Tumor In Situ Cu²⁺ Chelation Enhanced DSF Chemotherapy Combined With NIR-Ⅱ Photothermal Treatment For Breast Cancer

PART Ⅰ PREPARATION,CHARACTERIZATION AND PERFORMANCE OF DSF@PEG-HCuSNPsObjective To prepare DSF@PEG-HCuSNPs and test its basic physicochemical properties,photothermal performance and biodegradability.Methods HCuSNPs carriers were synthesized by sulfurization reaction that using Cu2O nanospheres as a template.The NH2-PEG2000 was modified on the surface of HCuSNPs by electrostatic adsorption,and then disulfiram(tetraethylthiuram disulfide,DSF)was loaded into the hollow structure of HCuSNPs to prepare DSF@PEG-HCuSNPs.Transmission electron microscopy,scanning electron microscopy,dynamic light scattering and zeta potential measurements,X-ray photoelectron spectroscopy,X-ray diffraction,specific surface area and pore size analysis,thermogravimetric analysis,Fourier transformed infrared spectra and UV-vis-NIR absorption spectra were used to detect the basic physicochemical properties of nanoparticles,including morphological structure,elemental composition,particle size,potential,Cu and S element valence,UV absorption spectrum,drug loading capacity,and other properties.808 and 1064 nm lasers were used to detect the photothermal performance of DSF@PEG-HCuSNPs under different concentrations and different power intensities in the first and second near-infrared biowindows.PEG-HCuSNPs were dispersed in SBF solutions at different pHs(6.5 and 7.4)and exposed to 1064 nm laser irradiation.The biodegradation behavior of PEG-HCuSNPs was directly investigated by TEM observation and quantified by ICP-OES analysis.Results TEM and SEM analysis revealed the morphology and size distribution of HCuSNPs,evidencing the spherical morphology and hollow nanostructure of HCuSNPs with the size of 220 nm.Their element mappings illustrated the homogeneous distribution of Cu and S elements in the hollow HCuSNPs framework.Also,the average hydrodynamic size of HCuSNPs was determined to be 240.6 nm by dynamic light scattering measurement,which conformed to the TEM and SEM results.X-ray diffractive(XRD)pattern of HCuSNPs displayed characteristic peaks were assigned to the crystal faces of hexagonal CuS crystals.The X-ray photoelectron spectroscopy(XPS)binding energy centers were indexed to the highresolution XPS peaks of Cu 2p3/2 and Cu 2p1/2,respectively.The binding energy centers of S 2p3/2 and S 2p1/2 implied the existence of sulfide and disulfide,respectively.The N2 absorption-desorption isotherms and poresize distribution analysis revealed that HCuSNPs manifested a high specific surface area and a uniform pore size of 285.5 m2 g-1 and 5.4 nm,respectively.All the results above demonstrated the successful preparation of HCuSNPs.After modification of NH2-PEG2000(PEG-HCuSNPs)and followed loading of DSF,the average hydrodynamic diameter was identified to be 262.0 nm and 263.7 nm,respectively.Moreover,the zeta potential of HCuSNPs,PEG-HCuSNPs,and DSF@PEG-HCuSNPs was determined to be-14.4±0.82,-4.64±0.70,and-1.67±0.26 mV,which was attributed to the surface modification and encapsulation of NH2-PEG2000 and DSF,respectively.The successful loading of DSF was further validated by Fourier transformed infrared(FTIR)analysis,which manifested the characteristic peaks of DSF.Additionally,thermogravimetric analysis(TGA)of HCuSNPs,PEG-HCuSNPs,and DSF@PEG-HCuSNPs was performed to confirm the efficient modification and loading of NH2-PEG2000 and DSF,and the modification and loading amount was calculated to be 6.72%and 40.2%,respectively.DSF@PEG-HCuSNPs exhibited similar absorbance with that of PEGHCuSNPs as observed by UV-Vis-NIR spectra of DSF@PEG-HCuSNPs and PEG-HCuSNPs,of which absorbance at 1064 nm elevated with the increasing concentrations.The results demonstrated the high potential application of DSF@PEG-HCuSNPs for NIR-Ⅱ laser-enabled photothermal conversion.After irradiated with 1064 nm laser,a visible temperature rises of DSF@PEG-HCuSNPs was correlated with concentration of nanoparticles,laser power,and irradiation time,showing a remarkable photothermal conversion efficiency(23.8%).Meanwhile,DSF@PEG-HCuSNPs also exhibited good photothermal performance under the irradiation of 808 nm laser.To validate that Cu2+ could be delivered and released from PEGHCuSNPs for self-supplying of Cu2+ at tumor sites upon NIR-Ⅱ laser exposure,the biodegradation behavior of PEG-HCuSNPs was examined by ICP-OES and TEM analysis after incubation in SBF at varying pHs upon NIR-Ⅱ laser illumination.The result illustrated that the degradation of PEGHCuSNPs was accelerated under mildly acidic condition.Notably,the accumulative release of Cu2+ increased significantly after laser irradiation under both acidic and neutral conditions.In contrast,PEG-HCuSNPs maintained their intact nanostructure at 96 h in neutral SBF solution in the absence of laser illumination.Conclusions The prepared HCuSNPs nanocarriers were spherical in shape,uniform in size,and good in dispersibility.NH2-PEG2000 was successfully modified on the surface of HCuSNPs and DSF was efficiently loaded in hollow mesoporous structure of HCuSNPs(named DSF@PEGHCuSNPs).DSF@PEG-HCuSNPs showed splendid performance in promoting conversion of NIR-Ⅱ laser into thermal energy effectively and accelerated its degradation in tumor tissue under NIR-Ⅱ laser irradiation,which thus reduced the off-target escape of DSF and promoted the formation of cytotoxic Cu(DTC)2 after NIR-Ⅱ laser irradiation under TME.PART Ⅱ BIODISTRIBUTION AND PHOTOACOUSTIC IMAGING OF DSF@PEG-HCuSNPsObjective To evaluate the photoacoustic imaging ability of DSF@PEG-HCuSNPs in vitro and in tumor-bearing nude mice.To investigate the effect of passive aggregation of DSF@PEG-HCuSNPs into tumor tissue through EPR effect.Methods According to the concentration of Cu,DSF@PEG-HCuSNPs were configured into aqueous solutions with concentrations of 100,200,400,600,and 800 μg·mL-1.A gel model was established,and the photoacoustic image and photoacoustic signal values were collected under the photoacoustic imaging system to observe the optimal imaging concentration of nanoparticles in vitro.4T1 cancer cells,growing in logarithmic phase,were subcutaneously administrated into the right back of the nude mice to establish animal tumor xenograft model.For in vivo PA imaging,the PA signal at tumor sites was monitored at varying time intervals(0,1,2,4,8,12,and 24 h)post injection and then performed quantitative analysis.After animal tumor xenograft model was established,nude mice bearing 4T1 tumors were administrated with intravenous injection of DSF@PEG-HCuSNPs.The main organs and tumors were dissected at varying time intervals(4,8,and 24 h).The Cu contents in organs and tumors were measured by ICP-OES.Results DSF@PEG-HCuSNPs exhibited excellent contrast enhancement in in vitro PA imaging,and the PA signals enhanced linearly with increasing concentration ranging from 100 to 800 ppm.The PA signal at tumor regions amplified in a time-dependent pattern,and the PA signal reached its maximum at 24 h after intravenous injection.Additionally,DSF@PEG-HCuSNPs passively accumulated into tumor tissues with a percentage of nearly 5.2%at 24 h due to the typical EPR effect after intravenous injection.Conclusions DSF@PEG-HCuSNPs represented good photoacoustic imaging properties and could effectively reached tumor tissues through the EPR effect,showing its broad prospects for application as photoacoustic imaging guide agents in the field of diagnosis and treatment in breast cancer.PART Ⅲ DSF@PEG-HCuSNPs FOR PHOTONIC HYPERTHEMIA-ENHANCED CHEMOTHERAPY ON TUMOR INHIBITIONObjective To evaluate biocompatibility of DSF@PEG-HCuSNPs.To investigate effect and mechanism of DSF@PEG-HCuSNPs in inducing hyperthermia-enhanced chemotherapy under NIR-Ⅱ laser irradiation.Methods 4T1 cells were dealt with varying doses of DSF(12.5,25,50,100,and 200 μg·mL-1),HCuSNPs(12.5,25,50,100,and 200 μg·mL-1),or DSF@PEG-HCuSNPs(12.5,25,50,100,and 200μg·mL-1)for 12,24,or 48 h,respectively.The CCK-8 assay was carried out to determine the cell viability.4T1 Breast cancer cells were irradiated by 1064 nm laser after coincubated with PEG-HCuSNPs and DSF@PEG-HCuSNPs.The therapeutic effects of pure photothermal therapy and hyperthermia synergistic/enhanced DSF chemotherapy on breast cancer 4T1 cells were qualitatively and quantitatively analyzed by CCK-8 method,flow cytometry and confocal laser microscopy.Female nude mice and Kunming mice(n=25 in each species)were intravenously injected with DSF@PEG-HCuSNPs solution(20 mg·kg-1),respectively.The blood and main organs were collected at 0,1,7,14,and 28 d post-injection for routine blood examination,serum biochemical analysis,and histological analysis by H&E staining.4T1 cells were subcutaneously administrated into the right back of the nude mice to establish animal tumor xenograft model.At a tumor volume of 50-80 mm3,the female 4T1 tumor-bearing nude mice(n=35)were randomly allocated into 7 groups(n=5 each):(1)control(PBS),(2)DSF only,(3)HCuSNPs only,(4)laser only,(5)DSF@PEG-HCuSNPs,(6)HCuSNPs+1064 nm laser,and(7)DSF@PEG-HCuSNPs+1064 nm laser groups.Among them,groups(2),(6)and(7)were irradiated by 1064 nm laser at the tumor site with a power of 1.5 W·cm-2 for 10 minutes after tail vein injection,and tumor-temperature elevations at varied treatment groups were recorded by an IR thermal imaging instrument.H&E,TUNEL,and Ki-67 antibody staining were further performed to further evaluate the therapeutic efficacy after various treatments.After treatment,the body weight and tumor size of nude mice were measured every 2 days,and the nude mice were sacrificed after 24 days of observation.One nude mouse was randomly selected from each group to take the main organs(heart,liver,spleen,lung,and kidney)for H&E staining histological analysis,which could further evaluate the biosafety.Results No evident cytotoxicity towards 4T1 cells was detected after incubation with HCuSNPs and DSF alone,even though the dose was increased to 200 μg·mL-1 and the treatment duration was prolonged to 48 h.In marked contrast,DSF@PEG-HCuSNPs exhibited considerable cytotoxicity against 4T1 cancer cells,and the survival rates decreased with increasing concentration of DSF@PEG-HCuSNPs and prolonging of incubation time.For instance,55.3%of 4T1 cancer cells were killed at a DSF@PEG-HCuSNPs concentration of 200 μg·mL-1 after incubation for 48 h.Furthermore,4T1 cells were incubated with elevated doses of DSF@PEGHCuSNPs and further subjected to 1064 nm laser exposure to evaluate NIRⅡ laser-triggered cytotoxicity of DSF@PEG-HCuSNPs.Only 10.7%of 4T1 cells kept alive after treated with 200 μg·mL-1 of DSF@PEG-HCuSNPs under NIR-Ⅱ laser irradiation,indicating that DSF@PEG-HCuSNPs could induce significant cell death via synergistic photonic hyperthermia and in situ activated chemotherapy.H&E staining images of main organs of the nude mice and Kunming mice after intravenous injection with DSF@PEG-HCuSNPs(20 mg·kg-1)for 0,1,7,14,and 28 days showed no obvious histopathological lesion.In addition,the routine blood analysis and biochemical indexes examination also indicated that DSF@PEG-HCuSNPs was almost nontoxic to the nude mice and Kunming mice during the whole observation period.The tumor temperatures in HCuSNPs+1064 nm laser and DSF@PEGHCuSNPs+1064 nm laser groups reached up to 51.9℃ and 51.7℃ within 10 min of 1064 nm laser illumination,respectively.In marked contrast,the tumor temperature after PBS administration only elevated by 5.5℃ under the identical irradiation milieu.The single employment of HCuSNPs,DSF@PEG-HCuSNPs,DSF,or 1064 nm laser exposure manifested no significant suppressive effect on tumor growth.Comparatively,tumor growth in HCuSNPs+laser group was completely suppressed with obvious neoplasm recurrence that could be found on day 14.Importantly,the integration of DSF@PEG-HCuSNPs with 1064 nm laser exposure induced a significant inhibitory effect on tumor proliferation,where the tumors were absolutely eradicated without further recurrence throughout the period of 24 days.H&E,TUNEL,and Ki-67 antibody staining displayed that tumor tissues in the DSF@PEG-HCuSNPs+1064 nm laser and HCuSNPs+1064 nm laser groups exhibited significant apoptosis/necrosis.However,the degree of apoptosis and necrosis in the DSF@PEG-HCuSNPs plus 1064 nm laser irradiation was significantly higher than that in other treatment groups.Therefore,it was concluded that photonic hyperthermia-enhanced DSFbased chemotherapy could provide the optimal therapeutic effect on tumor suppression.Moreover,no obvious tissue damage was observed from the H&E-stained images of the major organs in all treatment groups,confirming the high therapeutic biosafety of various treatments.Conclusions Hollow CuS NPs were fabricated to effectively deliver DSF into the tumor tissues,where DSF@PEG-HCuSNPs could be degraded at acidic microenvironment and rapidly release Cu2+ and DSF drug molecules.The released nontoxic DSF molecules transform into toxic Cu(DTC)2 complexes through in situ chelation of the released Cu2+,leading to augmented therapeutic efficacy towards tumor cells.The 1064 nm laser irradiation not only produced photothermal therapy effect on tumors,but also enhanced the degradation of HCuSNPs,promoted the release of Cu2+ and DSF and transformed into toxic Cu(DTC)2 complexes,achieving NIR-Ⅱphotonic hyperthermia enhanced DSF-based chemotherapy.Furthermore,biocompatibility analysis revealed that DSF@PEG-HCuSNPs presented high biosafety and possessed high potential in clinic application.This work not only represents a distinctive paradigm of TME-activated"nontoxicity-to-toxicity" transformation nanosystem for NIR-Ⅱ photonic hyperthermia-enhanced DSF-based chemotherapy but also provides an insight into repurposing FDA-approved drugs as versatile cancer therapeutics for effective cancer treatment.