Synthesis of gelatin nanoparticles by one-step desolvation method

This protocol is based on the following paper: Khramtsov, P.; Burdina, O.; Lazarev, S.; Novokshonova, A.; Bochkova, M.; Timganova, V.; Kiselkov, D.; Minin, A.; Zamorina, S.; Rayev, M. Modified Desolvation Method Enables Simple One-Step Synthesis of Gelatin Nanoparticles from Different Gelatin Types with Any Bloom Values. Pharmaceutics 2021, 13, 1537.


Text protocol:

1. Principle

Principle of the desolvation method is the addition of poor solvent (alcohols, acetone, or other water-miscible solvent) to the aqueous solution of gelatin. At a certain concentration of poor solvent solubility of gelatin decreases and protein molecules aggregate forming spherical nanoparticles. Nanoparticles are then stabilized by the addition of cross-linker (aldehydes, EDC, genipin etc.). In this video, gelatin molecules will be cross-linked by the glutaraldehyde. After that, nanoparticles are washed with water by centrifugation from organic solvent, free gelatin, and aldehyde. You can adjust synthesis conditions (concentrations, pH, solvent, volumes) and obtain larger or smaller nanoparticles with higher or lower yields. Resulting nanoparticles are stable in water for weeks and at pH 4-10 for at least several days. Nanoparticles can be autoclaved. Human blood mononuclear cells retained 90% viability in the presence of 1 mg/mL gelatin nanoparticles. 


2. Reagents

Herein, we will use gelatin B from bovine skin (type B) from Sigma (#G6650). Gel strength (bloom value) of this gelatin is 75. Gelatins from porcine skin (type A) as well as fish gelatin can also be used. Gelatins with higher and lower gel strength are compatible with the method. Isoelectric point of gelatin B is 4.7-5.3. We will adjust the pH of gelatin to 10, therefore gelatin molecules will be negatively charged. Concentration of gelatin in the video will be 20 mg/mL. You can use higher or lower concentration. In general, larger nanoparticles are formed at higher gelatin concentration. Note, that gelatin powder is not 100% gelatin. Moisture content can be more than 10%. Thus, in fact concentration of gelatin will be ABOUT 20 mg/mL. If you want better reproducibility and accuracy, prepare stock concentrated gelatin solutions and measure dry weight of gelatin gravimetrically. Stock solutions can be stored at +2... +8°C or filter-sterilized. We did not try food gelatins from local stores. They potentially may contain admixtures (e.g. salts) that can affect nanoparticle formation (e.g. aggregation). If such a gelatin does not work, try to dialyze it against water.


We will use isopropyl alcohol, >99.8%, because it provides higher yields in comparison with ethanol and methanol. Other organic solvents, which are miscible with water, can also be used. Glutaraldehyde is a widely used cross-linker for protein nanoparticles, which forms covalent bonds with primary amine bonds (e.g. lysines and terminal amines).

Reaction requires neutral or alkaline pH (see Migneault, I., Dartiguenave, C., Bertrand, M. J., & Waldron, K. C. (2004). Glutaraldehyde: behavior in aqueous solution, reaction with proteins, and application to enzyme crosslinking. BioTechniques, 37(5), 790–802. We use 50% glutaraldehyde from ITW (#A3166,0100). Commercial glutaraldehydes have different purity (can contain acids, polymerized forms), but we do not know how it can affect nanoparticle preparation.

Glutaraldehyde will be diluted to 0.8% before addition. Less concentrated preparations are OK. Concentrated glutaraldehyde should not be stored at room temperature. Prepare diluted glutaraldehyde rigth before the addition to nanoparticles.


3. Synthesis

Pre-warm isopropyl alcohol at +37 °C. We will use 5-fold excess of isopropyl alcohol (20 mL) in relation to gelatin volume (4 mL). 5:1 ratio provides good yield. The higher volume of alcohol gives higher yield, smaller nanoparticles (gelatin dilution effect), and larger overall volume; therefore, the choice of isopropyl alcohol volume depends on what you want. We did not study how nanoparticle size depends on temperature of solutions. However, we know that method works when all solutions are of room temperature. It still works if isopropyl alcohol is cooled to +4 °C before addition to gelatin. We use water bath and temperature control for better day-to-day reproducibility.


We weighed 500 mg of gelatin, added about 20 ml of water. Need several minutes with heating to dissolve. Volume was adjusted to 25 mL (to obtain 20 mg/mL of gelatin).

pH was adjusted to 10 with 1 M NaOH. Addition of neutral salts (such as NaCl) or buffers can lead to aggregation, because salts screen surface charges of gelatin molecules. However, addition of salt can be added if you want larger nanoparticles. Larger pH provides lower yield and smaller nanoparticles. Isoelectric points of gelatins are different. To avoid aggregation keep the pH away from isoelectric point. Neutral and alkaline pH provide faster cross-linking if you use glutaraldehyde as a cross-linker. Keep gelatin solution  in the water bath for 10-15 minutes.


Prepare about 1 mL of 0.8% glutaraldehyde. We add 16.2 uL of 50% glutaraldehyde to 1 mL of water. We did not adjust the pH of aldehyde, however it can shift the pH of suspension to more acidic values. When glutaraldehyde is added to desolvated gelatin, its local concentration is high. It can lead to the formation of aggregates. We dilute aldehyde to avoid this problem. Amount of glutaraldehyde depends on protein concentration. Approximately 10-fold excess of glutaraldehyde molecules (in relation to one pair of lysines) is a good starting point. Gelatin B contains approximately 11 lysine residues per 1000 amino acids (Hafidz, R.N.R.M., Yaakob, C.M., Amin, I., Noorfaizan, A., 2011. Chemical and functional properties of bovine and porcine skin gelatin. Int. Food Res. J. 18, 813-817). The mean molecular weight of a single amino acid is approx. 110 Da. The molecular weight of gelatin B with a bloom number of 75 is between 20 and 25 kDa. Therefore, each molecule of gelatin B contains 2-2.5 lysine residues. 4 mL of gelatin B solution (20 mg/mL) contains approx. 3.98 μM of protein (7.96 μM of lysines).


Add 4 mL of gelatin solution into 50-ml centrifuge tube. We use simple automatic pipette. For better accuracy weigh gelatin solution or use pipette with positive displacement.


KEY STEP. Carefully add isopropyl alcohol to gelatin. Dropwise addition is not necessary. Mixing should be very gentle. DO NOT USE VORTEX OR MAGNETIC STIRRER. High stirring speed results in aggregation. Stirring can be used only with high-bloom gelatins (say, 300 or more, see Geh, K.J.; Hubert, M.; Winter, G. Optimisation of one-step desolvation and scale-up of gelatine nanoparticle production. J. Microencapsul. 2016, 33, 595–604.) For better reproducibility, we use rotator and fixed speed mixing (10 rpm, 30 s, 5 rounds). Gentle manual mixing works well too.


Keep desolvated gelatin at +37 °C for 30 min. We did not optimize the duration of this incubation step, but noted that suspension become more turbid after incubation for 30 min


Add 0.9 mL of 0.8% glutaraldehyde quickly and immediately gently mix. Cross-link at +37 °C for 30 min. Cross-linking speed depends on pH and aldehyde-to-protein ratio, but at pH>7 it is usually rapid. Suspension become pinkish after cross-linking. Color depends on desolvating agent, gelatin type, and pH. Usually the color of nanoparticles is yellow or light-pink.


4. Purification

Transfer nanoparticles to centrifuge tube. Tube choice depends on the volume of suspension and available centrifuge. Centrifugation speed depends on volume of suspension and nanoparticle size. We centrifuged nanoparticles at maximum available speed (15557 g). We set time to 60 min, but nanoparticles settled completely even after the 15 min.

Nanoparticles of about 100 nm (measured by DLS) require speed of approx. 20000g and prolonged centrifugation time. Higher speed results in denser nanoparticle pellet, which can be difficult to disintegrate. After the first centrifugation, nanoparticles usually form large “film” on the wall of the tube. To recover all nanoparticles and make pellet softer, remove supernatant, add water (or other washing solution, e.g. buffer), wet the pellet and film, and leave for 10 minutes or more. Scrape and grind sediment with glass rod. Nanoparticles can be transferred to other tube, e.g. 1.5-mL. In our video sediment is very soft and detached from the wall easily. Usually it is elastic, something like chewing gum.

Free protein molecules can be entrapped between aggregated nanoparticles, therefore we sonicated them after each wash. We used VCX-130 sonicator (6 mm probe). 20-30 s is usually enough. Sonication power was 16 W (at 60% amplification). Avoid overheating. Nanoparticles are not very sensitive to heating (they withstand autoclaving), but boiling can result in change of nanoparticle volume. Repeat washing 2 more times. We washed nanoparticles with 20 mL of water, however volume of washing solution and number of washing cycles depend on desired resulting concentration of aldehyde and free protein in purified nanoparticles. Note that round-shaped soft pellet formed after 2nd and 3rd centrifugation. After the last centrifugation step transfer nanoparticles in thick-wall glass or plastic tube. Sonicate on ice for 10 min. Glass tubes can crack in the course of sonication, but they provide better cooling than plastic ones. Sonication time should be optimized as it depends on volume, nanoparticle concentration, sonication power and so on.


5. Size measurement

We measure size of nanoparticles by dinamyc light scattering technique. In our lab we use Zetasizer Nano ZS, very popular particle analyzer. Optimal concentration of nanoparticles for DLS measurements should be optimized. We know that concentration of our nanoparticles is about 2-4 mg/mL (according to our previous results). We dilute them 1:100, which gives concentration of 20-40 ug/mL, which is appropriate. Concentration of nanoparticles can be measured gravimetrically (Khramtsov, P., Kalashnikova, T., Bochkova, M., Kropaneva, M., Timganova, V., Zamorina, S., & Rayev, M. (2021). Measuring the concentration of protein nanoparticles synthesized by desolvation method: Comparison of Bradford assay, BCA assay, hydrolysis/UV spectroscopy and gravimetric analysis. International Journal of Pharmaceutics, 599, 120422.). Filter deionized water through 220 nm filter to remove dust and large particles, which interfere with measurement. Add 700 uL of water in each of 3 cuvettes, put 7 uL drop of nanoparticle suspension on the wall of the cuvette. Mix properly and cover the cuvettes.