Experimental Study Of Intratumoral Injection Of Carboplatin-loaded PLGA Microspheres Guided By Ultrasound

BachgroundCancer is a common disease that seriously threatens human healthy. The chemotherapy is one of most effective common methods in cancer therapy. But generally the chemotherapeutants have no specific selectivity between the normal cells and the tumor cells. The intratumoral drug concentration remains at a low level by the systemic administration, and side effects are usually serious. This limits the high dosage administration and clinical efficacy of the anticancer chemotherapeutants. The chemotherapeutants appear to inhibit tumor effectively in vitro, but not effective in vivo. The reasons for this are possibly due to some influential factors, like the form of prepared drugs, the administration methods and the internal environment. The therapeutic efficacy could be improved by changing the form of prepared drugs, the administration methods and prevention of the internal interference. All kinds of local chemotherapies can increase the drug concentration in the tumor and promote the clinical efficacies, especially the intratumoral injection chemotherapy. A high drug concentration of common form of prepared drugs in tumor maintained shortly, and the efficacy is still limited. So the microspheres, which are characterized by controlled release, sustained release and long-persistent effect, is regarded. The intratumoral injection of microspheres could maintain a high drug concentration in the tumor and continuously act on the tumor cells, which could improve the efficacy and reduce the side effects.ObjectiveThe carboplatin-loaded poly (D, L-lactic-co-glycolic) acid copolymer microspheres (CBP-PLGA-Ms) for ultrasound-guided intratumoral injection were prepared by optimal technical conditions. The releases of CBP-PLGA-Ms in vitro and in vivo were investigated. The efficacy and side effects on solid tumor were studied by changing the form of prepareddrugs and administration methods to find a novel therapeutic method of solid tumor. Methods1. CBP-PLGA-Ms were prepared by an acetone/mineral oil emulsion and solvent evaporation method. Various factors related with the drug loading and microparticle size were studied. The uniform experimental design (U15) was chosen to optimize the technology of microspheres preparation. The drug releases of CBP-PLGA-Ms in vitro were investigated.2. The tumor cell strain and the implant position was chosen by comparing the growth characterization of Sigo and H22 on different location. The subcutaneous implant tumor models were established by implanting tumor ascites. The tumor size and ultrasonography and color doppler flow imaging were examined to calculate the tumor volume and to draw the curve of tumor growth. The pathologic characterizations of tumor were observed.3. The concentration of carboplatin in the tumor, blood, liver and kidney was detected by inductively coupled plasma-atomic emission spectrometry on the relative time after the intratumoral injection of CBP-PLGA-Ms and free CBP. The index of pharmacokinetics and tissue distribution was analysed. The pharmacokinetics of CBP-PLGA-Ms in vivo was concluded.4. The tumor-bearing mice were divided into four groups: microsphere group, mixture group, carboplatin group and control group respectively. The drugs were injected into tumor guided by ultrasound. The tumor size and ultrasonography and color Doppler flow imaging were examined to calculate the tumor volume and the tumor growth double time and to draw the curve of tumor growth. The pathologic characterizations of tumor were observed.Results1. The best processing parameters for preparing microspheres were as following: the particle sizes of carboplatin were l~4um, the ratio of the carboplatin and PLGA was 1:7, the PLGA concentration was 10%, the ratio of internal and external phases was 1:8, the emulsifier concentration in the external phase was 8%, the stirring speed was 1 400 rpm, the emulsion time was 15min and the solvent evaporation time was 4 hr. The optimized microspheres of 100~200|um in size were global, smooth-filmed with 65.4% of yield and 11.6% of the drug loading. There is no burst effect of the microspheres in vitro; and it can release slowly and the release time continued in 15 days. The duration time necessary totransfer 63.2% of the administered drug into systemic circulation was 160.12hr, and carboplatin's was 0.1 lhr. The microspheres were stable after being sealed up.2. The mice models of subcutaneous tumor were set up by implanting H22 tumor cells under the skin of right latter leg of BALB/c mouse, and the tumor model was easy to implant. The tumor grew steadily with a natural survival time longer than 50 days. The tumor ultrasonography featured by clear boundary, abundant blood flow and solitary homogeneous nodule. The mean diameter of tumor could reach 11.78±0.74mm in 15 days and was suitable for intratumoral injection. The tumor ultrasonography presented a low homogeneous internal echo in 30 days. There were no necroses of the skin or ascites in mice. The model was ideal for comparative observation after the treatment.3. The tissue distribution and pharmacokinitical characterization of carboplatin in CBP-PLGA-Ms were studied in comparsion with free carboplatin as following: the percentage of relative tissue distribution of carboplatin in tumor of the microsphere group had been improved. The area under the concentration-time curve of microspheres was 2.98 time of free carboplatin group in tumor during the first 96 hr, indicating that the CBP-PLGA-Ms improved the bioavailability of carboplatin in tumor. The microspheres reduced the maximum concentration, prolonged the maximum time, half time of distribution and removal, mean risidence time obviously, meaning that the CBP-PLGA-Ms could be released considerably slow. The carboplatin concentration remained at a higher level in tumor for 15 days, which improved the efficacy of the microspheres group and the maximum concentration went on at a low level in blood, liver and kidney, this could greatly reduced the toxicity and side effects.4. The tumor growth was restrained persistently during the 15 days period in the microsphere group, and was restrained during the first 5 days in the mixture group and the carboplatin group, but was not restrained during the second and third 5 days according to the tumor volume, tumor growth curve and the tumor double time. The tumor ultrasonography found the feeding blood flow of the tumor reduced after the treatments and the ultrasonography of tumor of the microsphere group and the mixture group appeared to be inhomogeneous high echo but no changes happened in the carboplatin group and the control group. The pathological examinations showed that big coagulation necrosis masses were found in the tumors.Conclusions1. The major influential factors for the carboplatin-loaded PLGA microspheres preparation were as following: the particle sizes of carboplatin, the ratio of the carboplatin and PLGA, the PLGA concentration, the ratio of internal and external phases, the emulsifier concentration in the external phase, stirring speed, the emulsion time and the solvent evaporaton time. The microspheres properties could be controlled by preparative variables.2. The mouse solid tumor model with H22 can be made on right latter leg and its natural growth period is longer, its feature is steady and fits the study on intrarumoral injection.3. The carboplatin-loaded PLGA microspheres is characteristical in slowly release, persistent tumor restrain, and reduced the concentration in other tissues of the mouse. The purposes to prolong the release time of carboplatin, to reduce the side effect and to improve the efficacy of chemotherapy have been reached.4. The new therapy of intratumoral injection of carboplatin-loaded PLGA microspheres guided by ultrasound has a significant tumor growth inhibition. This method is easy to copy and the result is certain, and it paves a new way to treat the solid tumor.