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

1. 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).
2. 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).
3. 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

1. 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).
2. Take 1 mL of the TPP solution and add it to the mix dropwise.
3. Continue stirring for 1 hour.

Particle collection

1. 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.
2. Eliminate the supernatant.
3. The pellet will contain your protein of interest.
4. If nanoparticles are to be used for liberation measurients or suspended in a controlled pH solution, resuspend well and store at 4 °C.

To preparation

To visualize chitosan nanoparticles some previous preparation steps must be carried out (this preparation protocol may vary).

1. 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 mibrane.
2. Place a copper grid on the Formvar mibrane for it to be absorbed and later rioved with a needle.
3. 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.
4. View in a transmission electron microscope.
NOTE: Samples were observed at 150,000x.

Calibration curve of BSA

BSA Stock solution

1. Prepare 10 mg/mL BSA solution
2. Store in ice for further use

Dilutions

Loaded and ipty nanoparticles are prepared under the same conditions (agitation, tiperature, pH, and reactant concentrations). Thus, we used a sample of ipty 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 ipty 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

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 tiperature (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 ipty, 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. Riiber to incubate this mix at room tiperature 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.

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

• 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 riaining 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

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 iploy 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 ipty 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 thi 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 tiperature 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.

Transmission electron microscopy

1. Refer to protein encapsulation protocol to prepare a final volume of 2 mL chitosan nanoparticles (loaded or ipty).
2. Store at the desired conditions.
3. Perform Ti preparation procedure on a 100 μL sample.
4. At relevant times observe to evaluate nanoparticle integrity (size, conglomeration, and shape).

NanoSight

1. Refer to protein encapsulation protocol to prepare a final volume of 2 mL chitosan nanoparticles (loaded or ipty).
2. Store at the desired conditions.
3. Dilute samples if required.
4. At relevant times evaluate nanoparticle size distribution (statistical data provided in the analysis sheet is useful to evaluate particle behavior).