This likely indicates excessive crosslinking between the chitosan chains (via amine groups) and TPP (polyanionic phosphate). When this happens:

• Nanoparticles aggregate into larger structures.
• A macroscopic gel or network forms.
• These structures are too large or not mobile enough to be detected by DLS.
• UV signal disappears because particles are either aggregated or settled, not well-dispersed.

Troubleshooting and Suggestions:

1. TPP Concentration and Addition Rate

• Too much TPP leads to over-crosslinking and gelation.
• Try lowering the TPP concentration significantly (e.g., 0.25–0.5 mg/mL instead of ≥1 mg/mL).
• Add very slowly (dropwise over 30 min or more) under strong stirring or even vortexing.

2. Chitosan Concentration

• A high concentration of mPEG-chitosan (especially medium MW) promotes gelation.
• Try diluting your polymer solution to 0.1–0.2% w/v.

3. Gel Pellet Resuspension

• If it’s a dense gel, sonication alone may not break it into nanoparticles.
• You may need: Strong mechanical disruption (e.g., vortex + bath sonication). Diluting before sonication. Or simply start with lower TPP and chitosan concentrations to avoid gelation in the first place.

4. Storage Conditions

• Letting the system stand after TPP addition (e.g., overnight) may cause aggregation or gelation.
• Always analyze samples soon after preparation.

DLS & UV Analysis:

• DLS: Needs a stable, well-dispersed suspension of particles. If particles are stuck in a gel matrix, you won't detect them. DLS detects the motion to calculate the size.
• UV–Vis: Literature reports usually show a peak from mPEG-chitosan nanoparticles (often at ~200–220 nm) only if they’re dispersed. In your case, the absence of signal confirms aggregation or sedimentation.

References

Calvo, P. et al. (1997). Development of positively charged colloidal drug carriers: chitosan-coated polyester nanocapsules and submicron-emulsions. Colloids and Surfaces B.

Grenha, A. et al. (2005). Chitosan nanoparticles: a new vehicle for the improvement of the delivery of drugs to the lungs. European Journal of Pharmaceutical Sciences.

Agnihotri, S.A. et al. (2004). Recent advances on chitosan-based micro- and nanoparticles in drug delivery. Journal of Controlled Release.

Hanna Anop Thank you very much! I tried using 0.3 mg/mL of TPP and 0.5% w/v of the polymer. The gel formation still occurred, though the thickness varied compared to previous trials, but still I have NP form, I will try what you said also.