Formulation and in vitro-in vivo evaluation of black raspberry extract-loaded PLGA/PLA injectable millicylindrical implants for sustained delivery of chemopreventive anthocyanins

Abstract

Purpose: 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)).

Methods: Identification and quantitation of CS, CG, and CR in RE was performed by mass spectroscopy and HPLC. RE:triacetyl-beta-cyclodextrin (TA-beta-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-beta-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 degrees C) and after subcutaneous administration in male Sprague-Dawley rats, respectively. Anthocyanins were quantified by HPLC at 520 nm.

Results: 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, (1)H NMR and UV-visible spectroscopy indicated that the non-anthocyanin fraction molecules of RE formed an IC with TA-beta-CD, decreasing the hydrophilicity of RE. Formation of an IC with hydrophobic carrier, TA-beta-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-beta-CD IC/PLA implants) over 1 month, owing to reduced polymer water uptake and porosity.

Conclusion: PLA injectable millicylindrical implants loaded with RE:TA-beta-CD IC are optimal dosage forms for 1-month slow and continuous delivery of chemopreventive FBR anthocyanins.

Fig. 1

Identification of anthocyanins in RE. Mass spectrum of RE and reference CS, CG and CR. M⁺: molecular ion peak.

Fig. 2

Identification and determination of elution profiles of anthocyanins (CS, CG and CR) by HPLC with detection at 520 nm. a: A typical HPLC profile of RE (1 mg/mL in 1.5% (v/v) phosphoric acid solution). b: Chromatogram of CS, CG and CR.

Fig. 3

Identification and determination of elution profile of degradation products of reference CR by HPLC. HPLC chromatograms of CR solution (concentration = 18 μg/mL in PBST (pH 7.4)) at 0, 3, and 24 h of incubation at 37°C. Peaks I, II, and III are degradation products of CR in the order of formation.

Fig. 4

Chemical stability of RE anthocyanins (CS, CG and CR). a: Relative degradation profile of CS (filled square), CG (filled circle) and CR (filled triangle) in the medium used for release study (PBST (pH = 7.4)). b: Effect of pH (2.4 (open inverted triangle), 5.0 (open circle), and 7.0 (open triangle)) on the degradation rate of a highly unstable anthocyanin (CR). All the RE (0.1 mg/mL in PBST (a) or buffers (b)) solutions in sealed amber-colored ampoules were incubated at 37°C. Symbols represent mean ± SE (n=3).

Fig. 5

Effect of RE loading (theoretical) on cumulative anthocyanin (sum of CG and CR) release (a) and polymer water uptake (b) characteristics of PLGA 50:50 (i.v = 0.57 dl/g) implants millicylindrical implants. Polymer water uptake characteristics of blank (filled reverse triangle) and cumulative anthocyanin release and polymer water uptake characteristics of 5 (filled circle) and 10 (open circle) wt% RE-loaded implants. Studies were carried out in PBST (pH 7.4) at 37°C and symbols represent mean ± SE, n=3.

Fig. 6

Effect of RE loading (theoretical) on inner morphology of PLGA 50:50 (i.v = 0.57 dl/g) millicylindrical implants after 1 and 14 days of immersion in PBST at 37°C. SEM images are displayed for 0 (a), 5 (b), and 10 (c) wt% RE-loaded PLGA 50:50 millicylindrical implants.

Fig. 7

Effect of polymer lactide content on cumulative anthocyanin (sum of CG and CR) release (a) and polymer water uptake (b) characteristics of millicylindrical implants. Lactide:glycolide ratio was varied from 50:50 (filled circle), 85:15 (open circle), and 100:0 (filled reverse triangle). RE loading (theoretical) in all formulations was 10 wt%, and studies were carried out in PBST (pH 7.4) at 37°C. Inherent viscosity of PLGA 50:50, PLGA 85:15, and PLA polymer was 0.57, 0.61, and 0.58 dl/g, respectively. Symbols represent mean ± SE, n=3.

Fig. 8

Effect of polymer lactide content (50–100%) on inner morphology of millicylindrical implants after 1 and 14 days of immersion in PBST at 37°C. SEM images are displayed for 10 wt% (theoretical) RE-loaded PLGA 50:50 (i.v. = 0.57 dl/g) (a), PLGA 85:15 (i.v. = 0.61 dl/g) (b), and PLA (i.v. = 0.61 dl/g) (c) implants.

Fig. 9

X-ray diffraction of the formation of an inclusion complex of RE molecules with triacetyl-β-cyclodextrin (TA-β-CD) (RE:TA-β-CD IC).

Fig. 10

Analysis of the inclusion complex formation of RE molecules with triacetyl-β-cyclodextrin (TA-β-CD) (RE:TA-β-CD IC) by NMR. Examination of change in the chemical shift of RE molecules or TA-β-CD in the presence and absence of TA-β-CD and RE. ¹H NMR spectra is shown for RE, TA-β-CD, and RE:TA-β-CD IC. The NMR samples, concentration was 1 mg/mL in 1.5% (v/v) D₃PO₄ (RE) and 80/20 (v/v) CD₃CD₂OD/1.5% (v/v) D3PO4 (TA-β-CD and RE:TA-β-CD IC).

Fig. 11

Evaluation of the interaction of FBR anthocyanins with triacetyl-β-cyclodextrin (TA-β-CD). The UV-visible spectrum of 0.1 mg/mL RE in the absence and presence of TA-β-CD (0, 1, 2, 4, 8, and 9 mg/mL). RE or RE/TA-β-CD solutions were prepared in 80/20 ethanol/1.5% (v/v) phosphoric acid solution, and spectrum was obtained after 1 h of incubation at room temperature. The spectra of RE/TA-β-CD solutions were similar to RE solution, and hence naming of spectra could not be done distinctly.

Fig. 12

Effect of formation of hydrophobic inclusion complex (IC) of RE molecules with triacetyl-β-cyclodextrin (TA-β-CD) on cumulative anthocyanin (sum of CG and CR) release (a) and polymer water uptake (b) characteristics of millicylindrical PLA (i.v. = 0.58 dl/g) implants. Cumulative anthocyanin release and polymer water uptake characteristics of 10 wt% (theoretical) RE (filled circle) and 20 wt% (theoretical) RE:TA-β-CD IC (open circle)-loaded PLA implants. Studies were carried out in PBST (pH 7.4) at 37°C and symbols represent mean ± SE, n = 3.

Fig. 13

Effect of formation of hydrophobic inclusion complex (IC) of RE molecules with triacetyl-β-cyclodextrin (TA-β-CD) on inner morphology of PLA (i.v. = 0.58 dl/g) millicylindrical implants after 1, 14 and 28 days of immersion in PBST at 37°C (a and b) or subcutaneous implantation in male Sprague-Dawley rats (c). SEM images are displayed for 10 wt% (theoretical) RE (a) and 20 wt% (theoretical) RE:TA-β-CD IC (b and c)-loaded PLA millicylindrical implants.

Fig. 14

Comparison of in vitro and in vivo cumulative anthocyanin (sum of CG and CR) release (a) and polymer water uptake (b) characteristics of 20 wt% (theoretical) RE:TA-β-CD IC-loaded millicylindrical PLA (i.v. = 0.58 dl/g) implants. Studies were carried out in PBST (pH 7.4) at 37°C (in vitro) or after subcutaneous administration in male Sprague-Dawley rats (in vivo) and symbols represent mean ± SE, n=3 (in vitro) or 5 (in vivo). In vitro data are replotted from Fig. 12 for comparison. At each time point, there was no significant difference between in vitro and in vivo anthocyanin release and polymer water uptake (P>0.05).