Experiment Study On Polylactic Acid Microbubble Ultrasonic Contrast Agents For Drug Delivery

Cancer is one of chief diseases to threat the health of human being. Chemical therapy is an important method to treat it comparing with operation and radial therapy. Hydroxycamptothecin(HCPT) is an antitumor drag that has been used to treat a wide range of cancers. And the main medicament of HCPT is lyophilized power and has severe side effect. Polylactic acid (PLA) is under extensive research because of its unique and excellent biocompatibility and biodegradability, particularly in biomedical field. To solve the shortcoming of clinic medicament, we develop a novel medicament of PLA microbubble ultrasonic contrast agents loaded HCPT as targeting drag delivery systems.Ultrasonic locating and targeting microbubble Ultrasonic Contrast Agents (UCA) loaded drag is a kind of Targeting Drag Delivery System (TDDS) to release drag under the ultrasonic physical effect. The shells of microbubbles are made from biodegradable high molecular polymer and the microbubbles are prepared using ultrasonic emulsification method with SPG membrane emulsification technique and freeze-dry technique. The interior of microbubbles are filled with C₃F₈ gas. Reflection and scattering phenomenon is going to take place when the microbubbles are irradiated by ultrasonic, so we can see the image of them on the screen and locate them in vivo. Gas in the microbubbles will expand and contract periodically under ultrasonic influence, at last the microbubbles will burst and release drag in the area that irradiated by ultrasonic. Ultrasonic locating and targeting drag microbubbles could increase the drag concentration in targeting area and reduce the blood concentration, it is helpful to increase therapy effect and decrease adverse reaction. It is a novel physical targeting drag system and it will play a good part in thrombus and tumor therapy.In this paper, we use PLA as the drag carrier, HCPT as the model drag, and we study the preparation of microbubble ultrasonic contrast agents loaded model drag by combining ultrasonic emulsification method and SPG membrane emulsification technique. The contents are summarized as follows: 1. Preparation of PLA microbubble UCA by ultrasonic W/O/W double emulsion method and ultrasonic emulsion method with SPG membrane emulsification technique. As the result, by both of them we prepared good uniform-sized microbubble UCA, and ultrasonic emulsion method with SPG membrane emulsification technique is better than ultrasonic W/O/W double emulsion method.(1)We got the better preparation process and parameters by ultrasonic W/O/W double emulsion method: 1ml inner aqueous phase (W₁) was injected into 10ml outer oil phase (O) which contained 0.025g/ml PDLLA and 1.0%Span80 insonated by ultrasonic (80W·10S·10times), we got W₁/O emulsion. Then the W₁/O emulsion was injected into 100ml outer aqueous phase (W2) which contained 1.0% Tween80 insonated by ultrasonic (200W·10S·6times),so we got W₁/O/W₂ double emulsion. The double emulsion was poured into 1000ml 10% PVA solution and was churned up at 100rpm/min over night holding at 40℃on a magnetic force blender, and was centrifugated at 3000g for 10min. The microbubbles sediments were washed 3 times with distilled water after collected, and then were dried in vacuum refrigerant drier. By this method, the size distribution of the microbubbles was showed. 19.5% of the microbubbles were nearby peak 1 with the mean diameter of 238.1nm,and 34.3% of the microbubbles were nearby peak 2 of 1.713μm,and 46.2% of the microbubbles were nearby peak 3 of 3.961μm, and no microbubbles' diameters were bigger than 7μm.(2) We got the better preparation process and parameters by ultrasonic emulsion method with SPG membrane emulsification technique: 1ml inner aqueous phase (W₁) was injected into 10ml outer oil phase (O) which contained 0.025g/ml PDLLA and 1.0%Span80 insonated by ultrasonic (80W·10S·10times), we got W₁/O emulsion. The W₁/O emulsion was poured into the pressure cabin of SPG microporous membrane emulsifier. There was fixed a SPG membrane of 1.1μm diameter. Appropriate and equal 50kpa gas pressure was given by high purified N₂ to press the W₁/O emulsion through SPG microporous membrane into circularly flowing 1% PVA solution, and we got W₁/O/W₂ double emulsion. The W₁/O/W₂ double emulsion was churned up at 100rpm/min over night holding at 40℃on a magnetic force blender , and was centrifugated at 3000g for 10min. The microbubbles sediments were washed 3 times with distilled water after collected, and then were dried in vacuum refrigerant drier. By this method, the size distribution of the microbubbles was showed. 93.6% of the microbubbles were nearby peak 1 with the mean diameter of 4.124μm. 6.4% of the microbubbles were nearby peak 2 with the mean diameter of 274nm. So we got better uniform-sized microbubbles by this method than by ultrasonic W/O/W double emulsion method.2. Based on the better preparation process and parameters by ultrasonic emulsion method with SPG membrane emulsification technique, we studied the preparation of HCPT-PLA microbubbles UCA for drug delivery. We used different concentrations of HCPT and NaOH solutions as inner aqueous phase (W₁), 1.0%PVA and pH6.0 HCl solutions as outer aqueous phase (O). We studied the effects of drug loading and encapsulation efficiency with different concentrations of inner aqueous phase (W₁). In result, there was the maximum encapsulation efficiency 66.48% when the concentration of inner aqueous phase (W₁) was 4mg/ml; there was the maximum drug loading efficiency 1.084% when the concentration of inner aqueous phase (W₁) was4mg/ml. The SEM photos showed that the mean diameter of HCPT-PLA microbubbles UCA was about 5μm, and spherical shapes were formed, we got better uniform-sized microbubbles.3. In the condition of 100rpm/min, 37℃permanent temperature, HCPT-PLA microbubbles UCA were vibrant a week, the drug release experiments in vitro showed that the percentage of cumulative drug released was 64.14%. HCPT-PLA microbubbles UCA were insonated by ultrasonic (3.5MHz, MI1.0, 90s/per time×3) in vitro, and the percentage of cumulative drug released was 94.37%. Almost drug was released.4. The drug release experiments in vivo using New Zealand big rabbits as animal model showed there was higher blood drug concentration using HCPT-PLA microbubbles UCA than only HCPT by the same dosage. At the same time, there was higher blood drug concentration using HCPT-PLA microbubbles UCA for a longer time and better controlled-release effect. 5. The anti-H₂₂ cell tumor animal model experiments using Kunming small rats showed that the anti-H₂₂ cell tumor effect of HCPT-PLA microbubbles UCA with ultrasonic irradiation was better.6. The ultrasonic autoradiography experiments in vivo using New Zealand big rabbits as animal model showed the better heart autoradiography of the rabbit with HCPT-PLA microbubbles UCA, and the dosage was 20mg.Both of left and right cardiac ventricle showed better ultrasonic autoradiography. It showed that HCPT-PLA microbubbles UCA passed through lung circulation and was good uniform-sized.