Assessing tumor biopsy decellularization using contact angle method

Purpose: More than 30% of new female cancer cases each year are breast cancer, according to the National Cancer Institute, whose risk increases progressively with advancing age. To date, off-target toxicity remains a main drawback of anticancer therapy. Several drug delivery systems are developed to bypass this problem, and among these the nanoparticle-based delivery platforms have proved extremely promising. A successful example of this approach is liposome formulation which today is used to encapsulate drugs such as doxorubicin (DOXIL) and paclitaxel. A critical role in the development of anticancer drugs is played by preclinical testing, but animal testing is very expensive, time-consuming, and limited due to their different physiology. Using patient tissue biopsies may help identify promising drugs to advance to clinical trials, saving time and money. Extracellular matrix (ECM) in this context is well known to present an impact on anticancer drug efficacy by acting as a barrier between drug molecules and targeting cancer cells, but little has been done so far to quantify and characterize the behavior of drugs and formulations on it. ECM hydrophobicity, in particular, can affect drug penetration through tissue and tissue membranes changing drug absorption. We hypothesize that contact angle can be used as part of the pre-clinical screening of promising anti-cancer drug candidates by assessing ECM hydrophobicity and ECM-drug interactions. Here, we optimized a tissue decellularization process to test ECM hydrophobicity and interaction with liposomes using contact angle measurement.

Methods: An in-house modified optical goniometer instrument was used to measure the contact angle on glass slides and de-identified, commercially available breast cancer tissue sections (US Biomax Inc). Samples were deparaffinized by consecutive washing cycles in xylene, ethanol, and water. The slides were dried in the incubator for 60 minutes at 60 °C to ensure adequate tissue adherence. Tissues were then decellularized by up to three freeze-thaw cycles in water 0.1% w/v SDS (Sodium dodecyl sulfate). Contact angle measurements obtained at every step of the deparaffinization and decellularization process were used to determine the optimum tissue processing method. The contact angle of liposomal suspensions was then collected on glass and tissue sections that were processed accordingly.

Results: The optimum decellularization process for cancer biopsy sections was two freeze-thaw cycles using pure water; SDS was found to cause some tissue detachment from the glass slide. After each freezing cycle, contact angle was trending upwards. This indicates that tissue decellularization results in progressively more hydrophobicity, likely due to the removal of water-soluble proteins and polysaccharides. This effect was more pronounced in tumor tissue compared to healthy tissue from the same patient. Contact angles obtained using liposomal suspensions were lower than water contact angles and followed similar patterns.

Conclusions:

-Contact angle measurements can be used to quantify and optimize tissue decellularization.

• Decellularized biopsies are slightly more hydrophobic, an effect more pronounced in tumor tissue compared to healthy.

• Liposomes reduce contact angle due to their inherent surfactant-like properties.

• Contact angle may be used to distinguish how anti-cancer liposomes interact with tumor and healthy tissue.