Enzyme-Triggered Morphological Transition of Peptide

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Biological and Medical Applications of Materials and Interfaces

Enzyme-Triggered Morphological Transition of Peptide Nanostructures for Tumor-Targeted Drug Delivery and Enhanced Cancer Therapy Meiwen Cao, Sha Lu, Ningning Wang, Hai Xu, Henry Cox, Ruiheng Li, Thomas A. Waigh, Yuchun Han, Yilin Wang, and Jian R. Lu ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.9b03519 • Publication Date (Web): 16 Apr 2019 Downloaded from http://pubs.acs.org on April 16, 2019

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ACS Applied Materials & Interfaces

Enzyme-Triggered Morphological Transition of Peptide Nanostructures for Tumor-Targeted Drug Delivery and Enhanced Cancer Therapy

Meiwen Caoa,*, Sha Lua, Ningning Wanga, Hai Xua,*, Henry Coxb, Ruiheng Lib, Thomas Waighb, Yuchun Hanc, Yilin Wangc, and Jian R. Lub,*

a

State Key Laboratory of Heavy Oil Processing and Centre for Bioengineering and

Biotechnology, College of Chemical Engineering, China University of Petroleum (East China), 66 Changjiang West Road, Qingdao 266580, China b

Biological Physics Laboratory, School of Physics and Astronomy, University of

Manchester, Schuster Building, Oxford Road, Manchester, M13 9PL, U. K. c

Beijing National Laboratory for Molecular Sciences (BNLMS), Key Laboratory of

Colloid and Interface Science, Institute of Chemistry, Chinese Academy of Sciences , Beijing 100190, China

Corresponding Authors *

E-mail:

[email protected]

(M.C.);

[email protected]

1

ACS Paragon Plus Environment

[email protected]

(H.X.);

ACS Applied Materials & Interfaces 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ABSTRACT The use of smart drug carriers to realize cancer-targeted drug delivery is a promising method to improve the efficiency of chemotherapy and reduce its side effects. A surfactant-like peptide, Nap-FFGPLGLARKRK, was elaborately designed for cancer-targeted drug delivery based on an enzyme-triggered morphological transition of the self-assembled nanostructures. The peptide has three functional motifs: the aromatic motif of Nap-FF- to promote peptide self-assembly, the enzyme-cleavable segment of -GPLGLA- to introduce enzyme sensitivity, and the positively charged -RKRK- segment to balance the molecular amphiphilicity as well as to facilitate interaction with cell membranes. The peptide self-assembles into long fibrils with hydrophobic inner cores, which can encapsulate a high amount of anticancer drug doxorubicin (DOX). By having enzyme responsibility, these fibrils can be degraded into thinner ones by the cancer-overexpressed matrix metalloproteinase-7 (MMP7) at tumor sites and precipitate out to give sustained release of DOX, resulting in cancer-targeted drug delivery and selective cancer killing. In vivo antitumor experiments with mice confirm the high efficiency of such enzyme-responsive peptidic drug carriers in successfully suppressing the tumor growth and metastasis while greatly reducing the side effects. The study demonstrates the feasibility of using enzyme-sensitive peptide nanostructures for efficient and targeted drug delivery, which have great potential in biomedical cancer treatment.

KEYWORDS: peptide self-assembly; enzyme-sensitive; cancer therapy; drug carriers; targeted delivery

2


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1. INTRODUCTION Chemotherapy, a supplementary intervention of surgical operation, constitutes an essential part of the treatment of many cancers.1 However, conventional chemotherapy drugs usually disperse nonspecifically in body tissue, causing damage not only to tumor cells but to healthy ones as well. This always leads to severe systemic side effects.2 Successful chemotherapy also needs a high dose and a long circulation time of the drugs in the body, but most chemotherapy drugs have poor water-solubility and low physiological stability. It is hence crucial to find suitable strategies to specifically increase drug deposition and retention within tumors to improve chemotherapeutic efficacy while reducing the side effects.3 Tumor tissues can produce specific biological signals different to the normal host ones, such as lower pH, overexpressed enzymes, and active oxidation/reduction species. These signals can be used as cues to establish stimuli-responsive drug delivery systems for targeting and controlling anti-cancer drug delivery.4-8 By using smart nanocarriers to realize tumor-targeted drug delivery, therapeutic efficacy can be improved by a selective accumulation of therapeutics within tumors, reduced off-target effects due to the limited availability of cytotoxic drugs to normal host tissues, and an increased circulation time of a drug in the body.3,9 Self-assembled peptide nanostructures have been demonstrated to be excellent candidates as effective drug carriers.9-16 Firstly, with natural amino acid residues as the basic components, peptides have inherent biocompatibility and biodegradability, suitable for various bio-applications. Secondly, peptides can self-assemble into an array of distinct nanostructures including vesicles, tubes, fibrils, and tapes, which can provide hydrophobic inner cores and hydrophilic outer surfaces for incorporating both hydrophobic and hydrophilic drugs to improve their physiological stability.5,6,17-20 3



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Moreover, peptides have chemical versatility and can be easily designed to have specific

motifs

enzyme-triggered

with

stimuli-sensitivity

transition

of

the

and

target

self-assembled

specificity.10,21-23 peptide

The

supramolecular

nanostructures can be obtained by including an enzyme cleavable sequence in the peptide molecule.9,22-28 When cancer-specific enzymes are used as stimuli, smart peptide self-assembly systems with a capability of cancer-targeted drug delivery can be developed.5,6,10,24,29 Here we report the targeted use of matrix metalloproteinase-7 (MMP7), an extracellular protease that is frequently overexpressed in human cancer tissues,30,31 as an enzymatic stimulus to trigger the transition of peptide nanostructures for tumor-targeted drug delivery to achieve efficient cancer therapy. Three surfactant-like peptides of Nap-Phe-Phe-Gly-Pro-Leu-Gly-Leu-Ala-(Arg-Lys)n-CONH2 (n=1, 2, 3) have been designed and synthesized (Figure 1a). The peptides have three functional motifs: (1) Nap-Phe-Phe (Nap-FF), the aromatic segment promotes peptide self-assembly in aqueous solution by providing extensive aromatic-aromatic interactions and hydrophobic interactions.24,32,33 (2) Gly-Pro-Leu-Gly-Leu-Ala (GPLGLA) is the enzyme substrate that is cleaved by MMP7 to give the system enzymatic sensitivity.34 (3) (Arg-Lys)n ((RK)n) is a positively charged hydrophilic region which provides electrostatic interactions so as to balance the amphiphilicity of the molecule, as well as to facilitate the interaction with cell membranes. The peptides can self-assemble into long fibrils with hydrophobic inner cores that can encapsulate a high amount of anticancer drug of doxorubicin (DOX) (Figure 1b). These fibrils undergo a MMP7-triggered morphological transition when exposed to certain cancer cells, during which the drug molecules are released and accumulate in the cancerous regions to provide targeted delivery. Such a process does not occur when the 4


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drug-loaded fibrils are administrated to normal tissues, thus to achieve highly efficient selective cancer killing and in vivo tumor therapy (Figure 1c and d).

Figure 1. (a) The molecular structures of designed MMP7-sensitive peptides and the expected enzymatic hydrolysis reaction. (b) Schemes showing the peptide self-assembled nanostructures, the drug loading and enzymatic release processes. (c) Scheme illustrating the animal experiments for in vivo cancer therapeutic efficiency. (d) Schematic representation of cancer-targeted drug delivery and selective cancer killing. The drug-loaded fibrils retain their integrity at normal cell sites and do not 5



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release drug molecules. While at cancer cell sites the fibrils experience structural transition from well dispersed thicker fibrils to thinner fibrillar structures and disassembled aggregates due to MMP7 hydrolysis, leading to targeted release and the accumulation of drug molecules around the cancer cells.

2. Materials and Methods 2.1. Materials Protected

L-amino

acids

(Fmoc-Lys(Boc)-OH,

Fmoc-Arg(Pbf)-OH,

Fmoc-Phe-OH, Fmoc-Gly-OH, Fmoc-Pro-OH, Fmoc-Leu-OH and Fmoc-Ala-OH), O-Benzotriazole-N,N,N',N'-tetramethyluronium N-hydroxybenzotriazole

(HOBT),

hexafluorophosphate

N,N-diisopropyl

ethylamine

(HBTU), (DIEA),

Trifluoroacetic acid (TFA), Trisopropylsilane and Rink amide MBHA resin were from GL Biochem (Shanghai) Ltd and used as received. β-Naphthylacetic acid was from Sinopharm Chemical Reagent Co. Ltd (Shanghai). Solvents of piperidine, dichloromethane (DCM) and N, N-dimethyl formamide (DMF) were from Bo Maijie Technology (Beijing) and were redistilled before use. MMP7 was from ProSpec-Tany TechnoGene Ltd. Doxorubicin hydrochloride (DOX-HCl) was from TCI Shanghai. All water used was processed from a Milli-Q Biocel ultrapure water system with a resistivity of 18.2 MΩ cm. 2.2. Peptide Synthesis The peptides were synthesized on a microwave peptide synthesizer (CEM Liberty) following the standard Fmoc solid-phase synthesis strategy. Sample purification was performed as described in our previous work.35 The molecular structure and purity (>95%) were confirmed by both mass spectrometry and HPLC chromatography. 2.3. Preparation of Peptide Solutions and in Vitro MMP7 Treatment 6


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The peptide was dissolved in Tris(hydroxymethyl) aminomethane-buffered (50 mM, pH=7.4) solution. After sonication for 15 min the solution was incubated at room temperature for at least 2 days before it was characterized. For MMP7 treatment, 500 μL of peptide 2 (0.2 wt%) in Tris buffer was treated with 0.9 μL of human MMP7 (1.82 μg/μL) in the presence of 150 mM Na+ ions and 2 mM Ca2+ ions

and

incubated at 37 °C. 2.4. Reversed Phase High-Performance Liquid Chromatography (RP-HPLC) HPLC profiles of the peptides before and after treatment with MMP7 were recorded on a HPLC system (Waters 2695 Alliance) with a C18 reversed-phase column (4.6 mm  150 mm) at 25℃. Two eluents were used, that is, eluent A of 0.1% (v/v) TFA in water and eluent B of 0.1% (v/v) TFA in acetonitrile. After sample loading on the column, the following isocratic elution process was performed: 0→1 min, 95% eluent A and 5% eluent B; 1→40 min, a linear gradient elution of eluent A from 95% to 5% and eluent B from 5% to 95%; 40→45 min, a linear elution of 95% eluent A and 5% eluent B to restore the system. The eluent flow rate was set at 0.6 mL/min. The UV detector was set at 265 nm. 2.5. MALDI-TOF Mass Spectra (MS) A Bruker Microflex MALDI-TOF mass spectrometer was used to collect the mass spectra by using α-cyano-4-hydroxycinnamic acid (HCCA) as a matrix. The peptides and the HCCA were dissolved in a mixture of acetonitrile and water (30:70, v/v) containing 0.1% trifluoroacetic acid (TFA). About 1 μL of the resulting solution was deposited on a polished steel substrate and air-dried at room temperature. Mass spectra were acquired in reflection positive mode and used an accelerating voltage of 20 kV. 2.6. Zeta Potential (ζ) Measurements 7



The ζ measurements were carried out on a Malvern Nano-ZS instrument (ZEN3600, Malvern Instruments, Worcestershire, UK) at room temperature. A 4 mW He-Ne laser was equipped and set at 633 nm. A clear disposable capillary cell (DTS1060C) was used for all the measurements. 2.7. Encapsulation and Release of DOX The peptide 2 solution of 2.0 mM in Tris buffer was firstly prepared and incubated at room temperature for at least 2 days to produce the self-assembled nanostructures. Then DOX-HCl was added into the peptide solution to obtain a mixed solution of peptide 2 and DOX. After incubation with gentle shaking for one day the mixed solution was centrifuged at 13000 rpm for 1 h. A small volume of the supernatant was taken to measure the UV-Vis absorbance on a Shimadzu UV-2450 spectrophotometer to determine the DOX concentration. By carefully removing the supernatant, the DOX-loaded peptide 2 fibrils were collected as a pelleted residue at the bottom and then carefully washed to remove the unbound DOX. For the determination of the DOX concentration, a linear-fitted standard calibration plot was firstly obtained using the absorbance of the DOX solution with varying concentrations (from 1 to 20 μg/mL) at 485 nm, which was then used to determine the unknown DOX concentration in each system. For the DOX release experiments, a defined amount of the pelleted residue was dialyzed (MWCO = 14 kDa, MYM Biotech. Co., Ltd.) against 10 ml of the Tris buffer with or without MMP7 enzyme. The release reservoir was constantly stirred and at various time points 100 μL of the dialysate was taken out to measure the absorbance at 485 nm and an equal volume of fresh buffer was added to the reservoir. The supernatant DOX concentration was also determined from the above-mentioned standard calibration plot. The release percentage was calculated using the following 8


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formula: release (%) = 100 × (the cumulative amount of released DOX)/(the amount of fibrils-encapsulated DOX). 2.8. Circular Dichroism (CD) A MOS-450/AF-CD spectrophotometer (BioLogic, France) was used to collect the CD spectra at room temperature. A 0.2-mm quartz cell was used. Scans were obtained over a range of 190‒260 nm by taking points at 1.0 nm spacings. The results that are presented were averages of three repeats for each sample. 2.9. Fluorescence Measurements A Fluoro Max-P spectrophotometer (JOBIN YVON) was used to perform the fluorescence

measurements

at

room

temperature.

For

using

sodium

8-anilino-1-naphthalenesulfonate (ANS) binding assay to determine the internal polarity of the peptide 2 fibrils, the fluorescence emission spectra of aqueous solutions containing ANS were measured by excitation at 380 nm. To obtain the emission spectra of solutions containing DOX, the excitation wavelength was set at 480 nm. 2.10. Optical Microscopy The optical microscopy measurements were performed on a microscope (Leica DMI3000B) equipped with an oil-immersion objective lens (×100). The light sources were Argon ion and HeNe lasers. Images were acquired using a 405 nm laser for DOX. 2.11. Stochastic Reconstruction Microscopy (STORM) STORM experiments were performed on a custom built STORM microscope. The detailed equipment setup and experimental procedures can be found in our previous paper.36 An OxEA buffer was used for the STORM imaging.37 Images were reconstructed using the ThunderSTORM ImageJ plugin.38 9



2.12. Atomic Force Microscopy (AFM) AFM morphologies were captured on a Multimode Nanoscope IVa AFM system (Digital Instruments, Santa Barbara) under ambient conditions. For sample preparation, a drop of peptide or peptide/DOX mixed solution (~10 uL) was firstly deposited onto a freshly cleaved mica surface. After adsorption for ~30 sec, excess liquid was blown away with a nitrogen stream to dry the mica surface. TESP-V2 type Ohm-cm Antimony (n) doped Si probes (Bruker, Camarillo) with a nominal spring constant of 42 N/m were used to capture the tapping mode images at 512  512 pixels. The images were treated and analyzed using the vendor-provided software of NanoScope Analysis 1.40. 2.13. Transmission Electron Microscopy (TEM) TEM measurements were performed on a JEOL JEM-1400 TEM. The samples were prepared by the negative staining method. Firstly a drop of sample solution was placed onto a copper grid with carbon Formvar-coating. After adsorption for 5–10 min, the excess fluid was removed from the grid. The grid was then placed onto a drop of uranyl acetate solution (2% w/v) and stained for 2–5 min. The samples were imaged at an accelerating voltage of 200 kV. 2.14. Cytotoxicity Assay Cell viability was assessed by the tetrazolium reduction (MTT) test. Firstly, 100 μL of cells were cultured in 96-well plates at 1 × 105 cells/mL in complete medium and allowed to become adherent at 37 °C for 24 h. Then the medium was removed and 100 µL of fresh complete medium was added to each well. Consequently, 100 μL of peptide solution (or solution of DOX-loaded peptide fibrils) with varied concentration was added. The cells cultured in 100 μL of Tris buffer were used as a control. After incubation at 37 °C for 24 h, 20 μL of MTT reagent (5mg/mL) was 10


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added to each well and the plate was further incubated at 37 ºC. After 4 h, the supernatant was aspirated thoroughly and 150 μL of DMSO was added to each sample well and the four blank wells. Finally the plate was put in a microplate shaker for 10 minutes to ensure thorough mixing and the absorbance was then measured at 570 nm with a microplate reader. The cells’ survival ratios were calculated using the following formula: survival ratio = 1–(ATris – Apeptide)/(ATris – Ablank), where Ablank is the absorbance of DMSO, ATris is the absorbance of the complete medium in Tris buffer, and Apeptide is the absorbance of the mixed solution of peptide and complete medium. Results were presented as the mean and the standard deviation obtained from the 4 samples. For the MTT experiments with the peptide 2/DOX composites, a special experimental procedure was used. After cell culture in complete culture medium for 24 h and the renewal of culture medium, 100 μL of the peptide 2/DOX composited solution was added into each well. After incubation for 4 hours, the 96-well plates were centrifuged at 500 rpm/min for 10 min. The supernatant solutions were carefully removed from each well and 100 μL of fresh complete medium was added. After incubation for 48 h, MTT and DMSO treatments were sequentially performed and the absorbance was obtained. 2.15. Determination of MMP7 Secretion Levels of Different Cells The level of MMP7 secretion by various cells was assessed with a MMP7 ELISA Kit (MultiSciences, Lianke BiotechCo., Ltd.) at room temperature by following the vendor-provided standard protocols. The optical density was measured using a microplate reader (M2e, Molecular devices) at 450 nm. The amount of MMP7 was calculated based on a standard curve obtained according to the manufacturer’s instruction. Three repeats were performed for each cell line. 11



2.16. Subcutaneous Mouse Model Female BALB/c mice of 4−6 weeks and ~16 g body weight were purchased from Vital Laboratory Animal Centre (Beijing, China). 100 μL of HpeG2 cell suspension of 1.0 × 107 cells mL−1 was implanted into the oxter of healthy mice, in the right axillary region. HpeG2 was chosen since its metastasis level could indicate the efficiency of the drug. In accordance with the guidelines of international animal care, the mice were raised in a specific pathogen free (SPF) facility. Enough clean food and water was provided. The growth of tumors was monitored every day. After a week of feeding, the HpeG2 subcutaneous mouse model was well defined. 2.17. Antitumor Experiment in Vivo The mice were separated into six groups when the tumor size reached about 80 mm3, as shown in Table S1. Each group had five mice (n=5), which were injected through the tail vein with the corresponding formulation solution of fixed injection volumes once every other day. The living state, the body weight, and the tumor volume were recorded every day. The tumor volume was calculated with the equation, tumor volume = (length × width × width)/2. In accordance with the guidelines of international animal care, the mice of the blank group and the fiber group were sacrificed when the tumor size reached 1000 mm3. The other groups were tested for 28 days except when the mice died during the course of the experiment. After treatment the tumor of each mouse was obtained for comparison of the size. The liver was also obtained for evaluation of the tumor metastasis by recording the number of tumor sites on it. The tissues were washed twice using PBS, and then fixed with 4% formalin solution and embedded in paraffin. The dried paraffin blocks were chopped into slices with 5 mm side length and transferred onto clean glass slides. Then

12


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standard hematoxylin-eosin (H&E) staining was used. Later, the slides were observed through an optical microscope, and photos were taken at ×10 magnification.

3. Results and Discussion 3.1. Selection of a Suitable Drug Carrier The designed peptides were first evaluated by self-assembly and cellular toxicity to find the most suitable variety to be used as drug carrier (Figure S1, Supporting Information). Peptide 1 with one RK repeat showed quite low solubility (< 0.20 mM) in Tris buffer (pH 7.4), restricting its application in aqueous solutions. Peptide 3 with three RK repeats has six positive charges and self-assembled into ordered nanostructures at concentrations above 0.85 mM (Figure S2, Supporting Information). However, it killed 70‒90% of both HeLa and NIH 3T3 cells at 50 μM (Figure S1d, Supporting Information). Therefore, peptide 3 is also not a suitable drug carrier due to high cytotoxicity. Peptide 2 with two RK repeats was found to have optimal hydrophilic/hydrophobic balance. It has a high solubility (>5.0 mM) and a critical aggregation concentration (CAC) of ~0.12 mM in Tris buffer (Figure S1a, Supporting Information). The lower CAC value is advantageous for drug delivery as a lower dose of peptide is needed. Furthermore, it will form ordered nanostructures with high stability in dilute solution (similar to that in circulating blood), which should give hydrophobic inner cores for encapsulating drug molecules. Peptide 2 also showed lower toxicity toward both HeLa and NIH 3T3 cells at 0.25 mM, a concentration well above the CAC (Figure S1b, Supporting Information). All of these aspects make peptide 2 a suitable candidate as a drug carrier. 3.2. Enzyme-Triggered Morphological Transitions of Peptide Nanostructures

13



The self-assembled structures of peptide 2 before and after MMP7 treatment were characterized by transmission electron microscopy (TEM). The peptide formed long regular fibrils with diameters of 7.0 ± 1.2 nm in buffered solution (Figure 2a). However, after MMP7 treatment, the self-assembled structures retained their fibrillar profiles, but they became significantly thinner with diameters of only 3.0 ± 1.0 nm, about half of the diameters of the original peptide 2 fibrils. These thinner fibrillar structures readily formed bundles via lateral association (Figure 2b). AFM measurements (Figure S3, Supporting Information) also showed separate long fibrillar structures with heights of about 5.0 nm for peptide 2 and fibril bundles comprising of thinner fibrillar structures with height of ~2.5 nm for the MMP7-treated sample. The diameters derived from AFM section analysis are slightly smaller than the ones derived from the TEM results. The difference may be caused by the surface confinement and sample compression by the AFM tip during scanning. Some dot-like structures can also be observed for the MMP7-treated sample, which could be the aggregates from the cleaved peptide fragments.

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Figure 2. TEM images of the self-assembled structures of peptide 2 at 0.25 mM before (a) and after (b) MMP7 treatment. The insets are the magnified images with diameter distributions of the corresponding fibrillar structures. The size distribution in each case was calculated from at least 200 separate fibrils. The inset of (b) is the magnified image that shows the fibril bundles comprised of many thinner fibrillar structures. The thinner fibrillar structures were indicated by the red arrows. Mass spectra are shown for peptide 2 at 0.25 mM before (c) and after (d) MMP7 treatment, with the intensity plotted as a function of the mass/charge ratio (m/z). HPLC results of peptide 2 at 0.25 mM before (e) and after (f) MMP7 treatment where the intensity is plotted as a function of retention time. HPLC result of peptide Nap-FFGPLG-COOH (g) as a control where the intensity is plotted as a function of retention time.

15



In parallel with the morphological transition data (Figure 2), the CD spectra of the samples before and after MMP7 treatment also revealed significant changes in the secondary structure (Figure S4, Supporting Information). The CD spectrum from the peptide 2 solution had a positive peak at 197 nm and a negative peak at 218 nm, typical of a β-sheet.39 After treatment with MMP7, the negative peak at 218 nm greatly weakened and another negative peak at ~200 nm was observed, indicating a higher content of random coil conformations. Both the morphological transition and the secondary structural change indicate the enzymatic cleavage of the peptide 2 by MMP7. The molecular cleavage and product identification were further assessed using mass spectroscopy (MS) and high-performance liquid chromatography (HPLC). Peptide 2 has a theoretical molecular mass (M) of 1556.9. Therefore, the two signals of 1557.9 and 1579.8 in the MS spectrum can be ascribed to [M+H]+ and [M+Na]+, respectively (Figure 2c). In the HPLC spectrum peptide 2 shows a single peak at the retention time of ~22 min (Figure 2e). However, after MMP7 treatment, two new MS signals at 827.9 and 843.9 appear (Figure 2d). Noting that the cleaved segment of Nap-FFGPLG-COOH has a theoretical molecular mass (M') of 804.9, the two new signals can be attributed to [M'+Na]+ and [M'+K]+, respectively. The HPLC spectrum of MMP7-treated peptide gave a new peak at a retention time of ~27 min and a significantly decreased signal at 22 min (Figure 2f). This new peak is the same as that of the control, a chemically synthesized molecule of Nap-FFGPLG-COOH (Figure 2g), confirming the successful enzymatic cleavage of peptide 2. The enzymatic cleavage induced surface charge variation of the peptide 2 fibrils, was assessed by ζ potential measurements (Figure S5, Supporting Information). The fibrils showed positive ζ values of near +50 mV while the MMP7-treated fibrils gave 16


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negative ζ values of about ‒25 mV, showing that the enzymatic cleavage removed the positively charged residues of Lys and Arg from the fibril surface leaving negatively charged carboxyl groups. Simultaneously the molecular cleavage resulted in a structural change from thicker fibrils to thinner fibrillar structures, as displayed by the TEM and AFM results. The results also indicate that peptide 2 forms fibrils with a hydrophobic inner core and a hydrophilic outer surface, which is promising for encapsulating drug molecules. 3.3. Drug Loading into the Peptide Fibrils and Enzyme-Triggered Release Having established that peptide 2 can self-assemble into fibrils that are capable of undergoing a structural transition in response to MMP7, we next investigated the drug encapsulation within the nanostructures and its release upon MMP7 treatment. DOX, an effective antitumor drug that can inhibit the synthesis of nucleic acid and has no cellular selectivity, was selected as the model drug. Peptide 2 produced two categories of fibrils after incubation with DOX, which were measured with TEM (Figure 3a). One is the fibrils with diameters of 6.0 ± 1.0 nm, as indicated by the yellow arrows, which are the originally formed fibrils. The other is the much thicker fibrils with diameters of 10.5 ± 1.5 nm, as indicated by the red arrows. The AFM results also showed two kinds of fibrils with diameters of ~5.0 nm and ~9.0 nm, respectively (Figure 3b) in reasonable agreement with the TEM image. We speculate that the thicker fibrils indicate successful encapsulation of DOX molecules within them. Though some reports showed that the drug loading might not affect the morphology of the self-assembled structures,18 other studies have verified that the drug loading can change the carriers’ properties including morphology, size, and even stiffness.11,17,40 Some thicker fibrils show non-uniform diameters along the long axis, as indicated by the green arrows in Figure 3b. This indicates that DOX 17



molecules are heterogeneously embedded in the fibrils and the loading amount is high enough to result in an obvious change of the fibril diameters. Furthermore, by using stochastic reconstruction microscopy (STORM), a super-resolution fluorescence microscopy technique with a resolution of ~20 nm, many single peptide 2 fibrils with high aspect ratios can be observed (Figure 3c).36 This also provides good evidence for the successful encapsulation of DOX into the fibrils.41 The fluorescence spectra (Figure S6, Supporting Information) show that the DOX encapsulated in the fibrils exhibits a significant red shift of the characteristic peaks compared with free DOX. This indicates that the DOX molecules are hosted inside the hydrophobic inner cores of the fibrils,42,43 which has a polarity intermediate between methanol and ethanol, as determined by using 8-anilino-1-naphthalenesulfonate (ANS) as a fluorescent probe (Figure S7, Supporting Information). The DOX encapsulation affected the molecular arrangement in the fibrils, as evident from the CD spectrum of the DOX-loaded fibrils, which shows two negative peaks at 202 nm and 218 nm (Figure S8, Supporting Information). Compared to the spectrum of peptide 2 solution (Figure S4), the new peak at 202 nm indicates that some peptide molecules rearrange into random coiled structure due to DOX encapsulation. Here, both the fluorescence and CD results indicate that most of the DOX were encapsulated in the fibrils instead of just attaching on the fibril surface.

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Figure 3. (a) TEM, (b) AFM, and (c) STORM images of the DOX-loaded fibrils of peptide 2 at 1.0 mM. In (a) the red arrows point to the thicker fibrils loaded with DOX and the yellow arrows point to the thinner fibrils without DOX encapsulation. In (b) the green arrows point to the fibril areas with DOX distribution. Both TEM and AFM images show the bimodal size distribution of the fibril diameter, as displayed in the insets for (a) and (b). (d) The release profiles of DOX from the peptide 2 fibrils (1.0 mM) in solutions with and without MMP7 (6.0 μg/mL) at 37 ºC. The release profiles were obtained from the DOX-loaded peptide fibrils after centrifugation by the dialysis method.

To estimate the DOX loading efficiency in the peptide 2 fibrils, 12.5 mg of peptide was dissolved in 2.0 mL water and incubated for 2 days to allow the full assembly of the peptide molecules into fibrils. Then 5.0 mg DOX-HCl was added into the solution. After incubation under gentle shaking for 1 day the mixed solution was centrifuged at 13000 rpm for 1 h. The UV-Vis absorbance of the supernatant at 485 19



nm was measured to calibrate the DOX concentration (Figure S9, Supporting Information), which was about 1.95 mg/mL. This shows that about 1.1 mg DOX was encapsulated in the peptide 2 fibrils, giving a loading efficiency of approximately 8.8% by weight. Such a loading efficiency is relatively high in comparison with the reported values, which are typically about 2–5% by weight.44 The MMP7-triggered drug release from the DOX-loaded peptide fibrils was then investigated by dialysis (Figure 3d). For the control experiment without MMP7, an obvious DOX release occurred at early times, and the maximum release amount of about 20% of the total encapsulated DOX was reached after ~20 min. This is probably due to the release of the loosely bound DOX molecules on the fibril surface. Upon MMP7 treatment, a different DOX release profile was obtained, which reached a near plateau after ~90 min, giving a maximum release amount of >80%. The results show that MMP7 treatment can efficiently trigger the drug release. In a separate experiment using Nile red (NR) as the model drug (Figure S10, Supporting Information), the solution with NR-loaded peptide 2 fibrils was clearly red and transparent, indicating complete dissolution of the composite aggregates. However, MMP7 treatment resulted in a substantial mass of dark red precipitates settling at the bottom of the tube leaving a colorless supernatant. The precipitates were comprised of complexes of released NR molecules (very low water solubility) and bundles of the cleaved thinner fibrillar structures. The drug release mechanism is deduced as follows. MMP7 treatment resulted in successful cleavage and degradation of the peptide fibrils, as confirmed by the structural transition from thicker fibrils to thinner fibrillar structures and dot-like particles as well as the positive-to-negative transition of the surface charge. The successful degradation of the fibrils is believed to trigger the drug release. Moreover, we should note that DOX is positively charged at the studied pH. Some 20


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released DOX molecules might bind onto the thinner fibrillar structures with negatively charged surface after MMP7 cleavage. Though not dominating, this aspect may inhibit the drug release to some extent. 3.4. In Vitro Cancer-Targeted Drug Delivery and Selective Cancer Killing To test whether the DOX-loaded peptide 2 fibrils can be used to realize cancer-targeted drug delivery, MTT assays were performed for both normal cells (non-cancerous origins) and cancer cells (Figure 4a). For the two normal cells, COS7 and 293E, the cell viability remained above 75% up to a nominal DOX concentration of 10 μM. However, for the three cancer cell lines of HeLa, HepG2, and A549, the cell viability decreased to less than 30% at 2.0 μM. The peptide 2/DOX composites thus showed high selectivity in killing cancer cells. By using the MMP7 ELISA Kit we determined the MMP7 concentration in the culture medium of each cell (Figure 4b). The two normal healthy cells secreted MMP7 at concentrations of 0.5 μg/mL, which was over fifty times higher than that of the normal cells. A control experiment showed that the cancer-secreted level of MMP7 can induce efficient cleavage of peptide 2. Therefore, it is clear that the cell killing capacity correlates well with the MMP7 secretion level of the cells.

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Figure 4. (a) The cell survival ratios of different cells treated with DOX-loaded peptide 2 fibrils at different concentrations (measured by the DOX concentration) and cultured for 48 h (37 ºC). (b) The MMP7 secretion level of different cells as determined by the MMP7 ELISA Kit. Microscopic images of COS7 cells (c, d) and HpeG2 cells (e, f, g) after co-culturing with the peptide 2/DOX composites at 37 ºC and the nominal DOX concentration of 4.0 μM for 28 h. Images c and e are bright field images. Images d and f are fluorescence images. g is a composite image that combines e and f. The white arrows point to the apoptotic bodies produced by the cells.

Microscopic images of two representative cells of COS7 (Figure 4c, d) and HpeG2 (Figure 4e, f, g) after treatment with the peptide 2/DOX composites were 22


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taken. For COS7, the bright field image shows that most of the cells grow well with long spindled morphologies. The fluorescent image is dark in the whole view, indicating a very low concentration of free DOX in the cells. While for HpeG2, most cells are rounded in shape and give a bright red color, indicating a high uptake amount of DOX by the cells. The DOX molecules are mainly found in the cytoplasm rather than in the nucleus, as verified from the color distribution. Moreover, some cells produce apoptotic bodies as indicated by the white arrows, which is the sign of cellular death. Figure S11 shows similar results of 293E cells to COS7 cells and HeLa/A549 cells to HpeG2 cells. The differences between normal cells and cancer cells are thought to be linked to the variations in the level of MMP7 expression. Centrifugation and refreshment of the supernatant was performed during cell culturing. In the case of normal cells, the secreted MMP7 concentration was very low and did not degrade the DOX-loaded peptide fibrils. The fibrils remained well dispersed in the supernatant and were removed from the system during refreshment of the complete culture medium. During this process very little DOX was released and therefore it did not significantly affect the cell growth. While in the case of cancer cells, the high level of secreted MMP7 degraded the peptide fibrils and resulted in their precipitation. Then, the DOX-loaded fibrils were left in the centrifugation residue during refreshment of the complete medium, forming drug reservoirs near cancer cells.5 This process resulted in accumulation of DOX in the cancer cells, caused them to be stained and inhibited the cell growth. 3.5. In Vivo Antitumor Efficiency Encouraged by the excellent selectivity and efficiency of the DOX-loaded peptide 2 fibrils in killing cancer cells, animal experiments were further performed to confirm the in vivo therapeutic effect. In experiments, the mice implanted with HpeG2 tumors 23



were treated with either the peptide 2/DOX composites or solely the peptide 2 fibrils or DOX for comparative study (Table S1). The change of tumor volume, body weight and survival ratio of the mice with time were recorded every day and the results are shown in Figure 5 and Figure S12. For the mice treated with solely peptide 2, the tumor volume increased significantly with time, which was about 12 times larger than that before treatment at the 22th day. The trend of tumor volume change was quite similar to that of the blank control. The body weight almost kept constant during the period. The results indicated that peptide 2 had quite low toxicity to mice but it also had no therapeutic effect for the tumor. In the case of treatment with DOX, the tumor growth can be significantly suppressed, as confirmed by the slowing down of the increasing speed of tumor volume. However, DOX showed quite high physiological toxicity to mice, clearly inferred from the day-by-day decrease of the body weight. The toxicity was so severe that it ultimately caused death of the mice. The dosing amount of 5 and 12 mg/kg resulted in zero survival after 16 and 10 days, respectively (Figure 5b). Then, in the case of treatment with the peptide 2/DOX composites, the tumor growth was also successfully suppressed. In comparison with the blank control, the tumor volume growth speed was slowed down about 5–6 times. More excitingly, even for the higher dosing amount of the fibril-loaded DOX (12 mg/kg), the mice had 100% survival ratio and the body weight showed no significant variation during the entire experimental period. The results indicate that the composite formulation can greatly increase the safe dosing amount of DOX to enhance the therapeutic efficiency, while significantly reducing its toxicity. The successful suppression of tumor growth was also confirmed by images of the tumor sites of the live mice as well as the tumor tissues after dissection of the mice (Figure S12). Comparing with the DOX-5 mg/kg group, the mice treated with peptide 24


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2/DOX composites at DOX dosing amount of 5 mg/kg gave smaller tumors even after longer breeding time. More impressively, the peptide 2/DOX composites at higher DOX dosing amount of 12 mg/kg gave more dramatic suppression of tumor growth, as verified from the smallest tumor size. Furthermore, the tumor pathology results (Figure 5d) showed that, compared with the blank group and the peptide fibril group, the mice in the 12 mg/kg DOX group and the peptide/DOX composite groups gave significantly decreased tumor cell areas, again demonstrating efficient tumor suppression.

Figure 5. Variation of the tumor volume (a), survival ratio (b), and body weight (c) of the mice with time in different groups with the administration of different formulations, blank group: 0.9% NaCl solution; peptide fibril group: peptide fibrils (36 mg/kg); DOX group I: 5 mg/kg DOX; DOX group II: 12 mg/kg DOX; peptide/DOX group I: peptide fibril-loaded DOX at 5 mg/kg; peptide/DOX group II: 25



peptide fibril-loaded DOX at 12 mg/kg. (d) Pathological analysis of tumor tissues in different groups at the corresponding terminated breeding course.

The effect of drug administration on vital organs of the lung, heart, spleen, kidney and liver was also assessed by pathological analysis (Figure S13). For heart samples, 5 mg/kg DOX group reported inflammation, 12 mg/kg DOX group showed high level of cardiac edema, the peptide/DOX (12 mg/kg) group indicated slight cardiac edema, and the other groups reported no obvious toxicity. Clearly the DOX toxicity to the heart was greatly reduced by using the peptide fibrils as drug carriers. For kidney samples, the two peptide/DOX groups showed fewer renal tubular epithelial cells with acidophilic change. However, the mechanism of action is not clear. For lung samples, the 5 mg/kg DOX group reported obvious enlargements of the alveolar septum and inflammatory infiltration. Reddish brown particles could be observed in some macrophages, which probably resulted from interaction with the drugs. The other groups showed normal lung tissues. For spleen samples, all groups reported normal tissues. For liver samples, cancer cells can be observed in nearly all groups, as indicated by the red arrows in Figure 6a of the magnified images. In fact, such tumor metastasis can also be visualized as white dots on the liver tissue, as shown by the inset image of Figure 6b. The significantly lower number of metastasis sites (Figure 6b) in the cases of the DOX groups and the peptide/DOX groups clearly demonstrated the efficiency of DOX in inhibiting tumor metastasis.

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Figure 6. (a) A pathological analysis of liver in different groups at the corresponding terminated breeding course. The red arrows denote the cancer cells. (b) The number of tumor metastasis sites on liver in different groups. The inset is a representative photograph of a liver with the red arrows denoting the metastasis sites.

The animal experiments clearly show that the peptide/DOX composites can greatly enhance the therapeutic efficiency while reducing the side effects. Such an excellent performance is closely related to the use of peptide 2 fibrils as a drug carrier, which can not only improve the biocompatibility of the formulation system, but also greatly enhance the therapeutic efficiency.

4. Conclusions Highly efficient tumor therapy is achieved by using the self-assembled nanostructures of an MMP7-sensitive peptide as nanocarriers for cancer-targeted drug delivery. The peptides are designed to form long fibrils with hydrophobic inner cores that can encapsulate a high amount of the antitumor drug of DOX. The nanostructures with a high aspect ratio were reported to have advantages as drug carriers, e.g. 27



prolonged blood circulation time, improved targeting efficiency, and controlled cellular uptake.11,45 The cancer-targeted drug delivery is obtained by introducing an MMP7-sensitive sequence, GPLGLA, into the peptide molecules. The DOX-loaded peptide fibrils undergo a structural transition from thicker fibrils to thinner ones at cancer sites due to molecular cleavage by the cancer-overexpressed MMP7. This leads to cancer-targeted DOX delivery and release for selective cancer-killing. Such a mechanism makes these peptide nanostructures excellent drug carriers to realize successful in vivo cancer therapy, as confirmed by the animal experiments. The peptide 2/DOX composites can suppress the tumor growth and metastasis significantly while reducing the toxicity of DOX by a large amount. Such excellent performance is because that encapsulation of DOX molecules in the peptide 2 fibrils reduces their chances of direct contact with normal tissues, whilst only at tumor sites the DOX molecules can be released for cancer killing. The study successfully develops a tumor-targeted drug carrier to achieve highly efficient cancer therapy. It demonstrates the feasibility of using enzyme-sensitive peptide self-assembling nanostructures for efficient drug loading and smart release. It also highlights the concept of considering the cancer specific microenvironments for developing effective therapeutic materials.

Notes The authors declare no competing financial interest.

Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. 28


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Supporting Information Peptide CACs determination, cytotoxicity, TEM and AFM morphologies, CD spectra, zeta potential measurements, DOX fluorescence spectra, polarity evaluation of peptide fibrils, determination of drug loading amount, optical image of peptide precipitates after MMP7 treatment, peptide-mediated DOX delivery into cells, animal experiments set up, optical images of mice tumor growth and pathological analysis of vital organs. This material is available free of charge via the Internet at http://pubs.acs.org.

Acknowledgements This work was supported by the National Natural Science Foundation of China (21872173, 21473255), the Fundamental Research Funds for the Central Universities (17CX02050) and the open research funds from the Key Laboratory of Applied Surface and Colloid Chemistry (Shaanxi Normal University). JRL thanks UK Physical Sciences and Engineering Research Council for support.

References (1) Hirsch, H. A.; Iliopoulos, D.; Tsichlis, P. N.; Struhl, K. Metformin Selectively Targets Cancer Stem Cells, and Acts Together with Chemotherapy to Block Tumor Growth and Prolong Remission. Cancer Res. 2009, 69, 7507-7511. (2) Love, R. R.; Leventhal, H.; Easterling, D. V.; Nerenz, D. R. Side Effects and Emotional Distress During Cancer Chemotherapy. Cancer 1989, 63, 604-612. 29



(3) Chen, H.; Gu, Z.; An, H.; Chen, C.; Chen, J.; Cui, R.; Chen, S.; Chen, W.; Chen, X.; Chen, X.; Chen, Z.; Ding, B.; Dong, Q.; Fan, Q.; Fu, T.; Hou, D.; Jiang, Q.; Ke, H.; Jiang, X.; Liu, G.; Li, S.; Li, T.; Liu, Z.; Nie, G.; Ovais, M.; Pang, D.; Qiu, N.; Shen, Y.; Tian, H.; Wang, C.; Wang, H.; Wang, Z.; Xu, H.; Xu, J.-F.; Yang, X.; Zhu, S.; Zheng, X.; Zhang, X.; Zhao, Y.; Tan, W.; Zhang, X.; Zhao, Y. Precise Nanomedicine for Intelligent Therapy of Cancer. Sci. China Chem. 2018, 61, 1503-1552. (4) Lee, S. H.; Gupta, M. K.; Bang, J. B.; Bae, H.; Sung, H.-J. Current Progress in Reactive Oxygen Species (ROS)-Responsive Materials for Biomedical Applications. Adv. Healthc. Mater. 2013, 2, 908-915. (5) Kalafatovic, D.; Nobis, M.; Son, J.; Anderson, K. I.; Ulijn, R. V. MMP-9 Triggered Self-Assembly of Doxorubicin Nanofiber Depots Halts Tumor Growth. Biomaterials 2016, 98, 192-202. (6) Kalafatovic, D.; Nobis, M.; Javid, N.; Frederix, P. W. J. M.; Anderson, K. I.; Saunders, B. R.; Ulijn, R. V. MMP-9 Triggered Micelle-to-Fibre Transitions for Slow Release of Doxorubicin. Biomater. Sci.-UK 2015, 3, 246-249. (7) Callmann, C. E.; Barback, C. V.; Thompson, M. P.; Hall, D. J.; Mattrey, R. F.; Gianneschi, N. C. Therapeutic Enzyme-Responsive Nanoparticles for Targeted Delivery and Accumulation in Tumors. Adv. Materi. 2015, 27, 4611-4615. (8) Mura, S.; Nicolas, J.; Couvreur, P. Stimuli-Responsive Nanocarriers for Drug Delivery. Nat. Mater. 2013, 12, 991-1003. (9) Li, J.; Kuang, Y.; Shi, J.; Zhou, J.; Medina, J. E.; Zhou, R.; Yuan, D.; Yang, C.; 30


Page 30 of 36

Page 31 of 36 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Wang, H.; Yang, Z.; Liu, J.; Dinulescu, D. M.; Xu, B. Enzyme-Instructed Intracellular Molecular Self-Assembly to Boost Activity of Cisplatin against Drug-Resistant Ovarian Cancer Cells. Angew. Chem. 2015, 127, 13505-13509. (10) Habibi, N.; Kamaly, N.; Memic, A.; Shafiee, H. Self-Assembled Peptide-Based Nanostructures: Smart Nanomaterials toward Targeted Drug Delivery. Nano Today 2016, 11, 41-60. (11) Zhang, P.; Cheetham, A. G.; Lin, Y.-a.; Cui, H. Self-Assembled Tat Nanofibers as Effective Drug Carrier and Transporter. ACS Nano 2013, 7, 5965-5977. (12) Langer, R.; Tirrell, D. A. Designing Materials for Biology and Medicine. Nature 2004, 428, 487-492. (13) Li, S.; Zou, Q.; Li, Y.; Yuan, C.; Xing, R.; Yan, X. Smart Peptide-Based Supramolecular Photodynamic Metallo-Nanodrugs Designed by Multicomponent Coordination Self-Assembly. J. Am. Chem. Soc. 2018, 140, 10794-10802. (14) Li, S.; Xing, R.; Chang, R.; Zou, Q.; Yan, X. Nanodrugs Based on Peptide-Modulated Self-Assembly: Design, Delivery and Tumor Therapy. Curr. Opin. Colloid Interface Sci. 2018, 35, 17-25. (15) Fei, J.; Zhang, H.; Wang, A.; Qin, C.; Xue, H.; Li, J. Biofluid-Triggered Burst Release from an Adaptive Covalently Assembled Dipeptide Nanocontainer for Emergency Treatment. Adv. Healthc. Mater. 2017, 6, 1601198. (16) Sun, B.; Wang, L.; Li, Q.; He, P.; Liu, H.; Wang, H.; Yang, Y.; Li, J. Bis(pyrene)-Doped Cationic Dipeptide Nanoparticles for Two-Photon-Activated Photodynamic Therapy. Biomacromolecules 2017, 18, 3506-3513. 31



(17) Silva, R. F.; Araújo, D. R.; Silva, E. R.; Ando, R. A.; Alves, W. A. Diphenylalanine Microtubes As a Potential Drug-Delivery System: Characterization, Release Kinetics, and Cytotoxicity. Langmuir 2013, 29, 10205-10212. (18) Liu, J.; Liu, J.; Chu, L.; Zhang, Y.; Xu, H.; Kong, D.; Yang, Z.; Yang, C.; Ding, D. Self-Assembling Peptide of d-Amino Acids Boosts Selectivity and Antitumor Efficacy of 10-Hydroxycamptothecin. ACS Appl. Mater. Interfaces 2014, 6, 5558-5565. (19) Lim, Y.-b.; Lee, E.; Lee, M. Cell-Penetrating-Peptide-Coated Nanoribbons for Intracellular Nanocarriers. Angew. Chem. 2007, 119, 3545-3548. (20) Abbas, M.; Zou, Q.; Li, S.; Yan, X. Self-Assembled Peptide- and Protein-Based Nanomaterials for Antitumor Photodynamic and Photothermal Therapy. Adv. Mater. 2017, 29, 1605021. (21) Zhang, X.-X.; Eden, H. S.; Chen, X. Peptides in Cancer Nanomedicine: Drug Carriers, Targeting Ligands and Protease Substrates. J. Control. Rel. 2012, 159, 2-13. (22) Yang, Z.; Xu, K. M.; Guo, Z. F.; Guo, Z. H.; Xu, B. Intracellular Enzymatic Formation of Nanofibers Results in Hydrogelation and Regulated Cell Death. Adv. Mater. 2007, 19, 3152-3156. (23) Yang, Z.; Liang, G.; Guo, Z.; Guo, Z.; Xu, B. Intracellular Hydrogelation of Small Molecules Inhibits Bacterial Growth. Angew. Chem. Int. Ed. 2007, 46, 8216-8219. (24) Zhou, J.; Du, X.; Yamagata, N.; Xu, B. Enzyme-Instructed Self-Assembly of Small d-Peptides as a Multiple-Step Process for Selectively Killing Cancer Cells. J. 32


Page 32 of 36

Page 33 of 36 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Am. Chem. Soc. 2016, 138, 3813-3823. (25) Feng, Z.; Wang, H.; Zhou, R.; Li, J.; Xu, B. Enzyme-Instructed Assembly and Disassembly Processes for Targeting Downregulation in Cancer Cells. J. Am. Chem. Soc. 2017, 139, 3950-3953. (26) Feng, Z.; Wang, H.; Chen, X.; Xu, B. Self-Assembling Ability Determines the Activity of Enzyme-Instructed Self-Assembly for Inhibiting Cancer Cells. J. Am. Chem. Soc. 2017, 139, 15377-15384. (27) Shi, Y.; Hu, Y.; Ochbaum, G.; Lin, R.; Bitton, R.; Cui, H.; Azevedo, H. S. Enzymatic Activation of Cell-Penetrating Peptides in Self-Assembled Nanostructures Triggers Fibre-to-Micelle Morphological Transition. Chem. Commun. 2017, 53, 7037-7040. (28) Zhang, H.; Fei, J.; Yan, X.; Wang, A.; Li, J. Enzyme-Responsive Release of Doxorubicin from Monodisperse Dipeptide-Based Nanocarriers for Highly Efficient Cancer Treatment In Vitro. Adv. Funct. Mater. 2015, 25, 1193-1204. (29) Zhang, N.; Zhao, F.; Zou, Q.; Li, Y.; Ma, G.; Yan, X. Multitriggered Tumor-Responsive Drug Delivery Vehicles Based on Protein and Polypeptide Coassembly for Enhanced Photodynamic Tumor Ablation. Small 2016, 12, 5936-5943. (30) Ii, M.; Yamamoto, H.; Adachi, Y.; Maruyama, Y.; Shinomura, Y. Role of matrix Metalloproteinase-7 (Matrilysin) in Human Cancer Invasion, Apoptosis, Growth, and Angiogenesis. Exp. Biol. Med. 2006, 231, 20-27. (31) Junttila, M. R.; de Sauvage, F. J. Influence of Tumour Micro-Environment 33



Heterogeneity on Therapeutic Response. Nature 2013, 501, 346-354. (32) Yang, Z.; Liang, G.; Xu, B. Enzymatic Hydrogelation of Small Molecules. Acc. Chem. Res. 2008, 41, 315-326. (33) Zheng, W.; Gao, J.; Song, L.; Chen, C.; Guan, D.; Wang, Z.; Li, Z.; Kong, D.; Yang, Z. Surface-Induced Hydrogelation Inhibits Platelet Aggregation. J. Am. Chem. Soc. 2012, 135, 266-271. (34) Tanaka, A.; Fukuoka, Y.; Morimoto, Y.; Honjo, T.; Koda, D.; Goto, M.; Maruyama, T. Cancer Cell Death Induced by the Intracellular Self-Assembly of an Enzyme-Responsive Supramolecular Gelator. J. Am. Chem. Soc. 2014, 137, 770-775. (35) Xu, H.; Wang, J.; Han, S.; Wang, J.; Yu, D.; Zhang, H.; Xia, D.; Zhao, X.; Waigh, T. A.; Lu, J. R. Hydrophobic-Region-Induced Transitions in Self-Assembled Peptide Nanostructures. Langmuir 2008, 25, 4115-4123. (36) Cox, H.; Georgiades, P.; Xu, H.; Waigh, T. A.; Lu, J. R. Self-Assembly of Mesoscopic Peptide Surfactant Fibrils Investigated by STORM Super-Resolution Fluorescence Microscopy. Biomacromolecules 2017, 18, 3481-3491. (37) Nahidiazar, L.; Agronskaia, A. V.; Broertjes, J.; van den Broek, B.; Jalink, K. Optimizing Imaging Conditions for Demanding Multi-Color Super Resolution Localization Microscopy. PLoS One 2016, 11, e0158884. (38) Ovesný, M.; Křížek, P.; Borkovec, J.; Švindrych, Z.; Hagen, G. M. ThunderSTORM: A Comprehensive ImageJ Plug-in for PALM and STORM Data Analysis and Super-Resolution Imaging. Bioinformatics 2014, 30, 2389-2390. (39) Juban, M. M.; Javadpour, M. M.; Barkley, M. D. Circular Dichroism Studies of 34


Page 34 of 36

Page 35 of 36 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Secondary Structure of Peptides. In Methods in Molecular Biology; Shafer, W. M., Ed.; Humana Press Inc: Totowa, NJ, Chapter Antibacterial Peptide Protocols. (40) Soukasene, S.; Toft, D. J.; Moyer, T. J.; Lu, H.; Lee, H.-K.; Standley, S. M.; Cryns, V. L.; Stupp, S. I. Antitumor Activity of Peptide Amphiphile Nanofiber-Encapsulated Camptothecin. ACS Nano 2011, 5, 9113-9121. (41) Marchesan, S.; Waddington, L.; Easton, C. D.; Kushkaki, F.; McLean, K. M.; Forsythe, J. S.; Hartley, P. G. Tripeptide Self-Assembled Hydrogels: Soft Nanomaterials for Biological Applications. BioNanoScience 2013, 3, 21-29. (42) Marchesan, S.; Qu, Y.; Waddington, L. J.; Easton, C. D.; Glattauer, V.; Lithgow, T. J.; McLean, K. M.; Forsythe, J. S.; Hartley, P. G. Self-Assembly of Ciprofloxacin and A Tripeptide into An Antimicrobial Nanostructured Hydrogel. Biomaterials 2013, 34, 3678-3687. (43) Shao, H.; Parquette, J. R. Controllable Peptide-Dendron Self-Assembly: Interconversion of Nanotubes and Fibrillar Nanostructures. Angew. Chem. Int. Ed. Engl. 2009, 48, 2525-2528. (44) Lin, R.; Cheetham, A. G.; Zhang, P.; Lin, Y.-a.; Cui, H. Supramolecular Filaments Containing a Fixed 41% Paclitaxel Loading. Chem. Commun. 2013, 49, 4968-4970. (45) Geng, Y. A. N.; Dalhaimer, P.; Cai, S.; Tsai, R.; Tewari, M.; Minko, T.; Discher, D. E. Shape Effects of Filaments Versus Spherical Particles in Flow and Drug Delivery. Nature Nanotechnol. 2007, 2, 249-255.

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Enzyme-Triggered Morphological Transition of Peptide

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