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.

extraction-of-plant-based-capsules-for-microencapsulation-applications

Research

JoVE Journal

Bioengineering

Preparation of 3D Fibrin Scaffolds for Stem Cell Culture Applications

Stem cells are found in naturally occurring 3D microenvironments in vivo, which are often referred to as the stem cell niche 1. Culturing stem cells inside of 3D biomaterial scaffolds provides a way to accurately mimic these microenvironments, providing an advantage over traditional 2D culture methods using polystyrene as well as a method for engineering replacement tissues 2. While 2D tissue culture polystrene has been used for the majority of cell culture experiments, 3D biomaterial scaffolds can more closely replicate the microenvironments found in vivo by enabling more accurate establishment of cell polarity in the environment and possessing biochemical and mechanical properties similar to soft tissue.3 A variety of naturally derived and synthetic biomaterial scaffolds have been investigated as 3D environments for supporting stem cell growth. While synthetic scaffolds can be synthesized to have a greater range of mechanical and chemical properties and often have greater reproducibility, natural biomaterials are often composed of proteins and polysaccharides found in the extracelluar matrix and as a result contain binding sites for cell adhesion and readily support cell culture. Fibrin scaffolds, produced by polymerizing the protein fibrinogen obtained from plasma, have been widely investigated for a variety of tissue engineering applications both in vitro and in vivo 4. Such scaffolds can be modified using a variety of methods to incorporate controlled release systems for delivering therapeutic factors 5. Previous work has shown that such scaffolds can be used to successfully culture embryonic stem cells and this scaffold-based culture system can be used to screen the effects of various growth factors on the differentiation of the stem cells seeded inside 6,7.
 This protocol details the process of polymerizing fibrin scaffolds from fibrinogen solutions using the enzymatic activity of thrombin. The process takes 2 days to complete, including an overnight dialysis step for the fibrinogen solution to remove citrates that inhibit polymerization. These detailed methods rely on fibrinogen concentrations determined to be optimal for embryonic and induced pluripotent stem cell culture. Other groups have further investigated fibrin scaffolds for a wide range of cell types and applications - demonstrating the versatility of this approach 8-12.

This work details the preparation of 3D fibrin scaffolds for culturing and differentiating plutipotent stem cells. Such scaffolds can be used to screen the effects of various biological compounds on stem cell behavior as well as modified to contain drug delivery systems.

Stem cells are found in naturally occurring 3D microenvironments in vivo, which are often referred to as the stem cell niche 1. Culturing stem cells ...

Wideband Optical Detector of Ultrasound for Medical Imaging Applications

Optical sensors of ultrasound are a promising alternative to piezoelectric techniques, as has been recently demonstrated in the field of optoacoustic imaging. In medical applications, one of the major limitations of optical sensing technology is its susceptibility to environmental conditions, e.g. changes in pressure and temperature, which may saturate the detection. Additionally, the clinical environment often imposes stringent limits on the size and robustness of the sensor. In this work, the combination of pulse interferometry and fiber-based optical sensing is demonstrated for ultrasound detection. Pulse interferometry enables robust performance of the readout system in the presence of rapid variations in the environmental conditions, whereas the use of all-fiber technology leads to a mechanically flexible sensing element compatible with highly demanding medical applications such as intravascular imaging. In order to achieve a short sensor length, a pi-phase-shifted fiber Bragg grating is used, which acts as a resonator trapping light over an effective length of 350 &#181;m. To enable high bandwidth, the sensor is used for sideway detection of ultrasound, which is highly beneficial in circumferential imaging geometries such as intravascular imaging. An optoacoustic imaging setup is used to determine the response of the sensor for acoustic point sources at different positions.

Optical detection of ultrasound is impractical in many imaging scenarios because it often requires stable environmental conditions. We demonstrate an optical technique for ultrasound sensing in volatile environments with miniaturization and sensitivity levels appropriate for optoacoustic imaging in restrictive scenarios, e.g. intravascular applications.

Optical sensors of ultrasound are a promising alternative to piezoelectric techniques, as has been recently demonstrated in the field of optoacoustic ...

Designing Silk-silk Protein Alloy Materials for Biomedical Applications

Fibrous proteins display different sequences and structures that have been used for various applications in biomedical fields such as biosensors, nanomedicine, tissue regeneration, and drug delivery. Designing materials based on the molecular-scale interactions between these proteins will help generate new multifunctional protein alloy biomaterials with tunable properties. Such alloy material systems also provide advantages in comparison to traditional synthetic polymers due to the materials biodegradability, biocompatibility, and tenability in the body. This article used the protein blends of wild tussah silk (Antheraea pernyi) and domestic mulberry silk (Bombyx mori) as an example to provide useful protocols regarding these topics, including how to predict protein-protein interactions by computational methods, how to produce protein alloy solutions, how to verify alloy systems by thermal analysis, and how to fabricate variable alloy materials including optical materials with diffraction gratings, electric materials with circuits coatings, and pharmaceutical materials for drug release and delivery. These methods can provide important information for designing the next generation multifunctional biomaterials based on different protein alloys.

Blending is an efficient approach to generate biomaterials with a broad range of properties and combined features. By predicting the molecular interactions between different natural silk proteins, new silk-silk protein alloy platforms with tunable mechanical resiliency, electrical response, optical transparency, chemical processability, biodegradability, or thermal stability can be designed.

Fibrous proteins display different sequences and structures that have been used for various applications in biomedical fields such as biosensors, ...

In situ TEM of Biological Assemblies in Liquid

Researchers regularly use Transmission Electron Microscopes (TEMs) to examine biological entities and to assess new materials. Here, we describe an additional application for these instruments- viewing viral assemblies in a liquid environment. This exciting and novel method of visualizing biological structures utilizes a recently developed microfluidic-based specimen holder. Our video article demonstrates how to assemble and use a microfluidic holder to image liquid specimens within a TEM. In particular, we use simian rotavirus double-layered particles (DLPs) as our model system. We also describe steps to coat the surface of the liquid chamber with affinity biofilms that tether DLPs to the viewing window. This permits us to image assemblies in a manner that is suitable for 3D structure determination. Thus, we present a first glimpse of subviral particles in a native liquid environment.

In situ TEM of Biological Assemblies in Liquid

Here we describe a procedure to image viral complexes in liquid at nanometer resolution using a transmission electron microscope.

Researchers regularly use Transmission Electron Microscopes (TEMs) to examine biological entities and to assess new materials. Here, we describe an ...

Solid Lipid Nanoparticles (SLNs) for Intracellular Targeting Applications

Nanoparticle-based delivery vehicles have shown great promise for intracellular targeting applications, providing a mechanism to specifically alter cellular signaling and gene expression. In a previous investigation, the synthesis of ultra-small solid lipid nanoparticles (SLNs) for topical drug delivery and biomarker detection applications was demonstrated. SLNs are a well-studied example of a nanoparticle delivery system that has emerged as a promising drug delivery vehicle. In this study, SLNs were loaded with a fluorescent dye and used as a model to investigate particle-cell interactions. The phase inversion temperature (PIT) method was used for the synthesis of ultra-small populations of biocompatible nanoparticles. A 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenylphenyltetrazolium bromide (MTT) assay was utilized in order to establish appropriate dosing levels prior to the nanoparticle-cell interaction studies. Furthermore, primary human dermal fibroblasts and mouse dendritic cells were exposed to dye-loaded SLN over time and the interactions with respect to toxicity and particle uptake were characterized using fluorescence microscopy and flow cytometry. This study demonstrated that ultra-small SLNs, as a nanoparticle delivery system, are suitable for intracellular targeting of different cell types.

Solid Lipid Nanoparticles SLNs for Intracellular Targeting Applications

In this study, a method for synthesizing ultra-small populations of biocompatible nanoparticles was described, as well as several in vitro methods by which to assess their cellular interactions.

Nanoparticle-based delivery vehicles have shown great promise for intracellular targeting applications, providing a mechanism to specifically alter ...

Fabricating Superhydrophobic Polymeric Materials for Biomedical Applications

Superhydrophobic materials, with surfaces possessing permanent or metastable non-wetted states, are of interest for a number of biomedical and industrial applications. Here we describe how electrospinning or electrospraying a polymer mixture containing a biodegradable, biocompatible aliphatic polyester (e.g., polycaprolactone and poly(lactide-co-glycolide)), as the major component, doped with a hydrophobic copolymer composed of the polyester and a stearate-modified poly(glycerol carbonate) affords a superhydrophobic biomaterial. The fabrication techniques of electrospinning or electrospraying provide the enhanced surface roughness and porosity on and within the fibers or the particles, respectively. The use of a low surface energy copolymer dopant that blends with the polyester and can be stably electrospun or electrosprayed affords these superhydrophobic materials. Important parameters such as fiber size, copolymer dopant composition and/or concentration, and their effects on wettability are discussed. This combination of polymer chemistry and process engineering affords a versatile approach to develop application-specific materials using scalable techniques, which are likely generalizable to a wider class of polymers for a variety of applications.

Two- and three-dimensional superhydrophobic polymeric materials are prepared by electrospinning or electrospraying biodegradable polymers blended with a lower surface energy polymer of similar composition.

Superhydrophobic materials, with surfaces possessing permanent or metastable non-wetted states, are of interest for a number of biomedical and industrial ...

Fabrication of Mechanically Tunable and Bioactive Metal Scaffolds for Biomedical Applications

Biometal systems have been widely used for biomedical applications, in particular, as load-bearing materials. However, major challenges are high stiffness and low bioactivity of metals. In this study, we have developed a new method towards fabricating a new type of bioactive and mechanically reliable porous metal scaffolds-densified porous Ti scaffolds. The method consists of two fabrication processes, 1) the fabrication of porous Ti scaffolds by dynamic freeze casting, and 2) coating and densification of the porous scaffolds. The dynamic freeze casting method to fabricate porous Ti scaffolds allowed the densification of porous scaffolds by minimizing the chemical contamination and structural defects. The densification process is distinctive for three reasons. First, the densification process is simple, because it requires a control of only one parameter (degree of densification). Second, it is effective, as it achieves mechanical enhancement and sustainable release of biomolecules from porous scaffolds. Third, it has broad applications, as it is also applicable to the fabrication of functionally graded porous scaffolds by spatially varied strain during densification.

Bioactive and mechanically reliable metal scaffolds have been fabricated through a method which consists of two processes, dynamic freeze casting for the fabrication of porous Ti, and coating and densification of the Ti scaffolds. The densification process is simple, effective and applicable to the fabrication of functionally graded scaffolds.

Biometal systems have been widely used for biomedical applications, in particular, as load-bearing materials. However, major challenges are high stiffness ...

Light-mediated Formation and Patterning of Hydrogels for Cell Culture Applications

Click chemistries have been investigated for use in numerous biomaterials applications, including drug delivery, tissue engineering, and cell culture. In particular, light-mediated click reactions, such as photoinitiated thiol&#8722;ene and thiol&#8722;yne reactions, afford spatiotemporal control over material properties and allow the design of systems with a high degree of user-directed property control. Fabrication and modification of hydrogel-based biomaterials using the precision afforded by light and the versatility offered by these thiol&#8722;X photoclick chemistries are of growing interest, particularly for the culture of cells within well-defined, biomimetic microenvironments. Here, we describe methods for the photoencapsulation of cells and subsequent photopatterning of biochemical cues within hydrogel matrices using versatile and modular building blocks polymerized by a thiol&#8722;ene photoclick reaction. Specifically, an approach is presented for constructing hydrogels from allyloxycarbonyl (Alloc)-functionalized peptide crosslinks and pendant peptide moieties and thiol-functionalized poly(ethylene glycol) (PEG) that rapidly polymerize in the presence of lithium acylphosphinate photoinitiator and cytocompatible doses of long wavelength ultraviolet (UV) light. Facile techniques to visualize photopatterning and quantify the concentration of peptides added are described. Additionally, methods are established for encapsulating cells, specifically human mesenchymal stem cells, and determining their viability and activity. While the formation and initial patterning of thiol-alloc hydrogels are shown here, these techniques broadly may be applied to a number of other light and radical-initiated material systems (e.g., thiol-norbornene, thiol-acrylate) to generate patterned substrates.

We describe a sequential process for light-mediated formation and subsequent biochemical patterning of synthetic hydrogel matrices for three-dimensional cell culture applications. The construction and modification of hydrogels with cytocompatible photoclick chemistry is demonstrated. Additionally, facile techniques to quantify and observe patterns and determine cell viability within these hydrogels are presented.

Click chemistries have been investigated for use in numerous biomaterials applications, including drug delivery, tissue engineering, and cell culture. In ...

Rapid Mix Preparation of Bioinspired Nanoscale Hydroxyapatite for Biomedical Applications

Hydroxyapatite (HA) has been widely used as a medical ceramic due to its good biocompatibility and osteoconductivity. Recently there has been interest regarding the use of bioinspired nanoscale hydroxyapatite (nHA). However, biological apatite is known to be calcium-deficient and carbonate-substituted with a nanoscale platelet-like morphology. Bioinspired nHA has the potential to stimulate optimal bone tissue regeneration due to its similarity to bone and tooth enamel mineral. Many of the methods currently used to fabricate nHA both in the laboratory and commercially, involve lengthy processes and complex equipment. Therefore, the aim of this study was to develop a rapid and reliable method to prepare high quality bioinspired nHA. The rapid mixing method developed was based upon an acid-base reaction involving calcium hydroxide and phosphoric acid. Briefly, a phosphoric acid solution was poured into a calcium hydroxide solution followed by stirring, washing and drying stages. Part of the batch was sintered at 1,000 &#176;C for 2 h in order to investigate the products&#39; high temperature stability. X-ray diffraction analysis showed the successful formation of HA, which showed thermal decomposition to &#946;-tricalcium phosphate after high temperature processing, which is typical for calcium-deficient HA. Fourier transform infrared spectroscopy showed the presence of carbonate groups in the precipitated product. The nHA particles had a low aspect ratio with approximate dimensions of 50 x 30 nm, close to the dimensions of biological apatite. The material was also calcium deficient with a Ca:P molar ratio of 1.63, which like biological apatite is lower than the stoichiometric HA ratio of 1.67. This new method is therefore a reliable and far more convenient process for the manufacture of bioinspired nHA, overcoming the need for lengthy titrations and complex equipment. The resulting bioinspired HA product is suitable for use in a wide variety of medical and consumer health applications.

This paper describes a novel method for the rapid manufacture of high quality bioinspired nanoscale hydroxyapatite. This biomaterial is of great significance in the manufacture of a wide range of innovative medical devices for clinical applications in orthopedics, craniofacial surgery and dentistry.

Hydroxyapatite (HA) has been widely used as a medical ceramic due to its good biocompatibility and osteoconductivity. Recently there has been interest ...

Two Methods for Decellularization of Plant Tissues for Tissue Engineering Applications

The autologous, synthetic, and animal-derived grafts currently used as scaffolds for tissue replacement have limitations due to low availability, poor biocompatibility, and cost. Plant tissues have favorable characteristics that make them uniquely suited for use as scaffolds, such as high surface area, excellent water transport and retention, interconnected porosity, preexisting vascular networks, and a wide range of mechanical properties. Two successful methods of plant decellularization for tissue engineering applications are described here. The first method is based on detergent baths to remove cellular matter, which is similar to previously established methods used to clear mammalian tissues. The second is a detergent-free method adapted from a protocol that isolates leaf vasculature and involves the use of a heated bleach and salt bath to clear the leaves and stems. Both methods yield scaffolds with comparable mechanical properties and low cellular metabolic impact, thus allowing the user to select the protocol which better suits their intended application.

Here we present, and contrast two protocols used to decellularize plant tissues: a detergent-based approach and a detergent-free approach. Both methods leave behind the extracellular matrix of the plant tissues used, which can then be utilized as scaffolds for tissue engineering applications.

The autologous, synthetic, and animal-derived grafts currently used as scaffolds for tissue replacement have limitations due to low availability, poor ...