2 kda membrane

CNPs were synthesized by the ionic gelation method [51 ]. Briefly, 2.4 mg/mL of LMW Chitosan (50–190 kDa, deacetylation degree of 75–85%, CAS 9012-76-4, Sigma-Aldrich, St. Louis, MO, USA) was dissolved in acetic acid 2% v/v and left under magnetic stirring for 3 h to protonate the amine groups of monomers and consequently increase its solubility. The pH of the mixture was adjusted to 3.6 to induce a partial charge restoration. Chitosan chains were crosslinked with 1.2 μL of glutaraldehyde per milliliter of chitosan that was added dropwise and was left under stirring for 1 h to obtain the nanoparticles. The obtained CNPs were purified by dialyzing the reaction mixture against Type II water employing a 2 kDa membrane (Sigma-Aldrich, St. Louis, MO, USA) at room temperature for three days. Finally, the CNPs were lyophilized and stored at 4 °C until further use.

CNPs (100 mg) were resuspended in 70 mL of type II water, mixed with 2 mL of glutaraldehyde 2% v/v, activating the CNPs surface for 1 h. Afterward, 1 mg of the peptide was added and left to conjugate under agitation for two days. Rhodamine B was used as a fluorophore to label the CNPs. Before conjugating to the CNPs, 7 mg of EDC and 5 mg of NHS were mixed in 5 mL of type II water until complete dissolution. Next, 200 $\mathsf{\mu }$ L of DMF and 6 mg of Rhodamine B were added to the mixture and left to react at 40 °C for 15 min. Afterward, this activated Rhodamine B was mixed with the CNPs-peptide nanobioconjugates and left under agitation for 24 h at room temperature. To remove unconjugated rhodamine B, the mixture was dialyzed against Type II water using a 2 kDa membrane (Sigma-Aldrich, St. Louis, MO, USA). Finally, the labeled nanobioconjugates were lyophilized and stored at 4 °C until further use.

100 mg of CNPs were resuspended in 70 mL of type II water, mixed with 2 mL of glutaraldehyde 2% v/v, and left to react for 1 h. 1 mg of the peptide was then added and left to conjugate under continuous agitation for two days. To activate the fluorescent molecule rhodamine B, 7 mg of EDC and 5 mg of NHS were mixed in type II water (5 mL), followed by 200 µL of DMF and 6 mg of rhodamine B. The mixture was left to react at 40 °C for 15 min. Finally, activated rhodamine B was mixed with the CNPs-peptide nanobioconjugates and left under agitation for one day at room temperature. To remove excess rhodamine B, the mixture was dialyzed against Type II water aided by a 2 kDa membrane (Sigma-Aldrich, St. Louis, MO, USA). The labeled nanobioconjugates were lyophilized and stored at 4 °C until further use.