Proof of concept


Milestone Moments

We achieved several significant milestones in our project that highlighted the potential of our drug delivery system. Here's how we validated our approach.


Diving into Discovery: Doxorubicin and LLPS - A Perfect Match?

Our first big test was to see if liquid-liquid phase separation (LLPS) would still form when Doxorubicin (Dox) was conjugated to our system. While we were waiting for the production and purification our modified proteins, we decided to use the original proteins from our advisor, Yin, since we knew they formed LLPS for sure. We had previously explored conjugating Dox using EDC/NHS. [1] , and we thought, why not stick with that for this test? So, we conjugated Dox with Yin's original ADF-3 protein and performed dialysis. When we combined it with the MFP-1 protein, we were thrilled to see LLPS formation! This was a fantastic indication that Dox conjugation didn’t interfere with LLPS, marking our first milestone.

Figure 1: Formation of Liquid-Liquid Phase Separation (LLPS) with Modified ADF-33, MFP-1, and Doxorubicin-Conjugated Constructs

Second Win: Intein Ligation Success

Next, we shifted our focus to the intein system. We mixed our CfaC and CfaN inteins, which were attached to eGFP constructs, and then checked for successful ligation through SDS-PAGE analysis. To our excitement, we saw distinct bands that confirmed the intein ligation was working as intended! This means our payload with Dox and modified ADF-3 would effectively combine, another significant step forward in our project.

Figure 2: Assessing CfaC-CfaN Ligation through SDS-PAGE Analysis


Third Triumph: LLPS Formation with Conjugated Dox

For our final proof of concept, we combined the conjugated drug component (modified ADF3 with CfaN + Dox-conjugated CfaC-CGG8) with the modified MFP-1 (Tv1) to evaluate the system’s potential. This assembly was linked using EMCH (N-(ε-maleimidocaproic acid hydrazide)), a pH-sensitive linker. [2] EMCH ensures the stability of the drug conjugate under normal physiological conditions while facilitating targeted release in the acidic environment characteristic of cancer cells.

The successful formation of LLPS droplets confirmed the effectiveness of this strategy, demonstrating that our system could potentially deliver the drug effectively, representing a key milestone in our project by integrating all components into a functional drug delivery system.

Figure 3: Co-Assembly of MFP-1 and Doxorubicin-Conjugated ADF-3 Leading to LLPS


Looking Ahead: Future Perspectives

As we reflect on our achievements, we recognize that our work is not yet complete. There are still essential aspects of our system that require thorough testing and evaluation. Our drug delivery approach needs to be assessed for functionality, durability, and overall effectiveness. Unfortunately, due to time constraints, we were unable to access key areas before the project deadline. Moving forward, to ensure the success of our system, the following steps need to be taken.


Testing Dox Release: The pH Challenge

We haven't been able to test how effectively Dox is released from our system in relevant pH conditions using the pH-sensitive linker EMCH. This investigation is critical for ensuring that Dox can be released appropriately in environments that mimic tumor tissues, which is essential for its therapeutic efficacy.


Homing In: Peptide Binding Tests

Verifying the binding of our p160 tumor homing peptide to the MFP-1 target component on cancer cells is high on our priority list. Understanding the efficiency of this interaction will provide an understanding of our system's ability to effectively target tumor cells and improve the precision of our drug delivery mechanism.


Stability Check: Protecting Our LLPS

We will need to explore packaging methods to ensure our LLPS remains intact during delivery since it’s a very sensitive structure that is heavily affected by its environment. Testing how well the system holds up under conditions similar to the bloodstream is essential for maintaining stability until it reaches its target.


Dox Functionality Post-Release: Evaluating Effectiveness

We need to evaluate how well Dox performs after being released from LLPS. This assessment will help determine the minimal effective dosage required for achieving therapeutic outcomes and guide the optimization of our delivery strategy.


Fine-Tuning for Success

While our basic system has shown promise, there’s still plenty that needs fine-tuning. Working on these areas will be important for improving the effectiveness and reliability of our drug delivery approach. In the future, we also hope to adapt this system for not just cancer treatments but also other diseases and drugs. Although we've made significant progress, we understand that more work is necessary to fully tap into the potential of our system for enhancing treatment outcomes across various health challenges.

References

  1. Shankaranarayanan, J. S., Kanwar, J. R., Al-Juhaishi, A. J., & Kanwar, R. K. 2016. Doxorubicin conjugated to immunomodulatory anticancer lactoferrin displays improved cytotoxicity overcoming prostate cancer chemoresistance and inhibits tumour development in TRAMP mice. Scientific Reports 6:32062. DOI: https://doi.org/10.1038/srep32062.
  2. Yousefpour, P., Ahn, L., Tewksbury, J., Saha, S., Costa, S. A., Bellucci, J. J., Li, X., & Chilkoti, A. 2019. Conjugate of doxorubicin to albumin-binding peptide outperforms aldoxorubicin. Small (Weinheim an der Bergstrasse, Germany)15(12): e1804452. DOI: https://doi.org/10.1002/smll.201804452.