Surface-Engineered Breviscapine Nanocrystals for Enhanced Oral Absorption and Cardioprotection in Isoprenaline-Induced Myocardial Infarction

Figures

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  Figure 1. Schematic illustration of multifunctional R8-F127-VES@BRE-NCs for overcoming multiple oral absorption barriers. (A) Preparation of R8-F127-VES@BRE-NCs by high-pressure homogenization. (B) Proposed barrier-modulating process during intestinal absorption, including mucus penetration, R8-associated epithelial uptake, and VES-associated efflux modulation.

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  Figure 2. (A) FT-IR spectra and (B) 1H-NMR analysis of R8-F127-VES.

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  Figure 3. Appearance and morphology of R8-F127-VES@BRE-NCs. (A) Appearance of the suspensions: (i) BRE-raw; (ii) BRE-NCs; (iii) F127-VES@BRE-NCs; (iv) R8-F127-VES@BRE-NCs. (B) Appearance of the powders: (i) BRE-raw; (ii) BRE-NCs; (iii) F127-VES@BRE-NCs; (iv) R8-F127-VES@BRE-NCs. (C) SEM images of BRE-raw and the freeze-dried formulations: (i) BRE-raw; (ii) BRE-NCs; (iii) F127-VES@BRE-NCs; (iv) R8-F127-VES@BRE-NCs. Scale bars are indicated in the images.

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  Figure 4. Characteristics of R8-F127-VES@BRE-NCs. (A) Fourier transform-infrared spectroscopy. (B) Powder X-ray diffraction patterns. (C) Saturation solubility, data were expressed as mean ± SD (n = 3). Statistical analysis was performed using one-way ANOVA followed by Dunnett’s multiple comparisons test. Compared with BRE-raw, ****P<0.0001. (D) In vitro release evaluation data were expressed as mean ± SD (n = 3).

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  Figure 5. Storage stability of BRE-NCs, F127-VES@BRE-NCs, and R8-F127-VES@BRE-NCs. (A) Particle size changes of NCs formulations. (B) PDI changes of NCs formulations. (C) Zeta potential changes of NCs formulations. (D) Concentration changes of NCs formulations. All data were expressed as mean ± SD (n = 3).

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  Figure 6. In vitro mucus penetration evaluation of BRE-raw and NC formulations. (A) Particle size changes and (B) PDI changes after incubation with mucin for 0–4h. Data were expressed as mean ± SD (n = 3). (C) Schematic illustration of the PGM-coated Transwell mucus barrier model. The red arrow indicates the direction of transport from the apical (donor) chamber through the PGM mucus layer and porous membrane toward the basolateral (acceptor) chamber. (D) Apparent permeability coefficients across the mucus layer. Data were expressed as mean ± SD (n = 3). Statistical analysis was performed using one-way ANOVA followed by Dunnett’s multiple comparisons test. (compared with BRE-raw, *: p < 0.05).

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  Figure 7. (A) The effect of different concentrations of BRE-raw, BRE-NCs, F127-VES@BRE-NCs, R8-F127-VES@BRE-NCs on Caco-2 cell viability data were expressed as mean ± SD (n = 3). (B) Cellular uptake of BRE-raw, BRE-NCs, F127-VES@BRE-NCs, R8-F127-VES@BRE-NCs data were expressed as mean ± SD (n = 3). (C) Cell uptake mechanisms of BRE-raw, BRE-NCs, F127-VES@BRE-NCs, R8-F127-VES@BRE-NCs data were expressed as mean ± SD (n = 3) (I: Chlorpromazine, II: Colchicine, III: Verapamil, IV: Indomethacin) (compared with the corresponding formulation without inhibitor, &&&: p<0.001, &&&&: p<0.0001; compared to the BRE-raw, *: p<0.05, **: p<0.01, ****: p<0.0001; compared to the R8-F127-VES@BRE-NCs, ##: p<0.01, ###: p<0.001, ####: p<0.0001) (D) Schematic illustration of the Caco-2 cell monolayer Transwell model. The red arrow indicates the direction of transepithelial transport from the apical (AP) chamber across the Caco-2 cell monolayer toward the basolateral (BL) chamber. (E) The apparent permeability (Papp) of BRE-raw, BRE-NCs, F127-VES@BRE-NCs, and R8-F127-VES@BRE-NCs transport across cells data were expressed as mean ± SD (n = 3). (F) The TEER values of Caco-2 monolayers treated by NCs before and…

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  Figure 8. (A) The rate of weight change in rats data were expressed as mean ± SD (n = 6). (B) The cardiac index of different groups data were expressed as mean ± SD (n = 6). Activity of (C) CK (D) LDH (E) AST in rat serum data were expressed as mean ± SD (n = 6). Activity of (F) SOD (G) MDA (H) CAT in rat tissues data were expressed as mean ± SD (n = 6). For (B–H), statistical analysis was performed using one-way ANOVA followed by Tukey’s multiple comparisons test. (compared with the control group, &&&&: p< 0.0001; compared with the ISO group, *: p < 0.05, **: p < 0.01, ***: p < 0.001, ****: p < 0.0001; compared with the R8-F127-VES@BRE-NCs group, #: p < 0.05, ###: p < 0.001, ####: p < 0.0001).

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  Figure 9. (A) H&E staining of the (i) control group, (ii) ISO group, (iii) positive control group (metoprolol), (iv) BRE-raw group, (v) BRE-NCs group, (vi) F127-VES@BRE-NCs group, and (vii) R8-F127-VES@BRE-NCs group; scale bars: 50 μm. (B) Masson staining of the (i) control group, (ii) ISO group, (iii) positive control group (metoprolol), (iv) BRE-raw group, (v) BRE-NCs group, (vi) F127-VES@BRE-NCs group, and (vii) R8-F127-VES@BRE-NCs group; scale bars: 50 μm. (C) Infarct size of different groups data were expressed as mean ± SD (n=6). Statistical analysis was performed using one-way ANOVA followed by Tukey’s multiple comparisons test. (compared with the control group, &&&&: p< 0.0001; compared with the ISO group, ****: p < 0.0001; compared with the R8-F127-VES@BRE-NCs group, ##: p < 0.01, ####: p < 0.0001); (D) TTC staining of different groups.