| BackgroundUltrasound contrast agents(UCAs) are a kind of diagnostic agents which consist of high concentration of microobubbles that have the potential to enhance significantly the yield of Doppler-ultrasound examination and thus allow better imaging. Microbubble imaging agents can pass through the microcirculation after intravenous injection and have intensity ultrasonic scattering properties that can significantly enhance the ultrasonic echo signal thus allow better images of different organs and greatly improve the ultrasound diagnosis. Microbubbles can enhance the visualization of blood flow and improve the sensitivity and specificity of diagnosis, moreover, the development of ultrasound contrast agents have evolved diagnostic ultrasound from a routine scan mode into a contrast enhancement mode, which is called the third revolution in diagnostic ultrasound. More worthy of attention is that they will be the key technology that allow ultrasound effectively compete with MRI and CT.The preparation and application of ultrasound contrast agents have became hotspots of medicine ultrasound in recent years. Ultrasound contrast agents have been developed from the first generation that consists of air-filled microbubbles to the second generation that consist of perfluorocarbon-filled microbubbles, both of their stability and efficiency have received breakthrough progress. Nowadays, preparations of ultrasound contrast agents have headed at improving the outer membrane materials in order to make it more safety and have longer life span in the application. From the history of the ultrasound contrast agents, in the early1990s the contrast agents are mainly albumin microbubbles, and in the late1990s the contrast agents are mainly lipid-shelled microbubbles and poly material shelled microbubbles. The reasons are as follows: from the security consideration, human serum albumin is dangerous in the spread of AIDS、HBV and some other blood infectious diseases; From the quality of the contrast-enhanced ultrasound, the lipid-shell consist of macromolecules lipids, which made them more stable and flexible than albumin bubbles, thus can produce better images and better meet the clinical needs. Therefore, lipid microbubbles are the mainstream of the development of ultrasound contrast agents.The market of ultrasound contrast agent is emerging and flourishing, as the price of ultrasound contrast agents of foreign company is so expensive, so it’s significantly important to develop such agents in our country. Most of the technologies and patents were developed and dominated in US and Europe. SonoVue and Definity made by US and European companies are now sharing the world market of UCAs. Our groups have developed the albumin-coated microspheres named "dongguan parenteral solution" and "quanfuxian", and the acoustic activity of these microspheres was prior to the abroad preparations in many ways. But compared to albumin-coated microbubbles, lipid-shelled microbubbles have a clear advantage, in hence the subsequent develop of UCAs are mainly lipid microbubbles.Objects 1To explore the technique and formula of lipid-shelled microbubble agents, which have high concentration, suitable size and perform as stable, effective and safe preparations.2Assessment of the physicochemical of the lipid-shelled microbubbles, including the microbubbles appearance shape, concentration、size、size distribution、the analysis of perfluoropropane、pressure stability as well as its storage stability.3To further discussion on its clinical application value, the contrast effect of microbubbles in New Zealand rabbit’s kidney and liver was studied.Methods1Optimization of the technique and formula1.1Optimization of the techniqueThe lipid microbubbles were prepared by high speed shearing method, and the single-factor test was used to analysis the technological parameter of the preparation, including the shear velocity, the perfluoropropane ventilate time and the shearing time. The levels of each factor were as follows: shearing velocity(four shelves、five shelves、six shelves), perfluoropropane ventilate time(l5s、30s、45s、60s), shearing time (90s、110s、120s、130s、150s). The concentration of microbubbles was used as its index; optimum technological parameter was established by variance analysis.1.2Optimization of the formulaThe optimum technological parameter was employed to produce microbubbles that consist of the lipid shell and the perfluoropropane core. Bulk concentration of the lipid, ratio of DPPE-PEG5000, ratio of DPPA and the content of NaCl have great impact of the concentration and the particle diameter of the preparation, therefore such factors were used as independent variable, and the microbubble concentration was used as dependent variable in the orthogonal experimental test to ascertain the optimum formula of the preparation. 2Assessment of physicochemical of the lipid-shelled microbubbles2.1Observation of the microbubble appearanceTake a little microbubble preparation on the microscope slide and then cover with the cover glass, observe its appearance on the optical microscope.2.2Coulter counter analysisUsing the50μm pinhole tube,0.9%NaCl as electrolyte, the analyze volume was500μl, the counter particle diameter was from1.1μm～30μm. Take a precise20μl preparation into0.9%NaCl dilute into5000times, and then put into the Coulter counter for detection.2.3Determination of Perfluoropropane in the bubblesThe Perfluoropropane concentration was determined by means of gas chromatography coupled with mass spetrum/selected ion monitoring (GC-MS/SIM). Chromatographic conditions: ADB-5ms(30rn×250μm×0.25|im) was used as analysis column and the column temperature was set to35°C; Helium at constant rate of0.8ml/min was used as carrier gas; Mass spectrometry conditions: GC-MS/SIM mode and EI was used, the ion of monitor: m/z69、169. Sample preparation: precisely took2ml of the preparation and inject into an air package that contain500ml purity nitrogen, and then put it into the ultrasonic bath for10min to allow all the OFP had released from the microbubbles. A single of50μl injection of the sample was analyzed; the external standard method was used to calculate the content of OFP.2.4The stability about pressureFour milliliter suspension of microbubbles was taken by a5ml syringe, and connect to the eagle4000monitor by three-way tubes, and then150mmHg、300mmHg over-pressure were pressed for lmin、3min、5min、10min, respectively. Then measure the resulting samples’ concentration and diameter with Coulter counter to study their stability of anti-pressure. 2.5liquid preparation’s storage stabilityPrepare6baths of the lipid microbubbles and store in refrigerator at4°C±2°C. These samples’ concentration and diameter were measured with Coulter counter in0m,1m,2m,3m and6m, respectively.3Appraise the contrast effect3.1Dosage choice at acoustic testingThree New Zealand white rabbits were used to study, increasing dosages, from0.005、0.01、0.02、0.06、0.1ml/kg were injected through an angiography catheter inserted into the left ear vein respectively, and conduct kidney and liver ultrasonic test. The sequence of each test was randomly arranged, each dosage groups were repeated for5times, and the images were collected until it showed a significant decline. The Q-lab software of the instrument was used to analyze the images.3.2Acoustic effect appraisalsThree New Zealand white rabbits were used to study and a single dose of0.01ml/kg was given and then conduct kidney and liver ultrasonic image, respectively. Both the kidney and liver test repeat for3times, and the images were collected until the bubbles were clear away. The Q-lab software of the instrument was used to analyses the images.Results1The best technique of perfluoropropane-containing phospholipids-shelled microbubbles preparation was obtained according to the single-factor test, which the shear velocity was six shelves, the perfluoropropane ventilate time was30s and the shearing time was2min.2According to the orthogonal experimental test, the optimum formula of the preparation was that: the total concentration of lipid was1×10-3mol/L, the ratio of DPPE-PEG5000was8%, the ratio of DPPA was10%and the content of NaCl was 4.5mg/ml.3The best technique and formula was used to produce the perfluoropropane-containing phospholipids-coated microbubbles. The resulting preparation was white emulsion that contains microbubbles, with placement they would gradually layered, the top-layer was white microbubbles that about4mm～5mm thick. After gently turbid, the microbubbles can be dispersing as before. Under the microscope, they had uniform size and preferable dispersing and their appearance was a microsphere with bright midst. The concentration of the microbubbles was (2.99±0.19)×109/ml, the diameter was (2.46±0.05)μm and97%of them was smaller than7μm, the OFP content was (387.81±35.28) μg/ml.4The microbubbles had a good behavior at the anti-pressure test, After anti150mmHg, the concentration had no significantly difference of all the groups(P=0.051); After anti300mmHg, the concentration of5min and10min groups had significantly difference compare with the contrast group(0min)(P<0.001), but the concentration was still higher than2×109/ml, and still can fulfill with the contrast image.5Through6months observed to microbubbles stored in refrigerator at4±2°C, we found that the microbubbles can maintain stable within three months. The concentration and diameter had no significantly difference at the third month compare with original(P>0.05). At the6month, both the concentration and diameter had lightly decline, which had significant difference compared with original(0month)(P<0.001), but the concentration was still higher than2×109/ml, and the diameter was still suitable for the clinical used.6The result of contrast effect: The best dosage of the preparation was0.01ml/kg. Given0.01ml/kg microbubbles, all the New Zealand white rabbits had satisfactory ultrasound contrast images of rabbit’s kidney and liver. As for kidney images, the half life of the peak contrast time was603±47s, and the clearance time was726±6s; As for liver images, the half life of the peak contrast time was388±97, and the clearance time was718±89s, which can fulfill with the clinically application.Conclusions1The phospholipids-coated microbubbles with diameter less than7μm, concentration more than2.0×109个/ml and with relative stable property have been prepared by high speed shearing method with mixture of phospholipids and perfluoropropane.2The microbubbles concentration and diameter can be measured by Coulter counter, which is important to the quality control of the preparations.3OFP is the main component of the microbubbles, its content determination is a key index of the preparation’s quality. The OFP content is analyzed by GC-MS/SIM, which can be used to control the quality of ultrasound contrast agents.4Conserve at low temperature could help to make the microbubbles stable in a sense.5The microbubbles have a good anti-pressure quality, it’s believed that such preparation can also be stable in human microcirculation and behave as a good contrast media.6The resulting microbubbles can provide good contrast images of the rabbit’s kidney and liver, which suggest it can be a good contrast media in animal test. |
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