A Multifunctional Drug Delivery System Based on Switchable Peptide-Stabilized Emulsions

doi:10.21203/rs.3.rs-3121613/v1

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A Multifunctional Drug Delivery System Based on Switchable Peptide-Stabilized Emulsions

https://doi.org/10.21203/rs.3.rs-3121613/v1

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Emulsions are commonly used for drug delivery, yet they are usually limited to exclusively delivering either lipophilic compounds or hydrophilic compounds. This separation negates possible synergetic therapeutic roles between such compounds. Here, we introduce a novel design for a short peptide that can stabilize emulsions. Upon binding certain metal ions, the peptide acts as a molecular switch, changes conformation, and becomes amphiphilic. Spectroscopic methods, NMR, and molecular dynamics provide information on the mechanism of this complexation-triggered amphiphilicity. The stability of these unique emulsions is based on histidine-metal bonds, which break at low pH values, selectively releasing their contents at the extracellular pH of tumors. Paclitaxel-encapsulated emulsion demonstrated strong activity against HeLa cells with an IC₅₀ of 70 nM, possibly enhanced by the simultaneous release of Zn²⁺ ions. Importantly, the emulsion was easily functionalized with various hexahistidine-tagged motifs that can supply the emulsions with many functions beyond drug delivery.

Physical sciences/Nanoscience and technology/Nanobiotechnology

Physical sciences/Chemistry/Chemical biology/Peptides

Emulsion drug delivery systems are a common method for administering lipophilic drugs¹. They can be easily prepared and applied, and they have been extensively researched². However, these systems are limited to the delivery of hydrophobic drugs dissolved in the non-aqueous phase of oil-in-water (o/w) emulsions. Whereby water-soluble drugs can be encapsulated in water-in-oil (w/o) emulsions. This separation is detrimental to the potential of emulsions in drug delivery, as it negates possible synergetic therapeutic effects that water-soluble and -insoluble compounds may have. Synergistic therapy could originate from combinations of lipophilic and hydrophilic drugs³, combinations of drugs and stimuli-responsive materials^4–6, and combinations of drugs with metal ions^7–9.

Several anti-tumor drugs can be taken as an example for this problem. The efficacy of the chemotherapeutic drug paclitaxel (PTX) was shown to be enhanced by the presence of Zn²⁺ ions^10–12. Similarly, the anti-tumor effects of disulfiram and curcumin were also shown to be promoted by interactions with metal ions^13–16. Some of these metal ion-enhanced compounds are hydrophobic and cannot be dissolved in water, prohibiting their delivery concomitantly with metal ions.

Peptide amphiphiles (PAs) can assemble into various structures for different purposes, including targeted drug delivery and therapeutic applications^17–19. Stabilization of emulsion droplets by short PAs was demonstrated by their assembly into fibrous networks²⁰. Furthermore, PAs can be designed to incorporate metal-binding moieties^21,22. These moieties can ultimately act as triggers that alter the properties of the PAs or the PA-based structures. However, to our knowledge there are no publications describing peptide amphiphilicity that is triggered by complexation with metal ions.

To achieve simultaneous delivery of both compounds, we designed a simple and short metal-binding peptide that changes its conformation upon complexation with certain metal ions (Fig. 1a). This structural change can be viewed as a molecular switch that controls the peptide’s properties, as it turns the peptide into an amphiphilic complex. This complex is similar to Gemini surfactants in which two amphiphiles are connected at the polar groups, and it can stabilize emulsions²³. The metal ions on the surface of the emulsion droplets enable the functionalization of the droplets, as the ions can be used to bind various water-soluble compounds. By using this peptide to stabilize o/w emulsions, it is possible to simultaneously release the hydrophobic drugs, the surface-attached compounds, and the metal ions. This can prevent complications due to the stability of the drug-metal complex, or the metal ions in the serum, and the emulsion droplets can acquire various properties from their coating compounds^14,15,24.

Complexation-triggered amphiphilicity

We designed the peptide Trp-His-His-Trp (WHHW), which contains a metal-binding dihistidine moiety surrounded by two hydrophobic amino acids. When an aqueous solution of the peptide and certain metal ions are mixed with a non-aqueous phase, an emulsion is formed (Fig. 1b). This occurs only in presence of both the peptide and the metal ions, indicating that this emulsion is stabilized by the formation of a peptide-metal complex. Depending on the ratio between the aqueous and non-aqueous phases, o/w or w/o emulsions can be formed (Fig. 1c, Supplementary Fig. 1a). The peptide has weak inherent fluorescence, but its presence on the interface of the emulsion droplets was inferred by labeling it with FITC (Fig. 1d, Supplementary Fig. 1b).

Since the stability of the emulsion depends on the stability of the complex, it can be reversibly disassembled by lowering the pH, thus protonating the imidazole groups of the histidines and preventing their coordination to the metal ions (Supplementary Fig. 1c). By increasing the pH again, the emulsion can be reassembled. With chloroform as the non-aqueous phase, the emulsion has a critical micelle concentration of ~ 0.2 mM and it is stable for at least 4 months at room temperature (Supplementary Fig. 2a). Adding EDTA to remove the metal ions visibly disassembled the emulsion after ~ 24 h (Supplementary Fig. 2b).

The metal ions that favor the formation of stable emulsions are Cu⁺, Cu²⁺, Zn²⁺, Ag⁺, and Au³⁺ (Supplementary Fig. 3a). The reason for this selectivity may be that metal ions that are harder in nature (according to the 'hard and soft acids and bases' theory) such as Ni²⁺, Co²⁺, or Fe²⁺, have a stronger affinity to additional harder oxygen-based ligands, such as the peptide C-terminus²⁵. The same trend was observed in density-functional theory (DFT) simulations (Supplementary Fig. 4). In these simulations Cu⁺, Cu²⁺, Zn²⁺, Ag⁺, and Au³⁺ preferred to bind the histidines, whereas Ni²⁺, Co²⁺, and Fe²⁺ preferred the termini of the peptide. By interacting with one of the aromatic amino acids at the termini, these ions will form complexes that do not allow folding into an amphiphilic complex. Stable emulsions were also prepared using Eu³⁺ ions. These emulsion droplets showed stronger fluorescence, possibly derived from the incorporation of Eu³⁺ in the complex (Supplementary Fig. 3b)²⁶.

Using coarse-grained molecular dynamics (CGMD), we simulated the investigated peptide in an aqueous environment, in the presence of divalent cations, and in the presence of divalent cations as well as octane molecules (Fig. 2, Supplementary Fig. 5). Two derivatives of the peptide were also simulated: Trp-Ala-Ala-Trp (WAAW) and Ala-His-His-Ala (AHHA). These peptides were investigated to reveal the significance of the metal binding moiety and of the hydrophobic amino acids. Our results show that WHHW self-assembles into a fibrous structure in water, a spherical structure in the presence of ions, and it forms micelles with the hydrophobic phase (Fig. 2). These results agree with experimental observations of fibrous peptide structures in water, and spherical aggregates in the presence of Cu²⁺ ions (Supplementary Fig. 5a-b). In the case of AHHA, only a weak trend towards assembly in water was observed (Supplementary Fig. 5c) and an emulsion was not formed with octane. This may occur due to the lack of large hydrophobic groups that increase the peptide’s amphiphilicity. In the case of WAAW, the fibrous/spherical self-assembly relationship with the ions was reversed and only a loose emulsion was formed, whereby much of the hydrophobic phase was exposed to the water. The lack of a metal-binding moiety that can act as a lock, holding the peptide in an amphiphilic conformation, may cause this destabilization of the emulsion droplets.

Spectroscopic study of the complex

The peptide-Cu²⁺ complex displayed a red color with an absorbance peak at ~ 510 nm that increased with the addition of the metal ions up to 1 eq. (Fig. 3a). A Benesi-Hildebrand plot showed a good linear fit, indicating a 1:1 binding ratio between the peptide and the metal (Supplementary Fig. 6)^27,28. A Job’s plot was prepared as well with the peptide and Cu²⁺ ions (Fig. 3b). In the samples with excess Cu²⁺ ions (those above χ = 0.5) the absorbance increased without an increase in the complex concentration. This may be because of the formation of Cu(OH)₂ precipitates from the excess Cu²⁺ ions due to the alkaline pH. To negate this effect, the absorbance values of solutions that contained the concentration of the excess Cu²⁺ were subtracted from the original measurements. This yielded good linear fittings that indicate a 1:1 binding ratio.

The conformational change of the peptide’s structure upon addition of Cu²⁺ or Zn²⁺ ions can be inferred from circular dichroism (CD) measurements. The spectrum of a free peptide indicated a random coil conformation (Fig. 3c). The same conformation was obtained for the peptide in the presence of Fe²⁺ ions, which could not form an emulsion. In contrast, when the peptide was present with Cu²⁺ or Zn²⁺ ions, the CD spectra indicated a β-turn structure. This further supports the ability of these metal ions to fold the peptide into an amphiphilic complex by introducing a turn to the peptide’s structure, bringing the hydrophobic groups closer together.

To investigate the parts of the peptide that are necessary for the formation of the emulsion, we mixed different derivatives of the peptide with Cu²⁺ ions. The same absorbance peak at ~ 510 nm was also observed for the other dihistidine-containing tetrapeptides: Phe-His-His-Phe (FHHF), Tyr-His-His-Tyr (YHHY), and AHHA, suggesting the formation of similar coordination of the metal through the histidines (Supplementary Fig. 7a). Under the basic pH conditions of the experiment, the solution of Cu²⁺ ions formed Cu(OH)₂ precipitates that have an absorbance peak at ~ 650 nm²⁹. A similar spectrum was obtained for WAAW and for a single tryptophan amino acid, indicating the necessity of histidine for forming the complex. The mixtures of Cu²⁺ with a single histidine as well as with dihistidine show a peak around 590 nm as the metal ions interact with the imidazole moieties. Lastly, the peptide Trp-His (WH), displayed an absorbance peak similar to that of the Cu(OH)₂, with a shift towards that of the histidine and dihistidine peaks.

The same peptide derivatives in the presence of Cu²⁺ ions, were further mixed with chloroform and manually agitated to form emulsions (Supplementary Fig. 7b). The only peptide that formed emulsions other than WHHW was FHHF, albeit the emulsion was less stable. This suggests that to create stable emulsions, the termini amino acids have to be both hydrophobic and bulky, thus decreasing the distance between them in the amphiphilic complex.

Structural analysis

To further investigate the structural changes of the peptide, we performed 1D and 2D ¹H-NMR measurements. Titrating the peptide with Zn²⁺ ions corroborated that the metal binds through the imidazole groups, as shown from the difference between the values of the chemical shift (Δδ_a) of δ₂ and ε₁ of both histidines before and after binding the metal ions (Fig. 4a, Supplementary Table 1). Meanwhile, the tryptophan groups displayed little to no chemical shift changes during the titration. Adding more than 1 eq. of Zn²⁺ ions to the peptide did not cause further chemical shifts, as expected from the previous spectroscopic results. However, after adding ~ 15% (v/v) of CDCl₃, most of the polar groups displayed significant changes as indicated from the Δδ_b values (δ_emulsion- δ_bound). These included the ε₁ groups of both histidines, the HN group of the N-terminus, and the ε₁ groups of both tryptophans (Supplementary Fig. 8a). These results point to a significant rearrangement process that the peptide undergoes upon its repositioning at the water-chloroform interface. In this process, the polar groups will be the ones that are most repulsed by the non-aqueous medium, especially the N-terminal amine, which is adjacent to the hydrophobic indole group of the first tryptophan.

Interestingly, while the Δδ_a values of the δ₂ for both histidines are similar, the Δδ_a of ε₁ and of H_ß for the first histidine (His2) are 3 times higher than the values for the last histidine (His3) (Supplementary Table 1). This difference may indicate that the complex involves two different tautomeric forms of histidine in the peptide’s coordination to the Zn²⁺ ion. The deshielding effect for His2 may arise from the lower electron density around these protons because of their proximity to the metal³⁰. This suggests that the zinc coordination is carried out through Nδ₁ for His2 and through Nε₂ for His3.

2D ¹H-NMR correlated spectroscopy (COSY), total correlation spectroscopy (TOCSY), and rotating frame Overhauser enhancement spectroscopy (ROESY) measurements were performed to determine the structure of the peptide and of the complex under aqueous and emulsion conditions. The spectra were assigned according to Wüthrich and used to create ensembles of low energy conformations of the peptide (Fig. 4b-e)³¹. In the case of the free peptide, the histidines pointed to opposite directions (Fig. 4b, RMSD values: Backbone 0.86 Å, heavy atoms 2.36 Å, heavy atoms of His residues 1.39 Å). After adding 1 molar equivalent of Zn²⁺ ions, the histidines moved closer in order to coordinate the metal (Fig. 4c). A similar observation was derived from all-atom simulations of the peptide with and without Zn²⁺ ions, where the average dihedral angle was reduced from 50–60° to 0–10° upon addition of the metal (Supplementary Fig. 10). The structure of the ensemble was calculated using restraints derived from the bound structure but without the zinc-binding in the calculation (Fig. 4c, RMSD values: Backbone 0.90 Å, heavy atoms 1.83 Å, heavy atoms of His residues 1.39 Å). A similar structure was derived with zinc-binding in the calculation, based on the binding information derived from deviations in chemical shift upon binding (Fig. 4d, RMSD values: Backbone 0.78 Å, heavy atoms 2.13 Å, heavy atoms of His residues 0.77 Å). Both structures displayed freedom of motion in the tryptophan side chains. Contrarily, the ensemble determined for the Zn²⁺-bound peptide in the emulsion displayed a significantly more confined structure where the tryptophan side chains also showed limited motion (Fig. 4d, RMSD values: Backbone 0.32 Å, heavy atoms 0.71 Å, heavy atoms of His residues 0.04 Å). This constraint could originate from aromatic and hydrophobic intermolecular interactions between neighboring peptides at the CDCl₃-water interface as hypothesized in Fig. 4f.

Delivery of anti-cancer drugs

The ability to use these emulsions for drug delivery purposes was assessed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The assay was conducted on cervical adenocarcinoma HeLa cells with the widely used drug paclitaxel (PTX). For clinical use, PTX is usually dissolved in Kolliphor® EL (polyethoxylated castor oil, formerly Cremophor® EL) and ethanol, due to its low solubility³². This poses significant disadvantages due to stability issues of the drug that can precipitate when diluted, causing therapeutic failures and possibly harming the patients^11,20,33,34. Life-threatening hypersensitivity reactions to the vehicle are also possible, ranging from mild effects to systemic anaphylaxis, which can prove fatal^{24, 35–37,38}. One way to overcome these complications is to administer the drug in an emulsion. An additional advantage of the system proposed herein is that the disassembly of the emulsion droplets will release the metal ions of the complex in addition to the PTX. Since the efficacy of PTX is increased in the presence of Zn²⁺ ions, their release in proximity to the drug could enhance its therapeutic potential without introducing stability complications for the drug or the ions.

A crucial part of anti-cancer drug delivery through emulsions is their selectivity to cancer cells. The extracellular pH of tumors is found to be 6.2–6.9, which is lower than that of normal tissues (7.2–7.5)³⁹. The use of the histidine residues as the trigger for disassembling the emulsions and releasing the drugs is therefore beneficial due to the pK_a of the imidazole’s amine (~ 6). When the emulsion droplets approach the tumor, their stability will decrease, and they will release the drugs in a more selective fashion at the vicinity of the tumor. This behavior can be observed in Fig. 5a by turbidity measurements that can indicate the stability of the emulsion at different pH values. At pH 7.5, the emulsion was stable for several days, dropping below 60% turbidity after 1 month. At pH 6.9 the turbidity dropped below 50% after 2 days, and at pH 6.2 the emulsion completely disassembled after several minutes. This was further demonstrated in microscopy images as the emulsion remained stable for more than 1 h at pH values 6.9–7.5, whereas at pH 6.5 the emulsion completely disassembled after 10 min, and the emulsion droplets burst immediately in contact with the buffer at pH 6.2 (Supplementary Fig. 11).

For the drug delivery treatment of cancer cells, the size of the emulsion droplets was reduced by sonication in order to increase their ability to penetrate the cells. The size distribution of the emulsion as derived from dynamic light scattering (DLS) was centered at 120 nm, with a small fraction (1.6%) of droplets at the size of 2750 nm (Supplementary Fig. 12a). The inability of the larger droplets to enter the cells may be useful as they can increase the selectivity of the drug to cancer cells by bursting specifically at the lower extracellular pH of the tumor cells. Nonetheless, it is possible to filter out these larger droplets and further steps may be taken to affect the size of the droplets (Supplementary Fig. 12b).

The emulsion itself did not display any cytotoxicity (Fig. 5b). When the drug was tested on its own, it showed strong cytotoxic activity with a half maximal inhibitory concentration (IC₅₀) of 14 ± 5 nM. This is in accordance with the known values for this drug^35,40. The results for PTX encapsulated in the emulsion were slightly higher at 70 ± 10 nM. The decrease in the activity is expected, as it is possible that not all of the drug was encapsulated inside the droplets. Additionally, the drug may remain in the less dense castor oil phase after the emulsion disassembly, decreasing its contact with the cells attached to the bottom of the plate.

Overall, these results demonstrate that this peptide-stabilized emulsion can be used for drug delivery of the water-insoluble PTX alongside Zn²⁺ ions, with higher selectivity towards the tumor environment.

A multifunctional drug delivery system

As mentioned above, delivery via emulsions is mostly limited to the encapsulated material. The ability to easily coat emulsions droplets with different compounds by demand, can greatly expand the potential of this drug delivery system. This aim was investigated with the use of the simple and common metal-binding moiety hexahistidine. This sequence is frequently used in protein purification as it can coordinate to different metal ions including Cu²⁺ and Zn^2 + 41. Many different compounds are isolated with a hexahistidine tag, and more compounds are commercially available. By simply mixing the emulsion with hexahistidine-tagged compounds, the emulsion droplets can be functionalized for various purposes.

To prove this concept, we synthesized a hexahistidine peptide labeled with Rhodamine B (H₆-Rh). The coating of the emulsion droplets with this peptide was demonstrated by adding it to emulsions formed with the complex and with SDS at the same concentration as a control (Fig. 6a). The stronger fluorescence at the circumference of the droplets indicates the binding of the hexahistidine groups to the free coordination sites of the metal ions in the complex. In contrast, the SDS-stabilized emulsions did not display fluorescence surrounding the droplets, indicating the absence of the fluorescent peptide on the surface of the droplets.

To further widen the proof of concept, we utilized the horseradish peroxidase (HRP)-luminol system. In this system, a hexahistidine-tagged antibody conjugated to the enzyme HRP catalyzes the oxidation of luminol in presence of H₂O₂, bringing it to an excited state that produces fluorescence⁴². In a similar manner, the hexahistidine-tagged antibody was mixed with the peptide-stabilized emulsion and with an SDS-stabilized emulsion at the same concentration of the surfactant. When the reagents which include the H₂O₂ and luminol were added, the fluorescence was visible at the vicinity of the peptide-stabilized emulsion droplets, indicating the binding of the hexahistidine groups to the metal ions of the complex (Fig. 6b). Contrarily, with the SDS-stabilized emulsion, the fluorescence was weak and not localized at the droplets. This indicates that the enzyme did not bind the droplets and remained free in the solution, forming excited luminol that was dispersed in a much lower concentration at the aqueous solution.

These results were confirmed using ζ-potential measurements. The ζ-potential values for the emulsions with Zn²⁺ or with Cu²⁺ ions were − 51 ± 1 mV and − 75.3 ± 0.8 mV, respectively (Supplementary Fig. 12). After addition of ~ 0.2 eq. of the H₆-Rh peptide to the emulsion with Zn²⁺ ions, the charge changed to -11.9 ± 0.3 mV, in accordance with the positive charge of the rhodamine. After addition of 0.2 µg/mL of the HRP probe antibody to the emulsion with Cu²⁺ ions, the charge changed to -81 ± 2 mV. This is also expected due to the overall negative charge of HRP at pH ≥ 5⁴³.

In previous studies we synthesized gold nanoparticles (AuNPs) using peptides that contain the amino acid 3,4-dihydroxy-L-phenylalaninethe (DOPA)⁴⁴. These nanoparticles were shown to bind the gold surface through the N-terminal amine. To demonstrate that the emulsion can be covered with another type of compounds, we synthesized the metal-binding peptide DOPA-(Phe)₂-(His)₆ and used it to synthesize hexahistidine-capped AuNPs by using a similar method. We preformed scanning electron microscopy (SEM) imaging on a surface with a dry sample of the AuNPs-decorated emulsions stabilized with a peptide-Zn²⁺ complex or with SDS (Fig. 6c, Supplementary Fig. 13). The amount of AuNPs was visibly higher on the complex-stabilized emulsion droplets, as can also be inferred from the energy dispersive X-ray spectroscopy (EDS) line scan measurements. On the other hand, with the SDS-stabilized emulsion there appeared to be no localization of the AuNPs on the droplets. Overall, the ability to simply decorate the emulsions could provide them with numerous unique functions, such as attachment and delivery of hydrophilic drugs, labeling with fluorescent compounds, targeting through antibodies, and photothermal therapy properties via the AuNPs.

Outlook

We described herein a novel peptide that can be used to stabilize emulsions. These emulsions can be used as an intricate drug delivery system with the ability to deliver lipophilic compounds encapsulated inside the droplets, metal ions bound to the peptide, and hydrophilic compounds attached to the metal ions on the surface of the droplets. The unique complexation-triggered amphiphilicity of the peptide and the emulsions it forms could be applied for a multitude of drug delivery purposes, which were previously difficult to achieve.

Materials

2-Chlorotrityl chloride resin (1.0–1.6 mmol g − 1, 100–200 mesh) and N,N′-Diisopropylcarbodiimide (DIC) were purchased from Chem-Impex International, Inc. (Wood Dale, IL, United States). 1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate (HATU) was purchased from Matrix Innovation, Inc. (Saint-Hubert, QC, Canada). Triisopropylsilane (TIPS) and nile red were purchased from Tokyo Chemical Industry (Tokyo, Japan). Dimethylformamide (DMF), dichloromethane (DCM), piperidine, methanol, diethyl ether, N,N-Diisopropylethylamine (DIEA), acetonitrile, chloroform, and trifluoroacetic acid (TFA) were purchased from Bio-Lab (Jerusalem, Israel). Fmoc-Trp(Boc)-OH and the peptides Phe-His-His-Phe, Tyr-His-His-Tyr, Ala-His-His-Ala, Trp-Ala-Ala-Trp, and Trp-His were purchased from GL Biochem (Shanghai, China). EZ-ECL Enhanced Chemiluminescence Detection Kit for HRP was purchased from Biological Industries (Beit Haemek, Israel). His-probe (H-3) HRP sc-8036 was purchased from Santa Cruz Biotechnology (Dallas, TX, United States). Hydrogen tetracholoroaurate(III) hydrate, copper (I) chloride, and copper (II) chloride were purchased from Acros Organics (Fair Lawn, NJ, United States). Silver nitrate 99+%, castor oil, fluorescein isothiocyanate (FITC), and Fmoc-His(Trt)-OH were purchased from Alfa Aesar (Lancashire, United Kingdom). Zinc (II) chloride, histidine, tryptophan, and paraffin oil were purchased from Merck (Rahway, NJ, United States). Deuterated chloroform was purchased from Cambridge Isotope Laboratories (Tewksbury, MA, United States). Rink amide resin (0.48 mmol/g) was purchased from Matrix Innovations (Hoddesdon, United Kingdom). Ethyl cyanohydroxyiminoacetate (Oxyma) and hydroxybenzotriazole (HOBt) were purchased from Luxembourg Biotechnologies Ltd. (Rehovot, Israel). Rhodamine B was purchased from Thermo Fisher Scientific (Waltham, MA, United States).

Peptide synthesis

NH₂-Trp-His-His-Trp-OH was manually synthesized by Fmoc solid phase peptide synthesis on a 2-Chlorotrityl chloride resin (0.25 mmol). The amino acids at 5 eq. were activated by mixing with a DIEA/HATU mixture (4 eq. and 3.9 eq., respectively) for 4 min. The coupling of the amino acids was carried out for 1 h and confirmed using a Kaiser test. The Fmoc protecting groups were removed by mixing with 20% piperidine in DMF for 20 min. The resin was washed between steps twice with DMF, methanol, DCM, and DMF again. The cleavage reaction was performed by mixing with a TFA/TIPS/water mixture (38:1:1) for 3 h. The cleaved solution was evaporated, precipitated with diethyl ether, and centrifuged. The peptide product was dissolved in water and lyophilized.

The purity of the peptide was assessed using analytical reverse-phase high-performance liquid chromatography (HPLC) analysis (Waters Alliance) with UV detection (220 nm and 280 nm) and an XSelect C18 column (3.5 µm, 130 Å, 4.6 mm × 150 mm). The peptide was eluted using a linear gradient of ACN in TDW (0.1% TFA, 1 mL/min, 30°C). The mass of the peptide was determined using liquid chromatography mass spectrometry (LC/MS) with Agilent 6520 Q-TOF analyzer (Agilent Technologies, Santa Clara, CA, United States).

NH₂-(His)₆-Rhodamine B was synthesized on a Liberty Blue Microwave-Assisted Peptide Synthesizer using Fmoc amino acids and Oxyma/DIC/DIEA coupling reagents. The peptide was labeled prior to the cleavage by mixing it with 100 mg rhodamine B, 36 mg HOBT, and a mixture of DMF/DCM/DIEA/DIC (40.5:4.8:1.1:1) for 3 h in the dark. The peptide’s identity and purity were confirmed using MALDI TOF/TOF AutoFlex Speed (Bruker Daltonics Inc., Bremen, Germany).

Emulsion preparation

The emulsions were prepared by mixing a peptide solution with the metal ions to a final concentration of 1 mM each and adding NaOH to obtain pH = 10. The desired non-aqueous phase was added to this solution at 50% (v/v) and the two phases were manually agitated to form the emulsions. For the emulsions with chloroform, it was found that the best procedure was preparing a 50% (v/v) chloroform emulsion and then adding chloroform to 67% (v/v) and manually agitating again. To achieve smaller-sized emulsion droplets with castor oil, the same procedure was performed, following sonication for 1 h in an ultrasonic bath.

Emulsion characterization

For fluorescence microscopy, the castor oil was labeled with nile red prior to the emulsification. For labeling the peptide on the emulsion droplets, the prepared emulsion was mixed with FITC (4 µg/mL) for 1 h. The images were taken using an Axio Scope A1 fluorescence microscope (Zeiss, Oberkochen, Germany).

For the UV-vis titration, a peptide solution (1.5 mM) was titrated with a CuCl₂ solution (15 mM). The solutions were left for 5 min and the absorbance was measured using a UV–vis spectrophotometer (Shimadzu, UV-1650PC, Kyoto, Japan). For the Job’s plot assay, peptide solutions with different mole fractions of Cu²⁺ ions (up to 1 mM) were prepared at pH = 9 and their absorbance values were measured.

The far-UV CD spectra were recorded with a J-810 spectropolarimeter (JASCO Inc., Easton, MD, United States), using a 0.1 cm pathlength far-UV CD spectroscopy quartz cuvette (between 190–260 nm, 0.1 nm intervals).

The size distribution of the emulsion stabilized with peptide-Zn²⁺ was measured using a Zetasizer ZEN3600 (Malvern instruments, Malvern, UK).

NMR studies

The samples were prepared in 20 mM phosphate buffer (pH = 6.5, 10% D₂O) with the peptide (3 mM) and Zn²⁺ ions (3 mM) when mentioned. The measurements were taken at 298.1 K. For the emulsion spectra, a solution of zinc chloride (3 mM) was added to the peptide (3 mM). The solution was in phosphate buffer 20 mM, pH 6.5 with 10% D₂O. 100 µL of CDCl₃ was added to form an emulsion, where the excess chloroform settled to the bottom of the test tube below the line of the coils.

The NMR experiments were performed on a Bruker AVII 500 MHz spectrometer operating at the proton frequency of 500.13 MHz, using a 5-mm selective probe equipped with a self-shielded xyz-gradient coil at 25°C. The transmitter frequency was set on the water signal. COSY⁴⁵, TOCSY⁴⁶ using the MLEV-17 pulse scheme for the spin lock (150 ms)⁴⁵, and ROESY experiments (using an optimized mixing time of 400 ms)^45,47,48, were acquired using gradients for water saturation under identical conditions⁴⁹.

Spectra were processed and analyzed with TopSpin (Bruker Analytische Messtechnik GmbH) and NMRFAM SPARKY software⁵⁰. Resonance assignment followed the sequential assignment methodology developed by Wüthrich³¹.

The three-dimensional structures of the peptides were calculated using XPLOR-NIH (version 3.2)^51,52 by hybrid distance geometry-dynamical simulated annealing. Peak intensities were manually assigned as strong (2.5 Å), medium (3.5 Å), weak (4.5 Å) and very weak (5.5 Å) with a ± 0.5 Å error. Parameters were introduced using zinc geometry⁵³ and charges were derived from the Amber forcefields⁵⁴. Fifty initial structures were generated. The nuclear Overhauser effect (NOE) restraint energy was introduced as a square-well potential. Molmol⁵⁴ was used to create the final ensemble of structures. Low energy structures chosen for further analysis had no NOE violations, deviations from ideal bond lengths of less than 0.05 Å, and bond angle deviations from ideality of less than 5°. Figures were produced, using ChimeraX⁵⁵ from the University of California, San Francisco (supported by NIH P41 RR-01081)⁵⁶.

Molecular dynamic simulations

Coarse-grained molecular dynamics (CGMD) was performed using the GROMACS software package^57,58 using the MARTINI 3 forcefield⁵⁹ and a timestep of 20 fs. Velocity rescaling was used to hold the temperature around 310 K⁶⁰ and a Berendsen barostat was used to control the isotropic pressure at 1 bar⁶¹. Bond lengths within aromatic side chains were constrained using the LINCS algorithm⁶². Short range intermolecular interactions were evaluated using a Lennard-Jones algorithm with a shifted cut off at 1.1 nm and electrostatic interactions evaluated using the reaction-field algorithm with a cut off also at 1.1 nm⁶³. All the simulations were performed in aqueous conditions using the MARTINI small water (two water molecules to one bead). All systems were minimized using the steepest descent integrator prior to equilibration.

Cancer cell studies

The behavior of the emulsion at different pH values was performed by adding 1 µL of an emulsion stabilized with a peptide-Zn²⁺ complex to a glass slide with 10 µL of the phosphate buffers (10 mM) at different pH values.

The turbidity measurements were performed after dilution of 10 µL of the emulsion with 690 µL of the phosphate buffers and measuring the turbidity at 350 nm using a Biotek Synergy H1 plate reader (Lumitron), during several days. The emulsions were kept at 37°C between the measurements. The data were fitted to an exponential decay equation and the 95% prediction band is depicted.

The complete cell-culture medium was prepared by the addition of 10% fetal bovine serum (FBS), 1% L-glutamine, and 1% Penicillin- Streptomycin (all purchased from Sartorius), to Dulbecco's Modified Eagle's Medium (DMEM, Sigma Aldrich). Cervical adenocarcinoma HeLa (American Type Culture Collection) cancer cell lines were cultured as monolayers in complete cell culture medium at 37°C in a 5% CO₂ atmosphere. The cells were seeded in 96-well plates at a density of ca. 10,000 cells per well and allowed to attach overnight. On the following day, the tested emulsions were prepared at a 10 µM concentration in water, serially diluted to obtain a 10-concentration gradient, and added to the cells for a 72-h incubation. PTX was dissolved in DMSO to a 20 µM concentration, serially diluted in DMSO to obtain a 10-concentration gradient, consequently diluted 10 fold in medium to ensure a final concentration of 0.5% DMSO, and then added to the cells for 72 h. Afterwards, cytotoxicity was measured by the previously reported MTT method⁶⁴. In short, MTT was added to the wells (0.1 mg in 20 µL) for an additional 3 h incubation period. The medium was removed and replaced with isopropanol (200 µL), and upon complete dissolution of the formazan, the absorbance was measured at 550 nm on a Spark 10 M multimode microplate reader spectrophotometer (Tecan Group Ltd., Mannedorf, Switzerland). Each measurement was repeated at least 3 × 3 times: three repeats per plate, all repeated on at least three different days, giving at least nine repetitions for each experiment. The results for the highest diluted samples of PTX dissolved in DMSO were normalized to 100% cell viability. Relative IC₅₀ values and the standard error of means were determined by a nonlinear regression of a variable slope (four parameters) model using the GraphPad Prism 5.0 software.

Emulsion functionalization

Coating the emulsion droplets with the fluorescent H₆-Rh peptide was performed by diluting 1 µL of an emulsion stabilized with the peptide-Zn²⁺ complex with 9 µL water and adding the H₆-Rh at a concentration of 0.2 mg/mL. After 1 h, a drop from the solution was placed on a glass slide and the images were taken using the fluorescent microscope. The same procedure was followed for an SDS-stabilized emulsion.

For the coating with the HRP probe, 1 µL of the HRP probe (20 µg/mL) was added to 9 µL of a peptide-Cu²⁺-stabilized emulsion with castor oil. The solution was left at room temperature for 1 h and then a drop of 2 µL was placed on a glass slide. To this drop, 5 µL of a 1:1 mixture of both reagents from the HRP probe kit (which include the luminol and the H₂O₂) were added and the images were taken using the fluorescent microscope. The same procedure was followed for an SDS-stabilized emulsion.

The ζ-potential values of the emulsion samples were measured using a Nano-Zetasizer ZEN3600 (Malvern instruments, Malvern, UK) and folded capillary cells (DTS1070).

The gold nanoparticles were prepared by mixing an aqueous solution of HAuCl₄ (3 mM) with an aqueous solution of DOPA-(Phe)₂-(His)₆ (1 mM). The solution was stirred overnight at room temperature. The coating of the emulsion droplets with the nanoparticles was performed using emulsion stabilized with a peptide-Zn²⁺ complex with benzene as the non-aqueous phase. The emulsions (5 µL) were placed on clean silicon surfaces (cleaned by sonication in ethanol for 15 min, followed by washing with TDW) with 1 µL from the AuNPs solution. Benzene was chosen because upon drying, the emulsion droplets left visible impressions on the surface. The same procedure was followed for an SDS-stabilized emulsion.

The SEM images were taken using Apreo 2S analytical high resolution scanning electron microscope (Thermo Fisher Scientific), operating at 5 or 10 kV, and equipped with an UltraDry EDS Premium 60 mm² Silicon Drift detector (Thermo Fisher Scientific).

Acknowledgements

DB acknowledges the Center for Nanoscience and Nanotechnology of the Hebrew University for his Ph.D. fellowship. EYT is an incumbent of the Lester Aronberg Chair in Applied Chemistry. We thank Prof. Assaf Friedler for his help with the CD measurements, Prof. Raed Abu-Reziq for his help with DLS and ζ-potential measurements, and Ilya Torchinsky for his help with the SEM measurements.

Author Information

Authors and Affiliations

Institute of Chemistry, The Hebrew University of Jerusalem, Jerusalem, 9190401, Israel

Daniel Boas, Zohar Shpilt, Edit Y. Tshuva & Meital Reches

The Center for Nanoscience and Nanotechnology, The Hebrew University of Jerusalem, Jerusalem, 9190401, Israel

Daniel Boas & Meital Reches

WestCHEM, Department of Pure and Applied Chemistry, University of Strathclyde, Glasgow G1 1XL, UK

Alexander van Teijlingen & Tell Tuttle

Department of Pharmaceutical Engineering, Azrieli College of Engineering Jerusalem

Deborah E. Shalev

Wolfson Centre for Applied Structural Biology, the Hebrew University of Jerusalem

Deborah E. Shalev

Contributions

D.B. designed the materials, synthesized the peptides, performed the emulsion characterization experiments, analyzed the NMR results, and performed the MTT assays.

A.T. performed the molecular dynamics simulations.

Z.S. performed the MTT assays.

D.E.S. performed the NMR measurements and analyzed the results.

M.R., T.T., and E.Y.T. supervised the research.

The manuscript was written by D.B. and reviewed by M.R.

Corresponding author

Correspondence to Deborah E. Shalev, Edit Y. Tshuva, Tell Tuttle, or Meital Reches

Competing interests

The authors declare no competing interests.

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A Multifunctional Drug Delivery System Based on Switchable Peptide-Stabilized Emulsions

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Abstract

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Main

Complexation-triggered amphiphilicity

Spectroscopic study of the complex

Structural analysis

Delivery of anti-cancer drugs

A multifunctional drug delivery system

Outlook

Methods

Materials

Peptide synthesis

Emulsion preparation

Emulsion characterization

NMR studies

Molecular dynamic simulations

Cancer cell studies

Emulsion functionalization

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References

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Supplementary Files

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