Oral Bioavailability

  • Sanyog JainEmail author
  • Chamala Siva Kumar Reddy
  • Rajan Swami
  • Varun Kushwah
Research Article


Through current investigation, we presented a lucrative way to formulate amphotericin B loaded bile salt stabilized carbohydrate polymer i.e. chitosan nanoparticles (NPs) for enhancing gastrointestinal stability of NPs thereby increasing the oral bioavailability of the drug. NPs were prepared using ionic gelation method, and stabilized using bile salt to provide gastric pH stability to chitosan NPs. NPs were optimized on different parameters such as particle size, encapsulation efficiency and estimated for their in vitro and in vivo performance. Developed NPs presented a higher stability in gastrointestinal milieu, reduced haemolytic toxicity and significantly higher uptake in Caco-2 cell lines followed by increased bioavailability as compared to naive drug, marketed formulation i.e. Fungizone® and uncoated chitosan NPs. Biochemical parameters and histology further substantiated the lower toxicity. In nutshell, the present research explored the bioadhesive and higher uptake potential of cationic carbohydrate polymer at the same time along with bile salts for stabilization of NPs in gastric milieu.


amphotericin B chitosan bile salts oral bioavailability GI stability nephrotoxicity 



Amphotericin B


AmB loaded bile salt stabilized chitosan nanoparticles


Analysis of variance


Area under the curve


Blood urea nitrogen


Sodium cholate






High-performance liquid chromatography


Kilo dalton




Polydispersity index


Red blood cells


Rhodamine isothiocyanate


Scanning electron microscopy


Simulated gastric fluid


Simulated intestinal fluid


Sodium lauryl sulphate


Transmission electron microscope





The authors are thankful to Director, NIPER for providing the necessary infrastructure and facilities and Department of Science & Technology (DST), Government of India, New Delhi, and financial support. Rajan Swami is grateful to Science and Engineering Research Board (SERB), DST, GOI, New Delhi, for providing research fellowship. Varun Kushwah is appreciative to CSIR, GOI, New Delhi, for providing fellowships.

Compliance with Ethical Standards

Conflict of Interest

The authors report no financial interest that might pose a potential, perceived, or real conflict.

Supplementary material

12249_2018_1153_MOESM1_ESM.docx (25 kb)
ESM 1 (DOCX 24.8 kb)


  1. 1.
    Lipowsky R, Sackmann E. Structure and dynamics of membranes: I. from cells to vesicles/II. generic and specific interactions: Volume 1A, 1st Edition. New York: Elsevier; 1995.Google Scholar
  2. 2.
    Saravolatz LD, Ostrosky-Zeichner L, Marr KA, Rex JH, Cohen SH. Amphotericin B: time for a new “gold standard”. Clin Infect Dis. 2003;37(3):415–25.CrossRefGoogle Scholar
  3. 3.
    Gallis HA, Drew RH, Pickard WW. Amphotericin B: 30 years of clinical experience. Rev Infect Dis. 1990;12(2):308–29.CrossRefGoogle Scholar
  4. 4.
    Hiemenz JW, Walsh TJ. Lipid formulations of amphotericin B: recent progress and future directions. Clin Infect Dis. 1996;22(Supplement_2):S133–S44.CrossRefGoogle Scholar
  5. 5.
    Walsh TJ, Finberg RW, Arndt C, Hiemenz J, Schwartz C, Bodensteiner D, et al. Liposomal amphotericin B for empirical therapy in patients with persistent fever and neutropenia. N Engl J Med. 1999;340(10):764–71.CrossRefGoogle Scholar
  6. 6.
    Chaudhari MB, Desai PP, Patel PA, Patravale VB. Solid lipid nanoparticles of amphotericin B (AmbiOnp): in vitro and in vivo assessment towards safe and effective oral treatment module. Drug Deliv Transl Res. 2016;6(4):354–64.PubMedGoogle Scholar
  7. 7.
    Jain S, Valvi PU, Swarnakar NK, Thanki K. Gelatin coated hybrid lipid nanoparticles for oral delivery of amphotericin B. Mol Pharm. 2012;9(9):2542–53.CrossRefGoogle Scholar
  8. 8.
    Jain S, Yadav P, Swami R, Swarnakar NK, Kushwah V, Katiyar SS. Lyotropic liquid crystalline nanoparticles of amphotericin B: implication of phytantriol and glyceryl monooleate on bioavailability enhancement. AAPS PharmSciTech. 2018;19:1699–711.CrossRefGoogle Scholar
  9. 9.
    Adler-Moore JP, Gangneux J-P, Pappas PG. Comparison between liposomal formulations of amphotericin B. Sabouraudia. 2016;54(3):223–31.CrossRefGoogle Scholar
  10. 10.
    Hamill RJ. Amphotericin B formulations: a comparative review of efficacy and toxicity. Drugs. 2013;73(9):919–34.CrossRefGoogle Scholar
  11. 11.
    Kumar MNR. A review of chitin and chitosan applications. React Funct Polym. 2000;46(1):1–27.CrossRefGoogle Scholar
  12. 12.
    Artursson P, Lindmark T, Davis SS, Illum L. Effect of chitosan on the permeability of monolayers of intestinal epithelial cells (Caco-2). Pharm Res. 1994;11(9):1358–61.CrossRefGoogle Scholar
  13. 13.
    Rinaudo M. Chitin and chitosan: properties and applications. Prog Polym Sci. 2006;31(7):603–32.CrossRefGoogle Scholar
  14. 14.
    Liu L, Zhou C, Xia X, Liu Y. Self-assembled lecithin/chitosan nanoparticles for oral insulin delivery: preparation and functional evaluation. Int J Nanomedicine. 2016;11:761.CrossRefPubMedPubMedCentralGoogle Scholar
  15. 15.
    Khdair A, Hamad I, Alkhatib H, Bustanji Y, Mohammad M, Tayem R, et al. Modified-chitosan nanoparticles: novel drug delivery systems improve oral bioavailability of doxorubicin. Eur J Pharm Sci. 2016;93:38–44.CrossRefGoogle Scholar
  16. 16.
    Prego C, Garcia M, Torres D, Alonso M. Transmucosal macromolecular drug delivery. J Control Release. 2005;101(1):151–62.CrossRefGoogle Scholar
  17. 17.
    Harde H, Agrawal AK, Jain S. Tetanus toxoids loaded glucomannosylated chitosan based nanohoming vaccine adjuvant with improved oral stability and immunostimulatory response. Pharm Res. 2015;32(1):122–34.CrossRefGoogle Scholar
  18. 18.
    Harde H, Agrawal AK, Jain S. Development of stabilized glucomannosylated chitosan nanoparticles using tandem crosslinking method for oral vaccine delivery. Nanomedicine. 2014;9(16):2511–29.CrossRefGoogle Scholar
  19. 19.
    Jain S, Sharma RK, Vyas S. Chitosan nanoparticles encapsulated vesicular systems for oral immunization: preparation, in-vitro and in-vivo characterization. J Pharm Pharmacol. 2006;58(3):303–10.CrossRefGoogle Scholar
  20. 20.
    Agrawal AK, Urimi D, Harde H, Kushwah V, Jain S. Folate appended chitosan nanoparticles augment the stability, bioavailability and efficacy of insulin in diabetic rats following oral administration. RSC Adv. 2015;5(127):105179–93.CrossRefGoogle Scholar
  21. 21.
    Mukhopadhyay P, Bhattacharya S, Nandy A, Bhattacharyya A, Mishra R, Kundu P. Assessment of in vivo chronic toxicity of chitosan and its derivates used as oral insulin carriers. Toxicol Res. 2015;4(2):281–90.CrossRefGoogle Scholar
  22. 22.
    George M, Abraham TE. Polyionic hydrocolloids for the intestinal delivery of protein drugs: alginate and chitosan—a review. J Control Release. 2006;114(1):1–14.CrossRefGoogle Scholar
  23. 23.
    Jonassen H, Kjøniksen A-L, Hiorth M. Stability of chitosan nanoparticles cross-linked with tripolyphosphate. Biomacromolecules. 2012;13(11):3747–56.CrossRefGoogle Scholar
  24. 24.
    Sahariah P, Benediktssdóttir BE, Hjálmarsdóttir MÁ, Sigurjonsson OE, Sørensen KK, Thygesen MB, et al. Impact of chain length on antibacterial activity and hemocompatibility of quaternary N-alkyl and N, N-dialkyl chitosan derivatives. Biomacromolecules. 2015;16(5):1449–60.CrossRefGoogle Scholar
  25. 25.
    Sarmento B, Ribeiro A, Veiga F, Sampaio P, Neufeld R, Ferreira D. Alginate/chitosan nanoparticles are effective for oral insulin delivery. Pharm Res. 2007;24(12):2198–206.CrossRefGoogle Scholar
  26. 26.
    Azevedo MA, Bourbon AI, Vicente AA, Cerqueira MA. Alginate/chitosan nanoparticles for encapsulation and controlled release of vitamin B 2. Int J Biol Macromol. 2014;71:141–6.CrossRefGoogle Scholar
  27. 27.
    Wang Y, Qin F, Lu M, Gao L, Yao X. The screening and evaluating of chitosan/β-cyclodextrin nanoparticles for effective delivery mitoxantrone hydrochloride. Polym Sci Ser A. 2017;59(3):376–83.CrossRefGoogle Scholar
  28. 28.
    Moghimipour E, Ameri A, Handali S. Absorption-enhancing effects of bile salts. Molecules. 2015;20(8):14451–73.CrossRefGoogle Scholar
  29. 29.
    Mikov M, Fawcett J, Kuhajda K, Kevresan S. Pharmacology of bile acids and their derivatives: absorption promoters and therapeutic agents. Eur J Drug Metab Pharmacokinet. 2006;31(3):237–51.CrossRefGoogle Scholar
  30. 30.
    Chiang C-H, Lai J-S, Yang K-H. The effects of pH and chemical enhancers on the percutaneous absorption of indomethacin. Drug Dev Ind Pharm. 1991;17(1):91–111.CrossRefGoogle Scholar
  31. 31.
    Winuprasith T, Chantarak S, Suphantharika M, He L, McClements DJ. Alterations in nanoparticle protein corona by biological surfactants: impact of bile salts on β-lactoglobulin-coated gold nanoparticles. J Colloid Interface Sci. 2014;426:333–40.CrossRefGoogle Scholar
  32. 32.
    Yu J-n, Zhu Y, Wang L, Peng M, Tong S-S, Cao X, et al. Enhancement of oral bioavailability of the poorly water-soluble drug silybin by sodium cholate/phospholipid-mixed micelles. Acta Pharmacol Sin. 2010;31(6):759.CrossRefPubMedPubMedCentralGoogle Scholar
  33. 33.
    Jain S, Indulkar A, Harde H, Agrawal AK. Oral mucosal immunization using glucomannosylated bilosomes. J Biomed Nanotechnol. 2014;10(6):932–47.CrossRefGoogle Scholar
  34. 34.
    Käuper P, Forrest M, editors. Chitosan-based nanoparticles by ionotropic gelation. Switzerland: XIVth International Workshop on Bioencapsulation; 2006.Google Scholar
  35. 35.
    Vertzoni M, Fotaki N, Nicolaides E, Reppas C, Kostewicz E, Stippler E, et al. Dissolution media simulating the intralumenal composition of the small intestine: physiological issues and practical aspects. J Pharm Pharmacol. 2004;56(4):453–62.CrossRefGoogle Scholar
  36. 36.
    Swami R, Singh I, Jeengar MK, Naidu V, Khan W, Sistla R. Adenosine conjugated lipidic nanoparticles for enhanced tumor targeting. Int J Pharm. 2015;486(1):287–96.CrossRefGoogle Scholar
  37. 37.
    Mandal H, Katiyar SS, Swami R, Kushwah V, Katare PB, Meka AK, et al. ε-Poly-l-lysine/plasmid DNA nanoplexes for efficient gene delivery in vivo. Int J Pharm. 2018;542(1–2):142–52.CrossRefGoogle Scholar
  38. 38.
    Ma Z, Lim L-Y. Uptake of chitosan and associated insulin in Caco-2 cell monolayers: a comparison between chitosan molecules and chitosan nanoparticles. Pharm Res. 2003;20(11):1812–9.CrossRefGoogle Scholar
  39. 39.
    Ma O, Lavertu M, Sun J, Nguyen S, Buschmann MD, Winnik FM, et al. Precise derivatization of structurally distinct chitosans with rhodamine B isothiocyanate. Carbohydr Polym. 2008;72(4):616–24.CrossRefGoogle Scholar
  40. 40.
    Dora CP, Kushwah V, Katiyar SS, Kumar P, Pillay V, Suresh S, et al. Improved oral bioavailability and therapeutic efficacy of erlotinib through molecular complexation with phospholipid. Int J Pharm. 2017;534(1–2):1–13.CrossRefGoogle Scholar
  41. 41.
    Jain S, Chauhan D, Jain A, Swarnakar N, Harde H, Mahajan R, et al. Stabilization of the nanodrug delivery systems by lyophilization using universal step-wise freeze drying cycle. Indian Patent Application No 2011;2559.Google Scholar
  42. 42.
    Mattu C, Li R, Ciardelli G. Chitosan nanoparticles as therapeutic protein nanocarriers: the effect of ph on particle formation and encapsulation efficiency. Polym Compos. 2013;34(9):1538–45.CrossRefGoogle Scholar
  43. 43.
    Bhumkar DR, Pokharkar VB. Studies on effect of pH on cross-linking of chitosan with sodium tripolyphosphate: a technical note. AAPS PharmSciTech. 2006;7(2):E138–E43.CrossRefPubMedPubMedCentralGoogle Scholar
  44. 44.
    Kolhar P, Anselmo AC, Gupta V, Pant K, Prabhakarpandian B, Ruoslahti E, et al. Using shape effects to target antibody-coated nanoparticles to lung and brain endothelium. Proc Natl Acad Sci. 2013;110(26):10753–8.CrossRefGoogle Scholar
  45. 45.
    O'Donnell MD, McGeeney K, FitzGerald O. Effect of free and conjugated bile salts on α-amylase activity. Enzyme. 1975;19:129–39.CrossRefGoogle Scholar
  46. 46.
    Bhatia S, Kumar V, Sharma K, Nagpal K, Bera T. Significance of algal polymer in designing amphotericin B nanoparticles. Sci World J. 2014;2014:1–21.Google Scholar
  47. 47.
    Zhang C, Qu G, Sun Y, Wu X, Yao Z, Guo Q, et al. Pharmacokinetics, biodistribution, efficacy and safety of N-octyl-O-sulfate chitosan micelles loaded with paclitaxel. Biomaterials. 2008;29(9):1233–41.CrossRefGoogle Scholar
  48. 48.
    Italia J, Yahya M, Singh D, Kumar MR. Biodegradable nanoparticles improve oral bioavailability of amphotericin B and show reduced nephrotoxicity compared to intravenous Fungizone®. Pharm Res. 2009;26(6):1324–31.CrossRefGoogle Scholar

Copyright information

© American Association of Pharmaceutical Scientists 2018

Authors and Affiliations

  • Sanyog Jain
    • 1
    Email author
  • Chamala Siva Kumar Reddy
    • 1
  • Rajan Swami
    • 1
  • Varun Kushwah
    • 1
  1. 1.Centre for Pharmaceutical Nanotechnology, Department of PharmaceuticsNational Institute of Pharmaceutical Education and Research (NIPER)S.A.S. Nagar (Mohali)India

Personalised recommendations