(M1130-05-26) Effect of Tumor Grade and Type on ECM-Liposome Interactions Measured by Contact Angle

Purpose: In 2020, breast cancer accounted for over 2.26 million new cases and 685,000 deaths worldwide [1]. Despite advances in treatments and early diagnosis have contributed to reducing mortality, the annual incidence continues to increase and the mortality rate shows remarkable differences highlighting the presence of consistent health disparities [2]. These disparities are complex and influenced by several factors, including environmental, biological, and tumor composition factors. The extracellular matrix (ECM) plays a crucial role in tumor development [3] and has been identified as a biological factor contributing to these disparities. Variations in the ECM structure may impede the effectiveness of targeted tumor therapies, limiting their ability to reach the tumor site and reducing drug absorption. Our group has previously conducted research showing that contact angle measurements can characterize the tumor ECM, providing insights into its surface hydrophobicity [4]. In this study, we used our custom-made goniometer to characterize and quantify the impact of cancer types (metastatic, grade I, II, and III) on tumor ECM hydrophobicity through contact angle measurements. Furthermore, we will study the effect of liposomal formulations on the contact angle of tumor tissue ECM. Our goal is to quantify the physiochemical characteristics of both the liposomal structure and the tumor microenvironment (ECM), aiming to enhance targeted breast cancer therapy and reduce disparities in treatment outcomes.

Methods: Liposomes of Doxil-like compositions with varying polyethylene glycol (PEG) content, molecular weight, and end-cap group were prepared using the thin-film layer hydration method followed by sonication for particle size reduction. De-identified breast cancer tissue sections with demographic data were purchased from US Biomax Inc. Tissue sections were deparaffinized in xylene, ethanol, and water in consecutive washing cycles. Tissues were decellularized with three freeze-thaw cycles in water. Water contact angles were measured on glass slides and decellularized tissue sections using a customized optical goniometer. Tissue sections were treated with Doxil-like liposomes incubating them for 2 min in liposomal suspensions and rinsing them with DI water 3 times.

Results: The contact angle varies among different tissues, with fibrotic tissues like metastatic tumors tending to have a higher contact angle and thus higher hydrophobicity. Liposome incubation reduced the water contact angle on glass slides and tissue sections in a concentration-dependent and composition-dependent fashion. Doxil-like liposomes decrease the water contact angle for lipid concentrations higher than 0.5 mg/ml, with the reduction increasing as the concentration rises and reaching a plateau for lipid concentrations higher than 1.5 mg/m. PEG-free liposomes exhibited the lowest activity in contact angle reduction, suggesting PEG's central role in the surfactant-like activity of liposomes. Changes in PEG end-cap and molecular weight influence both, liposome surface activity and tissue retention. PEGylated liposomes are more retained by metastatic tissues and highly hydrophobic tissues, indicating that PEG enhances liposome adsorption in more hydrophobic environments.

Conclusion: The contact angle method for tumor ECM characterization developed in our laboratory enables the quantification of tumor ECM features through a liquid-solid dynamic interaction approach. Our data highlight the importance of PEG as a surface molecule on contact angle-measured interactions between anti-cancer liposomes and breast cancer tumors, consistent with clinical testing of Doxil® where the inclusion of PEG was crucial for therapeutic efficacy. In addition to liposome composition, tumor type (grade or metastatic) can be quantified with this method, permitting an increased understanding of the role that tissue surface properties play in the efficacy of anticancer nano-therapies. The modified contact angle method presented here can be used to quantify tumor interactions with liposomes presenting itself as a preclinical tool to screen drug delivery systems and predict their efficacy by quantifying their target tissue interactions.

References: [1] Global Cancer Statistics 2020: GLOBOCAN Estimates of Incidence and Mortality Worldwide for 36 Cancers in 185 Countries. CA Cancer J Clin 71, 209–249
[2] Yedjou CG, Sims JN, Miele L, Noubissi F, Lowe L, Fonseca DD, Alo RA, Payton M, Tchounwou PB. Health and Racial Disparity in Breast Cancer. Adv Exp Med Biol. 2019;1152:31-49. doi: 10.1007/978-3-030-20301-6_3. PMID: 31456178; PMCID: PMC6941147.
[3] Huang, J., Zhang, L., Wan, D. et al. Extracellular matrix and its therapeutic potential for cancer treatment. Sig Transduct Target Ther 6, 153 (2021). https://doi.org/10.1038/s41392-021-00544-0
[4] Rincon J, Kastellorizios M. Novel Method to Obtain Contact Angles of Tumor Biopsies. ACS Omega. 2023 Jul 17;8(30):26965-26972. doi: 10.1021/acsomega.3c01792. PMID: 37546668

Acknowledgements: This work is partially supported by National Institute on Minority Health and Health Disparities (Grant #2U54MD006882-06) and by the Pharmaceutical Research and Manufacturers of America Foundation, 2019 Research Starter Grant in Pharmaceutics


Water Contact angles of different breast tissue sections (Normal Adjacent, Cancer Adjacent, Invasive Ductal carcinoma, and Invasive Lobular carcinoma) before and after treatment with liposomes 100% and 0 % PEG
PEGylation significantly changes liposome-tissue interaction enhancing the adhesion of liposomes on the surface thus increasing water contact angle reduction. Remarkable differences in contact angle are observed between tissue types.


Difference in water contact angle on tissue sections before and after treatment with liposomes 100% and 0% PEG.




Cumulative graph of contact angle differences calculated by subtracting from the contact angle of pure water on glass (internal standard) the contact angle of liposomal suspensions at decreasing concentration (3\1.5\0.75\0.5\0.25\0.1 mg/ml) by varying the end-cap functionalization and the molecular weight(MW) of the DSPE-PEG present in the liposomal membrane in a Doxil-like ratio (HSPC/DSPE-PEG/Cholesterol ratio 3:1:1) (MW 2000-5000 and end-cap NH2, COOH, OCH3, Bis.DSPE)