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

15.7K 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

Silk Film Culture System for in vitro Analysis and Biomaterial Design

Silk films are promising protein-based biomaterials that can be fabricated with high fidelity and economically within a research laboratory environment 1,2 . These materials are desirable because they possess highly controllable dimensional and material characteristics, are biocompatible and promote cell adhesion, can be modified through topographic patterning or by chemically altering the surface, and can be used as a depot for biologically active molecules for drug delivery related applications 3-8 . In addition, silk films are relatively straightforward to custom design, can be designed to dissolve within minutes or degrade over years in vitro or in vivo, and are produce with the added benefit of being transparent in nature and therefore highly suitable for imaging applications 9-13. The culture system methodology presented here represents a scalable approach for rapid assessments of cell-silk film surface interactions. Of particular interest is the use of surface patterned silk films to study differences in cell proliferation and responses of cells for alignment 12,14 . The seeded cultures were cultured on both micro-patterned and flat silk film substrates, and then assessed through time-lapse phase-contrast imaging, scanning electron microscopy, and biochemical assessment of metabolic activity and nucleic acid content. In summary, the silk film in vitro culture system offers a customizable experimental setup suitable to the study of cell-surface interactions on a biomaterial substrate, which can then be optimized and then translated to in vivo models. Observations using the culture system presented here are currently being used to aid in applications ranging from basic cell interactions to medical device design, and thus are relevant to a broad range of biomedical fields.

Silk Film Culture System for in vitro Analysis and Biomaterial Design

Silk films are a novel class of biomaterials readily customizable for an array of biomedical applications. The presented silk film culture system is highly adaptable to a variety of in vitro analyses. This system represents a biomaterial design platform offering in vitro optimization before direct translation to in vivo models.

Silk films are promising protein-based biomaterials that can be fabricated with high fidelity and economically within a research laboratory environment ...

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, ...

Microfluidic Dry-spinning and Characterization of Regenerated Silk Fibroin Fibers

The protocol demonstrates a method for mimicking the spinning process of silkworm. In the native spinning process, the contracting spinning duct enables the silk proteins to be compact and ordered by shearing and elongation forces. Here, a biomimetic microfluidic channel was designed to mimic the specific geometry of the spinning duct of the silkworm. Regenerated silk fibroin (RSF) spinning doped with high concentration, was extruded through the microchannel to dry-spin fibers at ambient temperature and pressure. In the post-treated process, the as-spun fibers were drawn and stored in ethanol aqueous solution. Synchrotron radiation wide-angle X-ray diffraction (SR-WAXD) technology was used to investigate the microstructure of single RSF fibers, which were fixed to a sample holder with the RSF fiber axis normal to the microbeam of the X-ray. The crystallinity, crystallite size, and crystalline orientation of the fiber were calculated from the WAXD data. The diffraction arcs near the equator of the two-dimensional WAXD pattern indicate that the post-treated RSF fiber has a high orientation degree.

A protocol for the microfluidic spinning and microstructure characterization of regenerated silk fibroin monofilament is presented.

The protocol demonstrates a method for mimicking the spinning process of silkworm. In the native spinning process, the contracting spinning duct enables ...

Chemistry

Porous Silicon Microparticles for Delivery of siRNA Therapeutics

Small interfering RNA (siRNA) can be used to suppress gene expression, thereby providing a new avenue for the treatment of various diseases. However, the successful implementation of siRNA therapy requires the use of delivery platforms that can overcome the major challenges of siRNA delivery, such as enzymatic degradation, low intracellular uptake and lysosomal entrapment. Here, a protocol for the preparation and use of a biocompatible and effective siRNA delivery system is presented. This platform consists of polyethylenimine (PEI) and arginine (Arg)-grafted porous silicon microparticles, which can be loaded with siRNA by performing a simple mixing step. The silicon particles are gradually degraded over time, thereby triggering the formation of Arg-PEI/siRNA nanoparticles. This delivery vehicle provides a means for protecting and internalizing siRNA, without causing cytotoxicity. The major steps of polycation functionalization, particle characterization, and siRNA loading are outlined in detail. In addition, the procedures for determining particle uptake, cytotoxicity, and transfection efficacy are also described.

Delivery remains the main challenge for the therapeutic implementation of small interfering RNA (siRNA). This protocol involves the use of a multifunctional and biocompatible siRNA delivery platform, consisting of arginine and polyethylenimine grafted porous silicon microparticles.

Small interfering RNA (siRNA) can be used to suppress gene expression, thereby providing a new avenue for the treatment of various diseases. However, the ...

Reactive Inkjet Printing and Propulsion Analysis of Silk-based Self-propelled Micro-stirrers

In this study, a protocol for using reactive inkjet printing to fabricate enzymatically propelled silk swimmers with well-defined shapes is reported. The resulting devices are an example of self-propelled objects capable of generating motion without external actuation and have potential applications in medicine and environmental sciences for a variety of purposes ranging from micro-stirring, targeted therapeutic delivery, to water remediation (e.g., cleaning oil spills). This method employs reactive inkjet printing to generate well-defined small-scale solid silk structures by converting water soluble regenerated silk fibroin (silk I) to insoluble silk fibroin (silk II). These structures are also selectively doped in specific regions with the enzyme catalase in order to produce motion via bubble generation and detachment. The number of layers printed determines the three-dimensional (3D) structure of the device, and so here the effect of this parameter on the propulsive trajectories is reported. The results demonstrate the ability to tune the motion by varying the dimensions of the printed structures.

This protocol demonstrates the ability to utilize reactive inkjet printing to print self-motile biocompatible and environmentally friendly micro-stirrers for use in biomedical and environmental applications.

In this study, a protocol for using reactive inkjet printing to fabricate enzymatically propelled silk swimmers with well-defined shapes is reported. The ...

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 ...

Production of siRNA-Loaded Lipid Nanoparticles using a Microfluidic Device

The development of functional lipid nanoparticles (LNPs) is one of the major challenges in the field of drug delivery systems (DDS). Recently, LNP-based RNA delivery systems, namely, RNA-loaded LNPs have attracted attention for RNA therapy. In particular, mRNA-loaded LNP vaccines were approved to prevent COVID-19, thereby leading to the paradigm shift toward the development of next-generation nanomedicines. For the LNP-based nanomedicines, the LNP size is a significant factor in controlling the LNP biodistribution and LNP performance. Therefore, a precise LNP size control technique is indispensable for the LNP production process. Here, we report a protocol for size controlled LNP production using a microfluidic device, named iLiNP. siRNA loaded LNPs are also produced using the iLiNP device and evaluated by in vitro experiment. Representative results are shown for the LNP size, including siRNA-loaded LNPs, Z-potential, siRNA encapsulation efficiency, cytotoxicity, and target gene silencing activity.

Microfluidic-based lipid nanoparticle (LNP) production methods have attracted attention in drug delivery systems (DDSs), including RNA delivery. This protocol describes the fabrication, LNP (siRNA-loaded LNP) production, and LNP evaluation processes using our original microfluidic device named iLiNP.

The development of functional lipid nanoparticles (LNPs) is one of the major challenges in the field of drug delivery systems (DDS). Recently, LNP-based ...

Biochemistry

In Vivo Targeted Expression of Optogenetic Proteins Using Silk/AAV Films

The quest to understand how neural circuits process information in order to drive behavioral output has been greatly aided by recently-developed optical methods for manipulating and monitoring the activity of neurons in vivo. These types of experiments rely on two main components: 1) implantable devices that provide optical access to the brain, and 2) light-sensitive proteins that change neuronal excitability or provide a readout of neuronal activity. There are a number of ways to express light-sensitive proteins, but stereotaxic injection of viral vectors is currently the most flexible approach because expression can be controlled with genetic, anatomical, and temporal precision. Despite the great utility of viral vectors, delivering the virus to the site of optical implants poses numerous challenges. Stereotaxic virus injections are demanding surgeries that increase surgical time, increase the cost of studies, and pose a risk to the animal's health. The surrounding tissue can be physically damaged by the injection syringe, and by immunogenic inflammation caused by the abrupt delivery of a bolus of high-titer virus. Aligning injections with optical implants is especially difficult when targeting small regions deep in the brain. To overcome these challenges, we describe a method for coating multiple types of optical implants with films composed of silk fibroin and Adeno-associated viral (AAV) vectors. Fibroin, a polymer derived from the cocoon of Bombyx mori, can encapsulate and protect biomolecules and can be processed into forms ranging from soluble films to ceramics. When implanted into the brain, silk/AAV coatings release virus at the interface between optical elements and the surrounding brain, driving expression precisely where it is needed. This method is easily implemented and promises to greatly facilitate in vivo studies of neural circuit function.

In Vivo Targeted Expression of Optogenetic Proteins Using Silk/AAV Films

Here, we present a method for delivering viral expression vectors into the brain using silk fibroin films. This method allows targeted delivery of expression vectors using silk/AAV coated optical fibers, tapered optical fibers, and cranial windows.

The quest to understand how neural circuits process information in order to drive behavioral output has been greatly aided by recently-developed optical ...

Neuroscience

Formation and Attachment of FN-Silk Membranes to Inserts of Cell Culture 

Nanofibrillar Basement Membrane Mimic Made of Recombinant Functionalized Spider Silk in Custom-Made Tissue Culture Inserts

Replicating tissue barriers&#160;is critical for generating relevant in vitro models for evaluating novel therapeutics. Today, this is commonly done using tissue culture inserts with a plastic membrane, which generates an apical and a basal side. Besides providing support for the cells, these membranes come far from emulating their native counterpart, the basement membrane, which is a nanofibrillar, protein-based matrix. In this work, we show a simple way to considerably improve the biological relevance of the tissue culture inserts by replacing the plastic membrane with one made from a pure recombinant functionalized spider silk protein. The silk membrane forms through self-assembly and will spontaneously adhere to a membrane-free tissue culture insert, where it can provide support for cells. Custom-designed tissue culture inserts can be printed using a standard 3D printer, following the instructions provided in the protocol, or commercial ones can be purchased and used instead. This protocol shows how the culture system with silk membranes in inserts is set up and, subsequently, how the same cell culturing techniques that are used with traditional, commercially available inserts can be implemented.

Author Spotlight: Enhancing In Vitro Cell Culture Models with Recombinant Functionalized Spider Silk Membranes

Tissue culture inserts with plastic membranes are the golden standard in cell culture labs as permeable supports to establish cell layers and models of barrier tissues. Herein, we present a simple method to replace the plastic membrane with a more biologically relevant membrane made from a recombinant functionalized spider silk protein.

Replicating tissue barriers&#160;is critical for generating relevant in vitro models for evaluating novel therapeutics. Today, this is commonly done using ...

Preparation of Multifunctional Silk-Based Microcapsules Loaded with DNA Plasmids Encoding RNA Aptamers and Riboswitches

We introduce a protocol for the preparation of DNA-laden silk fibroin microcapsules via the Layer-by-Layer (LbL) assembly method on sacrificial spherical cores. Following adsorption of a prime layer and DNA plasmids, the formation of robust microcapsules was facilitated by inducing &#946;-sheets in silk secondary structure during acute dehydration of a single silk layer. Hence, the layering occurred via multiple hydrogen bonding and hydrophobic interactions. Upon adsorption of multilayered shells, the core-shell structures can be further functionalized with gold nanoparticles (AuNPs) and/or antibodies (IgG) to be used for remote sensing and/or targeted delivery. Adjusting several key parameters during sequential deposition of key macromolecules on silica cores such as the presence of a polymer primer, the concentration of DNA and silk protein, as well as a number of adsorbed layers resulted in biocompatible, DNA-laden microcapsules with variable permeability and DNA loadings. Upon dissolution of silica cores, the protocol demonstrated the formation of hollow and robust microcapsules with DNA plasmids immobilized to the inner surface of the capsule membrane. Creating a selectively permeable biocompatible membrane between the DNA plasmids and the external environment preserved the DNA during long-term storage and played an important role in the improved output response from spatially confined plasmids. The activity of DNA templates and their accessibility were tested during in vitro transcription and translation reactions (cell-free systems). DNA plasmids encoding RNA light-up aptamers and riboswitches were successfully activated with corresponding analytes, as was visualized during localization of fluorescently labeled RNA transcripts or GFPa1 protein in the shell membranes.

The protocol describes the formation of robust and biocompatible DNA-laden microcapsules as multiplexed in vitro biosensors capable of tracking several ligands.

We introduce a protocol for the preparation of DNA-laden silk fibroin microcapsules via the Layer-by-Layer (LbL) assembly method on sacrificial spherical ...

Manufacture and Drug Delivery Applications of Silk Nanoparticles

Summary

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Chapters in this video

Silk Film Culture System for in vitro Analysis and Biomaterial Design

Designing Silk-silk Protein Alloy Materials for Biomedical Applications

Microfluidic Dry-spinning and Characterization of Regenerated Silk Fibroin Fibers

Porous Silicon Microparticles for Delivery of siRNA Therapeutics

Reactive Inkjet Printing and Propulsion Analysis of Silk-based Self-propelled Micro-stirrers

Solid Lipid Nanoparticles (SLNs) for Intracellular Targeting Applications

Production of siRNA-Loaded Lipid Nanoparticles using a Microfluidic Device

In Vivo Targeted Expression of Optogenetic Proteins Using Silk/AAV Films

Nanofibrillar Basement Membrane Mimic Made of Recombinant Functionalized Spider Silk in Custom-Made Tissue Culture Inserts

Preparation of Multifunctional Silk-Based Microcapsules Loaded with DNA Plasmids Encoding RNA Aptamers and Riboswitches