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Biophysical Study of the Potential Antitumoral Activity of NA-CATH-ATRA-1-ATRA-1 with Model Membranes

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28 October 2024

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29 October 2024

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Abstract

Breast cancer is the most commonly diagnosed cancer in women in both developed and developing countries. Due to the increasing alarms in the detection of cases every year, it is considered a priority to evaluate new molecules as potential antitumoral agents. Among these molecules, cationic peptides have emerged as promising therapeutic agents with a membrane-based mechanism of action very hard to counteract by the cancer cells. This biophysical study seeks to offer preliminary insights into how NA-CATH:ATRA-1-ATRA-1, a synthetic cationic peptide derived of the peptide NA-CATH, isolated from the species Natra aja or Chinese cobra, is a potential antitumoral compound at the membrane level on breast cancer cell lines. The interaction of the NA-CATH-ATRA-1-ATRA-1 with two lipid systems representative of tumoral and non-tumoral membranes was followed by differential scanning calorimetry and Fourier-transform infrared spectroscopy. The results showed a strong interaction of NA-CATH-ATRA-1-ATRA-1 with the lipids present in the eukaryotic membranes, especially phosphatidylserine, suggesting a mechanism based on the electrostatic interaction of the peptide with the anionic lipids of the tumoral cancer membranes. The biophysical studies are essential to understand the mechanism of action of a potential pharmaceutics.

Keywords: 
NA-CATH-ATRA-1-ATRA-1
;  
breast cancer
;  
lipid&ndash
;  
peptide interactions
;  
differential scanning calorimetry
;  
Fourier-transform infrared spectroscopy 
;  
Subject: 
Biology and Life Sciences  -   Biophysics

1. Introduction

Bioactive peptides (BAPs) are a heterogeneous group of molecules, generally between 5-50 aminoacids with a broad-spectrum of activities, expressed throughout the tree of life, from bacteria to humans [1]. One interesting source of BAPs if the snake venoms, they are mixtures of a great many distinct proteins and peptides that display an amazing diversity of structure and function [2]. NA-CATH:ATRA1-ATRA1 (NA) is a cationic (+15) and amphipathic peptide build of 34 amino acids (KRFKKFFKKLKNSVKKRAKKFFKKPKVIGVTFPF). This peptide is a designed synthetic sequence from the modification of the NA-CATH cathelicidin identified in the venom of the elapid snake Naja atra, and the substitution of the motif ATRA1 (KRFKKFFKKLK) in the structure [3]. NA has demonstrated to present a potent antimicrobial activity (EC50 = 0.51 μg/mL or 0.09 μM) against Staphylococcus aures (S. aureus) cell cultures and biofilms [3].
BAPs are mainly known for their direct interactions with microbial membranes, but in the last decade their potential activity against cancer cells has make them interesting pharmaceutical source [4]. Their most accepted mechanism of action is based on their ability to alter the membranes of target cells through complex molecular interactions between the peptide and the lipid environment of the microbial or cancer membrane [5]. BAPs mechanism involves several stages, beginning with electrostatic interactions between the positively charged residues of the peptide and the negatively charged lipids of the target membrane. In the case of cancer cells, the presence of phosphatidylserine (PS) in the outer membrane enhances the electrostatic interactions with BAPs [6,7], in contrast to normal cell membranes, which are predominantly neutral due to their zwitterionic lipid composition [8,9]. Following this, hydrophobic interactions between the phospholipid acyl chains and non-polar residues of the peptide facilitate the integration of the peptide into the membrane bilayer through various modes, such as barrel-stave, carpet detergent, and toroidal pore formations, leading to cell death [10].
The activity of BAPS makes them a promising avenue for breast cancer treatment, as they can help mitigate resistance mechanisms developed by cancer cells [11]. While traditional chemotherapeutics must penetrate cancer cells to be effective, often leading to resistance, bioactive peptides (BCPs) have the advantage of acting from outside the membrane—a mechanism that cancer cells struggle to counteract [12,13]. Previous results of our group demonstrated that NA presented antitumoral activity against two molecular subtypes of breast cancer, the MCF-7 (IC50 = 13.4 µM) and the MDA-MB-23 (IC50 = 6.4 µM) cells [14]. However, there is no information reported about the biophysical interaction of NA and lipid models representative of cancer cells. The study of the interactions responsible of the biological activity are important to understand and design new therapeutic molecules. For this reason, in this study we investigated the potential antitumoral mechanism of NA against two representative lipid models of MCF-7 and the MDA-MB-23 cell membranes. Additionally, a comparative study was performed with a non-tumoral model system representative of immortalized human keratinocytes (HaCaT) cells. The quantification of the lipid composition of the three cell lines was previously reported by Klaiss-Luna and co-workers [15]. The lipid systems were employed to investigate the lipid–peptide interaction using differential scanning calorimetry (DSC) and infrared spectroscopy (FT-IR). This study was focused on the thermodynamic properties, structure, and dynamics of both tumor and non-tumor model membranes. Additionally, the study examined the secondary structure–activity relationship of the peptide in specific aqueous and lipid environments. The findings offer valuable insights into how the lipid profile affects membrane–peptide interactions and help elucidate the antitumoral mechanism of action of NA.

2. Results

The following biophysical results about the potential antitumoral activity of NA come as a combination of techniques that studied the lipid-peptide interaction from the dynamics and structure of both, model membranes and peptide. The below mentioned with the aim to get a more comprehensive compilation of NA potential effect.

2.1. Thermotropic Phase Behavior by DSC

2.1.1. Model Membranes

Model membranes MCF-7 PC/PE/SM/PS 47:35:5:13 (w/w); MDA-MB-231 PC/PE/SM/PS 43:33:4:20 (w/w) and HaCaT PC/PE/SM 47:27:5 (w/w), were constructed from the determined phospholipid class composition after performing lipid extraction and high-performance thin-layer chromatography (HPTLC), as reported previously [16]. DSC measurements lead to the analysis of the thermotropic phase behavior along with the changes associated with lipid-peptide interactions. Each heating endotherm evidenced the melting process where lipids changed from the lamellar gel phase (Lβ) to the lamellar liquid-crystalline phase (Lα) and provided fundamental thermodynamic parameters such as main transition temperature (Tm), the temperature at which the melting process occurs, and phase transition enthalpy (∆H), the amount of energy involved in the calorimetric event.
Figure 1a illustrates the heating endotherm of breast cancer model membrane MCF-7. The broad peak represents the change from the lipid metastable gel phase to the liquid-crystalline phase with a Tm = 53.86 °C and a corresponding transition enthalpy of 38.51 kJ mol−1, as reported in Table 1. The presence of the peptide seems to generate a remarkable effect on peak height reduction as peptide concentration was increased. The strongest effect was evidenced at 10 mol% where NA reduced enthalpy to ΔH = 24.17 kJ mol−1 and induced the formation of peptide-poor lipid domains in the calorimetric trace, manifested with a shoulder at 46.19 °C and the shift of the main peak transition temperature to 54.72 °C.
The thermotropic behavior of breast cancer model membrane MDA-MB-231 is represented in Figure 1b by a broad endothermic peak with a maximum at 54.47 °C and a transition enthalpy ΔH = 41.47 kJ mol−1, reported in Table 1. The interaction with NA at 1 mol% induced phase separation of the lipid mixture with an additional peak at 42 °C, and at 5 and 10 mol%, the appearance of shoulders indicates the formation of peptide-poor lipid domains. NA caused an effect on the width of all peaks, making them sharper and more cooperative transitions, as well as increasing the enthalpy and temperature compared to the control peak.
The heating thermogram of HaCaT, the human keratinocytes model membrane, is presented in Figure 1c. The calorimetric trace registered at 42.93 °C a DPPC microdomain, and at 53.79 °C the main transition peak with an enthalpy ΔH =36.94 kJ mol−1. Upon incubation with the peptide, their interaction caused phase separation at 5 and 10 mol%, having a stronger effect at the highest evaluated concentration, in which NA induced a more cooperative transition and lowered the system's temperature to 53.10 °C.
To comprehensively analyze the peptide effect in the thermodynamic parameters, the maximum main transition temperature and molecular heat capacity were graphed as a function of peptide concentration in all model membranes, represented in Figure 2a and Figure 2b, respectively. The outcome evidenced that the more susceptible model to the presence of the peptide was the breast cancer model MCF-7, having a net change of Tm = 0.86 °C and ∆H = 13.82 kJ mol−1. Results revealed no clear tendency between the parameters and NA concentration. However, it suggests that the peptide exerts a concentration-dependent effect on model membranes.

2.1.2. Individual Phospholipids

Since model membranes are multicomponent lipid systems, we evaluated the interaction between the peptide and individual lipid components DPPC, DPPE, DPPS, SM to understand better and complement the abovementioned results. Starting with the most abundant phospholipid in the built models, the thermogram of pure DPPC vesicles shown in Figure 3a exhibited the lipid bilayer pretransition that corresponds to the change from the gel phase (Lβ) to the ripple phase (Pβ) represented by a small endothermic peak at 35.84 °C with ΔH = 2.84 kJ mol−1 The melting process from the ripple to the liquid-crystalline phase (Lα) is also evidenced by the sharp peak at 42.20 °C, indicating a cooperative transition with higher enthalpy ΔH = 32.37 kJ mol−1 (see Table 2). The interaction of NA with DPPC vesicles caused the abolishment of the ripple phase at 5 and 10 mol %. It altered the cooperative chain-melting transition, significantly reducing the peak height and enthalpy to ΔH = 24.99 kJ mol−1 with minor changes in Tm.
The heating endotherm of DPPE, the second most abundant lipid in the models, is presented in Figure 3b. Its calorimetric trace evidenced a highly cooperative transition by the single sharp peak at 65.04 °C with an enthalpy ΔH = 35.60 kJ mol−1. The incubation of NA and DPPE vesicles remarkably reduced the original peak height of the DPPE transition at all evaluated concentrations, as reported in Table 2. However, the Tm of the pure lipid was not significantly affected. Furthermore, the heating endotherm obtained for the anionic lipid, DPPS, is illustrated in Figure 3c with a single sharp transition peak centered at 54.69 °C and a transition enthalpy ΔH = 35.48 kJ mol−1. Results highlight that the presence of NA in DPPS liposomes caused significant decrease of the original peak enthalpy in a concentration-dependent manner, also the concomitant reduction in lipid transition temperature was observed until 47.6 °C (ΔH = 20.18 kJ mol−1) at 10 mol%.
Finally, the SM thermogram is represented in Figure 3d with a broad endothermic peak centered at 39.74 °C and a transition enthalpy ΔH = 42.00 kJ mol−1, reported in Table 2. SM seems to be dramatically affected by the presence of NA peptide. Its peak height was reduced 1.2 times at 1 mol%, and at the highest concentrations, the phase transition of SM liposomes was almost completely abolished.
The maximum values of transition temperature and molecular heat capacity as a function of peptide concentration in mol % are shown in Figure 4a and 4b, respectively. Results evidence that NA-CATH-ATRA1-ATRA1 did not significantly affect the transition temperature of DPPC and DPPE liposomes; however, it slightly increased the Tm of SM vesicles from 5 mol%. The strongest effect was noticed with DPPS vesicles at 10 mol%, resulting in a net change ∆Tm = 7.04 °C compared to the control. In the case of the specific heat capacity, there was a clear tendency to decrease when peptide concentration was increased. Figure 4b highlights the effect in SM and DPPS liposomes with a net change ΔH = 22.67 and 15.3 kJ mol−1, respectively. The above suggests a selectivity of NA peptide towards anionic PS and sphingolipid SM lipid systems.

2.2. Lipid Order and Interfacial Hydration by Infrared Spectroscopy

Membrane structure insights were acquired using Infrared Spectroscopy in ATR mode to follow the lipid phase transition and the peptide effect on this process by analyzing the vibrational bands that offer information about lipid order and interfacial hydration, which are the change in the maximum methylene symmetric stretching (ʋsCH2) and the ester carbonyl stretching (ʋsC=O) vibrational modes. The melting process from the metastable lamellar gel phase to the liquid-crystalline phase is seen for the hydrophobic core of the membrane around 2850-2853 cm−1 due to the methylene isomerization trans to gauche, meanwhile for the interfacial hydration is seen between 1840 - 1835 cm−1 due to the incorporation of water molecules at the carbonyl backbone.

2.3. Lipid Order and Interfacial Hydration by Infrared Spectroscopy

Membrane structure insights were acquired using Infrared Spectroscopy in ATR mode to follow the lipid phase transition and the peptide effect on this process by analyzing the vibrational bands that offer information about lipid order and interfacial hydration, which are the change in the maximum methylene symmetric stretching (ʋsCH2) and the ester carbonyl stretching (ʋsC=O) vibrational modes. The melting process from the metastable lamellar gel phase to the liquid-crystalline phase is seen for the hydrophobic core of the membrane around 2850-2853 cm−1 due to the methylene isomerization trans to gauche, meanwhile for the interfacial hydration is seen between 1840-1835 cm−1 due to the incorporation of water molecules at the carbonyl backbone.
Figure 5a-c represents the peptide effect on the maximum symmetric stretching CH2. For the breast cancer model membranes NA-CATH-ATRA1-ATRA1 showed to significantly shift the initial Tm of the MCF-7 model to higher temperatures as seen in Figure 5a and data in Supplementary A1. The incubation of the peptide increased the rigidity of the MCF-7 bilayer until a Tm = 55.1 °C (ΔTm = 2.5 °C) with a significant effect in wavenumbers associated to liquid-crystalline phase. This finding could suggest that NA-CATH-ATRA1-ATRA1 strongly binds through an electrostatic interaction with the negatively charged headgroups, causing a relief of charge repulsion and the consequent closer fatty acid chain packing, noteworthy in the gel phase. MDA-MB-231 and NA-CATHATRA1- ATRA1 interaction in Figure 5b evidenced the increase in wavenumbers of both Lβ and Lα phases, as the concentration was increased and inducing transition temperature change ΔTm = 1.2 °C to higher temperatures, making also a more rigid system. The opposite was evidenced with the non-tumoral HaCaT model membrane, Figure 5c illustrates the increase in wavenumbers of the Lβ and Lα phases as the peptide concentration increased, aside from the Tm shift to lower temperatures (see supplementary data). Thus, could be interpreted as a NA fluidizing effect on the HaCaT model membrane, which was stronger at 10 mol%, giving a net change ΔTm = 0.8 °C.
Figure 5d-f represents the peptide effect on the maximum symmetric stretching C=O. The interaction between MCF-7 supported lipid bilayers (SLBs) and NA-CATH-ATRA1-ATRA1 evidenced in Figure 5d a strong exclusion of water molecules effect with the increasing wavenumbers as the concentration was also increased; indeed, the effect was more favorable in the Lα phase. The peptide effect on the MDA-MB-231 model membrane represented in Figure 5e reflected a dehydration effect of the carbonyl group when the peptide concentration increased. Lasting the incubation of HaCaT SLBs with increasing concentration of NA peptide resulted in a shift towards higher wavenumbers. These results indicate an overall weakening of hydrogen bonds that could suggest that peptide binds and releases water molecules from the lipid surface.

2.4. Peptide Conformational Analysis

The secondary structure of NA-CATH-ATRA1-ATRA1 in solution was studied using infrared spectroscopy. The BPROT1 internal calibration of the peptide spectra suggested that NA is unordered in aqueous buffer and in presence of POPC neutral small unilamellar vesicles. However, it adopted a slightly α-helix conformation in the presence of multicomponent lipid vesicles. The conformational change with the HaCaT model membrane was negligible but more pronounced with tumoral model membranes, higher in the presence of MCF-7 vesicles. The most significant α-helical content was evidenced in the interaction with anionic lipid vesicles POPS giving a 62% α-helix adoption. Results suggest that NA-CATH-ATRA1-ATRA1 is an environment-sensitive peptide to negatively charged and hydrophobic environments that stabilize its conformational structure.
Table 3. Conformational analysis of NA-CATH-ATRA1-ATRA1 peptide in aqueous and hydrophobic lipid environments at 37 °C.
Table 3. Conformational analysis of NA-CATH-ATRA1-ATRA1 peptide in aqueous and hydrophobic lipid environments at 37 °C.
SUVs α-helical content (%)
Hepes* -
POPC -
NA-CATH-ATRA1-ATRA1 + POPS 62.2
MCF-7 4.2
MDA-MB-231 1.2
HaCaT 0.3
*Buffer described in Section 4.4.

3. Discussion

Bioactive peptides are emerging as prospective pharmaceutical sources to treat several types of human diseases. The BAP of interest for this research was the NA-CATH-ATRA1-ATRA1 (NA), KRFKKFFKKLKNSVKKRFKKFFKKLKVIGVTFPF, a synthetic peptide designed and modified from the Chinese cobra Naja atra host defense peptide NA-CATH, KRFKKFFKKLKNSVKKRAKKFFKKPKVIGVTFPF, by substituting alanine to phenylalanine at position 18 and proline to leucine at position 25, with the aim to increase its hydrophobic character and improve the antimicrobial activity already reported from the parental peptide against Staphylococcus aureus [3]. Dean and collaborators reported that NA evidenced greater antimicrobial and antibiofilm potency against the pathogen S. aureus and associated it to a possible peptide structure factor, especially in the presence of anionic membrane environment [3]. Since BAPs commonly report a broad spectrum activity, specially a concomitant antimicrobial-antitumoral activity [17], our research group has been interested in evaluating the antitumoral activity of NA-CATH-ATRA1-ATRA1. Previous results concerning the half maximal inhibitory concentration in breast cancer cell lines MCF-7, MDA-MB-231 evidenced a promising antitumoral activity [14], however how it exerts its activity is still unknown. The present paper contributes to study the interaction of NA and lipid systems at the membrane level and explores its potential antitumoral molecular mechanism through the biophysical techniques: Differential Scanning Calorimetry and Infrared Spectroscopy using lipid models of biological cell membranes in established conditions.
The outcome of this work is discussed in this section based on the physicochemical properties of the peptide and the lipid composition of model membranes. The latter emphasizes in the presence of phosphatidylserine in the cancer model membranes to resemble the negatively charged environment at membrane level caused by the overexpression of PS in the exoplasmic leaflet of tumoral membranes, and the higher presence of anionic molecules like heparin sulfates, sialylated gangliosides and O-glycosylated mucins [18,19]. DSC results indicate that model membranes of breast cancer cells were significantly affected by the presence of NA, the above was expected due to the strong electrostatic interaction between the +15 cationic peptide and the negatively charged vesicles. However, how the interactions occurred seemed to be in different ways considering the effects on the thermodynamic parameters reported in Section 2.1.1. We suggest that in the vesicles, which are model membranes of MCF-7, NA generates peptide-poor lipid domains that might reflect a partial insertion of the peptide between fatty acids in a concentration-dependent manner, while with MDA-MB-231 model it is proposed that the peptide induced the stabilization of the gel phase due to the increase in Tm. In addition, NA potentially contributes to diminishing the electrostatic repulsion from negatively charged phosphatidylserine in the MDA-MB-231 model, which reduces the distance between lipid molecules and improves the van der Waals interactions between acyl chains [20]. On the opposite, NA evidenced a fluidizing effect in the HaCaT model membrane with no significant changes in enthalpy, leading to consider a superficial membrane interaction with lipid polar headgroups. Based on the described findings, NA-CATH-ATRA1-ATRA1 is able to alter the physical state of model membranes exerting an effect that is dependent not only on peptide concentration but also on the membrane lipid composition.
Since multicomponent lipid systems exhibited broad chain-melting endotherms, we decided to evaluate the individual interactions with PC, PE, PS and SM vesicles to complement the previous information. Our results indicate that NA strongly interacts with phosphatidylcholine headgroup at the highest evaluated concentrations, perturbing the physical properties of the lipid vesicles. In fact, there are reports in the literature showing the frequent binding of cationic molecules to PC headgroup by electric surface charge that change the direction of the dipole between N+ and water molecules, affecting the orientation of phosphate group [21], which explains the abolishment of the ripple phase transition in PC thermogram. The NA effect on PC might predict a cytotoxic effect since phosphatidylcholine is the most abundant phospholipid [22], indeed, there are reports about the low cytotoxicity from NA in host cells [3,23,24]. The outcome interaction with DPPE vesicles possibly implies a lipid surface aggregation and primary hydrophobic interaction, similar to the report by Willumeit and collaborators with NK-2 peptide [25].
Although all phospholipids seem to be affected by the presence of the peptide, a closer look at the thermodynamical parameters from Table 2 reveals a remarkable selective effect they have towards PS and SM lipid systems. Our findings demonstrated a strong and favorable electrostatic interaction between NA and phosphatidylserine that ended up destabilizing the vesicles. The peptide induced lipid microheterogeneity and phase separation to which we suggest NA could be intercalated between fatty acids chains perturbing the lipid packing by reducing the Van der Waals interactions.
Finally, since the phase transition of SM was abolished in the presence of the peptide, we believe it is due to the destabilization of lipid structure following NA binding to the sphingosine moiety through several intra- and intermolecular hydrogen bonds at the interfacial level.
Furthermore, the lipid-peptide interaction assays analyzed by FTIR-ATR are in concordance with DSC evidence, the NA and MCF-7 interaction reflects the strong binding that causes a release of water molecules and at the same time a relief of charge repulsion with a consequent increase in lipid packing [26]. Spectra portrayed the generation of a more dehydrated and rigid MDA-MB-231 system in presence of NA where the lipid-peptide interaction is more favorable in the liquid-crystalline phase. Taking into consideration that membranes at physiological conditions are in the fluid state, it would be expected a remarkable potential antitumoral activity [27,28]. In addition, the peptide incubation with HaCaT SLBs highlights the fluidizing effect caused by their binding and the consecutive release of water molecules from the interfacial region [29].
Most biological activities of peptides have been related to their structure. Accordingly, Dean and coworkers stated that the increased helical nature of NA generates greater antimicrobial activity [3]. We observed that negatively charged environments promote the disordered to ordered transition of NA during incubation with PS liposomes until having a proper helical folding of a peptide. Although NA seems to generate a conformational change upon binding, the disordered segment should be considered essential to exert its potential antitumoral activity.
Finally, after assembling the described above we conclude and suggest a peptide localization and interaction, with the breast cancer model membranes MCF-7 and MDA-MB-231 after the initial electrostatic attraction peptide seems to reduce the electrostatic repulsion from PS in the models while it acquires a slightly helical conformation that might promote insertion in the lipid bilayer, having a stronger insertion in the MDA-MB-231 model. On the other hand, NA peptide in the control model HaCaT appears to be located on the lipid surface and exerting an electrostatic interaction with lipid headgroups, thus giving energetically unfavorable conditions for the peptide to fold. These biophysical results are in agreement with previous biological assays and offer insights into the potential antitumoral activity of NA-CATH-ATRA1-ATRA1, however, more studies using complementary techniques and biological assays are required to elucidate its mechanism of action.

4. Materials and Methods

4.1. Peptide and Phospholipids

The Naja atra derived peptide NA-CATH-ATRA1-ATRA1 (KRFKKFFKKLK-NSVKKRFKKFFKKLKVIGVTFPF, Lot. U037QFC180-11/PE6100) was acquired from GenScript (Piscataway Township, NJ, USA), synthesized by the solid-phase method. The 97.5 % purity and molecular weight of the peptide were determined using HPLC and MALDI–TOF mass spectrometry, respectively. 16:0 and 16:0/18:1 acyl chain length phospholipids were purchased from Avanti Polar Lipids (Alabaster, AL, USA). 1,2-Dipalmitoyl-sn-glycero-3-phosphocholine (DPPC, Lot. 160PC-318), 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphocholine (POPC, Lot. 850457P-500MG-A-211), 1-,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE, Lot. 160PE-106), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE, Lot. 850757P-500MG-B-151), 1-,2-dipalmitoyl-sn-glycero-3-phospho-L-serine sodium salt (DPPS, Lot. 840037P-500MG-A-078), 1-palmitoyl-2-oleoyl-sn-glycerol-3-phospho-L-serine sodium salt (POPS, Lot. 840034P-25MG-397 A-250), sphingomyelin egg chicken (SM, Lot. 860061P-25MG-A-116). HEPES, EDTA, NaCl, and other analytical grade reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA).

4.2. Phase Transition Measurements by DSC

Individual stock solutions of phospholipids were prepared at 10 mM, DPPC and SM in chloroform, while DPPE and DPPS in chloroform:methanol (70:30 v/v). 1 mM multilamellar vesicles (MLVs) were prepared by adding the exact volume from the individual stock solutions into a test tube to resemble the multi-component phospholipid composition reported for the following breast cancer model membranes, MCF-7, DPPC/DPPE/SM/DPPS 47:35:5:13 (w/w); MDA-MB-231, DPPC/DPPE/SM/DPPS 43:33:4:20 (w/w), and as non-tumoral model membrane HaCaT DPPC/DPPE/SM 59:35:6 (w/w) [16]. Solvent was evaporated under N2 stream, and the dried lipid film was hydrated with buffer 10 mM HEPES, 500 mM NaCl, and 1 mM EDTA pH 7.4. MLVs were formed after 6 min of several vortex-hot water immersion cycles at a temperature above the transition temperature of the lipid system and 1 min sonication. To evaluate the peptide effect on the MLVs, the lipid film was hydrated by adding a NA-CATH-ATRA1-ATRA1 solution prepared at 1, 5, and 10 molar% in the same buffer.
Microcalorimeter Nano DSC Series III platinum capillary cell (TA Instruments, New Castle, DE, USA) was used to perform the calorimetric experiments. The reference cell was loaded with 400 µL of buffer, while the sample cell with lipid or lipid-peptide suspension. After sealing, the calorimeter was pressurized at 0.3 MPa and cells were equilibrated for 10 min at the initial temperature. Heating scans were performed at 1 °C min−1 in the temperature intervals 15 °C to 60 °C for DPPC, 40 °C to 80 °C for DPPE, 35°C to 75 °C for DPPS, 15 °C to 50 °C for SM, 15 °C to 75 °C for breast cancer model membranes MCF-7 and MDA-MB-231, and, from 15 °C to 65 °C for the non-tumoral model membrane HaCaT. Main phase transition temperature (Tm) and main phase transition enthalpy (∆H) were analyzed and calculated using the NanoAnalyze 3.12.0 software (TA Instruments, New Castle, DE, USA). The accuracy was ± 0.1 °C for Tm, and ± 1 kJ.mol−1 for ∆H. DSC data was plotted using the software Origin Pro 8.0 (OriginLab Corporation, Northampton, MA, USA).

4.3. Infrared Spectroscopy Experiments

Phospholipid masses were weighed and added into a glass test tube to achieve a 20 mM stock solution of the breast cancer model membranes MCF-7, DPPC/DPPE/SM/DPPS 47:35:5:13 (w/w); MDA-MB-231, DPPC/DPPE/SM/DPPS 43:33:4:20 (w/w), and the non-tumoral model membrane HaCaT, DPPC/DPPE/SM 59:35:6 (w/w). The dried mixture was dissolved in chloroform and stored at -20 °C. For the gel to liquid crystalline phase measurements 20 mM Supported Lipid Bilayers (SLBs) were prepared in situ on the silicon crystal of the BioATR II cell coupled to the Tensor II spectrometer (Bruker Optics, Ettlingen, Germany) with an MCT (Mercury, Cadmium, Tellurium) detector and connected to a circulating water bath Huber Ministat 125 (Huber, Offenburg, Germany) that offers ± 0.1 °C temperature accuracy. SLBs were formed after adding 20 µL of the stock, removing the solvent, and hydrating the lipid film 15 min above the main transition temperature with the same volume of buffer 10 mM HEPES, 500 mM NaCl, and 1 mM EDTA pH 7.4 in the absence or presence of peptide at 1, 5, 10 molar%. Infrared spectra were taken at a temperature range of 43 to 63 °C for MCF-7, 45 to 65 °C for MDA-MB-231, and 40 to 60 °C for HaCaT. The final spectra were the average of 120 consecutive scans per temperature after the buffer subtraction with a resolution of 4 cm−1.
Lipid phase transition temperature was evaluated through the sensitive marker of lipid order, the symmetric stretching vibration of the methylene band (2970 to 2820 cm−1), and the interfacial hydration by the sensitive marker of lipid hydration, the ester carbonyl stretching vibration band (1725 to 1740 cm−1). Data analysis was performed using the OPUS 3D software (Bruker Optics, Ettlingen, Germany); vibrational bands of interest were cut from final spectra with subsequent baseline correction using the Rubberband method with 20% sensitivity. The pick-picking tool indicated the maximum wavenumber for the symmetric extensions per temperature, leading to graph wavenumber as a function of temperature. Tm was determined as the inflection point of the sigmoidal curve that was fitted into a Boltzmann function using the iterative algorithm Levenberg Marquardt by Origin Pro 8.0 software (Origin Lab Corporation, USA).

4.4. Peptide Conformational Analysis

3 mg. mL−1 peptide solution was prepared in buffer (10 mM HEPES, 500 mM NaCl, 1mM EDTA, pH 7.4) in the absence or presence of small unilamellar vesicles (SUVs). 6 mM SUVs were prepared following the composition of the model membranes MCF-7, POPC/POPE/SM/POPS 47:35:5:13 (w/w); and MDA-MB-231, POPC/POPE/SM/POPS 43:33:4:20 (w/w), and HaCaT POPC/POPE/SM 59:35:6 (w/w). Each lipid mass was weighed, mixed, and dissolved in chloroform. The solvent was removed with N2 stream, lipid films were hydrated with buffer and sonicated for 30 min, 50/60 Hz at a temperature above the main phase transition temperature of the lipid model. Peptide and vesicles were mixed to attain a 15 molar% peptide concentration, and its mixture was incubated for 5 min at 37 °C before measurements. The final spectra of the peptide were acquired as the average of 124 scans taken at 37 °C by the Tensor II spectrometer (Bruker Optics, Ettlingen, Germany) using the integrated AquaSpec Transmission Cell. The secondary structure elements alpha-helix, and beta-sheet with ± 4.4 % deviation were predicted by the predefined method BPROT1 from the Confocheck FT-IR system after internal calibrations with the data set of 43 proteins.

5. Conclusion

This biophysical study offers information about the molecular bases that govern the potential antitumoral activity of NA-CATH-ATRA1-ATRA1 evidenced in previous biological assays from the research group with breast cancer cell lines. We suggest that the cationic and hydrophobic character, and the secondary structure of the peptide is essential to be considered, but also the lipid composition of the biological membrane involved. In this research, tumoral model membranes MCF-7 and MDA-MB-231 evidenced greater effect after binding NA due to the strong electrostatic and hydrophobic interaction that apparently embedded the peptide into the hydrocarbon core, while in the non-tumoral model membrane it is suggested that peptide localization is on lipid surface. Secondary structure experiments demonstrated a possible structure-activity correlation.

Supplementary Materials

The following supporting information can be downloaded at the website of this paper posted on Preprints.org.

Author Contributions

M.C.K.-L. writing-original draft preparation; M.J.-R experimental design, writing-review and editing; M.M.-M. funding acquisition, project administration, writing-review and editing; K.S. funding acquisition, formal analysis. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by MinCiencias Research Grant Cod. 111584467189, RC 946-2019, and by the Faculty of Biochemistry, Biophysics, and Biotechnology at Jagiellonian University. The European Regional Development Fund (contract No. POIG.02.01.00-12-167/08, Project Malopolska Centre of Biotechnology) supported purchasing the DSC instrument for measurements. .

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The original data presented in the study about the lipid composition of model membranes are openly available at [10.3390/ijms242216226]. The original contributions presented in the study are included in the article/supplementary material; further inquiries can be directed to the corresponding authors.

Acknowledgments

M.C.K.-L. and M.M.-M. acknowledge the Faculty of Biochemistry, Biophysics, and Biotechnology at Jagiellonian University for the provided accommodation during the research stay from July to September 2022.

Conflicts of Interest

The authors declare no conflict of interest.

Sample Availability

Samples of the compounds are available from the authors.

Appendix A

Table A1. Main phase transition temperature (Tm) of the MCF-7, MDA-MB-231, and HaCaT model membranes in the presence and absence of the peptide NA-CATH-ATRA1-ATRA1 at 1, 5, 10 mol %. Standard deviation from Infrared Spectroscopy is ± 0.1 °C.
Table A1. Main phase transition temperature (Tm) of the MCF-7, MDA-MB-231, and HaCaT model membranes in the presence and absence of the peptide NA-CATH-ATRA1-ATRA1 at 1, 5, 10 mol %. Standard deviation from Infrared Spectroscopy is ± 0.1 °C.
Tm (°C)
Lipid system Control NA 1% NA 5% NA 10%
MCF-7 52.6 52.5 53.3 55.1
MDA-MB-231 53.2 53.5 54.2 54.4
HaCaT 50.2 50.4 49.4 49.5

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Figure 1. DSC heating scans of 1 mM multilamellar vesicles representative of breast cancer model membranes (a) MCF-7, (b) MDA-MB-231, and (c) HaCaT, human keratinocytes, in the absence and presence of different concentrations of NA-CATH-ATRA1-ATRA1.
Figure 1. DSC heating scans of 1 mM multilamellar vesicles representative of breast cancer model membranes (a) MCF-7, (b) MDA-MB-231, and (c) HaCaT, human keratinocytes, in the absence and presence of different concentrations of NA-CATH-ATRA1-ATRA1.
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Figure 2. Change of the (a) main phase transition temperature and (b) maximum specific heat capacity of MCF-7 (■), MDA-MB-231 (▲) and HaCaT (♦) model membranes as a function of peptide concentration.
Figure 2. Change of the (a) main phase transition temperature and (b) maximum specific heat capacity of MCF-7 (■), MDA-MB-231 (▲) and HaCaT (♦) model membranes as a function of peptide concentration.
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Figure 3. DSC heating endotherms of 1 mM multilamellar vesicles of pure (a) DPPC, (b) DPPE, (c) DPPS, and (d) SM in the absence and presence of different concentrations of NA-CATH-ATRA1-ATRA1.
Figure 3. DSC heating endotherms of 1 mM multilamellar vesicles of pure (a) DPPC, (b) DPPE, (c) DPPS, and (d) SM in the absence and presence of different concentrations of NA-CATH-ATRA1-ATRA1.
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Figure 4. Change of (a) the main phase transition temperature and (b) maximum specific heat capacity of pure DPPC (■), DPPE (●), DPPS (▲), and SM (▼) multilamellar vesicles as a function of peptide concentration.
Figure 4. Change of (a) the main phase transition temperature and (b) maximum specific heat capacity of pure DPPC (■), DPPE (●), DPPS (▲), and SM (▼) multilamellar vesicles as a function of peptide concentration.
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Figure 5. Peptide concentration effect on the maximum symmetric CH2 (a-c) and C=O (d-f) peak positions of MCF-7 (■), MDA-MB-231 (▲) and HaCaT (♦) model membranes as temperature was increased. NA was evaluated at 1 (●), 5 (★), and 10 (Preprints 137687 i001) mol%.
Figure 5. Peptide concentration effect on the maximum symmetric CH2 (a-c) and C=O (d-f) peak positions of MCF-7 (■), MDA-MB-231 (▲) and HaCaT (♦) model membranes as temperature was increased. NA was evaluated at 1 (●), 5 (★), and 10 (Preprints 137687 i001) mol%.
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Table 1. Main phase transition temperature (Tm), enthalpy (∆H), and entropy (∆S) from DSC heating endotherms of breast cancer MCF-7, MDA-MB-231, and human keratinocytes HaCaT multilamellar liposomes including mixtures with NA at 1, 5, 10 mol %.
Table 1. Main phase transition temperature (Tm), enthalpy (∆H), and entropy (∆S) from DSC heating endotherms of breast cancer MCF-7, MDA-MB-231, and human keratinocytes HaCaT multilamellar liposomes including mixtures with NA at 1, 5, 10 mol %.
Heating
Tm [°C] ΔH [kJ mol−1] ΔS [kJ mol−1 K−1]
MCF-7 53.86 38.51 0.12
+ 1 mol% NA 53.86 32.46 0.10
+ 5 mol% NA 53.36 32.85 0.10
+ 10 mol% NA 54.72 24.17 0.07
MDA-MB-231 54.47 41.47 0.13
+ 1 mol% NA 54.42 44.49 0.14
+ 5 mol% NA 54.66 43.57 0.13
+ 10 mol% NA 55.01 42.63 0.13
HaCaT * 42.93 and 53.79 36.94 0.11
+ 1 mol% NA 53.69 36.74 0.11
+ 5 mol% NA 53.93 41.54 0.13
+ 10 mol% NA 53.10 38.59 0.12
Table 2. Main phase transition temperature (Tm), enthalpy (∆H), and entropy (∆S) from DSC heating endotherms of pure DPPC, DPPE, DPPS, and SM multilamellar liposomes including mixtures with NA-CATH-ATRA1-ATRA1 at 1, 5, 10 mol %.
Table 2. Main phase transition temperature (Tm), enthalpy (∆H), and entropy (∆S) from DSC heating endotherms of pure DPPC, DPPE, DPPS, and SM multilamellar liposomes including mixtures with NA-CATH-ATRA1-ATRA1 at 1, 5, 10 mol %.
Heating
Tm [°C] ΔH [kJ mol−1] ΔS [kJ mol−1 K−1]
DPPC 42.20 32.37 0.10
+ 1 mol% NA 42.10 28.74 0.09
+ 5 mol% NA 42.02 27.76 0.09
+ 10 mol% NA 42.03 24.99 0.08
DPPE 65.04 35.60 0.11
+ 1 mol% NA 64.97 22.44 0.07
+ 5 mol% NA 64.98 21.81 0.06
+ 10 mol% NA 65.06 22.22 0.07
DPPS 54.69 35.48 0.11
+ 1 mol% NA 54.25 27.36 0.08
+ 5 mol% NA 54.21 22.35 0.07
+ 10 mol% NA 47.65 20.18 0.06
SM 39.73 42.00 0.13
+ 1 mol% NA 39.29 33.01 0.11
+ 5 mol% NA 41.46 26.79 0.09
+ 10 mol% NA 41.84 19.33 0.06
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