Folate-bearing Doxorubicin-loaded Magnetic Poly(Nisopropylacrylamide) Microsphere As A New Strategy For Cancer Therapy

Chemotherapy is a very important way for cancer treatment.However, Chemotherapy drugs can destroy all the cells of hunman body.Finding a way to solve the problem become a popular study in clinic.Recently numerous strategies have been employed to enhance efficacy of drug delivery for cancer treatment.For example,by modifying poly-microsphere,a nano-carrier,makes it more effective. Conjugation with folic acid increases specific targeted drug delivery towards folate receptor-bearing cancer cells to improve anticancer effectiveness by increasing the tissue's local concentration of drugs.Doxorubicin(DOX) is a classic anti-cancer agent.It can inhibit the synthesis of RNA and DNA.In many tumors,it has a good effect.However,it has strong cytotoxic.So,how to enhance the effect of anti-cancer and reduce the cytotoxic at the same time become the focus.In the current studies, we synthesized Folate-bearing,DOX loaded,Magnetic,Poly(N-isopropylacrylamide) Micropheres(FDMPM) to treat breast cancer cells(Human SKBR-3).At the same time,we also synthesized MPM,FMPM,DMPM for the contrast.We chosed human breast cancer cells in our study because the cells highly expressed folate receptors which was reported by the papers and our experiment results.Otherwise we chosed MDA-435 human breast cancer cells which don't expressed folate receptors for the contrast.This study includes two parts:1.Synthesis the new nano anti-cancer drug by physic and chemistry process.In the process of synthesis,we found efficiency of drug encapsulation very high(95%) of FDMPM at pH above 7.4.And efficiency of drug load was above 50%at the same pH.2.RT-PCR showed that cancer cells highly expressed folate receptors.Confocal laser scanning microscopy and flow cytometry revealed internalization of the carrier by SKBR-3 in treatments with FDMPM,which was not the case with any other combination for drug delivery(MPM,FMPM,and DMPM).Similarly,SKBR-3 cell growth was inhibited more(assessed by MTT and trypan blue exclusion assays) when treated with FDMPM than with any other combination.In conclusion,Current results confirm our predication and demonstrate that FDMPM has potential as a new targeting strategy in cancer therapy. Ecto-5'-nucleotidase(EC3.1.3.5,CD73,ecto-5'-NT) is a glycosyl phosphatidylinositol(GPI)-linked,membrane-bound glycoprotein which hydrolyzes extracellular nucleoside monophosphates into bioactivenucleoside intermediates. Recent studies showed that mice tissues was highly damaged by inhibiting the activity of CD73.Our experiments also suggested kidney of the mice in CD73^-/- C57BL/6 mice were more obvious than CD73^+/+ mice.Therefore,CD73 plays an important role in the progress of kidney ischemia-reperfusion.However,it is remained unknown how CD73 affects it.In this study,firstly,we isolated and cultured the model of mice kidney ischemia-reperfusion,then studied the effects of CD73 on kidney ischemia-reperfusion test in vitro;Secondly,we observed kidney ischemia-reperfusion using morphology and biochemistry detection in CD73^+/+ and CD73^-/- C57BL/6 mice respectively.Our project studied the relationship between CD73 and kidney ischemia-reperfusion,tried to put forward a new molecule correlated with protect ischemia-reperfusion tissues.This study include three parts:1.Identification of CD73^+/+ and CD73^-/- C57BL/6 mice.We detected the genetype of mice by PCR and confirmed CD73 activity by enzyme histochemistry.188bp band is the product for CD73^+/+ C57BL/6 mice with positive stainings in the liver, heart and kidney;280bp band is the product for CD73^-/- C57BL/6 mice without staining in above tissues.2.Culture the kidney ischemia-reperfusion of CD73^+/+ and CD73^-/- C57BL/6 mice.Then using HE stain and tissue EB detect to observe the different of CD73^+/+ and CD73^-/- C57BL/6 mice:The tissues of the kidney ischemia-reperfusion of CD73^+/+ and CD73^-/- C57BL/6 mice were markedly different.In the HE stain,we can see damage of CD73^-/- kidney was much stronger than CD73^+/+mice.Morever,EB in the CD73^-/- kidney was much more than CD73^+/+ mice showed that the injury of CD73^-/- mice was dramatically higher than CD73^+/+mice.Thus,CD73 probably protected ischemia-reperfusion kidney tissues.3.Using of HPLC and fluorescent probe,we detected many factors in ischemia-reperfusion kidney tissues and found that in CD73^+/+ C57BL/6 mice the AMP,free radical and MDA were higher than CD73^-/-.AMP is a part of ATP.In reperfusion,AMP is decomposed to hypoxanthine and then uric acid.At the progress, a lot of negatons are diliveryed.Dioxygen combined with these negatons and convert to free radical.Tissues can be injuryed by these free radical.CD73 can convert AMP to adenosine.Maybe it can demonstrate the reason that damage of CD73^-/- kidney was much stronger than CD73^+/+mice in ischemia-reperfusion.In conclusion,CD73 can protect ischemia-reperfusion kidbey tissues and the protection is concerned with to reduce the AMP and free radical.This study offers new thought for ischemia-reperfusion protection.