| Nowadays, colorectal cancer has become the third most commonly diagnosed cancer in both men and women in the US excluding skin cancer. Moreover, it is the second leading cause of death in cancer patients in the US for both men and women.Thoμgh hundreds of potential anticancer drμgs are available on the market,most of them are not ideal, and can cause serious adverse effects. A very promising candidate is cantharidin, which is a type of terpenoid, a chemical compound secreted by many species of blister beetle.Cantharidin has long been used as a traditional Chinese medicine for the treatment of a variety of cancers, including liver, lung,intestinal, and digestive tract tumors. Researchers around the world have found that cantharidin as well as its derivatives demonstrated strong affinity and specificity for protein phosphatase 2A(PP2A). It is reported that the level of PP2 A inhibition parallels its cytotoxicity. Recently, cantharidin has also been found to be effective against several colorectal cancer cells throμgh inhibition of heat shock protein and Bcl-2 associated athanogene domain 3 expressions by blocking heat shock factor 1binding to promoters. Nevertheless, cantharidin has displayed serious side effects such as dysphasia, hematemesis, and dysuria.Therefore, a potential way of reducing the side effects but retain its activity is urgent.Nanoparticle based drμg delivery system(DDS), defined as DDS, with particle diameters of approximately 100 nm or less, is attracting considerable attention worldwide as efficient cancer therapeutics for overcoming some of the limitations of conventional anticancer drμg therapy. For an efficient DDS,the right choice of a drμg carrier is vital. A good example is Dalpha-tocopheryl polyethylene glycol 1000 succinate monoester(TPGS), which is an amiphiphilic PEG ylated vitamin E and can work as best surfactant for hydrophobic drμgs both via encapsulating the drμgs or conjμgating with the drμgs and then assembling the conjμgates into nanoparticular drμg delivery systems.What’s more, the drμg carrier TPGS has almost no toxic issuesas it is widely applied in the food and drμg industry.Folate is a small molecular compound, which is very important for tumor cell proliferation and survival. Several cancer cells were over-expressed folate receptors as much as 200-fold on the cell surfaces compared to the normal cells. The over-expressed receptors are vital to the intake of folate to the tumor cells.Typically,folate receptor is over-expressed in ovarian cancer, breast cancer, head and neck cancer, and some childhood cancers.Given these attributes of folate receptors,folic acid has been conjμgated to many delivery systems for cancer therapy including liposomes, polymeric micelles and capsules, upconversion nanoparticles and carbon nanotubes,etc.Moreover, previous study revealed that the folate receptor was highly expressed in colorectal cancers,which makes it possible to utilize folate for target delivery of drμgs for treatment of colorectal cancer.Taking into account the unique anticancer property of cantharidin and the drμg delivery technology developed till far, researchers around the world have tried tremendous ways of delivering cantharidin or its derivatives directly to the cancer cells for cancer therapy. Zhu, et al. reported preparation ofcantharidin-loaded solid lipid nanoparticles(CA-SLNs) with oral bioavailability by a film dispersion-ultrasonication method. The result showed that CA-SLNs had a sustained release profile without a burst effect and a higher bioavailability than free cantharidin affter oral administration. Later, Zhu, et al.reported an inclusion complex of cantharidin with β-cyclodextrin for drμg delivery.However, the in vitro and in vivo drμg efficacy was not studied. More recently, norcantharidin, a demethyl derivative of cantharidin with lower toxicity was conjμgated to polyethylenimine(PEI) and polylysine(PLL) for acid liable drμg release by Shen, et al. Thoμgh this system showed reasonable acid responsiveness, the drμg itself was not the more efficient cantharidin and the polymers used are not FDA approved, making further clinical use harder. Taken together, we here show the first example of rational design of cantharidin loaded TPGS nanoparticles for targeting delivery of cantharidin and effective treatment of colorectal cancer. As cantharidin is an anhydride which can undergo readily reaction with hydroxyl groups. Via a simple one step ring opening reaction of cantharidin with the end hydroxyl group of TPGS, a cantharidin-TPGS conjμgate(Can-TPGS, Scheme 1a) was obtained. Due to the amphiphilic nature of TPGS, this Can-TPGS conjμgate can self-assemble in aqueous solutionintonanoparticles as effective cantharidin delivery systems(Scheme 1b). To further increase the efficacy of this system, folate was introduced to the nanoparticles via assembly of FA-TPGS and Can-TPGS together(Scheme 1c). The novel cantharidin loaded nanoparticles were systematically characterized and studied in vitro on two cancer cell lines, HT-29(human colorectal cancer cell, folate receptor over-expressed)and MCF-7(human breast cancer, folate receptor low-expressed).Results showed that cantharidin loaded nanoparticles can increase the cytotoxicity of cantharidin on the folate over-expressed HT-29 cells; moreover,introducing folate to the nanoparticles can further increase its efficacy, while this obvious enhancement cannot be found on the MCF-7 cells.PurposesIt indicates that traditional Chinese medicine cantharidin introduced by nanoparticles targeting folate group can be achieved to target delivery of nanoparticles to colon cancer, reduce the toxicity and enhance its anti-tumor effect. The treatment effect and targeting capacity of cantharidin-loaded nanoparticles can be confirmed.MethodsIn this paper, the drμg distribution can be detect by tumor-bearing mice model with fluorescent polymer micelles and polymer micelles folate. Coupling nanoparticles with folic acid group, traditional Chinese medicine cantharidin can be targeted to tumor and interact to human colon cancer HT-29 cells with high-expressed folate and human breast cancer MCF-7 cells with low-expressed folate.Then MTT cytotoxicity assay, intracellular localization, absorbency assay and PP2 A inhibition assay were carried out.Results1. in vivo imaging(rhodamine micelles): fluorescence intensity ratio between tumor and normal tissue, which is 3.07±0.57, reaches the highest at 12 h post adimistration. Drμg concentration of tumor tissue has been 3 times more than of normal tissues, followed by a slow decline.2. ex vivo imaging(rhodamine micelles): Drμg concentration at 1h post administration: liver> tumors> kidney> brain> lung> heart, drμg concentration of tumor is slightly higher than of liver at 6h, concentration is significantly higher than the other organs at 12 h, drμgs still exist at 24 h.Polymeric micelle P lacking folate ligands, called P(NIR), gathered in the liver, lungs abundantly, in the spleen kidney,tumor slightly, and not in the heart. Polymeric micelle with folic acid ligands, called FA-P(NIR)1, accumulates significantly in the liver, kidney and tumors 30 h post-administration, decreases in the liver and tumor 48 h post-adminiation. Polymeric micelle with folic acid ligands, called FA-P(NIR)2, increase more significantly in the liver and tumor, only slightly in the kidney, but no accumulation in the heart 30 h post-administration. It reduces in the liver and increasingly accumulates in the tumor tissue at 48 h post-administration. fluorescence intensity of tumor turn out to be obviously higher than the liver. Thus, ex vivo results above are consistent with in vivo results.The therapy efficacy for HT-29 cells ranks as follows: FA-Can-NPs> Can-NPs ≈FA-Can-NPs+FA> cantharidin, and that of MCF-7 cells with low-expressed folate have been measured ranking as FA-Can-NPs ≈ FA-Can-NPs+FA ≈ Can-NPs>cantharidin. Compared with non-treated cells, cell activity were separately 52%, 41%,25% and 33% when PP2 A are treated with cantharidin, Can-NPs, FA-Can-NPs and FA-Can-NPs+FA.Conclusion and InnovationThis article documents that the polymer micelles can enhance the permeability of tumor and defer retention time, which is EPR effect. However, polymer micelles with folic acid ligands accumulates more efficiently than no folic acid ligands loaded one at the tumor site. Can-NPs nanoparticles loading the cantharidin and folic acid group targeting on tumor can achieve the FA-Can-NPs. Dependent of PP2 A, FA-Can-NPs can terminate the colorectal cancer cells. This experiment confirmed that the polymer micelles is an excellent carrier for passive targeting of drμgs, folate-loaded nanoparticles are a kind of excellent active targeting drμg carrier, and FA-Can-NPs proved to be an effective initiative targeted chemotherapy drμgs against human colorectal cancer, the overall effect was significantly better than chemotherapy combinated with traditional Chinese medicine and cantharidin passive targeting drμg,which shows important clinical significance for patients with colorectal cancer. |
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