Peptide-based delivery vectors with pre-defined geometrical locks

Abstract

Design of peptide-based targeted delivery vectors with attributes of specificity and selective cellular targeting by fixing their topology and resulting electrostatic fingerprint is the objective of this study. We formulated our peptide design platform by utilizing the possibilities of side-chain induced geometric restrictions in a typical peptide molecule. Conceptually, we locked the conformation of the RGD/NGR motif of tumor homing peptides (THPs) by mutating glycine in these motifs with d-proline and tailed the peptides with a syndiotactic amphipathic segment for cellular penetration. The designed peptides were synthesized, characterized, and tested in vitro on various cell lines, including breast cancer (MDA-MB-231), cervical cancer (HeLa), osteosarcoma (U2-OS) and non-cancer mammary epithelial cells (MCF-10A), by flow cytometry and confocal microscopy. The results showed differential cellular uptake in different cell types, as a result of the distinct electrostatic fingerprint encoded in their design. The uptake of serum pre-treated peptides by cells reveals the retention of peptide activity even after the incubation with serum. In addition, peptide–methotrexate (MTX) conjugates compared to the methotrexate drug showed enhanced apoptotic cell death in MTX-resistant MDA-MB-231 cells, indicating the increase in MTX bioavailability.

Design of topologically fixed heterochiral peptide-based delivery vectors for selective cellular targeting, drug delivery and biocompatibility under serum treatment conditions.

Introduction

Chemotherapy is one of the most effective therapeutic options for the treatment of cancer. The efficacy of such systemically administered drugs is often limited by their non-selectivity, poor water solubility, and low therapeutic index. The purpose of using a drug delivery vector is to transport drugs to the site of action with high specificity. This report focuses on the design and development of a delivery vector with tumor homing and cell-penetrating peptide segments conjugated with a drug molecule.

Peptides, the short polymers of amino acids, fall between small chemical compounds and large proteins in their size distribution profile. Peptides were known to function as hormones, signaling molecules, carriers, and supplements. The use of insulin, a peptide hormone for diabetes, opened the science of peptides as therapeutics.¹ Later, when chemical synthesis and sequence identification became plausible, many peptides have emerged as promising theranostic molecules in nanomedicine as targeting agents, nanovectors, drug delivery vehicles, and cytotoxic molecules.^2,3

The development of peptides for targeted drug delivery is very evident from the receptor-based studies of the cilengitide, iRGD, and NGR peptides.^4–6 The RGD (arginine–glycine–aspartic acid) and NGR (asparagine–glycine–arginine) motifs are known for their ability to selectively home in on tumor vasculature by targeting α_vβ₃/α_vβ₅ integrins and aminopeptidase N (CD13) receptors, respectively.^7–10 RGD polymeric peptides conjugated with pro-apoptotic peptides,¹¹ matrix metalloprotease (MMP-9) cleavable sequences,¹² and drugs like paclitaxel¹³ have also been reported for effective tumor targeting. The preferential affinity of these peptides for cancer cells through receptor targeting has introduced the concept of tumor homing peptides (THPs).¹⁴ On the other hand, cell-penetrating peptides have the ability to translocate cargoes ranging from small molecules to large proteins into the cells.^15–18

Homing peptides are known to enhance the accumulation of drugs on tumor sites⁵ and have been widely tested in various clinical studies.¹⁹ However, none of them succeeded in getting FDA approval. The major limiting factors for their development in the clinics include proteolytic stability, cell specificity, and response variations in in vitro, in vivo and clinical studies.²⁰ Herein, we present a plausible strategy to address the first two limitations by focussing on the design and development of a delivery vector with tumor homing and cell-penetrating motifs conjugated with a drug molecule. The third limitation can only be confirmed after in vivo studies and clinical trials.

Results

Peptide design strategy

Our efforts for the rational peptide design through the principles of the Ramachandran map incorporate stereochemically varied residues to produce enriched THPs. Earlier studies have shown that if glycine (G) in an RGD or NGR motif is mutated, then their efficacy is largely compromised.²¹ This observation directly points to the possibility of glycine assuming a (ϕ, ψ) dihedral angle combination, which falls in the D-region of the Ramachandran map (Fig. 1A).^22,23d-Proline is one residue with which we can fix this geometry firmly in the designated region in the (ϕ, ψ) map. So, we intentionally mutated glycine with d-proline in the RGD/QGR motifs to produce a topologically specific, yet constrained peptide, based on a reported homing peptide RGDPAYQGRFL²⁴ (Fig. 1A). We have earlier shown that design directives such as stereochemistry, amphipathicity, and electrostatics can be suitably incorporated in modulating the cell penetration of a peptide sequence with a good degree of selectivity.²⁵ In this study, we have combined the homing and penetration motifs in one sequence, in an attempt to design an optimum carrier for targeted delivery of small molecules. In the design, the tumor homing segment was followed by a short tail of a syndiotactic (peptide chain with alternating l and d stereochemistry), amphipathic sequence (Table 1). Amphipathicity in the tail sequence was achieved by designing lysine and leucine side-chains protruding in opposite directions resulting in two distinct zones of different polarities (Fig. 1B).

Fig. 1. Design of peptide-based delivery vectors. (A) Informed walk across the sterically allowed regions of the Ramachandran plot to fix the geometry of the RGD and NGR motifs. The highlighted text represents the conformational basins to introduce the topological fixation in the RGD/QGR motifs of peptides. (B) Electrostatic potential distribution of the designed peptides signifying the differences in potential values obtained as an effect of the change in stereochemistry and amphipathicity. (C) CD spectra of the designed peptides indicating the design directed structural disparities in secondary structure. (D) FTIR spectra with the C O bond signature peak, suggesting extended conformation.

Peptide sequences and mass characterization.

Peptide code	Amino acid sequence	Expected molecular mass			Observed molecular mass
		UN	CF	MTX	UN	CF	MTX
RG201	RpDPAYQpRFK	1373.7	1731.7	1809.7	1376	1734	1812
RG202	RpDPAYQpRFKLK	1614.8	1972.8	2050.8	1617	1975	2053
RG203	RpDPAYQpRFKkL	1614.8	1972.8	2050.8	1616	1975	2053
RG204	RpDPAYQpRFKkLlK	1856	2214	2292	1858	2217	2295

Electrostatic fingerprinting of the designed delivery vectors

Electrostatic interactions have a pivotal role in the specificity and efficacy of homing and cell penetration. The electrostatic potential distribution of each peptide was calculated through the finite difference Poisson–Boltzmann (FDPB) equation using DelPhi software.²⁶ Keeping the amino acid composition identical up to the eleventh residue in all four sequences, the extended tail with Lys-Leu-Lys (KLK) in RG202, Lys-(d-Lys)-Leu (KkL) in RG203 and Lys-(d-Lys)-Leu-(d-Leu)-Lys (KkLlK) in RG204 has significantly altered the electrostatic fingerprint of the parent sequence (Fig. 1B). Thus, each designed molecule has its own electrostatic signature, which directly modulates its functional properties, as discussed in the following sections.

Characterization of the peptide-based delivery vectors

The designed peptides were synthesized using solid-phase peptide synthesis and characterized by reverse-phase high-performance liquid chromatography (RP-HPLC) (Fig. S2†) and mass spectrometry (MALDI-TOF-MS) (Table 1 and Fig. S3†). N-Terminus modifications with 5(6)-carboxyfluorescein (CF) or methotrexate (MTX) were aimed to investigate their efficacy and specificity upon membrane interaction. Further, the design incorporated structural changes were verified by circular dichroism (CD) spectroscopy, and Fourier transform infrared (FTIR) spectroscopy. CD spectral analysis suggests that the designed peptides adopted different conformations at room temperature, suggesting no typical secondary signatures (Fig. 1C). FTIR experiments also indicate a major peak at 1635 cm⁻¹, which corresponds to C O bond stretching (Fig. 1D).²⁷ The CD and FTIR spectroscopy results broadly suggest that the designed peptide sequences are mainly in the extended conformation.

Molecular dynamics (MD) simulation

From our previous study, we have observed that l-Pro residues conform to a set of backbone dihedral angles in the Ramachandran plot.²⁸ In the present peptide design platform, we used d-Pro with the assumption that d-Pro will help in fixing the conformation of the RGD and NGR motifs in the range, which mirrors that for l-Pro.

RG201–RG204 peptides have two d-Pro residues at the second and eighth positions. The RGDPAYQGRFL peptide has Gly residues at each of the two mentioned positions. Therefore, the MD simulations were analyzed for the distribution of backbone dihedrals for d-Pro and Gly residues in the RG201–RG204 peptides and RGDPAYQGRFL, respectively (Fig. S1†). The backbone dihedral distributions of d-Pro residues in the RG201–RG204 peptides were in the range which can be assigned as the region corresponding to the mirror/inverse regions allowed for l-Pro. However, the dihedral angle distributions for Gly residues in the RGDPAYQGRFL peptide were spread over the Ramachandran map (Fig. S1†). This suggests that the use of d-Pro assists in fixing the conformation of the RGD and NGR motifs. Further, the Ramachandran map distributions on the peptide structure level in the MD simulations suggest irregular/disordered overall conformations for the designed peptides. This observation is concurrent with the CD spectroscopy experiment for the designed peptides.

Cellular binding or uptake of the designed peptides

The comparative uptake of peptides was quantitatively verified through flow cytometry in breast cancer (MDA-MB-231), cervical cancer (HeLa), LAMP-RFP transfected osteosarcoma (U2-OS) and mammary epithelial (MCF-10A) cells. The obtained results suggest that we could accomplish the two broad objectives envisaged in the design: (i) differential uptake between cancerous and non-cancerous cells and (ii) differential uptake between different cancer cell types. Peptide uptake in all cell lines is higher compared to that of the positive control RGDPAYQGRFL peptide. In particular, the uptake in MCF-10A cells is nearly four times higher, whereas this increment of peptide uptake varies between two to four times in HeLa and up to six times in MDA-MB-231, and doubles in U2-OS cells (Fig. 2). Also, we observed the maximum cell-associated fluorescence of the designed peptides in the U2-OS cell line followed by the MDA-MB-231 and HeLa or MCF-10A cell lines. Interestingly, in HeLa and MCF-10A cells, the peptide fluorescence signals are almost similar and are 2–3 times less than that observed in U2-OS cells (Fig. 2). This observation of lower cell-associated fluorescence in cervical cancer cells and mammary epithelial cells markedly represents the differential uptake and selective targeting of the designed peptides. This observation underlines an earlier report by Roland Brock and coworkers, clearly pointing out that the cell surface binding and internalization are two distinct mechanisms with separate structure–activity relationships involved.²⁹

Fig. 2. Cellular uptake of the peptides. The comparative uptake of the peptides in breast cancer (MDA-MB-231), cervical cancer (HeLa), LAMP-RFP transfected osteosarcoma (U2-OS), and mammary epithelial (MCF-10A) cells using flow cytometry. All cells were treated with 10 μM of CF-tagged peptides (RG201–RG204) in serum-free DMEM for 4 h at 37 °C. After treatment, the cells were washed, extracted, and analyzed by flow cytometry. Peptide ‘RGDPAYQGRFL,’ a known tumor homing peptide, was taken as the positive control.²⁴ Corrected MFI represents mean fluorescence intensity values obtained after subtracting the autofluorescence of cells. The results are presented as mean ± SD of three independent experiments. One-way analysis of variance (ANOVA) with a P-value <0.05 was used to calculate statistical significance. The asterisks without bars show the significance with respect to non-cancerous MCF-10A cells of the same peptide treatment group.

The cellular uptake of peptides was qualitatively assessed using confocal laser scanning microscopy in the same cell types. Strikingly, fluorescence imaging observations show that the uptake of the designed peptides varies with the cell types (Fig. 3 and S4–S7†). We observed that the peptides RG203 and RG204 show penetration into MDA-MB-231 cells, while in the case of U2-OS cells, they are observed to be mostly membrane-bound. Similarly, only RG202 molecules enter HeLa cells, unlike in MDA-MB-231 cells, where the penetration was observed for all the designed molecules (RG201–RG204). These observations clearly reflect the manifestation of differential electrostatic signature and stereochemical modifications encoded in the design of peptides to interact differently with the membranes of different cell types.

Fig. 3. Peptide binding/uptake using confocal laser scanning microscopy. Cellular uptake of CF-tagged peptides in (A) MDA-MB-231 cells, (B) HeLa cells, (C) MCF-10A cells, and (D) LAMP-RFP transfected U2-OS cells in serum-free DMEM for 4 h at 37 °C. Blue indicates Hoechst 33342 staining; green indicates peptide uptake; red shows lysosomes, and ‘merged’ shows the cellular uptake. Scale bars: 50 μm (A) and 20 μm (B–D).

The other reason for differential uptake can be attributed to the cell membrane heterogeneity and resting membrane potential of cells. Cancer cells have a more negatively charged plasma membrane compared to non-cancerous cells due to the presence of phosphatidylserine on the outer membrane of cancer cells. For example, MDA-MB-231 cells are hypo-polarized (resting membrane potential ≥20 mV) compared to MCF-10A (resting membrane potential ≥50 mV).^30–32 More negative charge, hypo-polarized resting membrane potential, and variable receptor density could be the other contributing factors affecting the peptide uptake in cancer cells.

Biocompatibility and hemotoxicity of the peptides

Peptide compatibility in biological fluids like serum is of foremost concern for bioavailability in the physiological system, and therefore, their functional activity was verified in the presence of serum. The cellular uptake of the designed peptides was tested using serum pre-treated peptides and untreated peptides by flow cytometry. There is almost 90% retention in peptide function after treatment with serum irrespective of the cell types. The similar cellular uptake of the peptides under serum treated and untreated conditions confirms their biocompatible nature (Fig. S8†).

In addition to this, we have also performed the serum stability assay of the designed peptides using HPLC.³³ The results indicate that the peptides RG203 and RG204 retained their stability after serum treatment (Fig. S9†). Further, their therapeutic safety was assessed in mammalian red blood corpuscles (RBCs). We observed that the peptides, along with the peptide–MTX conjugates, are non-hemolytic in nature, as they showed less than three percent toxicity to mammalian RBCs up to a concentration of 100 μM (Fig. S10†).

Cytotoxicity assessment

The designed peptides were also conjugated with an anticancer drug, methotrexate (MTX), at the N-terminus to investigate their cellular toxicity as a drug delivery vector. The cytotoxic activity of these peptides, along with free MTX and the MTX conjugated peptides, was evaluated in MTX-resistant MDA-MB-231,^34,35 and MCF-10A cells using the tetramethylrhodamine methyl ester (TMRM) based assay. The release of TMRM dye from mitochondria indicates apoptotic cell death due to the depolarization of mitochondrial membrane potential. In our study, the cells with TMRM loss and karyopyknosis were considered undergoing programmed cell death and hence, considered for analysis. As compared to MTX alone, the peptides and peptide–MTX conjugates possess augmented toxicity to MDA-MB-231 cells, compared to MCF-10A cells, thereby supporting the earlier observation of higher uptake rates in cancer cells (Fig. 4 and S11 and S12†). In addition to this, the similar toxicity of the peptides and peptide–MTX conjugates indicates them as probable anticancer peptides.

Fig. 4. Cytotoxicity of the designed peptides. Analysis of peptide toxicity in MDA-MB-231 cells and MCF-10A cells by peptide treatment employing the TMRM based assay. MDA-MB-231 and MCF-10A cells were treated with MTX (Xn), the peptide vectors (Pn) and peptide–methotrexate (PXn) conjugates for 48 h at three different concentrations (n = 25 μM, 50 μM, and 100 μM) for 48 h at 37 °C. The cells with TMRM loss and nucleus condensation indicate apoptosis.

Caspase-3 activation

To confirm the cell death through apoptosis, MDA-MB-231 cells having the stable expression of a CFP–YFP FRET-based caspase sensor (DEVD) were used for apoptosis detection. This probe is composed of a cyan fluorescent protein (ECFP) and yellow fluorescent protein (EYFP or Venus), which are fused by the linker having a caspase-3 cleavage site (DEVDG). The increase in the CFP/YFP ratio as a result of the loss of fluorescence resonance energy transfer (FRET) indicates caspase-3 activation.³⁶ These transfected cells were treated with MTX, the peptides, and peptide–MTX conjugates at different concentrations. After treatment, the loss of FRET in cells is observed by using the ratiometric fluorescence imaging.³⁶ The obtained results present high caspase-3 activation in cells treated with the peptide–MTX conjugates, compared to cells treated with only MTX or the peptides alone (Fig. 5 and S13†).

Fig. 5. Validation of apoptotic cell death. To confirm the cell death by apoptosis, MDA-MB-231 cells having the stable expression of the CFP–YFP FRET-based caspase sensor, DEVD, were treated with MTX, the peptides (RG20X–UN) and peptide–MTX (RG20X–MTX) conjugates for 48 h. The loss of FRET in cells was measured in terms of their difference (increase) in the CFP–YFP ratio, and the cells with FRET loss were taken for analysis. The graph represents the ratio (mean ± SD) of cells undergoing cell death by caspase-3 activation.

Verification of peptide aggregation propensity

The self-assembly of peptides is driven by the synergistic effect of the involved interactions like metal-ion coordinations, non-covalent interactions, stacking-pair interactions, etc. The composition of amino acids in a peptide chain is the major decisive factor for the nature of peptide self-assembly formation. Peptide nano-assemblies are crucial in imparting or augmenting the specified function of peptides like the anti-tumor effect.^37–39 Looking into this fact, we have verified the self-assembly formation of peptides by using standard methods like scanning electron microscopy (FE-SEM), right-angle scattering, and the ThT fluorescence assay. Interestingly, the results from all the experiments confirm that the designed peptides are non-aggregating in nature and do not form any defined nano-structures, even in the solution form (Fig. S14 and S15†). This suggests that the observed differential behavior of the peptides towards different cell types is not due to the peptide nano-assembly formations but only due to the fundamental properties of the designed peptides attained by their amino acid composition.

Discussion

Tumor homing peptides have RGD/NGR trimers as common motifs with glycine as the middle residue; without glycine, the motif generally loses its activity except in a few cases such as RHD and RYD.^40,41 This observation compelled us to understand the specific selection of the glycine amino acid in these motifs. This study started with the hypothesis that glycine occupies the ϕ, ψ dihedral angle combinations typical of a d-amino acid, to provide flexibility for recognition of cognate receptors of these motifs. To verify this, we mutated glycine with d-proline to fix the topology with clearly defined and constrained dihedral angles, and also verified this using molecular dynamics simulation (Fig. 6A and S1†). We calculated the geometrical parameters like the Euclidean distance and the angle between the terminal residues of homing domains to observe the effect of d-proline mutation (Fig. 6B). We designed a second segment to the peptide, which is cationic or amphipathic in its charge distribution (Fig. 6C–E). Our earlier studies on antimicrobial peptides and cell-penetrating peptides have shown that the properties like amphipathicity and syndiotacticity add good value to the overall penetrative ability.^25,42 It was also reported that the alternating ldldld stereochemical sequence has a natural tendency to form a gramicidin helix.^43,44 Using this information, we designed a peptide system with a homing domain and a penetrating segment loaded with a typical anticancer drug molecule. The designed peptides were synthesized by solid-phase peptide synthesis using Fmoc chemistry, and N-terminally conjugated with a fluorophore and an anticancer drug for imaging and cytotoxicity studies. CD and FTIR spectroscopic studies provided the necessary information on the extended secondary structures of the synthesized peptides.

Fig. 6. Schematic illustration of the evolved concept for vector design. (A) Ramachandran plot with the highlighted region to depict the fixed geometry of d-proline in the RGD and QGR motifs. (B) Geometrical parameters of d-proline fixation showed in peptide RG201. (C–E) The amino acid sequence of designed peptides RG202, RG203, and RG204, respectively, specifically indicating the tailed cationic (blue) and hydrophobic (grey) residues. (F) The syndiotactic segment of the RG204 peptide, forming a gramicidin helix with a distinct cationic zone highlighted by the yellow arc. l- and d-amino acids are mentioned as upper case and lower case letters, respectively.

The attempt of sequential addition of cationic and hydrophobic residues after the homing segment in the designed peptides alters their electrostatic potential and, consequently, functional properties like selectivity and penetrative ability. This has been verified by evaluating their preferential uptake, biocompatibility in serum, and selective toxicity against cancer cells. In confocal imaging, we observed that the cellular uptake of each peptide varies with the cancer cell types. Peptide RG202 with a poly-l tail penetrates into MDA-MB-231 cells and HeLa cells, whereas peptides RG203 and RG204 with a syndiotactic (LDLD) extension penetrate only into MDA-MB-231 cells. Flow cytometry experiments suggest the preferential cellular uptake or binding of peptides in breast cancer MDA-MB-231 and osteosarcoma U2-OS cells, compared to non-cancerous mammary epithelial MCF-10A cells. We consider that the syndiotactic tail of RG204 forms the gramicidin helix (Fig. 6F) and aids in the penetration, whereas the homing domain provides selectivity.

The principal aim of any delivery vector is to enhance the therapeutic index. We observed a two–three fold difference in the cytotoxicity of the peptide–MTX conjugates compared to that of MTX alone. The selective toxicity to MDA-MB-231 cells of all the peptides, compared to that for non-cancerous MCF-10A cells, points to the possibility of developing the designed molecular system as a specific vector for breast cancer. However, the similar toxicity of the peptides and peptide–MTX conjugates indicates that the designed peptides can also be used as anticancer peptides and can be improved further by conjugating them with drugs like paclitaxel and doxorubicin. Furthermore, the ratiometric fluorescence assay of caspase-3 activation confirms the apoptotic cell death, which is a preferred feature for any such lead candidates.

The designed molecules retained their functional activity after serum treatment indicating their biocompatible nature, and their negligible toxicity against mammalian RBCs indicates their therapeutic safety. Taken together, our approach of vector design can be helpful in developing potential peptide-conjugated drug delivery vehicles for selective targeting of different cancer types.

Conclusion

The scope of peptide therapeutics is evident from their current status in the pharmaceutical industry. The synergism of peptide–cargo conjugations has offered better solutions to the present therapeutics. We have conceived this project with the aim of developing a comprehensive therapeutic agent with three distinct motifs: a tumor homing segment, a cell-penetrating sequence, and a cytotoxic drug molecule. The conceptual development of a tumor homing peptide designed by mutating glycine of the RGD and NGR motifs with d-proline was found to be very effective in homing of cancer cells preferentially, and to a greater extent, introduce selectivity between cancer cell types. Incorporation of the amphipathic syndiotactic tail could translocate the cargo (MTX) into the cell, inducing apoptosis. The developed peptides offer selective membrane targeting or cellular penetration or both, depending on cell types. The two to three-fold difference observed in the drug conjugates in cytotoxicity experiments clearly suggest the possibility of a corresponding difference in their therapeutic index. The strategy of encoding topology with RpD and QpR motifs, along with the amphipathic cell-penetrating tail, worked well in inducing selectivity and penetration. The tuning of the structure by stereochemical sequence selection and tuning of electrostatics by chemical sequence selection were the guiding principles for the design of these delivery vectors. This work will be further extended with in vivo and clinical studies to graduate from a proof of concept investigation to a therapeutic solution.

Experimental

Peptide synthesis

The designed peptides were synthesized by solid-phase peptide synthesis (SPPS) on HMPA resin using Fmoc chemistry. All solvents, activators, Fmoc protected amino acids and resin were purchased from Sigma-Aldrich and Merck with 99% purity. Fmoc protected amino acids were coupled on HMPA resin as a solid support. For coupling, a three-fold excess of amino acids was dissolved in N,N-dimethylformamide (DMF), and activated using hydroxy benzotriazole (HOBt), 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU) and N,N-diisopropylethylamine (DIPEA). The activated mixture was added to HMPA resin pre-soaked in DMF and stirred on a rotor for 1 h at room temperature. The excess activating mixture was washed with DMF, and the second coupling of the same amino acid was performed for 30 minutes to ensure complete attachment at all resin sites. Before subsequent amino acid attachment, Fmoc deprotection of the attached amino acid was performed with 20% piperidine for 20 min. The resin was washed with DMF till neutral pH was achieved. This cycle was repeated until the last N-terminus residue of the peptide. Post synthesis, peptides were conjugated with 5(6)-carboxyfluorescein (CF) and methotrexate (MTX) at the N-terminus employing the same protocol. However, after CF attachment, the resin was washed twice with 20% piperidine for ten and forty minutes, respectively, with intermittent DMF washes, till the attainment of neutral pH.⁴⁵ The synthesized peptides were cleaved from the solid support using a cocktail of m-cresol, thioanisole, 1,2-ethanedithiol (EDT), and trifluoroacetic acid (TFA) in a ratio of 2 : 2 : 1 : 20, and precipitated in cold diethyl ether. Crude peptides were purified by reverse phase high-performance liquid chromatography (RP-HPLC; Shimadzu, LC 20 AD) with a semi-preparative C-18 column and 10 to 100% acetonitrile gradient in water with 0.1% trifluoroacetic acid (TFA). Purified peptides were characterized by mass spectrometry (MALDI-TOF MS; Bruker, Autoflex Speed) using an α-cyano-4-hydroxycinnamic acid (HCCA) matrix. All the peptides and their conjugates used were more than 90% pure.

Secondary structure characterization

The design incorporated structural changes in the peptides were monitored by circular dichroism (CD) spectroscopy (Jasco J-1500 spectropolarimeter) and Fourier transform infrared (FTIR) spectroscopy (Shimadzu IR Affinity-1S FTIR spectrophotometer). Peptide solutions were prepared in water, and a 10 μM peptide concentration was used to conduct both studies. Far-UV CD spectra were recorded in a 0.1 cm path length cuvette at a scan rate of 100 nm min⁻¹ and converted into mean residue ellipticity, as described in our earlier work.²⁵ FTIR spectra of the same peptide samples were recorded in attenuated total reflection (ATR) mode at a resolution of 4 cm⁻¹.

Electrostatic profiling of the designed peptides

The designed peptides with varied syndiotactic tails were mapped for estimating electrostatic potential by solving the finite difference Poisson–Boltzmann equation using Delphi software.²⁶ The electrostatic potential values distinctly represent each designed molecule with a unique electrostatic signature. The obtained signature profiles were plotted using PyMOL in three-dimensional space.

Molecular dynamics simulation

Peptide structure coordinate files were generated using Ribosome software provided by George D. Rose. The chirality of d-proline residues was verified using ProChiral.⁴⁶ All molecular dynamics (MD) simulations were performed on GROMACS,⁴⁷ a molecular dynamics simulation suite using the GROMOS96 43a1 force field,⁴⁸ for 20 nanoseconds (ns) at 300 K. The structures were energy minimized in a water solvated system using the steepest descent algorithm. A production run of 20 ns was completed with an integration step of 2 × 10⁻¹² s. Bond lengths were constrained with 10⁻⁴ geometrical accuracy with the LINCS algorithm, and non-bonded interactions were spaced at 0.8–1.1 nanometers (nm). The Berendsen thermostat was used for temperature coupling of the peptide, and initial velocities were calculated as per the Maxwell distribution at 300 K.

Cell culture

The MDA-MB-231 (breast cancer; purchased from NCI, USA), HeLa (cervical cancer; procured from ATCC) and U2-OS (osteosarcoma; obtained from ATCC) cell lines were cultured in Dulbecco's modified Eagle media (DMEM) supplemented with fetal bovine serum (FBS) (10% v/v), 1% antibiotic solution (10 000 units per mL penicillin and 10 mg mL⁻¹ streptomycin and 25 μg mL⁻¹ amphotericin B) and NaHCO₃ salt. MCF-10A cells (non-cancerous mammary epithelial cells; purchased from Sigma) were cultured in mammary epithelial cell growth medium (MEGM) with BPE, hEGF, insulin, hydrocortisone, and GA-1000. The cells were grown as sub-confluent monolayers in T-flasks at 37 °C under a water-saturated environment with 5% CO₂. Mammalian expression vector, Lamp1-RFP, was procured from Addgene, USA (Plasmid #1817) to transfect U2-OS cells and FRET caspase-3 sensor expression vector ECFP-VENUS SCAT-3 was gifted by Dr. Masayuki Miura, RIKEN Brain Science Institute, Japan.

Cellular uptake of the designed peptide-based delivery vectors

MDA-MB-231, HeLa, U2-OS, and MCF-10A cells were seeded in 96-well glass-bottom plates at a density of 10 000 cells per well and incubated overnight. The cells were incubated with CF-tagged peptides for 4 h at 37 °C in serum-free DMEM. After 4 h, the cells were washed twice and stained with Hoechst 33342 (1 μg mL⁻¹) to stain nuclei. The cells were washed again twice after Hoechst staining to remove the excess stain and replenished with phenol-red and serum-free DMEM to acquire fluorescence images using a confocal laser scanning microscope (NikonA1R).

For quantitative experiments, the four cell types (MDA-MB-231, HeLa, U2-OS, and MCF-10A) were seeded in 24-well plates at a density of 20 000 cells per well. After overnight incubation, the cells were treated with 10 μM of CF-tagged peptides in serum-free DMEM for 4 h at 37 °C. Post-treatment cells were washed twice with 1× PBS and extracted by treatment with 0.06% EDTA in 1× PBS. After extraction, again, the cells were washed twice with 1× PBS using centrifugation at 1200 rpm for 5 minutes and stored on ice. The cells were analyzed using a flow cytometer (FACS Aria III, BD Biosciences). Cells without treatment were considered as a negative control in the experiment.

Cytotoxicity assessment

The TMRM assay was used to investigate the cytotoxicity of MTX, the peptides, and peptide–MTX conjugates. MDA-MB-231 and MCF-10A cells were seeded in the 96-well glass-bottom plates and cultured, as explained in section 2.5. After 24 h, the cells were washed and stained with Hoechst 33342 (1 μg mL⁻¹) and 100 nM TMRM at 37 °C. The cells were rewashed and replenished with peptide treatment in 20 nM TMRM phenol-red and serum-free DMEM at different concentrations. The montage images (2 × 2) were captured in blue and red channels using a Pathway Bio-imager 435 (BD Biosciences, San Jose, CA, USA) after 48 h using a dry 20× objective with NA 0.7. After the acquisition, images were analyzed for karyopyknosis and TMRM loss.

Validation of apoptosis

The apoptotic effect of MTX, the peptides, and peptide–MTX conjugates was confirmed using the cell-based assay.³⁶ MDA-MB-231 cells with stable expression of the FRET-based caspase sensor (DEVD) were seeded in 96-well glass-bottom plates. After incubation for 24 h, the cells were treated with the peptides in phenol-red and serum-free DMEM at various concentrations for 48 h. The ratiometric imaging of CFP/YFP FRET was performed using the high-throughput Pathway Bio-imager 435 (BD Biosciences, San Jose, CA, USA) in different channels. Montage images (2 × 2) were acquired and later stitched as a single image for each well and used to generate quantitative data.

Biocompatibility of the designed peptides

CF-tagged peptides were incubated with fetal bovine serum (FBS) in equal volume for 1 h at 37 °C. MDA-MB-231, HeLa, U2-OS, and MCF-10A cells were seeded in 24-well plates at a density of 30 000 cells per well and incubated overnight. Then, the cells were washed and treated with serum pre-treated and untreated peptides for 4 h at 37 °C. Post-treatment, the cells were rewashed and harvested to quantify the uptake under both conditions using flow cytometry, as described previously.

Serum stability of the designed peptides

The serum stability assay of the designed peptides was performed using HPLC to verify their stability in serum.³³ For this experiment, 100 μl of peptides (1 mM concentration) were mixed with 100 μl of 100% human serum (sterile; Sigma) and incubated for 2 h at 37 °C in a shaking incubator. After incubation, 100 μl of 0.6% trichloroacetic acid (TCA) was added to precipitate serum proteins. The samples were kept at 4 °C for 15 min and then centrifuged at 13 000 rpm for 5 min. The supernatant was collected, and 20 μl of it was injected in a HPLC instrument (Shimadzu, LC 20 AD). Similar treatment was given to conditions having only serum and only peptides, respectively.

Hemotoxicity estimation of the peptides

The hemotoxicity of the peptides and their MTX conjugates was assayed by using fresh human blood samples collected in a vacutainer blood collection tube. The collected blood sample was centrifuged at 800g for 5 min and washed thrice with PBS to obtain an erythrocyte pellet. The pellet was resuspended to obtain 10% hematocrit, and was subsequently incubated with an equal volume of peptides of 200 μM concentration for 2 h at 37 °C in a shaking incubator. After incubation, the samples were centrifuged at 800g for 5 min, and the supernatant was collected. The absorbance of the supernatant was recorded at 540 nm to quantify heme release after RBC lysis. The data were normalized with the value obtained from complete lysis, i.e., with 0.5% of Triton X-100. This study was approved by the Institutional Human Ethics Committee (IHEC) of the Indian Institute of Technology Guwahati, as per the guidelines of the Govt. of India. Informed consent was obtained from the human participants of this study.

Right angle scattering of the designed peptides

The self-assembly formation of the peptides in solution was monitored by performing the 90° scattering assay. Time course measurement spectra were recorded for all the peptides at 0 h and 24 h, respectively, at 450 nm using a spectrofluorometer (Jasco FP 8500; slit width = 2.5 nm).

Thioflavin T (ThT) fluorescence assay

The assembly formation of peptides was evaluated by the ThT binding assay. Freshly prepared ThT dye solution was added to the peptide samples of 0 h and 24 h, respectively. Fluorescence spectra (Ex/Em: 450/485 nm; slit width Ex/Em: 2.5/5 nm) were recorded on the spectrofluorometer (Jasco FP 8500) at 25 °C using a quartz cuvette (Helma, Sigma Aldrich) with a 1 cm path length.

Field emission scanning electron microscopy (FESEM)

Peptide samples (30 μl) were drop-cast on a clean glass surface and allowed to air dry. The coated glass samples were loaded on the FESEM sample holder with carbon tape and uniformly coated with platinum to enhance the conductivity. The images were acquired using a JSM-7610F electron microscope (JEOL).

Statistical analysis

The one-way analysis of variance test was used to analyze the data. A P-value < 0.05 was considered as the threshold for statistical significance. All experiments were performed with a minimum sample size of three per experiment. Results are presented as mean ± standard deviation (SD).

Author contributions

V. R. conceived the idea and directed the project. V. R. and T. R. S. K. designed the experiments. R. G. performed most of the experiments; A. C. helped in bioimaging experiments; G. J. contributed in design development. R. G., T. R. S. K. and V. R. analyzed the data and wrote the manuscript.

Conflicts of interest

There are no conflicts to declare. However, a patent of this work has been filed with IPO Kolkata (patent number TEMP/E-1/36058/2019-KOL dated 23.08.2019).