| Ultrasound contrast agents (UCA) 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.However, the ultrasound contrast agents mainly are made of gas at present, the sizes are general on micron level. Microbubble ultrasound contrast agent (UCA) can only image and exert therapy within tumor vessel and there existed many problems of nanoscale ultrasound contrast agent such as poor imaging and can not cure. To solve the problem mentioned above, base on the study of microbubble and ultrasound contrast agents, tumor-targeted ultrasound contrast agent which was sensitive to ultrasound was investigated in this paper.Curcumin is a polyphenol compound with the beta diketone structure that is extracted from turmeric rhizome zingiberaceae plants. In recent years, it is found that curcumin has a range of pharmacological effects, and low toxicity, it has the function of the anti-tumor, anti-inflammatory, anti-oxidation,reducing blood fat and protecting the liver and kidney. It has became a hotspot in the research, so we choose the curcumin as the model drug.The ultrasound contrast agent was prepared with curmine (CUR) as the model drug, PEG-PCL as film-former, perfluoropentane (PFP)(boiling point29℃) as the imaging gas.The morphological observation was performed using optical microscope and transmission electron microscopy (TEM), then its behaviors in vitro and in vivo were investigated. The main contents were described in details as follows:1. Preparation and characterization of CUR-loaded micelles:dialysis method and solvent evaporation method were used to preparation for nano-micelles, the good biocompatibility of polyethylene glycol, poly caprolactone amphiphilic(PEG-PCL) was used as film-forming material, the anticancer curcumin was used as the model drug. HPLC analysis method was established in vitro, the formulation and preparation method of CUR micelles were optimized, using the particle size distribution, loading amount and encapsulation efficiency as the evaluation indexs. Negative staining TEM imaging showed the structure of micelles was globular and core-shell, with hydrophobic domain forming core and hydrophilic domain forming shell. The average size was (64±1.52) nm, drug loading capacity was (8.5±0.15)%and the encapsulation efficiency was (93.9±1.46)%.2. The preparation and structural characteristics of the ultrasound contrast agent: The UCAs were prepared by the ultrasound injection method. Its size distribution was uniform with the average size400nm and the technique reproducibility was good. After incubated in37℃water,the structure of the UCA was observed by light microscope,and the results showed that it was core-shell and CUR was localized in the hydrophobic compartment.3. Characteristics of CUR-loaded ultrasound contrast agent:The temperature sensibility, ultrasound sensibility and dilution stability were investigated, the size and the morphology were used as the evaluation indexs. The results showed that the size of ultrasound contrast agent changed with the temperature. Ultrasound could make nanobubbles incorporate into microbubbles, and microbubbles disrupted when ultrasound power was increased, thus releasing CUR rapidly. Ultrasound contrast agent was stable for a few days when stored in4℃, ultrasound contrast agent was incubated in37℃water for10min, only0.89%CUR was released from the CUR-loaded ultrasound contrast agent. The stability was good.4. Release experiments of CUR-loaded ultrasound contrast agent in vitro: We took the same volume of nanoscale ultrasound contrast agent, then divided them into two groups.They were dissolved in the10ml PBS respectively. The first group was dealed with the ultrasonic treatment in the pool at the constant temperature of37℃; the second group was placed in the constant temperature pool without any ultrasonic treatment processing. The ultrasound contrast agents in vitro ultrasonic environment cumulative release dosage of90.04%, far higher than the control group without any ultrasonic processing of polymer nano ultrasonic contrast agents, initial in vitro release quantity0.89%, experimental results showed that UCA release characteristics without ultrasound irradiation was consistent with the release of sustained-release preparation and ultrasonic control drug release effect was obvious.5. CUR-loaded ultrasound contrast agent imaging experiments in vivo and in vitro:In vitro experiment, we set the blank degassed water group and contrast group, using the philips IU22ultrasound diagnostic apparatus, L9-3type probe, MI=0.6, the Depth of3.5cm,we could clearly observe group with the polymer nano ultrasonic contrast agents have obvious echoes, imaging effect is remarkable, and the duration was longer. In vivo experiments, after the tail vein injection of polymer nano ultrasonic contrast agents, adjust the relevant index for liver ultrasound imaging, record the image data until angiography image is clear,we could observe the polymer nano ultrasonic contrast agents have obvious echoes, imaging effect is remarkable, and the duration is longer.The liver parenchyma imaging was strengthened after30mins and increased up to peak after50mins,the liver liver parenchyma imaging could last for3hours.6. Pharmaeodynamics of CUR-loaded ultrasound contrast agent:Subcutaneous tumors model was established using Kunming mice. Pharmacodynamics of CUR-loaded ultrasound contrast agents were investigated using tumor-bearing mice. The tumor weight differentiated signifieantly between the control and experimental group (P<0.01). There were distinguished differences between the ultrasound group and non-ultrasound group (P<0.01). The results showed that CUR-loaded ultrasound contrast agent had good passive targeting action and ultrasound can facilitate it into tumor cells.Tumor targeting and ultrasound-sensible CUR-loaded ultrasound contrast agents with uniform size distribution and good stability when stored in4℃were prepared in this paper. Its core-shell structure was demonstrated and CUR was localized in the hydrophobic wall of the ultrasound contrast agent. Ultrasound imaging experiments in vivo and in vitro showed its good development effect. It had good tumor targeting for tumor-bearing mice after iv, and also exerted good pharmacodynamic action when using ultrasound. |
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