Hybrid Peptides as Platform for Synchronized Combination Therapy

Abstract

Combination therapy, where two or more therapeutic agents are combined to target different cellular pathways, is an effective tool in cancer treatment but often difficult to execute. Here we present the collagen peptide-based platform that allows for synchronous and colocalized cellular delivery of three different agents. The peptide is a hybrid between collagen and cell penetrating peptide (CPP) that assembles into a heterotrimer helix and forms fully organic, high aspect ratio nanoparticles. The validity of the approach was tested with three chemically different agents (Paclitaxel, Doxorubicin, and 5-Fluorouracil; a combination used in clinical treatment of (ER)-positive and (PR)-positive breast cancer) conjugated to N-terminus of the peptide. The design of this peptide-based drug delivery system provides several advantages: it avoids drug loading problems; removes the need for orthogonal synthesis; and allows for colocalized delivery of up to three drugs (which leads to the same biodistribution for each drug). In addition, hybrid collagen/CPP peptides are known to enhance cellular uptake and improve solubility of drugs. The synergistic effect, in terms of enhanced efficacy, of the Paclitaxel-Doxorubicin-5-Fluorouracil combination was also calculated. We envision self-assembling peptides as a platform for drug codelivery that can be expanded into a library of personalized combinations that may also include other functionalities like targeting or imaging.

1. Introduction

Combination therapy for cancer treatment has been envisioned since the early sixties. The use of more than one drug in cancer therapy enables targeting different cellular survival and adaptive resistance pathways and may lead to more successful clinical outcomes [1]. There are several potential benefits of combination therapy: (1) lower the therapeutic dose of a single drug, thus decreasing systemic effects, (2) avoid the emergence of drug resistance, and (3) improve efficacy in solid, heterogeneous tumors [2,3]. The application of drugs combination in cancer was proven to be particularly beneficial when drugs were interreacting in the synergistic mode [4,5].

There are still many challenging aspects of combination therapy. The pharmacokinetics of administered combination can affect the pharmacokinetics of a single agent, and drug interactions in combination can result in harmful effects. Furthermore, if administered in a codelivery system, the drug ratios can be difficult to control [6,7]. While the drug combinations acting in the synergistic mode are in highest demand, synergistic effects are difficult to predict [8]. Recently, in an effort to quantify these effects the two new methodologies were proposed: “CombiPlex” platform and “Drug Atlas” approach [9,10]. Even if the combination of agents acts in a synergistic manner, the mode of drug administration could affect its action [11]. Due to differences in pharmacokinetics, biodistribution, and heterogeneity of the tumor cells, ensuring the controlled delivery of multiple agents proves challenging [12].

One method to guarantee that the therapeutic combination of drugs has the same biodistribution, is to combine them in a single codelivery vehicle. The nanoparticles (lipid micelles, polymer micelles, liposomes, and liposome composites) are the most common platform for coformulations of chemically dissimilar agents, but the polymer drug conjugates (PDCs) are quickly gaining popularity as carrier materials for combination therapy [12, 13]. In most polymer systems (HPMA, PEG, PG, Polysaccharide) a single drug is conjugated to a specific polymer and assembled either as a block co-polymer or as a self-assembled mono-polymer to form specific drug combination [14].

In many of the co-delivery systems one or more agents are loaded based on the partition coefficient or adsorption [15]. Thus, the control over the simultaneous loading process is difficult and, in many cases, the desired drug ratio cannot be achieved. An alternative approach is to conjugate the selected agents to a single delivery vehicle [16]. In that case, to control the ratio, each agent must have a unique conjugation chemistry; therefore, orthogonal synthetic routes must be applied [17,18]. The application of orthogonal reactions complicates synthesis, purification, and release of the agents. For example, in the PG polymer, greater therapeutic benefit has been demonstrated of the PG-PTX-DOX conjugate compared to the physical mixture of PG-PTX and PG-DOX; however, the orthogonal synthetic route had to be applied and the ratio of the drugs was not stoichiometrically exact [19].

Here we present a peptide-based platform that can combine up to three agents in a well-controlled equimolar ratio. The peptide carrier design is based on the collagen sequence combined with the short cell-penetrating peptide (CPP) sequence. The cargo is conjugated to N-terminus of peptide. The collagen domain folds (self-assemble) into triple helix resulting in the combination of up to three different agents in a single delivery module. The synthetic collagen heterotrimers were introduced by the Hartgerink group and expanded by others [20-23]. In general, collagen peptide sequence can be generalized as (POG)_n sequence (P = proline and O = hydroxyproline). If n>6, the peptide folds into stable homotrimer helix at room temperature. The substitution of P and O for charged amino acids (+/−) in the opposite strands promotes intermolecular charge-charge interactions. These interactions guide peptide folding, thus allowing for the formation of heterotrimers with a different sequence in each strand [20]. It was shown that the sequence of each strand can be chosen to form selectively an ABC-heterotrimer and avoid formation of homotrimers, and other combinations of peptides [22].

The peptide-based platform proposed here avoids complications related to partition-based drug co-loading and development of orthogonal synthetic pathways; each agent is conjugated via N-terminus using well established peptide protocols, including standard cleavable linkers. In this work we are testing the validity of the proposed approach with the combination of three agents Paclitaxel (PTX), Doxorubicin (DOX), and 5-Fluorouracil (5FU) that is used in the clinical treatment of (ER)-positive and (PR)-positive breast cancer [24,25]. The chemical, physical, and biological characteristics of each agent are very different (Table 1), and to the best of our knowledge, the co-delivery of these three agents was not previously reported.

Table 1.

Properties of agents used in combination [26-32]

Property	Paclitaxel	Doxorubicin	5-Fluorouracil
molecular weight [g/mol]	854	580	130
solubility in water [mg/ml] ([mM])	sparingly	52 (90)	12.5 (96)
solubility in methanol [mg/ml]	50	sparingly	18
logP (octanol/water)	4	0.5 to 1.3	−0.46 to −0.83
IC50 (range for human cancer cell lines)	2.5 to 7.5 nM	0.6 to 8.5 μM	2 to 20 μM

Upon conjugation of PTX, DOX, and 5FU to three different “complementary” peptides, the peptides self-assemble into a single nanoparticle. The application of self-assembly eliminates the need for complex co-loading protocols and facilitates the same biodistribution of the three drugs in equimolar ratio.

2. Materials and Methods

2.1. Peptides

All peptides were purchased from Tufts University Core Facility (Table 2) with a free amine group at N-terminus to allow conjugation with cargo: Paclitaxel, Doxorubicin, or 5-Fluorouracil. All peptides have a C-terminus blocked by amidation to prevent cross reactions. The detailed description of the peptide sequence design is listed in 3.1 and MALDI TOF spectra (S1-S3) in Supplementary Materials.

Table 2.

Peptide sequences and charges. “O” represents hydroxyproline. Color legend: blue/red/green indicates collagen folding domain, where blue is positive region, red is negative region, and green is a complementary (no charge) region; black is cell penetrating domain, purple is a spacer for cargo conjugation.

Name	Peptide Sequence	+ Charge	− Charge	Net Charge	Net Charge Homotrimer
HT1	GG-(PRG)5-(POG)5-POGRRG	7	0	+7	+21
HT2	GG-(POG)5-(EOG)5-RRGRRG	4	5	−1	−3
HT3	GG-(EOG)5-(PRG)5-RRGRRG	9	5	+4	+12
HT123	Folded HetroT: HT1•HT2•HT3	20	10	+10	N/A

2.2. Bioconjugation of Doxorubicin to HT1 peptide

Cis-aconityl-Doxorubicin.

Following the synthesis process of Kakinoki et al. [33], 27 mg (173.1 μmol) cis-aconitic anhydride was dissolved in 1 mL dioxane. Doxorubicin 30 mg (51.7 μmol) was dissolved in 10 mL 0.1 M Na₂HPO₄ solution and placed in an ice bath. Cis-aconitic anhydride solution was added dropwise to the cold doxorubicin solution. The pH was adjusted to 8.9- 9.0 using 0.5 M NaOH. After stabilizing the pH, the solution was stirred for 15 min at 4°C and an additional 15 min at room temperature. The pH was adjusted to 7.0-7.4 and the mixture was placed in an ice bath. The reaction was allowed to proceed for an additional 60 min, then the pH was adjusted to 2.5-3.0 using ice cold 1 M HCl. The solution was extracted using ethyl acetate (200 mL total). Organic layers were collected and washed with saturated NaCl solution. The organic phase was dried using sodium sulfate. Solution was filtered through glass wool and evaporated to a dry red powder in a large vial (yield: 60.0%). The formation of Cis-aconityl-Doxorubicin was confirmed with NMR spectroscopy (Bruker Ascend ^™ 400 MHz NMR). ¹H NMR (DMSO-D6, 400 MHz): δ 14.015 (1 H, -OH), 13.250 (1 H, -OH) 7.881 (1H, -NH-), 7.642 (m, aromatic 1,2,3 positions), 6.416 (m, -CO-CH=C-), 5.469 ( 9-OH), 5.257 (1’), 4.941(7), ~4.910 (14 OH), 4.588 (14), 4.225 (5’), 4.044 (-OCH3), 3.573 (-C-CH2-CO-OH), ~3.540 (4’), 3.143 (3’), 2.976 ( 10), 2.508 (DMSO), 2.199 (8), 1.915 (2’), 1.180 (6’)

Conjugation of cis-aconityl-Doxorubicin to HT1 peptide.

53.98 mg Cis-aconityl-Doxorubicin was dissolved in 1 mL dimethylformamide (DMF) under Argon. Once the solution was homogenous, 27.9 μL N, N-Diisopropylethylamine (DIEA) was added and stirred for 1 h at room temperature. Next, 36.04 mg O-(7-Azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate (HATU) was added to the reaction mixture and stirred for 30 min. In a separate flask, 53.54 mg HT1 peptide was dissolved in 500 μL DMF and added dropwise to the reaction mixture. The reaction was stirred at 50°C for 24 h. The mixture was then diluted with 2 mL water and extracted with 5 mL ethyl acetate 3 times. The aqueous phase was collected and purified by dialysis (MW cutoff of 1kDa) against water for 72 h with water exchanged every 24 h. The dialyzed compound was lyophilized and yielded a red fluffy product (yield: 33.8%). The conjugation was confirmed with MALDI-TOF: HT1- cis-aconityl-Doxorubicin (C₁₈₈H₂₇₇N₆₁O₅₇; Calc. Mass: 4317.1) peak found was fragment (C₁₅₅H₂₄₈N₆₀O₄₄) Calc Mass: 3656.1, Peak found [X+K]⁺ 3698.6 m/z.

2.3. Bioconjugation of 5-Fluorouracil to HT2 peptide

5-fluorouracil-1-succinic acid.

Following the synthesis procedure of Dong et al. [34], 100 mg (0.789 mmol) 5-Fluorouracil was dissolved in 87.3 μL 37% formaldehyde solution (1.079 mmol) and the mixture was stirred at 60°C for 24 h. The solvent was removed in vacuo. Without further purification, the crude product, N-1-hydroxymethyl-5-fluorouracil, was dissolved in anhydrous acetonitrile. The 100 mg (1.0 mmol) succinic anhydride, 5.26 mg (0.043 mmol) 4-Dimethylaminopyridine (DMAP), and 77.7 μL (0.446 mmol) N, N-Diisopropylethylamine (DIEA) were added. The reaction was stirred at 50°C for 24 h. The solvent was removed in vacuo. The crude product, 4-((5-fluoro-2,4-dioxo-3,4-Dihydropyrimidin -1(2H)-yl) methoxy)-N1-methoxy)-4-oxobutanoic acid (5FU-1-N-SA), was recrystallized from methanol (white crystals, yield: 26%). The formation was confirmed with NMR and ESI-MS spectroscopy: ¹H NMR (DMSO-D₆, 400 MHz): δ 12.218 (1 H, br -COOH), 8.094-8.078 (d, 1H, -CH=CF), 5.572 (s, 2H NOCH₂), 2.544-2.471 (m, 4H ─COCH₂CH₂-COOH). ESI-MS (Advion expression CMS) of 5FU-1-N-SA (C₉H₉FN₂O₆; Calc. Mass: 260.04) found: [M+Na]⁺ 283.0 m/z Calc. Mass 283.03 m/z, found: [2M+K]⁺ 559.0 m/z Calc. Mass 559.45 m/z.

Conjugation of 5-fluorouracil-1-succinic acid to HT2 peptide.

In a large vial, 83.70 mg 5-fluorouracil-1-succinic acid was dissolved in 1mL dimethylformamide (DMF) followed by the addition of copious amounts of argon. After dissolution, 27.9 μL N, N-Diisopropylethylamine (DIEA) was added and stirred for 1 h at room temperature. 33.08 mg O-(7-Azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate (HATU) was added to the reaction vial and stirred for 30 min. Separately 93.06 mg HT2 peptide was dissolved in 500 μL of DMF and then added dropwise to the reaction vial. The reaction was stirred at 50°C for 24 h, and reaction progress after 24 hours was monitored with TLC/Ninhydrin test. After no color was observed, the solution was diluted with 2 mL water and extracted with 5 mL ethyl acetate 3 times. The aqueous phase was collected and purified by dialysis (MW cutoff of 1kDa) against water for 72 h with water replacement every 24 h. The dialyzed compound was lyophilized and yielded a white fluffy product (yield: 30.0%). The conjugation was confirmed with MALDI-TOF: HT2-1-succynil-5-fluorouracil (C₁₆₀H₂₄₀N₅₃O₆₃F) Calc. Mass: 3930.7 Peak found was fragment of HT2-1-succynil-O (C₁₅₆H₂₃₈N₅₁O₆₁) Calc. Mass: 3804.7; Peak found [X+H]⁺ 3807.1 m/z and [X+Na]⁺ 3830.1 m/z.

2.4. Bioconjugation of Paclitaxel to HT3 peptide

2’-O-succinyl Paclitaxel.

Following Deutsch’s method [35], Paclitaxel (0.05 mmol, 43 mg, MilliporeSigma) and succinyl anhydride (0.6725 mmol, 67 mg, Thermo Scientific) were dissolved in 1 mL pyridine. The reactants were mixed at room temperature for 24 h. The reaction mixture was concentrated in vacuo and 2 mL water was added to the sample yielding a slurry of water and white precipitate. The solution was stirred for 30 min before vacuum filtration. The filtrate was collected and dissolved in 2 mL acetone. Ice cold water was added dropwise to the solution until white crystals stopped forming. The crystals were washed, filtered, and dried (yield: 98%). The formation of 2’-O-succinyl Paclitaxel was confirmed with NMR spectroscopy by the presence of the multiple peaks at 2.61-2.68 identified as the succinyl linker.

¹H NMR (CDCl₃, 400MHz) : δ = 1.132 [s, ¹⁶CH₃], 1.227 [s, ¹⁵CH₃], 1.679 [s, ¹⁸CH₃], 1.917 [s, ¹⁷CH₃], 2.218 [m, OAc], 2.441 [m, OAc], 2.517-2.782 [m, ^2’C-OOC-CH₂-CH₂-COOH], 3.796 [d, ³CH], 4.188-4.322 [d, ¹⁹CH₂], 4.430 [dd, ⁷CH], 4.983 [d, ⁹CH], 5.543 [d, ^2’CH], 5.675 [d, ²CH], 5.995 [dd, ^3’CH], 6.241 [t, ¹³CH], 6.290 [s, ¹⁰CH], 7.018 [d, NH], 7.250 [s, 3’-Ph], 7.400 [m, 3’-NBz], 7.513 [ m, 2-OBz], 7.776 [d, 3’-NBz], 8.130 [d, 2-OBz] ppm.

¹³C NMR (CDCl₃): δ = 203.79, 174.4 [^2’C-OOC-CH₂-CH₂-COOH] , 171.2 [^2’C-OOC-CH₂-CH₂-COOH], 171.0, 169.9, 168.7, 167.2, 167.0, 166.0, 142.7, 136.8, 133.7, 133.5, 132.7, 132.0, 130.2, 129.2, 129.1, 128.7, 128.6, 127.2, 126.5, 81.1, 79.1, 75.6, 58.5, 52.7, 45.6, 43.1, 35.5, 28.9 [^2’C-OOC-CH₂-CH₂-COOH], 26.8, 22.1, 20.8, 14.8, 9.6 ppm.

ESI-MS (Advion expression CMS) of 2’-Succinyl-Paclitaxel C₅₁H₅₅NO₁₇ [M+H]⁺ found: 953.5 m/z Calc. Mass: 954.35 m/z.

Conjugation of 2’-Succinyl-Paclitaxel to HT3 peptide.

83.84 mg 2’-O-succinyl Paclitaxel was dissolved in 800 μL dimethylformamide (DMF) under argon. After the solution was homogenous, 27.9 μL N, N-Diisopropylethylamine (DIEA) was added and stirred for 1 h at room temperature. 37.53 mg O-(7-Azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate (HATU) was added and stirred for 30 min. Separately 70.07 mg of HT3 peptide was dissolved in 500 uL of DMF and then added dropwise to the reaction vial. The reaction was stirred at 50°C for 4 h. The reaction was monitored with TLC/Ninhydrin; it was deemed finished when no color appeared. The mixture was then diluted with 2 mL water and extracted with 5 mL ethyl acetate 3 times. The aqueous phase was collected and purified by dialysis (MW cutoff of 1kDa) against water for 72 h, with water exchanged every 24 h. The dialyzed compound was lyophilized and yielded a white fluffy product (final yield: 20.4%). The conjugation was confirmed with MALDI-TOF: (C₂₀₈H₃₁₁N₆₇O₆₉); Calc. Mass: 4851.28 Peak found [M+H]+ 4853.0 m/z and fragment of succinyl-HT3 (C₁₆₁H₂₆₂N₆₆O₅₅) Calc Mass: 3999.97. Peak found [X+H]+ 3999.2 m/z.

2.5. Circular Dichroism

Circular dichroism (CD) measurements were performed using a JASCO J-810 spectropolarimeter (JASCO Inc., Easton, MD, USA) equipped with a Peltier temperature control system containing a quartz cell (path length 0.2 cm). Prior to each measurement, the peptides were thermally annealed: peptide solutions in 0.1X Phosphate Buffer (PBS) (1× 10⁻⁴ M) were pre-heated at 80°C for 10 min and slowly cooled to 4°C, then incubated for 24 h. To record the spectra, the peptide solutions were transferred to a CD cell and equilibrated for 30 min at initial scan temperature. A scan speed of 50 nm/min was used, and 4 scans per sample were acquired. A reference spectrum containing buffer was subtracted from the final peptide spectrum.

Thermal unfolding curves were obtained by monitoring the decrease in ellipticity in a 10-80°C temperature range (dependent on the peptide) at a wavelength where the CD spectra show a positive maximum (224 nm) at a heating rate of 0.1°C/min. The derivative of the plotted unfolding curve was calculated using the JASCO Spectra Manager II software (JASCO Inc.). The minimum of the derivative indicates the steepest slope of the unfolding process and determines the helix-to-coil transition temperature (Tm) under the described conditions. All experiments were performed in duplicate or triplicate.

2.6. Cell Culture

The MCF-7 breast cancer cell line (ER-positive and PR-positive) was purchased from ATCC and cultured according to recommended protocols. In short, MCF-7 cells were grown in a T-75 cell culture flask at 37°C and humidified 5% CO₂ incubator. The cells were cultured in Eagle’s Minimum Essential Medium (Mediatech Inc.) supplemented with 10% fetal bovine serum (Cell Grow) and 1% penicillin streptomycin glutamine (Fisher Scientific). The cells were passaged every 2 wk and all experiments were performed on passage 2-10. All the experiments were performed once cells reached 70-80% confluency.

2.7. Cell Viability Assay

After reaching 70-80% confluency, MCF-7 cells were detached from the cell culture flask with Accutase (Gibco), passed through a 20 μm cell strainer to remove the cell clusters and counted (Luna II). Cells were seeded into a 96-well plate at 5x10³ cells/well and incubated for 1 h. The 50 μL solution containing peptide-drug conjugate in variable concentrations was added to each well and after 72 h incubation, the viability assay was performed. In short, 100 μL of CellTiter-Glo 2.0 (Promega) reagent was added to each well, the plate was inserted into a plate reader (BioTek Synergy H1) and mildly shaken for 2 min. The readout was set for 10-min delay and read in luminescence mode. From the intensity readings, the average intensity of each concentration and cell fraction was calculated. The sigmoidal line fit equation is defined as:

$$y=x_{\max }+\frac{(x_{\text{min}}-x_{\max })}{(1+{10}^{(\log }(I{C}_{50})-c{)}^{{\ast }_m)})}$$

where c is the constant and m is the slope. All experiments were performed in 6 replicates and error calculated as standard deviation.

2.8. Data Analysis

Prism 9 (GraphPath) and Kaleidagraph (Synergy) were used for statistical analysis of data and IC₅₀ was determined from curve fitting (sigmoidal) parameters. Mathematica (Wolfram) was used to plot prodrug interaction planes.

3. Results

3.1. Heterotrimer Hybrid Peptide Design

The single peptide strands were synthesized, purified, and characterized (HPLC, MS) at Tufts University Core Facility. The sequences of peptides were designed to form a nanoparticle (7.5 nm long, Ø1.5 nm; size calculated based on crystal structure of collagen peptides) based on heterotrimer assembly (Table 2). We applied the rational design methodology, developed by Hartgerink group, to guide the self-assembly of collagen peptides into monodisperse heterotrimers [22]. The folding motif (Table 2) was placed in the middle of the sequence, leaving peptide terminals “unzipped” for the functionalization of the nanoparticle. In the sequence design, additional consideration was given to maintain high water solubility of the folded peptide.

The N-terminus was left uncapped to facilitate the bioconjugation with cancer drugs via cleavable linker and GG spacer. The linkers were chosen to promote peptide release upon delivery [36]. The C-terminus was modified with the short cell-penetrating peptide (CPP) with sequences dependent on the collagen sequence of the given peptide (Table 2). The choice of CPP sequence was dictated by the net charge of the single peptide strand; the charge should not exceed +8, to avoid uptake of unfolded peptide, but the total particle charge (after peptide self-assembly) should be larger than +9, to facilitate cellular uptake [37].

The self-assembly of the peptide into the triple helix is temperature-dependent and can be controlled by the sequence and the length of the peptide [38]. The sequence of the heterotrimer was designed to ensure particle stability (helix) at 37°C. While there are models that predict the helix to coil transition temperature (Tm), they agree well with experimental values for homotrimers and sequences with no permanent charge. Thus, they were not used in our system.

In addition to the factors mentioned above, having multiple charged amino acids like E, R or K in the sequence, lowers thermal stability of the nanoparticle (lower Tm), while the conjugation of the cargo to N-terminus, especially in case of hydrophobic molecules, stabilizes the folded nanoparticle [39]. Thus, we based the peptide design on the previously studied heterotrimers and our past work with highly charged homotrimers [37,39].

3.2. Bioconjugation of Paclitaxel (PTX), Doxorubicin (DOX), and 5-Fluorouracil (5FU) to peptide carrier

The peptides were synthesized and characterized using solid phase synthesis by Tufts University Core Facility (commercially available). To conjugate the cargo to peptide the N-terminus was presented as primary amine group with the GG spacer between the collagen folding domain and the cargo attachment. There is no other primary amine group present in the sequence available for conjugation. The C-terminus is blocked by amidation to prevent cross reaction. The conjugation of each tested cancer drug was performed by first modifying the drug with a linker that contained the carboxyl group to ultimately form the peptide bond with the N-terminus (Scheme 1a).

Scheme 1.

The schematic representation of coupling reaction between the linker-modified drug and peptide (a) and drug modification with cis-aconitic (b) and succinic (c,d) linker.

The linkers used in the current study were previously shown to undergo effective cleavage after delivery, resulting in successful release of the active cargo. DOX was modified with cis-aconitic anhydride resulting in the formation of amid bond at the C3’-NH₂ position according to known procedure [34]. The Cis-aconityl anhydride linker is acid labile and in pH ≤ 6 effectively releases active DOX (Scheme 1b). The 5FU and PTX were modified with succinyl anhydride via known procedure [35]. To modify 5FU with a succinic linker, the 5FU was first converted to N-1- hydroxymethyl-5FU in the presence of formaldehyde and subsequently reacted with succinic anhydride to form 5-fluorouracil-1-succinic acid (Scheme 1c) [34]. We have previously described the PTX modification with an esterified succinate linker at the C2’-OH position to form the accessible carboxylic acid group (Scheme 1d) [39].

3.3. Self Assembly of Peptide Heterotrimer With and Without Cargo

The self-assembly of peptides was monitored with Circular Dichroism (CD) spectroscopy. Collagens have characteristic transitions in CD spectroscopy: ππ* amid transition at 197 nm and positive nπ* transition at 224 nm (Figure 1a). By measuring the intensity of the positive peak at 224 nm with respect to temperature (Figure 1a), the helix-to-coil transition temperature (Tm) can be detected. The Tm strongly depends on the peptide sequence, length, and modifications; thus, we can use the Tm value to identify the peptide trimers. In Table 3 we list Tm values for peptide homotrimers that were measured by taking the first derivative of the molar ellipticity recorded at 224 nm with respect to temperature (the unfolding curves). The thermal stability of the peptides that have glutamic acid residues (negatively charged), HT2 and HT3, is much lower than a peptide that has only positive charge originating from arginine residues. In the case of HT3, the homotrimer was not formed at the studied range of temperatures (4-80°C).

Figure 1.

Circular Dichroism Spectroscopy: (a) spectra of HT123 heterotrimer recorded at different temperatures. The 224nm peak used to monitor the helix-to-coil transition is indicated with the arrow, (b) First derivative of HT123-DOX-5FU-PAX unfolding before (red) and after (black) annealing.

Table 3.

Peptides helix-to-coil transition temperatures (Tm) measured by the first derivative of unfolding curve with CD at 224 nm. The standard deviation is ± 1°C and is measured in triplicates.

Homotrimers
Peptide	Tm [°C]
HT1	47.3
HT2	24.3
HT3	N/A

Peptide/Cargo	Tm [°C]
HT1-DOX	64.5
HT2-5FU	28.7
HT3-PTX	58.6

Heterotrimers
Peptide/Cargo	Tm [°C]
HT123	51.1
HT123-DOX	51.8
HT123-5FU	40.7
HT123-PTX	58.7
HT123-DOX-5FU-PTX	41.3

The conjugation of the cargo to the peptide has a large stabilizing effect on homotrimer formation. The effect is very large in case of conjugation of PTX to HT3 peptide; without PTX we are not able to detect Tm, and with PTX, Tm is 58.6 ± 1°C. The smallest effect on Tm was observed with a conjugation of 5FU to HT2 were Tm rises only from 24.3 to 28.7 ± 1°C. The conjugation of 5FU to HT2 fails to produce a homotrimer that is stable at 37°C, thus, the peptide nanoparticle is not formed at physiological conditions. The heterotrimers were formed by combining the HT1, HT2, and HT3 peptides in the equimolar ratio, annealing the mixture at 80°C for 10 min and slowly cooling to room temperature. Subsequently, peptides were incubated at 4°C for 24 h before the CD analysis. The electrostatic interactions between the peptide strands lead to the formation of heterotrimer nanoparticle, HT123, with higher thermal stability (Tm = 51.1 ± 1°C) than any of the homotrimers. To study the heterotrimers as carriers for a single drug, the combination of one peptide conjugated to the drug, and two non-conjugated, complementary peptides were combined in the equimolar ratio (a) TH1-DOX, HT2, HT3, (b) HT1, HT2-5FU, HT3 and (c) HT1, HT2, HT3-PTX. The mixture was annealed and cooled, as described above, before the CD measurement. The Tm for all the heterotrimers (Table 3) is above the physiological temperature, thus, the carrier is folded into nanoparticle during the cellular uptake. The thermal stability of heterotrimer nanoparticle HT123 changes with the drug conjugation: PTX (+7.2°C), 5FU (−10.8°C), DOX (+0.7°C). The large stabilizing effect of conjugation is often observed in homotrimer assemblies when conjugation of hydrophobic cargo significantly increases the Tm of peptide. Because PTX is the most hydrophobic of the three drugs, this effect was expected.

To assembly the nanoparticle with all three drugs, the HT1-DOX, HT2-5FU and HT3-PTX was combined in equimolar ratio. The unfolding curve was recorded with CD before and after annealing of the peptide solution (Figure 1b). Before annealing the solutions contained the mixture of homotrimers which is observed as the broad distribution in the unfolding curve (Figure 1b, red), with several poorly resolved peaks. The most pronounced peak at 62°C was identified as peak representing the HT1-DOX homotrimer. After the annealing process (Figure 1b, black), one narrow peak is observed, with the unique Tm = 41.3°C. Because we observed only one, narrow peak with the unique Tm, we concluded that the heterotrimer with three different drugs was successfully self-assembled without other hetero- or homotrimer combinations present (within the detection limit by CD).

3.4. Efficacy of DOX, 5FU, PTX and Their Combination Delivered with Heterotrimer Hybrid Peptide to MCF-7 Breast Cancer Cell Line.

We have shown before that the collagen/CPP hybrid constructs without the drug modifications have very low cytotoxicity [36,39]. The HT123 heterotrimer was tested using the same method as peptide drug conjugate and shows small decrease in cell viability above 30 μM concentration (Figure 2i).

Figure 2.

Effect of (a) HT1-DOX, (b) HT2-5FU, (c) HT3-PTX (d) HT123-DOX, (e) HT123-5FU, (f) HT123-PTX, HT123-DOX-5FU-PTX heterotrimer (g) not annealed (h) annealed, (i) HT123 heterotrimer on MCF-7 cell survival. Error bars represent standard deviation calculated from at least six measurements.

To measure cell viability with the peptide-drug conjugate, the MCF-7 cells were incubated with solution of peptide conjugated with the drug for 72h and the viability assay (CellTitter Glo 2.0) was performed in the 96-well plate. While the peptide uptake is completed in about 30 min, the cells were analyzed after 72h because this is the optimal time for one of the drugs, 5FU, to show effectiveness [29,36]. Figure 2a-c illustrates the cell viability of MCF-7 cells treated with different concentrations of the homotrimers that carry three molecules of the same drug. The measured efficacy of HT1-DOX homotrimer nanoparticle (Figure 2a) expressed as IC50 (6.6 ± 0.1 μM) was calculated on the bases of the dose-response (sigmoidal) curve. The IC50 value indicates that the homotrimer can cross the cellular membrane and release DOX with similar effectiveness as free DOX which is between 0.6-8.5 μM for the range of human cancer cells [26-28]. As seen in Figure 2b, the HT2-5FU has no effect on survival of MCF-7 cells. The HT2 homotrimer is only partially folded at 37°C (Figure 2i, Tm = 28.7 °C) and even though the cell penetrating peptide sequence is a part of the HT2 sequence, the net charge of a single strand is negative (−1); thus, the carrier (−3) with 5FU cannot cross the cell membrane. The homotrimer HT3-PTX potency (Figure 2c, IC₅₀ = 198 ± 1 nM) is significantly lower than the free PTX (low nanomolar range in human cancer cells). Because the HT3-PTX homotrimer is not optimized for cellular delivery, we expected the IC50 to be higher than literature value for pure drug.

To test their optimization for cellular delivery, the HT123 heterotrimers with only single drug molecule conjugated to either HT1, HT2, or HT3 were self-assembled and tested on MCF-7 cells (Figure 2d-f). In all three cases, the IC50 for the heterotrimer conjugated to the specific drug were close to expected literature values for the free drug: IC50 (HT123-DOX) = 5.9 ± 0.1μM (free drug 0.6-8.5 μM), IC50 (HT123-5FU) = 20.4 ± 0.4 μM (free drug 2-20 μM), and IC50 (HT123-PTX) = 42.5 ± 0.7 nM (free drug 2.5-7.5 nM).

The efficacy of self-assembled peptide nanoparticles that carry the combination of all three drugs: DOX, 5FU and PTX in equimolar ratio was measured the same way as for single drug heterotrimer. In Figure 2g, h, we compare the effect of the equimolar mixture of homotrimers-drug conjugates (not annealed heterotrimer) and assembled heterotrimer (annealed nanoparticle). The IC50 in the latter case is 23.6 nM, while the random combination of homotrimer conjugates is 47.5 nM. In both cases the efficacy of the combination of drugs is higher than the efficacy of the single drug, including the very potent PTX.

4. Discussion

Collagen homotrimers have been used in the past for drug delivery, but their sequence must be modified by hybridization with a short cell penetrating peptide (CPP) sequence to improve the efficiency of cellular uptake and endosomal escape [36]. Adding a CPP sequence (in most cases short peptide containing multiple R and/or K in sequence) to collagen peptide has a strong, negative effect on Tm; thus, CPP addition may lead to thermally unstable nanoparticles, partially unfolded peptide at physiological conditions. To facilitate the synchronized delivery (simultaneous delivery with the same biodistribution) of three different drugs, we used the collagen heterotrimer. To promote self-assembly into the monodisperse nanoparticles, electrostatic guidance had to be used during self-assembly. Therefore, each of the three peptides in the heterotrimer carries a charge. Initially, we examined homotrimers assembled from the complementary heterotrimer sequences. Surprisingly, HT1 homotrimer, which has the largest positive charge (+7/strand), is also the most thermally stable (Tm = 47.3 ± 1°C). HT2 and HT3 homotrimers incorporate the CPP sequence (RRGRRG) that we used in the past [33,35], but they also have the (EOG)₅ sequence used to guide the folding of the collagen domain into heterotrimer. Even though the net charges are much smaller than HT1 homotrimer, HT2 homotrimer (−1/strand) has much lower Tm = 24.3 ± 1°C, and we are unable to measure Tm for HT3 homotrimer (+4/strand). We concluded that destabilizing effect of (EOG)₅ sequence (−5/strand), is much greater than (PRG)₅ sequence (+5/strand) in formation of homotrimer nanoparticles.

Conjugation of the cargo to the N-terminus of the peptide often results in thermal stabilization of nanoparticles, particularly if the cargo has hydrophobic characteristics. To avoid the large thermal stabilization of homotrimers, we conjugated the most hydrophobic drug (PTX) to the least stable peptide (HT3), what resulted in the Tm = 58.6°C of HT3-PTX homotrimer. During conjugation 2’-O-succinyl-PTX was activated (HATU) before addition to HT3 peptide to avoid cross-coupling with glutamic acid side groups. To confirm the conjugation the SDS-PAGE (16%, tricine buffer) analysis was performed (Figure S7). We also observed a large stabilizing effect by conjugating the DOX to HT1 (ΔTm = +17.2°C). The conjugation of least hydrophobic and smallest 5FU resulted only in ΔTm = +4.4°C thermal stabilization. PTX and 5FU were conjugated to the peptide via a succinate linker (amide bond) that in the past showed effective cleavage after the delivery and, thus, effective release of active drugs. However, when we used the succinate linker to conjugate DOX to peptide, upon delivery the activity of DOX was significantly diminished. Thus, we used cis-aconityl anhydride linker, which is acid labile, and it has been shown to release active DOX upon delivery [33]. Due to the intense red color of HT1-DOX conjugate, the presence of DOX is apparent, but MALDI analysis didn’t show the molecular peak of the HT1-DOX (see 2.2). We concluded that the main reason for the missing molecular peak was our use of the DHB matrix (recommended for short peptides analysis) that results in the cleavage of DOX from the peptide. To confirm the conjugation, SDS-PAGE (16%, tricine buffer) analysis was performed (Figure S8) and gel was imaged with Coomassie and in fluorescence mode, since DOX is a fluorophore. MALDI analysis of other conjugates directly confirmed the conjugation; HT3-PTX showed molecular peak and HT2-5FU showed fragment HT2-linker, that can only be present if the 5FU cleavage occur after conjugation.

The assembly of homo- and hetero- trimers was observed with circular dichroism spectroscopy. Based on the previous work of Hartgerink, the presence of the pairwise interactions between the hetero strands lead to a large shift toward the formation of ABC-type heterotrimer [22]. The reduction in other assemblies can be promoted by incorporation of amino acids in the X and Y position that were not proline (X) or hydroxyproline (Y), with the most effective being residues that carry permanent charge. The stability of the heterotrimer HT123-DOX-5FU-PTX expressed as Tm was lower than the two of the homotrimers but based on CD (Figure 2b), it was the only product of self-assembly. This indicates that the heterotrimer H123-DOX-5FU-PTX was the lowest energy state, and the system was at the thermodynamic equilibrium. If we consider other available states, for example homotrimer HT3-PTX, the energetic cost of assembly of this most thermally stable product would force the other peptides to assemble in several mixtures like HT112-DOX-DOX-5FU or HT221-DOX-5FU-5FU etc.; thus, the energy of the system would have to be viewed as the combination of all those states. If the overall energy of those states is higher than the single state energy of heterotrimer HT123-DOX-5FU-PTX, the heterotrimer will be the predominant, if not single, product of the peptide self-assembly. In the case of heterotrimer HT123-DOX-5FU-PTX assembly, in addition to guided charge pairing within the collagen domain, each peptide also had the CPP domain containing arginine residues, that disrupt the high thermal stability of homotrimers, thus pushing the energetics of the assembly towards the heterotrimer HT123-DOX-5FU-PTX formation. The lack of HT2-5FU toxicity (IC50) was additional evidence that the trimer assembly is necessary for the prodrug to enter the cell. For the detailed discussion see [36, 39].

Drug efficacy is often expressed as IC50, but in the case of prodrugs, measurement of IC50 reflects not only the potency of free drug, but also the effectiveness of the transport process: delivery and release [39]. The mechanism of collagen/CPP hybrid peptide cellular uptake, localization, and the endosomal escape has been discussed in detail in [37, 39]. The conjugation of HT1 to DOX and HT3 to PTX results in the formation of nanoparticle (homotrimer) thermally stable at 37°C. Based on the IC50 (Figure 2a, c), the homotrimer nanoparticles with DOX and PTX crossed the cellular membrane and were released in the cells. The DOX delivery and release via homotrimer is more effective than PTX delivery. The measured IC50 for DOX with homotrimer nanoparticle delivery was in the expected range of the DOX activity, 6.6 μM, but the delivery of PAX with HT3 homotrimer was significantly less effective than its free form, IC50 = 198 nM vs < 8 nM. Because the Tm of HT1 and HT3 homotrimers were similar, the nanoparticle folding fraction is similar at 37°C during the uptake of the peptide. However, the net charge of folded HT1 and HT3 nanoparticles is +21 and +12 respectively, thus the uptake of HT1 is most likely more effective at the same folding fraction. In addition, the more labile linker is employed in DOX delivery than PTX. It appears that the net charge of nanoparticle is very important for effective uptake; the IC50 of 5FU conjugated to HT2 homotrimer could not be measured, because the prodrug exhibited no cytotoxicity. The net charge of the HT2 homotrimer was −3 (Table 2); thus, it is unlikely that the nanoparticle could cross the negatively charged cell membrane. We have also considered that the low Tm of HT2-5FU homotrimer (28.7°C) contributed the smaller “effective concentration” of homotrimer, as only 18.6% of the peptide was folded (FF@37), and only folded peptides can cross the cellular membrane [37]. However, if a peptide (positively charged) can cross the membrane, the uptake is detectable when folded fraction is above 8% [37]. Thus, we should be able to detect some toxicity of 5FU delivered by HT2 homotrimer. Because we did not observe this effect, we concluded that the net charge of the nanocarrier is the main factor in determining the effectiveness of cellular uptake.

To be able to compare the drugs uptake via heterotrimer HT123, we assembled the peptide with single drug, HT1-DOX with two complementary peptides, HT2 and HT3 without the drug, and forming HT123-DOX. We used an analogous approach to form HT123-5FU, and HT123-PTX and measured the IC50 for single drug heterotrimer (Figure 2d-f). The measured IC50 was much closer to literature values for each of the free drugs with comparison to the homotrimers, which suggests better delivery. The heterotrimer with all three drugs HT123-DOX-5FU-PTX was also assembled, and the measured IC50 (Figure 2h) was lower than for any single drug heterotrimer, 23.6 nM.

The combination of DOX, 5FU and PTX is used in the clinical treatment, but the drugs are not delivered simultaneously [24]; PTX is more potent than DOX and 5FU, thus the last two are administered in much higher doses. Moreover, PTX is highly insoluble, and must be formulated to increase its bioavailability, which often creates additional issues [40]. The heterotrimer assembly provided the unusual platform to deliver equimolar ratios of the three drugs and possibility to search for synergistic effects at very low doses of DOX and 5FU. One way to compare the effectiveness of the peptide platform was to apply the Loewe Additivity Model [8], expanded to 3 drugs, and calculate the combination indices (CI) defined as:

$$CI=\frac{C_{DOX}}{C{S}_{DOX}}+\frac{C_{5FU}}{C{S}_{5FU}}+\frac{C_{PTX}}{C{S}_{PTX}}$$

Where C_n is the concentration of each drug in the heterotrimer combination that shows 50% cell viability, and CS_n is the concentrations of a single drug in the trimer that are necessary to obtain the same effect as the drug combination. The calculated CI in the heterotrimer system HT123-DOX-5FU-PTX was 0.561 which indicates that the combination of the three drugs delivered simultaneously had synergistic effects (values less than 0.9 indicate synergistic system, close to 1 an additive system, and greater than 1.1 an antagonism between the drugs). In this calculation we used only the assembled heterotrimer nanoparticles. To visualize the simplified interaction planes, we didn’t consider the drug pair interactions and plotted (Figure 3) C_n plane (blue) and CS_n plane (orange) in the logarithmic (-log) scale [41]. Since there is no plane intersection and CS_n plane is below the C_n plane the drug combination delivered in heterotrimer nanoparticle indicating synergy between the drugs.

Figure 3.

Interaction planes in logarithmic scale of C_n (blue) and CS_n (orange)

We also calculated CI for the homotrimers and their equimolar mixture that was not assembled (Figure 2g), CI = 0.247. We suspect that the larger synergistic effect measured for homotrimer reflects the fact that in the mixture of homotrimers we cannot prevent assembly of some peptides into the heterotrimers because the sequences were designed to promote that assembly and the CS_5FU cannot be determined (very large) due to lack of prodrug uptake. Thus, we can only approximate the CI for homotrimers, as some of the drugs is carried to the cell as heterotrimer. In this case, we view the CI value as an indication of effectiveness of delivery and release. This conclusion is also supported by the measured values of IC50: for PTX in the homotrimer assembly, it is about 5 times larger than in heterotrimer assembly. When combined in the mixture with homotrimers containing DOX and 5FU in the equal, nanomolar ratio (much lower than IC50 for both DOX and 5FU), the IC50 of this combination was almost as effective as PTX in heterotrimer assembly.

In conclusion, we presented a peptide-based delivery system that can carry three chemically different cargos in equimolar ratio to the cell and release them simultaneously with the same biodistribution; the described platform ensured the delivery of each drug to the same cell. The combination of drugs was used for proof of concept, DOX, 5FU, PTX are used in clinical treatment of breast cancer, and we observed the synergistic increase in potency when delivered simultaneously in equimolar ratio. The concentration of DOX and 5FU used in the combination was much lower than the IC50 of the free drug. Most likely, the synergistic effect will be varied for different cell lines and different drug combinations, what would have to be explored in future studies. We envision that this approach has the potential to allow multidrug treatment, where the peptide-conjugated drug library can be used to co-deliver formulations based on the exact needs of the individual patients. In addition, this peptide system can be applied to deliver multi-functional nanoparticles modified with targeting ligands, drugs, imaging agents etc. This functionality can greatly improve the specificity of drug delivery.