Mitochondria-specific drug release and reactive oxygen species burst induced by polyprodrug nanoreactors can enhance chemotherapy

Cancer cells exhibit slightly elevated levels of reactive oxygen species (ROS) compared with normal cells, and approximately 90% of intracellular ROS is produced in mitochondria. In situ mitochondrial ROS amplification is a promising strategy to enhance cancer therapy. Here we report cancer cell and mitochondria dual-targeting polyprodrug nanoreactors (DT-PNs) covalently tethered with a high content of repeating camptothecin (CPT) units, which release initial free CPT in the presence of endogenous mitochondrial ROS (mtROS). The in situ released CPT acts as a cellular respiration inhibitor, inducing mtROS upregulation, thus achieving subsequent self-circulation of CPT release and mtROS burst. This mtROS amplification endows long-term high oxidative stress to induce cancer cell apoptosis. This current strategy of endogenously activated mtROS amplification for enhanced chemodynamic therapy overcomes the short lifespan and action range of ROS, avoids the penetration limitation of exogenous light in photodynamic therapy, and is promising for theranostics.

Anhydrous acetone (0.7 mL) and 4-mercapto-butyric acid (1.5 g) were mixed in CHCl 3 (10 mL), then added with 0.3 equivalent TiCl 4 at -10 °C. 3 Instantly, the solution turned yellowish formed with precipitate. The resulting reaction mixture was allowed to warm-up slowly to room temperature and stirred overnight. White precipitate was formed and washed thoroughly with diethyl ether. Finally, colorless solid was obtained and dried under reduced pressure to afford TK (1.24 g). 1

Synthesis of HEMA-TK
TK (1.0 g) and DMAP (0.49 g) in anhydrous DCM (30 mL) was added 2-hydroxyethyl methacrylate (HEMA) (0.52 g) at room temperature. 4 After stirring for 10 min, EDC (1.2 g) dissolved in anhydrous DCM (5 mL) was slowly added to the above solution under N 2 atmosphere. The reaction was performed under nitrogen atmosphere for 24 h at room temperature. After that, the reaction mixture was then extracted third with an equal volume of brine. The organic layer was separated, dried over anhydrous Na 2 SO 4 , filtered and concentrated on a rotary evaporator. The crude product was purified by column chromatography using an eluent of 1:1 hexane: ethyl acetate, yielding colorless solid. 1

Synthesis of ROS-cleavable CPT prodrug monomer (CPTSM)
Briefly, a mixture of CPT (0.5 g), HEMA-TK (0.6 g) and DMAP (0.18 g) were suspended in anhydrous DCM under N 2 atmosphere, then EDC (0.42 g) dissolved in anhydrous DCM (5 mL) was added slowly under N 2 atmosphere and stirring for 10 min at room temperature. 5 The reaction was performed under nitrogen atmosphere for 24 h at room temperature. After that, the reaction mixture was then extracted brine, and the organic layer was separated, dried over anhydrous Na 2 SO 4 , and concentrated on a rotary evaporator. The solid residues were purified by column chromatography using an eluent gradient from the mixture of hexane and ethyl acetate to afford the resulting CPTSM (0.82 g). The chemical structure of CPTSM was verified by 1 H NMR and 13 C NMR analysis ( Figure S1). 1
The reaction was performed under nitrogen atmosphere for 12 h at room temperature.
After that, the reaction mixture was concentrated on a rotary evaporator. The solid residues were purified by column chromatography using an eluent gradient from 100% EtOAc to 100% acetone to afford CPPA-TPP as an orange solid (0.22 g). 1

Synthesis of hydrophilic PDMA
Reversible addition-fragmentation chain transfer (RAFT) polymerization was employed for the synthesis of PDMA hydrophilic polymer. 6 Typically, CPPA-TPP (30 mg), DMA (639 mg), and AIBN (3.54 mg) were charged into a glass ampoule containing 1,4-dioxane (1.2 mL). The ampoule was then degassed via three freeze-pump-thaw cycles and flame-sealed under vacuum. It was then immersed into an oil bath thermostated at 70 o C to start the polymerization. After 9 h, the ampoule was quenched into liquid nitrogen to terminate the polymerization. The mixture was precipitated into an excess of diethyl ether to generate light pink residues, the residues were dissolved in DCM and precipitated into diethyl ether. The final product was dried in a vacuum oven overnight at room temperature, affording TPP-PDMA a light pink solid powder (654.9 mg). Following similar procedures, other polyprodrug amphiphiles were also synthesized. The structural parameters of all polyprodrug amphiphiles were summarized in Supplementary Table 1.

Conjugation of cRGD to the polyprodrug
Amine functionalized cRGD was conjugated to the PDMA-b-P(CPTSM-co-RhB) polyprodrug amphiphiles using an EDC/sulfo-NHS technique. 7 PDMA-b-P(CPTSMco-RhB) (40 mg) were suspended in 2 mL anhydrous DCM with EDC (0.7 mg) and NHS (0.4 mg) at room temperature for 36 min. The NHS-activated PDMA-b-P(CPTSM-co-RhB) polyprodrug amphiphiles were allowed to react with amine-terminated cRGD (2 mg) for 24 h under magnetic stirring. The cRGD functionalized polyprodrug amphiphiles was precipitated into an excess of diethyl ether to generate red residues, the residues were dissolved in DCM and precipitated into diethyl ether. The final product was dried in a vacuum oven overnight at room temperature, yielding a red solid powder (39.5 mg). shot under vigorous stirring. The colloidal dispersion was further stirred, followed by