Pharmaceutical Research

, Volume 34, Issue 2, pp 438–452 | Cite as

Bromelain-Functionalized Multiple-Wall Lipid-Core Nanocapsules: Formulation, Chemical Structure and Antiproliferative Effect Against Human Breast Cancer Cells (MCF-7)

  • Catiúscia P. OliveiraEmail author
  • Willian A. Prado
  • Vladimir Lavayen
  • Sabrina L. Büttenbender
  • Aline Beckenkamp
  • Bruna S. Martins
  • Diogo S. Lüdtke
  • Leandra F. Campo
  • Fabiano S. Rodembusch
  • Andréia Buffon
  • Adalberto PessoaJr
  • Silvia S. Guterres
  • Adriana R. PohlmannEmail author
Research Paper



This study was conducted a promising approach to surface functionalization developed for lipid-core nanocapsules and the merit to pursue new strategies to treat solid tumors.


Bromelain-functionalized multiple-wall lipid-core nanocapsules (Bro-MLNC-Zn) were produced by self-assembling following three steps of interfacial reactions. Physicochemical and structural characteristics, in vitro proteolytic activity (casein substrate) and antiproliferative activity (breast cancer cells, MCF-7) were determined.


Bro-MLNC-Zn had z-average diameter of 135 nm and zeta potential of +23 mV. The complex is formed by a Zn-N chemical bond and a chelate with hydroxyl and carboxyl groups. Bromelain complexed at the nanocapsule surface maintained its proteolytic activity and showed anti-proliferative effect against human breast cancer cells (MCF-7) (72.6 ± 1.2% at 1.250 μg mL−1 and 65.5 ± 5.5% at 0.625 μg mL−1). Comparing Bro-MLNC-Zn and bromelain solution, the former needed a dose 160-folds lower than the latter for a similar effect. Tripan blue dye assay corroborated the results.


The surface functionalization approach produced an innovative formulation having a much higher anti-proliferative effect than the bromelain solution, even though both in vitro proteolytic activity were similar, opening up a great opportunity for further studies in nanomedicine.


bromelain lipid-core nanocapsules MCF-7 human breast cancer cells metal-chitosan complex surface-functionalized nanoparticles 





One-way analysis of variance


Acridine orange base


Bromelain-functionalized multiple-wall lipid-core nanocapsules


Caprylic/capric triglyceride


Center of Nanoscience and Nanotechnology at the Federal University of Rio Grande do Sul


AO-labeled chitosan


Median diameter


Particle diameter at percentile 90 under the particle size distribution curves




Dulbecco’s modified Eagle’s medium




Dimethyl sulfoxide


N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride


Fetal bovine serum


Human epidermal growth factor receptor


Lipid-core nanocapsule


Cationic lipid-core nanocapsules/lecithin-chitosan-polysorbate 80-coated lipid-core nanocapsules


Lecithin-polysorbate 80-coated lipid-core nanocapsules

log D

Distribution coefficient


Multi-wall lipid-core nanocapsules


Mammalian target of rapamycin


3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide


Nanoparticle tracking analysis




Rhodamine B-poly(ε-caprolactone) conjugate


Polydispersity index




Phenylalanine-functionalized multi-wall lipid-core nanocapsules


Particle number density


Rhodamine B


Surface area


Trichloroacetic acid


Transmission electron microscopy



Catiúscia Padilha de Oliveira and Willian Andrade de Prado thanks the Brazilian Agency CAPES for their fellowships. The authors thank the Grants from Brazilian Agencies: CNPq/Brasilia/Brazil, CAPES/MEC and FAPERGS. The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Catiúscia P. Oliveira and Willian A. Prado contributed equally. The authors report no conflicts of interest in this work.

Supplementary material

11095_2016_2074_MOESM1_ESM.docx (3.1 mb)
ESM 1 (DOCX 3197 kb)


  1. 1.
    Marin E, Briceno MI, Caballero-George C. Critical evaluation of biodegradable polymers used in nanodrugs. Int J Nanomed. 2013;8:3071–91.Google Scholar
  2. 2.
    Pardeike J, Hommoss A, Muller RH. Lipid nanoparticles (SLN, NLC) in cosmetic and pharmaceutical dermal products. Int J Pharm. 2009;366:170–84.CrossRefPubMedGoogle Scholar
  3. 3.
    Estanqueiro M, Amaral MH, Conceição J, Lobo JM. Nanotechnological carriers for cancer chemotherapy: the state of the art. Colloids Surf B. 2015;126:631–48.CrossRefGoogle Scholar
  4. 4.
    Pohlmann AR, Fonseca FN, Paese K, Detoni CB, Coradini C, Beck RCR, et al. Poly(ε-caprolactone) microcapsules and nanocapsules in drug delivery. Expert Opin Drug Delivery. 2013;10:623–38.CrossRefGoogle Scholar
  5. 5.
    Oliveira CP, Venturini CG, Donida B, Poletto FS, Guterres SS, Pohlmann AR. An algorithm to determine the mechanism of drug distribution in lipid-core nanocapsule formulations. Soft Matter. 2013;9:1141–50.CrossRefGoogle Scholar
  6. 6.
    Bender EA, Adorne MD, Colomé LM, Abdalla DSP, Guterres SS, Pohlmann AR. Hemocompatibility of poly(Ɛ-caprolactone) lipid-core nanocapsules stabilized with polysorbate 80-lecithin and uncoated or coated with chitosan. Int J Pharm. 2012;426:271–9.CrossRefPubMedGoogle Scholar
  7. 7.
    Bender EA, Cavalcante MF, Adorne MD, Colomé LM, Guterres SS, Abdalla DSP, et al. New strategy to surface functionalization of polymeric nanoparticles: one-pot synthesis of scFv anti-LDL(−)-functionalized nanocapsules. Pharm Res. 2014;31:2975–87.CrossRefPubMedGoogle Scholar
  8. 8.
    Mayer FQ, Adorne MD, Bender EA, de Carvalho TG, Dilda AC, Beck RCR, et al. Laronidase-functionalized multiple-wall lipid-core nanocapsules: promising formulation for a more effective treatment of mucopolysaccharidosis type I. Pharm Res. 2015;32:941–54.CrossRefPubMedGoogle Scholar
  9. 9.
    Gil EMC. Targeting the PI3K/AKT/mTOR pathway in estrogen receptor-positive breast cancer. Cancer Treat Rev. 2014;40:862–71.CrossRefGoogle Scholar
  10. 10.
    Baselga J, Campone M, Piccart M, Burris III HA, Rugo HS, Sahmoud T, et al. Everolimus in postmenopausal hormone-receptor–positive advanced breast cancer. N Engl J Med. 2012;366:520–9.CrossRefPubMedGoogle Scholar
  11. 11.
    Fabi A, Mottolese M, Segatto O. Therapeutic targeting of ERBB2 in breast cancer: understanding resistance in the laboratory and combating it in the clinic. J Mol Med. 2014;92:681–95.CrossRefPubMedGoogle Scholar
  12. 12.
    Königsberg R, Maierhofer J, Steininger T, Kienzer G, Dittrich C. Long-term remission of a Her2/neu positive primary breast cancer under double monoclonal antibody therapy with trastuzumab and bevacizumab. Radiol Oncol. 2014;48:184–8.CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Mamounas EP, Bryant J, Lembersky B, Fehrenbacher L, Sedlacek SM, Fisher B, et al. Paclitaxel after doxorubicin plus cyclophosphamide as adjuvant chemotherapy for node-positive breast cancer: results from NSABP B-28. J Clin Oncol. 2005;23:3686–96.CrossRefPubMedGoogle Scholar
  14. 14.
    Tu Y, Hershman DL, Bhalla K, Fiskus W, Pellegrino CM, Andreopoulou E, et al. A phase I-II study of the histone deacetylase inhibitor vorinostat plus sequential weekly paclitaxel and doxorubicincyclophosphamide in locally advanced breast cancer. Breast Cancer Res Treat. 2014;146:145–52.CrossRefPubMedGoogle Scholar
  15. 15.
    Beuth J, Bernhard O, Abolghassem P, Rethfeldt E, Bock PR, Hanisch J, et al. Impact of complementary oral enzyme application on the postoperative treatment results of breast cancer patients – results of an epidemiological multicentre retrolactive cohort study. Cancer Chemother Pharmacol. 2001;47:S45–54.CrossRefPubMedGoogle Scholar
  16. 16.
    Bhui K, Tyagi S, Prakash B, Shukla Y. Pineapple bromelain induces autophagy, facilitating apoptoticresponse in mammary carcinoma cells. Biofactors. 2010;36:474–82.CrossRefPubMedGoogle Scholar
  17. 17.
    Kalra N, Bhui K, Roy P, Srivastava S, George J, Prasad S, et al. Regulation of p53 nuclear factor kappaB and cyclooxygenase-2 expression by bromelain through targeting mitogen-activated protein kinase pathway in mouse skin. Toxicol Appl Pharmacol. 2008;226:30–7.CrossRefPubMedGoogle Scholar
  18. 18.
    Bhui K, Tyagi S, Srivastava AM, Singh M, Roy P, Singh R, et al. Bromelain inhibits nuclear factor Kappa-B translocation, driving human epidermoid carcinoma A431 and melanoma A375 cells through G2/M arest to apoptosis. Mol Carcinog. 2012;51:231–43.CrossRefPubMedGoogle Scholar
  19. 19.
    Romano B, Fasolino I, Pagano E, Capasso R, Pace S, De Rosa G, et al. The chemopreventive action of bromelain, from pineapple stem (Ananas comosus L.), on colon carcinogenesis is related to antiproliferative and proapoptotic effects. Mol Nutr Food Res. 2014;58:457–65.CrossRefPubMedGoogle Scholar
  20. 20.
    Amini A, Ehteda A, Moghaddam SM, Akhter J, Pillai K, Morris AL. Cytotoxic effects of bromelain in human gastrointestinal carcinoma cell lines. OncoTargets Ther. 2013;6:403–9.Google Scholar
  21. 21.
    Pillai K, Ehteda A, Akhter J, Chua TC, Morris DL. Anticancer effect of bromelain with and without cisplatin or 5-FU on malignant peritoneal mesothelioma cells. Anti-Cancer Drugs. 2014;25:150–60.CrossRefPubMedGoogle Scholar
  22. 22.
    Tysnes BB, Maurer HR, Porwol T, Probst B, Bjerkvig R, Hoover F. Bromelain reversibly inhibits invasive properties of glioma cells. Neoplasia. 2001;3:469–79.CrossRefPubMedPubMedCentralGoogle Scholar
  23. 23.
    Pillai K, Akhter J, Chua TC. Anticancer property of bromelain with therapeutic potential in malignant peritoneal mesothelioma. Cancer Invest. 2013;31:241–50.CrossRefPubMedGoogle Scholar
  24. 24.
    Batkin S, Taussig SJ, Szekerezes J. Antimetastatic effect of bromelain with or without its proteolytic and anticoagulant activity. J Cancer Res Clin Oncol. 1988;114:507–8.CrossRefPubMedGoogle Scholar
  25. 25.
    Dhandayuthapani S, Perez HD, Paroulek A, Chinnakkannu P, Kandalam U, Jaffe M, et al. Bromelain-induced apoptosis in GI-101A breast cancer cells. J Med Food. 2012;15:344–9.CrossRefPubMedGoogle Scholar
  26. 26.
    Bhatnagar P, Gupta KC. Oral administration of Eudragit coated bromelain encapsulated PLGA nanoparticles for effective delivery of bromelain for chemotherapy in vivo. Biomed. Eng. Conf (SBEC), 2013 29th Southern. 2013; 47–8.Google Scholar
  27. 27.
    Armarego WLF. In Purification of Laboratory Chemicals. 5th ed. Cornwall: Elsevier Academic Press; 2003.Google Scholar
  28. 28.
    Rodembusch FS, Leusin FP, Medina LFC, Brandelli A, Stefani V. Synthesis and spectroscopic characterisation of new ESIPT fluorescent protein probes. Photochem Photobiol Sci. 2005;4:254–9.CrossRefPubMedGoogle Scholar
  29. 29.
    Dick PF, Coelho FL, Rodembusch FS, Medina LFC. Amphiphilic ESIPT benzoxazole derivatives as prospective fluorescent membrane probes. Tetrahedron Lett. 2014;55:3024–9.CrossRefGoogle Scholar
  30. 30.
    Poletto FS, Fiel LA, Lopes MV, Schaab G, Gomes AMO, Guterres SS, et al. Fluorescent-labeled poly(ε-caprolactone) lipid-core nanocapsules: synthesis, physicochemical properties and macrophage uptake. J Colloid Sci Biotechnol. 2012;1:1–10.CrossRefGoogle Scholar
  31. 31.
    Zelenka K, Borsig L, Alberto R. Trifunctional 99mTc based radiopharmaceuticals: metal-mediated conjugation of a peptide with a nucleus targeting intercalator. Org Biomol Chem. 2011;9:1071–8.CrossRefPubMedGoogle Scholar
  32. 32.
    Kobara H, Wakisaka A, Takeuchi K. Preferential Solvation of Na + in N, N-Dimethylformamide-Water Binary Mixture. J Phys Chem B. 2003;107:11827–9.CrossRefGoogle Scholar
  33. 33.
    Soares PAG, Vaz AFM, Correia MTS, Pessoa Jr A, Carneiro-da-Cunha MG. Purification of bromelain from pineapple wastes by ethanol precipitation. Sep Purif Technol. 2012;98:389–95.CrossRefGoogle Scholar
  34. 34.
    Mosmann T. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods. 1983;65:55–63.CrossRefPubMedGoogle Scholar
  35. 35.
    Hansen MB, Nielsen SE, Berg K. Re-examination and further development of a precise and rapid dye method for measuring cell growth/cell kill. J Immunol Methods. 1989;119:203–10.CrossRefPubMedGoogle Scholar
  36. 36.
    Rodembusch FS, Leusin FP, Campo LF, Stefani V. Excited state intramolecular proton transfer in amino 2-(2′-hydroxyphenyl)benzazole derivatives: effects of the solvent and the amino group position. J of Luminescence. 2007;126:728–34.CrossRefGoogle Scholar
  37. 37.
    Kumirska J, Czerwicka M, Kaczynski Z, Bychowska AK, Thoming J, Stepnowski P. Application of spectroscopic methods for structural analysis of chitin and chitosan. Mar Drugs. 2010;8:1567–636.CrossRefPubMedPubMedCentralGoogle Scholar
  38. 38.
    Brugnerotto J, Lizardi J, Goyoolea FM, Argüelles-Monal W, Desbrières J, Rinaudo M. An infrared investigation in relation with chitin and chitosan characterization. Polymer. 2001;42:3569–80.CrossRefGoogle Scholar
  39. 39.
    Prasad V, Semwogerere D, Weeks ER. Confocal microscopy of colloids. J Phys Condens Matter. 2007;19:1–25.CrossRefGoogle Scholar
  40. 40.
    Barros FCF, Cavalcante RM, Carvalho TV, Dias FS, Queiroz DC, Vasconcellos LCG, et al. Produção e caracterização de esfera de quitosana modificada quimicamente. Rev Iberoamericana de Polímero. 2006;7:232–46.Google Scholar
  41. 41.
    Lucena GL, Silva AG, Honório LMC, Santos VD. Remoção de corantes têxteis a partir de soluções aquosas por quitosana modificada com tioacetamida. Rev Ambiente Agua. 2013;8:144–54.Google Scholar
  42. 42.
    Hari K, Pichaimani A, Kumpati P. Acridine orange tethered chitosan reduced gold nanoparticles: a dual functional probe for combined photodynamic and photothermal therapy. RSC Adv. 2013;3:20471–9.CrossRefGoogle Scholar
  43. 43.
    Qi L, Xu Z, Jiang X, Li Y, Wang M. Cytotoxic activities of chitosan nanoparticles and copper-loaded nanoparticles. Bioorg Med Chem Lett. 2005;15:1397–9.CrossRefPubMedGoogle Scholar
  44. 44.
    Du WL, Niu SS, Xu YL, Xu ZR, Fan CL. Antibacterial activity of chitosan tripolyphosphate nanoparticles loaded with various metal ions. Carbohydr Polym. 2009;75:385–9.CrossRefGoogle Scholar
  45. 45.
    Wang Y, Li Y, Zhou Z, Zu X, Deng Y. Evolution of the zinc compound nanostructures in zinc acetate single-source solution. J Nanopart Res. 2011;13:5193–202.CrossRefGoogle Scholar
  46. 46.
    Sakohara S, Tickanen LD, Anderson MA. Luminescence properties of thin zinc oxide membranes prepared by the sol–gel technique: change in visible luminescence during firing. J Phys Chem. 1992;96:11086–91.CrossRefGoogle Scholar
  47. 47.
    Sakohara S, Ishida M, Anderson A. Visible luminescence and surface properties of nanosized ZnO colloids prepared by hydrolyzing zinc acetate. J Phys Chem B. 1998;12:10169–75.CrossRefGoogle Scholar
  48. 48.
    Sumanta KP, Rojalin S, Vadivelu M. Synthesis, structure, thermal studies on Zn(II), Cd(II) complexes of N-(2-pyridylmethyl)pyridine-2-carbaldimine and N-(2-pyridylmethyl)pyridine-2-methylketimine. Polyhedron. 2008;27:805–11.CrossRefGoogle Scholar
  49. 49.
    Ajlouni AM, Mhaidat I, Momani WA, Hijazi AK, Taha ZA, Zouby MA. Synthesis, characterization and antibacterial activity of new Cu(II) and Zn(II) complexes of schiff bases derived from 9-H-Fluoren-9-one. Jordan J Chem. 2013;8:225–36.CrossRefGoogle Scholar
  50. 50.
    Harrach T, Eckert K, Schutze-Foster K, Nuck R, Grunow D, Maurer HR. Isolation and partial characterization of basic proteinases from stem bromelain. J Protein Chem. 1995;14:41–52.CrossRefPubMedGoogle Scholar
  51. 51.
    Zeng Y, Liu Z, Wu W, Xu F, Shi J, Micropor. Combining scanning electron microscopy and fast Fourier transform for characterizing mesopore and defect structures in mesoporous materials. Mesopor Mat. 2016;220:163–7.CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  • Catiúscia P. Oliveira
    • 1
    • 2
    Email author
  • Willian A. Prado
    • 3
  • Vladimir Lavayen
    • 3
  • Sabrina L. Büttenbender
    • 3
  • Aline Beckenkamp
    • 1
  • Bruna S. Martins
    • 3
  • Diogo S. Lüdtke
    • 3
  • Leandra F. Campo
    • 3
  • Fabiano S. Rodembusch
    • 3
  • Andréia Buffon
    • 1
  • Adalberto PessoaJr
    • 4
  • Silvia S. Guterres
    • 1
  • Adriana R. Pohlmann
    • 1
    • 2
    • 3
    Email author
  1. 1.Programa de Pós-Graduação em Ciências Farmacêuticas, Faculdade de FarmáciaUniversidade Federal do Rio Grande do SulPorto AlegreBrazil
  2. 2.Departamento de Química Orgânica, Instituto de QuímicaUniversidade Federal do Rio Grande do SulPorto AlegreBrazil
  3. 3.Programa de Pós-Graduação em Química, Instituto de QuímicaUniversidade Federal do Rio Grande do SulPorto AlegreBrazil
  4. 4.Faculdade de Ciências FarmacêuticasUniversidade de São PauloSão PauloBrazil

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