Name: manufacture and drug delivery applications of silk nanoparticles
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Description: Watch this Scientific Journal Video about Manufacture and Drug Delivery Applications of Silk Nanoparticles at JoVE.com

This research was supported by a Marie Curie FP7 Career Integration Grant 334134 within the seventh European Union Framework Program.

1. Preparation of a Reverse-engineered Silk Solution from Bombyx mori Cocoons
NOTE: This methodology is based on protocols described elsewhere 12,27.
<ol>
	<li>Cut 5 g of dried cocoons with scissors into 5 mm x 5 mm pieces. Remove any soiled layers.</li>
	<li>Weigh out 4.24 g of sodium carbonate and add this carefully to 2 L of boiling distilled water. 
	NOTE: This yields a 0.02 M sodium carbonate solution.</li>
	<li>Add the cut cocoon pieces to the boiling sodium carbonate solution and ...

<ol>
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L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Physical+and+chemical+aspects+of+stabilization+of+compounds+in+silk.">Physical and chemical aspects of stabilization of compounds in silk.</a> Biopolymers. 97 (6), 479-498 (2012).</li><li>Seib, F. P., Pritchard, E. M., Kaplan, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Self-Assembling+Doxorubicin+Silk+Hydrogels+for+the+Focal+Treatment+of+Primary+Breast.">Self-Assembling Doxorubicin Silk Hydrogels for the Focal Treatment of Primary Breast.</a> Adv. Funct. Mater. 23 (1), 58-65 (2013).</li><li>Seib, F. P., Kaplan, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Doxorubicin-loaded+silk+films:+drug-silk+interactions+and+in+vivo+performance+in+human+orthotopic+breast+cancer.">Doxorubicin-loaded silk films: drug-silk interactions and in vivo performance in human orthotopic breast cancer.</a> Biomaterials. 33 (33), 8442-8450 (2012).</li><li>Seib, F. P., Coburn, 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=Focal+therapy+of+neuroblastoma+using+silk+films+to+deliver+kinase+and+chemotherapeutic+agents+in+vivo.">Focal therapy of neuroblastoma using silk films to deliver kinase and chemotherapeutic agents in vivo.</a> Acta. Biomater. 20, 32-38 (2015).</li><li>Coburn, J. M., Na, E., Kaplan, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modulation+of+vincristine+and+doxorubicin+binding+and+release+from+silk+films.">Modulation of vincristine and doxorubicin binding and release from silk films.</a> J. Control. Release. 220, 229-238 (2015).</li><li>Seib, F. P., Jones, G. T., Rnjak-Kovacina, J., Lin, Y., Kaplan, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=pH-dependent+anticancer+drug+release+from+silk+nanoparticles.">pH-dependent anticancer drug release from silk nanoparticles.</a> Adv. Healthc. Mater. 2 (12), 1606-1611 (2013).</li><li>Wongpinyochit, T., Uhlmann, P., Urquhart, A. J., Seib, F. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PEGylated+Silk+Nanoparticles+for+Anticancer+Drug+Delivery.">PEGylated Silk Nanoparticles for Anticancer Drug Delivery.</a> Biomacromolecules. 16 (11), 3712-3722 (2015).</li><li>Seib, F. P., Kaplan, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Silk+for+Drug+Delivery+Applications:+Opportunities+and+Challenges.">Silk for Drug Delivery Applications: Opportunities and Challenges.</a> Isr. J. Chem. 53 (9-10), 1-12 (2013).</li><li>Yucel, T., Lovett, M. L., Kaplan, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Silk-based+biomaterials+for+sustained+drug+delivery.">Silk-based biomaterials for sustained drug delivery.</a> J. Control. 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Various methods are available to produce silk nanoparticles, including polyvinyl alcohol blending20, spray drying21, salting out22, capillary microdot printing23, supercritical CO2 precipitation24 and nanoprecipitation16,25 (reviewed in reference26). However, nanoprecipitation, due to its overall simplicity, is the most popular technique for generating silk nanoparticles. Therefore, the purpose of this study was to apply nanoprecipita...

Nano-sized drug delivery systems are often used to control drug release and to deliver a diverse set of therapeutic payloads &#8211; for example, proteins, peptides and small molecular weight drugs &#8211; to target cells and tissues. These therapeutic payloads are often incorporated into various macromolecular drug carriers, such as liposomes, water soluble polymers (including dendrimers), and micro- and nanoparticles1. Nanoparticles (typically in a size range of 1 nm to 1,000 nm) are being widely explored as potential drug carriers, particularly for anticancer drug delivery2. The successful introduction of Abraxane (120 nm sized albumin-based nanoparticles loaded with paclitaxel) into routine clinical practice3 has catalyzed the field, so that many more nanoparticles for drug delivery are now entering clinical trials4. Solid tumors generally show poor lymphatic drainage and have leaky blood vessels which means that nanoparticles of up to 200 nm will be passively targeted to these tumors following intravenous administration. This passive targeting phenomenon is called the enhanced permeability and retention (EPR) effect and was first reported in 19865. The EPR effect can lead to a 50- to 100-fold increase in drug concentrations within the tumor microenvironment for a given drug dose when the drug payload is delivered using a macromolecular drug carrier approach rather than the free drug without the carrier. Drug-loaded nanoparticles designed for anticancer drug delivery have to reach the tumor microenvironment and often must enter a specific intracellular compartment, usually by endocytic uptake, for the drug to achieve its desired therapeutic effect3. Nanoparticles designed for intracellular drug delivery exploit endocytosis as a gateway into the cell as well as a route to overcome drug resistance mechanisms. Drug release from nanoparticles is often specifically designed to occur in lysosomes (i.e., lysosomotropic drug delivery)6 where the pH responsiveness of the nanoparticle carrier (lysosomal pH approximately 4.5) can serve as trigger for drug release or lysosomal enzymes that liberate the payload from the carrier7.
Many different classes of materials can be used to generate nanoparticles (e.g., metals and many organic and inorganic materials). However, biopolymers are emerging as attractive materials because of their known biocompatibility, biodegradability and low toxicity8. Many biopolymers are being explored, including albumin, alginate, chitosan and silk. Of these, silk has emerged as a promising contender for development into drug delivery systems9. Silks of various types are produced by a number of arthropods, including spiders (e.g., Nephila clavipes) and silkworms (e.g., Bombyx mori). Silkworm silk is used far more extensively than spider silk because the silkworm is fully domesticated and its silk thus represents a reproducible starting material. Silkworm silk is a Food and Drug Administration (FDA) approved material for human use, particularly as a suture material; it has a robust safety record in humans and is known to degrade in vivo10. The degradation profile of silk can be fine-tuned to range from hours (low crystalline silk) to 12 months or more (high crystalline silk). Silk degradation products are non-toxic and are metabolized in the body10. The silk structure imparts the ability to bind small molecular weight compounds and macromolecular protein drugs11, making it a good material for controlled drug release. Protein drugs (e.g., antibodies) are susceptible to denaturation, aggregation, proteolytic cleavage and clearance by the immune system. However, silk stabilizes therapeutic proteins due to the buffering capacity of its nanocrystalline regions and its ability to tailor water content at the nanoscale11. These unique features provide physical protection and reduce payload mobility11 and are typically not seen with other (bio)polymers. Many anticancer drug delivery systems, for example silk-based hydrogels12, films13-15 and nanoparticles16,17, have now been developed to exploit these features (reviewed in references18,19 )
Here, silk nanoparticles were characterized by determining their size and charge over an extended time frame. Doxorubicin, a clinically relevant anticancer drug, was used as a model drug for drug loading and cytotoxicity studies in triple negative human breast cancer cells treated with drug-loaded silk nanoparticles.

<table border="0" cellpadding="0" cellspacing="0" width="859">
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		<col />
	</colgroup>
	<tbody>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Acetone</td>
			<td style="width:235px;">VWR International, Radnor, PA, USA</td>
			<td style="width:119px;">20066.33</td>
			<td style="width:291px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Automated Critical Point Dryer</td>
			<td style="width:235px;">Leica Microsystems, Wetzlar, Germany</td>
			<td style="width:119px;">EM CPD300</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Balancing&#160;</td>
			<td style="width:235px;">Mettler Toledo, Greifensee, Switzerland</td>
			<td style="width:119px;">NewClassic MS</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Black polystyrene microplate , 96 well</td>
			<td style="width:235px;">Sigma-Aldrich, St. Louis, MO, USA</td>
			<td style="width:119px;">3991</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Capillary cell (DTS 1070)</td>
			<td style="width:235px;">Malvern Instrument, Worcestershire, UK</td>
			<td style="width:119px;">DTS107</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Carbon adhesive disc</td>
			<td style="width:235px;">Agar Scientific, Essex, UK</td>
			<td style="width:119px;">G3347N</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Centrifuge&#160;</td>
			<td style="width:235px;">Hermle Labortechnik, Wehingen, Germany</td>
			<td style="width:119px;">Z323K</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Centrifuge&#160;</td>
			<td style="width:235px;">Beckman Coulter, Brea, CA, USA</td>
			<td style="width:119px;">Avanti J-E, Rotor: J20</td>
			<td></td>
		</tr>
		<tr height="80">
			<td height="80" style="height:80px;width:216px;">Centrifuge&#160;</td>
			<td style="width:235px;">Beckman Coulter, Brea, CA, USA</td>
			<td style="width:119px;">Optima L-70K, Rotor: 50.2 Ti, Adaptor 303392</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Coater, low vacuum</td>
			<td style="width:235px;">Leica Microsystems, Wetzlar, Germany</td>
			<td style="width:119px;">EM ACE200</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Cuvettes, polystyrene, disposable</td>
			<td style="width:235px;">Fisher Scientific, Waltham, MA, USA</td>
			<td style="width:119px;">FB55147</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Doxorubixin&#160;</td>
			<td style="width:235px;">LC Laboratories, Boston, MA, USA</td>
			<td style="width:119px;">D4000</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Electronic pipetting, Easypet&#160;</td>
			<td style="width:235px;">Eppendorf, Hamburg, Germany</td>
			<td style="width:119px;">N/A</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">FE-SEM</td>
			<td style="width:235px;">Hitachi High-Technologies, Krefeld, Germany</td>
			<td style="width:119px;">SU6600</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Fetal Bovine Serum</td>
			<td style="width:235px;">Thermo Scientific, Waltham, MA, USA</td>
			<td style="width:119px;">16000-044</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Freeze dryer</td>
			<td style="width:235px;">Martin Christ, Osterode, Germany</td>
			<td style="width:119px;">Epsilon 2-4</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Heat inactivated Bombyx mori silk cocoons</td>
			<td style="width:235px;">Tajima Shoji, Kanagawa, Japan</td>
			<td style="width:119px;">N/A</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Hotplate with Stirrer</td>
			<td style="width:235px;">Bibby Scientific, Stanffordshire, UK</td>
			<td style="width:119px;">US 152</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Incubator</td>
			<td style="width:235px;">Memmert, Schwabach, Germany</td>
			<td style="width:119px;">INB 200</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Insulin, human recombinant, zinc solution</td>
			<td style="width:235px;">Thermo Scientific, Waltham, MA, USA</td>
			<td style="width:119px;">12585-014</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Lithium bromide</td>
			<td style="width:235px;">Acros Organics, Geel, Belgium</td>
			<td style="width:119px;">AC199870025</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">MDA-MB-231</td>
			<td style="width:235px;">ATCC, Manassas, VA, U.S.A</td>
			<td style="width:119px;">N/A</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Micropipette and tips</td>
			<td style="width:235px;">Eppendorf, Hamburg, Germany</td>
			<td style="width:119px;">N/A</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Microplate Reader</td>
			<td style="width:235px;">Molecular devices, Sunnyvale, CA, USA</td>
			<td style="width:119px;">SpectraMax M5</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Oak Ridge High-Speed Centrifuge Tubes, 50 ml</td>
			<td style="width:235px;">Thermo Scientific, Waltham, MA, USA</td>
			<td style="width:119px;">N/A</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Open-Top Thickwall Polycarbonate tube, 4 ml</td>
			<td style="width:235px;">Beckman Coulter, Brea, CA, USA</td>
			<td style="width:119px;">355645</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Penicilin/streptomycin&#160;</td>
			<td style="width:235px;">Thermo Scientific, Waltham, MA, USA</td>
			<td style="width:119px;">15140-122</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">RPMI medium</td>
			<td style="width:235px;">Thermo Scientific, Waltham, MA, USA</td>
			<td style="width:119px;">11875-093</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Serological pipettes, 5 ml</td>
			<td style="width:235px;">Sigma-Aldrich, St. Louis, MO, USA</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Silicon wafers</td>
			<td style="width:235px;">Agar Scientific, Essex, UK</td>
			<td style="width:119px;">G3391</td>
			<td></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:216px;">Slide-A-Lyzer Dialysis cassettes, 3.5K MWCO, 15 ml</td>
			<td style="width:235px;">Thermo Scientific, Waltham, MA, USA</td>
			<td style="width:119px;">87724</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Sodium carbonate anhydrous</td>
			<td style="width:235px;">Fisher Scientific, Waltham, MA, USA</td>
			<td style="width:119px;">S/2840/62</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Specimen stubs for SEM</td>
			<td style="width:235px;">Agar Scientific, Essex, UK</td>
			<td style="width:119px;">G301</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Ultrasonic homogenizer</td>
			<td style="width:235px;">Bandelin, Berlin, Germany</td>
			<td style="width:119px;">Sonoplus HD 2070</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">UV transparent microplate, 96 well</td>
			<td style="width:235px;">Sigma-Aldrich, St. Louis, MO, USA</td>
			<td style="width:119px;">3635</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Vortex</td>
			<td style="width:235px;">IKA, Staufen, Germany</td>
			<td style="width:119px;">Genius 3</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Zetasizer</td>
			<td style="width:235px;">Malvern Instrument, Worcestershire, UK</td>
			<td style="width:119px;">Nano ZS</td>
			<td></td>
		</tr>
		<tr height="20">
			<td colspan="2" height="20" style="height:20px;">Zetasizer Software version 7.11</td>
			<td style="width:119px;"></td>
			<td>DLS software</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Micro Modulyo</td>
			<td style="width:235px;">&#160;Thermo Fisher</td>
			<td style="width:119px;">230</td>
			<td>Freeze drying system&#160;</td>
		</tr>
	</tbody>
</table>

manufacture and drug delivery applications of silk nanoparticles

Silk is a promising biopolymer for biomedical and pharmaceutical applications due to its outstanding mechanical properties, biocompatibility and biodegradability, as well its ability to protect and subsequently release its payload in response to a trigger. While silk can be formulated into various material formats, silk nanoparticles are emerging as promising drug delivery systems. Therefore, this article covers the procedures for reverse engineering silk cocoons to yield a regenerated silk solution that can be used to generate stable silk nanoparticles. These nanoparticles are subsequently characterized, drug loaded and explored as a potential anticancer drug delivery system. Briefly, silk cocoons are reverse engineered first by degumming the cocoons, followed by silk dissolution and clean up, to yield an aqueous silk solution. Next, the regenerated silk solution is subjected to nanoprecipitation to yield silk nanoparticles &#8211; a simple but powerful method that generates uniform nanoparticles. The silk nanoparticles are characterized according to their size, zeta potential, morphology and stability in aqueous media, as well as their ability to entrap a chemotherapeutic payload and kill human breast cancer cells. Overall, the described methodology yields uniform silk nanoparticles that can be readily explored for a myriad of applications, including their use as a potential nanomedicine.

Data were statistically analyzed as detailed previously17. The Student&#39;s t-test was used for sample pairs and one-way analysis of variance (ANOVA) followed by Bonferroni&#39;s multiple comparison post hoc test for multiple samples. An asterisk denotes statistical significance as follows: *P &#60;0.05 and **P &#60;0.001. All data are presented as mean values &#177; standard deviation (SD) and the numbers in brackets indicate the number of independent experiments.
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Watch this Scientific Journal Video about Manufacture and Drug Delivery Applications of Silk Nanoparticles at JoVE.com

Manufacture and Drug Delivery Applications of Silk Nanoparticles

Nanoparticles are emerging as promising drug delivery systems for a broad range of indications. Here, we describe a simple yet powerful method to manufacture silk nanoparticles using reverse engineered Bombyx mori silk. These silk nanoparticles can be readily loaded with a therapeutic payload and subsequently explored for drug delivery applications.

Silk is a promising biopolymer for biomedical and pharmaceutical applications due to its outstanding mechanical properties, biocompatibility and ...

Research

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Bioengineering

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