This is our experiment section. Here we compile important protocols for the development of
TecTissue, ranging from our bacterial transformation procedures to our cell proliferation
assays.
We also address cell culture maintenance and protein loaded chitosan nanoparticles.
Here you may find the protocol for our growth factor delivery to damaged cells and how much
harm can be inflicted in vitro.
Protocols
Chitosan nanoparticles
Protein encapsulation protocol
Reactants
Chitosan low molecular weight from Sigma-Aldrich
TPP from Sigma-Aldrich
NaOH 1M
Acetic acid 1M
Distilled water
Protein of interest (10 mg/mL)
Procedure
Stock solutions
In a 15 mL Falcon tube add 30 mg of chitosan and 10 mL of distilled water (to get a
solution with a concentration of 3 mg/mL).
Add 10 microliters of acetic acid for each mL of chitosan solution to solubilize the
chitosan.
To adjust the pH acetic acid and NaOH should be used.
NOTE: the pH should be adjusted depending on your protein of interest, taking into account
the isoelectric point, always maintaining the chitosan solution positively charged (pH <
6.5)
and the protein of interest negatively charged (preferred).
In another 15 mL falcon
tube add 10 mg of TPP and 10 mL of distilled water (to get a concentration of 1 mg/mL).
Nanoparticle preparation
In a 20 mL beaker add 1 mL of chitosan solution and 100 uL of your protein, stir the
mix at 1100 rpm with a magnetic stirrer (the size of nanoparticles is affected by rpm
value; for smaller nanoparticles use higher rpm).
Take 1 mL of the TPP solution and add it to the mix dropwise.
Continue stirring for 1 hour.
Particle collection
Transfer the mix to 2 1.5 mL Eppendorf tubes.
NOTE: If nanoparticles are to be extracted centrifuge the tubes at 20,000 rpm for 30
minutes at 4°C.
Eliminate the supernatant.
The pellet will contain your protein of interest.
If nanoparticles are to be used for liberation measurements or suspended in a
controlled pH solution, resuspend well and store at 4 °C.
TEM preparation
To visualize chitosan nanoparticles some previous preparation steps must be carried out
(this preparation protocol may vary).
A film of Formvar has to be previously prepared and used to coat a glass slide for the
creation of an 80-120 μm thick membrane.
Place a copper grid on the Formvar membrane for it to be absorbed and later removed
with a needle.
Add 20 μL of your solution of interest into the grid and let it be absorbed. Add a
solution of 1% (w/v) phosphotungstic acid until the sample dries.
View in a transmission electron microscope.
NOTE: Samples were observed at 150,000x.
Protein encapsulation efficiency protocol
This protocol will evaluate and standardize encapsulation efficiency when working with a specific protein. It is highly recommended to work with a highly purified protein sample, so as to get the most reliable quantification. Measurements are performed according to the Bradford assay.
Detection range: 0.1-1.4 mg/mL
NOTE: Bradford reactant must be at room temperature and shaken gently before starting the protocol.
Calibration curve of BSA
BSA Stock solution
Prepare 10 mg/mL BSA solution
Store in ice for further use
Dilutions
Loaded and empty nanoparticles are prepared under the same conditions (agitation, temperature, pH, and reactant concentrations). Thus, we used a sample of empty encapsulation supernatant as a blank to construct a standard curve to estimate protein encapsulation efficiency. Volumes of supernatant were mixed with volumes of BSA stock solution to obtain dilutions of known protein concentrations.
Refer to encapsulation protocol to prepare empty chitosan nanoparticles.
Prepare encapsulation solution aliquots.
Centrifuge samples after splitting the initial encapsulation volume at 13,400 rpm for 30 min.
Supernatant will be used to derive the curve. Do not discard.
Prepare dilutions according to the following table and label each tube. A small-scale procedure was adapted from Sigma Aldrich to perform Bradford assay on the prepared dilutions.Table 1. Dilutions to derive a standard curve for encapsulation efficiency quantification
Dilution
BSA concentration (mg/mL)
Volume of BSA stock solution 10 mg/mL (uL)
Volume of empty nanoparticle encapsulation supernatant (uL)
Final volume (uL)
0
0
0
100
100
1
0.26
2.6
97.4
100
2
0.52
5.2
94.8
100
3
0.78
7.8
92.2
100
4
1.04
10.4
89.6
100
5
1.4
14
86
100
Experimental procedure
Place 6.5 μL of each pattern of BSA (0 mg/mL to 1.4 mg/mL) in sterile 0.6 mL tubes.
Add 193.5 μL of Bradford reagent to each tube. The final volume is 200 μL.
Vortex gently.
Incubate 5-45 min at room temperature (until a change in color is noticeable).
Transfer 50 μL to a spectrophotometer cell.
Blank with the tube of null BSA concentration + Bradford reagent.
Take absorbance at 595 nm for the samples and record it.
Derive a standard curve for protein concentration in encapsulation supernatant.
Note: There shouldn’t be a time difference higher than 10 minutes between each read.
Efficiency quantification
Refer to protein encapsulation protocol here using BSA.
NOTE: Calculate initial protein concentration before stirring and record it.
Experimental procedure
Prepare eight 1 mL aliquots of loaded chitosan nanoparticles: four empty, four containing the protein of interest.
Centrifuge aliquots at 13,400 rpm for 30 min.
Take 6.5 μL of the supernatant and measure absorbance at 595 nm with 193.5 μL of Bradford reagent. Remember to incubate this mix at room temperature 5-45 minutes (until a change of color is noticeable).
Record reads and estimate protein concentration in the supernatant using the previously derived standard curve.
Calculate encapsulation efficiency at this initial time as follows.
