Artificial Tyroserleutide Transporters for Synergistic Cancer Therapy

Abstract

Peptide transporters are integral membrane proteins responsible for the cellular uptake of dipeptides and tripeptides from the extracellular environment, which play pivotal roles in nutrient absorption, antigen presentation, and cellular signaling. Despite their essential biological functions, the development of artificial peptide transporters capable of efficiently transporting charge-neutral peptides, which are highly polar and prone to aggregation, remains a significant challenge. Herein, we introduce a novel class of peptide transporters involving the integration of anion and cation transport functionalities. Notably, 5F–C12, which functions as a molecular tweezer, forms a stable 1:1 complex with the charge-neutral peptide tyroserleutidean anticancer agent currently in Phase III clinical trialsand actively facilitates its transmembrane transport by shielding it from the membrane’s hydrophobic core, achieving an EC₅₀ value of 7.5 μM. For the first time, 5F–C12 could remarkably enhance the peptide’s bioavailability and exhibit a pronounced enhanced anticancer effect against MCF-7 breast cancer cells both in vitro and in vivo.

Introduction

In nature, peptide transporters are integral membrane proteins responsible for the uptake of di- and tripeptides from the extracellular environment into cells, playing a crucial role in the physiological regulation of various biological processes, including nutrient absorption, antigen presentation, and cellular signaling. The most well-characterized peptide transporters belong to the proton-coupled oligopeptide transporter (POT) family, including PEPT1 and PEPT2, which are predominantly expressed in the small intestine and kidney, respectively. , The proper functioning of peptide transporters is essential for maintaining metabolic balance and ensuring the effective absorption of essential nutrients. Dysfunction in peptide transporters, however, can lead to a range of pathological conditions. For instance, defects in the PEPT1 transporter are associated with malabsorption syndromes, leading to nutritional deficiencies, while abnormalities in PEPT2 have been linked to renal disorders. −

Theoretically, conditions arising from peptide transporter dysfunction could be managed by administering functional peptide transporters. However, the practical application of natural peptide transporters in clinical settings is hindered by challenges such as limited availability and inadequate stability. To tackle these challenges, considerable efforts have been devoted toward the development of artificial peptide transporters that mimic the essential functions of their natural counterparts. − These artificial systems not only offer valuable models for elucidating the molecular mechanisms of peptide translocation across membranes but also hold therapeutic potential for addressing disorders related to impaired peptide transport. Furthermore, peptide transporters are among the most abundant drug transporters in the intestinal epithelium, representing a critical pathway for the delivery of peptide-based therapeutics. Therefore, the development of artificial peptide transporters might offer innovative strategies to enhance the bioavailability of peptide-based drugs that are typically poorly absorbed, thereby improving their therapeutic efficacy through a synergistic effect.

Despite significant advancements in the field of artificial transporters for ions and water, − artificial peptide transporters remain notably underexplored. This gap is largely due to the fact that peptides predominantly exist as highly polar zwitterions and tend to aggregate under physiological pH conditions, which severely impedes their ability to cross the nonpolar environment of lipid membranes. To date, the transport of cationic peptides has been achieved using counterion activators, such as anionic/polyanionic transporters, − anionic calixarene amphiphiles, − and superchaotropic cluster anions, , which function by forming charge-neutralized complexes to enhance membrane permeability. To the best of our knowledge, the transport of charge-neutral peptides, which present greater challenges owing to their increased propensity for aggregation and weaker interactions with the phosphate head groups of lipids compared to cationic peptides, has only been achieved using superchaotropic cluster anions, albeit with relatively limited efficacy. Consequently, the development of artificial transporters capable of efficiently mediating the transport of charge-neutral peptides remains a significant and unresolved challenge in the field.

To address this gap, we herein introduce a novel class of peptide transporters capable of effectively facilitating the transmembrane transport of a charge-neutral peptide such as tyroserleutide (YSL). Inspired by ambiphilic receptors that are tailored to recognize ion pairs, − our transporters are engineered through the integration of anion and cation transport functionalities connected via alkyl chains. Functioning as molecular tweezers, these transporters form stable 1:1 complexes with YSL, effectively shielding the peptide from the hydrophobic core of the membrane and enabling its transport. Notably, transporter 5F–C12 facilitates YSL transport with an EC₅₀ value of 7.5 μM. Fascinatingly, 5F–C12 significantly boosts the cellular uptake of YSL. When applied at a concentration of 0.1 μM, the cellular uptake of YSL in MCF-7 breast cancer cells can be enhanced by 90-fold. This dramatic increase in uptake translates to a 16.1-fold enhancement in the anticancer potency of YSL at a concentration of 4 μM, where 5F–C12 exhibits minimal toxicity. This unprecedented synergistic anticancer effect was further validated in vivo, underscoring the potential of artificial peptide transporters to revolutionize peptide-based therapeutic strategies and improve the efficacy of peptide therapeutics.

Results and Discussion

Molecular Design

Designing effective peptide transporters requires overcoming the challenges induced by the zwitterionic charges from the carboxylate and amino groups, as well as peptide aggregation that impedes membrane transport. To tackle these challenges, we proposed a strategy that integrates anion and cation transport functionalities connected by hydrophobic hydrocarbon side chains. As illustrated in Figure a,b, this architecture allows the transporters to act as molecular tweezers to shield peptides from the membrane’s nonpolar environment and facilitate their transmembrane transport. In pursuit of this goal, we initially designed a series of anion transporters3F, 5F, and 7Fbased on a bis­(carboxylic amides) scaffold decorated with fluorocarbon side chains. Concurrently, we designed a series of cation transportersC8, C10, C12, and C14derived from 18-crown-6 decorated with alkyl side chains. This design framework enables us to evaluate the influence of ion binding strength and hydrophobicity of the transporters on peptide transport efficiency, facilitating the rapid identification of favorable transporter combinations. Given that the formation of a complex with peptides necessitates a specific arrangement of individual components, which reduces their degrees of freedom and incurs an entropic penalty, we hypothesized that integrating anion and cation transporter elements into a single, unified transporter might further boost peptide transport efficacy.

1.

(a) Chemical structures of the peptide transporters derived through the integration of anion and cation transporters. (b) Illustration of the transmembrane transport of tyroserleutide (YSL) facilitated by peptide transporters, demonstrating the enhanced cellular uptake and anticancer efficacy.

The binding affinities of the anion transporters 3F, 5F, and 7F for the carboxylate group were assessed via UV–vis titration experiments in acetonitrile at 25 °C. Specifically, a 100 μM solution of anion transporters was titrated with the N-Boc-glycine tetrabutylammonium salt (Boc-Gly-COO^–TBA⁺), resulting in a notable increase in the UV–vis absorbance of the anion transporters, indicative of a binding interaction with carboxylate groups (Figure S1). Job’s plot analysis, based on absorbance changes during titration, confirmed the formation of a 1:1 complex between the anion transporters and Boc-Gly-COO^–TBA⁺. The association constants calculated for 3F, 5F, and 7F were (0.66 ± 0.10) × 10³, (2.08 ± 0.60) × 10³, and (3.04 ± 0.83) × 10³ M^–1, respectively, showing a binding trend of 7F > 5F > 3F. This trend could be attributed to the enhanced electron-withdrawing effect of the side chains, which increase the acidity of the amide protons and, consequently, strengthen the carboxylate binding capacity.

In parallel, the binding affinities of cation transporters C8, C10, C12, and C14 for amino groups were evaluated using a similar approach. The addition of the tert-butyl ester of glycine hydrochloride (NH₂-Gly-COOtBu·HCl) to solutions of cation transporters gave rise to a marked increase in UV–vis absorbance, supporting the effective recognition of amino groups by crown ether moieties in the transporters (Figures S2 and S3). Job’s plot analysis further confirmed the formation of 1:1 complexes. The association constants determined for C8, C10, C12, and C14 were (3.58 ± 1.41) × 10³, (5.39 ± 1.62) × 10³, (6.96 ± 0.94) × 10³, and (6.31 ± 1.02) × 10³ M^–1, respectively, with the binding affinity following the order C12 ≈ C14 > C10 > C8. This trend suggests that crown ethers with longer hydrophobic side chains generally exhibit higher affinities toward amino groups, likely due to enhanced hydrophobic interactions that stabilize the binding complexes.

Transmembrane Transport Activity Study

The robust binding affinities of the anion and cation transporters for carboxylate and amino groups prompted us to investigate their efficacy in mediating the transport of zwitterions, both individually and in combination, utilizing a Cu²⁺-calcein fluorescence assay outlined by Gale and colleagues, with glycine serving as a simple zwitterion model. As depicted in Figure a, this assay involves the preparation of large unilamellar vesicles (LUVs) encapsulating calcein and CuSO₄. Within these LUVs, free Cu²⁺ ions quench the calcein fluorescence. Glycine or peptides transported into LUVs mediated by transporters will compete with calcein for binding to Cu²⁺, leading to the restoration of the calcein fluorescence. The extent of this fluorescence enhancement quantitatively reflects the transporters’ ability to mediate zwitterion transport.

2.

(a) Illustration of the Cu²⁺-calcein assay for assessing the capability of transporters in mediating the transport of glycine (Gly), which serves as a simple zwitterion model (λ_ex = 495 nm, λ_em = 515 nm). (b) Fluorescence intensity changes of calcein upon the addition of various transporters at 10 μM (10 mol % relative to lipid). (c) Comparison of glycine transport activity between transporter pairs and unimolecular transporter at 10 μM (10 mol % relative to lipid). (d) Illustration of the ¹³C NMR assay for monitoring Gly-¹³C transport and partial ¹³C NMR spectra of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) vesicle suspensions with external Gly-¹³C (100 mM) before and after addition of MnSO₄ (0.5 mM). (e) Partial ¹³C NMR spectra of POPC vesicle suspensions with external Gly-¹³C (100 mM) in the absence and presence of various transporters at 0.5 mM (0.13 mol % relative to lipid).

We performed assays to evaluate the glycine transport activity of two anion transporters5F and 7Fand four cation transportersC6, C8, C10, C12, and C14. 3F was excluded from the tests due to its insufficient binding affinity for the amino group. As expected, the individual application of these transporters at a concentration of 10 μM (i.e., 10 mol % relative to lipid) did not induce a significant enhancement in calcein fluorescence intensity after background correction, suggesting their limited capacity for glycine transport when acting alone (Figure b and S8). In contrast, the combination of anion transporters and cation transporters yielded remarkable synergistic effects, particularly observed in the pairs5F with C12, 5F with C14, 7F with C12, and 7F with C14with enhancements in calcein fluorescence (ΔI) measured at 110.9, 52.9, 68.3, and 101.5, respectively. These enhancements were significantly greater than the sum of the fluorescence changes observed when anion transporters and cation transporters were tested independently (ΔI _5F = 14.7, ΔI _7F = 1.2, ΔI _C12 = 13.8, and ΔI _C14 = 15.2).

Subsequently, we synthesized four unimolecular transporters, denoted as 5F–C12, 5F–C14, 7F–C12, and 7F–C14, by integrating the anion transporters 5F and 7F with the cation transporters C12 and C14. Similarly, we assessed their glycine transport activity using a Cu²⁺-calcein fluorescence assay. As shown in Figure c, the unimolecular transporters consistently demonstrated superior performance compared with their corresponding combinations of anion and cation transporters. Among them, 5F–C12 and 7F–C14 demonstrated the highest glycine transport activity, thereby highlighting the effectiveness of this integrated approach in enhancing the transport capabilities for zwitterions.

In addition to the Cu²⁺-calcein fluorescence assays, we employed ¹³C NMR spectroscopy of ¹³C-labeled Gly (Gly-¹³C) to obtain more direct evidence of glycine transport. As depicted in Figure d, the binding of paramagnetic Mn²⁺ ions to glycine causes the broadening of the ¹³C NMR signal corresponding to Gly-¹³C. This distinctive feature is utilized to differentiate between the Gly-¹³C in the extravesicular and intravesicular compartments. The facilitated influx of Gly-¹³C, triggered by transporters, is indicated by the appearance of a distinct ¹³C NMR signal. As illustrated in Figure e, when Gly-¹³C was incubated with 5F or C12 at a concentration of 0.5 mM (0.13 mol % relative to lipid) for 1 h, only minimal diffusion was observed, resembling the control conditions. This suggests that individual ion transporters, 5F and C12, are inefficient in mediating the transport of glycine on their own. Following the incorporation of transporter pairs 5F + C12 at the same concentration, the ¹³C NMR spectra displayed a small but distinct signal peak at 172.4 ppm. In contrast, the presence of unimolecular transporters 5F–C12 yielded a pronounced sharp signal peak, highlighting that the unimolecular transporters significantly outperform the transporter pairs. A similar phenomenon was also observed for 7F–C14 (Figure S9).

Following these promising results, we were keen to investigate the potential of transporters in mediating the transport of charge-neutral peptides, with a special focus on therapeutic peptides such as tyroserleutide (YSL). YSL, a tripeptide composed of tyrosine, serine, and leucine, has emerged as a promising candidate for cancer therapy. , This peptide exhibits significant anticancer effects by inducing apoptosis in cancer cells, inhibiting their proliferation, and suppressing angiogenesis, as demonstrated in both in vitro and in vivo models. Currently, YSL is undergoing Phase II and Phase III clinical trials to evaluate its therapeutic efficacy and safety across various cancer types. , However, the zwitterionic nature and strong aggregation propensity of YSL under physiological conditions limit its membrane permeability, often necessitating higher concentrations to achieve therapeutic efficacy. Consequently, the development of peptide transporters for YSL holds the potential to significantly enhance its bioavailability and demonstrate synergistic therapeutic efficacy in cancer therapy (Figure b).

To assess the binding affinity of transporters toward YSL, we first examined the capacity of 5F–C12 to extract solid YSL into a THF-d ₈/DMSO-d ₆ (9:1, v:v) solution. Solid YSL was suspended in the solvent mixture, sonicated for 5 min, and subsequently analyzed by ¹H NMR spectroscopy. As illustrated in Figure a, the limited solubility of YSL in this solution yielded only weak signals in the ¹H NMR spectra. Additionally, the amide protons in YSL presented broad signals, likely resulting from the aggregation of YSL molecules due to their intermolecular H-bonding interactions. However, upon the addition of 25 mM 5F–C12, the intensity of the YSL proton signals was markedly enhanced, suggesting the effective recognition of YSL by 5F–C12 in solution. Signal integration analysis implied the formation of a 1:1 complex between 5F–C12 and YSL, which was further validated by high-resolution mass spectroscopy (Figure S10). Importantly, the initially broad amide proton peaks of YSL became sharp in the presence of 5F–C12, indicating that 5F–C12 promoted the disaggregation of YSL molecules in the formation of the complex.

3.

(a) The chemical structure of 5F–C12·YSL complex and partial ¹H NMR spectra demonstrating the ability of 5F–C12 to extract solid YSL into a THF-d ₈/DMSO-d ₆ (9:1, v:v) solution. (b) UV absorbance of 5F–C12 (20 μM) in tetrahydrofuran in the presence of varying concentrations of YSL at 25 °C (λ = 234 nm). (c) Job’s plot analysis by measuring the UV absorbance of a mixture containing 5F–C12 with YSL at varying molar ratios, confirming the formation of a 1:1 complex. (d) The computationally optimized structure of the 5F–C12·YSL complex, obtained from B3LYP/6-31G.

The binding affinities of 5F–C12 and 7F–C14 for YSL were evaluated by using UV–vis titration experiments in tetrahydrofuran (THF) at 25 °C. As depicted in Figure b and S4, the addition of YSL to solutions of 5F–C12 and 7F–C14 led to a prominent increase in UV–vis absorbance, indicating a host–guest interaction between the transporters and YSL. Job’s plot analysis further supported the formation of a 1:1 complex (Figure c). The association constants for the binding of 5F–C12 and 7F–C14 to YSL were determined to be (6.43 ± 2.54) × 10⁴ and (1.41 ± 0.86) × 10⁴ M^–1, respectively, reflecting their strong affinities for YSL. Notably, 5F–C12 exhibits a relatively higher binding strength.

The computationally optimized structure of the 5F–C12·YSL complex, displayed in Figure d, reveals significant stabilization upon complex formation, with an energy decrease of 56.5 kcal/mol at the B3LYP/6-31G level. The optimized structure highlights the formation of multiple intermolecular H-bonds that contribute to the stability of the complex. Specifically, an H-bonding interaction occurs between the oxygen atoms in the crown ether moiety of 5F–C12 and the amino protons of YSL. Additionally, protons in the bis­(carboxylic amide) moiety form hydrogen bonds with the oxygen atoms in the carboxylate group of YSL. Further stabilization is provided by interactions between one carbonyl oxygen atom in the bis­(carboxylic amide) moiety and YSL’s phenolic proton. These intermolecular interactions collectively enhance the affinity and structural integrity of the 5F–C12·YSL complex, underscoring the role of hydrogen bonding in complex stabilization.

The strong binding affinities of transporters 5F–C12 and 7F–C14 with YSL prompted further investigation of their transport capabilities across the membrane using the Cu²⁺-calcein assay (Figure a). We initially evaluated YSL transport activities for the anion transporters (5F, 7F) and the cation transporters (C12, C14) at 10 μM (10 mol % relative to lipid) as controls. As expected, neither the anion nor the cation transporters were capable of mediating YSL transport independently. Next, we assessed the YSL transport activities for 5F–C12 and 7F–C14 and their corresponding transporter pairs (5F + C12 and 7F + C14). The results revealed a clear difference in the transmembrane transport capabilities of the unimolecular transporters compared with their paired counterparts. As depicted in Figure b and S11, the transporter pairs 5F + C12 and 7F + C14, when applied at a concentration of 10 μM, produced only a slight fluorescence enhancement, yielding ΔI values of 9.2 and 8.2 after background correction. This minimal increase suggests that the transporter pairs are inefficient at facilitating YSL transport. In contrast, 5F–C12 and 7F–C14 induced a pronounced increase in fluorescence intensity, with ΔI values reaching 59.4 and 39.5, respectively, indicative of substantially higher transport activities. The superior performance of the unimolecular transporters over the transporter pairs may arise from more favorable binding interactions and structural alignment within the simpler complex, which results in a reduced entropic penalty.

4.

(a) Illustration of the Cu²⁺-calcein assay for assessing the capability of transporters in mediating the transport of YSL (λ_ex = 495 nm, λ_em = 515 nm). (b) Fluorescence intensity variations of calcein upon the addition of various transporters at 10 μM (10 mol % relative to lipid). (c) Fluorescence intensity changes of calcein in response to 5F–C12 at varying concentrations. (d) Observed influx rate constants (K _obs) for YSL calculated at varying concentrations of 5F–C12, indicating that 5F–C12 facilitates YSL transport through a single-molecule mechanism. (e) Molecular dynamic simulation of 5F–C12·YSL complex within a simulation box 66 Å (w) × 66 Å (w) × 72 Å (h), comprising 128 POPC molecules and 2368 water molecules on each side of the membrane.

To quantitatively evaluate the transport efficiencies of 5F–C12 and 7F–C14, the fractional fluorescence at 10 min was plotted against the transporter loading and subjected to Hill analysis. As shown in Figure c, the addition of 5F–C12 induced a concentration-dependent increase in fluorescence intensity, reaching saturation at 20 μM. The maximum fluorescence intensity increase obtained under this condition (ΔI _max) was used as a calibration reference. The effective transport loadings required to achieve 50% transport (EC₅₀) for 5F–C12 and 7F–C14 were determined to be 7.5 and 12.3 μM, respectively, suggesting that 5F–C12 is more efficient in mediating the transport of YSL than 7F–C14, likely due to its stronger binding affinity for YSL (Figure c and S13). Next, the observed influx rate constants (K _obs) for YSL at various transporter concentrations were derived using nonlinear fitting of the time-course curves acquired from the Cu²⁺-calcein assay. The data presented in Figure d and S14 unveil a clear linear correlation between K _obs and transporter concentration, implying that 5F–C12 operates in a single-molecule manner in mediating the transport of YSL.

To elucidate the relationship between the structural features of transporters and their YSL transport efficiency, we systematically investigated the influence of the hydrophobic chain length on both YSL binding affinity and transport efficiency. In addition to 5F–C12 and 5F–C14, we synthesized 5F–C8 and 5F–C10 with C8 and C10 hydrocarbon chains, respectively. As shown in Figure S5a–d, extending the length of the hydrophobic chain from C8 to C12 significantly enhanced the binding affinity toward YSL, with the association constant increasing from 1.18 × 10³ to 6.43 × 10⁴ M^–1. However, further extension to C14 resulted in a notable decrease in the binding affinity (1.54 × 10⁴ M^–1). This decline may stem from the enhanced conformational flexibility with the longer chain, which could compromise the cooperative interaction between the anion and cation recognition modules. Transport activity evaluated using the Cu²⁺-calcein assay revealed a strong correlation between binding affinity and YSL transport efficiency, identifying C12 as the optimal chain for YSL transport (Figure S11b).

We further explored the role of fluorocarbon chains and the synergistic interaction between anion and cation recognition modules by assessing 3F–C12 and 7F–C12. Increasing the fluorocarbon chain length in the anion recognition module enhanced YSL binding, with the association constant increasing from 8.52 × 10³ to 7.04 × 10⁴ M^–1, likely due to the increased acidity of amide protons, as depicted in Figure S5e,f. Interestingly, transport efficiency also improved when the trifluorocarbon chain was replaced with a pentafluorocarbon chain, suggesting enhanced synergy between the anion and cation recognition modules (Figure S11b). However, further extension of the fluorocarbon chain led to a decline in transport activity, despite a slight increase in binding affinity. This effect is likely a result of the enhanced lipophobicity associated with longer fluorocarbon chains, which reduces membrane compatibility and impairs effective membrane insertion and transport. Collectively, these findings demonstrate that both the length of hydrophobic and fluorocarbon chains and the synergistic interaction between anion and cation recognition modules play critical roles in determining the transporters’ overall efficacy for YSL transport.

Given that peptides with diverse structures can exhibit substantial differences in their binding affinities and transport efficiencies with 5F–C12, we further explored the binding affinity and transport efficiency of 5F–C12 with the tripeptide Gly-Gly-Gly (GGG). As shown in Figure S6a, the binding constant of 5F–C12 for GGG in THF was determined to be 1.02 × 10³ M^–1, markedly lower than its affinity for YSL. This disparity likely arises from the absence of key intermolecular interactions, such as H-bonding and hydrophobic interactions, present in the 5F–C12·YSL complex. Consistently, transport studies using the Cu²⁺-calcein assay demonstrated that 5F–C12 was unable to efficiently facilitate the transport of GGG, further highlighting the critical role of binding affinity in determining transport efficiency (Figure S12a). We also examined the performance of 5F–C12 with Ile-Arg (IR), a therapeutic dipeptide with known antioxidant, anti-inflammatory, and signaling-modulatory effects. A similar correlation between binding affinity and transport activity was observed, reaffirming the importance of specific transporter–peptide interactions (Figure S12b).

Furthermore, we conducted a self-quenching assay utilizing 5(6)-carboxyfluorescein (CF) to assess membrane integrity in the presence of various peptide transporters. CF, with dimensions of 1.0 × 1.0 nm, exhibits high fluorescence at low concentrations but experiences significant self-quenching at the elevated concentrations. As depicted in Figure S16, the addition of transporters at 10 μM (10 mol % relative to lipid)a concentration sufficient to induce notable activities in our transport assayscaused no detectable CF leakage. In contrast, melittin, a peptide known to form nanopores in membranes, induced 57% CF efflux at 0.5 μM, serving as a positive control and suggesting that these transporters do not compromise the integrity of the LUV membranes. Furthermore, we employed dynamic light scattering (DLS) to monitor the LUV size distribution before and after the incorporation of transporters. As illustrated in Figure S17, treatment with Triton-X at concentrations of 0.2–1.0 mM caused substantial fragmentation of LUVs in a concentration-dependent manner, indicative of membrane disruption. However, incorporation of 5F–C12 or 7F–C14 at concentrations ranging from 10 to 30 μM did not alter the LUV size distribution, further supporting the idea that the lipid bilayer remains intact upon the addition of these transporters.

To shed some light on the formation of the 5F–C12·YSL complex and its role in mediating YSL transport across the lipid membrane, molecular dynamic (MD) simulation employing the CHARMM program, enhanced with the particle mesh Ewald (PME) method for electrostatic calculations and the SHAKE algorithm, was performed. The initial structure of the 5F–C12·YSL complex optimized at the B3LYP/6-31G level was incorporated into a lipid bilayer composed of 128 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), surrounded by 2368 water molecules on either side within a simulation box with dimensions of 66 Å (w) × 66 Å (w) × 72 Å (h). After equilibrating the system, a 40 ns production MD simulation was carried out to assess the stability of the complex within the membrane. Analysis of the final 10 ns trajectory indicated robust H-bonding interactions between crown ether oxygen atoms and the amino protons of YSL, as well as between the amide protons of YSL and carboxylate oxygen atoms, thereby forming a stable complex in the hydrophobic region of the membrane to facilitate the diffusion of YSL across the membrane (Figure e).

Cellular Uptake and Anticancer Activity Study

Following the successful transport of YSL mediated by 5F–C12 in the LUV-based assay, we extended our study to assess its efficacy in facilitating the transmembrane delivery of YSL in living cells. Initially, confocal fluorescence microscopy was employed to evaluate the intracellular uptake of a pyrene-labeled YSL (YSL-py) in MCF-7 cells. Figure b unveils that when YSL-py (0.2 mM) was incubated with MCF-7 cells in the absence of 5F–C12 for 12 h, an almost negligible fluorescence signal was detected in the cytosol, attributed to the poor membrane permeability of the peptide. In contrast, the presence of 5F–C12 at 0.2 μM significantly enhanced the intracellular delivery of the peptide, as evidenced by a substantial increase in cytoplasmic fluorescence. Flow cytometry analysis (Figure c) further quantified these observations, confirming a 3-fold increase in the level of uptake of YSL-py in cells in the presence of 5F–C12 at 0.2 μM.

5.

(a) Chemical structure of YSL-py. (b) Confocal images of MCF-7 cells treated with YSL-py (0.2 mM) in the absence and presence of 5F–C12 (0.2 μM) for 12 h, followed by staining with rhodamine phalloidin. The red and blue channels were merged using a ZEISS ZEN 3.8. (c) Quantitative analysis of the enhanced cellular uptake of YSL-py upon the addition of 5F–C12 (0.2 μM) for 12 h using flow cytometry. (d) The cellular uptake rate of YSL (100 μM) in MCF-7 cells after 4 h incubation, in the presence of 5F, C12, 5F + C12, and 5F–C12 at 0.1 μM, measured by HPLC-MS. (e) The cellular uptake rate of YSL (100 μM) in MCF-7 cells after 4 h incubation with varying concentrations of 5F–C12 (0, 0.025, 0.05, 0.1, and 0.2 μM), measured by HPLC-MS. Two-tailed Student’s t-test was used for statistical significance, and data are presented as means ± SEM (n = 3) biologically independent samples. p Values (5F–C12) < 0.0001. Symbols **** stand for significant differences between the control group and other groups, with p < 0.0001. Symbols ns stand for no difference between the control group and other groups. Results were analyzed using GraphPad Prism 8.0.1. (f) IC₅₀ values and cytotoxicity enhancement of YSL against MCF-7 cells in the presence of varying concentrations of 5F–C12 (0.1, 0.2, 0.5, 1, 2, and 4 μM).

Encouraged by the findings from the fluorescence study, we proceeded to quantitatively evaluate the cellular uptake of YSL (100 μM) in the presence of various transporters. We incubated MCF-7 cells with 5F, C12, 5F + C12, and 5F–C12 at a concentration of 0.1 μM for 4 h. After incubation, cells were quenched with cold methanol and detached from the culture dish, and the cell pellets were collected by centrifugation and washed with methanol. The concentration of YSL in the resulting cell extracts was then analyzed via high-performance liquid chromatography–mass spectrometry (HPLC-MS). The results depicted in Figure d and S18 revealed that the 5F, C12, and 5F + C12 groups did not significantly enhance the cellular uptake of YSL compared to the control. In contrast, 5F–C12 led to a remarkable 9.1% YSL uptake rate, which was 90 and 53 times higher than that of the control and 5F + C12 group, respectively. Furthermore, a dose-dependent increase in YSL uptake was observed as the concentration of 5F–C12 was elevated, underscoring its ability to effectively enhance the transmembrane transport and cellular uptake of YSL in vitro (Figure e and Table S1).

To elucidate the mechanism underlying the efficient transport of YSL in the presence of various biologically relevant zwitterionic competitors, we first conducted a series of binding studies using representative zwitterionic molecules found in cells and culture medium, including an amino acid (glutamine, Gln), a dipeptide (alanine–glutamine, Ala-Gln), and a zwitterionic metabolite (phosphorylated serine, pSer). The results revealed that 5F–C12 exhibited measurable binding affinities for these zwitterionic molecules, with association constants of 3.58 × 10⁴, 1.32 × 10⁴, and 0.87 × 10⁴ M^–1, respectively. Although 5F–C12 exhibited a binding preference for YSL, transport assays indicated that its transport efficiencies for Gln, Ala-Gln, and pSer were comparable to that of YSL, suggesting 5F–C12 exhibits limited selectivity in transporting YSL over other zwitterions in simplified systems with equal concentrations (Figure S15).

Using HPLC-MS, we analyzed the concentrations of these zwitterionic molecules in both extracellular (culture medium) and intracellular compartments in the presence and absence of 5F–C12 at 0.2 μM (Figure S19). Notably, Gln and Ala-Gln showed no significant concentration gradient across the cell membrane, and 5F–C12 did not induce substantial changes in their intracellular levels. For pSer, which is absent from the culture medium, a slight decrease in intracellular concentration from 4.3 to 3.8 μM was observed upon treatment with 5F–C12. Therefore, the efficient delivery of YSL by 5F–C12 in the presence of other zwitterionic competitors arises from two key factors: (1) the absence of endogenous YSL in cells, creating a steep inward concentration gradient, and (2) the lack of significant transmembrane gradients for competing zwitterions (e.g., Gln and Ala-Gln) or their inherently low abundance (e.g., pSer).

The therapeutic potential of YSL in cancer treatment has been well established, yet its clinical application still faces challenges due to its zwitterionic nature and propensity to aggregate at physiological pH. These features hamper its membrane permeability, requiring higher doses to achieve sufficient antitumor effects. The efficient transport of YSL mediated by 5F–C12 prompted further investigation into its potential for enhanced anticancer therapy both in vitro and in vivo. Using the MTT cell viability assay, we assessed the anticancer efficacy of YSL alone and in combination with varying concentrations of 5F–C12 against human breast cancer cells (MCF-7). Figure S20 demonstrates that YSL by itself exhibits limited anticancer activity, with an IC₅₀ value of 4.34 mM. However, coadministration with 5F–C12 at concentrations of 0.1–4.0 μMconcentrations demonstrating negligible toxicity to MCF-7 cellsresulted in a significant enhancement in YSL’s anticancer effect (Figure f and S21). This concentration-dependent enhancement aligned well with the trends observed in the calcein assay, indicating a clear correlation between the concentration of 5F–C12 and the increased anticancer efficacy. Notably, in the presence of 4.0 μM 5F–C12, the anticancer activity of YSL was remarkably potentiated, lowering the IC₅₀ to 0.27 mM, corresponding to an improvement of 16.1-fold. The Chou–Talalay method, which uses the combination index (CI) to classify the interaction between two or more anticancer agents as synergistic (CI < 1), additive (CI = 1), or antagonistic (CI > 1), was employed to evaluate the combination anticancer effect of 5F–C12 and YSL. The corresponding calculated CI value of 0.18 provided clear evidence of a synergistic interaction between the two compounds in cancer therapy.

To verify that the enhanced cytotoxicity results from 5F–C12-mediated intracellular delivery of YSL, we evaluated apoptosis in MCF-7 cells using Annexin V/PI staining and Caspase-3 quantification. Treatment with 0.2 μM 5F–C12 alone exhibited no apoptotic effects or Caspase-3 downregulation, confirming its biocompatibility at this concentration (Figures S22 and S23). In contrast, YSL alone induced moderate apoptosis (4.3% at 0.2 mM and 6.6% at 0.4 mM) and Caspase-3 downregulation. Notably, cotreatment with 5F–C12 dramatically amplified YSL-induced apoptosis (13.6% and 20.2%, respectively) and Caspase-3 downregulation, confirming that the observed cytotoxicity is specifically due to apoptosis triggered by YSL, whose delivery is facilitated by 5F–C12.

Building on the encouraging in vitro synergy between 5F–C12 and YSL, we extended our investigation to an in vivo proof-of-concept study using a tumor-bearing mouse model (Figure a). Mice were randomly assigned to six groups (n = 5) and received intratumoral injections of saline, YSL (8 mg/kg), and 5F–C12 at either 0.8 or 1.6 mg/kg, or combination treatments of YSL (8 mg/kg) with either dose of 5F–C12. Injections were administered every 2 days over a two-week period. Tumor growth was monitored throughout, with saline-treated controls exhibiting rapid tumor progression. Notably, while 5F–C12 alone had a minimal impact on tumor growth inhibition, YSL treatment resulted in moderate suppression of approximately 36.4%. However, the coadministration of YSL and 5F–C12 produced a significant enhancement in antitumor activity, evidencing strong synergistic effects. In the YSL (8 mg/kg) + 5F–C12 (0.8 mg/kg) group, tumor inhibition increased markedly, reaching approximately 65.7% (Table S2). Impressively, mice treated with YSL (8 mg/kg) combined with a higher dose of 5F–C12 (1.6 mg/kg) exhibited an even more pronounced tumor growth inhibition of 81.1% (Figure b,d).

6.

In vivo synergistic anticancer efficacy of YSL and 5F–C12 on female BALB/c nude mice models. (a) Schematic illustration of the treatment schedule involving intratumoral administration of the anticancer agents. (b) Volume of MCF-7 tumors in nude mice (n = 5 per group) subjected to various treatments. (c) Monitoring of body weight in nude mice (n = 5 per group) across treatment groups to assess systemic toxicity. (d) Representative images of excised tumors from each treatment group at day 14 postadministration. (e) Histological analysis of tumors through H&E, Ki-67, and TUNEL staining after various treatments. Data are presented as means ± SEM (n = 5 biologically independent samples). Statistical analysis was conducted using GraphPad Prism 8.0.1. The statistical significance is analyzed by one-way ANOVA with a Tukey post hoc test. Symbols **** stands for significant differences between the G0 group and other groups, with p < 0.0001.

Further histopathological evaluation, including Ki-67 immunostaining and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining for apoptotic cells, corroborated these findings. The combination treatment consistently yielded extensive tumor cell apoptosis and necrosis, confirming the enhanced efficacy observed in tumor volume reduction (Figure e). Ki-67 immunostaining highlighted reduced proliferation in the combined treatment group, indicating effective tumor growth control. Meanwhile, TUNEL staining revealed a substantial increase in apoptotic cell death, particularly in the combination-treated tumors, underscoring the potent synergistic effects between YSL and 5F–C12 in vivo.

In addition to efficacy, the safety profile of these treatment regimens was evaluated. Throughout the study, no significant weight loss was recorded in any of the treatment groups, suggesting the absence of systemic toxicity (Figure c). Histopathological staining of major organs, including the heart, liver, spleen, lungs, and kidneys, showed no detectable histopathological abnormalities or treatment-related damage across all groups, further supporting the biosafety of these therapeutic agents (Figure S24). This favorable safety profile, along with the observed high efficacy, underscores the promise of artificial peptide transporters 5F–C12 in enhancing the therapeutic effects of peptide drugs through synergistic combinations.

Conclusion

In conclusion, we have developed a novel class of artificial peptide transporters that integrate anion and cation transport functionalities through alkyl side chains. These transporters function as molecular tweezers to form stable 1:1 complexes with a charge-neutral peptide such as YSL, thereby shielding peptides from the hydrophobic core of the membrane and enabling their efficient transmembrane transport. In particular, 5F–C12 demonstrated superior performance in mediating YSL transport, with an EC₅₀ value of 7.5 μM (7.5 mol % relative to lipid). Remarkably, 5F–C12 enhanced the cellular uptake of YSL by 90-fold in MCF-7 breast cancer cells at 0.1 μM and improved its anticancer potency by 16.1-fold at 4 μM, where 5F–C12 exhibited minimal toxicity. The synergistic anticancer effect was further validated in vivo, marking the first demonstration of such therapeutic potential between artificial peptide transporters and therapeutic peptides in a living system. This study addresses the longstanding challenge of transporting charge-neutral peptides and provides a facile yet robust strategy to enhance the bioavailability and efficacy of peptide-based drugs, opening new avenues for peptide delivery systems and advancing the treatment of diseases that benefit from improved peptide absorption.

Materials and Method

Cu­(II)-Calcein Assay for YSL Transport

Large unilamellar vesicles (LUVs) of POPC were prepared as follows. POPC powder (25.0 mg) was dissolved in 1 mL of anhydrous CHCl₃, and the solvent was removed under reduced pressure at 35 °C. The resulting lipid film was dried under vacuum overnight. Following this, the lipid film was rehydrated by vigorous mixing with a buffer solution containing Cu²⁺ and calcein (pH 7.4, 20 mM HEPES, 100 mM Na₂SO₄, 0.2 mM CuSO₄, and 0.2 mM calcein), followed by incubation at 300 rpm on a rotary shaker for 1 h to achieve a uniform suspension. The suspension was subjected to 10 freeze–thaw cycles, each consisting of 1 min in liquid nitrogen and subsequent 2 min heating at 55 °C in a water bath. The mixture was then extruded through a 200 nm polycarbonate membrane 25 times at 50 °C to obtain a homogeneous dispersion. Unencapsulated Cu²⁺ and calcein were removed via gel filtration on a Sephadex G-25 column with an eluent consisting of a buffer solution (pH 7.4, 20 mM HEPES, 100 mM Na₂SO₄). The final product was a suspension of LUVs with a diameter of approximately 200 nm at a concentration of 6.6 mM, which was stored at 4 °C overnight.

For each assay, the LUVs are mixed with an external solution containing Cu²⁺ and YSL (pH 7.4, 20 mM HEPES, 100 mM Na₂SO₄, 0.2 mM CuSO₄, 30 mM YSL) to reach a final lipid concentration of 0.2 mM in a 2 mL volume. A 20 μL solution of the transporter in DMSO was added to initiate the measurement of YSL transport, and the resulting changes in fluorescence emission (λ_ex = 495 nm, λ_em = 515 nm) were monitored over a period of 10 min using a fluorescence spectrophotometer (Hitachi, Model F-7100, Japan). We utilized 5F–C12 and 7F–C14 to determine the maximum change in fluorescence intensity using eq .

$$\mathrm{\Delta }{I}_{\mathrm{m}\mathrm{a}\mathrm{x}}=I_{\mathrm{m}\mathrm{a}\mathrm{x}}-I_0$$ 1

where I _max = the maximum fluorescence intensity achieved postaddition of transporters, and I ₀ = the initial fluorescence intensity.

This value was employed for calibration purposes. The fractional fluorescence intensity, which serves as an approximation of the YSL influx percentage relative to the maximum transport capacity, was calculated by employing the subsequent eq .

$$I_{\mathrm{f}}=(I_t-I_0)/\mathrm{\Delta }{I}_{\mathrm{m}\mathrm{a}\mathrm{x}}$$ 2

where I _t = the fluorescence intensity at time t, while I ₀ = the fluorescence intensity at the initial time point.

By fitting the fractional peptide transport activity R against the transport concentration to determine the EC₅₀ values and the Hill coefficient n using eq .

$$R=1/(1+{({\mathrm{EC}}_{50}/[\mathrm{transporter}])}^n)$$ 3

The raw data were analyzed in accordance with the established protocol described by Smith et al. Fitting of the individual peptide transport curves to eq allowed us to determine the observed influx rate constants for YSL (K _obs). The equation is as follows:

$$I_{\mathrm{f}1}=A-\exp (-k\times t)$$ 4

where I _f1 = the fractional emission intensity, A = the top plateaus in units of fractional emission intensity, and k = the observed influx rate constants for YSL at time t.

Measurement of Intracellular YSL Content

A total of 1 × 10⁶ MCF-7 cells were seeded in each well of a 6-well plate and incubated at 37 °C with 5% CO₂ for 24 h. Following incubation, the cells in the plate were treated with YSL (100 μM) and various transporters (0.1 μM) and further incubated for 4 h. A control group received treatment with YSL (100 μM) under the same conditions. After incubation, the cells were washed with 1× PBS three times. Subsequently, the cells were quenched by using cold methanol (HPLC grade) before being gently detached from the culture dish.

The methanol solution containing the quenched cells was snap-frozen in liquid nitrogen. The samples were thawed on ice for 2 min, vortexed for 15 s, and sonicated for 2 min. Then, the samples were centrifuged at 3000 × g for 5 min at 4 °C, and the supernatant was collected. The cell pellet was washed twice with cold methanol. The methanol of the combined supernatants was removed, and the residual solutions were freeze-dried. The cell extracts were kept at −80 °C for further analysis. The concentration of YSL in the cell extracts was measured by high-performance liquid chromatography–mass spectrometry (HPLC-MS).

HPLC-MS analysis was performed on a Thermo Scientific Dionex UltiMate 3000 system coupled to a high-resolution Thermo Scientific Q-Exactive Orbitrap mass spectrometer. The samples were dissolved in methanol for further analysis using a reversed-phase XBridge C18 column (5 μm particle size, 4.6 × 250 mm) and maintained at 35 °C. The mobile phases were deionized water containing 0.1% formic acid (mobile phase A) and methanol (mobile phase B). The flow rate was 1 mL/min. The gradient elution was as follows: 30% B was maintained for 5 min and then linearly increased to 100% B from 5 to 13 min, and 100% B was maintained for 2 min. Finally, the mobile phase was returned to 30% B in 15.5 min, followed by equilibration at 30% B for 5 min.

To prepare the standard calibration curve, the YSL solution was serially diluted to concentrations of 0.5, 1, 2, 4, 6, 8, and 10 μM using methanol (HPLC grade) and then subjected to HPLC-MS analysis. The standard calibration curve was generated by plotting the mean ion peak area of the feature peaks with the corresponding YSL concentrations. The concentrations of YSL in cell extracts were determined by measuring the characteristic ion peak and substituting the mean ion peak area for the standard calibration curve. Samples were appropriately concentrated or diluted to ensure that their mean peak areas fell within the calibration range. Each sample was analyzed in triplicate to obtain the mean ion peak area. The cellular uptake rates of YSL in MCF-7 cells were calculated using eq .

$$\mathrm{Uptake\; ratio}=\frac{[\mathbf{YSL}]\mathrm{in\; cell\; extracts}}{[\mathbf{YSL}]\mathrm{in\; cell\; medium}}\times 100\%$$ 5

MTT Assay

Human breast cancer cells (MCF-7) were seeded into 96-well plates and cultured to a 60% confluence level for conducting a cell viability assay. Solutions of 5F–C12 and YSL at various concentrations were made in DMEM and then applied to the cells for a duration of 24 h. After this period, 20 μL of an MTT solution (5 mg/mL in PBS) was added to each well, and the cells were further incubated for an additional 4 h at 37 °C. The formed formazan product was dissolved using DMSO, and its absorbance was measured at 490 nm with a microplate reader. The cell viabilities were determined using the eq .

$$\%\mathrm{Cell\; viability}=\frac{{\mathrm{OD}}_{490}(\mathbf{compound})}{{\mathrm{OD}}_{490}(0.5\%\mathrm{DMSO})}\times 100\%$$ 6

The collected data were plotted against the logarithm of the concentrations of the compounds, and the IC₅₀ values were calculated using a nonlinear regression curve fit in GraphPad Prism 8.0.1.

The Combination Index (CI), formulated by Chou and Talalay, serves as a robust quantitative framework for analyzing drug interactions in combination therapies. Its mathematical definition is expressed as eq :

$$\mathrm{CI}=D_{\mathrm{A}}/{\mathrm{IC}}_{50,\mathrm{A}}+D_{\mathrm{B}}/{\mathrm{IC}}_{50,\mathrm{B}}$$ 7

where D _A = the experimental concentrations of drug A, D _B = the experimental concentrations of drug B, IC_50, A = the half-maximal inhibitory concentrations of drug A, and IC_50, B = the half-maximal inhibitory concentrations of drug B.

In Vivo Anticancer Efficacy of YSL and 5F–C12

The study adhered strictly to the Guide for the Care and Use of Laboratory Animals and was approved by the Institutional Animal Care and Use Committee of Xiamen University (approval number: XMULAC20220041). To establish a breast cancer model, 30 SPF-grade female BALB/c nude mice (6–7 weeks old, 20 ± 2 g) were subcutaneously inoculated with MCF-7 cells (1 × 10⁷ cells/mouse). Once tumors exceeded 50 mm³ in volume, the mice were randomized into six groups (n = 5 per group) according to the random number table method: (i) G0: saline group, receiving intratumoral injections of phosphate-buffered saline; (ii) G1: low-dose 5F–C12 group (0.8 mg/kg); (iii) G2: high-dose 5F–C12 group (1.6 mg/kg); (iv) G3: YSL treatment group (8 mg/kg); (v) G4: YSL (8 mg/kg) + low-dose 5F–C12 (0.8 mg/kg); and (vi) G5: YSL (8 mg/kg) + high-dose 5F–C12 (1.6 mg/kg). Treatments were administered via intratumoral injection every 2 days for 14 days. Throughout the study, the general condition of the mice (daily activity, mental state, and body weight) was carefully monitored. Tumor dimensions were measured using calipers, and volumes were calculated as V = πabc/6 (where a, b, and c are the tumor dimensions). Data were presented as means ± SEM (n = 5 biologically independent samples). Statistical analysis was conducted using GraphPad Prism version 8.01. Statistical significance was analyzed by one-way ANOVA with a Tukey post hoc test. Symbols **** stands for significant differences between the control group and other groups, with p < 0.0001. After 14 days, the mice were euthanized, and selected organs were snap-frozen in liquid nitrogen for subsequent analysis.

The tumor inhibition rate (TIR) is a quantitative measure of the antitumor efficacy of therapeutic agents. It is calculated using eq :

$$\mathrm{TIR}(\%)=1-\frac{\mathrm{Mean\; tumor\; volume\; of\; treatment\; group}}{\mathrm{Mean\; tumor\; volume\; of\; control\; group}}\times 100\%$$ 8

Statistical Analysis

All data were presented as mean ± standard deviation, and the ANOVA t-test was used for significance analysis between the two groups. A p-value below 0.05 was considered a significant difference, where *** represents p < 0.001 and **** represents p < 0.0001.

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/jacsau.5c00204.

• Synthetic procedures and a full set of characterization data, including ¹H NMR, ¹³C NMR, MS, peptide transport studies, and biological studies (PDF)

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D.L., C.J., and X.Z. contributed equally to this work.

The authors declare no competing financial interest.