Ultrasound (US) imaging is a safe medical tool for disease diagnosis with the advantages of non-invasiveness, non-ionizing radiation, low cost and real-time visualization. To improve imaging quality and resolution, ultrasound contrast agents have been developed, such as gas-filled microbubbles (MBs) stabilized by proteins, lipids, polymers, and inorganic nanoparticles, which can increase ultrasound reflectivity and prolong the transmission of ultrasound signals. Organ residence time. The ultrasonic signal of MBs is greatly affected by the physical and chemical properties of the shell material. Although cross-linking can effectively strengthen the polymer shell and enhance the stability of MBs, its compressibility will be further reduced and nonlinear oscillation will be greatly reduced. Large suppression, weakening the effect of contrast . Therefore, how to resolve the contradiction between the long-term stability and contrast effect of MBs ultrasound contrast agents remains a difficult challenge.
Based on this, Professor Liu Shiyong of the University of Science and Technology of China Professor , Professor Hu Jinming , Professor Shen Aizong of the First Affiliated Hospital of the University of Science and Technology of China Professor Shen Aizong and Professor He Nian'an collaborated to synthesize a series of amphipathic copolypeptides and used them as stable Shell material for MBs filled with perfluorocarbon (PFC). Among them, the hydrophilic polyethylene glycol (PEG) block and the fluorinated perfluoropolyoxymethylene block constitute the outer and inner layers of the MBs shell respectively. Fluorinated blocks with lower interfacial tension and lower Laplace pressure can improve the stability of MBs when in contact with internally loaded octafluoropropane (C3F8). At the same time, the authors also synthesized a copolypeptide containing a cross-linkable diacetylene block in the side chain. UV-induced cross-linking can further improve the stability of MBs, but the US signal decreases because the nonlinear oscillation is suppressed. In order to solve this problem, the authors synthesized a copolymer with maleimide (MI) at the end. After covalently coupling with plasma proteins such as albumin through in situ Michael addition reaction, the MI-modified MBs Nonlinear oscillation is significantly enhanced.
Figure 1 Synthesis of amphiphilic copolypeptides and preparation of MBs Preparation and characterization of
MBs
copolypeptides were hydrated in PBS buffer to obtain a translucent suspension, which was further stirred at 50 oC for 48 h to obtain a micellar dispersion. Then, C3F8 was added to it and the temperature was lowered to 0oC, and C3F8-loaded MBs were obtained under ultrasound, and the size of MBs could be controlled by changing the ultrasound time. After being placed in 4oC for 14 days, the size and number of C3F8-loaded MBs did not change significantly, while the particle size of commercial SonoVueTMMBs increased significantly and the number decreased significantly, which may be due to the leakage of the load gas. After being left for 14 days, the copolypeptide-stabilized MBs could completely recover their macroscopic appearance by hand shaking, with the size slightly increasing from ~2.4 μm to ~3 μm.
Figure 2 Characterization of copolypeptide-stabilized MBs
Ultrasound imaging performance of copolypeptide-stabilized MBs in vitro
Then, the authors studied the contrast-enhanced ultrasound imaging (CEUS) performance of the prepared MBs under different BSA concentrations. The original gray value of the image is 98. When the BSA concentrations are 1, 3 and 10 g/L, after incubation for 1 minute, the gray value is enhanced to 135, 170 and 198 respectively. In addition to enhancing signal strength, covalent binding of BSA can reflect second harmonic signals, greatly increasing the US imaging time window. In CEUS mode, the signal intensity of copolypeptide-stabilized MBs was only weakened by 20% within 300 s after incubation with 10 g/L BSA, while the signal of SonoVueTMMBs was significantly weakened under the same incubation time. In vitro imaging experiments show that SonoVueTMMBs loses signal after 120 seconds. In contrast, copolypeptide-stabilized MBs only dimmed at the bottom of the tube, while the top remained bright after 300 s, indicating that these MBs have higher signal intensity and longer imaging windows.
Figure 3 In vitro ultrasound imaging performance of copolypeptide-stabilized MBs
In vivo ultrasound imaging performance of copolypeptide-stabilized MBs
Next, the authors further evaluated the in vivo US imaging performance of copolypeptide-stabilized MBs in rabbit . After intravenous injection of copolypeptide-stabilized MBs, rabbit livers and kidneys were harvested for B-mode and CEUS-mode US imaging.The contrast improvement effect of MBs stabilized by copolypeptide BP1 was not obvious, while in CEUS imaging mode, the signals of MBs stabilized by copolypeptide BP5 and the commercial product SonoVueTMMBs were improved, indicating that BP5-stabilized MBs have the ability to serve as an efficient ultrasound Potential of contrast agents. Compared with BP1-stabilized MBs, the US imaging ability of BP5-stabilized MBs in vivo was significantly enhanced, which may be attributed to the in situ binding of MI groups to plasma proteins, further indicating that the binding of albumin can significantly enhance the US imaging ability of BP5-stabilized MBs. US signal. Then, the authors studied the time-dependent changes in gray value of rabbit liver and kidney in B mode and CEUS mode after intravenous injection of BP5-stabilized MBs and SonoVueTMMBs. The B-mode signal of BP5 stabilized MBs decays relatively slowly, and the first harmonic enhancement at 100 s is twice that of SonoVueTMMBs. In CEUS mode, the gray value of BP5 stable MBs is always higher than SonoVueTMMBs. Compared with BP1-stabilized MBs, the image intensity of BP5-stabilized MBs in B mode is similar, while its gray value in CEUS mode (~120) is 1.7 times that of BP1-stabilized MBs (~70). This is because plasma proteins can be in situ coupled to BP5-stabilized MBs through the MI group, improving the nonlinear oscillation of MBs.
Figure 4 Classic B mode and CEUS mode imaging performance
Summary: The authors of synthesized block copolypeptides and used them to stabilize perfluorocarbons to obtain MBs with controllable size and narrow size distribution, which can be further used as ultrasound contrast agents. Due to the lower interfacial tension, the stability of MBs is greatly improved. In addition, cross-linking can further enhance the stability of MBs, allowing for a longer imaging time window compared with SonoVueTMMBs on the market. More importantly, the authors proposed an in-situ surface modification method to covalently couple albumin to the surface of MBs to increase the second harmonic signal intensity of MBs, solving the problem of long-term stability and long-term stability of ultrasound contrast agents based on MBs. The paradox of short duration contrast enhancement.
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Original link:
https://onlinelibrary.wiley.com/doi/10.1002/anie.202209610
Source: Frontiers of Polymer Science
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