Dear Adriana

I suggest using medium molecular weight (MMW) chitosan and reducing the TPP: chitosan ratio (or testing different ratios).

Precipitation is a pain, I've been there with chitosan nanoparticles too. Seeing it the next day with your pH 5.5 TPP crosslinking setup probably means the system's not quite stable. Here are a few directions you could explore to adjust things:

• Try lowering the chitosan concentration. If the concentration is too high, the nanoparticles tend to aggregate. You could start by halving your current chitosan concentration and gradually decrease it to see if that helps.
• The TPP concentration is also pretty crucial. Too much or too little can both be problematic. I'd recommend optimizing the ratio of TPP to chitosan. For instance, try halving or doubling the molar ratio you're currently using and observe the effect.
• Pay attention to the stirring speed and duration. During synthesis, both the stirring speed and time can influence the size and uniformity of the nanoparticles. Make sure you're stirring evenly, but also avoid overly vigorous stirring that might disrupt the nanoparticle structure once it's formed.
• Fine-tune the pH. While you're aiming for pH 5.5, the actual pH of your system might deviate slightly. It's best to use a pH meter to precisely measure it and then try making small adjustments within the 5.0 to 6.0 range to see if you can find a more optimal pH value.

Also, making sure your reagents are of good quality is really important. When I was doing experiments, I always paid extra attention to the purity of the reagents to ensure reliable results. For things like chitosan and TPP, using higher quality reagents gives you more peace of mind. Thermo Fisher, MedChemExpress, BOC Sciences, for example, they have some pretty good quality reagents, you might want to check them out.

Hope these suggestions are helpful!