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Fluorescent Leakage Assay to Investigate Membrane Destabilization by Cell-Penetrating Peptide
Published on: December 19, 2020
Biophysical Characterization of LTX-315 Anticancer Peptide Interactions with Model Membrane Platforms: Effect of
Dong Jun Koo1, Tun Naw Sut1, Sue Woon Tan1
1School of Chemical Engineering and Translational Nanobioscience Research Center, Sungkyunkwan University, Suwon 16419, Korea.
Abstract:
LTX-315 is a clinical-stage, anticancer peptide therapeutic that disrupts cancer cell membranes. Existing mechanistic knowledge about LTX-315 has been obtained from cell-based biological assays, and there is an outstanding need to directly characterize the corresponding membrane-peptide interactions from a biophysical perspective. Herein, we investigated the membrane-disruptive properties of the LTX-315 peptide using three cell-membrane-mimicking membrane platforms on solid supports, namely the supported lipid bilayer, intact vesicle adlayer, and tethered lipid bilayer, in combination with quartz crystal microbalance-dissipation (QCM-D) and electrochemical impedance spectroscopy (EIS) measurements. The results showed that the cationic LTX-315 peptide selectively disrupted negatively charged phospholipid membranes to a greater extent than zwitterionic or positively charged phospholipid membranes, whereby electrostatic interactions were the main factor to influence peptide attachment and membrane curvature was a secondary factor. Of note, the EIS measurements showed that the LTX-315 peptide extensively and irreversibly permeabilized negatively charged, tethered lipid bilayers that contained high phosphatidylserine lipid levels representative of the outer leaflet of cancer cell membranes, while circular dichroism (CD) spectroscopy experiments indicated that the LTX-315 peptide was structureless and the corresponding membrane-disruptive interactions did not involve peptide conformational changes. Dynamic light scattering (DLS) measurements further verified that the LTX-315 peptide selectively caused irreversible disruption of negatively charged lipid vesicles. Together, our findings demonstrate that the LTX-315 peptide preferentially disrupts negatively charged phospholipid membranes in an irreversible manner, which reinforces its potential as an emerging cancer immunotherapy and offers a biophysical framework to guide future peptide engineering efforts.
Insights
The anticancer peptide LTX-315 selectively disrupts negatively charged cancer cell membranes, irreversibly damaging them without changing peptide structure. This supports its potential in cancer immunotherapy.
Area of Science:
- Biophysics
- Molecular Biology
- Cancer Therapeutics
Background:
- LTX-315 is a peptide anticancer therapeutic.
- Current understanding of LTX-315 mechanism is based on cell assays.
- Need for direct biophysical characterization of membrane-peptide interactions exists.
Purpose of the Study:
- Investigate the membrane-disruptive properties of LTX-315.
- Characterize LTX-315 interactions with cell membranes using biophysical methods.
- Determine factors influencing LTX-315 membrane disruption.
Main Methods:
- Utilized supported lipid bilayers, vesicle adlayers, and tethered lipid bilayers.
- Employed quartz crystal microbalance-dissipation (QCM-D) and electrochemical impedance spectroscopy (EIS).
- Conducted circular dichroism (CD) and dynamic light scattering (DLS) experiments.
Main Results:
- LTX-315 preferentially disrupted negatively charged phospholipid membranes.
- Electrostatic interactions were key, with membrane curvature as a secondary factor.
- EIS showed irreversible permeabilization of cancer cell membrane mimics; CD indicated no peptide conformational change.
Conclusions:
- LTX-315 irreversibly disrupts negatively charged phospholipid membranes.
- Findings support LTX-315's potential as a cancer immunotherapy agent.
- Provides a biophysical framework for future peptide engineering.
