| Objective:Thyroid cancer(TC)is a very common malignant tumor,the incidence of thyroid cancer is much higher than that of other endocrine organs.Evidence has proved that the incidence rate of TC has been the first among all malignant tumors,and its incidence has been nearly 3 times,and there is still an increasing trend,so TC has attracted more and more people’s attention.At present,papillary carcinoma of thyroid occupies the majority(80%-90%)of all known pathological types of TC.According to the latest pathological classification of TC,it is divided into two types,high aggressive and low aggressive,and a total of 14 subtypes are divided.The highly invasive subtypes of papillary thyroid carcinoma(PTC)have more active biological behavior,and most of the tumors show the growth characteristics of malignant tumors,strong invasion ability,high probability of lymph node metastasis,and easy to appear distant metastasis.Such patients have a high recurrence rate even after radical surgery,and some of the highly aggressive subtypes of PTC even progress further to iodine-resistant PTC,which is often the cause of death in patients.The treatment of TC has been going on for many years.At present,the main treatment plan is still surgical treatment,supplemented by endocrine therapy,iodine-131 therapy,chemotherapy,targeted therapy,immunotherapy and traditional Chinese medicine therapy.Chemotherapy is one of the important means of systemic therapy.However,previous treatment experience tells us that for those refractory and highly aggressive PTC,chemotherapy often leads to drug resistance soon,unable to inhibit tumor growth,and accompanied by many side effects.In recent years,the appearance of aptamer has brought a lot of hope to many refractory malignant tumors.It can combine with target molecules highly selectively to attack target tumor cells,providing a new direction for clinical treatment of malignant tumors.The aptamer we selected is AS1411,which is more stable than ordinary aptamer and has strong targeting and affinity.The surface chemistry of novel nanoparticles(NPs)enables targeted therapy and controlled drug release,promising to reduce systemic toxicity and adverse reactions.The selected gold nanoparticles(Au NPs)also have photothermal therapeutic effect(PTT),which can have synergistic effect with chemotherapy drugs and has great potential value.At present,there are still few nanomaterials with good targeting and anti-tumor properties for the treatment of TC.This study intends to design a multifunctional nano drug delivery system with high targeting and excellent killing ability for highly invasive PTC.Methods:1.AS1411/Au NPs/Dox drug delivery system with targeting and photothermal effect was prepared.After the successful synthesis of Au NPs,isomolar AS1411 and Hairpin DNA were combined with gold nanoparticles respectively,and a control group(Polyt-modified Au NPs)was prepared.Then Hairpin DNA was used to carry DOX.2.The morphology of Au NPs and AS1411/Au NPs/Dox were detected and confirmed by transmission electron microscopy(TEM)and ultraviolet spectrophotometer,and the particle size and zeta potential were also determined.3.Using the characteristics of Au NPs photothermal effect,the fluorescence intensity of DOX(λ=590nm)in solution under different p H values of 7.4 and 5.0 under different irradiation conditions and solution temperature at different irradiation conditions and different irradiation time were detected under the condition of near infrared laser irradiation(808nm).4.TPC-1 cells were cultured,AS1411/Au NPs/Dox and Poly T/Au NPs/Dox were labeled with FAM,the fluorescence intensity of FAM was detected by flow cytometry,and the binding capacity of AS1411/Au NPs/Dox with TPC-1 cells was calculated.To confirm the targeting of AS1411.5.TPC-1 cells and L929 cells of the control group were cultured,and DOX and AS1411/Au NPs/Dox were added,respectively.The endocytosis was observed by confocal microscopy.6.TPC-1 cells were cultured,and different concentrations of free DOX or AS1411/Au NPs/Dox were added,respectively,to calculate IC50 values and cell survival rate under different drug concentrations.7.TPC-1 cells and L929 cells were cultured and added with DOX,AS1411/Au NPs/Dox and Poly T/Au NPs/Dox(equivalent DOX concentration was 2.5μg/ml).The killing ability of TPC-1 cells and L929 cells was detected,and the difference was compared.8.TPC-1 cells were cultured with AS1411/Au NPs/Dox and Poly T/Au NPs/Dox(equivalent DOX concentration of 2.5μg/ml),irradiated with 808nm laser(1W/cm~2or 2W/cm~2)for 10min,absorbance values were determined,and cell survival rate was calculated.To show the killing ability of TPC-1 cells.9.The model of subcutaneous and right abdominal graft tumor in mice with TPC-1 cells was established.Free DOX,0.9%normal saline,AS1411/Au NPs/Dox,Poly T/Au NPs/Dox and increased laser irradiation conditions were injected,respectively.Changes in tumor volume and final tumor weight were recorded.10.The expressions of BCL-2,Caspase-3 and Caspase-9 genes were determined by Real-time PCR to confirm the apoptosis of thyroid cancer tissues induced by AS1411/Au NPs/Dox drug delivery system in vivo.11.The body weight of mice in each group was monitored,and the blood of mice was collected to detect the changes of creatine kinase and liver and kidney function.The safety of AS1411/Au NPs/Dox drug delivery system in mice was systematically studied.Results:1.Au NPs were successfully prepared with a particle size of about 17.5nm.The particle size of AS1411/Au NPs/Dox was obviously larger than that of Au NPs through TEM.The characteristic absorption peaks of Au NPs and AS1411/Au NPs/Dox were detected by UV-spectrophotometry.The redshift of the characteristic absorption peaks of Au NPs and Au NPs/Dox were 520 nm and 529 nm respectively.The Zeta potential test also confirmed the successful preparation of AS1411/Au NPs/Dox,and the Au NPs/Au NPs/Dox potential was-36.5m V and-19.1m V,respectively.2.Detection of the photothermal effect of the nanoborne drug system:Our results confirm that the laser power is 2 W/cm~2,the irradiation time is 10min,and the temperature reaches about 38℃.With the extension of irradiation time,the solution temperature no longer increased significantly.DOX fluorescence signal(λ=590nm)could not be detected basically at p H7.4,while obvious DOX fluorescence signal could be detected at p H 5.0.The fluorescence signal of DOX was enhanced when the near infrared laser irradiation(808nm),and the release of DOX was increased under the condition of brightening laser irradiation.In terms of illumination intensity,the DOX fluorescence signal released by2W/cm~2was stronger than that by 1W/cm~2.The DOX fluorescence intensity was the strongest when laser irradiation of 2W/cm~2was applied at p H 5.0,which also proved that laser irradiation and acidic environment could synergically promote DOX release.3.The fluorescence intensity of FAM labeled AS1411/Au NPs/Dox and Poly T/Au NPs/Dox in TPC-1 cells was detected by flow cytometry.The fluorescence intensity of FAM in AS1411/Au NPs/Dox group was significantly higher than that in Poly T/Au NPs/Dox group,which confirmed the good targeting of AS1411.4.Confocal microscopy showed that Dox fluorescence signal(red)in TPC-1 cells treated with AS1411/Au NPs/Dox was significantly stronger than that in L929 cells.Under near-infrared laser irradiation(808nm),AS1411/Au NPs/Dox entered the cells and released a large number of DOX(red).Meanwhile,the experimental results showed that the red fluorescence intensity of cells irradiated by 2W/cm~2was stronger than that irradiated by 1W/cm~2.5.Under the action of different drug concentrations,with the increase of drug concentration,the cell survival rate of free DOX group and AS1411/Au NPs/Dox group gradually decreased,and the IC50 value was obtained.6.There was no significant difference in survival rate between TPC-1 and L929 cells after DOX or Poly T/Au NPs/Dox treatment.The survival rate of TPC-1 cells treated with AS1411/Au NPs/Dox was significantly lower than that of L929 cells.In conclusion,AS1411/Au NPs/Dox releases DOX in cells and plays a killing role in the absence of infrared laser irradiation.After infrared laser irradiation,the survival rate of TPC-1 cells was further decreased by PTT in coordination with DOX chemotherapy,and the survival rate of TPC-1 cells was even lower under the irradiation condition of 2W/cm~2.7.The subcutaneous and right abdominal graft tumor model of TPC-1 cell mice was successfully constructed,and treated with free DOX,0.9%normal saline,AS1411/Au NPs/Dox,Poly T/Au NPs/Dox,and laser irradiation(2W/cm~2)was increased in the latter two groups.The results showed that AS1411/Au NPs/Dox could significantly inhibit the growth of transplanted tumors under laser irradiation,and both tumor volume and tumor weight were significantly lower than those in other groups.It is suggested that it has a good antitumor effect,and also proves the targeting ability of AS1411.8.Real-time PCR results suggested that AS1411/Au NPs/Dox down-regulated the expression of anti-apoptotic gene Bcl-2 and up-regulated the expression of pro-apoptotic genes caspase-3 and caspase-9,suggesting that AS1411/Au NPs/DOX could increase the apoptotic level.The treatment of thyroid cancer has a more significant effect.9.The weight of mice in each group after medication was measured,which showed that the weight of mice in free DOX group decreased significantly compared with other groups.Creatine kinase(CK)concentration in each group was detected by blood collection.The CK value in free DOX treatment group was the highest,while that in Poly T/Au NPs/Dox treatment group and AS1411/Au NPs/Dox treatment group was within the normal range.The liver and kidney function tests of each group were basically in the normal range.This also demonstrated the targeting of AS1411 and the safety of Au NPs carrying drugs,which achieved the effect of reducing DOX side effects.Conclusion:We successfully prepared the AS1411/Au NPs/Dox drug delivery system with a novel active targeting technique in addition to the passive targeting of"high permeability and retention"(EPR)effects.In addition,under near infrared light(808nm),the synergistic effect of PTT and drug release can be demonstrated to target TPC-1 cells,which has been confirmed in vivo experiments in mice.At the same time,the drug delivery system also has the advantages of high safety and low side effects,which provides a new therapeutic idea for the clinical treatment of highly invasive PTC. |
PDF Full Text Request