Doxorubicin-loaded Liquid-solid Phase Transitive Gel For Subcutaneous Residual VX₂Tumor After High Intensity Focused Ultrasound

Background: High intensity focused ultrasound （HIFU） has become animportant auxiliary therapy for several solid tumors. However, highrecurrence rates were observed in HIFU single-using therapy for solidtumors, which may be related to residual tumor from incompletely ablationof HIFU. In order to enhance the therapeutic effect of HIFU and decreaserelated tumor recurrence rates, chemotherapy was applied to combine withHIFU. Although this combination reduced the tumor recurrence rate tosome extent, serious side effects from traditional systemic chemotherapywas observed which limited the application of this combination. Injectablebiodegradable in-situ forming gel implant （ISFI） which possessedadvantages like local drug delivery, extending the drug release period,reduction of adverse drug reactions and relatively simple process and so on,is drawing increasing attentions, which was widely used in dentistry andophthalmology. However, the application of it in therapy for tumors wasseldom reported. Doxorubicin （DOX） was a clinical commonly usedantharcycline and broad-spectrum anti-tumor drug which was effective in treatment for tumors. But serous side-effect limited its using dose. Thus, inour research, doxorubicin-loaded liquid-solid phase transition in-situforming gel （DOX gel） was structured. Its characteristics of phase inversionin vitro and drug-releasing in vivo were investigated. Then we applied it totreat VX₂subcutaneous tumor in rabbit after uncompleted ablation fromHIFU, in order to decrease tumor recurrence rates, and search for newtherapeutic strategy of reoccurrence of tumor after HIFU ablation.Objective:（1） to structure Doxorubicin-loaded liquid-solid phase transitionin-situ forming gel （DOX gel）, investigating its phase inversion in vivo andsustained-release characteristics in vitro.（2） To evaluate the effect ofDoxorubicin-loaded liquid-solid phase transition in-situ forming gel （DOXgel） injection for residual VX₂tumor after high intensity focusedultrasound ablation and discuss this therapeutic application.Method:（1） DOX gel was structured by carrier PLGA and menstruumNMP. DOX was applied as model medicine. And its7-days drugcontinuous release rate was measured by high pressure liquidchromatography （HPLC）. And its process of phase inversion was observedby ultrasound imaging.（2） Rabbit VX₂subcutaneously transplanted tumormodel was established, then we injected DOX gel into the tumors afterHIFU incomplete ablation.4days and8days after treatment, in blankgroup （without any treatment）, control group （HIFU+Blank gel） and experimental group （HIFU+DOX gel）, tumor volumes were measured andgrowth rate was calculated. Tumor tissues pathological changes wereobserved under H.E. staining. Proliferation and apoptosis of tumor cellswas revealed by PCNA immunohistochemistry （IHC） and TUNEL methods,PI （proliferative index） and AI （apoptosis index） were calculated. Bcl-2andBax protein levels were detected by Western Blot.Result:（1） DOX gel was structured successfully which showed a low burstrelease effect. Cumulative release rate in vitro was78.92±4.68%. A steadyreleasing curve and slight burst effect were observed in7days. ObviouslyDOX gel sustained-release effect was observed in DOX gel cumulativedrug release curve. In vitro, phase inversion of DOX gel started at the timeof contact with water, the average gray scale values rise along the time andreached the maximum at the1stday, then began to decrease. In vivo,hyperecho appeared at the time of DOX gel was injected into the tumor, theaverage gray scale values increased and reached the maximum at the4thhour, then showed decreasing. Implantation materials were swelling andcross-sectional area was increasing while the phase inversion in vitro and invivo, then reached the maximums at the7thday.（2）4days and8days aftertreatment, the lowest mean tumor volume was observed in experimentalgroup when compared with other two groups （p>0.05）. Mean tumorvolume in Blank group showed a highest growing speed: mean growth rates were1.55（to4thday） and3.11（to8thday）, however a lowest meantumor volume growing speed was observed in experimental group:0.74（onthe4thday） and0.77（on the8thday）. Ablation necrotic area and a smallamount of tumor cells were observed under H.E. staining. IHC and TUNELshowed that PI in experimental group was significant lower than both blankgroup and control group （p<0.05）. AI in experimental group was3.53±0.26,much higher than other two groups （p<0.05） also. Western Blot showedthat protein Bcl-2was highly expressed in both control group and blankgroup, but a low expression was observed in experimental group. However,protein Bax showed an opposite expressive characteristic in these threegroups.Conclusion:（1） successfully structured DOX gel showed a low burstreleasing effect and a well DOX controlled-release characteristic.Liquid-solid phase inversion characteristic was observed both in vivo andin vitro which could be a well form of sustained release in-suit formingimplant.（2） DOX gel showed an effective treatment for residual tumorafter HIFU ablation which promised to be a new treatment method andrelapse prevention for residual tumor after HIFU ablation.