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.

Protein encapsulation protocol

Procedure

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

1. Prepare 10 mg/mL BSA solution
2. 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.

1. Refer to encapsulation protocol to prepare empty chitosan nanoparticles.
2. Prepare encapsulation solution aliquots.
3. Centrifuge samples after splitting the initial encapsulation volume at 13,400 rpm for 30 min.
4. 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

1. Place 6.5 μL of each pattern of BSA (0 mg/mL to 1.4 mg/mL) in sterile 0.6 mL tubes.
2. Add 193.5 μL of Bradford reagent to each tube. The final volume is 200 μL.
3. Vortex gently.
4. Incubate 5-45 min at room temperature (until a change in color is noticeable).
5. Transfer 50 μL to a spectrophotometer cell.
6. Blank with the tube of null BSA concentration + Bradford reagent.
7. Take absorbance at 595 nm for the samples and record it.
8. 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

1. Prepare eight 1 mL aliquots of loaded chitosan nanoparticles: four empty, four containing the protein of interest.
2. Centrifuge aliquots at 13,400 rpm for 30 min.
3. 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).
4. Record reads and estimate protein concentration in the supernatant using the previously derived standard curve.
5. 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

1. Use Bradford assay to derive a standard curve for a standard protein (BSA, for instance).
2. 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

3. 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).
4. Read the absorbance of the remaining dilutions using dilution 0 as blank.
5. Graph absorbance reads vs concentration.
6. Use a linear trend to get the equation to compute protein concentration evaluating correlation coefficient.

Protein release behavior

1. Refer to protein encapsulation protocol to prepare enough loaded-nanoparticles for six 1 mL aliquots (some volume is lost in every transfer).
2. Centrifuge the total encapsulation volume at 20000 rpm for 20 minutes.
3. Discard supernatant.
4. 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.
5. Prepare aliquots as previously stated.
6. Refer to protein encapsulation protocol to prepare enough empty nanoparticles for six 1 mL aliquots (some volume is lost in every transfer).
7. Centrifuge the total encapsulation volume at 20000 rpm for 20 minutes.
8. Discard supernatant.
9. Resuspend pellet in a volume of PBS pH 7.4 equal to the original volume.
10. Prepare aliquots as previously stated.
11. Label all aliquots to measure them at time 0, 2, 4, 6, 12, 24, and 48 h. Store at 37 °C and 100 rpm.
12. At the right time, centrifuge the aliquots at 20000 rpm for 20 minutes.
13. Take 193.5 μL of Bradford reagent and mix with 6.5 μL of centrifugation supernatant. Vortex gently.
14. Incubate tube at room temperature for 20 minutes.
15. Transfer 50 μL to a spectrophotometer cell.
16. 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.