Antitumor Paclitaxel/Tetra Carboxyl Zinc Phthalocyanine-Loaded PH-Sensitive Polymeric Micelles

Photodynamic therapy(PDT) indicates that a photosensitizer is irradiated by a certain beam to achieve its active state that transports enegy to surrounding oxygens after attenuation. Reactive oxygen species(ROS) are then produced and damage surrounding biomacromolecules, leading to cell death. PDT is mainly used for therapy of diseases on the body surface, such as psoriasis, yellow spot disease, and bladder cancer.The second generation of photosensitizers mainly includes phthalocyanines that are insoluble in water so that the biomedical applications are limited. In this study, the nanoscale drug delivery systems—polymeric micelles, were used to entrap photosensitizers and chemotherapeutics. They targeted melanoma based on the enhanced penetration and rentention(EPR) effect. Furthermore, the micelles had pH sensitive property,improving drug release to tumor tissues. After a certain laser light was performed, the photosensitizers were irradiated. Therefore, the PDT and chemotherapy were simultaneously performed with the micelles to achieve high antitumor effect.In this study, a p H-sensitive copolymer, mPEG-pDEA-PCL(PDP), and another copolymer mPEG-p DEA(PD), previously synthesized in our lab, were used to prepare polymeric micelles that entrap zinc phthalocyanine(ZnPC4, also previously synthesized in our lab) and an antitumor drug, paclitaxel. The photophysical properties of ZnPC4, photodynamic antimicrobial therapy(PACT), in vivo and in vitro antitumor effect of the PTX/ZnPC4-loaded p H-sensitive polymeric micelles were explored.1. Photophysical properties of ZnPC4The molar coefficient(?) of ZnPC4 was determinated using the spectrophotometric method. The fluorescence quantum yield(?F/ZnPC4) was detected with the fluorescent spectrophotometry. DMA(9,10-dimethyllanthracene), an active oxygen probe, was used to quantify the singlet oxygen quantum yield of ZnPC4. ZnPC4 showed the maximal absorbance at 680 nm with the maximal fluorescence excitation wavelength at 616 nm and the maximal emission wavelength at 686 nm. After triggered by a laser, ZnPC4 had a higher singlet oxygen quantum yield than traditional photosensitizers. ZnPC4 could be a promising photosensitizer with high biological penetration due to high excitation wavelength.2. Photodynamic antimicrobial therapy with ZnPC4In this work, three microbial solutions were co-cultured with ZnPC4 under dark for 45 min. The solutions were lighted with 660 nm lasers once, respectively. The solutions were diluted with PBS followed by spreading on agar plates to invesitigate the in vitro PACT for Staphylococcus aureus, Escherichia coli,and Candida albicans. ZnPC4 showed siginificant PACT for S. aureus with the light dark toxicity. The PACT effect was moderate for Escherichia coli and the PACT for Candida albicans was a little better than the former. This study gave evidence to show ZnPC4 was a good PACT agent.3. Preparation and properties of drug-loaded pH-sensitive polymeric micellesZnPC4, paclitaxel(PTX), and their mixture were used as model drugs, respectively. Drug-loaded PDP/PD polymeric micelles were prepared with the tetrahydrofuran(THF) sonication method. After removing organic solvents, homogeneous micellar suspensions were obtained. According to the transmission electron microscopy(TEM), the micelles were nanoscale. Sizes and zeta potentials of the micelles were measured using the Zetasizer Nano ZS under different pHs(4.5-8.0). The mean sizes and zeta potentials were highly pH dependent, wherein the larger sizes and positive charges were shown under the weak acidic condition and the smaller sizes and negative charges were shown under the neutral and weak basic condition. The unique property favored drug encapsulation and release under the acidic condition of tumors. Entrapment efficiency(EE)and drug loading efficiency(DL) were used to screen the optimal formulation of micelles as follows:(1) PDP:Zn PC4:PTX, 5:1:1(w/w/w) with PTX's EE of 55.60%and DL of 6.05%;(2) PD:ZnPC4:PTX, 5:1:0.5(w/w/w) with PTX's EE of 76.81% and DL of 6.05%. The high EE and DL could satify the in vivo and in vitro investigation.4. Analytical method of PTX and ZnPC4Content of Paclitaxel was analyzed by HPLC, the method validation was performed. Content of ZnPC4 was determined by pectrophotometric method,linear range and degree of precision was investigated meamwhile.5. In vitro pharmacodynamics of drug-loaded pH-sensitive micellesHepG2 and B16-F10 cells was used to investigate photodynamic therapy of drug-loaded p H-sensitive micelles. Cells were co-cultured with free drug(PTX,ZnPC4), drug-loaded or blank micelles for 4h, then thesolutions were lighted with 660 nm laser for 60s(9J), respectively. MTT and CCK8 was used to detect cell viability. PDP and PD blank polymeric micelles show low toxicity to both cells. Free PTX and free ZnPC4 with 6J light show lower antitumor efficiency than PTX or ZnPC4-loaded micelles which testify the copolymer a good drug delivery material.nder certain drug concentration and ZnPC4 showed low dark toxicity. Light alone did not affect the growth of cells. Furthermore we investigate the mechanism of PDT by polymeric micelles. We found that cells swallow the micelles and reach a platform after 4h,660 nm laser was performed at this time,which the sharp decrease of mitochondria membrane potential, leading to the death of cells.We found synergistic effect of PDT and chemotherapy, laying the very basis for in vivo antitumor study.6. In vivo pharmacodynamics of pH-sensitive micellesSubcutaneouly transplantation melanoma model was established with B16-F10 cells. Small animals living imager was empolied at different times to moniter the drug distribution after iv. Administration. Drug distribution in tumor was higher than in heart 4h after injection. Tumor-bearing mice were grouped randomly firstly. Drug-loaded pH-sensitive micelles were given through vein every two days for 4 times.Physical state of mice were observed everyday, body weight and tumor volume was monitored. Eight days later the mice were managed euthanasia, then the tumor tissues were dissercted and weighed. PDP co-delivery group show higher tumor inhibitiory ratio than PTX or ZnPC4-loaded micelles and a similar ratio with positive drug control group, howerer the positive drug was too toxict to induce the death of mice.