| Background and ObjectionAccording to the World Health Organization (WHO) statistics, every year around the world at least 1000 million people suffering from cancer, each year about 600 million people died of cancer, mortality rates are among the highest in the second, after cardiovascular system diseases, accounted for 12% of deaths worldwide. In recent years, the incidence of cancer has been increasing rapidly with the deterioration of environment and the change of people’s life style. Expected in 2020 the world’s annual new cases of cancer will reach more than 20 million, the death of 12 million. In China, there are nearly 3 million new cases of cancer each year, and nearly 2 million of the deaths. Statistics show that the main cause of death of urban and rural residents in 2008 released by the Ministry of health, the city has become the first cause of death in China, the second cause of death in rural areas. According to the National Cancer Institute, malignant melanoma is one of the most common cancers, with more than 65000 new cases in the United States each year, and 8650 deaths. The incidence of white was higher than that of other skin color. Queensland, Australia and the United States of Southern Arizona for regions with a high incidence of malignant melanoma, the morbidity rate is forty per 10 million, thirty per 10 million. Asia incidence rate is low, the morbidity rate of male is 1.7%, female 1.3%.90% of Malignant melanoma occurs in the skin, cutaneous malignant melanoma is a malignant tumor originating from neural crest melanoma, which is developed by the development of pigmented moles or stains. The prognosis of the disease is poor, the mortality rate as high as 75%, second only to lung cancer, is the cause of death in most skin cancer patients. In the past twenty years, the morbidity and mortality of malignant melanoma have increased at a faster rate compared with other malignant tumors.The treatment of cancer is difficult for clinicians and cancer research experts, treatment methods include surgery, chemotherapy, radiation therapy, gene therapy, adjuvant therapy, and so on. In the chemical treatment of cancer, the drugs enter the body, mainly through the blood circulation in the human body and diffuse distribution, only small part of the drug really accumulate to the site of disease and plays an important role in the efficacy, the majority are excreted as metabolites, biological utilization rate is low, at the same time normal body organs and cells suffered great damage, these shortcomings greatly affect the treatment effects. In general, a chemotherapeutic drug is more effective, it is more toxic, which brings serious side effects and even lead to chemotherapy failure. It is commonly believed that many problems in chemotherapy and the failure of chemotherapy are mainly caused by the pharmacokinetic characteristics, drug forms, toxicity and drug resistance of cancer cells.Drug loaded nanoparticles preparation from biodegradable polymer copolymer attracts more and more attention because that they can not only solve the problem of medicine, but also solve the other cytotoxic chemotherapy, cancer cell drug resistance and pharmacokinetic dynamics. Nanoparticle is the particle size of 1-1000nm particles with small volume and relatively large surface area, and has a unique advantage in the field of drug delivery. Nanoparticles matrix used in chemotherapy is generally a natural or synthetic polymer with good biocompatibility, biodegradability, and Approved by the food and Drug Administration of the United States. After enter the body The anticancer drug which is dispersed in the polymer matrix or attached to the polymer is released through permeation and diffusion with the polymer eroding and degradating in the body. While the drug release and play a therapeutic effect, as a drug carrier matrix polymer is decomposed into small molecules which is harmless to human body so as to avoid the possible serious adverse reactions caused by adjuvant. After appropriate modification the biodegradable polymers can make chemotherapy drugs to achieve targeted continuous control in a number of weeks or even months. Modified polymer also can make drug accurately act on cancer cells without harming the surrounding normal cells, avoiding the common side effects of chemotherapy in great extent. In addition, because of its small size, nanoparticles is easy to pass through various physiological barrier without restriction, will not lead to thrombosis. Altogether, nanoparticles has the advantages of small volume, Easy absorption, reducing uptake of the mononuclear phagocyte system, improving blood drug concentration, reducing the side effects caused by drugs or adjuvant, controlled drug release, so as to improve the efficacy of drugs.The treatment of malignant melanoma includes surgery, chemotherapy, immunotherapy and adjuvant therapy, and so on. Surgery is the firsrt choice of treatment for cases with indications of surgery. Rapid progression of malignant melanoma clinically, many patients were found late, or rapid progressing after irregular treatment, lost the chance of operation, then chemotherapy has become the important means of treatment. Chemotherapeutic drugs sensitive to malignant melanoma are Daba C, temozolomide, paclitaxel, platinum, Changchun methylcytisine, Fumo Stine etc.. Paclitaxel is a natural antitumor drug originating plant, has significant therapeutic effect on a variety of cancers.1992 FDA of the United States approved paclitaxel can be used in the treatment of ovarian cancer, then paclitaxel is used in the treatment of a variety of tumors. Listed in just a few years, the whole world sales volume of paclitaxel formulations have exceeded $1 billion, to obtain a single anti-cancer drug sales champion. This kind of medicine is the most popular and most promising new type of anticancer drug in the past twenty years.Paclitaxel is derived from Taxus chinensis. Taxus is ancient Quaternary glacial relict world of rare and endangered plants. It has 250 million years of history in earth which is very rare and has a reputation of "Shenshu" in China. Yew can only survive in the 2500-3000 meters above sea level in the mountain forests, timber need 50 to 250 years. Due to its special growth conditions,slow growth in nature and poor ability to regenerate, any country has’t formed a large scale of the raw material base all over the world for a long time. China has classified yew as the first class national protective keystone plant in the rare dangerous plants. The United Nations has long been explicitly prohibit the harvesting. The scarcity of resources has led to high price of the paclitaxel anti-cancer drugs. Very low water solubility and biological utilization also lead to increased demand. Because of the shortage of resources which is difficult to overcome researchers focused on the development of new dosage forms, improving the bioavailability of drugs. Because advantages of the small volume, the large surface area, easily absorbed degradable polymers loaded drug nanoparticles gradually become the new darling of the drug delivery system.Poly(e-caprolactone) (PCL), as a biodegradable copolymers, is noncytotoxicity, thermal properties, and biocom-patibility and its degradation is lower than PGA, PLGA, PLA as its molecular contains longer methylene. Moreover, PCL is suitable for the long-term efficacy system, because it needs about 1 year to be absorbed and metabolized completely, and the molecular is larger, the time is longer. Poly(ethylene glycol, PEG), is Polyether polymer materials and very useful in lots of fields. PEG has good water-solubility because of its plenty of ethoxy, which can form hydrogen bond with H2O. Furthermore, PEG is one of the most commonly used water soluble carrier, increase the bioavailability of drugs with promote the solubility rate of them without the side-effects of carcinogenic, teratogenic and gene mutation. MPEG is the molecular of PEG containing methoxy. MPEG, as an initiator caprolactone monomers are polymerized to form an amphiphilic block copolymer MPEG-b-PCL, can promote the degradation rate of polycaprolactone and the formed drug-loaded nanoparticles captured by the reticu-loendothelial system (RES) when they are injected into the blood stream. Therefore, this kind of nano-drugs can play cycle long-term efficacy and increase the bioavailability to make the drug-loaded nanoparticles be changed into a wide rang of applications. TEGS, being the derivative of vitamin-E and the excellent emulsifier, has amphiphilic to prepare a uniform dispersion of nano particle drugs. TEGS can also inhibit P-glycoprotein (P-gP) to overcome the tolerance and inhibit the proliferation of tumor cell. All in all, TEPS was used to prepare the emulsifier of nano particle drugs.Although PEG has many advantages such as biocompatibility, antigenicity, and lack of toxicity, as well as the fact that PEGylation increases circulation longevity, it has been reported that PEGylated NPs do not completely avoid cumulative uptake by MPS cells, and they cannot mostly eliminate complement system activation for their corresponding naked NPs. It was reported that PEG showed a rather high urinary excretion and peripheral distribution volume. Besides, the molecular structure of PEG as a general polymeric modifier does also not readily allow the addition of new functions along its chain. To surmount these challenges, we use a simple and versatile surface modification method based on polydopamine (PDA).PDA, a mimic of the specialized adhesive foot protein Mefp-5 (mytilus edulis foot protein-5) secreted by mussels, can modify almost all the drug-loaded polymeric NPs. PDA coating displays excellent biocompatibility and low cytotox-icity, making it a versatile platform for applications, including biomolecule immobilization, molecular surface imprinting for protein recognition, cells adhesion, and encapsulation. Furthermore, PDA is also a major pigment of naturally occur-ring eumelanin. PDA displays a lot of striking properties of naturally occurring eumelanin in terms of electrical, optical, and magnetic properties. Another valuable advantage of PDA lies in its chemical structure that possesses many functional groups such as amine, catechol, and imine. These functional groups could serve as the starting points for covalent modification with the targeting ligand for drug delivery. Due to these benefits, PDA has been rapidly changed into a wide range of applications across the biological, chemical, and nanomedicine fields. MethodsIn this study, to enhance the therapeutic efficacy and minimize the side effects of malignant melanoma treatment, we prepared novel PTX-loaded methoxy poly(ethylene glycol)-b-poly(ε-caprolactone) NPs (PTX-loaded MPEG-b-PCL NPs) and used surface modification with PDA as drug carrier. MPEG-b-PCL was obtained by ring-opening polymerization, and characterized by proton nuclear magnetic resonance (H-NMR) and gel permeation chromatography.PTX-loaded nano particles was prepared by advanced nano-precipitation technique. The PTX-loaded nano particles and PDA modified PTX-loaded nano particles were characterized by particle size and particle size distribution, zeta potential, surface morphology, drug loading and encapsulation efficiency. The in vitro drug release, cellar uptake, in vitro cell viability, in vitro antitumor efficacy and side effect were researched in order to illustrate whether this nano particles can be used in clinical.ResultsThe results are as follows:1) The in vitro drug release results suggested that the chemotherapeutic drug PTX of PDA-modified NPs was almost totally preserved during the preparation procedure. The NPs showed steady, continuous release patterns, with ini-tial burst releases. The drug release from PTX-loaded NPs@PDA was found to be 53.6% and 88.2% of the encapsulated drug in the first 5 days and after 30 days, respectively.2) The cellular uptake results implied that the C6-loaded NPs@PDA had been internalized into the A875 cells. Furthermore, the fluorescence of C6-loaded NPs@PDA in the cytoplasm was much brighter than that of C6-loaded NPs.3) In vitro cell viability and cytotoxic results displayed four conclusions. ①the cytotoxicity of the three PTX formulations increased with increasing drug dose and incubation time. ② the cytotox-icity of the three formulations is very similar in the first 12 hours of incubation. However, after 24 hours and 48 hours of incuba-tion, the PTX-loaded NPs@PDA showed significantly higher cytotoxicity than Taxol(?). ③the surface-modified PTX-loaded NPs@PDA are most destructive to A875 cells. ④ the synthesized copolymer MPEG-b-PCL and PDA seemed to be nontoxic in cell culture because no significant cytotoxicity was found for the drug-free NPs at various concentrations. After 12 hours of incubation at a drug concentration of 2.5 μg/mL, the cell viability was 85.61% for Taxol(?),86.32% for PTX-loaded NPs, and 83.45% for PTX-loaded NPs@PDA. However, after 24 hours and 48 hours of incubation, the cyto-toxicity of PTX-loaded NPs@PDA against A875 cells was, respectively,27.48% and 39.17% higher than that of Taxol(?). The PTX-loaded NPs@PDA showed better cytotoxicity effi-cacy against A875 cells than PTX-loaded NPs and commercial Taxol(?).4). In vivo antitumor efficacy and side effect analysis demonstrated:PTX-loaded MPEG-b-PCL NPs@PDA was investigated in human melanoma cell line A875 cell-bearing nude mice; the clinical drug Taxol(?) and physiological saline were used for comparison. The tumor size of the saline group increased obviously. However, tumor growths in the groups treated with Taxol(?), PTX-loaded NPs, and PTX-loaded NP s@PDA were significantly inhibited. The melanoma tumor inhibitory effects were in the following order:PTX-loaded NPs@PDA. PTX-loaded NPs> Taxol(?)>saline. The results of the antitumor efficacy experiment demonstrated that PTX-loaded NPs having surfaces modified with PDA could be released from the NPs effectively and that the released PTX main-tained its bioactivity for melanoma treatment. Safety profiles of PTX formulations were evaluated by analyzing the changes in the body weights of animals. The results shown that the body weight loss was only found at the end of Taxol(?) therapy. However, the body weight of the groups injected with PTX-loaded NPs and PTX-loaded NPs@PDA grew steadily. Meanwhile, the mice treated with PTX-loaded NPs@PDA remained healthy and vigorous throughout the therapy period. These observations demonstrated that the side effects in mice injected with PTX-loaded NPs@PDA were fewer than those after administration of commercial Taxol(?).In other words, the PTX-loaded MPEG-b-PCLNPs having surface modification with PDA could retain the pharmacological activity of PTX and they significantly inhibited tumor growth compared to the effect of Taxol(?) at the same dose.ConclusionIn the study, a novel of type of PTX-loaded MPEG-b-PCL NOs modified with PDA, was prepared for malignant melanoma treatment. The PTX-loaded NPs were manufactured by a modified nanoprecipitation method and subsequent surface modification with PDA. The hydrodynamics size of PTX-loaded NPs @PDA is about 140nm and it has smooth surface. The PTX-loaded NPs@PDA and the PTX-loaded NPs@PDA and precursor PTX-loaded NPs had similar drug load LC, Encapsulation efficiency EE and the drug release profiles. The drug-loaded NPs@PDA possessed higher celluar uptake activity and greater cytotoxic compared to the precursor PTX-loaded NPs.The in vitro and in vivo antitumor effects of the PTX-loaded NPs@PDA were obviously more potential than those of PTX-loaded NPs and Taxol(?).In a word, the novel PTX-loaded NPs@PDA displayed excellent high therapeutic effects and low side effects in treatment of malignant melanoma. |
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