The Study Of Norcantharidin-loaded Poly (Ethylene Glycol)-poly(Caprolactone) Block Copolymeric Micelles

Background:Poly (ethylene glycol)-poly (caprolactone)(PEG-PCL), as one of the amphiphilic block copolymers materials, composed of hydrophilic chain (PEG) and hydrophobic chain (PCL), has attracted much attention owing to its biocompatibility and degradability. In aqueous media, PEG-PCL self-assembles into copolymeric micelles which contain hydrophobic core and hydrophilic shell. In recent years, there has been a growing interest in polymeric micelles as a potential carrier for anticancer drug delivery. The internal hydrophobic core provides a storeroom for loading hydrophobic drug and the hydrophilic shell allows the stability of copolymeric micelle in aqueous environment. Just because of the special stucture of micelles, polymeric micelles have been considered important for enhancing drug stability in vitro and in vivo, increasing drug solubility and improving transport properties of pharmaceutical molecules. Copolymeric micelles also demonstrate other attractive properties as drug delivery system, such as having good biocompatibility, increasing drug bioavailability, minimizing undesirable side-effects and targeting the tumor areas that is because copolymeric micelles with small size are able to spontaneously accumulate in pathological areas due to the damaged ("leaky") vasculature of these areas via enhanced permeability and retention (EPR) effect.In this paper, PEG-PCL was used to encapsulate anticancer drug-norcantharidin (NCTD). NCTD, a demethylation derivative of cantharidin which is an active constituent obtained from the dried body of the Chinese blister beetle (mylabris), is one of the new chemotherapy agents that has exhibited a very effective activity against cancer and a wide tumor spectrum, including primary hepatic carcinoma, colorectal cancer, oral cancer, lung cancer, ovarian cancer, breast cancer and so on. The anti-cancer mechanisms of NCTD have been illuminated by the evidence showing its anti-proliferation, pro-apoptotic and anti-migratory effects on the tumor cells. However, the clinical application of NCTD has been limited because of its poor water-solubility and irritation to urinary organs.Drug solubility is an important factor affecting the release and absorption of the drug. The solubility of NCTD is not satisfactory. The maximum solubility of NCTD in water is2.5mg/mL at pH6.0and this increases to9.5mg/mL at pH9.5. Clinically, NCTD is mostly given by injection of its sodium salt at a pH of about9.0. The high pH is an important cause of this irritation following intravenous injection. Although the toxicity of NCTD has been reduced after demethylation, the irritation to urinary organs still exists. Once increase dose or extend treatment time, adverse action produces because drug widely distributes in various organizations. This is another reason to limit clinical application. So developing of new drug delivery systems aimed to enhance solubility, decrease side-effects and improve treatment efficacy of NCTD should be placed in the first.The aim of this study was to investigate the possibility of producing NCTD-loaded PEG-PCL diblock copolymer micelles and optimize the preparation process. We intented to confirm the forming of micelles by transmission electron microscop (TEM) and verify NCTD loaded in hydrophobic core factually via infrared analysis (IR). The critical micellization concentration (CMC) of PEG-PCL was detected to elucidate anti-dilution of micelles. The stability of lyophilized sample and NCTD were compared. In addition, in vitro drug release properties, and anti-cancer effect of copolymeric micelles both in vitro and in vivo were explored to illustrate its potential clinical application value.Chapter1. Preparation of norcantharidin-loaded poly (ethylene glycol)-poly (caprolactone) diblock copolymeric micelles and optimizion of the preparation processDialysis method and volatilization dialysis method were adopted to encapsulate NCTD. The quantification of NCTD entrapped in PEG-PCL micelles was measured by HPLC. Factorial design was used to optimize the preparation process and the orerall desirability(OD) of drug loading content, encapsulation efficiency and particle size was as the index. The results of single factor showed that the three factors (preparative method, the type of organic solvent and feed weight ratio of NCTD to polymer) had notable influence on OD values. So we used the three factors as investigated factors.The results of factorial design showed the optimal micelles with size of (95.6±10.1)nm, drug loading content of (6.0±0.3)%and encapsulation efficiency of (79.1±0.8)%, emerged from volatilization dialysis, tetrahydrofuran and feed weight ratio of0.0625:1.Chapter2. characterizationMeasurement of critical micellization concentration(CMC):The most common method to determine CMC of polymer is using pyrene fluorescence probe. A known amount of pyrene in acetone was added to a series of blank micelles with different concentrations. At emission wavelength of393nm, fluorescence excitation spectra of all samples were measured at excitation wavelength of335nm and338nm, respectively. The graph with ratio of pyrene fluorescence intensity in338nm and335nm vs. logarithm of the copolymer concentrations was drew and the intersection point of these two segments gave CMC values that was0.3×10-6mol·L-1. CMC values in a10-6range was very important for keeping the stability of micelles in vitro and keeping their intact structure upon dilution with body liquid in vivo.Size determination:The size and distribution of blank micelles and drug-loaded micelles were measured by dynamic light scattering(DLS). The mean diameters of blank micelles and drug-loaded micelles were (126.55±0.28)nm and (87.12±0.17)nm, respectively. We found that micelles with drug had a smaller size than micelles without drug under the same preparation conditions. Once NCTD got into the hydrophobic core of micelles, it was covalently combined to polycaprolactone under the role of Van der Waals force or electrostatic attraction, so that the space of micelles core was smaller than blank micelles.Observation of micelles morphology:Micelles shape and size were observed under TEM. The samples should be dripped on a copper grid coated with film and then negatively stained with2%phosphotungstic acid before observation. There were not significant difference between drug-loaded micelles and drug-free micelles except the particle size. TEM microphotograph showed the spherical shape and shell-core structure of micelles. The inner hydrophobic core with light color was clearly distinguished from the outer hydrophilic dark ring that had absorbed phosphotungstic acid. The results from TEM revealed that core-shell structure had been formed completely during the micelles fabrication.Infrared spectrum (IR) analysis:After the samples were dried thoroughly, KBr should be added to press into thin slices. IR was utilized to determine the structures of blank micelles, NCTD-loaded micelles and mixture of NCTD and PEG-PCL. NCTD-loaded micelles had different bands and signals compared with mixture but the same as the blank micelles. Characteristic absorptions of NCTD-loaded micelles and blank micelles appertained to the IR spectrum characteristic of PEG-PCL. The mixture of NCTD and PEG-PCL had absorptions of NCTD and PEG-PCL. According to IR, the conclusions could be clearly got:IR spectra certified the free drug being not loaded in the micelles had been removed completely. It was confirmed that IR was an excellent method to control the purity of copolymeric micelles.Chapter3. Stability testThe choice of lyoprotectant:Galactose, mannitol and trehalose were used to be lyoprotectants in order to test the effect of lyoprotectant on the morphology, size and distribution of micelles. The conclusion has been got that the lyophilized sample had the best morphology and the minimal size when trehalose was used as lyoprotectant.Stability:The conservation rates of NCTD and lyophilized samples would be checked after placed on40℃,60℃and4500Lx light irradiation at1,3,5,10d. According to collection points, the rates of weight change of NCTD and lyophilized samples were checked after placed in the humid of75%and92.5%, respectively. Result:1. After heated10d at40℃, the conservation rate of lyophilized sample was higher than NCTD.2. Lyophilized sample began to melt at60℃, so it is necessary to avoid high temperature for lyophilized sample.3.There were not significant difference for conservation rate of NCTD and lyophilized sample after10d light irradiation.4NCTD is unstability of wet, but when it was entrapmented in micelles, its stability increased.The stability of lyophilized samples at room temperature:Lyophilized samples were placed hermetically at room temperature. Size and encapsulation efficiency were determined on the first, second, third month. After three months, size was not bigger and encapsulation efficiency was not decreased significantly. Chapter4. In vitro drug release properties and cytotoxic activityIn vitro drug release properties:A certain micelles solution in a dialysis bag was put into a tube containing fixed amount of phosphate buffer solution (pH6.5,7.0,7.4) with1%w/v sodium lauryl sulfate (SDS). The tube was placed in an orbital shaker at the rate of100r/min with water bath of37℃. At scheduled time intervals, original PBS was taken out and replaced by fresh PBS solution. After that, tube was put back to the shaker for continuous measurement. The assay of NCTD was also by HPLC. After72h oscillation, the amounts of NCTD released in the buffers of pH6.5,7.0and7.4were (83.4±2.5)%,(80.0±1.6)%and (72.0±1.5)%, respectively. That was to say, more drug was released in tumor cells in which pH (pH=6.5to7.0) was less than that in normal tissue (pH=7.4).Cytotoxic activity:cytotoxic activity was determined using MTT [3-(4,5-dimethyl)-2,5-diphenyl-2H-tetrazolium bromide]) assay. Human hepatoma (HepG2) cell line, human lung cancer (A549) cell line,varian cancer (A2780) cell line and normal liver cell line(LO2)were chosen as target cells to evaluate the cytotoxic activity of NCTD and drug-loaded micelles. The graph with cell viability vs.drug concentrations was drew. Cancer cell growth inhibitory effects depended on the change of drug concentrations and incubation time. Cancer cells were more sensitive to copolymer micelles than NCTD. Compared with NCTD, the enhanced inhibition to A549and A2780of micelles was strongger than to HepG2. IC50from SPSS could also illustrate the above conclusions.The inhibition effect of micelles to LO2was also trongger than NCTD.Chapter5. In vivo antineoplastic activityS180mouse sarcoma cells suspension were injected subcutaneously into the right hindlimb of30Kunming mice. After inoculation, the mice were assigned at random to different experimental groups, including control group (physiological saline) and medication administration groups (NCTD, NCTD-loaded micelles with low dose, NCTD-loaded micelles with moderate dose and NCTD-loaded micelles with high dose).24h later, mice were given an tail intravenous injection of various formulations for continous8days, and tumor size was measured daily with a vernier caliper. The ninth day, the subcutaneous tumor blocks were stripped and weighed after killing the mice and the inhibition rate on tumor weight (IRw) was tested. Tumor inhibition effects of NCTD-loaded micelles were better than that of NCTD (P <0.05). The inhibition rate of NCTD-loaded micelles with high dose was up to77.63%. We concluded that the dilevery of NCTD in micelles could improve tumor-inhibitory activity in vivo because polymeric micelles made NCTD accumulate in the tumor tissue though passive targeting strategies and EPR effect. The finding on the basis of the above results could have important clinical application value.