Drug Delivery and Translational Research

, Volume 9, Issue 5, pp 997–1007 | Cite as

Design and evaluation of anti-fibrosis drug engineered resealed erythrocytes for targeted delivery

  • Piyali Dey
  • Subham Banerjee
  • Santa Mandal
  • Pronobesh ChattopadhyayEmail author
Original Article


Resealed erythrocytes (RSE) are potential, site-specific carrier system for drug delivery with prolonged drug release activity. In this study, erythrocytes obtained from Wistar albino rats were loaded with ambroxol hydrochloride (AH) with the focus to convenience the lung targeting possibility of the carrier erythrocytes. AH loading in erythrocytes using preswell dilution technique with glutaraldehyde (GA) as a cross-linking agent was evaluated and validated. Drug-loaded erythrocyte was characterized in terms of in vitro drug release followed by osmotic fragility study which showed amplified drug entrapment efficiency (DEE) and hemoglobin content values as well. In vivo lung fibrosis study, rats were sensitized to egg albumin by intraperitoneal (i.p.) injection and then inhalation in a whole body inhalation chamber. A sign of inflammation, airway sub-mucosal fibrosis, hypertrophy, and hyperplasia was observed. A series of in vivo studies were carried out to describe the effect of AH-loaded RSE including measurement of cytokines in Bronchoalveolar Lavage (BAL) fluid and histopathology study. AH showed a stepwise reduced level of cytokines in BAL at a different time interval after being injected of AH-loaded RSE. Furthermore, in vivo lung distribution experiments were performed for optimized formulation, and degree of distribution of the drugs inside the targeted organ was found to be satisfactory.


Resealed erythrocytes Ambroxol hydrochloride Preswell dilution technique Glutaraldehyde Lung fibrosis 



The authors are thankful to Director, DRL, Tezpur, Assam, India for providing all necessary facility to bearing this research work. Sincere thanks also to the scientists and all staff of Division of Pharmaceutical Technology Division DRL, Tezpur, Assam, for cooperation while carrying out the work.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.


  1. 1.
    Beeh KM, Beier J, Esperester A, Paul LD. Antiinflammatory properties of ambroxol. Eur J Med Res. 2008;13(12):557–62.PubMedGoogle Scholar
  2. 2.
    Mezzetti M. A pharmacokinetics study on pulmonary tropism of ambroxol on patients under thoracic surgery. Surg Intensive Care. 1990;13:179–85.Google Scholar
  3. 3.
    Houtmeyers E, Gosselink R, Gayan-Ramirez G, Decramer M. Effects of drugs on mucus clearance. Eur Respir J. 1999;14(2):452–67.Google Scholar
  4. 4.
    Nowak D, Antczak A, Król M, Bialasiewicz P, Pietras T. Antioxidant properties of ambroxol. Free Radic Biol Med. 1993;16:517–22.Google Scholar
  5. 5.
    Yang B, Yao DF, Ohuchi M, Ide M, Yano M, Okumura Y, et al. Ambroxol suppresses influenza-virus proliferation in the mouse airway by increasing antiviral factor levels. Eur Respir J. 2002;19(5):952–8.Google Scholar
  6. 6.
    Naranjo TW, Lopera DE, Diaz-Granados LR, Duque JJ, Restrepo MA, Cano LE. Combined itraconazole-pentoxifylline treatment promptly reduces lung fibrosis induced by chronic pulmonary paracoccidioidomycosis in mice. Pulm Pharmacol Ther. 2011;24:81–91.Google Scholar
  7. 7.
    Jadhav KR, Sankpal SV, Gavali SM, Sawant SV, Kadam VJ. Drug, enzyme and peptide delivery using erythrocytes as drug carrier. Int J Pharm Sci Rev Res. 2012;12:79–88.Google Scholar
  8. 8.
    Azarmi S, Roa WH, Löbenberg R. Targeted delivery of nanoparticles for the treatment of lung diseases. Adv Drug Deliv Rev. 2008;60(8):863–75.Google Scholar
  9. 9.
    Vyas SP, Khar R. Targeted and controlled drug delivery – novel carrier systems. New Delhi: CBS Publishers and Distributors; 2002.Google Scholar
  10. 10.
    Nicholas B. Retrometabolic approaches to drug targeting, Membrane and Barriers. NIH Publications; 1995:1-6.Google Scholar
  11. 11.
    Jaitely V, Kanaujia P, Venkatesan N, Jain S, Vyas SP. Resealed erythrocytes: drug carrier potentials and biomedical applications. Indian Drugs. 1996;33(12):589–94.Google Scholar
  12. 12.
    Hamidi M, Tajerzadeh H, Dehpour AR, Rouini MR, Ejtemaee-Mehr S. In vitro characterization of human intact erythrocytes loaded by enalaprilat. Drug Deliv. 2001;8(4):223–30.Google Scholar
  13. 13.
    Tajerzadeh H, Hamidi M. Evaluation of hypotonic preswelling method for encapsulation of enalaprilat in intact human erythrocytes. Drug Dev Ind Pharm. 2000;26(12):1247–57.Google Scholar
  14. 14.
    Mishra PR, Jain NK. Biotinylated methotrexate loaded erythrocytes for enhanced liver uptake.‘a study on the rat’. Int J Pharm. 2002;231(2):145–53.Google Scholar
  15. 15.
    Rossi L, Serafini S, Cappellacci L, Balestra E, Brandi G, Schiavano GF, et al. Erythrocyte-mediated delivery of a new homodinucleotide active against human immunodeficiency virus and herpes simplex virus. J Antimicrob Chemother. 2001;47(6):819–27.Google Scholar
  16. 16.
    Noël-Hocquet S, Jabbouri S, Lazar S, Maunier JC, Guillaumet G, Ropars C. Erythrocytes as Carriers of New Anti-Opioid Prodrugs: In Vitro Studies. Use Resealed Erythrocytes as Carriers Bioreact [Internet]. Boston: Springer US; 1992. p. 215–21.Google Scholar
  17. 17.
    Eichler HG, Rameis H, Bauer K, Korn A, Bacher S, Gasić S. Survival of gentamicin-loaded carrier erythrocytes in healthy human volunteers. Eur J Clin Investig. 1986;16(1):39–42.Google Scholar
  18. 18.
    Gothoskar AV. Resealed erythrocytes : a review. Pharm Technol. 2004;28:140–55.Google Scholar
  19. 19.
    Mishra PR, Jain S, Jain NK. Engineered human erythrocytes as carriers for ciprofloxacin. Drug Deliv. 1996;3(4):239–44.Google Scholar
  20. 20.
    Shavi GV, Doijad RC, Deshpande PB, Manvi F, Meka SR, Udupa N, et al. Erythrocytes as carrier for prednisolone: in vitro and in vivo evaluation. Pak J Pharm Sci. 2010;23(2).Google Scholar
  21. 21.
    Briones E, Colino CI, Lanao JM. Study of the factors influencing the encapsulation of zidovudine in rat erythrocytes. Int J Pharm. 2010;401(1–2):41–6.Google Scholar
  22. 22.
    Hamidi M, Zarrin AH, Foroozesh M, Zarei N, Mohammadi-Samani S. Preparation and in vitro evaluation of carrier erythrocytes for RES-targeted delivery of interferon-alpha 2b. Int J Pharm. 2007;341(1–2):125–33.Google Scholar
  23. 23.
    Dacie JV, Lond MB, Vaughan JM, Oxon DM. The fragility of the red blood cells: its measurement and significance. J Pathol Bacteriol. 1938;46(2):341–56.Google Scholar
  24. 24.
    Jain S, Jain NK. Engineered erythrocytes as a drug delivery system. Indian J Pharm Sci. 1997;59(6):275.Google Scholar
  25. 25.
    Khar RK, Diwan M. Targeted delivery of drugs. Adv. Control. Nov. drug Deliv. New Delhi: CBS Publishers and Distributors; 2001. p. 420–56.Google Scholar
  26. 26.
    Abo-zeid Y, Irving W, Thomson B, Garnett M. P22: nanoparticle delivery systems for HCV treatment: do nanoparticles avoid uptake by erythrocytes? J Viral Hepat. 2013;20:28–9.Google Scholar
  27. 27.
    Fan Y, Chen G, Li D, Luo Y, Lock N, Jensen AP, et al. Highly selective Deethylation of rhodamine B on TiO2 prepared in supercritical fluids. Int J Photoenergy. 2012;2012:1–7.Google Scholar
  28. 28.
    Field WN, Gamble MD, Lewis DA. A comparison of the treatment of thyroidectomized rats with free thyroxine and thyroxine encapsulated in erythrocytes. Int J Pharm. 1989;51(2):175–8.Google Scholar
  29. 29.
    Kim SE, Kim JH, Min BH, Bae YM, Hong ST, Choi MH. Crude extracts of Caenorhabditis elegans suppress airway inflammation in a murine model of allergic asthma. PLoS One. 2012;7(4):e35447.Google Scholar
  30. 30.
    Layachi S, Rogerieux F, Robidel F, Lacroix G, Bayat S. Effect of combined nitrogen dioxide and carbon nanoparticle exposure on lung function during ovalbumin sensitization in Brown Norway rat. PLoS One. 2012;7:e45687.Google Scholar
  31. 31.
    Kenyon NJ, Ward RW, Last JA. Airway fibrosis in a mouse model of airway inflammation. Toxicol Appl Pharmacol. 2003;186(2):90–100.Google Scholar
  32. 32.
    Sanz S, Lizano C, Luque J, Pinilla M. In vitro and in vivo study of glutamate dehydrogenase encapsulated into mouse erythrocytes by a hypotonic dialysis procedure. Life Sci. 1999;65(26):2781–9.Google Scholar
  33. 33.
    Pinilla M, Jordan JA, Diez JC, Luque J. In vitro stability properties of crosslinked rat red blood cells. Adv Biosci. 1994;92:7–16.Google Scholar
  34. 34.
    Pedroza M, Schneider DJ, Karmouty-Quintana H, Coote J, Shaw S, Corrigan R, et al. Interleukin-6 contributes to inflammation and remodeling in a model of adenosine mediated lung injury. PLoS One. 2011;6(7):e22667.Google Scholar
  35. 35.
    Fox CH, Johnson FB, Whiting J, Roller PP. Formaldehyde fixation. J Histochem Cytochem. 1985;33:845–53.Google Scholar
  36. 36.
    Werner M, Chott A, Fabiano A, Battifora H. Effect of formalin tissue fixation and processing on immunohistochemistry. Am J Surg Pathol. 2000;24(7):1016–9.Google Scholar
  37. 37.
    Dale GL, Villacorte DG, Beutler E. High-yield entrapment of proteins into erythrocytes. Biochem Med. 1977;18(2):220–5.Google Scholar
  38. 38.
    Bhaskaran S, Dhir SS. Resealed erythrocytes as carriers for salbutamol sulphate. Indian J Pharm Sci. 1995;57(6):240.Google Scholar
  39. 39.
    Millán CG, Castañeda AZ, López FG, Marinero ML, Lanao JM, Arévalo M. Encapsulation and in vitro evaluation of amikacin-loaded erythrocytes. Drug Deliv. 2005;12(6):409–16.Google Scholar
  40. 40.
    Richieri GV, Mel HC. Temperature effects on osmotic fragility, and the erythrocyte membrane. Biochim Biophys. 1985;813(1):41–50.Google Scholar
  41. 41.
    Sawant KK, Soni HN, Murthy RR. Investigation on resealed erythrocytes as carriers for 5-fluorouracil. Indian J Pharm Sci. 2001;63(2):105.Google Scholar
  42. 42.
    Tanaka H, Masuda T, Tokuoka S, Komai M, Nagao K, Takahashi Y, et al. The effect of allergen-induced airway inflammation on airway remodeling in a murine model of allergic asthma. Inflamm Res. 2001;50(12):616–24.Google Scholar
  43. 43.
    Sugahara K, Iyama KI, Kuroda MJ, Sano K. Double intratracheal instillation of keratinocyte growth factor prevents bleomycin-induced lung fibrosis in rats. J Pathol. 1998;186(1):90–8.Google Scholar
  44. 44.
    Su X, Wang L, Song Y, Bai C. Inhibition of inflammatory responses by ambroxol, a mucolytic agent, in a murine model of acute lung injury induced by lipopolysaccharide. Intensive Care Med. 2004;30(1):133–40.Google Scholar
  45. 45.
    Camacho MT, Totapally BR, Torbati D, Wolfsdorf J. Pulmonary and extrapulmonary effects of increased colloid osmotic pressure during endotoxemia in rats. Chest. 2001;120:1655–62.Google Scholar
  46. 46.
    Sinha B, Mukherjee B, Pattnaik G. Poly-lactide-co-glycolide nanoparticles containing voriconazole for pulmonary delivery: in vitro and in vivo study. Nanomed Nanotechnol Biol Med. 2013;9:94–104.Google Scholar

Copyright information

© Controlled Release Society 2019

Authors and Affiliations

  • Piyali Dey
    • 1
  • Subham Banerjee
    • 2
  • Santa Mandal
    • 1
  • Pronobesh Chattopadhyay
    • 1
    Email author
  1. 1.Division of Pharmaceutical TechnologyDefence Research LaboratoryTezpurIndia
  2. 2.Department of PharmaceuticsNational Institute of Pharmaceutical Education and Research (NIPER)GuwahatiIndia

Personalised recommendations