The overall goal of this procedure is to manufacture silk nanoparticles from a reverse-engineered aqueous silk solution, and subsequently use these silk nanoparticles for drug-delivery applications. So this method utilized nano-precipitation to generate silk-based particle, which can be used for a broad range of applications, such as drug delivery. The main advantage of this technique is simplicity and reproducibility to deliver uniform silk nanoparticles that are stable, and of a narrow-side distribution.
To begin this procedure, bring two liters of distilled water to a boil. Then, carefully add 4.24 grams of sodium carbonate. Next, use scissors to cut five grams of dried cocoons into five-millimeter-by-five-millimeter pieces.
Add the cocoon pieces to the sodium carbonate solution, and boil for 60 minutes to de-gum the silk fibers, stirring occasionally. Once boiling is complete, removed the de-gummed silk. Wash with one liter of distilled water for 20 minutes, three times.
Then, squeeze the washed silk to remove excess liquid, untie the silk by hand, and place it in a fume hood to air dry overnight. After drying is complete, tightly pack five grams of air-dried silk fibers into the bottom of a 50-milliliter beaker. Then, prepare a fresh 9.3-molar solution of lithium bromide.
Dissolve the silk in lithium bromide, adding four milliliters of lithium bromide per gram of silk. Cover the beaker with aluminum foil to prevent evaporation. Allow the silk to dissolve for four hours at 60 degrees Celsius, stirring occasionally.
After dissolution is complete, wet a dialysis cassette in water for five minutes. Then, transfer 15 milliliters of the silk-lithium bromide solution into the dialysis cassette. Use a needle and syringe to remove any air bubbles.
Dialyze against one liter of distilled water. Change the water at one, three, and six hours. Change the beaker with new water the following morning and evening, and one final time on the morning of the third day.
After dialysis is complete, collect the silk solution from the cassette and centrifuge at 9, 500 times g for 20 minutes at five degrees Celsius. Recover the supernatant and repeat the centrifugation twice more. Then, remove, dry, and determine the total dry weight and concentration of the silk solution as outlined in the text protocol.
Next, add a 5%weight-per-volume silk solution drop-wise to acetone, maintaining the acetone volume-per-volume concentration over 75. Fill the tubes completely to ensure that they do not collapse during centrifugation. Centrifuge the precipitate at 48, 000 times g for two hours at four degrees Celsius.
Dislodge the pellet with a spatula and add 20 milliliters of distilled water. Use pipette tips to remove the pellet from the spatula and vortex it for 20 seconds. Then, sonicate the pellet using an ultrasonic probe at 30%amplitude for 30 seconds, two times.
After sonication is complete, top up the centrifuge tube to capacity with distilled water. Repeat the centrifugation and resuspension at least two more times. Then, store at four degrees Celsius for future use.
Begin by preparing a Doxorubicin solution as outlined in the text protocol. Then, mix two milliliters of a 0.2 micromole-per-milliliter Doxorubicin solution with 200 microliters of silk nanoparticles at 10, 30, or 50 milligrams per milliliter in a two-milliliter tube. Incubate the silk-Doxorubicin suspension at room temperature overnight on a rotating mixer.
After drug-loading is complete, centrifuge the suspension at 194, 000 times g for 30 minutes. Wash the Doxorubicin-loaded silk nanoparticles with distilled water and repeat the centrifugation twice. Next, pull the supernatant and note the total volume.
Pipette 200 microliters of the supernatant into a black microtiter plate. Using a fluorescence microplate reader, set the excitation wavelength to 485 nanometers and the emission wavelength to 590 nanometers, and record the fluorescence values. After culturing and seeding MDA-MB-231 cells, add freely diffusable Doxorubicin, silk nanoparticles, and silk nanoparticles loaded with Doxorubicin into the respective 96-well plate.
Then, add MTT at 72 hours to determine cell viability and the half-maximal inhibitory concentration. Incubate for five hours. After incubation is complete, carefully drain the wells with a pipette.
Add 100 microliters of dimethyl sulfoxide to dissolve the formazan. Using an absorbance microplate reader, measure the absorbance at 560 nanometers. Gather this data for three independent experiments.
In this procedure, silk nanoparticles are manufactured from Bombyx mori cocoons. Doxorubicin is used as a clinically relevant chemotheraputic model drug for drug loading and in vitro cytotoxicity studies. The representative results for the Doxorubicin encapsulation efficiency for 2, 6, and 10 milligrams of silk nanoparticles can be seen here.
The ability of drug-loaded silk nanoparticles to deliver Doxorubicin and subsequently kill cancer cells is assessed in vitro with human breast cancer MDA-MB-231 cells. At equivalent drug doses of 0.1 micrograms, freely diffusable Doxorubicin and Doxorubicin-loaded silk particles cause significant decreases in cell viability. Freely diffusable Doxorubicin shows a greater cytotoxicity than loaded silk nanoparticles due to differences in cell uptake.
The quantitative measurements are corroborated by qualitative scanning-electron-microscope imaging. Control cultures show higher cellular density and a predominating mesenchymal phenotype. Similar results are seen in cultures exposed to silk nanoparticles.
Cultures exposed to Doxorubicin show a markedly different phenotype, with a broad and spread-out morphology. Exposure to either freely diffusable Doxorubicin or Doxorubicin-loaded silk nanoparticles results in a substantial reduction of cell numbers. So one master delivers engineered silk solution, can be delivered within three days.
And a silk nanoparticle can be manufactured within a day. Where attempting this procedure, is it important to remember to use an acetone-resistant centrifuge to insure nanoprecipitation and sample work-up. Furthermore, insure that the centrifuge tube are filled completely so that they don't collapse during the centrifugation.
After watching this video, you should have a good understanding of how to manufacture a reverse-engineered silk solution and use this to generate the uniform silk nanoparticle with a narrow-side distribution.
