| Pet tumors have become a common clinical condition,causing great suffering to pets.Among them,breast cancer has become one of the malignant tumors because of its strong proliferation and metastasis.In clinical,the incidence of breast cancer reaches 50% of the total cases.Breast cancer treatment mainly relies on surgery,chemotherapy and radiation therapy,but general anesthesia is required during animal treatment,which is very expensive.In addition,the side effects during chemotherapy are large and prone to drug resistance.Photodynamic therapy(PDT)has received much attention due to its high efficacy,minimal side effect and noninvasiveness,and it kills cancer cells by converting intra-tumoral oxygen into reactive oxygen species(ROS)through light exposure to photosensitizers.However,oxygen is consumed during PDT treatment,resulting in a severe hypoxic environment and further inducing an increase in Hypoxia inducible factor-1(HIF-1α)expression,which hinders the therapeutic effect of PDT and further induces tumor metastasis.YC-1,3-(5-hydroxymethyl-2-furanyl)-1-phenylmethylindazole)is a HIF-1α inhibitor that both blocks HIF-1α transcription and has various biological activities such as antivascular and anti-cancer.However,the poor solubility of YC-1 severely prohibit its in vivo application.In recent years,nanodrugs have been widely used due to their advantages in improving drug solubility,bioavailability and reducing toxic side effects.To maximize antitumor efficacy,researchers have explored various stimuli-responsive nanoparticles(NPs)for a specific release of drugs through endogenous or exogenous stimulatory responses,such as p H,ROS,redox and hypoxic.Therefore,based on the ability of the PDT to exacerbate intratumoral hypoxic conditions,this paper designed a hypoxia-responsive nanocarrier that co-encapsulation Ce6(photosensitizer)and YC-1(HIF-1α inhibitor)to form a synergistic anti-tumor nanodrug delivery system(HC/PI@YC-1 NPs).The illumination promotes dissociation of NPs and release of drug(YC-1)to achieve synergistic anti-tumor therapy and prevention of tumor metastasis.This experiment provides a novel and efficient therapeutic strategy for animal tumor treatment,while overcoming the drawbacks of PDT.Details of the study are shown below.1.The first part,preparation of nanoparticles with hypoxia sensitivity and characterization of nanoparticles.First,synthesized alkylated 2-nitroimidazole(NI)conjugated polyethyleneimine(PEI)and Ce6-modified hyaluronic acid(HA-Ce6),which is able to self-assemble to form NPs and encapsulate YC-1(HC/PI@YC-1 NPs).It was shown that In Vitro light irradiate(660 nm,5 m W/cm2),which produces high levels of ROS to eliminate tumors and causes a severe hypoxic environment can cause the conversion of hydrophobic 2-nitroimidazole to hydrophilic 2-aminoimidazole(AI),triggering the dissociation of NPs.The particle size of HC/PI@YC-1 NPs in normal environment was 180 nm,while in hypoxic environment,the particle size of HC/PI@YC-1 NPs increased dramatically to 800 nm.Meanwhile,the release of YC-1was measured before and after light exposure,and the release rate of YC-1 in the hypoxic environment was 70% in 48 h compared with the normal environment.Moreover,the particle size of NPs had minimal alteration after incubation with serum for 3 days,suggesting that the NPs had ideal serum stability.2.The second part evaluates the targeting efficiency of NPs and the synergistic anti-tumor efficacy by 4T1 cells(mouse breast cancer cells).Since HA is able to bind to the CD44 receptor overexpressed on the surface of tumor cells,binding HA to the nanoparticle surface can effectively increase endocytosis.The demonstrated that nanoparticle uptake was significantly increased in 4T1 cells compared to L929 cells(mouse fibroblasts).With the endocytosis of HC/PI@YC-1 NPs into cells under light(660 nm,5 m W/cm2),a large amount of ROS was generated to induce cell death,while the resulting hypoxic environment could further promote YC-1 release,thus achieving synergistic antitumor efficacy between PDT and PDT-activated YC-1.The results showed that the half-maximal inhibitory concentrations(IC50)of both YC-1(7.6 μg/m L)and Ce6(1.4 μg/m L)in HC/PI@YC-1 NPs were lower than those in other single treatment groups,and the CI values were less than 1.This indicated that HC/PI@YC-1NPs could achieve synergistic antitumor therapy.3.The third part the 4T1 xenograft tumor-bearing mice were used to evaluate the in vivo antitumor efficacy.During the 18 d treatment period,HA/PI@YC-1 NPs(single drug treatment group)and HC/PI NPs(single PDT treatment group)showed only partial inhibition of tumor growth,while HC/PI@YC-1 NPs produced significant inhibition of tumor growth and showed high survival rate at day 52.In addition,there were no significant changes in mouse body weight and histopathological sections of mice,demonstrating the high tissue safety of HC/PI@YC-1 NPs.4.Part four to verify the anti-metastatic activity of HC/PI@YC-1 NPs,4T1 metastatic cancers were established in BALB/c mice by orthotopic injection of Luc-4T1 cells in mammary pad,and monitored by the in vivo imaging system during five weeks treatment period.The results of the trial showed that lung metastases started to appear at week 4 in the PBS group versus the single PDT group.And the single PDT group had the strongest bioluminescence signal in the lungs at week 5,indicating that treatment with PDT alone may cause severe lung metastasis.In contrast,HC/PI@YC-1 NPs showed significantly lower bioluminescence signals throughout the body of the mice at week 5.Moreover,both lung pathology sections and lung tumor nodule data of HC/PI@YC-1 NPs showed a significant reduction in tumor lesion area.This demonstrated that HC/PI@YC-1 NPs had significant efficacy in preventing tumor metastasis.In summary,in this paper,we successfully constructed a hypoxia-responsive nanoparticle(HC/PI@YC-1 NPs)co-encapsulation with YC-1 and Ce6,which induced cell death by generating a large amount of ROS under light.In addition,causing a hypoxic environment can promote nano-carrier dissociation and specific release of YC-1,thus achieving synergistic anti-tumor and tumor metastasis prevention of PDT and YC-1.This study provides a new experimental basis and idea for animal tumor treatment. |
