| Background and ObjectiveUltrasound contrast agents (UCA) are the materials which can enhance visualization of blood flow and improve sensitivity and specificity of diagnosis in ultrasound image. In the past 40 years ultrasound contrast agents have been developed from the second generation of shell membrane-encapsulated, inner air-filled microbubbles to the third-generation of targeted microbubbles binding with specific ligands and nanometer scale bubbles taking the imaging of pathologic tissues through neovascular endothelium. However, this new generation agent should meet with several pivotal problems when applied in clinic, one of which is surface modification, that is, how to make high specific and active ligands linked with microbubbles surfaces firmly and effectively. Currently, the contrast agents are not easy to link with ligands for the lack of active groups, which result in the decreasing rate of combination, and can't meet the require of the developing of targeted agents because their diameter is too large to through neovascular endothelium. Therefore, developing a new type of UCA with good modifiability, submicron or nanometer in size and better stability will be of great investigative and applied value. Lots of studies have been shown that polymer and polysaccharide are comparatively low-cost and have plenty of active and modifiable groups, which are prove to have the broadest prospect in UCA.Chitosan set polymer and polysaccharide merits in a body, and have fine surface activity. It is not only non-toxic, non-irritating, of good biocompatibility and biodegradability, but also has a unique physiology and pharmacology activeness. Therefore, it has already been widely applied in each biomedicine domain. Because of poor water-solubility, chitosan should be modified or added with a specific crosslinking agent if served as shell membrane materials. It has been reported that N-carboxymethyl chitosan used as UCA membrane materials easily produced the remains of the crosslinker glutaric dialdehyde and N-hexane, and the preparation craftwork was too complex. Gang Biao-Jiang et al. introduced the carboxymethyl to the chitosan, and then prepared amphiphilic derivation N-palmitoyl chitosan (PLCS), which had fine solubility, emulsification and self-assembly capability, and therefore was a kind of ideal shell membrane materials.The current study aimed to prepare new types of microbubbles and nanobubbles using N-palmitoyl chitosan as shell membrane materials, and the bubbles size, morphology, concentration and electric potential micrographs were respectively determined by optical microscope, Coulter counter and Zeta potential. The visualization effects of agents were evaluated in animal organs. Moreover, the doses of auxiliary materials were altered to optimize the compound of bubbles, and the functions, mechanisms of these materials in the preparations were studied.Methods1. Optimization experiments of PCMB1.1 Preparation of PCMB:Microbubbles emulsion were prepared with N-palmitoyl chitosan, octadecyl glycidylether and PEG-4000. The membrane materials were firstly dissolved in 15ml distillation water while being stirred under the condition of water-bath at 50℃for 90 min. The mixture was then transferred into a 50mL-syringe after being settled to the volume of 20ml. Perfluoropropane gas was slowly infused into the syringe from the bottom. The sheering stick was placed in 2.5cm under the surface of the liquid, and then high-speed shearing was supplied. The prepared microbubbles were then sealed and stored for preservation in refrigerator at 4℃.1.2. Optimization designs of particle size of PCMB:According to the methods mentioned above, PEG-4000 was added to the four shares of PCMB with fixed and equal dosage of PLCS at the concentration of 0%,0.5%,1%,5%, respectively (Group PEG 0%,Group PEG 0.5%,Group PEG 1%,Group PEG 5%) to study the effects of PEG-4000 on PCMB. For a second time, PEG was replaced respectively by Octadecyl glycidylether (OGE) at the quality of Og,0.0015g,0.003g and 0.006g (Group OGE Og, Group OGE 0.0015g, Group OGE 0.003g, Group OGE 0.006g) to learn the influence of OGE on PCMB. Each group was made in 6 batches.1.3 The determination of physico-chemical property:One small drop of samples of each group was added onto the blood cell counting chamber to observe the morphology, size of PCMB with the microscope.50μl of PCMB diluted with 1000 times was determined with Coulter counter to gain the concentrations and average diameter of microbubbles; 100μl diluted with 50 times was tested with a zeta-sizer to analyze its potential value.2. Examination of optimized PCMB:2.1 Preparation of optimized PCMB:0.03g PLCS,0.003g Octadecyl glycidylether and 0.2g Polyethylene glycol-4000 were used to prepare N-palmitoyl chitosan microbubbles in the same way described in part (1.1).2.2 The determination of physico-chemical property of optimized PCMB:To study the stability of N-palmitoyl chitosan microbubbles, the shape, size and distribution of the microbubbles after optimization were measured with Coulter counter 24 hours,2 months and 6 months after preparation, respectively.2.3 In vivo ultrasound contrast imaging:The N-palmitoyl chitosan microbubbles were divided into three group:Group PCMB1 (24 hours after preparation), Group PCMB2 (6 months kept in 4℃after preparation), Quanfuxian was serviced as the control. Three kinds of agents were given to each rat in order by intravenous bolus-injection. The contrast-enhanced images of left ventricle, liver and kidney of each rat were required during intravenous bolus-injections. Injection interval of the microbubbles was over 15 min so as to avoid interference.Sequoia512 Ultrasonic diagnostic apparatus was used. The ultrasound probe was fixed on the surface of the rat to get satisfactory two-dimensional images of left ventricle, liver and kidney. Kept the position of the probe, instrument gain and scan depth of the apparatus invariable. CPS mode on Sequoia512 Ultrasonic diagnostic apparatus was used to verify the contrast effect of the prepared agents in the rats'left ventricular, liver and kidney. The peak intensity, time to peak intensity, half peak intensity and visual opacification time were analyzed offline with Siemens Syngo ACQ system.3. Preparation and examination of N-palmitoyl chitosan nanobubbles3.1 Preparation of nanobubbles:N-palmitoyl chitosan was added into 15ml distillation water to prepare nanobubbles in the way described in part (1.1).3.2 Characteristics and stability of nanobubbles:Characteristics and stability of nanobubbles were examined at 1 day,45 days and 90 days respectively. Both optical micrography and scanning electron micrography were performed to determine the bubbles size and morphology. The mean diameter, concentration and zeta potential of the bubbles were measured by cell counting chamber, Malvern lazer particle analyzer and zeta-sizer.3.3 In vivo ultrasound contrast imaging:0.1ml nanobubbles were given to each rat by intravenous bolus-injection. The contrast-enhanced images of left ventricle, liver and kidney for each rat were required in the way described in part (2.3).Results1. Optimization of N-palmitoyl chitosan Microbubbles1.1 Effect of PEG-4000 on PCMB:PCMB without PEG were nanobubbles with narrow particle-size distributions, and the diameter and concentration of microbubbles were gradually increased by the addition of PEG. While PEG concentration was 1%, the concentration of PCMB can reach to the highest, (8.63±0.74)x108/ml, with the mean diameter of (1277±31) nm. However, continuing to increase the dose of PEG didn't enhance the diameter and concentration of microbubbles. The higher the dose of PEG, the lower the potential value of PCMB.1.2 Effect of OGE on PCMB:PCMB without OGE had the highest concentration of (4.52±0.48)×108/ml and greatest diameter. With the addition of a small dose of OGE (0.0015g), the concentration of PCMB had no significant change, while the mean diameter was (1315±24) nm, which was significantly greater than microbubbles without crosslinker. On the contrary, further increasing the dose of OGE would reduce the concentration and diameter of PCMB. Additionally, crosslinker had no significant impact on the potential value of PCMB.2. Examination of optimized PCMB2.1 Determination of physico-chemical property of optimized PCMB:PCMB were well-distributed with a mean concentration of (2.88±0.44)×109/ml, a mean diameter of(1.31±0.07)μm and a surface potential of (55.3±2.1)mV, and 99.98% of them were less than 8μm. All of these parameters didn't change significantly in 1-day,2-months and 6-months(P>0.05). The concentration of PCMB was obviously larger than Quanfuxian[(2.88±4.49)×109/ml vs. (1.34±0.34)×109/ml, (P>0.05)], while its mean diameter was obviously smaller than Quanfuxian, [(1.31±0.07)μm vs.(2.87±0.40)μm, (P>0.05].2.2 In vivo ultrasound contrast imaging2.2.1 PCMB vs. Quanfuxian: comparison of satisfactory ultrasound contrast images of the left ventricle, liver and kidney were obtained after PCMB. TP and TTP of PCMB were similar to Quanfuxian(P> 0.05), while HPT and the visual opacification-time of PCMB were obviously longer than Quanfuxian, (P< 0.001).2.2.2 PCMB at 6-month vs. PCMB just prepared:There were no significant difference in peak intensity and peak time of both agents. The half peak intensity and visual opacification-time of both in kidney were close, whereas the former was shorter than the latter in hearts and livers [in hearts, (245.9±21.8)s vs.(316.0±23.9)s, (399.9±22.7)s vs. (545.4±18.1)s); in livers, (233.6±17.5) s vs. (336.4±22.9)s, (394.6±23.0) s vs. (489.9±29.4)s, totally P<0.001].3.1 Characteristics and stability of nanobubbles:The nanobubbles were spherical, vacuous, of diffused distribution and uniformity observed by optical microscope and scanning electron microscope. The nanobubbles with nice round-shape and uniform site-distribution were demonstrated. The mean diameter, concentration and zeta potential of the nanobubbles were(612±14)nm, (7.1±0.5)×109/ml and (52.3±0.5)mV at the 1-day, and all of parameters did not change significantly in 45-day and 90-day(P>0.05). 3.2 In vivo ultrasound contrast imaging:A significant contrast-enhancement was noted on heart and kidney during infusion of nanobubbles. VI on both tissues was (15-17) GU and (26-30) GU. The visual contrast-enhancement last up to about 10 mm.Conclusions1,A novel ultrasound microbubble was successfully prepared by N-palmitoyl chitosan, Octadecyl glycidylether and PEG-4000, with the mean diameter of (1.31±0.07)μm. It had been demonstrated that this novel microbubble can present good and prolonged ultrasound imaging in hearts, livers and kidneys of rats.2,The ideal qualitative ratio of Octadecyl glycidylether and PLCS to optimize cross-linking effect was 1:10. And increasing or reducing dose that resulted in decreased diameter of microbubbles, had no significantly effect on potential value of PCMB. PEG-4000 was contributed to increase the diameter of mircobubbles and reduce its potential value.3,A novel ultrasound nanobubbles prepared by N-palmitoyl chitosan shell membrane, with the mean diameter of (612±14) nm and the concentration of (7.1±0.5)×109/ml, preformed ideal ultrasound imaging in hearts and kidneys of rats. |
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