Formulation and In Vitro-In Vivo Evaluation of Black Raspberry Extract-Loaded PLGA/PLA Injectable Millicylindrical Implants for Sustained Delivery of Chemopreventive Anthocyanins
- 432 Downloads
The objective of this study was to formulate and evaluate freeze-dried black raspberry (FBR) ethanol extract (RE) loaded poly(DL-lactic-co-glycolic acid) (PLGA) and poly(DL-lactic acid) (PLA) injectable millicylindrical implants for sustained delivery of chemopreventive FBR anthocyanins (cyanidin-3-sambubioside (CS), cyanidin-3-glucoside (CG) and cyanidin-3-rutinoside (CR)).
Identification and quantitation of CS, CG, and CR in RE was performed by mass spectroscopy and HPLC. RE:triacetyl-β-cyclodextrin (TA-β-CD) inclusion complex (IC) was prepared by a kneading method and characterized by X-ray diffraction (XRD), nuclear magnetic resonance spectroscopy (NMR) and UV-visible spectroscopy. RE or RE:TA-β-CD IC-loaded PLGA or PLA implants were prepared by a solvent extrusion method. In vitro and in vivo controlled release studies were conducted in phosphate-buffered saline Tween-80 (pH 7.4, 37°C) and after subcutaneous administration in male Sprague-Dawley rats, respectively. Anthocyanins were quantified by HPLC at 520 nm.
The content of CS, CG, and CR in RE was 0.2, 1.5, and 3.5 wt%, respectively. The chemical stability of anthocyanins in solution was determined to be pH-dependent, and their degradation rate increased with an increase in pH from 2.4 to 7.4. PLGA/PLA millicylindrical implants loaded with 5 or 10 wt% RE exhibited a high initial burst and short release duration of anthocyanins (35–52 and 80–100% CG + CR release after 1 and 14 days, respectively). The cause for rapid anthocyanins release was linked to higher polymer water uptake and porosity associated with the high osmolytic components of large non-anthocyanin fraction of RE. XRD, 1H NMR and UV-visible spectroscopy indicated that the non-anthocyanin fraction molecules of RE formed an IC with TA-β-CD, decreasing the hydrophilicity of RE. Formation of an IC with hydrophobic carrier, TA-β-CD, provided better in vitro/in vivo sustained release of FBR anthocyanins (16–24 and 97–99% CG + CR release, respectively, after 1 and 28 days from 20 wt% RE:TA-β-CD IC/PLA implants) over 1 month, owing to reduced polymer water uptake and porosity.
PLA injectable millicylindrical implants loaded with RE:TA-β-CD IC are optimal dosage forms for 1-month slow and continuous delivery of chemopreventive FBR anthocyanins.
KEY WORDSblack raspberry anthocyanins chemoprevention controlled release inclusion complex injectable millicylindrical implants microencapsulation oral cancer PLA PLGA Triacetyl-β-cyclodextrin
This study was supported by NIH R01 CA95901 and NIH R01 CA129609. We thank Dr. Scott Woehler, College of Pharmacy, University of Michigan, for the technical assistance with the NMR analysis.
- 7.Rodrigo KA, Rawal Y, Renner RJ, Schwartz SJ, Tian QG, Larsen PE, et al. Suppression of the tumorigenic phenotype in human oral squamous cell carcinoma cells by an ethanol extract derived from freeze-dried black raspberries, Symposium on Berries in Cancer Prevention. Lahti: Lawrence Erlbaum Assoc Inc; 2004. p. 58–68.Google Scholar
- 11.Tian QG, Giusti MM, Stoner GD, Schwartz SJ. Screening for anthocyanins using high-performance liquid chromatography coupled to electrospray ionization tandem mass spectrometry with precursor-ion analysis, product-ion analysis, common-neutral-loss analysis, and selected reaction monitoring. J Chromatogr A. 2005;1091:72–82.CrossRefPubMedGoogle Scholar
- 16.Hecht SS, Huang CS, Stoner GD, Li JX, Kenney PMJ, Sturla SJ, et al. Identification of cyanidin glycosides as constituents of freeze-dried black raspberries which inhibit anti-benzo[a]pyrene-7, 8-diol-9, 10-epoxide induced NF kappa B and AP-1 activity. Carcinogenesis. 2006;27:1617–26.CrossRefPubMedGoogle Scholar
- 29.Knobloch TJ, Casto BC, Kresty LA, Stoner GD, D’Ambrosio SM, Mallery SR, et al. Bench to bedside: chemoprevention of oral cancer by black raspberries. Cancer Epidem Biomar. 2005;14:2751S–2.Google Scholar
- 32.Shahidi F, Naczk M. Phenolics in food and nutraceuticals. Boca Raton: CRC; 2004.Google Scholar
- 36.Fischer W, Klokkers K. Crystalline cyclodextrin complexes of ranitidine hydrochloride, process for their preparation and pharmaceutical compositions containing the same. US Patent:5,665,767; 1997.Google Scholar
- 37.Rao KR, Bhanumathi N, Yadav JS, Krishnaveni NS. Inclusion complex of rifampicin, an anti-tubercular drug, with β-cyclodextrin or 2-hydroxypropyl-β-cyclodextrin and a process thereof. US Patent:7,001,893; 2006.Google Scholar
- 39.Amato ME, Lipkowitz KB, Lombardo GM, Pappalardo GC. High-field NMR spectroscopic techniques combined with molecular dynamics simulations for the study of the inclusion complexes of α- and β-cyclodextrins with the cognition activator 3-phenoxypyridine sulphate (CI-844). Magn Reson Chem. 1998;36:693–705.CrossRefGoogle Scholar
- 41.Fernandes CM, Carvalho RA, da Costa SP, Veiga FJB. Multimodal molecular encapsulation of nicardipine hydrochloride by β-cyclodextrin, hydroxypropyl-β-cyclodextrin and triacetyl-β-cyclodextrin in solution. Structural studies by 1H NMR and ROESY experiments. Eur J Pharm Sci. 2003;18:285–96.CrossRefPubMedGoogle Scholar