Efficacy Of Magnetic Carbon Nanotubes Loaded With Doxorubicin For Treatment Of Breast Cancer With Axillary Metastasis

1. BackgroundThe breast cancer is one of the most common malignant tumor among women.The morbidity accounts for7%to10%of kindsofmalignant tumor among women.Every year about1.36million women are dignosed as breast cancer while about0.34million women are died of the breast cancer.Axillary lymph node status is the important factors that affects the prognosis of breast cancer, the surgery cleaning of the remains of the axillary lymph node, and the low rate of drug content in thetraditonal chemotherapy which lead to uncomplete treatment and the relapse. So in recent years, the tumor chemotherapy to the metastasis of lymph node are taken more seriously, the lymph nodes have become a taget organ which has much potential in tumor chemotherapy.Lymphatic system can endocytosismacromolecular substances and particles, it will combine chemotherapy with carrier to keep the local balance of the medcine and at the same time slowly release the effective drug conceration to kill the tumor cells in lymphatic system and restore the function of the lymphocyte, helping to eliminate the remaining tumor cells, reducing tumor metastasis through lymphaticsystem;Also, it will control the amout of the drug that enter blood circulation and reduce the side effects of the chemotherapy.Nowdays, carriers used to lymph target therapy has many kinds such as activated carbon, lipsome and nanoparticles, etc.The reaseaches of these carriers has gained much progress.These carriers can make drugs arrive at the targeted lymhatic system more accurately, release slowly and reduce the side effects.However, they have some disadvantages such as the little amount that the drugs can becarried, the unstablity of the particle size and weakness of the targeting.So it counts in lymph-targeted-chemotherapy that selecting a carrier whose particle size is stable and which has good compatibility with the human body as well as release drugs slowly, meanwhile which is harmless to the body.2. Objective2.1Preparing a carrier which has a strong adsorption and good lymphatic-targeting magnetic multi-walled carbon nanotubes(O-mMWNT-PEG), and understanding basic physical characteristics and lymphatic tracer.2.2Discussing the efficacy of killing of DOX-O-mMWNT-PEG on breast cancer cells in vitro and in vivo treatment of breast cancer with lymph node metastasis.3.Materials and MethodsEstablish BALB/c mouse model of breast cancer with lymph node metastasis and prepare a strong absorption and good lymphatic-targeting magnetic multi-walled carbon nanotubes(mMWNT), and adsorbing doxorubicin to make into mMWNT-D suspension.Inject the micewith breast cancer lymph node metastasis with the drug which containthe same concentration of doxorubicin, doxorubicin, magnetic multi-walled carbon nanotubes doxorubicin, magnetic multi-walled carbon nanotube dispersion.At the sametime, observe the inbibitional effect of mMWNT-D on tumor cells. MTT method assay the inhibiting effect of these three groups on EMT-6cell in vitro.3.1The preparation and testing of DOX-O-mMWNT-PEG.In order to increase the water-soluble ablity of multi-walled carbon nanotubes, firstly use H2SO4and HNO3to oxidize the multi-walled carbon nanotubes (MWNT) to get oxidized multi-walled carbon nanotubes (Oxidized-MWNT, O-MWNT), then through the π-π adsorption join O-MWNT and DSPE-PEG2000together to obtain the O-MWNT-PEG. O-mM WNT-PEG was synthesized with a layer of magnetite Fe3O4nanoparticles on the surface of multi-walled carbon nanotube though chemical methods, for the preparation of magnetic particles loading the drug. Finally, make the magnetic particles adsorb the Doxorubicin(Dox) to preparedO-mMWNT-PEG-Dox.Particleswere characterizedvisually by transmissioneleetronmicroscopy(TEM), and then at room temperature obeserve it for the suspension stability and mangnetic responsiveness.Use theZetasizer Nano ZS to meansure the hydration size and the Zeta potential of the O-mMWNTs-PEG and DOX-O-mMWNTs-PEG.Using UV-Vis-NIRto meansure the DOX concentration, and confirm the absorption wavelength of DOX is480nm. The drug is caculated in accordance with the formula (DL). And study the O-mMWNT-PEG tracer of the lymph nodes in mice.3.2The inhibition effect on EMT-6cells in vitroMTT(3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay the determination of drugs on lethality to cell, MTT method is widely used for the determination of cell proliferation and cytotoxicity experimentaly. It is the principle that MTT (3-(4,5dimethyl-thiazol-2)-2,5-diphenyl tetrazolium bromide) may mitochondrial dehydrogenase with the occurrence of the reduction reaction of the purple-crystals product.In this dissolving agent, the product may be dissolved in purple.Then measure its absorbance at a wavelength of570nm by a microplate reader, the higher the absorbance, the more proliferation, and lower cytotoxicity.vice versa. The experiment detected the cytotoxic effect of EMT-6mouse breast cancer cells in vitro through MTT.3.3The construction of BALB/c mouse model that has breast cancer lymph node metastasisand the obeservation of in-vivo drug efficacy3.3.1The Construction of the animal modelTake the EMT-6mouse breast cancer cells during the logarithmic growthphase, live cells>95%, wash the sample3times with1X PBS liquid.Take1X PBS resuspended cells, and adjust cell concentration of1×108·mL-to prepare a cell suspension on4℃ice. On the left footpad of BALB/c mice inoculated cell suspension0.2mL/per. Observed the growth tumor and inguinal lymph node metastasis.Those models five weeks later the left groin where the lymph nodes were the size of soybean are qualified animal models.The left popliteal lymph node and tumor tissue of three of those qualified models were collected and embedded in paraffin and cut into slices and dyed in HE.At lastobserve under light microscopy examination.3.3.2Animal grouping and observation of drug efficacySelect36qualified models five weeks later after the inoculation of tumor cell, which were randomly divided into six groups, each in6.Separately, In group A which is saline group:saline is subcutaneously injected in the left hind limb of mice;In Group B which is O-mMWNTs-PEG emptyvector group:O-mMWNT-PEG is suspension subcutaneously injected in the left hind limb of mice.In group C which is DOX monotherapy group:DOX solution subcutaneously injected in mice left hind;In group D which is lower concentrations DOX-O-mMWNTs-PEG group without a magnetic field, the low concentration DOX-O-mMWNTs-PEG suspension is injected subcutaneously in mice left hind, and permanent magnets are fixed at the part which has lymph nodes metastasis;In E group:low concentrations of DOX-O-mMWNTs-PEG and magnetic field group applied, low concentrations of DOX-O-mMWNTs-PEG suspension is injected subcutaneously in the left hind leg of mice.And permanent magnets are fixed in the parts of the lymph nodes metastasis;In groupF:high concentrations of DOX-O-mMWNTs-PEG and magnetic field group applied, high concentrations of DOX-O-mMWNTs-PEG suspension wassubcutaneously injected in the left hind limb of mice, and permsnent magnets are fixed in parts of the lymph nodes metastasis. Observe the effect and side effects of different treatments.Operate pathological examination on lymph nodes and tumor tissue, including HE-staining examination and use the TUNEL to assay if there is any apoptosisi of lymph nodes or tumor tissue of tumor cells.4. Results4.1Identification of O-mMWNT-PEGO-mMWNTs-PEG suspension was black, distributed evenly, slightly sticky.It has good stability at room temperature, also with a significant magnetic response. mMWNTs has a good ablity of tracer of the lymph, migarating with lymph through the lymph nodes, and mainly distributed in the cortex.The local skin on footpad with the injection of mMWNTs has no signs of allergies and toxicity. Histological examination showed no vital organs in rats have gathered carbon nanotubes.4.2The determination of the amount of drugUsing UV spectrophotometry determine the DOX concentration, according to the formula the rate of drug carring was85%.The original DOX concentration is1g/ml, the rate of drug carrying was85%, The DOX concentration of the prepared DOX-O-mMWNTs-PEG was0.85mg/ml.4.3The inhibition to EMT6cells in vitroWith PBS as a negative control, take a series of different concentrations of DOX groups, O-mMWNTs-PEG groups and DOX-O-mMWNTs-PEG groups as experimental groups.The results of MTT show that even in larger O-mMWNTs-PEG concentration, the toxicity to breast cancer cells EMT6is still small. The inhibition of DOX on breast cancer cells is in a dose effect dependent rate.DOX group and DOX-O-mMWNTs-PEG group containing the same rate of DOX have no obvious difference in the inhibition to EMT-6cells. All these suggests that during the DOX-O-mMWNTs-PEG preparation process, the durg activity of DOX was not affected, so the main component which has inhibitory effect on the EMT-6cell is DOX in vitro.4.4The identification of Animal ModelKill three tumor-bearing mice, and then carry pathological examination on tumor tissue and metastatic lymph tissue, progressed with HEstaining.Under the light microscope, tumor cells could be seen in various size and shape, and hyperchromatic nuclei, heteromorphism, nest shape, obviously glandular messy, and accompanied by necrosis.The biopsy of metastatic lymph node showed that cancer cells firstly entermarginal sinusas to form the scatteredmetastases, then tumor cells pileinto apparent metastases.4.5Observation of drug efficacy After O-mMWNTs-PEG carrying DOX. in the magnetic field, the anti effect of lymph node metastasis and tumor growth was better compared with no magnetic field.The efficacy of high-dose DOX group is obviously better than the low-dose group.With the role of O-mMWNTs-PEG, the inhibition effect to lymphatic metastasis and tumor growth in mice was significantly enhanced, while mMWNT group compared with the control group, it did not show any significant anti effect on tumor lymphatic metastasis and anti effect on tumor.No matter mMWNT carrying or not carrying DOX, after administration, the skin of injected site though still has black dye, but no redness, irritation or ulcers, but also have no effect on the health of the mice.And kinds of the blood and biochemical indicators are located normal range, while DOX-only group had leukopenia, transaminase rise, and the rest showed no significant difference compared with other groups.5. ConclusionsThe experiment took chemical wayto combine nano Fe3O4and Oxidized-MWNT-PEG to form a magneticoxidized MWCNTs (O-mMWNT-PEG), for the preparation of magnetic particles carrying the drug. Then make the magnetic particles adsorb Doxorubicin (Dox) and then make it into DOX-O-mMWNT-PEG complex.Magnetic targeted drug delivery system(MTDS) is to combine the drug with magnetically active ingredient, in a sufficiently strong external magnetic field, the carrier will be positioned in the target region, which position the containing drugto focus on the lesion to be effective. At home and abroad the researchers study MTDS as a model drug targeting system for the treatment of malignant tumor.Compared to conventional chemotherapy, the drug combined with external magnetic field enhanced selectivity for tumor, systemic adverse reactions are small, as well as good efficacy. Recent studies have demonstrated that the magnetic field in vitro is in inhibition of tumor itself. O-mMWNT-PEG is a powerful weapon in magnetically-targeted drug delivery system, and it has a lymphatic tracer which can be used to guide the surgical operation.This experiment in vitro can clearly demonstratemagnetic nanotubes drug-delivery system can effectively inhibit the proliferation of breast cancer EMT6cells, which play a major component of tumor suppression is DOX, which explains the the magnetic nanotubes play a facilitating role in inhibitory effect; Vivo experiments indicates that the magnetic MWCNTs can effectively gather in lymph nodes and tumors in vivo, and release the drug for a long time, thus inhibiting the growth and metastasis of breast cancer lymph nodes.O-mMWNT-PEG is a new types of targeted drug carriers, but also has the advantages of lymphatic trends and magnetic targeted drug delivery, and it is a powerful weapon in magnetic targeting drug delivery system.In addition to the guidance in surgery operation, this carrier can not only raise the effect of anti-tumor in chemotherapy and the effect of anti-lymphatic metastasis in targeted therapy, but also reduce the toxicity of the drug. As such advantages, the application prospect of such a carrier is very broad.