The overall goal of this procedure is to establish a streamlined process to extract Sporopollenin Exine Capsules, or SECs for microencapsulation applications. This method can quickly provide clean micro-capture for food and healthcare applications. The main advantage of this technique is that the SEC extraction process time can be significantly reduced to produce clean, intact SECs.
Though this method can provide insight into the streamlined SEC's extraction process, and extensive step-wise characterization, it can also be used to explore fundamental microstructural and morphological evaluations of different plant-based biomaterials. Generally individuals new to this method will struggle because of the conventional 8 to 10 days extraction process, involving highly corrosive acids at elevated temperatures. To begin this procedure, prepare a reflux set-up in a fume hood with a glass condenser, a water circulation system, and a water bath.
Since the SEC extraction process involves use of highly corrosive acids and organic solvent the process should be performed with proper personal protective equipment such as goggles, lab coat, face mask, and gloves with hazard notification. Carefully weigh 100 grams of L clavatum spores without creating dust and away from any ignition source. Then, slowly transfer the spores to a one-liter PTFE round bottom flask with a magnetic stirring rod.
Add 500 milliliters of acetone to the spores and shake the flask gently to form a homogenous suspension. Now place the flask in a water bath set at 50 degrees Celsius and connected to the reflux condenser. Heat the suspension to reflux for six hours with stirring at 200 RPM.
After allowing the suspension to cool at room temperature, filter it using filter paper under vacuum and collect the defatted spores in large glass petri dishes. Dry the spores at room temperature by covering the petri dishes with aluminum foil with holes for solvent evaporation. After transferring the dried, defatted dry spores to one-liter PTFE flask, add 500 milliliters of 85%phosphoric acid solution.
Place the flask in a water bath set at 70 degrees Celsius and connect it to the reflux condenser. Heat the suspension to reflux for 30 hours with stirring. Once the suspension has cooled to room temperature, dilute it with 500 milliliters of warm deionized water, and collect the SECs by vacuum filtration using filter paper.
Following this, transfer the SECs to a three-liter glass beaker to perform a series of washings. In the fume hood add 800 millimeters of hot deionized water to the SECs and gently stir for 10 minutes. Then, filter the suspension using filter paper under vacuum.
Transfer the SECs to a clean three-liter glass beaker. After repeating the previous steps five times add 600 milliliters of hot acetone to the SECs, and gently stir for 10 minutes. Collect the SECs by filtration under vacuum and transfer them to a clean three-liter glass beaker.
After repeating the previous steps with the appropriate solvents, transfer the clean SECs to six large glass petri dishes, and dry in a fume hood at room temperature for 24 hours to remove all water. Next, dry the SECs in a vacuum oven at 60 degrees Celsius and one millibar of vacuum for 10 hours, or until constant weight. Then, transfer the dried SECs to a polypropylene bottle for storage in a dry cabinet.
Prepare 1.2 milliliters of a Bovine Serum Albumin, or BSA solution in deionized water, and transfer it to a 50 milliliter polypropylene tube. Add 150 milligrams of SECs to the BSA solution. Vortex the mixture for 10 minutes to form a homogenous solution.
After covering the tube with filter paper, transfer it to a freeze dryer flask and dry for four hours with vacuum at one millibar. Once the BSA loaded SECs are dry, remove the agglomerates with a mortar and pestle. Now transfer the BSA loaded SECs to a 50 milliliter tube and add two milliliters of deionized water to remove the residual BSA.
Collect the SECs by centrifugation at 4, 704 for five minutes. When finished, discard the supernatant. Repeat the washing with deionized water.
Following this, cover the tube containing the BSA loaded SECs with filter paper. Then, transfer the SECs to a freezer for one hour. Freeze dry the SECs for 24 hours.
Store the SECs in the freezer until further characterization. Use five milligrams of the BSA loaded SECs in a two-milliliter polypropylene tube. Add 1.4 milliliters of PBS and vortex for five minutes.
Following this, probe sonicate the suspension at 40%amplitude for three cycles of ten seconds. When finished, filter the solution using 0.45 micron polyethersulfone filter. After performing the previous steps with the placebo SECs, measure the absorbents of the BSA loaded SECs at 280 nanometers using placebo as a blank.
Finally, quantify the amount of BSA encapsulated using a BSA standard curve in PBS. SEM images indicate intact spores with a unique micro-structure and before processing micron scale sporoplasmic organelles were observed in the cross-sectional image. With spore defatting and alkali treatment, no morphological and structural changes were observed.
SEM images after acidolysis show intact SECs devoid of sporoplasmic materials. No substantial proteinaceous material removal is observed in defatted and alkali treated SECs by CHN analysis. The majority of proteinaceous materials is removed with acidolysis.
DIPA analysis shows the uniform size distribution of SECs after acidolysis for up to 30 hours. SEC's structural integrity decreases with prolonged acidolysis. SEM and DIPA data show that after the defatting step, acidolysis produced intact SECs with a uniform sized distribution.
CLSM data indicates that untreated SECs exhibit auto fluorescence due to sporoplasm constituents inside the spore cavity. After acidolysis only processing all sporoplasmic contents are removed. After encapsulation of FITC-BSA green florescence is observed inside the SECs.
The SEM images show intact micro-ridges after BSA loading. No substantial changes were observed in diameter, circularity, and aspect ratio after BSA loading into SECs. BSA quantification from encapsulated SECs confirms the consistency of the microencapsulation process with 0.170 plus or minus 0.01 grams of BSA loading per gram of SECs.
Once mastered this streamlined SEC extraction process can be done in 48 hours if it is performed properly. While attempting this procedure for various plant spores and pollen, it is important to remember step-wise evaluation of structural morphological and elemental analysis to achieve intact and clean capsules. Following this procedure, other extraction processes involving alkali enzymes can be performed in order to check the feasibility of the optimized process for diverse plant-based capsules.
After its development, this technique paved the way for researchers in the field of microencapsulation, drug delivery, and tissue engineering to explore diverse plant-based SECs as next generation biomaterials. After watching this video, you should have a good understanding of how to extract and characterize SECs from plant spores and pollen grains, as well as perform encapsulation of drugs and biomacromolecules into SECs.
