Intratumorally Injectable Photothermal Drug-loaded PH-sensitive Photodynamic Nanocarriers

Malignant tumor is one of the most serious diseases that endanger human health and the main cause of human death at present.The treatment for malignant tumors including surgery,chemotherapy,radiotherapy and immunotherapy,but these methods can not obtain good curative effect for most cancers.Chemotherapy and radiotherapy may have serious side effects.In particular,if the chemotherapy drugs are administered systemically,not only the distribution of the tumor site is less,but also the damage to the normal tissue is large.After long-term use of drugs,the tumor cells are susceptible to drug resistance.In recent 10 years,tumor light therapy has gained the attention of basic and clinical researchers.It uses light emitted from external instruments to stimulate drugs in vivo to kill cancer cells.Photodynamic therapy(PDT)is that photosensitizers irradiated by laser beams to deliver energy to the surrounding oxygen,and the resulting ROS can kill nearby tumor cells.Photothermal therapy(Photothermal therapy,PTT)is that photothermal agents irradiated by lasers can transmits energy to the surrounding tissues in thermal form by means of vibrational relaxation or other nonradiative transitions.Because the tumor cells are not resistant to heat,they are easily killed by high heat.In this paper,based on the systemic toxicity and systemic distribution produced by chemotherapy,drug resistance of cancer cells and other issues,intratumoral injection of photodynamic nanocarrier containing photothermal agent was designed and prepared.The detailed in vitro and in vivo researches were carried out,showing it's high anti-tumor effect.Then we explored its mechanism.1.Preparation and properties of photothermal drugDBEC was designed and synthesized based on photothermal drug indocyanine green(ICG)as a stable hydrophobic photothermal agent.The structure was confirmed by 1H NMR and ESI-MS.DBEC is insoluble in water while soluble in organic solvents.The photophysical properties of DBEC are as follows:(1)The maximum absorption wavelength of DBEC is 830nm;(2)The maximum wavelength of fluorescence emission is 830nm.The fluorescence quantum yield is 0.27;(3)The stability of light is pretty good.The absorbance at 830nm decreased by 35%with 830nm(1 W/cm2)laser irradiation after 32min;(4)Photothermal effect is strong,when the concentration of DBEC was 50?g/mL,the temperature quickly rose to 41.3 ? after 830nm(1.0W/cm2)laser irradiation.DBEC has good photothermal properties and fluorescence emission properties providing the basis for vivo research.2.Preparation and property of pH sensitive zinc phthalocyanine nanocompositesBased on the former study of our laboratory,we designed a pH-sensitive triblock copolymer which hydrophilic fragment is mPEG750(PCL-pDEA-mPEG750).PH sensitive zinc phthalocyanine copolymer(ZPPDP)was prepared with the zinc-(3-tetra-(4-carboxyphenoxyl)phthalocyanine(ZnPC4)and PCL-pDEA-mPEG750.ZPPDP soluble in organic solvents was dissolved in water to form a single molecule micelle.The photochemical properties of ZPPDP were determined:(1)The maximum visible absorption wavelength was 674nm;(2)The maximum excitation wavelength was 666nm.The maximum wavelength of fluorescence emission was 681nm,and the fluorescence quantum yield was 0.26;(3)Singlet oxygen yield ?=0.47.ZPPDP was prepared in water by ultrasonic injection and reverse injection using tetrahydrofuran(THF)as the solvent.The average particle size was 60 nm and the polydispersity was 0.264.The Zeta potential of the nanocarrier was pH-dependent from-6.04 mV(pH 7.4)to 18.57 mV(pH 5).Transmission electron microscopy showed that the nanocarriers were spherical.Therefore,ZPPDP could produce strong reactive oxygen species,can be used as photodynamic reagents,and a drug nanocarrier.3.Preparation of pH sensitive zinc phthalocyanine copolymer nanocarrier containing photothermal drugThe pH sensitive zinc phthalocyanine copolymer nanocarriers containing DBEC(DBEC@ZPPDP)were prepared by reverse injection.The average particle size was 76.6nm,and the polydispersity was 0.134.The Zeta potential was 44.2mV.Free DBEC and DBEC@ZPPDP were separated by ultrafiltration tube.The optimal prescription was ZPPDP:DBEC(w/w)= 2:1 with the drug loading and the encapsulation efficiency as the evaluation index.The encapsulated rate was 65.9%and the drug loading was 25.6%.The zeta potential of DBEC@ZPPDP was pH-dependent from-17.9 mV(pH 7.4)to 44.2 mV(pH 5).TEM observation showed that DBEC@ZPPDP was spherical and the particle size increased with the decrease of pH value.Release of DBEC@ZPPDP was also pH-dependent.With the pH of release media decreased,the release of DBEC increased.The DBEC@ZPPDP has a strong ability of photothermal:When the concentration of DBEC was 50?g/mL,the temperature quickly rose to 41.6 ? by 830nm(1W/cm2)laser irradiation.The rise of temperature is proportional to the DBEC concentration and the irradiation time.Hemolysis tests showed that DBEC and ZPPDP were almost insoluble.DBEC@ZPPDP has high drug loading and high heat production effect,could be used for photothermal/photodynamic therapy.4.Drug analysis methodThe content determine methods of DBEC and ZnPC4 were established by UV-Vis spectrophotometry.The HPLC method was used to determine the content of MIT.All methods are validated by methodology.5.In vitro antitumor efficacy of nanocarrier containing thermal drugThe phototherapy effect of DBEC@ZPPDP was investigated by using mouse melanoma cells(B16-F10)and mouse breast cancer cell(4T1)as model.Laser irradiation at 660 nm(200 mW/cm2)and 830 nm(1.0 W/cm2)alone did not inhibit the growth of B16-F10 and 4T1 cells.Free DBEC has a certain cell killing effect.After dark incubation for 2h the cell survival rate was about 80%,an additional laser irradiation using 830nm for 3min could produce stronger cell killing effect.DBEC@ZPPDP was added to the two cell models.After 660nm and/or 830nm laser irradiation,strong cell killing was achieved.The cell viability was 8.1%.After 4h of incubation of the vector and B16-F10 cells,flow cytometry showed that the phagocytosis of cells were saturated.These results provide a basis for the study of antitumor studies in DBEC@ZPPDP.6.Infrared thermal imaging and near infrared fluorescence imaging of DBEC@ZPPDP on tumor-bearing animal modelsTo observe the performance of DBEC@ZPPDP on infrared thermal imaging and near infrared fluorescence imaging,we build the tumor-bearing animal models of B16-F10 and 4T1 cells.After the intratumoral injection of DBEC@ZPPDP(containing DBEC 5?g),the tumor temperature is higher than 43 ? with 1W/cm2(830nm)laser irradiation for 3min showing photodynamic effect.The effect of near infrared fluorescence imaging of DBEC@ZPPDP was investigated on the tumor-bearing mice model of 4T1.After injection,DBEC@ZPPDP was found to be clearly imaged in the animal using a small animal fluorescence imaging device.Compared with tail vein injection,the concentration of drug in the tumor site is higher with a long time for more than 24h and less distribution in other tissues.After tail vein injection,DBEC@ZPPDP distributed all of body,and no specific distribution in the tumor tissue.7.Study on pharmacodynamics of nanocarrier containing photothermal drugBalb/c nude mice subcutaneous B16-F10 melanoma model was established.The mice were injected with control drug mitoxantrone,DBEC,ZPPDP and DBEC@ZPPDP.The body weight rate,tumor volume,tumor inhibition rate,histopathology and cytokine expression of tumor were measured.Firstly intratumor inject the DBEC@ZPPDP.After 4h of injection,irradiate the tumor with 830nm(1.0 W/cm2)laser for 3min.After 1h of the above action,irradiate tumor with 660nm(200mW/cm2)laser for 5min.The above method could kill over the tumor completely,and there was no recurrence.In this paper,we designed and prepared a pH-sensitive zinc phthalocyanine copolymer nanocarrier contained photothermal drug.This nano system produced high-efficiency anti-tumor effect under long-wavelength laser irradiation with intratumoral injection.The optimal treatment regimen can completely cure malignant tumor with no side effects occur except for the local self-repairable trauma.This article provides a new idea for tumor therapy,which avoids the problem of extremely limited tumor targeting after intravenous injection of nanosized vector,and the light treatment mechanism is effective for various tumors.DBEC@ZPPDP is also expected to become a new type of highly effective anti-tumor nano preparations.