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 boil for 60 min to degum the silk fibers. Stir the silk occasionally to ensure homogenous sample processing.</li>
	<li>Remove the degummed silk and wash with 1 L of distilled water for 20 min; repeat the washing step at least 3 times.</li>
	<li>Remove the washed silk and squeeze it well to remove excess liquid and then untie/pull the silk by hand. Place the untied silk in a fume hood to air dry overnight. This typically yields 3.6 g of degummed silk fibers.</li>
	<li>The next day, weigh out 5 g air-dried degummed silk fibers and pack the silk tightly into the bottom of a 50 ml beaker.</li>
	<li>Prepare a fresh 9.3 M LiBr solution. Dissolve the silk fibers in LiBr using a 1 g silk to 4 ml LiBr ratio. Cover the silk LiBr sample with aluminum foil to prevent evaporation and allow the silk to dissolve completely at 60 &#176;C. This step takes up to 4 hr and is aided by occasionally stirring.</li>
	<li>Wet a dialysis cassette (molecular weight cut-off of 3,500 Da) in water for 5 min. Inject 15 ml the silk LiBr solution into the 15 ml dialysis cassette and use a needle and syringe to remove any air bubbles.</li>
	<li>Dialyze against 1 L of distilled water and change the water at 1, 3 and 6 hr (i.e., 3 changes on the first day) and again on the next morning and evening (i.e., 2 changes on the second day), and again on the following morning (i.e., 1 change on the third day).</li>
	<li>Collect the silk solution from the dialysis cassette and centrifuge the solution for 20 min at 5 &#176;C at 9,500 x g. Recover the supernatant and repeat this centrifugation process twice more.</li>
	<li>Determine the weight of an empty weighing boat (W1) and add 1 ml of the silk solution. Record the weight again (W2) and then dry the sample by leaving the weighing boat at 60 &#186;C overnight. Next, determine the total dry weight (W3) (dried silk and weighing boat). The concentration of the silk solution (w/v) is: % = (W3-W1/W2-W1) x 100.</li>
</ol>
2. Preparation of Silk Nanoparticles from Reverse-engineered Silk Solution
<ol>
	<li>Add a 5% (w/v) silk solution dropwise to acetone while maintaining a &#62;75% (v/v) acetone solution. For example, add 9 ml of a 5% (w/v) silk solution dropwise (10 &#181;l/drop at a rate of 50 drops/min) to 34 ml acetone.</li>
	<li>Centrifuge the precipitate at 48,000 x g for 2 hr at 4 &#176;C.</li>
	<li>Aspirate the supernatant and resuspend the pellet in distilled water by first dislodging the pellet with a spatula and then adding 20 ml distilled water. Use pipet tips to remove the pellet from the spatula. After vortexing for 20 sec followed by two sonication cycles using an ultrasonic probe at 30% amplitude for 30 sec, top up the centrifuge tube to capacity with distilled water.</li>
	<li>Repeat the centrifugation and resuspension step at least twice.</li>
	<li>Resuspend the pellet in 6 ml distilled water, as detailed in 2.3 and store at 4 &#176;C until use. For cell culture studies, silk nanoparticle stocks can be gamma irradiated17.</li>
</ol>
3. Determination of Silk Nanoparticle Concentration
<ol>
	<li>Centrifuge silk nanoparticles at 48,000 x g for 2 hr at 4 &#176;C.</li>
	<li>Collect all nanoparticles in 3 ml of distilled water, followed by two sonication cycles at 30% amplitude for 30 sec.</li>
	<li>Divide the 3 ml stock of silk nanoparticles into 2 ml and 1 ml lots and transfer into pre-weighed 2 ml tubes. Record the total weight of the 2 ml sample. Store the 1 ml lot at 4 &#176;C until use; this 1 ml sample will be used to generate a calibration curve.</li>
	<li>Snap freeze and then lyophilize the 2 ml silk nanoparticle lot in a freeze dryer overnight. After freeze-drying, reweigh the 2 ml tube and calculate the amount of silk nanoparticles (mg) that was originally present in the 2 ml sample.</li>
	<li>Dilute the 1 ml silk nanoparticle stock with distilled water to generate a 5-point calibration curve (0.04-7 mg/ml). Ensure that samples do not exceed the absorbance maximum.</li>
	<li>Determine the absorbance of each standard dilution at 600 nm. This is best done using a 96-well plate setup. Plot absorbance versus concentration (mg/ml) for the standard curve. Then use this standard curve routinely to determine the concentration of silk nanoparticles in suspensions.</li>
</ol>
4. Preparation of Doxorubicin-loaded Silk Nanoparticles
<ol>
	<li>Preparation of the Doxorubicin Solution
	<ol>
		<li>Dissolve 1.2 mg of doxorubicin HCl in 8 ml distilled water.</li>
		<li>Make up to 10 ml with distilled water to yield a working stock of 116 &#181;g/ml (0.2 &#181;mol/ml).</li>
	</ol>
	</li>
	<li>Preparation of Doxorubicin-loaded Silk Nanoparticles
	<ol>
		<li>Mix 2 ml of 0.2 &#181;mol/ml doxorubicin solution with 200 &#181;l of 10, 30 or 50 mg/ml of silk nanoparticles in a 2 ml tube.</li>
		<li>Incubate the silk-doxorubicin suspension at room temperature (25 &#176;C) overnight on a rotating mixer.</li>
		<li>Next, centrifuge the silk-doxorubicin suspension at 194,000 x g for 30 min. Wash the doxorubicin-loaded silk nanoparticles with distilled water and repeat this procedure twice more.</li>
		<li>Pool the supernatant and note the total volume (this sample is used to determine the encapsulation efficiency).</li>
		<li>Resuspend the doxorubicin-loaded silk nanoparticles in distilled water, protect from light and store at 4 &#186;C until use.</li>
	</ol>
	</li>
	<li>Determination of Encapsulation Efficiency and Drug Loading
	<ol>
		<li>Pipet 200 &#181;l of the supernatant from step 4.2.4 into a black microtiter plate.</li>
		<li>Use a fluorescence microplate reader to measure doxorubicin-associated fluorescence at a fixed photomultiplier setting.</li>
		<li>Set the excitation wavelength to 485 nm and emission wavelength to 590 nm and record the fluorescence values.</li>
		<li>Generate a doxorubicin calibration curve. Ensure measurements are acquired with identical instrument settings (i.e., with a fixed photomultiplier setting). Using the calibration curve, calculate the doxorubicin concentration in the combined supernatant. Repeat this measurement in three independent experiments.</li>
		<li>Use equation (1) to determine encapsulation efficiency: 
		<img alt="Equation 1" src="/files/ftp_upload/54669/54669equation1.jpg" /></li>
	</ol>
	</li>
</ol>
5. Characterization of Silk Nanoparticles
<ol>
	<li>Assessment of the Size and Zeta Potential of Freshly Prepared and Stored Silk Nanoparticles.
	<ol>
		<li>Store silk nanoparticles in distilled water at 4 &#176;C and 25 &#176;C.</li>
		<li>Measure the size and zeta potential of the silk nanoparticles on days 0, 14 and 28 using dynamic light scattering (DLS). Set refractive indices to 1.33 for distilled water and 1.60 for protein17. Calculate particle sizes with the user interface software.</li>
	</ol>
	</li>
	<li>Morphological Assessment of Silk Nanoparticles by Scanning Electron Microscopy (SEM).
	<ol>
		<li>Place a carbon adhesive disc onto an SEM stub and subsequently attach a silicon wafer.</li>
		<li>Dilute silk nanoparticles to a concentration of 1 mg/ml. Pipette 10 &#181;l of the sample onto a silicon wafer, freeze the sample at -80 &#176;C and lyophilize overnight using a freeze drying system as per manufacturer&#39;s instructions.</li>
		<li>Coat the sample with a gold layer up to 20 nm thick using a low vacuum sputter coater. 
		NOTE: Instrument settings vary between models. The model used here is fully automated and operates by thickness only.</li>
		<li>Image samples with a scanning electron microscope at 5 kV and a 40,000-fold magnification.</li>
	</ol>
	</li>
</ol>
6. In Vitro Cytotoxicity of Control and Doxorubicin-loaded Silk Nanoparticles
<ol>
	<li>Cell Viability Following Exposure to Silk Nanoparticles.
	<ol>
		<li>Culture MDA-MB-231 cells in RPMI 1640 with 10% v/v FBS. Plate cells on tissue culture treated polystyrene and incubate in a humidified 5% CO2 atmosphere at 37 &#176;C. Routinely subculture at 80% confluence every 2&#8722;3 days.</li>
		<li>Plate MDA-MB-231 cells at a density of 2 x 104 cells/cm2 in 96-well plates. Allow the cells to recover overnight.</li>
		<li>Add (i) 0.001-1 &#181;g freely diffusible doxorubicin, (ii) 0.001-0.5 mg silk nanoparticles and 0.1 mg silk nanoparticles loaded with 0.001-1 &#181;g doxorubicin into 96-well plates (final volume 100 &#181;l per well).</li>
		<li>Determine cell viability and the half maximal inhibitory concentration (IC50) by adding (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT 5 mg/ml in PBS) at 72 hr. Incubate for 5 hr, carefully drain the wells with a pipette and dissolve the formazan with 100 &#181;l of dimethylsulfoxide. Measure the absorbance at 560 nm. Repeat this measurement in three independent experiments. 
		NOTE: The absorbance values of untreated controls serve as a reference value for 100% cell viability.</li>
	</ol>
	</li>
	<li>SEM of Cells Exposed to Silk Nanoparticles.
	<ol>
		<li>Seed MDA-MB-231 cells on sterile glass coverslips at a density of 2 x 104 cells/cm2. Allow the cells to recover overnight. Expose the cells to the desired treatment conditions for 72 hr.</li>
		<li>Fix the cells with 2% v/v glutaraldehyde in PBS for 30 min, wash with distilled water twice, dehydrate with an ethanol series, and critical point dry the samples, as detailed elsewhere28.</li>
		<li>Sputter coat the samples with a gold layer up to 20 nm thick using a low vacuum sputter coater. 
		NOTE: Instrument settings vary between models. The model used here is fully automated and operates by thickness only.</li>
		<li>Image the samples by SEM using an electron acceleration of 5 kV and 700-fold magnification.</li>
	</ol>
	</li>
</ol>

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The authors have nothing to disclose.

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">
	<colgroup>
		<col />
		<col />
		<col />
		<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>
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		<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 ...

manufacture-and-drug-delivery-applications-of-silk-nanoparticles

Research

JoVE Journal

Bioengineering

15.6K Views.  University of Strathclyde. 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.

Video: Manufacture and Drug Delivery Applications of Silk Nanoparticles

Evaluation of Polymeric Gene Delivery Nanoparticles by Nanoparticle Tracking Analysis and High-throughput Flow Cytometry

Non-viral gene delivery using polymeric nanoparticles has emerged as an attractive approach for gene therapy to treat genetic diseases1 and as a technology for regenerative medicine2. Unlike viruses, which have significant safety issues, polymeric nanoparticles can be designed to be non-toxic, non-immunogenic, non-mutagenic, easier to synthesize, chemically versatile, capable of carrying larger nucleic acid cargo and biodegradable and/or environmentally responsive. Cationic polymers self-assemble with negatively charged DNA via electrostatic interaction to form complexes on the order of 100 nm that are commonly termed polymeric nanoparticles. Examples of biomaterials used to form nanoscale polycationic gene delivery nanoparticles include polylysine, polyphosphoesters, poly(amidoamines)s and polyethylenimine (PEI), which is a non-degradable off-the-shelf cationic polymer commonly used for nucleic acid delivery1,3 . Poly(beta-amino ester)s (PBAEs) are a newer class of cationic polymers4 that are hydrolytically degradable5,6 and have been shown to be effective at gene delivery to hard-to-transfect cell types such as human retinal endothelial cells (HRECs)7, mouse mammary epithelial cells8, human brain cancer cells9 and macrovascular (human umbilical vein, HUVECs) endothelial cells10.
A new protocol to characterize polymeric nanoparticles utilizing nanoparticle tracking analysis (NTA) is described. In this approach, both the particle size distribution and the distribution of the number of plasmids per particle are obtained11. In addition, a high-throughput 96-well plate transfection assay for rapid screening of the transfection efficacy of polymeric nanoparticles is presented. In this protocol, poly(beta-amino ester)s (PBAEs) are used as model polymers and human retinal endothelial cells (HRECs) are used as model human cells. This protocol can be easily adapted to evaluate any polymeric nanoparticle and any cell type of interest in a multi-well plate format.

A protocol for nanoparticle tracking analysis (NTA) and high-throughput flow cytometry to evaluate polymeric gene delivery nanoparticles is described. NTA is utilized to characterize the nanoparticle particle size distribution and the plasmid per particle distribution. High-throughput flow cytometry enables quantitative transfection efficacy evaluation for a library of gene delivery biomaterials.

Non-viral gene delivery using polymeric nanoparticles has emerged as an attractive approach for gene therapy to treat genetic diseases1 and as a ...

Self-reporting Scaffolds for 3-Dimensional Cell Culture

Culturing cells in 3D on appropriate scaffolds is thought to better mimic the in vivo microenvironment and increase cell-cell interactions. The resulting 3D cellular construct can often be more relevant to studying the molecular events and cell-cell interactions than similar experiments studied in 2D. To create effective 3D cultures with high cell viability throughout the scaffold the culture conditions such as oxygen and pH need to be carefully controlled as gradients in analyte concentration can exist throughout the 3D construct. Here we describe the methods of preparing biocompatible pH responsive sol-gel nanosensors and their incorporation into poly(lactic-co-glycolic acid) (PLGA) electrospun scaffolds along with their subsequent preparation for the culture of mammalian cells. The pH responsive scaffolds can be used as tools to determine microenvironmental pH within a 3D cellular construct. Furthermore, we detail the delivery of pH responsive nanosensors to the intracellular environment of mammalian cells whose growth was supported by electrospun PLGA scaffolds. The cytoplasmic location of the pH responsive nanosensors can be utilized to monitor intracellular pH (pHi) during ongoing experimentation.

Biocompatible pH responsive sol-gel nanosensors can be incorporated into poly(lactic-co-glycolic acid) (PLGA) electrospun scaffolds. The produced self-reporting scaffolds can be used for in&#160;situ monitoring of microenvironmental conditions whilst culturing cells upon the scaffold. This is beneficial as the 3D cellular construct can be monitored in real-time without disturbing the experiment.

Culturing cells in 3D on appropriate scaffolds is thought to better mimic the in vivo microenvironment and increase cell-cell interactions. The resulting ...

Models and Methods to Evaluate Transport of Drug Delivery Systems Across Cellular Barriers

Sub-micrometer carriers (nanocarriers; NCs) enhance efficacy of drugs by improving solubility, stability, circulation time, targeting, and release. Additionally, traversing cellular barriers in the body is crucial for both oral delivery of therapeutic NCs into the circulation and transport from the blood into tissues, where intervention is needed. NC transport across cellular barriers is achieved by: (i) the paracellular route, via transient disruption of the junctions that interlock adjacent cells, or (ii) the transcellular route, where materials are internalized by endocytosis, transported across the cell body, and secreted at the opposite cell surface (transyctosis). Delivery across cellular barriers can be facilitated by coupling therapeutics or their carriers with targeting agents that bind specifically to cell-surface markers involved in transport. Here, we provide methods to measure the extent and mechanism of NC transport across a model cell barrier, which consists of a monolayer of gastrointestinal (GI) epithelial cells grown on a porous membrane located in a transwell insert. Formation of a permeability barrier is confirmed by measuring transepithelial electrical resistance (TEER), transepithelial transport of a control substance, and immunostaining of tight junctions. As an example, ~200&#160;nm polymer NCs are used, which carry a therapeutic cargo and are coated with an antibody that targets a cell-surface determinant. The antibody or therapeutic cargo is labeled with 125I for radioisotope tracing and labeled NCs are added to the upper chamber over the cell monolayer for varying periods of time. NCs associated to the cells and/or transported to the underlying chamber can be detected. Measurement of free 125I allows subtraction of the degraded fraction. The paracellular route is assessed by determining potential changes caused by NC transport to the barrier parameters described above. Transcellular transport is determined by addressing the effect of modulating endocytosis and transcytosis pathways.

Many therapeutic applications require safe and efficient transport of drug carriers and their cargoes across cellular barriers in the body. This article describes an adaptation of established methods to evaluate the rate and mechanism of transport of drug nanocarriers (NCs) across cellular barriers, such as the gastrointestinal (GI) epithelium.

Sub-micrometer carriers (nanocarriers; NCs) enhance efficacy of drugs by improving solubility, stability, circulation time, targeting, and release. ...

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, ...

Drug-induced Sensitization of Adenylyl Cyclase: Assay Streamlining and Miniaturization for Small Molecule and siRNA Screening Applications

Sensitization of adenylyl cyclase (AC) signaling has been implicated in a variety of neuropsychiatric and neurologic disorders including substance abuse and Parkinson&#39;s disease. Acute activation of G&#945;i/o-linked receptors inhibits AC activity, whereas persistent activation of these receptors results in heterologous sensitization of AC and increased levels of intracellular cAMP. Previous studies have demonstrated that this enhancement of AC responsiveness is observed both in vitro and in vivo following the chronic activation of several types of G&#945;i/o-linked receptors including D2 dopamine and &#956; opioid receptors. Although heterologous sensitization of AC was first reported four decades ago, the mechanism(s) that underlie this phenomenon remain largely unknown. The lack of mechanistic data presumably reflects the complexity involved with this adaptive response, suggesting that nonbiased approaches could aid in identifying the molecular pathways involved in heterologous sensitization of AC. Previous studies have implicated kinase and Gb&#947; signaling as overlapping components that regulate the heterologous sensitization of AC. To identify unique and additional overlapping targets associated with sensitization of AC, the development and validation of a scalable cAMP sensitization assay is required for greater throughput. Previous approaches to study sensitization are generally cumbersome involving continuous cell culture maintenance as well as a complex methodology for measuring cAMP accumulation that involves multiple wash steps. Thus, the development of a robust cell-based assay that can be used for high throughput screening (HTS) in a 384&#160;well format would facilitate future studies. Using two D2 dopamine receptor cellular models (i.e.&#160;CHO-D2L and HEK-AC6/D2L), we have converted our 48-well sensitization assay (&#62;20 steps 4-5 days) to a five-step, single day assay in 384-well format. This new format is amenable to small molecule screening, and we demonstrate that this assay design can also be readily used for reverse transfection of siRNA in anticipation of targeted siRNA library screening.

Persistent activation of inhibitory G protein-coupled receptors results in sensitization of adenylyl cyclase signaling. To identify the essential molecular pathways, nonbiased approaches are necessary; however, this strategy requires the development of a scalable cell-based cAMP sensitization assay. Herein, we describe a sensitization assay for small molecule and siRNA screening.

Sensitization of adenylyl cyclase (AC) signaling has been implicated in a variety of neuropsychiatric and neurologic disorders including substance abuse ...

Biofunctionalized Prussian Blue Nanoparticles for Multimodal Molecular Imaging Applications

Multimodal, molecular imaging allows the visualization of biological processes at cellular, subcellular, and molecular-level resolutions using multiple, complementary imaging techniques. These imaging agents facilitate the real-time assessment of pathways and mechanisms in vivo, which enhance both diagnostic and therapeutic efficacy. This article presents the protocol for the synthesis of biofunctionalized Prussian blue nanoparticles (PB NPs) - a novel class of agents for use in multimodal, molecular imaging applications. The imaging modalities incorporated in the nanoparticles, fluorescence imaging and magnetic resonance imaging (MRI), have complementary features. The PB NPs possess a core-shell design where gadolinium and manganese ions incorporated within the interstitial spaces of the PB lattice generate MRI contrast, both in T1 and T2-weighted sequences. The PB NPs are coated with fluorescent avidin using electrostatic self-assembly, which enables fluorescence imaging. The avidin-coated nanoparticles are modified with biotinylated ligands that confer molecular targeting capabilities to the nanoparticles. The stability and toxicity of the nanoparticles are measured, as well as their MRI relaxivities. The multimodal, molecular imaging capabilities of these biofunctionalized PB NPs are then demonstrated by using them for fluorescence imaging and molecular MRI in vitro.

This protocol describes the synthesis of biofunctionalized Prussian blue nanoparticles and their use as multimodal, molecular imaging agents. The nanoparticles have a core-shell design where gadolinium or manganese ions within the nanoparticle core generate MRI contrast. The biofunctional shell contains fluorophores for fluorescence imaging and targeting ligands for molecular targeting.

Multimodal, molecular imaging allows the visualization of biological processes at cellular, subcellular, and molecular-level resolutions using multiple, ...

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 ...

Mesenchymal Stromal Cell Culture and Delivery in Autologous Conditions: A Smart Approach for Orthopedic Applications

Human Mesenchymal Stromal Cells (hMSCs) are cultured in vitro with different media. Limits on their use in clinical settings, however, mainly depend on potential biohazard and inflammation risks exerted by xenogeneic nutrients for their culture. Human derivatives or recombinant materials are the first choice candidates to reduce these reactions. Therefore, culture supplements and materials of autologous origin represent the best nutrients and the safest products.
Here, we describe a new protocol for the isolation and culture of bone marrow hMSCs in autologous conditions &#8212;&#160;namely,&#160;patient-derived serum as a supplement for the culture medium and fibrin as a scaffold for hMSC administration. Indeed, hMSC/fibrin clot constructs could be extremely useful for several clinical applications. In particular, we focus on their use in orthopedic surgery, where the fibrin clot derived from the donor&#39;s own blood allowed effective cell delivery and nutrient/waste exchanges. To ensure optimal safety conditions, it is of the utmost importance to avoid the risks of hMSC transformation and tissue overgrowth. For these reasons, the approach described in this paper also indicates a minimally ex vivo hMSC expansion, to reduce cell senescence and morphologic changes, and short-term osteo-differentiation before implantation, to induce osteogenic lineage specification, thus decreasing the risk of subsequent uncontrolled proliferation.

Culturing human Mesenchymal Stromal Cells (hMSCs) with autologous serum, reduces the risks of rejection by xenogeneic material and other negative effects. It also allows for the recovery of a subset of mesodermal progenitors, which can deliver fresh hMSCs. Embedding hMSCs in an autologous fibrin clot enables easy handling and effective surgical implantation.

Human Mesenchymal Stromal Cells (hMSCs) are cultured in vitro with different media. Limits on their use in clinical settings, however, mainly depend on ...

Synthesis of Functionalized 10-nm Polymer-coated Gold Particles for Endothelium Targeting and Drug Delivery

Gold nanoparticles (AuNPs) have been used extensively in medical research due to their size, biocompatibility, and modifiable surface. Specific targeting and drug delivery are some of the applications of these AuNPs, but endothelial extracellular matrices&#39; defensive properties hamper particle uptake. To address this issue, we describe a synthesis method for ultrasmall gold nanoparticles to improve vascular delivery, with customizable functional groups and polymer lengths for further adjustments. The protocol yields 2.5 nm AuNPs that are capped with tetrakis(hydroxymethyl)phosphonium chloride (THPC). The replacement of THPC with hetero-functional polyethylene glycol (PEG) on the surface of the AuNP increases the hydrodynamic radius to 10.5 nm while providing various functional groups on the surface. The last part of the protocol includes an optional addition of a fluorophore to allow the AuNPs to be visualized under fluorescence to track nanoparticle uptake. Dialysis and lyophilization were used to purify and isolate the AuNPs. These fluorescent nanoparticles can be visualized in both in vitro and in vivo experiments due to the biocompatible PEG coating and fluorescent probes. Additionally, the size range of these nanoparticles render them an ideal candidate for probing the glycocalyx without disrupting normal vasculature function, which may lead to improved delivery and therapeutics.

We describe a method of synthesizing biocompatible 10-nm gold nanoparticles, functionalized by coating poly-ethylene glycol onto the surface. These particles can be used in vitro and in vivo for delivering therapeutics to nanoscale cellular and extracellular spaces that are difficult to access with conventional nanoparticle sizes.

Gold nanoparticles (AuNPs) have been used extensively in medical research due to their size, biocompatibility, and modifiable surface. Specific targeting ...

Comprehensive Evaluation of the Effectiveness and Safety of Placenta-Targeted Drug Delivery Using Three Complementary Methods

No effective treatments currently exist for placenta-associated pregnancy complications, and developing strategies for the targeted delivery of drugs to the placenta while minimizing fetal and maternal side effects remains challenging. Targeted nanoparticle carriers provide new opportunities to treat placental disorders. We recently demonstrated that a synthetic placental chondroitin sulfate A binding peptide (plCSA-BP) could be used to guide nanoparticles to deliver drugs to the placenta. In this protocol, we describe in detail a system for assessing the efficiency of drug delivery to the placenta by plCSA-BP that employs three separate methods used in combination: in vivo imaging, high-frequency ultrasound (HFUS), and high-performance liquid chromatography (HPLC). Using in vivo imaging, plCSA-BP-guided nanoparticles were visualized in the placentas of live animals, while HFUS and HPLC demonstrated that plCSA-BP-conjugated nanoparticles efficiently and specifically delivered methotrexate to the placenta. Thus, a combination of these methods can be used as an effective tool for the targeted delivery of drugs to the placenta and development of new treatment strategies for several pregnancy complications.

We describe a system that utilizes three methods to evaluate the safety and effectiveness of placenta-targeted drug delivery: in vivo imaging to monitor nanoparticle accumulation, high-frequency ultrasound to monitor placental and fetal development, and HPLC to quantify drug delivery to tissue.

No effective treatments currently exist for placenta-associated pregnancy complications, and developing strategies for the targeted delivery of drugs to ...