液态氟碳纳米脂质微粒与全氟丙烷纳米脂质微泡的物理特性及体外显影效果比较

An in vitro comparative study of the physical and imaging enhancement characteristics between two nano-scale ultrasound contrast agents： PFOB lipid particles and C3Fs lipid microbubbles

目的制备液态氟碳（PFOB）纳米脂质微粒与全氟丙烷气体（c3F8）纳米脂质微泡，比较两者一般理化性质及体外显影效果。方法分别制备PFOB纳米脂质微粒与C3F8纳米脂质微泡，检测2种造影剂形态、粒径、表面电位、浓度及稳定性，并进行比较。制备生物素化（外膜标记生物素）及空白（外膜未标记生物素）的PFOB脂质微粒及C，R微泡造影剂。以高频探头观察各组造影剂加入亲和素前后的显影效果，并运用Matlab软件获得图像平均灰度值，再进行统计分析。2组间比较采用两样本t检验，同一样本多个时间点观察比较采用重复测量方差分析，组内不同时间点间两两比较采用Bonferroni法，同一样本加入亲和素前后的显影强度比较采用配对t检验。结果（1）PFOB脂质微粒与C3F8脂质微泡粒径分别为（152．30±35．99）nm和（774．59±108．59）nm，前者明显小于后者（t=-24．327，P〈0．001）；表面电位分别为（-40．90±6．51）mV和（-14．80±3．97）mV，前者明显大于后者（t=-15．308，P〈0．001）。（2）PFOB脂质微粒造影剂的浓度及粒径在整个观察期间内无明显改变[c0h：（2．28±0．64）×10^11／ml，C（2．06±0．53）×10^11／ml；D0h：（152．30±35．99）nm，D。月：（178．80±63．07）nm]。c3F8脂质微泡造影剂制备后放置12h，浓度[c0h：（4．08±0．96）X10^10／ml，C㈨：（3．25±1．02）X10^10／nil]尚未发生明显改变，而放置24h、2d、4d及1周后浓度明显减低[白h：（z28±Q73）X10”／ml，c2d：（1．56±n54）X10^10／ml，qd：（1．03±n37）X10^10／ml，C1目：（0．74±0．24）×10^10／ml；F=78．515，P〈0．01]；c3F8微泡造影剂放置2d内粒径[Dnh：（774．59±108．59）nm，D2d：（1020．68±223．64）nm]未见明显变化，放置4d及1周后粒径明显增大[D4d：（1391．67±268．65）nm，Dl（1532．41±326．25）nm，F=50．772，P〈0．01]。（3）加入亲和素前，空白及生物素化PFOB脂质微粒均未见明显显影，而空白及生物素化C3F8脂质微泡均有较高的显影强度（31．34±7．03与28．75±7．18）；加入亲和素之后，生物素化PFOB脂质微粒造影剂显影明显增强（2．18±0．71和82．19±15．68，t=-15．698，P〈0．001），而空白PFOB脂质微粒及2组脂质C3F8微泡显影情况无明显改变。结论与C3F8脂质微泡造影剂相比，以PFOB为核心的纳米脂质微粒造影剂在粒径优化和稳定性方面具有更好的可控性，且具有独特的显影特性，有利于制备靶向功能的超声造影剂。

Objective To prepare two nano-scale ultrasound contrast agents, lipid particles and microbubbles, and to compare their physical and imaging enhancement characteristics. Methods Lipid nanopartieles were prepared with phospholipid and liquid perfiuorocarbon （PFOB）. Lipid microbubbles were prepared with phospholipid and perfluoropropane （C3 Fs ）. Physical and chemical properties of the two ultrasound contrast agents were measured, including morphology, size, zeta potential, concentration and stability. Both lipid nanoparticles and microbubbles with/without biotin were imaged using a 12.0 MHz linear-array transducer with/without avidin. The imaging data were analyzed by using Matlab software. Comparative data between the two groups were statistically analyzed by two-sample t test. Repeated measures analysis of variance was used among the observations at multiple time points within the same sample and Bonferroni method was used for post hoc analysis. Comparison of the signal intensity with and without avidin of the same sample was performed by paired t test. Results （ 1 ） Microscopically, both PFOB lipid particles and C3Fs microbubbles were in form of spheres. Their particle diameters were （152.30 ± 35.99） nm and （774. 59 ± 108.59） nm, respectively （t = - 24. 327, P 〈 0. 001 ）. Their zeta potential was （ - 40.90± 6. 51 ） mV and （ - 14.80 ± 3.97） mV, respectively （ t = - 15. 308, P 〈 0.001 ）. （2） Concentration and size of PFOB lipid particles remained stable during the whole observation period （ Co h = （2. 28 ± 0.64） X 10n/ml, Cw = （2.06 ± 0.53） xl0n/ml; Doh= （152.30 ±35.99） nm, Dw= （178.80 ±63.07） nm）. Concentrations of lipid microbubbles were stable within 12 h （ Co h = （ 4. 08 ± 0. 96 ） X lO/ml ; Ct2 h = （3.25 ± 1.02） X 10/ml） ; and then reduced significantly after 24 h, 2 d, 4 d and 1 week （ Ch ： （2.28 ± 0.73 ） X 10^10/ml ; C2 d ： （ 1.56 ± O. 54） x 101/ml ; C4 d ： （ 1.03 ± 0.37） X 10^10/ml ; G w ： （0. 74 ± 0.24） X 10/ml; F =78. 515, P 〈0.01 ）. Diameters of the particles remained stable within 2 d （DO h = （774.59 ：t： 108. 59 ） rim, D2 d = （ 1020.68 ± 223.64） nm） ; and then increased after 4 d and 1 week （D4 d ： （ 1391.67 ± 268.65 ） nm ; D1 ： （ 1532.41 ± 326.25） nm, respectively, F = 50. 772, P 〈 0.01 ）. （3） Neither control nor biotinylated PFOB particles were visible without avidin. With the addition of avidin, significantly enhanced images were generated by biotinylated PFOB particles （2.18 ±0.71, 82.19 ± 15.68, t = - 15. 698, P 〈 0. 001 ） but not with the control PFOB particles. Both control and biotinylated C3Fs microbubbles showed no significant difference in their signal intensity with/without avidin （29.16 ± 6.84 and 31.34 ± 7.03, 33.69±8.24 and28.75± 7.18, t=0.653, -1.393, bothP〉0.05）. Conclusion Compared to C3 Fs microbubbles,PFOB particles have smaller size, greater stability, higher SNR ratio and better imaging enhancement characteristics, therefore, more suitable for use as the target contrast agent for ultrasound imaging.