Background: Many dyes or radioactive markers used for sentinel lymph node (SLN) have the shortcomings of false positive and radiation injury. Indocyanine green (ICG) seems to have a lower false positive rate and tissue damage, without a clear field of vision during the operation., Methods: For the shortcomings, we successfully synthesized three anionic pullulan materials, changed the degree of hydrophobic for size controlling (< 50nm) to prepare CHP nanoparticles (NPs) and changed the succinyl degree to prepare CHPC NPs with different negative surface potential., Results: The size of those NPs were less than 50nm under (transmission electron microscope) TEM, with hydrodynamic size of 90.67 ± 2.2 nm of CHP, 105.8 ± 1.7 nm of CHPC1 and 115.9 ± 2.3 nm of CHPC2. Moreover, the Zeta potential of CHP, CHPC1 and CHPC2 were -1.9 ± 0.2 mV, -9.6 ± 0.3 mV and -19.4 ± 0.7 mV. The size of ICG-loading CHP, CHPC1 and CHPC2 NPs increased to 109.4 ± 2.7 nm, 113.8 ± 1.2 nm and 30.6 ± 3.5 nm, as the zeta potential decreased to -2.7 ± 0.4 mV, -12.5 ± 1.6 mV and -23.1 ± 1.2 mV. With the increasing degree of succinyl, the size increased and the zeta potential decreased. At the same time, the higher degree of succinyl drug-loading NPs have lower release and have increased the stability of ICG. We found that the blank-NPs had no significant toxicity to normal cells (HSF), as the ICG@CHP group had larger toxicity than the CHPCs and control. Moreover, the cellular uptake was decreased with the increased degree of succinyl., Conclusion: In this study, we successfully prepared CHPC2 carriers with the maximum negative surface charge, for follow-up research and providing new ideas for SLN., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)