Pluronic®/casein micelles for ophthalmic delivery of resveratrol: In vitro, ex vivo, and in vivo tests
Abstract
Ocular health may strongly benefit from the supply of antioxidant agents that counteract free radicals and reactive oxygen species responsible for long-term eye diseases. Additionally, natural antioxidants like resveratrol can inhibit bacteria growth and restore natural microbiota. However, their use is hindered by limited solubility, fast degradation, and low ocular permeability. This work aimed to overcome these limitations by preparing single and mixed micelles of Pluronic® F127 and casein that serve as resveratrol nanocarriers. Single and mixed (0.1 % casein) micelles (0.0 to −17.0 mV; 2.4 to 32.7 nm) increased 50-fold resveratrol solubility, remained stable for one month at 4 °C, withstood fast dilution, underwent sol-to-gel transitions in the 23.9–27.1 °C range, and exhibited potent antioxidant properties. All formulations successfully passed the HET-CAM assay but showed Pluronic®-casein dose-dependent toxicity in the zebrafish embryo model. Resveratrol-loaded single and mixed micelles (10–15 mM Pluronic® F127) displayed antimicrobial activity against S. aureus and P. aeruginosa. The micelles favored resveratrol accumulation in cornea and sclera, but mixed micelles showed larger lag times and provided lower amount of resveratrol permeated through sclera. In vivo (rabbit) tests confirmed the safety of resveratrol-loaded single micelles and their capability to supply resveratrol to anterior and posterior eye segments.
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SUPPLEMENTARY MATERIAL Pluronic®/casein micelles for ophthalmic delivery of resveratrol: in vitro, ex vivo, and in vivo tests Maria Vivero-Lopez1, Chiara Sparacino1, Ana Quelle-Regaldie2, Laura Sánchez2,3, Eva Candal4, Antón Barreiro-Iglesias4, Fernando Huete-Toral5, Gonzalo Carracedo5, Ana Otero6, Angel Concheiro1, Carmen Alvarez-Lorenzo1,* 1Departamento de Farmacología, Farmacia y Tecnología Farmacéutica, I+D Farma (GI-1645), Facultad de Farmacia, Instituto de Materiales (iMATUS) and Health Research Institute of Santiago de Compostela (IDIS), Universidade de Santiago de Compostela, 15782 Santiago de Compostela, Spain 2Departamento de Zooloxía, Xenética y Antropoloxía Física, Facultade de Veterinaria, Universidade de Santiago de Compostela, 27002 Lugo, Spain 3Preclinical Animal Models Group, Health Research Institute of Santiago de Compostela (IDIS), 15706 Santiago de Compostela, Spain 4Department of Functional Biology, CIBUS, Faculty of Biology, Universidade de Santiago de Compostela, 15782 Santiago de Compostela, Spain 5Ocupharm Research Group, Faculty of Optics and Optometry, University Complutense of Madrid, C/Arcos del Jalon 118, 28037 Madrid, Spain 6Departamento de Microbiología y Parasitología, Facultad de Biología, Edificio CIBUS, Universidade de Santiago de Compostela, 15782 Santiago de Compostela, Spain Table S1. Size (by number), polydispersion index (PDI), Z-potential and pH of single and mixed micelles in PBS pH 7 at 10 ºC before being loaded with resveratrol (mean values ± standard deviations). Formulation Size (nm) PDI Z-Pot (mV) pH P2.5 3.1 ± 0.2 0.6 ± 0.3 -3.4 ± 1.6 7.3 ± 0.1 P5 3.2 ± 0.4 0.6 ± 0.2 -1.3 ± 0.4 7.3 ± 0.1 P7.5 2.6 ± 0.0 0.6 ± 0.1 -4.2 ± 1.2 7.3 ± 0.1 P10 2.2 ± 0.2 1.5 ± 0.1 -1.0 ± 0.4 7.4 ± 0.0 P15 2.2 ± 0.2 0.7 ± 0.2 -1.1 ± 0.6 7.4 ± 0.1 PC2.5 11.9 ± 19.0 0.9 ± 0.2 -11.4 ± 1.4 7.0 ± 0.0 PC5 80.8 ± 128.3 0.7 ± 0.2 -7.1 ± 0.7 7.0 ± 0.1 PC7.5 206.6 ± 136.2 0.6 ± 0.0 -5.1 ± 0.8 7.1 ± 0.0 PC10 239.0 ± 84.1 0.7 ± 0.2 -4.4 ± 0.8 7.2 ± 0.0 PC15 639.3 ± 271.1 0.5 ± 0.1 -2.5 ± 0.6 7.3 ± 0.0 C 73.1 ± 31.1 0.5 ± 0.0 -6.1 ± 0.8 7.0 ± 0.0
SUPPLEMENTARY MATERIAL Table S2. Size (by number), polydispersion index (PDI), Z-potential and pH of single and mixed micelles in PBS pH 7 at 35 ºC with and without resveratrol (mean values ± standard deviations). Formulation Size (nm) PDI Z-Pot (mV) pH P2.5R 8.1 ± 3.6 0.2 ± 0.1 0.2 ± 0.4 7.00 ± 0.01 P5R 5.8 ± 0.9 0.3 ± 0.1 0.0 ± 0.3 6.98 ± 0.01 P10R 4.2 ± 0.2 0.5 ± 0.1 0.0 ± 0.3 7.03 ± 0.01 PC2.5R 18.2 ± 3.1 0.7 ± 0.1 -12.6 ± 1.0 6.92 ± 0.02 PC5R 5.6 ± 0.5 0.7 ± 0.0 -8.4 ± 0.9 6.93 ± 0.01 PC10R 5.0 ± 0.5 1.0 ± 0.0 -2.7 ± 0.4 6.96 ± 0.01 P2.5 8.4 ± 3.9 0.2 ± 0.1 -0.5 ± 0.4 7.03 ± 0.02 P5 5.4 ± 1.4 0.3 ± 0.0 -0.4 ± 0.6 7.04 ± 0.03 P10 4.3 ± 0.3 0.4 ± 0.2 0.0 ± 0.3 7.11 ± 0.01 PC2.5 13.9 ± 14.6 0.7 ± 0.1 -10.2 ± 0.4 6.89 ± 0.01 PC5 5.3 ± 0.6 0.8 ± 0.1 -6.8 ± 0.6 6.91 ± 0.00 PC10 4.6 ± 0.4 1.3 ± 0.0 -1.9 ± 0.2 6.99 ± 0.00 Table S3. Detachment force and mucoadhesion work recorded for the formulations tested in cornea and sclera. The experiments were carried out by subjecting each tissue to repeated compression cycles placing alternatively a drop of formulation or a drop of PBS pH 7 (control) between the tissue and the substrate. Mean values and standard deviations (n=5). Two independent experiments were carried out for each tissue and formulation. Formulation Cornea Sclera Fmax (N) Work (N·mm) Fmax (N) Work (N·mm) P10R-test1 0.382 ± 0.043 0.070 ± 0.015 0.359 ± 0.063 0.060 ± 0.015 PBS-test1 0.212 ± 0.028 0.032 ± 0.006 0.316 ± 0.055 0.059 ± 0.040 P10R-test2 0.245 ± 0.018 0.068 ± 0.010 0.520 ± 0.156 0.086 ± 0.016 PBS-test2 0.154 ± 0.008 0.037 ± 0.002 0.456 ± 0.154 0.071 ± 0.016 PC10R-test1 0.240 ± 0.035 0.066 ± 0.006 0.629 ± 0.247 0.096 ± 0.033 PBS-test1 0.173 ± 0.048 0.043 ± 0.007 0.507 ± 0.178 0.078 ± 0.020 PC10R-test2 0.350 ± 0.072 0.080 ± 0.024 0.308 ± 0.079 0.051 ± 0.011 PBS-test2 0.251 ± 0.038 0.044 ± 0.009 0.277 ± 0.079 0.040 ± 0.016
SUPPLEMENTARY MATERIAL Table S4. Resveratrol levels (µg/mL) in tear fluid after one drop (50 µL) instillation of P10R micelles in the right eye of male New Zealand white rabbits. Individual values (n= 4) and mean values ± standard deviations (s.d.). Time (h) Eye 1 Eye 2 Eye 3 Eye 4 Mean ± s.d. 0.083 176.94 138.00 3230.14 1476.73 1255 ± 1456 0.25 1265.38 16.50 144.87 192.70 404 ± 578 0.50 154.59 197.68 69.29 56.00 119 ± 68 1 206.91 31.78 43.75 11.31 73.4 ± 89.9 2 48.51 7.77 22.90 7.20 21.6 ± 19.4 3 29.76 10.94 13.78 5.93 15.1 ± 10.3 4 23.93 1.95 4.86 5.15 8.97 ± 10.07 5 101.03 5.73 4.19 1.34 28.1 ± 48.7 6 10.39 5.76 8.89 2.24 6.82 ± 3.61 7 10.35 5.44 2.38 6.06 6.06 ± 3.28 8 4.08 8.57 6.24 0.70 4.90 ± 3.35 Table S5. Resveratrol levels (ng/g) in eye tissues 8 hours after one drop (50 µL) instillation of P10R micelles in the right eye of male New Zealand white rabbits. Individual values (n= 4) and mean values ± standard deviations (s.d.). Tissue Eye 1 Eye 2 Eye 3 Eye 4 Mean ± s.d. Cornea 8.24 47.96 53.11 63.39 43.17 ± 24.15 Aqueous humor 0.883 0.158 n.d. 0.643 0.56 ± 0.37 Lens 0.118 0.531 0.025 1.922 0.65 ± 0.88 Sclera 0.711 1.937 n.d. 2.272 1.64 ± 0.82 Vitreous humor 0 0.076 0 0.212 0.07 ± 0.10 Retina 2.373 2.428 0.217 0.068 1.27 ± 1.31
SUPPLEMENTARY MATERIAL Figure S1. Size distribution by intensity (%) at 10 ºC for single (A, C) and mixed (B, D) micelles before (A, B) and after (C, D) being loaded with resveratrol. Figure S2. Size distribution by number (%) at 10 ºC for single (A, C) and mixed (B, D) micelles before (A, B) and after (C, D) being loaded with resveratrol. Intensity (%) P2.5 P5 P7.5 P10 P15 PC2.5 PC5 PC7.5 PC10 PC15 C Size (nm) 110 100 1000 Intensity (%) P2.5R P5R P7.5R P10R P15R Size (nm) 110 100 1000 PC2.5R PC5R PC7.5R PC10R PC15R CR AB CD Number (%) P2.5 P5 P7.5 P10 P15 PC2.5 PC5 PC7.5 PC10 PC15 C Size (nm) 110 1000 Number (%) P2.5R P5R P7.5R P10R P15R Size (nm) 110 100 1000 PC2.5R PC5R PC7.5R PC10R PC15R CR AB CD
SUPPLEMENTARY MATERIAL Figure S3. Size (nm) of all formulations loaded (A) and not (B) with resveratrol at 0, 15 and 30 days storage at 4 °C protected from light. Figure S4. PDI of all formulations loaded (A) and not (B) with resveratrol at 0, 15 and 30 days storage at 4 °C protected from light. P2.5R P5R P7.5R P10R P15R PC2.5R PC5R PC7.5R PC10R PC15R Size (nm) 1 10 100 1000 0 days 15 days 30 days P2.5P5 P7.5 P10 P15 PC2.5 PC5 PC7.5 PC10 PC15 AB P2.5R P5R P7.5R P10R P15R PC2.5R PC5R PC7.5R PC10R PC15R PDI 0.0 0.5 1.0 1.5 2.0 0 days 15 days 30 days P2.5P5 P7.5 P10 P15 PC2.5 PC5 PC7.5 PC10 PC15 AB
SUPPLEMENTARY MATERIAL Figure S5. Z-potential of all formulations loaded (A) and not (B) with resveratrol at 0, 15 and 30 days storage at 4 °C protected from light. Figure S6. pH values of all formulations loaded (A) and not (B) with resveratrol at 0, 15 and 30 days storage at 4 °C protected from light. P2.5R P5R P7.5R P10R P15R PC2.5R PC5R PC7.5R PC10R PC15R Z-potential (mV) -25 -20 -15 -10 -5 0 0 days 15 days 30 days P2.5P5 P7.5 P10 P15 PC2.5 PC5 PC7.5 PC10 PC15 AB P2.5R P5R P7.5R P10R P15R PC2.5R PC5R PC7.5R PC10R PC15R pH values 0 2 4 6 8 10 0 days 15 days 30 days P2.5P5 P7.5 P10 P15 PC2.5 PC5 PC7.5 PC10 PC15 AB
SUPPLEMENTARY MATERIAL Figure S7. Evolution of the absorbance of resveratrol-loaded micelles after 100-fold dilution in simulated lachrymal fluid pH 7.5. Time (min) 0 5 10 15 20 25 30 Absorbance (305 nm) 0.0 0.5 1.0 1.5 2.0 2.5 3.0 P2.5R P5R P7.5R P10R P15R PC2.5R PC5R PC7.5R PC10R PC15R CR
SUPPLEMENTARY MATERIAL Figure S8. Images of the HET-CAM test. Formulation codes as in Table 1 (R indicates formulations loaded with resveratrol). Controls (-Control and +Control) refer to 0.9% NaCl and 0.1 N NaOH solutions.
SUPPLEMENTARY MATERIAL Figure S9. Antioxidant activity of resveratrol-loaded Pluronic® F127 single micelles (up) compared to the non-loaded counterparts (down). Figure S10. Antioxidant activity of resveratrol-loaded Pluronic® F127/casein mixed micelles (up) compared to the non-loaded counterparts (down). P2.5R P5R P7.5R P10R P15R P2.5 P5 P7.5 P10 P15 PC2.5R PC5R PC7.5R PC10R PC15R PC2.5 PC5 PC7.5 PC10 PC15