Biophysical Characterization of LTX-315 Anticancer Peptide Interactions with Model Membrane Platforms: Effect of Membrane Surface Charge

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.

Keywords: LTX-315; anticancer peptide; electrochemical impedance spectroscopy; membrane-peptide interactions; oncolytic; peptide; quartz crystal microbalance-dissipation.

Figure 1

Overview of LTX-315 anticancer peptide and experimental strategy: (A) amino acid structures, sequence, and 3D molecular model of LTX-315 peptide. Hydrophobic (Trp and Dip) and cationic (Lys) amino acids are depicted in yellow and blue, respectively; (B) proposed biological mechanism of how LTX-315 peptide exhibits anticancer activity based on cancer cell membrane disruption; and (C) experimental strategy to track membrane-peptide interactions using the supported lipid bilayer (low curvature), intact vesicle (high curvature), and tethered bilayer lipid membrane platforms with different membrane surface charges. Measurements were conducted using the quartz crystal microbalance-dissipation (QCM-D) and electrochemical impedance spectroscopy (EIS) techniques.

Figure 2

DLS characterization of LTX-315 peptide effects on suspended lipid vesicles with different membrane surface charges. The size distribution of solution-phase lipid vesicles was obtained before (blue lines) and after incubating lipid vesicles with LTX-315 peptide (red lines) by dynamic light scattering (DLS) measurements. Corresponding changes in the size distribution are presented as Gaussian profiles for 70/30 DOPC/DOPS (top), 100 DOPC (middle), and 70/30 DOPC/DOEPC (bottom) lipid vesicles.

Figure 3

QCM-D tracking of LTX-315 peptide interactions with intact vesicle adlayer depending on membrane surface charge: (A) schematic illustration of intact vesicle adlayer on TiO₂-coated sensor surface before and after peptide addition; (B–F) corresponding QCM-D measurement kinetics for peptide addition to (B) 70/30 DOPC/DOPS, (C) 85/15 DOPC/DOPS, (D) 100 DOPC, (E) 85/15 DOPC/DOEPC; and (F) 70/30 DOPC/DOEPC lipid vesicle adlayers. In each panel, the QCM-D Δf (top, blue squares) and ΔD (bottom, red triangles) shifts are presented as a function of time and the initial baseline signals correspond to the intact vesicle adlayer. Stages 1, 2, and 3 correspond to intact vesicle platform alone, during peptide addition, and during buffer washing, respectively. Arrows i and ii denote peptide addition and buffer washing steps, respectively.

Figure 4

Summary of QCM-D measurement responses for LTX-315 peptide interactions with intact vesicle adlayers. Maximum responses of the (A) Δf and (B) ΔD shifts are presented based on the data in Figure 3 and reported as the mean ± standard deviation from n = 3 measurements.

Figure 5

QCM-D tracking of LTX-315 peptide interactions with supported lipid bilayers depending on membrane surface charge: (A) schematic illustration of supported lipid bilayer on SiO₂-coated sensor surface before and after peptide addition; (B–D) corresponding QCM-D measurement kinetics for peptide addition to (B) 70/30 DOPC/DOPS, (C) 100 DOPC, and (D) 70/30 DOPC/DOEPC supported lipid bilayers. In each panel, the QCM-D Δf (top, blue squares) and ΔD (bottom, red triangles) shifts are presented as a function of time and the initial baseline signals correspond to the supported lipid bilayer platform. Arrows i and ii denote peptide addition and buffer washing steps, respectively; (E,F) summary of QCM-D measurement responses for LTX-315 peptide interactions with supported lipid bilayers. In panels (B–D), stages 1, 2, and 3 correspond to supported lipid bilayer platform alone, during peptide addition, and during buffer washing, respectively. Maximum responses of the (E) Δf and (F) ΔD shifts are presented based on the data in panels (B–D) and are reported as the mean ± standard deviation from n = 3 measurements.

Figure 6

EIS tracking of LTX-315 peptide interactions with tethered lipid bilayers depending on membrane surface charge: (A) time-dependent changes in conductance (G_m) and capacitance (C_m) signals upon LTX-315 peptide addition to a 70/30 DOPC/DOPS tBLM platform. LTX-315 peptide was added to the tBLM platform starting at t = 10 min (arrow i), followed by a buffer washing step from t = 30 min onward (arrow ii); corresponding EIS data for (B) 85/15 DOPC/DOPS, (C) 100 DOPC, (D) 85/15 DOPC/DOEPC, and (E) 70/30 DOPC/DOEPC tBLM platform; and (F) summary of G_m and C_m shifts upon LTX-315 peptide addition (treatment) and after buffer washing (post-wash) for tBLMs with different membrane surface charges. The data are reported as the mean ± standard deviation from n = 3 measurements.

Figure 7

Schematic summary of LTX-315 peptide interactions with lipid membranes and relevant mechanistic factors. The two main tested parameters were membrane surface charge and nanoarchitecture. In general, LTX-315 preferentially disrupts negatively charged membranes and demonstrates enhanced attachment to curved membranes over planar membranes. It should be noted that peptide attachment is a necessary but insufficient step for triggering membrane disruption, which was strongly related to the lipid composition.