| BachgroundTargeting to special organ or tissue and delayed drug releasing, two of the characteristics of microspheres containing drugs, have been the hot spots of the studies on the controlled release of drugs in recent years. It is accordant to the principle of targeting, prolonged action, high performance and low toxicity to prepare the microspheres loading Carboplatin, a broad-spectrum anticarcinogen of second generation. In the former experiments, our team has made primary researches on the preparation technology of the microspheres and their curative effect and suitable dosage to the transplanted liver tumor and pancreatic tumor. The research on the drug releasing of microspheres in vitro can reflect the progress in vivo and the feasible experiment of release degree in vitro is also important to evaluate its safety and efficacy. At present, the detection of release degree in vitro is made mostly by imitating the conditions in vivo, including temperature, stirring, pH value of mediator and enzymes, etc. This research will estimate the drug releasing mechanisms of the Carboplatin-loaded PLGA Microspheres farther in vitro and thus to find out its correlation in vivo. Apoptosis is the progress of self extinction of live tissues or cells regulated by genes or some other factors, the disorder of which is related with the morbility of tumor. Apoptosis-controlling genes can be divided into the inhibitive and the activating. Bcl-2 is one of the inhibiting genes, which can be suppressed by a heterodimer formed when it combined with Bax. So whether the intratumorally injected Carboplatin-loaded PLGA microspheres can activate the apoptosis? Since the microspheres are better than the active compounds, could the reason be that their effect to inducing apoptosis of the microspheres is stronger? Therefore we start the research from the point of apoptosis inducing.Objectives To observe the ultramicrostructure changes of Carboplatin-loaded PLGA microspheres during the releasing progress in vitro by scanning electron microscope, estimate the mechanisms of drug releasing, and deduce their state in vivo. To discuss the mechanisms of Carboplatin-loaded PLGA microspheres to induce apoptosis of the subcutaneous transplanted tumor in mice and prove further that Carboplatin-loaded PLGA microspheres are superior to the active compounds.Methods1. The research on the degradation of Carboplatin-loaded PLGA microspheres in vitroThe ultramicrostructure of the microspheres was observed by scanning electron microscope at various time points (3, 7, 14 and 12d) and the mechanisms of drug releasing were discussed.2. The establishment of the mice models of subcutaneous transplanted tumorWalker-256 tumor cell lines were cultured and passaged in vitro and the model was established by ascites inoculation method. The tumors were observed dynamically with 2-dimentional ultrasonography and color Doppler flow imaging (CDFI) and their sizes and doubling times were calculated, with which the natural growth curves were drawn.3. The observation to the curative effects of intratumorally injected Carboplatin-loaded PLGA microspheres guided by ultrasound in the mice models of subcutaneous transplanted tumor.The mice were divided into groups of microspheres, Carboplatin, saline, and the control. Intratumorally injecting therapy was performed at the proper drug dosages, while the tumors were monitored by 2-dimentional sonography and CDFI dynamically. The growth curves of the tumors were made according to the changes of the size with the time. The pathological characteristics were observed.4. Carboplatin-loaded PLGA microspheres'induction to the apoptosis of the subcutaneous transplanted tumor in mice and their effects on the expression of apoptosis-associated proteins bcl-2 and bax.The expression of Bcl-2 and Bax were detected by immunohistochemistry and TUNEL method.Results1. The experiment of drug releasing in vitro showed that there was no sudden releasing effect during the initial stage. The drug releasing started after the first day, kept stable from 2 to 12d, speeded up at 13d, and then slow down to a stable level again at 14d.Observed under scanning electron microscope, the surface of Carboplatin-loaded PLGA microspheres was smooth and had no pore. After the spheres were put into the mediator of PBS fluid, some folds and pores appeared on the surface at 7d, aggravated at 14d, and became more extensive at 21d.2. The mice models of subcutaneous transplanted tumor were established successfully by implanting Walker-256 tumor ascites under the skin of right latter leg of mouse. The tumor model was easy to make and the tumor grew steadily with a natural survival time longer than 50 days. There was no skin ulceration and ascites during the 30 days after implantation. The tumor ultrasonography featured by clear boundary, abundant blood flow and solitary homogeneous nodule. The mean diameter of tumor could reach 11.59±1.28mm at the 7th day.3. With the tumor size, doubling time and growth curve compared between groups, the growth pf tumor in microsoheres group was inhibited continuously during the observation period of 21d while there was a obvious inhibiting effect in Carboplatin group only at the first 7 days and disappeared gradually thereafter, The microspheres showed a continuous inhibition during all the time, which was weaker than that of Carboplatin at the first 3 days, stronger from 4th to 7th day but without statistic significance, and stronger obciously from 7th to 21st d while the difference between Carboplatin, saline and control has no statistic significance. The results implied that the microspheres'inhibition could last for a long time and improve the curative effect. The sonography showed that the blood signals reduced in the curative groups, the tumor presented uneven high-level echo and showed colliquation and necrosis at the later stage in microsphere group, and the echogenecity in tumor didn't change obviously in Carboplatin and control group. Pathological examination showed that there was a great deal of coagulation necrosis in the tumors in the curative goups.4. With the detection by TUNEL'method, the apoptosis index rose with the time in the microsphere group while there was a adverse tendency in the Carboplatin group. The expression of Bcl-2 in tumor of microsphere group was lower than those of the other groups significantly while it's adverse for Bax。 Conclusion1. Carboplatin-loaded PLGA microspheres have an ability of controlled drug releasing. The encapsulated Carboplatin was released by the effects of dissolution and permeation through"pores".2. The mice models of subcutaneous transplanted tumor can be established by implanting Walker-256 tumor ascites under the skin of right latter leg of mouse.3. Intratumorally injected Carboplatin-loaded PLGA microspheres guided by ultrasound have stronger inhibiting effect than the Carboplatin only in the mice models of subcutaneous transplanted tumor. It's easy and effective, which may be a new way to treat solid tumor.4. Carboplatin-loaded PLGA microspheres can induce the apoptosis and inhibit the growth of tumor at the same time, which may be the reason why it's superior to the active compound of Carboplatin. |
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