I prepared chitosan nanogels with chitosan and TPP by ionic gelation method. I concentrated nanogels by ultrafiltration centrifugation. The result concentrated solutions (pH 5) was diluted with RIPM 1640 to treat BMDCs. While the diluted chitosan nanogels were instability which particle size would be 1μm, perhaps it's because the pKa of chitosan is 6.5. Did the CLSM picture show the chitosan nanogels aggregate outer of cells? (red-lyso, green-FITC chitosan nanogel, Blue-Hoechst) But I saw some published paper can treat cells well, is my method of diluting chitosan nanogels wrong? How can I solve this problem?