Introduction

Crohn’s disease (CD) affects patients by abdominal pain, diarrhea, bowel obstruction, and fistulas leading to multiple surgeries and emotional stress. Thus, early diagnostic assessment of acute disease episodes is essential to treat before severe complications occur. However, current diagnostic methods, like colonoscopy MRI or CT, have disadvanatges in terms of invasiveness or limited sensitivity¹. Here, molecular ultrasound imaging using poly(butyl cyanoacrylate) microbubbles (PBCA MB) functionalized with c(RGDfK) might be a promising alternative, which are currently prepared for a first clinical application. These probes identify α_vβ₃-integrins^2–4, which are overexpressed on endothelial cells of intestinal segments affected by CD⁵. To achieve their clinical translation for CD assessment, long-term stability and sterility must be ensured. Therefore, the lyophilization, storage, and sterilization were evaluated and their impact on the MB properties was analyzed. 

Materials and Methods

Non-targeted and targeted MBs were lyophilized and subsequently stored for 16 and 4 weeks respectively. Additionally, targeted MBs were irradiated with 25 kGy γ-rays for sterilization. The influence of these processes on the MBs was evaluated by analyzing the MB concentration and size using Coulter Counter. Ultrasound imaging in non-linear contrast mode was performed to measure their mean signal intensity. Finally, binding properties were analyzed by a self-established binding assay using immunoprecipitation. 

Results 

Lyophilization of non-targeted MB could be successfully performed without substantially changing the MB concentration and size (Fig. 1 A, B). Altough the mean signal intensity decreased by 50 % after lyophilization, the obtained intensity of I = 1000 was still high enough for the MBs to be well visible (Fig. 1 C, H).  Storage of lyophilized MB did not substantially influence their properties, which was confirmed by Coulter Counter and US-imaging (Fig. 1 A-C). Targeted MBs could also be successfully lyophilized (Fig. 1 D-G). Their concentration decreased by about 20-30 % (Fig. 1D), which may be increased by further optimizing the lyophilization cycle. Also, MB size and binding ability slightly decreased after lyophilization (Fig. 1 D, G). The binding assay confirmed that targeting ability can be retained during lyophilization since 50 % of MBs could still bind to α_vβ₃-coated magnetic particles. Comparatively, only 10 % of non-targeted MBs bound to the coated particles. In the case of targeted MBs, lyophilization did not change US signal intensity significantly (Fig. 1 F, I). Storage, transport, and sterilization did not further impact the MBs’ acoustical or binding properties (Fig. 1 D-G).

Conclusion

It could be shown that it is possible to lyophilize, sterilize, store, and transport integrin-targeted c(RGDfK)-PBCA MBs without substantially changing their properties. After GMP production, the regulatory required safety and toxicity tests will be performed in order to advance the MBs towards clinical translation to diagnose CD. 

Acknowledgment

This project is supported by the Federal Ministry of Education and Research (Germany) (01EK2201A). 

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Image/Figure Caption:

Figure 1: Influence of the lyophilization and storage time (1 day – 16 weeks) of non-targeted PBCA MB, with a concentration of c = 1 x 10⁹ MB/mL (A-C) and of the lyophilization, storage, transport, and sterilization of integrin-targeted MBs with a concentration of c = 2 x 10⁹ MB/mL (D-G) on their properties. MBs were characterized regarding their concentration c, mean diameter d, mean signal intensity during ultrasound imaging in non-linear contrast mode I(4%) and the amount of MB that bound during the binding assay to α_vβ₃-integrins. Values are represented as mean ± standard deviation. B-Mode and non-linear contrast mode images of non-targeted (H) and targeted (I) MBs. 

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