Name: extraction of plant-based capsules for microencapsulation applications
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Description: Watch this Scientific Journal Video about Extraction of Plant-based Capsules for Microencapsulation Applications at JoVE.com

This work was supported by the National Research Foundation (NRF-NRFF2011-01) and the National Medical Research Council (NMRC/CBRG/0005/2012).

1. Extraction of Sporopollenin Exine Capsules (SECs) from L. clavatum Spores
Note: The SEC extraction process involves a flammable powder (L. clavatum), hot corrosive acids, and flammable solvents, hence proper personal protective equipment (goggles, face mask, gloves, lab coat), approved risk assessment on usage, and disposal of chemicals by authorized laboratory personnel is essential.
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	<li>Spore Defatting
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		<li>Prepare a reflux set-up in a fume hood by using a gla...

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	<li>Paunov, V. N., Mackenzie, G., Stoyanov, S. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sporopollenin+micro-reactors+for+in-situ+preparation,+encapsulation+and+targeted+delivery+of+active+components.">Sporopollenin micro-reactors for in-situ preparation, encapsulation and targeted delivery of active components.</a> J. Mater. Chem. 17 (7), 609-612 (2007).</li><li>Barrier, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Physical and chemical properties of sporopollenin exine particles. Doctoral dissertation thesis. , (2008).</li><li>Beckett, S. T., Atkin, S. L., Mackenzie, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dosage+Form.">Dosage Form.</a> US Patent. , (2009).</li><li>Lorch, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MRI+contrast+agent+delivery+using+spore+capsules:+controlled+release+in+blood+plasma.">MRI contrast agent delivery using spore capsules: controlled release in blood plasma.</a> Chem. Comm. (42), 6442-6444 (2009).</li><li>Wakil, A., Mackenzie, G., Diego-Taboada, A., Bell, J. G., Atkin, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhanced+bioavailability+of+eicosapentaenoic+acid+from+fish+oil+after+encapsulation+within+plant+spore+exines+as+microcapsules.">Enhanced bioavailability of eicosapentaenoic acid from fish oil after encapsulation within plant spore exines as microcapsules.</a> Lipids. 45 (7), 645-649 (2010).</li><li>Barrier, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sporopollenin+exines:+A+novel+natural+taste+masking+material.">Sporopollenin exines: A novel natural taste masking material.</a> LWT-Food Sci. Technol. 43 (1), 73-76 (2010).</li><li>Barrier, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Viability+of+plant+spore+exine+capsules+for+microencapsulation.">Viability of plant spore exine capsules for microencapsulation.</a> J. Mater. Chem. 21 (4), 975-981 (2011).</li><li>Hamad, S. A., Dyab, A. F., Stoyanov, S. D., Paunov, V. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Encapsulation+of+living+cells+into+sporopollenin+microcapsules.">Encapsulation of living cells into sporopollenin microcapsules.</a> J. Mater. Chem. 21 (44), 18018-18023 (2011).</li><li>Diego-Taboada, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sequestration+of+edible+oil+from+emulsions+using+new+single+and+double+layered+microcapsules+from+plant+spores.">Sequestration of edible oil from emulsions using new single and double layered microcapsules from plant spores.</a> J. Mater. Chem. 22 (19), 9767-9773 (2012).</li><li>Diego-Taboada, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+free+microcapsules+obtained+from+plant+spores+as+a+model+for+drug+delivery:+Ibuprofen+encapsulation,+release+and+taste.">Protein free microcapsules obtained from plant spores as a model for drug delivery: Ibuprofen encapsulation, release and taste.</a> Mater. Chem. B. 1 (5), 707-713 (2013).</li><li>Atwe, S. U., Ma, Y., Gill, H. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pollen+grains+for+oral+vaccination.">Pollen grains for oral vaccination.</a> J. Control. Release. 194, 45-52 (2014).</li><li>Mackenzie, G., Beckett, S., Atkin, S., Diego-Taboada, A., Gaonkar, A. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ch+24.">Ch 24.</a> Microencapsulation in the Food Industry: A Practical Implementation Guide. , 283-297 (2014).</li><li>Diego-Taboada, A., Beckett, S. T., Atkin, S. L., Mackenzie, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hollow+Pollen+Shells+to+Enhance+Drug+Delivery.">Hollow Pollen Shells to Enhance Drug Delivery.</a> Pharmaceutics. 6 (1), 80-96 (2014).</li><li>Ma, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+a+novel+rape+pollen+shell+microencapsulation+and+its+use+for+protein+adsorption+and+pH-controlled+release.">Preparation of a novel rape pollen shell microencapsulation and its use for protein adsorption and pH-controlled release.</a> J. Microencapsul. 31 (7), 667-673 (2014).</li><li>Archibald, S. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+does+iron+interact+with+sporopollenin+exine+capsules?+An+X-ray+absorption+study+including+microfocus+XANES+and+XRF+imaging.">How does iron interact with sporopollenin exine capsules? An X-ray absorption study including microfocus XANES and XRF imaging.</a> J. Mater. Chem. B. 2 (8), 945-959 (2014).</li><li>Southworth, D., et al., Blackmore, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ch+10.">Ch 10.</a> Microspores Evolution and Ontogeny: Evolution and Ontogeny. , 193-212 (2013).</li><li>Stanley, R. G., Linskens, H. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Pollen: Biology, Biochemistry, Management. , 307 (1974).</li><li>Ariizumi, T., Toriyama, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+regulation+of+sporopollenin+synthesis+and+pollen+exine+development.">Genetic regulation of sporopollenin synthesis and pollen exine development.</a> Annu. Rev. Plant Biol. 62, 437-460 (2011).</li><li>Mundargi, R. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Natural+Sunflower+Pollen+as+a+Drug+Delivery+Vehicle.">Natural Sunflower Pollen as a Drug Delivery Vehicle.</a> Small. 12 (9), 1167-1173 (2015).</li><li>Mundargi, R. 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Chem. 461 (1), 89-109 (1928).</li><li>Zetschke, F., Kaelin, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Untersuchungen+&#252;ber+die+membran+der+sporen+und+pollen+v.+4.+Zur+autoxydation+der+sporopollenine.">Untersuchungen &#252;ber die membran der sporen und pollen v. 4. Zur autoxydation der sporopollenine.</a> Helv. Chim. Acta. 14 (1), 517-519 (1931).</li><li>Zetzsche, F., Vicari, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Untersuchungen+&#252;ber+die+Membran+der+Sporen+und+Pollen+III.+2.+Picea+orientalis+Pinus+silvestris+L.,+Corylus+Avellana+L.">Untersuchungen &#252;ber die Membran der Sporen und Pollen III. 2. Picea orientalis Pinus silvestris L., Corylus Avellana L.</a> Helv. Chim. 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Mitteilung &#252;ber die Membran der Sporen und Pollen.</a> Journal f&#252;r Praktische Chemie. 148 (9-10), 267-286 (1937).</li><li>Shaw, G., Yeadon, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemical+studies+on+the+constitution+of+some+pollen+and+spore+membranes.">Chemical studies on the constitution of some pollen and spore membranes.</a> Grana. 5 (2), 247-252 (1964).</li><li>Shaw, G., Yeadon, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemical+studies+on+the+constitution+of+some+pollen+and+spore+membranes.">Chemical studies on the constitution of some pollen and spore membranes.</a> J. Chem. Soc. C Org. , 16-22 (1966).</li><li>Dom&#236;nguez, E., Mercado, J. A., Quesada, M. A., Heredia, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pollen+sporopollenin:+degradation+and+structural+elucidation.">Pollen sporopollenin: degradation and structural elucidation.</a> Sex. Plant Reprod. 12 (3), 171-178 (1999).</li><li>Mundargi, R. C., Tan, E. L., Seo, J., Cho, N. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Encapsulation+and+controlled+release+formulations+of+5-fluorouracil+from+natural+Lycopodium+clavatum+spores.">Encapsulation and controlled release formulations of 5-fluorouracil from natural Lycopodium clavatum spores.</a> J. Ind. Eng. 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R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lycopodine+triggers+apoptosis+by+modulating+5-lipoxygenase,+and+depolarizing+mitochondrial+membrane+potential+in+androgen+sensitive+and+refractory+prostate+cancer+cells+without+modulating+p53+activity:+signaling+cascade+and+drug-DNA+interaction.">Lycopodine triggers apoptosis by modulating 5-lipoxygenase, and depolarizing mitochondrial membrane potential in androgen sensitive and refractory prostate cancer cells without modulating p53 activity: signaling cascade and drug-DNA interaction.</a> Eur. J. 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In this work, a systematic analysis of SEC extraction from L. clavatum spores is presented and this report shows that it is possible to produce higher quality capsules while also achieving a significant streamlining of the pre-existing commonly used protocol.11 In contrast to the existing protocol requiring a high process temperature (180 &#176;C) and a long process duration (7 days),11 the current SEC extraction processing optimization was primarily focused on reducing the temperature and ...

There is significant interest in natural plant-based capsules obtained from plant spores and pollens for use in microencapsulation applications.1-15 In nature, spores and pollen provide protection for sensitive genetic materials against harsh environmental conditions. The basic structure of plant spores and pollen typically comprises an outer shell layer (exine), an inner shell layer (intine), and the internal cytoplasmic material. The exine is comprised of a chemically robust biopolymer1,9,10,13,16 referred to as sporopollenin and the intine is comprised primarily of cellulosic materials.16-18 Empty capsules can be isolated by various processes7,9 for removing cytoplasmic material, proteins, and the intine layer.2,12,16 These sporopollenin exine capsules (SECs) provide a compelling alternative to synthetic encapsulants due to their narrow size distribution and uniform morphology.7,9,13,19,20 The development of standardized processes to obtain SECs from various plant species, such as Lycopodium clavatum, opens the potential for a wide range of microencapsulation applications in the fields of drug delivery, foods, and cosmetics.6,10-13,21
In order to obtain SECs, researchers first treated spores and pollen with organic solvents and refluxed in alkaline solutions to remove cytoplasmic contents.22-25 However, the remaining capsule structure was determined to still contain the cellulosic intine layer. In order to remove this, researchers explored the use of prolonged acidic hydrolysis processing using hydrochloric acid, hot sulfuric acid, or hot phosphoric acid over several days,22-25 with phosphoric acid becoming the preferred method of SEC intine removal.2 However, ongoing research over the years has shown that various spores and pollens have differing degrees of resilience to the harsh processing methods commonly used.26,27 Some spores and pollens are completely degraded and lose all structural integrity in strong alkaline solutions, or become heavily damaged in strong acidic solutions.16 The variability in SEC response to treatment conditions is due to subtle variations in the chemical structure and exine morphology of the sporopollenin exine material between species.28 Due to the variability in the robustness of sporopollenin exine capsules (SECs), it is necessary to optimize the processing conditions for each species of spore and pollen.
Plant spores from the species L. clavatum have become the most widely studied single source of SECs. It is proposed that this is primarily due to its widespread availability, low cost, monodispersity, and chemical robustness.9,29 The spores can be easily harvested and contain sporoplasmic contents in the form of groupings of 1 - 2 &#181;m cellular organelles and biomolecules.11 L. clavatum spores have been used as a natural powder lubricant,30,31 a base for cosmetics,30 and in herbal medicine32-36 for a wide range of therapeutic applications. The SECs obtained from L. clavatum have been shown to be more resilient to processing than SECs from other species of spores and pollen.2 After processing, the resulting SECs have been shown to retain their intricate microridge structures and high morphological uniformity while providing a large internal cavity for encapsulation.7 Studies indicate that L. clavatum SECs can be used for the encapsulation of drugs,10,13 vaccines,11 proteins,7,14 cells,8 oils,5-7,9 and food supplements.5,15 Observed SEC loading efficiencies are relatively high in comparison to conventional encapsulation materials.7 There are also a number of reported benefits to SEC encapsulation such as the ability to mask tastes,6,10 and to provide some degree of natural protection against oxidation.12 In the existing studies, the most commonly used SEC extraction method for L. clavatum is based on four main steps. Step one is solvent refluxing in acetone for up to 12 hr at 50 &#176;C to defat the spores.11 Step two is alkaline refluxing in 6% potassium hydroxide for up to 12 hr at 120 &#176;C to remove cytoplasmic and proteinaceous materials.11 Step three is acid refluxing in 85% phosphoric acid for up to 7 days at 180 &#176;C to remove the cellulosic intine material.11 Step four is a comprehensive washing process using water, solvents, acids, and bases to remove all remaining non-exine material and chemical residues.
The main goals of SEC extraction in relation to encapsulation applications are to produce capsules which are empty of cytoplasmic material, free from potentially allergenic proteins, and morphologically intact.2,37 However, from an industrial manufacturing perspective, it is also important to consider additional economic and environmental factors, such as, energy efficiency, production duration, safety, and resulting waste. With regards to energy efficiency, both high temperatures and long processing times affect production costs as well as environmental footprint. Production duration and turnaround time are key factors influencing processing profitability. Of particular concern is that high temperature phosphoric acid processing increases safety issues and is known to result in corrosive scaling which leads to significant increases in infrastructure maintenance and delays in batch turnaround times.38-40 Where possible, minimizing the number of steps required may lead to significantly reducing the waste produced. However, the commonly used four-step process of L. clavatum SEC extraction has simply evolved from decades of research and has had little actual process optimization. Recently, Mundargi et al.,41 made a significant contribution to the ongoing work in this field by systematically evaluating and optimizing one of the most commonly reported SEC extraction techniques.
In the first section of this study: spore defatting is demonstrated utilizing acetone processing at 50 &#176;C for 6 hr; sporoplasm and intine removal procedures are demonstrated utilizing 85% phosphoric acid processing at 70 &#176;C for 30 hr; extensive washing with water, solvents, acid, and base is used to demonstrate the removal of residual sporoplasmic contents; and SEC drying is demonstrated utilizing convection drying and vacuum oven drying. In the third section, SEC vacuum loading is demonstrated utilizing vacuum loading of a model protein, bovine serum albumin (BSA), followed by BSA-loaded-SEC washing and lyophilization. In the fourth section, the determination of the BSA encapsulation efficiency is demonstrated utilizing centrifugation, probe sonication, and UV/Vis spectrometry.

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	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">Lycopodium clavatum spores (s-type)</td>
			<td style="width:235px;">Sigma</td>
			<td style="width:171px;">19108-500G-F</td>
			<td style="width:171px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">Bovine serum albumin</td>
			<td style="width:235px;">Sigma</td>
			<td>A2153-50G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">FITC-conjugated BSA</td>
			<td style="width:235px;">Sigma</td>
			<td style="width:171px;">A9771-250MG</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">Phosphoric acid (85 % w/v)</td>
			<td style="width:235px;">Sigma</td>
			<td style="width:171px;">438081-2.5L&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">Hydrochloric acid</td>
			<td style="width:235px;">Sigma</td>
			<td style="width:171px;">V800202&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">Sodium hydroxide</td>
			<td style="width:235px;">Sigma</td>
			<td style="width:171px;">S5881-1KG&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">Acetone</td>
			<td style="width:235px;">Sigma</td>
			<td style="width:171px;">V800022</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">Ethanol</td>
			<td style="width:235px;">AcME&#160;</td>
			<td style="width:171px;">C000356</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">Deionized water</td>
			<td style="width:235px;">Millipore purified water&#160;</td>
			<td style="width:171px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:319px;">Qualitative filter paper (grade No. 1, cotton cellulose)</td>
			<td style="width:235px;"></td>
			<td style="width:171px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">Polystyrene microspheres (50 &#177; 1 &#181;m)&#160;</td>
			<td style="width:235px;">Thermoscientific (CA, USA)</td>
			<td style="width:171px;">4250A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">Vectashield&#160;</td>
			<td style="width:235px;">Vector labs (CA, USA)</td>
			<td style="width:171px;">H-1000</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:319px;">Sticky-slides, D 263 M Schott glass, No.1.5H (170 &#956;m, 25 mm x 75 mm) unsterile glass slide</td>
			<td style="width:235px;">Ibidi GmbH (Munich, Germany)</td>
			<td style="width:171px;">10812</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">Commercial Lycopodium SECs (L-type)</td>
			<td style="width:235px;">Polysciences, Inc. (PA, USA)</td>
			<td style="width:171px;">16867-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">Heating plates</td>
			<td style="width:235px;">IKA, Germany</td>
			<td style="width:171px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">Scanning electron microscope</td>
			<td style="width:235px;">Jeol, Japan</td>
			<td style="width:171px;">&#160;JFC-1600</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">Elemental analyzer&#160;</td>
			<td style="width:235px;">Elementar, Germany</td>
			<td style="width:171px;">VarioEL III</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">FlowCam: The benchtop system</td>
			<td style="width:235px;">Fluid Imaging Technologies, USA</td>
			<td>FlowCamVS</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">Confocal laser scanning microscope</td>
			<td style="width:235px;">Carl Zeiss, Germany</td>
			<td style="width:171px;">LSM710</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">Freeze dryer</td>
			<td>Labconco, USA&#160;</td>
			<td style="width:171px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">UV Spectrometer</td>
			<td>Boeco, Germany</td>
			<td style="width:171px;">S220</td>
			<td></td>
		</tr>
	</tbody>
</table>

extraction of plant-based capsules for microencapsulation applications

Microcapsules derived from plant-based spores or pollen provide a robust platform for a diverse range of microencapsulation applications. Sporopollenin exine capsules (SECs) are obtained when spores or pollen are processed so as to remove the internal sporoplasmic contents. The resulting hollow microcapsules exhibit a high degree of micromeritic uniformity and retain intricate microstructural features related to the particular plant species. Herein, we demonstrate a streamlined process for the production of SECs from Lycopodium clavatum spores and for the loading of hydrophilic compounds into these SECs. The current SEC isolation procedure has been recently optimized to significantly reduce the processing requirements which are conventionally used in SEC isolation, and to ensure the production of intact microcapsules. Natural L. clavatum spores are defatted with acetone, treated with phosphoric acid, and extensively washed to remove sporoplasmic contents. After acetone defatting, a single processing step using 85% phosphoric acid has been shown to remove all sporoplasmic contents. By limiting the acid processing time to 30 hr, it is possible to isolate clean SECs and avoid SEC fracturing, which has been shown to occur with prolonged processing time. Extensive washing with water, dilute acids, dilute bases, and solvents ensures that all sporoplasmic material and chemical residues are adequately removed. The vacuum loading technique is utilized to load a model protein (Bovine Serum Albumin) as a representative hydrophilic compound. Vacuum loading provides a simple technique to load various compounds without the need for harsh solvents or undesirable chemicals which are often required in other microencapsulation protocols. Based on these isolation and loading protocols, SECs provide a promising material for use in a diverse range of microencapsulation applications, such as, therapeutics, foods, cosmetics, and personal care products.

Streamlined Extraction Process for Sporopollenin Exine Capsules 
The L. clavatum SEC extraction was achieved by three main steps: (1) Defatting using acetone; (2) Acidolysis using phosphoric acid 85% (v/v); and (3) Extensive SEC washing using solvents. The flow of the streamlined SEC extraction process is presented in Figure 1 A - I. Briefly, the process involves a defatting step with a...

Watch this Scientific Journal Video about Extraction of Plant-based Capsules for Microencapsulation Applications at JoVE.com

Extraction of Plant-based Capsules for Microencapsulation Applications

This protocol/manuscript describes a streamlined process for the production of sporopollenin exine capsules (SECs) from Lycopodium clavatum spores and for the loading of hydrophilic compounds into these SECs.

Microcapsules derived from plant-based spores or pollen provide a robust platform for a diverse range of microencapsulation applications. Sporopollenin ...

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.

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