In Vitro And In Vivo Evaluation Of The Antitumor Effect Of Drug-loaded Nanoparticles Against Murine Malignant Melanoma

Objectives:Malignant melanoma is a common skin cancer, it can be treated with operation therapy at early period. But it often metaptosis easily, and has little effect for lately or metaptosis tumor with operation therapy. Chemotherapeutics have low effect, and significant side effects . There isn’t any very effect drug. Find new effect and safe durgs is imminent. Docetaxel(DOC) and Curcumine can be used to treat many types cancers. Recently, both of them were used to treat malignant melanoma, some effects have been found. But they have less solubility in water, many severe hypersensitive reaction, short effect time in vivo; toxicity to other organs. So many disadvantages limit their clinic useness. Nanoparticle carrier system possess special targeting character、delayed release character and high-stability、dimish side effects and toxicity. PEG-PCL core shell nanoparticle has bilateral solubility, our study use PEG-PCL to encapsulate DOC and Curcumine, investigate its in vivo and in vitro effects and side effects, study nanoparticle’s size、solubility、Drug loading content (DLC) and encapsulation efficiency (EE)、targeting character、sustained release effect、in vitro cytotoxicity、in vivo antitumor efficacy.Methods:(1) mPEG-PCL block copolymers were synthesized by a ring opening copolymerization. Preparation of docetaxel-loaded nanoparticles(DOCNP). Morphological examination of the nanoparticles was conducted with transmission electron microscope (TEM). Atomic force microscope (AFM) was used to study the surface morphology of nanoparticles in a greater detail. Mean diameter, size distribution and Zeta potential of the nanoparticles were measured by DLS. Doc loading content in nanoparticles was determined by HPLC.(2) In vitro release of Doc from the DOCNP was evaluated using a dialysis bag diffusion technique. The difference in release from dialysis bag for both free and loaded docetaxel was compared, to study nanopartice’s influence on DOC effect time. Nanoparticle uptake by tumor cells were imaged with a fluorescence microscope. Cytotoxicity of Doc-loaded nanoparticles against B16 cells was assessed by MTT assay.(3) ICR mice implanted with B16 cell line were used to qualify the relative efficacy of Doc-loaded nanoparticles through intravenous injection. Tumor volume and mice weight were measured every other day.(4) ICR mice implanted with B16 cell line were used to qualify the relative efficacy of Doc-loaded nanoparticles through intratumoral administration. Tumor volume and mice weight were measured every other day.(5) Preparation of Curcumine -loaded nanoparticles(Curcumine NP), Morphological examination of the nanoparticles was conducted with transmission electron microscope (TEM). Atomic force microscope (AFM) was used to study the surface morphology of nanoparticles in a greater detail. Mean diameter, size distribution and Zeta potential of the nanoparticles were measured by DLS. Doc loading content in nanoparticles was determined by HPLC.(6) In vitro release of Curcumine from the CurcumineNP was evaluated using a dialysis bag diffusion technique. The difference in release from dialysis bag for both free and loaded Curcumine was compared, to study nanopartice’s influence on Curcumine effect time. Nanoparticle uptake by tumor cells were imaged with a fluorescence microscope. Cytotoxicity of Curcumine-loaded nanoparticles against B16 cells was assessed by MTT assay.(7) ICR mice implanted with B16 cell line were used to qualify the relative efficacy of Curcumine -loaded nanoparticles through intratumoral administration. Tumor volume and mice weight were measured every other day.Results(1) DOCNP has a solubility in NS up to 20 mg/ml, which is much better than that of free Doc. The solution of Doc-loaded nanoparticles is bluish in color. The morphology images of the DOCNP obtained from TEM and AFM observation indicate that the nanoparticles, estimated less than about 100nm in size, are in spherical shape with a smooth surface. Meanwhile, LDA measurement of DOCNP showed that the zeta potentials exhibit a negative value (-5mv), which is much lower than polyester nanoparticles without the PEG coating surface (?35 mV). Resulting in a reduction of cell repulsion to the nanoparticles. The highest drug loading content of Doc in mPEG-PCL nanoparticles was 19.4±2.4%. The encapsulation efficiency is more than 90%.(2) It is obvious that free docetaxel released much faster than DOCNP. The difference in release from dialysis bag for both free and loaded docetaxel is evidently attributed to the prolonged release function of the nanoparticle. The observed coumarin-6 fluorescence was mainly located inside the cells, which indicates that coumarin-6 was mainly transported into the cells and localized around the nuclei in the cytoplasms, conclude that Doc, encapsulated into the nanoparticles together with coumarin-6, was also transferred into the cells by the nanoparticles as well. DOCNP could induce slightly more cell death of B-16 than that of free Doc at most corresponding incubation times, implying that DOCNP show higher or, at least, the same cytotoxicity against B-16 cell with free Doc at equivalent doses.(3) DOCNP showed significant antitumor effect in B16 xenografts against equivalent doses of free DOC through intravenous injection. Mean bodyweights of mice treated with free DOC were obviously lower than mice treated with DOCNP.(4) All the tumor volumes treated with different doses of DOCNP were smaller than that treated with the same dose of free Doc (p<0.05) by intratumoral delivery. The extent of weight loss was much smaller than those induced by free Doc.(5) Curcumine NP has a solubility in NS up to 15mg/ml, which is much better than that of free Curcumine. The solution of Curcumine -loaded nanoparticles is bluish in color. The morphology images of the Curcumine NP obtained from TEM and AFM observation indicate that the nanoparticles, estimated less than about 100nm in size, are in spherical shape with a smooth surface. Meanwhile, LDA measurement of Curcumine NP showed that the zeta potentials exhibit a negative value (-7.6mv). Resulting in a reduction of cell repulsion to the nanoparticles. The highest drug loading content of Curcumine in mPEG-PCL nanoparticles was 16.3±1.9%. The encapsulation efficiency is more than 89%.(6) It is obvious that free Curcumine released much faster than Curcumine NP. The difference in release from dialysis bag for both free and loaded Curcumine is evidently attributed to the prolonged release function of the nanoparticle. Curcumine NP could induce more cell death of B-16 than that of free Doc at most corresponding incubation times, implying that Curcumine NP show higher cytotoxicity against B-16 cell than free Doc at equivalent doses.(7) All the tumor volumes treated with different doses of Curcumine NP were smaller than that treated with the same dose of free Curcumine (p<0.05) by intratumoral delivery. the extent of weight loss was much smaller than those induced by free Curcumine.Conclusions:(1) DOCNP strengthens the stability of the drug delivery system.Doc in DOCNP can be released slowly and kept at a constant concentration in long period both in vitro and in vivo. It has targeting effect. DOCNP show higher or, at least, the same cytotoxicity against B-16 cell with free Doc at equivalent doses. DOCNP showed stronger anti-melanoma effects than free DOC by intravenous delivery with no obivous bodyweight loss. DOCNP intratumoral administration can more effectively inhibit tumor growth, but the extent of weight loss in DOCNP group was much smaller than those induced by free Doc.(2) Curcumine NP strengthens the stability of the drug delivery system. Curcumine in Curcumine NP can be released slowly and kept at a constant concentration in long period both in vitro and in vivo. It has targeting effect. Curcumine NP show higher cytotoxicity against B-16 cell than free Curcumine at equivalent doses. Curcumine NP intratumoral administration can more effectively inhibit tumor growth,the extent of weight loss in Curcumine NP group was much smaller than those induced by free Curcumine.