Spatiotemporal Controllable Sono‐Nanovaccines Driven by Free‐Field Based Whole‐Body Ultrasound for Personalized Cancer Therapy

Abstract Therapeutic cancer vaccines fail to produce satisfactory outcomes against solid tumors since vaccine‐induced anti‐tumor immunity is significantly hampered by immunosuppression. Generating an in situ cancer vaccine targeting immunological cold tumor microenvironment (TME) appears attractive. Here, a type of free‐field based whole‐body ultrasound (US)‐driven nanovaccines are constructed, named G5‐CHC‐R, by conjugating the sonosensitizer, Chenghai chlorin (CHC) and the immunomodulator, resiquimod (R848) on top of a super small‐sized dendrimeric nanoscaffold. Once entering tumors, R848 can be cleaved from a hypoxia‐sensitive linker, thus modifying the TME via converting macrophage phenotypes. The animals bearing orthotopic pancreatic cancer with intestinal metastasis and breast cancer with lung metastasis are treated with G5‐CHC‐R under a free‐field based whole‐body US system. Benefit from the deep penetration capacity and highly spatiotemporal selectiveness, G5‐CHC‐R triggered by US represented a superior alternative for noninvasive irradiation of deep‐seated tumors and magnification of local immune responses via driving mass release of tumor antigens and “cold‐warm‐hot” three‐state transformation of TME. In addition to irradiating primary tumors, a robust adaptive anti‐tumor immunity is potentiated, leading to successful induction of systemic tumor suppression. The sono‐nanovaccines with good biocompatibility posed wide applicability to a broad spectrum of tumors, revealing immeasurable potential for translational research in oncology.


Extraction of chlorophyll a
One hundred grams of spirulina platensis powders originated from Chenghai Lake were soaked in 500 mL of acetone, and transferred into a 1 L three-necked flask.The three-necked flask was placed in an oil bath equipped with an electrical stirring device and a reflux condenser.
The oil bath was heated to 65°C.Once the solvent in the three-necked flask reached a temperature of 56°C, it started refluxing.Kept the reflux for 2 h.Subsequently, stopped heating and kept stirring until the solution in the three-necked flask naturally cooled down to 30°C.
Vacuum filtration was used for solid liquid separation.The residue was washed with acetone until the filtrate exhibited a light colour.The afore-mentioned extraction steps were repeated thrice, and the resulting filtrates were combined and subjected to rotary evaporation to obtain a spirulina-derived chlorophyll extract.

Preparation of Chenghai Pheophorbide a (CHP)
The spirulina-derived chlorophyll extract was dissolved in 300 mL of ethyl ether and transferred into a 1 L three-necked flask.Installed with a mechanical stirring device and drip funnel, nitrogen gas was introduced, and stirring was started while the mixture was gradually cooled down to -10°C in an ice-salt bath.Added 12 M HCl (150 mL) to the solution slowly at -10°C.Controlled the rate of adding HCl to maintain the temperature of the reaction mixture below 0°C.The resultant mixture was then warmed up to room temperature and stirred at the same temperature until chlorophyll a was consumed (TLC analysis, petroleum ether/ethyl acetate = 3:1).The reaction mixture was transferred into a 5 L separatory funnel and extracted thrice with petroleum ether (350 mL).The water layer was collected.
A saturated Na2CO3 solution was added dropwise to the collected solution to adjust pH to 4.0.The solid precipitate in dark green was separated via vacuum filtration using a Büchner funnel and washed with 1 vol.%aqueous propanoic acid solution.Subsequently, the obtained solid precipitate was placed in a vacuum for drying at 25°C.The dried solid was dissolved in dichloromethane (DCM) mixed with methanol in a ratio of 10:1 and transferred into an eggplant flask.When the solvent was removed via rotary evaporation, Chenghai Pheophorbide a (CHP, 1.08 g) was obtained (total yield, 1.1%).
A small amount of the magnesium-free chlorophyllin a was placed in an eppendorf tube and dissolved in chromatography-grade methanol (1.5 mL).The solution was drawn into a 2 mL syringe, filtered through a 0.22 μm membrane filter, and injected into a liquid sample vial.
Subsequently, the purity of the compound was measured using a high-performance liquid chromatography (HPLC) system (Waters Alliance e2695 HPLC) equipped with a 2489 ultraviolet/visible detector.Methanol and ammonium acetate buffered salt solution, 100 mM were used as the mobile phase (detection wavelength, 400 nm; flow rate of the mobile phase, 1 mL min -1 ; separation column, C18 reversed-phase column; injection volume, 20 μL).The purity of Chenghai Pheophorbide a (CHP) was 70.8%.

Synthesis of Chenghai chlorin (CHC)
CHC was synthesized through a two-step process using CHP as the raw material.Step one: The extracted CHP (500 mg) was accurately weighed and thoroughly dissolved in 40 mL of 5% sulphuric acid in methanol.Subsequently, the solution was transferred into a 250 mL eggplant flask and stirred at 25°C for 4 h under nitrogen.The reaction progress was monitored via thin-layer chromatography (TLC).After the reaction was completed, the solvent was removed via rotary evaporation, and the residue was transferred into a separatory funnel.
Extraction was performed thrice with deionized water (20 mL) and once with a saturated aqueous NaCl solution.The lower-layer solution was collected and dried over with anhydrous Na2SO4.Subsequently, the solvent was removed via rotary evaporation to obtain the carboxyl methyl esterified product of CHP.
The carboxyl methyl esterified product of CHP was dissolved in methanol (25 mL) followed by the addition of sodium methoxide (1.5 mL).The obtained solution was transferred into a 50 mL eggplant flask and reacted at 25°C for 12 h under nitrogen.Reaction progress was monitored using TLC.Upon the reaction completion, formic acid (0.3 mL) was added to the system, and the solvent was removed via rotary evaporation.After dissolution in DCM, the solution was transferred into a separatory funnel.Extraction was performed thrice with deionized water and once with a saturated aqueous NaCl solution.The organic layer solution was collected, and dried over with anhydrous Na2SO4.Finally, the rotary evaporation of the solvent and column chromatographic separation were performed to obtain 117 mg of the product. 1 H NMR (400 MHz, CDCl3) δ 9.68 (s, 1H), 9.54 (s, 1H), 8.73 (s, 1H), 8.04 (dd, J =   Step two: To a solution of CHC-TME (45.5 mg) in THF, added 1 M KOH solution and refluxed reaction under nitrogen.Reaction progress was monitored via TLC.After 12 h of reaction, the organic solvent was removed via rotary evaporation.Thereafter, a 1 M aqueous hydrochloric acid solution was added to the reaction mixture to adjust its pH to approximately 4.0.The solid product was obtained via vacuum filtration using a Büchner funnel and washed with a 1 vol.%aqueous propanoic acid solution.Subsequently, the solid was dried in a vacuum drying oven and dissolved in a mixture of DCM and methanol with a 1:1 (v/v) ratio.After the solvent removal via rotary evaporation, 34.4 mg of CHC was ultimately obtained (yield: 81%). 1 H NMR (400 MHz, DMSO-d6) δ 9.74 (d, J = 28.2Hz, 2H), 9.10 (s, 1H), 8.30 (dd, J = 17.9, 11.6 Hz, 1H), 6.45 (d,J = 16.3 Hz,1H),6.18 (d,J = 11.6 Hz,1H),2H)    Synthesis of compound 2: Compound 2 (0.4114 g, 2.0 mmol) was dissolved in 10 mL of MeOH.NaBH4 (0.1513 mg, 4 mmol) was added, and the reaction mixture was stirred in an ice bath while TLC was used to monitor the reaction.Following completion of the reaction, the solvent MeOH was removed using a rotary evaporator.Water (10 mL) was added and the mixture was extracted three times with ethyl acetate.Na2SO4 was used to dry the composite organic layer, and the solvent was removed using a rotary evaporator.The crude product was purified by a flash chromatography on silica gel (ethyl acetate:hexane = 1:1) to obtain a white solid of compound 2 (0.4012 g, 97% yield).Synthesis of compound 3: Compound 2 (0.0414 g, 0.2 mmol) was dissolved in DCM (4 mL).TFA was added (80 µL) and stirred for 30 minutes in an ice bath.To this solution was added 4-nitrophenyl chloroformate (0.0605 g, 0.3 mmol) and monitored using a TLC spot plate.
Without further purification, the product of the reaction was used directly for the next step of synthesis.
Synthesis of compound 4: To a solution of Compound 3 in DCM (5 mL) added TEA (150 µL).To this solution was added R848 (0.0628 g) in DCM/DMF (4 mL /1 mL, V/V), and the mixture was stirred at room temperature for 12 h.The reaction was monitored using a TLC, and the solvent was evaporated using a rotary evaporator to yield a yellow oil.The crude product was purified by a flash chromatography on silica gel (dichloromethane:methanol = 50:1) to obtain a pale yellow solid (compound 4, 0.0828 g).

Figure
Figure S3. 1 H NMR spectrum of CHC-TME in CDCl3

Figure
Figure S10. 1 H NMR spectrum of compound 2 in CDCl3.

Figure
Figure S21.a) Absorption spectra of DPBF in the presence of G5-CHC-R with ultrasound irradiation for varied durations.b) Comparison of relative intensity of DPBF in free CHC and G5-CHC-R treated groups in the presence of US irradiation up to 10 min.Data are presented mean ± SD (n=3).

Figure S27 .
Figure S27.In vivo biodistribution of free CHC, G5-CHC and G5-CHC-R.Representative in vivo real-time fluorescence images of 4T1-bearing BALB/c mice after intravenous injection of free CHC, G5-CHC and G5-CHC-R at dosage of 15 mg CHC kg -1 bodyweight.Images were taken at 1, 4, 8, and 12 h post-injection.The ex vivo tissue images were taken at 12 h post-injection (Ex 665 nm, Em 700 nm).

Figure S28 .
Figure S28.Representative CRT western blot image and quantify the expression of CRT by blot images.Data represented mean ± SD (n=2 or 3).

Figure S30 .
Figure S30.The ex vivo fluorescent images of major organs of mice at 12, 24, 48 and 72 h post-injected with G5-CHC-R.

Figure S31 .
Figure S31.Histological analysis of different organs in Pan02 tumor-bearing mice after various treatments.No obvious signs of organ damage appeard in different groups treated mice.Scale bars, 100 μm.

Figure S34 .
Figure S34.Representative dot plots of CD8 + T cells in tumor tissues in the orthotopic pancreatic cancer model.

Figure S36 .
Figure S36.Representative fluorescent images of the excised lymph nodes (LNs) from normal mouse (left) and the tumor draining lymph nodes (TDLNs) from tumor-bearing mice (right) after i.v.administration of G5-CHC-R.

Figure S37 .
Figure S37.Tumor accumulation efficiency of the sono-nanovaccines.After three doses, the appearance of primary tumors of G5-CHC-R+US treated group were in dark green (left), but not Free CHC+US group (right).

Figure S39 .
Figure S39.Dynamic body weights of 4T1-bearing mice in different groups during treatment.Data represented mean ± SD (n=5).

Figure S40 .
Figure S40.Histological analysis of different organs in 4T1 tumor-bearing mice after various treatments.No obvious signs of organ damage appeared in different groups treated mice.Scale bars, 100 μm.