Full-text (PDF). Available from: Jeongeun Seo, Nov 03, 2015 ... Hitomi Shirahama, Jae Ho Lee, Jeongeun Seo, and Nam-Joon. Cho*. 1 ..... Cosmetics Company.
Copyright WILEY‐VCH Verlag GmbH & Co. KGaA, 69469 Weinheim, Germany, 2015.
Supporting Information
for Small, DOI: 10.1002/smll.201500860
Natural Sunflower Pollen as a Drug Delivery Vehicle Raghavendra C. Mundargi, Michael G. Potroz, Soohyun Park, Hitomi Shirahama, Jae Ho Lee, Jeongeun Seo, and Nam-Joon Cho*
Copyright WILEY-VCH Verlag GmbH & Co. KGaA, 69469 Weinheim, Germany, 2013.
Supporting Information Natural Sunflower Pollen as a Drug Delivery Vehicle Raghavendra C. Mundargi, Michael G. Potroz, Soohyun Park, Hitomi Shirahama, Jae Ho Lee, Jeongeun Seo, Nam-Joon Cho*
1. Methods 1.1. Microencapsulation techniques to load macromolecule (BSA) into natural sunflower pollen grains: 1.1.1. Passive filling: Dissolve 75 mg BSA into 0.5 mL purified water in a 1.5 mL polypropylene tube and suspend 150 mg natural pollen grains into BSA solution. Mix the suspension by vortex (VWR, Singapore) for 5 min and transfer the tube to thermoshaker (Hangzhou Allsheng Inst. Singapore) at 4°C, 500 rpm for 2 h. Stop the process and collect the BSA-loaded particles by centrifugation at 12000 rpm for 4 min. Wash using 0.5 ml water and centrifuge to remove surface adhered BSA. Freeze the pollen grains in a freezer at -70°C for 30 min and freeze dry for 24 h. The resultant particles are stored in -20°C for further characterization. Prepare the placebo pollen grains with the same procedure without BSA and preserve in -20°C. 1.1.2. Compression filling: Fill 150 mg natural sunflower pollen grains into 12 mm die and compress to form a tablet of around 13 mm diameter under hydraulic press to provide 5 ton load for 20 sec in 132.75 mm2 area using FTIR pellet maker. The pellet formed in this method is soaked in 0.5 mL of 75 mg BSA containing aqueous solution in 20 mL flat glass bottle for 2 h at 4°C to allow swelling of pollen grains thereby BSA is entrapped in the pollen grains. Stop the process and collect the BSA-loaded particles by centrifugation at 12000 rpm for 4 min. Wash using 0.5 ml water and centrifuge to remove surface bound BSA. Freeze the pollen grains in a freezer at -70°C for 30 min and freeze-dry for 24 h. The resultant particles are stored in -20°C for further characterization. Prepare the placebo pollen grains with the same procedure without BSA and preserve in -20°C. 1.1.3. Vacuum filling: Dissolve 75 mg BSA into 0.5 mL purified water in a 1.5 mL centrifuge tube and suspend 150 mg pollen grains and vortex for 5 min to homogenize. Apply a vacuum at 2 mbar for 2 h using a freeze-drier. Stop the process and collect BSA-loaded pollen grains by centrifugation at 12000 rpm for 4 min. Wash using 0.5 ml water and centrifuge to remove surface bound BSA, then freeze the pollen grains in a freezer at -70°C for 30 min and freeze 1
dry for 24 h. The resultant particles are stored in -20°C for further characterization. Prepare the placebo pollen grains with the same procedure without BSA and preserve in -20°C. In order to predict localization of BSA into natural pollen grains, FITC-conjugated BSA was encapsulated by three different techniques as mentioned in section 1.1. with a batch size of 22.5 mg containing 7.5 mg FITC-BSA per batch of natural pollen grains.
1.2. Characterization of natural and macromolecule-loaded natural pollen grains 1.2.1. Dynamic image particle analysis by FlowCam®: FlowCam VS benchtop system (FlowCam® VS, Fluid Imaging Technologies, Maine, USA) was equipped with a 200 μm flow cell (FC-200), a 20X magnification lens (Olympus®, Japan) and controlled by the visual spreadsheet software version 3.4.11. The system was flushed with 1 mL deionized water (Millipore, Singapore) at a flow rate of 0.5 ml/min and flow cell cleanliness was monitored visually before each sample run. Natural sunflower pollen grains and macromolecule-loaded grains of 0.5 mL (2 mg/ml) with a pre-run volume of 0.5 mL (primed manually into the flow cell) were analyzed with a flow rate of 0.1 ml/min and a camera rate of 10 frames/s leading to a sampling efficiency of about 9 %. A minimum of 10,000 particles were fixed as the count for each measurement and three separate measurements were performed and data analysis was carried out using 1000 well focused pollen grains segregated by edge gradient. The instrument was calibrated using polystyrene microspheres (50 ± 1 µm, Thermoscientific, USA) Representative data was plotted as a histogram and fitted with a Gaussian curve and values are reported with standard deviations. The DIPA uses a high-resolution digital camera and objective lens to capture images of the particles flowing through a thin transparent flow cell. Particle size uniformity data is then generated based on digital signal processing of the images. Besides size determination, the digital particle images allow us to obtain additional information including edge gradient, circularity, and the shape of pollen grains. Size, edge gradient and circularity analysis by FlowCam was performed with an initial particle count of 10,000 pollen grains for all the batches of pollen formulations and images were processed using the FlowCam software to obtain 1000 well focused pollen grains.
1.2.2. Surface morphology evaluation by scanning electron microscopy (SEM): SEM imaging performed using FESEM 7600F (JEOL, Japan). Samples were coated by platinum with a thickness of 10 nm by using JFC-1600 (JEOL, Japan) (20mA, 60 sec) and images were recorded by employing FESEM with an acceleration voltage of 5.00 kV at different magnifications to observe morphological characteristics. 2
1.2.3. Confocal laser scanning microscopy analysis of natural and macromolecule loaded natural pollen grains: Confocal laser scanning micrographic analysis was performed using a Carl Zeiss LSM700 (Germany) confocal microscope equipped with three spectral reflected/fluorescence detection channels, six laser lines (405/458/488/514/543/633 nm) and connected to Z1 inverted microscope (Carl Zeiss, Germany). Natural and macromoleculeloaded pollen grains were mounted on sticky slides (Ibidi, Germany), a drop of mounting medium (Vectashield®) was added and pollen grains were covered with another sticky slide. Images were collected immediately under the following conditions: laser excitation lines 405 nm (6.5 %), 488 nm (6 %) and 633 nm (6 %) with DIC in an EC Plan-Neofluar 100x 1.3 oil objective M27 lens. Fluorescence from natural and macromolecule loaded pollen grains were collected in photomultiplier tubes equipped with the following emission filters; 416-477, 498550, 572-620. The laser scan speed was set at 67 sec per each phase (1024x1024: 84.94 µm2 sizes) and plane mode scanning with 3.15 µsec of pixel dwell. The iris was set as optimal for the sample conditions and all images were captured at the mid region of the particle (optical sections) and other settings were fixed the same for all samples and at least three images were captured for each different sample and all images were processed and converted under the same conditions using software ZESS 2008 (ZEISS, Germany). 1.3. Encapsulation efficiency: Suspend 5 mg BSA-loaded SP in 1.6 mL PBS vortex for 5 min and probe sonication for 10 sec (3 cycles, 40 % amplitude). Filter the solution to collect extracted BSA using 0.45 µm PES syringe filters. Measure the absorbance at 280 nm using a placebo extract as a blank to compute the amount of BSA in the pollen grains as below: Amount of BSA (mg) = Absorbance x dilution factor Slope (standard curve) x 1000 % Loading = Amount of BSA x 100 Weight of pollen grains % Encapsulation efficiency = Practical loading x 100 Theoretical loading
1.4. In-vitro drug release evaluation in simulated gastric fluid (0.1 M HCl, pH 1.2): Suspend 5 mg BSA-loaded pollen grains and placebo in pH 1.2 solution. Incubate at 37°C at 50 rpm and collect the release samples at 5 min, 15 min and 30 min by centrifugation at 14000 rpm for 30 sec, replenish with fresh release media and continue the release study. Filter the release sample using PES membrane filters and measure absorbance at 280 nm using a placebo release sample as a blank. Compute the amount of BSA released using a BSA standard curve. 3
1.5. In-vitro drug release evaluation in simulated intestinal fluid (PBS pH 7.4): Suspend 5 mg BSA-loaded pollen grains and placebo in pH 7.4 solution. Incubate at 37°C at 50 rpm and collect the release samples at 5 min, 15 min and 30 min by centrifugation at 14000 rpm for 30 sec, replenish with fresh release media and continue the release study. Filter the release sample using PES membrane filters and measure absorbance at 280 nm using a placebo release sample as a blank. Compute the amount of BSA released using a BSA standard curve.
1.6. Formulation of macromolecule-loaded natural pollen grains into alginate beads: In order to control the macromolecule release from natural pollen grains, these are incorporated into alginate beads. 150 mg of BSA-loaded (vacuum loading) pollen grains were mixed homogeneously with 1.5 ml alginate (2 %) in water. The resulting suspension was slowly added using blunt 18 G needle to a 10 ml calcium chloride (8 %) solution under magnetic stirring, the stirring was continued for 5 minutes and the calcium chloride was removed by centrifugation (1500 rpm, 2 minutes) followed by washing twice with 1.5 ml water and freeze-drying for 24 h. All the formulations were stored in -20°C for further studies. 1.7. Statistical analysis: Statistical analysis was performed using two-tailed t –tests and P
