Eradication of solid tumors by chemodynamic theranostics with H2O2-catalyzed hydroxyl radical burst

Activatable theranostics, integrating high diagnostic accuracy and significant therapeutic effect, holds great potential for personalized cancer treatments; however, their chemodynamic modality is rarely exploited. Herein, we report a new in situ activatable chemodynamic theranostics PAsc/Fe@Cy7QB to specifically recognize and eradicate cancer cells with H2O2-catalyzed hydroxyl radical (•OH) burst cascade. Methods: The nanomicelles PAsc/Fe@Cy7QB were constructed by self-assembly of acid-responsive copolymers incorporating ascorbates and acid-sensitive Schiff base-Fe2+ complexes as well as H2O2-responsive adjuvant Cy7QB. Results: Upon systematic delivery of PAsc/Fe@Cy7QB into cancer cells, the acidic microenvironment triggered disassembly of the nanomicelles. The released Fe2+ catalyzed the oxidation of ascorbate monoanion (AscH-) to efficiently produce H2O2. The released H2O2, together with the endogenous H2O2, could be converted into highly active •OH via the Fenton reaction, resulting in enhanced Fe-mediated T1 magnetic resonance imaging (MRI). The synchronously released Cy7QB was activated by H2O2 to produce a glutathione (GSH)-scavenger quinone methide to boost the •OH yield and recover the Cy7 dye for fluorescence and photoacoustic imaging. Conclusion: The biodegradable PAsc/Fe@Cy7QB designed for tumor-selective multimodal imaging and high therapeutic effect provides an exemplary paradigm for precise chemodynamic theranostic.

. The mechanism for generation of hydroxyl radicals (•OH).

Synthesis of compound 1 and 2
Salicylaldehyde (0.025 mol) and paraformaldehyde (0.015 mol) were added to a three-necked flask with 15 mL of concentaated. HCl. The mixture was stirred, and 1.1 mL of POCl3 were gradually added dropwise within 1 h. The mixture was reacted at room temperature for 18 h. The white solid was filtered, washed with 3% NaHCO3 solution and distilled water, dried and recrystallized in petroleum ether. A mixture of methacrylic acid (0.4 g, 0.005 mol), sodium hydroxide (0.2 g, 0.005 mol) and water (5 mL) was stirred at room temperature for 30 min. Compound 1 (0.85 g, 0.005 mol), potassium iodide (0.15 g, 0.0003 mol) and toluene (1 mL) were added successively to the above aqueous solution. The reaction mixture was heated to 50 °C and stirred overnight. The mixture was extracted with ethyl acetate (50 mL × 3), and the organic extract was dried over anhydrous Na2SO4. After filtration, the volatiles were removed by using a rotary evaporator. The residue was purified by column chromatography on silica gel with petroleum ether/dichloromethane (9:1) to obtain compound 2 as a white solid with a yield of 68.5%.

Synthesis of compound 3
A solution of 2, 4-pentanedione (1.09 mL, 10.5 mmol) in dichloromethane (8 mL) was added dropwise to a solution of 1, 2-diaminoethane (1.36 mL, 20.1 mmol) in dichloromethane (8 mL). The resulting mixture was heated under reflux for 1 h. The excess 1, 2-diaminoethane was removed from the resulting mixture by rotary evaporation under vacuum at 60 °C for 30 min. The yield was 87%.

Synthesis of compound 4
Immobilized Candida antarctica lipase and ascorbic acid (ascorbate, Asc) were dried under high vacuum in a desiccator with phosphorous pentoxide for 24 h prior to reaction. The reaction was an enzymatic transesterification, in which the primary hydroxyl group of ascorbic acid was regioselectively acylated by 2, 2, 2-trifluoroethyl methacrylate via the acyl enzyme complex. In a typical reaction, ascorbic acid (5.0 g, 28.4 mmol), 2,2,2-trifluoroethyl methacrylate (6.07 mL, 42.6 mmol), and C. antarctica lipase (3.3 g, immobilized) were stirred in 100 mL of anhydrous dioxane at 60 °C . Then, 83.0 mg of 2, 6-ditertbuty-l,4-methylphenol were added to the reaction mixture to avoid vinyl polymerization at 60 °C , functionalized poly(methyl methacrylate) and during solvent evaporation. Reactions were monitored by thin layer chromatography. The enzyme was filtered; the product was washed thoroughly with dioxane and the solvent was evaporated by rotary evaporation under reduced pressure.
The yield was 90%.

Synthesis of compound 5
Typically, mPEG (10.0 g, 2 mmol), CPADB (0.57 g, 2 mmol), DMAP (0.062 g, 0.5 mmol) and DCC (1.16 g, 5.63 mmol) were dissolved in 20 mL anhydrous methylene chloride. The solution was stirred at room temperature for 48 h. After filtration of the precipitate, the solution was poured into 200 mL cold ethyl ether, yielding a pink precipitate. The pink precipitate was redissolved in methylene chloride and precipitated again in cold ethyl ether. After washing two additional times with ethyl ether, the pink product mPEG-CPAD was finally obtained, and the yield was 92%.

Synthesis of compound PAsc-PDPA
Compound 5 (150 mg), compound 4 (206.6 mg, 0.8 mmol), DPAMA (1.5 mg) and AIBN (2.0 mg) were dissolved in 2 mL 1, 4-dioxane/methanol (v/v, 3:1). The mixture was deaerated by applying three freeze-pump-thaw cycles. Afterwards, the tube was immerged in a 65 °C oil bath and stirred for 24 h. After cooling to room temperature, the mixture was precipitated in cold ethyl ether. A slight pink precipitate was collected and washed twice with ethyl ether. The product was obtained after vacuum drying for 24 h with a yield of 81%.

Synthesis of compound PAsc-PFHMA
PAsc-PDPA (150 mg), compound 2 (114.28 mg, 0.16 mmol) and AIBN (2.0 mg) were dissolved in 2 mL 1, 4-dioxane/methanol (v/v, 3:1). The mixture was deaerated by applying three freeze-pump-thaw cycles. Afterwards, the tube was immersed in a 65 °C oil bath and stirred for 24 h. After cooling to room temperature, the mixture was precipitated in cold ethyl ether. A slight yellow precipitate was collected and washed twice with ethyl ether. The product was obtained after vacuum drying for 12 h with a yield of 85%.

Synthesis of compound PAsc-PS
PAsc-PDPA (150 mg) and compound 3 (22.5 mg, 0.16 mmol) were dissolved in 2 mL of 1, 4-dioxane/methanol (v/v, 3:1). The mixture was deaerated by applying three freeze-pump-thaw cycles. Afterwards, the tube was immersed in a 50 °C oil bath and stirred for 24 h. After cooling to room temperature, the mixture was precipitated in cold ethyl ether. A slight pink precipitate was collected and washed twice with ethyl ether. The product was obtained after vacuum drying for 12 h with a yield of 80%.

Synthesis of compound PAsc-PSFe
FeCl2 (20.28 mg, 0.16 mmol) in 50 mL of ethanol was added dropwise to a solution of 150 mg PAsc-PS in 50 mL of ethanol, which was stirred in a round -bottomed flask.
To avoid oxidation of Fe 2+ , a few drops of glacial acetic acid were added. The

Preparation of PAsc/Fe@Cy7QB
The Cy7QB was synthesized according to previous similar protocol and successfully characterized by 1 H NMR. Then Cy7QB (2 mg) dissolved in CH2Cl2 was added to a deionized water (2 mL) solution of PAsc-PSFe (10 mg). The mixture was then stirred at 25 °C for 4 h. PAsc/Fe@Cy7QB was obtained after dialysis in a cellulose dialysis bag (MWCO 3500 Da) overnight. The resulting solution was freeze-dried for 6 h, and the product was obtained. The dry product was re-dispersed in deionized distilled water (pH 7.4) to produce the PAsc/Fe@Cy7QB solution. The PAsc/Fe@Cy7QB solution was filtered through a 0.22 μm filter to sterilize the sample before being used in cells and mice.

pH-Dependent Fe 2+ release
To check the valence states and release of Fe 2+ , potassium ferricyanide and potassium thiocyanate were applied. Briefly, PAsc-PSFe was dispersed in a buffer solution with pH values of 5.0 and 7.4, and the PAsc-PSFe were concentrated into 12-well plates. Then potassium ferricyanide and potassium thiocyanate were added, respectively, and the solutions were incubated for 24 h. After centrifugation to remove undissolved PAsc-PSFe, photos of the products were taken.

pH-Responsive magnetic resonance imaging (MRI) in solution
PAsc/Fe@Cy7QB was dispersed into acidic buffer solutions (pH = 7.4, 6.5, 5.5 and 5.0) separately to measure the T1 relaxivity. Then, the shaken solution transferred into Sample Jet tubes for MRI scanning. In vitro MRI of PAsc/Fe@Cy7QB NPs at different concentrations was carried out on a Niumag 1.0 T whole-body magnetic resonance imaging scanner (NM42-040H-I, Niumag, China) at 35 °C. The array was embedded in a phantom consisting of a water tank to allow appropriate image acquisition. The following parameters were adopted: repetition time (TR) = 500 ms, echo time (TE) = 20 ms, number of averages = 3. The relaxivity value (r1, L mM -1 s −1 ) was calculated using T1 measurements with a series of dilutions of the nanoparticle dispersions in 0.9% saline.

PA measurement in solution
For in vitro measurement, The PA signal intensity of PAsc/Fe@Cy7QB (50 μg/mL) incubated in solution with different H2O2 concentrations was recorded by a 10 MHz, 10 mJ cm -2 , 384-element ring ultrasound array. The multi-element transducer has a center frequency of 2.5 MHz with a nominal bandwidth of 70%.

Flow cytometry analysis
To study cell apoptosis induced by PAsc/Fe@Cy7QB, a flow cytometric assay involving Annexin V-FITC and PI costaining was carried out. The cells were harvested, rinsed in PBS, resuspended, and determined by flow cytometry (Becton Dickinson, Mountain View, CA, USA). All the experiments detected at least 10,000 cells, and the data were analyzed using the FCS Express V3. For live/dead assay, HepG2 cells were incubated with various samples for 4 h, following by staining with Calcein AM and PI by laser scanning confocal microscopy.