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Multifunctional carbon dots with near-infrared absorption and emission for targeted delivery of anticancer drugs, tumor tissue imaging and chemo/photothermal synergistic therapy

Cancer therapy faces considerable challenges related to improving treatment efficiency and overcoming damage to healthy cells. To address these concerns, a strategy for tumor microenvironment-induced cancer imaging/drug release and synergistic chemo–photothermal therapy (chemo/PTT) is proposed in this study. Carbon dots with near-infrared (NIR) absorption and emission, referred to as RCDs, were first prepared and covalently coupled with a Pt(iv) prodrug to form a complex, referred to as RCD–Pt(iv). The surface of the prepared complex was then coated with the polyethylene glycol–chitosan–2,3-dimethylmaleic anhydride polymer (PEG–CS–DA) to obtain a tumor-targeted multifunctional nanoprobe (RCD–Pt(iv)/PEG–CS–DA). When the nanoprobe RCD–Pt(iv)/PEG–CS–DA entered the tumor cells, the acidic environment of the tumor cells allowed rapid PEG–CS–DA hydrolysis and RCD–Pt(iv) release. High levels of glutathione (GSH) in cancer cells reduced Pt(iv) to Pt(ii) and released the RCDs, resulting in cancer tissue imaging and targeted Pt(ii) release. Meanwhile, Pt(ii) collected in the tumor tissues could realize targeted chemotherapy, and the RCDs in the tumor tissues absorbed light energy under NIR light irradiation to produce a large amount of heat to quickly eliminate cancer cells. Thus, cancer tissue imaging/targeted drug release and synergistic chemo/PTT were achieved simultaneously.


Synthesis of the RCDs
Osmanthus seed skin (15 g) was shredded, added to 30 mL of an anhydrous ethanol solution, and transferred to a 50 mL round bottom flask. The flask was sealed with nitrogen protection and fixed to an electric heating sleeve heated to 100 °C. The content was stirred at this temperature and under a nitrogen atmosphere for 12 h. After the reaction product solution was naturally cooled to room temperature, the supernatant was filtered via a 0.22 μm filter membrane. Up to 30 mL of the filtrate was transferred to a 50 mL beaker to which 0.2 g of H 2 N-PEG-NH 2 anhydrous ethanol solution was added. The mixture was heated to 120 °C and then agitated for 10 h under a nitrogen atmosphere.
The resulting supernatant was then filtered via a 0.22 μm filter membrane. The filtrate was dialyzed with a dialysis bag (MWCO 1000) in pure water for 48 h to obtain pure RCDs. The purified product was ultimately removed from the solvent by rotary evaporation under vacuum when dried for 24 h at 65 °C and then stored in a refrigerator at 4 °C.

Preparation and Characterization of RCD-Pt(IV)/PEG-CS-DA
Synthesis of the Pt(IV) prodrugs: the synthesis of the Pt(IV) prodrugs is illustrated in Fig.S7. Approximately 300.0 mg of Cis-Pt(NH 3 ) 2 Cl 2 was suspended in 7.5 mL water in a water bath set to 50 °C. Subsequently, 10.5 mL of H 2 O 2 (wt: 30%) was added dropwise and then stirred for 2 h under this condition. The reaction product solution was cooled to room temperature. The solvent was then concentrated to about 2 mL by rotary evaporation, and the resulting solution was stored overnight at 0 °C. The pale yellow crystals were washed and filtered with cold water and diethyl ether solution, and the product was obtained, which was then stored in a vacuum dryer; 120.0 mg of the product and 36.0 mg of succinic anhydride were dissolved in 2.0 mL dimethylformamide and agitated for 24 h at room temperature. The reaction product was freeze-dried, and the

In vitro Drug Release Experiments
Approximately 15.0 mg of RCD-Pt(IV)/PEG-CS-DA was dissolved in 1 mL of PBS buffer solution (pH 7.4 or 6.8) containing GSH at 0, 5 µM, and 10 mM. The mixed solution was transferred to a dialysis tube (MWCO:1 kDa), and dialyzed at 37 °C for 1, 2, 4, 6, 8, and 10 h by using a PBS buffer solution containing 0, 5 µM and 5 mM GSH (69 mL, pH 7.4 or 6.8). The content of Pt in the dialysate was determined by inductively coupled plasma mass spectrometry (ICP-MS).

Photothermal Effects of RCD-Pt(IV)/PEG-CS-DA
Approximately 20 μg of RCDs, 105 μg of RCD-Pt(IV)/PEG-CS-DA, and 155 μg of RCD-Pt(IV)/PEG-CS-DA were dissolved in 1 mL of PBS (pH 6.8) solution, Pt(II), and PBS solution, which were used as blank controls. The aforementioned solution was placed in a 1.5 mL centrifuge tube and continuously irradiated with 680 nm laser (1.5 W/cm2) for 10 min. Changes in the temperature of the solution were recorded at the 0, 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 6, 7, 8, 9, and  Tumor sites in the mice were then irradiated with a 680 nm laser (1.5 W/cm2). The infrared thermal imager (optical PI) was used to record infrared thermal imaging images at different times and analyze the change in temperature at tumor sites.

Chemo-Photothermal Synergistic Therapy for Tumor-Bearing Mice
In this experiment, the tumor-bearing mice were randomly divided into experimental groups: PBS, Pt(II), RCDs, and RCD-Pt(IV)/PEG-CS-DA. Each group underwent a control experiment with and without laser irradiation. Female nude mice (18-20 g) with 60-70 mm3 tumor volume were selected as an experimental object after T24 cells implantation for about 7 days. The 200 µL PBS, Pt(II), RCDs, and RCD-Pt(IV)/PEG-CS-DA solutions were administered to the mice via tail vein injection; each nude mouse had S5 the same Pt content (10 mg/kg). The tumor site in the mice in the laser irradiation group was irradiated with 680 nm laser (1.5 W/cm2) for 3 min after the treatment agent was injected for 10 h via the tail vein. The same treatment lasted for 14 days, and the tumor volume of the nude mice was measured daily during treatment. After the tumor treatment experiment was completed, the mice were anesthetized, and the tumor tissue was removed for H&E staining. Histopathological experiments were conducted in accordance with standard operating procedures. After H&E staining, the pathological sections were observed and photographed using an optical microscope.