| Objective1. To prepare glycolic acid-ethylcellulose microspheres loaded with glycolic acid.2. To evaluate the therapeutic effects of glycolic acid-ethylcellulose microspheresinfused via hepatic artery on hepatocarcinoma in rabbits.Materials and Methods1. Preparation of glycolic acid-ethycellulose microspheres.(1)Determinate the preparation technicsThe glycolic acid microspheres coated with ethylcellulose as the shell will beprepared using the volatile solvent and ultrasonic W/O/W method.(2)Select the optimized preparative conditions of glycolic acid-ethycellulosemicrospheres.The factors such as the amount of glycolic acid and ethylcellulose have the mostimportant effect on the size and size distribution of glycolic acid-ethycellulosemicrospheres.The optimum conditions were investigated in different conditions.(3)Measure glycolic acid loading and encapsulation efficiency of glycolicacid-ethycellulose.2ml dichloromethane added100g freeze-dried microspheres were placed in10mlcentrifuge tube, dissolved under ultrasound, extract with2ml distilled water,and thencentrifuge5minutes. Collect the supernatant solution and pipet into10ml volumetric flaskextract5times and settle to permit. Calculated drug loading and encapsulation efficiencywith criterion curvilinear equation. The encapsulation efficiency of microsphere and GAloading were calculated with formula described below：Encapsulation efficiency=amount of GA in microspheres/GA initially added×100%GA loading=amount of GA in microspheres/amount of microspheres×100%(4)Glycolic acid-ethycellulose microspheres size and size distribution measuredThe glycolic acid-ethycellulose microspheres size and size distribution were measured using dynamic light scattering.(5) The shape morphology of glycolic acid-ethycellulose microspheresThe morphologies of the microspheres were observed using a scanning electronmicroscope. The microspheres microspheres were mounted onto brass stubs andsputter-coated with gold prior to examination under microscope.(6) In vitro release of glycolic acid from microspheresMicrospheres added PBS buffer (pH7.4) were placed in centrifuge tube, then placedin oscillator at37±0.5℃,100r/min.At setting time, supernatant solution was determined byHPLC at the suitable wave length (220nm) to measure the drug content in the solution. Thepercentage of GA cumulative release rate was investigated.2. Hepatic Arterial Embolization with GlycolicAcid-Ethylcellulose Microspheres in aRabbit Liver Tumor Model(1)AnimalsIn this experiment，30Newsland rabbits weighted at2.5±3.5kg were used．All rabbitswere buying from the animal center of Second military medical university．Prepare for theliver metastasis model．(2) Development of the animal modelsFor successful implantation of the Vx-2tumor into the liver,the tumor was firstgrown for2weeks on the hind leg of a carrier rabbit. Each carrier rabbit was used tosupply tumor cells for implantation into the left lobe of the liver of two separate rabbits.All the rabbits, carriers and recipients, were anesthetized with sodium pentobarbital(2.5mg/kg; German) administered i.v.access was gained via a marginalear vein.Theabdomen of each rabbit was shaved and prepped with iodine tincture. The liver wasexposed with a linea mediana ventralis incision, and clumps of tumor (about1mm3) fromthe harvested minced tumors were implanted directly into the left lobe of the exposed liverof recipient rabbits. The abdomen was closed in two layers, and proper aseptic techniquewas rigorously executed during each implantation.After surgery,the animals were placedin cages and monitored in the animal laboratory under direct supervision. Benzylpenicillin Sodium (5000u/kg) was administered i.m for3days after surgery.(3)Group separatedBecause results of TACE can be influenced by tumor volume, animals withsimilar-size tumors were distributed evenly to each group. Group A (n=10) were treatedwith an intra-arterial injection of the glycolic acid-ethylcellulose micropheres (1ml), groupB (n=10) received an intra-arterial injection of iodinated oil alone (1ml). As a controlgroup, Group C (n=10) were treated with sodium chloride alone (1ml).Microspheres(100-300μm) contained106μg drug/mg. A dose of0.2g microspheres wassuspended in10ml Iopromide Injection for10minutes before administration.(4) TACEprocedure.Twoweeks after implantation of VX2carcinoma in the liver,TACEwas performedwith fluoroscopic guidance (Innova4100, GE,America).Preanesthetics were administered,and anesthesia was carried out as described above, and an incision was made to expose theright femoral artery.Withuse of a3-F microcatheter (Cook, Bloomington, Ind), celiacangiography was performed to identify the hepatic arterial anatomy and the feeder artery ofthe tumor. The left hepatic artery, which exclusively supplies blood flow to the tumor, wascatheterized selectively. When the catheter was advanced to an adequate position in the lefthepatic artery, the drug was injected carefully to avoid effluxion of the embolic materialsout of the artery. After completion of the infusion, the catheter was removed, and thecommon femoral artery was ligated to obtain hemostasis. During the therapy, aseptictechnique was rigorously executed. The animals were returned to cages afterBenzylpenicillin Sodium was administered.(5) Observation item①Blood collection Before and at7days after treatment, Whole blood (3mL) wascollected from heart with blood taking needle in all animal groups. Blood samples werecollected to determine liver function (liver function test).②Follow-up CT was performedat7days after Teethe growth ratio was calculated by comparing the tumor volumesobtained before (V1) and at7days after (V2) treatment with the following formula:(V2/V11)×100%.③5rabbits were randomly chose from each group to observe thesurvival time which was accounted from treatment. The life prolonged ratio was calculatedby the mean survival time of Group C with the following formula:(Mx/Mc1)×100%. ④Other rabbits were sacrificed for histologic investigation with an overdose ofpentobarbital sodium at7days after TACE. Tissue slices were immunohistochemicallystained for specific proliferating cell nuclear antigen (PCNA) and vascular endothelialgrowth factor (VEGF) staining kit.(6)Statistical analysis.SPSS11.0was used as analyzing software．ANOVA analyzed the difference amongdifferent groups. Differences were considered statistically significant for P <0.05.Results1. The optimized preparative condition: The embolization microspheres used in thisstudy were composed of ethylcellulose that has the capability of tolerating corrosion ofglycolic acid. Microspheres were prepared by using water-in oil-water (W1/O/W2) doubleemulsion solvent diffusion method. The initial W/O emulsion was prepared by addingglycolic acid to the mixed solvent system consisting of ethylcellulose anddichloromethane(0.18g:2ml) while emulsifying using a mechanical emulsifyer at400W for3minuts.This W/O primary emulsion was slowly added to20ml of the second watersphase containing1%PVA which contain glycolic acid (concentration:0.04112g/ml)whilestirring by a paddle propeller at900rpm for10minuts,then stirring was continued at400rpm. After4h, microspheres were collected by centrifugation, washed with distilledwater, and lyophilized in fridge.2. The surface of microspheres observed by SEM was spherical and smooth. Particlesize analysis showed that the microspheres were homogeneous in size with200um averagediameter.The encapsulation efficiency and drug content of microspheres were74%and106μg/mg respectively.3. The glycolic acid-ethylcellulose microspheres continuously released glycolic acidfor72hours in vitro.4. The volumes of tumors ranged from0.71to3.21cm3(2.00cm3±0.87[mean±SD]) in group A, from1.33to4.66cm3(2.28cm3±1.15) in the group B, and from1.00to2.91cm3(1.95cm3±0.70) in the group C. Tumor volumes were not statistically differentamong the three groups (P=.68, ANOVA).5. There was no significant difference among the3groups before interventionaltreatment. Compare the parameters difference between pre-and post-interventionaltreatment in3groups, there was no difference among the3groups in ALB; ALT in Group A and B were higher than that in Group C（respectively,P=.037, P=.007, Game-Howelltest）,there was no difference between GroupA and Group B;there was significantdifference between GroupA and C（P=.011,Game-Howell test）.The tumor growth ratio inGroup A was much slower than that of Group B and C (respectively,P=.022, P=.001,Game-Howell test). Compared with Group B and C, the survival time of Group A wasprolonged significantly. Pathological examination showed large necrotic area in tumors inGroup A and B.The necrotic areas in Group A were larger than that of Group B. TheVEGF expression and proliferation activity in Group A were weaker than that of Group B.Conclusions1. The glycolic acid-ethylcellulose microspheres continuously released glycolic acidfor72hours, the glycolic acid ethyl cellulose microspheres have good sustained releaseprofile.2. The glycolic acid-ethylcellulose microspheres transcatheter infusion to liver caninhibit the growth of liver cancer and prolong the survival of the rabbits, and thus is aneffective chemoembolization agent. |
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