The EPR Effect Enhancement And Therapy Study Of Multimodal Imaging-guided Polymer Drug Delivery System Photodynamics On Breast Cancer

Objective:Breast cancer is currently the tumor with the highest incidence in Chinese women.Conventional treatment approaches for breast cancer are the combination of surgical resection,radiotherapy and chemotherapy.Photodynamic therapy（PDT）,a relatively novel treatment for solid tumors gets more and more attention recently.Chlorin e6（Ce6）and Pyropheophorbide-a（Ppa）are the two widely used small molecule photosensitizers for tumor PDT.However,their disadvantages such as poor water solubility,short blood circulation time and poor tumor targeting may limited their application.The use of polymer drug delivery systems to carry photosensitizers can increase the water solubility of the photosensitizers,improve tumor targeting,and prolong blood circulation time,which is conducive to apply photodynamic therapy and handle the therapeutic time window.The permeability of tumor blood vessel is higher than that of normal blood vessel.Meanwhile,the tumor lacks lymphatic drainage.Therefore,some macromolecular substances can enter the tumor through the highly permeable blood vessels and stay in the tumor for a long time.This effect is called enhanced permeability and retention effect（EPR effect）.Traditional PDT mainly achieves the therapeutic effect by directly attacting tumor cells.However,while the therapeutic time window and dose are well-timed,the main function of PDT can be increasing the permeability of tumor blood vessels,which is conducive to improve the EPR effect of tumor,then increase the concentration of polymer drug in tumor.At present,there are seldom reports about the application of combining EPR effect increasing photodynamics with other polymer drug delivery system to treat breast cancer.2-Methoxyestradiol（2-ME）,an apoptosis inducer and HIF-1αinhibitor,is an endogenous metabolite of 17β-estradiol and has effective anti-tumor activity.At present,there is no report about the combination of polymer delivery system based EPR effect increasing photodynamics with 2-ME to treat breast cancer.Enhanced magnetic resonance imaging is the most commonly used imaging method for tumor diagnosis in clinical practice,and fluorescence imaging and magnetic resonance imaging is complementary.However,the most widely used clinical small molecule gadolinium contrast agents have the disadvantages of short blood circulation time,low relaxivity,and low sensitivity.The fluorescent contrast agent also has shortcomings such as poor water solubility,short blood circulation time,and aggregation quenching effect.Moreover,because the EPR effect of tumors is for macromolecular substances,enhanced imaging with small molecule contrast agents cannot reflect the EPR effect of tumors.The polymer drug delivery system can carry both water-soluble gadolinium and fat-soluble fluorescent contrast agent and is able to form micelles in the aqueous solution,then constructing a magnetic resonance/fluorescence dual-modal contrast agent.This dual-modal contrast agent has increasing realxivity,long blood circulation time and water solubility.In view of the above problems,firstly,we constructed a magnetic resonance/fluorescence dual-modal contrast agent based on a polymer drug delivery system for magnetic resonance/fluorescence dual-modal imaging to observe the imaging of tumors,major tissues,organs and blood vessels.Then,a polymer drug delivery system-based photosensitizer was constructed and applied on EPR effect enhancement photodynamics.Their synergistic anti-tumor effect with polymer drug delivery system-based tumor cell attacking PDT was estimated.Finally,the same polymer drug delivery system carrying photosensitizer and 2-ME was used for combination anti-tumor therapy.EPR effect enhancement photodynamics firstly improved the EPR effect of the tumor,so that the subsequent polymer drug delivery system-based photosensitizer and 2-ME can be more concentrated on the tumor site,so as to achieve a better anti-tumor effect.In addition,multi-modal images were used to guide treatment and observe curative effects.Materials and Methods:1.We have successfully synthesized an amphiphilic polymer,Gd-DOTA-TPBP,a magnetic resonance/fluorescence contrast agent.Cytotoxicity test and blood compatibility test were used to preliminarily evaluate the biological safety of Gd-DOTA-TPBP.The fluorescence aggregation-induced emission of Gd-DOTA-TPBP was detected by fluorescence spectrophotometer.A clinical MRI3T scanner was applied to measure the relaxivity of Gd-DOTA-TPBP.Finally,Gd-DOTA-TPBP was used as contrast agent in MRI vascular enhancement imaging,MRI tumor enhancement imaging,MRI imaging of the main organs,and two-photon fluorescence confocal angiography in vivo.2.Polymer drug delivery system-based photosensitizer Ce6@PLGA was constructed by wrapping Ce6 with PLGA and the ability of reactive oxygen species producing in aqueous solution and cells was detected with SOSG（Singlet Oxygen Sensor Green）and DCFH-DA（2’,7’-Dichlorodihydrofluorescein diacetate）respectively.The cellular uptake of Ce6@PLGA was observed using flow cytometry.CCK8 was used to detect the cytotoxicity of Ce6@PLGA before or after light exposure.BALB/c mice were used to establish a 4T1 subcutaneous tumor model,and MR tumor enhancement scan was performed using Gd-DOTA-TPBP as a contrast agent to verify the photodynamics enhancement of EPR effect.Finally,we used a mouse 4T1 tumor subcutaneous model to verify the efficacy of Ce6@PLGA tumor EPR effect enhancement photodynamics combined with tumor cell attack PDT.3.Two amphiphilic block copolymers:2-ME-Ppa and GFLG-2-ME-Ppa,which carrying both 2-ME and Ppa were synthesised and Bz-Ppa,a copolymer carrying Ppa alone,was used as a control.The in vitro release rates of 2-ME by 2-ME-Ppa,GFLG-2-ME-Ppa were studied by HPLC.SOSG and DCFH-DA were used to detect whether those micelles can produce reactive oxygen species in aqueous solution and in cells.Flow cytometry was used to detect cellular uptake of micelles and CCK8assay was performed to detect the cytotoxicity of micelles before or after laser irradiation.A 4T1 orthotopic tumor model was established and the expression of HIF-1αwas detected using immunofluorescence staining.Western-Blot was conducted to detect the expression of HIF-1αin 4T1 cells.The distribution of the three micelles within the mouse tumor before and after irradiation was study using IVIS Spectrum.And magnetic resonance tumor enhancement scan was performed using Gd-DOTA-TPBP as a contrast agent to verify the improvment of photodynamics enhancement of EPR effect by 2-ME-Ppa,GFLG-2-ME-Ppa and Bz-Ppa.Finally,we used 2-ME-Ppa,GFLG-2-ME-Ppa and Bz-Ppa to perform EPR effect enhancement photodynamics and combined with 2-ME to treat 4T1 tumor.The therapeutic efficiency was evaluated with multimodal imaging.Results:1.There was no significant cytoctoxicity and hemolysis are found with Gd-DOTA-TPBP.In terms of fluorescence imaging,we observed that the contrast agent had fluorescence aggregation-induced emission.As for MRI,the relaxivity of Gd-DOTA-TPBP was 7.19 m M^-1S^-1,which was about twice that of the clinical small molecule contrast agent Gd-DTPA.MR angiography showed that Gd-DOTA-TPBP had a longer blood circulation time compared with Gd-DTPA and the imaging time of cerebrovascular and tumor blood vessels was also longer than that of Gd-DTPA.MRI tumor enhancement experiments showed that enhancing with Gd-DOTA-TPBP had longer and more obvious tumor imaging that made it easier to distinguished the tumor from surrounding normal tissue.The enhancement MRI of the main organs showed that Gd-DOTA-TPBP was metabolized in the liver and cleared from the body through the kidney.Confocal imaging experiments showed that Gd-DOTA-TPBP can be used for in vivo two-photon confocal cerebral blood vessel,tumor blood vessel and tumor cell imaging.2.We successfully used PLGA to wrap Ce6 to prepare Ce6@PLGA.The results showed that Ce6@PLGA can produce reactive oxygen species in aqueous solution and cells after being irradiated with 660 nm laser.Flow cytometry showed that Ce6@PLGA can be taken up by the cells.Cytotoxicity experiments showed that Ce6@PLGA and free Ce6 had significant cytotoxicity to 4T1 and HUVEC cells only under laser irradiation.MRI tumor enhancement scans with Gd-DOTA-TPBP proved that Ce6@PLGA EPR effect enhancement photodynamics can improve the EPR effect of tumors.The results of tumor treatment experiments in tumor-bearing mice showed that compared with the use of free Ce6 for combination therapy,and the use of Ce6@PLGA for single PDT against tumor cells,the use of Ce6@PLGA for tumor EPR effect enhancement photodynamics combined with tumor cell attack PDT has a better tumor suppression effect,and even one treatment can make the tumor disappear.However,this method cannot inhibit tumor recurrence and lung metastasis.3.To tackle the problem of lung metastasis,we constructed and successfully produced amphiphilic block copolymers 2-ME-Ppa,GFLG-2-ME-Ppa and Bz-Ppa carrying both 2-ME and Ppa,or Ppa alone.Among them,the critical micelle concentration of GFLG-2-ME-Ppa was the highest.The in vitro release experiment showed that GFLG-2-ME-Ppa released 2-ME more efficiently than that of 2-ME-Ppa in the acidic environment with cathepsin B.The three kinds of micelles can produce reactive oxygen species in the aqueous solution and cells after being irradiated by the660 nm laser.In the cellular uptake experiment,2-ME-Ppa was the easiest to enter cells,and Bz-Ppa can also be taken up by cells,but GFLG-2-ME-Ppa hardly entered cells during the 6 h incubation time.The cytotoxicity test showed that the toxicity of2-ME-Ppa and GFLG-2-ME-Ppa to 4T1 and HUVEC cells was not significantly different.We established a 4T1 orthotopic tumor model and used immunofluorescence staining to prove that the model highly expressed HIF-1α.Western-Blot showed that 2-ME-Ppa and GFLG-2-ME-Ppa can inhibit the expression of HIF-1αin 4T1 cells.In vivo fluorescence imaging experiments showed that the three kinds of micelles can accumulate in the tumor and the accumulation can be increased after EPR effect enhancement photodynamics.We also used Gd-DOTA-TPBP to perform MRI tumor enhancement scan to prove that EPR effect enhancement photodynamics can effectively improve the accumulation of polymer contrast agent in tumor.Finally,we used the mouse 4T1 tumor orthotopic model for therapy.The results showed that the average tumor volume of the 2ME-Ppa+L group is lower than that of 2ME-Ppa-L,GFLG-2ME-Ppa+L,Saline and Saline+L groups,but there is no significant difference compared with the Bz-Ppa+L group.CT scans and pathological sections confirmed that the lung metastasis of the 2ME-Ppa+L group was less than that of the Bz-Ppa+L group.Conclusions:1.The polymer drug delivery system-based MR/fluorescence dual-modal contrast agent Gd-DOTA-TPBP has good biosafety,good water solubility,high relaxivity,long blood circulation time and high degree of tumor aggregation.It has the characteristics of fluorescence aggregation-induced emission and can be used for two-photon imaging,so that Gd-DOTA-TPBP can be used for MRI enhancement scanning of blood vessels,tumors and organs.It is also suitable for in vivo confocal fluorescence imaging.2.The polymer drug delivery system-based Ce6@PLGA EPR effect enhancement photodynamics can increase the accumulation of polymer contrast agents in the tumor.Ce6@PLGA EPR effect enhancement photodynamics combined with tumor cell attack PDT has a better inhibiton on tumor volume than that of traditional tumor cell attack PDT,but it fails to inhibit tumor lung metastasis.3.The use of amphiphilic block polymers equipped with 2ME and Ppa for EPR effect enhancement photodynamics can improve the EPR effect of tumors,so that more macromolecular drugs would subsequently accumulate in the tumors.Among the two amphiphilic block polymers that carry both 2ME and Ppa,the inhibition of tumor growth with 2ME-Ppa is better than that of GFLG-2ME-Ppa,which probably due to suitable particle size and good cellular uptake of 2ME-Ppa.Although the Bz-Ppa without 2ME could achieve good tumor volume inhibition,the lung metastasis was more serious than that of 2ME-Ppa and GFLG-2ME-Ppa.The results show that the EPR effect enhancement photodynamics combined with 2ME therapy not only helps to increase the accumulation of macromolecular drugs in the tumor to exert a greater tumor volume inhibiton,but also reduces the risk of tumor lung metastasis.In summary,we have successfully constructed photosensitizers based on a polymer drug delivery system and used it for the EPR effect enhancement photodynamics.In combination with tumor cell attack PDT or 2ME based polymer drug delivery system,the EPR effect enhancement photodynamics is carried out in the first step to improve the EPR effect of tumors then make subsequent polymer drug delivery system-based photosensitizer or 2ME accumulate more at the tumor site.Gd-DOTA-TPBP,a dual-modal magnetic resonance/fluorescence contrast agent based on a polymer drug delivery system,was successfully constructed,combined with a multi-modal imaging mode,to evaluate the changes of tumor EPR effect and the treatment efficacy.This study confirms that the polymer drug delivery system-based EPR effect enhancement photodynamics can effectively improve the tumor EPR effect,and the combination with the tumor cell attack PDT or 2ME based on the polymer drug delivery system can effectively improve the tumor suppressive effect.Among them,the combination with 2ME can inhibit lung metastasis at the same time.The multimodal imaging combined with the new contrast agent Gd-DOTA-TPBP successfully guided tumor treatment and evaluated the treatment efficacy.