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.
Protein liberation and stability protocol
This protocol will assess the protein release and nanoparticle stability in aqueous solutions. We quantified protein liberation by Bradford assay.
Materials
PBS pH 7.4
Bradford reagent
Standard curve derivation
Use Bradford assay to derive a standard curve for a standard protein (BSA, for instance).
Prepare 6 dilutions from a 10 mg/mL stock solution of the standard protein according to the table. A small-scale procedure was adapted from Sigma Aldrich to perform Bradford assay on the prepared dilutions.
Table 1. Dilutions for standard curve derivation using PBS
Dilution
Standard protein concentration (mg/mL)
Volume of stock solution 10 mg/mL (uL)
Volume of PBS pH 7.4 (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
For each dilution mix 6.5 uL of the sample and 193.5 uL of Bradford reactant for a final volume of 200 uL and leave it react for 20 minutes (Sigma Aldrich suggests 5-45 minutes).
Read the absorbance of the remaining dilutions using dilution 0 as blank.
Graph absorbance reads vs concentration.
Use a linear trend to get the equation to compute protein concentration evaluating correlation coefficient.
Protein release behavior
Refer to protein encapsulation protocol to prepare enough loaded-nanoparticles for six 1 mL aliquots (some volume is lost in every transfer).
Centrifuge the total encapsulation volume at 20000 rpm for 20 minutes.
Discard supernatant.
Resuspend pellet in a volume of PBS pH 7.4 equal to the original volume.
NOTE: Given the low solubility of chitosan in neutral pH solutions, some protocols employ mild to moderate sonication to disrupt possible non-dissolved pellet.
Prepare aliquots as previously stated.
Refer to protein encapsulation protocol to prepare enough empty nanoparticles for six 1 mL aliquots (some volume is lost in every transfer).
Centrifuge the total encapsulation volume at 20000 rpm for 20 minutes.
Discard supernatant.
Resuspend pellet in a volume of PBS pH 7.4 equal to the original volume.
Prepare aliquots as previously stated.
Label all aliquots to measure them at time 0, 2, 4, 6, 12, 24, and 48 h. Store at 37 °C and 100 rpm.
At the right time, centrifuge the aliquots at 20000 rpm for 20 minutes.
Take 193.5 μL of Bradford reagent and mix with 6.5 μL of centrifugation supernatant. Vortex gently.
Incubate tube at room temperature for 20 minutes.
Transfer 50 μL to a spectrophotometer cell.
Measure absorbance and calculate protein concentration in the supernatant using the previously derived standard curve. Blank should be PBS pH 7.4 + Bradford reagent as stated above.
NOTE: to achieve a time-efficient protocol, a previous standardization of protein encapsulation efficiency is strongly suggested (refer to protein encapsulation efficiency protocol). Since you already know your protein encapsulation efficiency, protein liberation calculations may be performed as follows.
Nanoparticle stability
When studying the nanoparticle behavior in a certain environment several studies are carried out to assess particle stability throughout time. Such procedures comprise Z potential measurement and visual examination of size, shape, and particle physical integrity. A stability monitoring is suggested as particles may change their shape, degrade, and conglomerate when subjected to different stimuli. Such a study is helpful to predict the behavior of the created nanoparticles throughout time and greatly improves the design of drug release experiments. Here we include a suggested simple procedure to visually evaluate particle sizes and integrity. You can also use a Z potential measurement equipment, or NanoSight NS300, as we did.
