Engineering Anticancer Amphipathic Peptide-Dendronized

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Biological and Medical Applications of Materials and Interfaces

Engineering Anticancer Amphipathic Peptide-Dendronized Compounds for Highly-Efficient Plasma/Organelle Membrane Perturbation and Multidrug Resistance Reversal Xiao Zhang, Yachao Li, Cheng Hu, Yahui Wu, Dan Zhong, Xianghui Xu, and Zhongwei Gu ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b07917 • Publication Date (Web): 08 Aug 2018 Downloaded from http://pubs.acs.org on August 9, 2018

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ACS Applied Materials & Interfaces

Engineering Anticancer Amphipathic PeptideDendronized Compounds for Highly-Efficient Plasma/Organelle Membrane Perturbation and Multidrug Resistance Reversal Xiao Zhang,†,‡ Yachao Li,‡ Cheng Hu,‡ Yahui Wu,‡ Dan Zhong,‡ Xianghui Xu*, †,‡ and Zhongwei Gu*, †,‡ † College of Materials Science and Engineering, Nanjing Tech University, Nanjing, Jiangsu 210009, P.R. China ‡ National Engineering Research Center for Biomaterials, Sichuan University, Chengdu, Sichuan 610064, P.R. China KEYWORDS amphipathic dendronized compounds, antitumor nanoaggregates, membrane perturbation, membrane dysfunctions, multidrug resistance reversal

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ABSTRACT

Discovering new strategies for combating drug-resistant tumors becomes a worldwide challenge. Thereinto, stubborn drug-resistant tumor membrane is a leading obstacle on chemotherapy. Herein, we report a novel tumor-activatable amphipathic peptide-dendronized compound, which could form nanoaggregates in aqueous solutions, for perturbing tumor plasma/organelle membrane and reversing multidrug resistance. Distinguished from classical linear amphipathic peptide drugs for membrane disturbance, dendritic lysine-based architecture is designed as a multivalent scaffold to amplify the supramolecular interactions of cationic compound with drugresistant tumor membrane. Moreover, arginine-rich residues as terminal groups are hopeful to generate multiple hydrogen bonding and electrostatic interactions with tumor membrane. On the other hand, antitumor molecule (doxorubicin) is devised as a hydrophobic moiety to intensify the membrane-inserting ability owing to the prominent interactions with hydrophobic domains of drug-resistant tumor membrane. As expected, these amphipathic peptide-dendronized compounds within the nanoaggregates could severely disturb the both structures and functions of tumor plasma/organelle membrane system, thereby resulting in the rapid leakage of many critical biomolecules, highly-efficient apoptotic activation and anti-apoptotic inhibition. This strategy on tumor membrane perturbation demonstrates a bran-new antitumor activity with high contributions to cell cycle arrest (at the S phase), strong apoptosis-inducing ability and satisfying cytotoxicity to a variety of drug-resistant tumor cell lines.


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1. INTRODUCTION

Throughout the living nature, cellular membrane systems perform a pivotal role in the formation of well-organized biological compartmentalization, which is of great importance to homeostasis, signal transduction, transport and metabolism, finally affecting cell fates (such as cell cycle and apoptosis).1-3 For malignant tumor cells, membrane systems act as solid barriers against internalization of antitumor drugs, leading to poor chemotherapeutic index.4 Attractively, many researchers discover many membrane-perturbing compounds (such as peptides, lipids and nucleic acids) to disturb plasma membrane and hence exert cytotoxic effects.5-7 Likewise, disturbance on organelle membranes (e.g., mitochondrial membrane and endoplasmic reticulum membrane) is capable of provoking organelle dysfunctions and tumor cell death.8-12 However, drug-resistant tumor membrane systems have quite different lipid compositions and biophysical properties from the drug sensitive tumor membranes (such as much more sphingolipid and cholesterol, higher packing density, less fluidity and stronger rigidity), accompanying with very lower molecular permeability.13, 14 More evidences indicate that many intricate multidrug resistance mechanisms (including ultralow drug bioavailability, strong apoptotic inhibition and anti-apoptotic activation) are closely related to deviant tumor membrane systems.15-19 Yet until now, only a few membrane-perturbing therapeutic compounds have been reported for perturbing the drug-resistant tumor membrane and surmounting multidrug resistance.20 Consequently, rapid development of highly-efficient membrane-perturbing compounds is a challenging and promising research to hasten the settlement of multidrug resistance for cancer treatments. With inspiration from natural membrane lipids, mimicking amphipathic molecular structure is essential for developing robust membrane-perturbing compounds, since the similar structure will



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help bioactive compounds efficiently squeeze into membrane bilayer.21-23 Thereinto, hydrophilic segment of compounds should be in the charge of the firm attachment on electronegative surface of tumor membrane system via strong interactions.23-25 And cationic peptides (such as lysine-rich or arginine-rich peptides) are widely used as hydrophilic heads of membrane-perturbing compounds, owing to their multiple potential interactions with tumor membrane, wide spectrum and high efficacy.21-23, 26 Meanwhile, hydrophobic segment is designed for facilitating powerful insertion of the bioactive compound into membrane bilayer, hence peptides (usually containing leucine and phenylalanine) and lipids (such as phospholipid and fatty acid) are explored as common building blocks of hydrophobic segment.9, 27-30 In addition, endowing the therapeutic compounds with tumor selectivity is an important consideration to enhance drug efficacy and reduce side effects.25, 31-33 In order to defeat the condensed and rigid membranes of drug-resistant tumors, rational design of new amphipathic entity compound is urgently needed to create the ultraviolent membrane-perturbing potentials for inducing tumor cell death and overcoming multidrug resistance. In this work, we demonstrate a membrane-perturbing strategy based on de novo design of tumor-activatable amphipathic peptide-dendronized compounds (APDCs) for tumor plasma/organelle membrane perturbation and multidrug resistance reversal (Figure 1). Instead of classical linear molecular structures, dendronized lysine-based architecture is utilized as a multivalent scaffold to amplify the interactions between cationic segments and tumor membrane systems.34-38 Moreover, arginine residues as dendritic terminal groups are hopeful to form multiple hydrogen bonding and electrostatic interactions on membrane surface.39-41 On the other hand, we plan to explore therapeutic agents as hydrophobic segments to promote the membraneinserting ability, since some recent findings suggest that antitumor drugs such as doxorubicin


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(DOX) and paclitaxel possess the prominent supramolecular interactions with hydrophobic domains of tumor membrane systems, especially stubborn drug-resistant tumor membrane systems.13, 14, 42, 43 To realize tumor-specific perturbation, the peripheral abundant arginine residues of dendronized compounds (Compound 1) are sheltered with tumor extracellular pHdemountable groups (2,3-dimethylbut-2-enedioic acid, DEA) to obtain unactivated APDCs (Compound 6).44 A weakly acidic condition is able to remove the chemical inactivation and arouse the membrane-perturbing ability of amphipathic peptide-dendronized compounds. Of course, the unactivated APDCs would spontaneously form the nanoaggregates (NAs) via supramolecular hydrophobic effects in an aqueous solution.45 It is reasonable to hypothesize that once these unactivated APDCs within NAs recognize the pH condition nearby tumor cells (pH 7.0 ~ 6.5) and launch the membrane-perturbing capacity,46 i) both the structures and functions of tumor plasma/organelle membranes will be acutely destroyed, ii) cell homeostasis will be heavily disturbed, leading to abnormal distribution of certain critical biomolecules, and iii) multiple tumor apoptotic activations and anti-apoptotic inhibition will induce the drug-resistant tumor cell death. 2. MATERIALS AND METHODS 2.1. Synthesis of Amphipathic Peptide-Dendronized Compounds. All amino acids and condensing agents were obtained from GL Biochem LTD (Shanghai, China). The arginine-rich peptide dendrons were prepared using a divergent approach, and the detailed synthetic routes and procedures can be found in the Supporting Information. The core of peptide dendrons was decorated with the hydrazine hydrate (Aladdin Reagents Company, China) for dynamic conjugation of hydrophobic drug (DOX, Hisun Pharmaceutical, Zhejiang, China) and the Compound 1 was obtained. Finally, arginine residues on Compound 1 were masked with 2,3-



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dimethylbut-2-enedioic acid (DEA, Aladdin Reagents Company, Shanghai, China) to obtain tumor-activatable amphipathic peptide-dendronized compounds (Compound 6). The chemical characterizations of synthetic compounds were also illustrated in the Supporting Information. 2.2. Preparation of Biomimetic Bilayer Membranes (BMs). 1-palmitoyl-2oleoylphosphatidylcholine (POPC) and 1-palmitoyl-2-oleoylphosphatidylglycerol (POPG) (POPC: POPG = 3:7) were dissolved in CHCl3 solution in a glass bottle. The dry lipid film was obtained after entire remove of CHCl3 under vacuum overnight. After resuspension and hydration in phosphate buffered solutions (PBS) (ionic strength = 0.01 M) with two vortex cycles, the lipid film was subjected with five freeze (-20 °C)/thaw (65 °C) cycles and extruded through a polycarbonate membrane filter (pore size, 100 nm) for 19 times to form BMs. The size distribution and zeta potential of BMs were detected by dynamic light scattering (DLS). 2.3. Circular Dichroism (CD) Spectroscopy. To monitor the secondary structure transformation, the activated APDCs within NAs (20.0 μM) was incubated with BMs from molar ratio (BMs/APDCs) 0 to 30 for 1 h in PBS at 25 °C prior measurement. CD spectra were recorded from 190 to 300 nm at 25 °C using a Jasco J-1500 CD spectrophotometer, and secondary structure composition was analyzed with CDNN software. 2.4. Isothermal Titration Calorimetry (ITC) Experiments. ITC measurements were performed with a VP-ITC (MicroCal, Northampton, MA). The sample cell was loaded with PBS (ionic strength = 0.01 M) containing 150 μM Compound 1 within NAs-1, Compound 2 within NAs-2, Compound 3 within NAs-3, mixture of Compound 4 and Compound 5, unactivated APDCs within NAs and activated APDCs within NAs respectively and BMs (6.0 mM) was placed in injection syringe. After degassing for 10 min under vacuum, BMs (10 μL per time) were titrated into sample cell containing different formulation solutions with 4 min intervals for


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120 min at 25 °C under constant stirring at 260 rpm. Resulting data were analyzed with a one site model using origin software provided by MicroCal, and the molar enthalpy change for the binding (ΔH) and binding constant (K) were obtained. The dissociation constant (Kd) was calculated by the equation: Kd = 1/K. The free energy (ΔG) was calculated by the equation: ΔG = −RT ln K. The entropy (TΔS) was calculated by the equation: TΔS = (ΔH –ΔG). 2.5. Cell Lines. Drug-resistant human ovarian (SKOV3/R) cancer cells were obtained from Chongqing Medical University (Chongqing, China) and cultured in modified RMPI-1640 with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin at 37 °C in a humidified atmosphere containing 5% CO2. 2.6. Confocal Laser Scan Microscopy (CLSM) Imaging. To study the membrane-perturbing ability of APDCs, SKOV3/R cells were cultured at a density of 8000 cells/well to glassbottomed dishes for 24 h. After treatment with 2.0 μM activated APDCs from NAs for 0.5 h, cells were washed with PBS and separately stained with CellMask Deep Red (Ex: 630 nm; Em: 650 nm), Lyso-Tracker Blue (Ex: 373 nm, Em: 422 nm), MitoView 633 (Ex: 622 nm; Em: 648 nm) and ER-Tracker Red (Ex: 587 nm; Em: 615 nm) in the dark. Then the stained cells were washed with PBS for CLSM observation. 2.7. Lactate Dehydrogenase (LDH) Level Assay. SKOV3/R cells at a density of 1 × 104 cells/well were cultured in 96-well microplates for 24 h. DOX, unactivated APDCs from NAs, activated APDCs from NAs and DOX.HCl (2.0 μM) were added to replace the medium in sextuplicate. After treatment with certain time, cytoplasmic LDH level of cells was measured with LDH Assay Kit (Dojindo, Japan) according to the manufacture’s protocol. PBS and RIPA lysis buffer groups were employed as negative and positive control, respectively. The relative



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cytoplasmic LDH level was calculated by the equation: relative cytoplasmic LDH level = 100% (ODsample - ODnegative) / (ODpositive - ODnagative) × 100%. 2.8. Cytoplasmic Calcium (Ca2+) Level Assay. SKOV3/R cells were cultured at a density of 1 × 104 cell/well in 96-well plates for 24 h. After treatment with 2.0 μM DOX, unactivated APDCs within NAs, activated APDCs within NAs and DOX.HCl for 24 h in triplicate, cells were stained with 5 μM Ca2+-sensitive dye Fluo 4-AM (Dojindo, Japan) for 1 h at 37 °C. Then Fluo-4 AM containing medium was removed and PBS containing 1% FBS was added to cells for another 1 h incubation at 37 °C. Cytoplasmic Ca2+ level was detected by by a microplate reader (Thermo Fisher Scientific, USA) using a 494 nm excitation and 516 nm emission. 2.9. Cathepsin B Intracellular Distribution. To evaluate the leakage of cathepsin B from lysosome to cytoplasm, SKOV3/R cells were cultured at a density of 8000 cell/well to glassbottomed dishes for 24 h. Then the cells were incubated with 2.0 μM DOX, unactivated APDCs, activated APDCs and DOX.HCl for 24 h. Magic Red Cathepsin B Detection Kit (Immunochemistry Tech, USA) and Hoechst 33342 (Dojindo, Japan) were used to separately stain the cathepsin B and nucleus for CLSM observation. 2.10. Intracellular Cytochrome C (Cyt C) Distribution. SKOV3/R were cultured in 6-well plates at a density of 5 × 105 cells/well and treated with 2.0 μM DOX, unactivated APDCs within NAs, activated APDCs within NAs and DOX.HCl for 24 h in triplicate. Mitochondrial and cytosol fractionation from SKOV3/R cells were performed using Cell Mitochondria Isolation Kit (Beyotime, Shanghai, China). After addition of 200 μL RIPA lysis buffer at 4 °C, mitochondrion and cytoplasm lysates were gathered. Intracellular Cyt C distribution was quantitated using a Human Cytochrome c Elisa Kit (R&D Systems, USA) following the manufacturer’s instructions.


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2.11. Mitochondrial Membrane Potential (ΔΨm) Assay. SKOV3/R cells were cultured at a density of 8000 cells/well in glass-bottomed dishes for 24 h. After treatment with 2.0 μM DOX, unactivated APDCs, activated APDCs and DOX.HCl for 24 h, cells were stained with MitoView 633 for CLSM observation. On the other hand, cells were cultured in 6-well plate with abovementioned agents for fluorescence-activated cell sorter (FACS) quantification. The MitoView 633 fluorescence intensity represents the ΔΨm level. 2.12. Intracellular Reactive Oxygen Species (ROS) Assay. Fluorometric Intracellular ROS Kit (Green, Sigma-Aldrich, USA) was used to detect the intracellular ROS level. In brief, SKOV3/R cells were seeded at a density of 1× 104 cells per well to 96-well plates for 24 h. Then cells were incubated with 2.0 μM DOX, unactivated APDCs from NAs, activated APDCs from NAs and DOX.HCl for certain time. After washing with PBS and addition of ROS detection reagent for 1 h incubation at 37 °C, cells were lysed through RIPA buffer and detected by a microplate reader using a 490 nm excitation and 525 nm emission for quantitative analysis. 2.13. Western Blot Assay. SKOV3/R cells were seeded into 6-well plates (5 × 105 cells per well) and treated with 2.0 μM DOX, unactivated APDCs, activated APDCs and DOX.HCl for 48 h. Cells were washed with ice-cold PBS 3 times and total cell lysates were gathered after addition of 100 μL RIPA lysis buffer at 4 °C. Protein extracts were isolated from cell lysates after centrifugation and quantified using BCA Protein Assay Kit (Thermo Fisher Scientific, USA). An equal amount of protein (30 μg) from each sample was separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride (PVDF) membrane. Membranes were blocked at room temperature for 1 h in blocking buffer containing 5% non-fat dry milk to prevent nonspecific binding and then separately incubated with anti-Bcl-2, anti-caspase 3, anti-p53 and anti-β actin primary antibodies



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(Abcam, UK) at 4 °C overnight. After washing with Tris-buffered saline containing 0.4% Tween-20 (TBST) for 30 min and incubation with secondary antibodies (goat anti-rabbit IgG HPR, Abcam, UK) for 2 h, the membranes were visualized with ECL detection kit (Thermo Fisher Scientific, USA) by a UV illuminator (Bio-Rad ChemiDoc XRS+). 2.14. Quantitative Polymerase Chain Reaction (qPCR) Analysis. SKOV3/R cells were seeded into 6-well plates (5 × 105 cells per well) and treated with 2.0 μM DOX, unactivated APDCs, activated APDCs and DOX.HCl for 48 h. Total RNA was extracted from cells using Trizol reagent and cDNA was synthesized by reverse transcription from mRNA using the iScript cDNA Preparation Kit (Bio-Rad Laboratories, Hercules, CA). Amplification reactions were carried out in 96-well plates in CFX96 real time PCR detection system (Bio-Rad Laboratories, CA). Each 20 µL reaction contained 1 µL of DNA template, 0.5 µL of primer-F (10 µM), 0.5 µL of primer-R (10 µM), 8 µL of distilled deionized water and 10 µL of supermix. qPCR was performed using SsoFast EvaGreen Supermix (Bio-Rad Laboratories, CA). 2.15. In Vitro Antitumor Assay. SKOV3/R, MCF-7/R and LoVo/R cell lines were seeded at a density of 8000 cells/well to 96-well plates respectively. Then, culture media were replaced by fresh medium containing various compounds at different concentrations for 48 h incubation. The medium was removed and washed with PBS for three times. The SKOV3/R cells were incubated with 100 μL of FBS-free medium containing 10% cell counting kit-8 (CCK-8, Dojindo, Japan) for another 2 h at 37 °C. The absorbance was measured at a wavelength of 450 nm using a microplate reader. The relative cell viability was calculated by the equation: cell viability = (ODsample - ODbackground) / (ODcontrol - ODbackground) × 100%. 2.16. Cell Cycle Distribution. SKOV3/R cells were cultured in 6-well plates (1 × 106 cells per well) for 24 h. After treatment with 2.0 μM DOX, unactivated APDCs, activated APDCs within


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NAs and DOX.HCl for 24 h, cells were collected and fixed with 70% precooled ethanol at 4 °C overnight. Ethanol-fixed cells were washed with PBS and incubated with DAPI for 30 min in the dark. The cell cycle distribution was measured by flow cytometry. 2.17. Apoptosis Assay. SKOV3/R cells were seeded into 6-well plates (5 × 105 cells per well) and treated with 2.0 μM DOX, unactivated APDCs within NAs, activated APDCs and DOX.HCl for 48 h, respectively. After treatment with trypsin/EDTA, SKOV3/R cells were washed with PBS and collected by refrigerated centrifugation. After addition of the Annexin V-APC/7-AAD Apoptosis Detection Kit (KeyGEN, Nanjing, China), the cells were incubated for another 20 min at room temperature. Stained cells were collected and immediately analyzed by FACS. 3. RESULTS AND DISCUSSION 3.1. Supramolecular Interactions between APDCs and BMs. First, we synthesized the target compound of Compound 1 with the precise structure (molecular weight 1567.4, Figure S4) using divergent approach, and Compound 1 could be controllably decorated with DEA into Compound 6 according to some our previous reports.47-52 To disclose the advantages of our design, we also accurately construct a lysine-rich peptide-dendronized compound without arginine (Compound 2), an arginine-rich peptide-dendronized compound based on a hydrophobic oleylamine (Compound 3), mixture of the arginine-rich cationic dendron (Compound 4) and lipophilic fluorescent DOX (Compound 5) as control compounds. Their synthetic procedures and characterizations can be found in the Supporting Information (Scheme S1 to S6, Figure S1 to S5). Weakly acid conditions corresponding to tumor pH microenvironment (pH 6.5) was capable of reversing unactivated APDCs (Compound 6) into activated APDCs for membrane perturbation, through complete removal of masked groups (DEA) from Compound 6 (Figure S6). Self-assembling these amphipathic peptide-dendronized compounds into nanoaggregates is a



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spontaneous process, and atomic force microscope (AFM) result suggested the Compound 6 of unactivated APDCs could assemble into spherical NAs in an aqueous solution (Figure S7). We also found that low critical aggregation concentrations (CAC) of Compound 6 at pH 7.4 and pH 6.5 aqueous solutions were 0.58 μg/mL and 0.87 μg/mL, respectively (Figure S8). Moreover, NAs had an average size of ~ 150 nm both at the pH 7.4 and pH 6.5 solutions, while the zeta potentials of NAs would reverse from -30.3 mV (pH 7.4) into +18.8 mV (pH 6.5) due to pHtriggered cleavage of DEA modification (Table S1). The results indicated unactivated APDCs and activated APDCs maintained amphipathic molecular structures for supramolecular aggregation both at the normal physiological condition and tumor microenvironment. In addition, the amphipathic Compound 1, Compound 2 and Compound 3 could self-assemble into the nanoaggrates-1 (NAs-1, ~160 nm, +18.3 mV), nanoaggrates-2 (NAs-2, ~155 nm, +17.8 mV) and nanoaggrates-3 (NAs-3, ~150 nm, +18.1 mV) as well (Table S1). To disclose the membrane-perturbing capacity of APDCs, we built an anionic unilamellar vesicle to mimic natural membrane system including similar phospholipid composition, electronegative surface and bilayer structures (zeta potential of about -30 mV and an average size of about 100 nm, Figure S10).53 CD spectroscopy was employed to investigate the interactions between the activated APDCs and BMs.6, 54 We first verified that the unactivated condition (pH 7.4) and activated condition (pH 6.5) showed no obvious impact on the secondary structure of amphipathic dendronized NAs (Figure S11), which well agreed with the unchanged CAC and size of NAs in the different pH conditions. However, once the activated NAs composing of APDCs met the BMs, the secondary structures of NAs including 9.6% α-helix, 62.4% β-sheet and 18.0% β-turn dramatically transformed (Figure 2A). With the addition of BMs from BMs/APDCs molar ratio (MR) 1 to 30, the ordered folding structures (α-helix, β-sheet and β-


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turn) decreased but random coil content increased from 10.0% to 21.2%. These results suggested that the APDCs within activated NAs would spontaneously interact with BMs, and remarkable interactions between APDCs and BMs influenced the original secondary structure of nanoaggregates. Next, ITC analysis was used to evaluate supramolecular interactions between synthetic compounds and BMs,35, 55 according to standard titration of amphipathic dendronized nanoaggregates or mixture with a total concentration of 150 μM into the BM solution (6.0 mM) at 25 °C. As shown in Figure 2B, Compound 1 within NAs-1 revealed a high binding affinity to the BMs with a Kd value of 1.4 μM at a phosphate buffered solution (PBS, pH 6.5). Strong interactions between Compound 1 and BMs should be actuated by beneficial contributions of enthalpy (ΔH = -3.5 kcal mol-1, Table 1) and entropy (TΔS = 4.5 kcal mol-1). The calculated free energy of binding (ΔG = -8.0 kcal mol-1) reflected favourable membrane disturbance of Compound 1 to BMs. However, Compound 2 from NAs-2 having a lysine-rich peptide dendron only was detected a very high Kd value of 1063.8 μM in ITC analysis (Figure 2C), suggesting that arginine-rich peptide dendron played a crucial role in multiple membrane binding (hydrogen bonding and electrostatic interactions). When the hydrophobic DOX moiety was replaced by oleylamine, the binding affinity of Compound 3 within NAs-3 (Figure 2D) to the BMs significantly decreased (Kd value was 66.7 μM), confirming antitumor drug indeed could strengthen the interactions of APDCs with BMs. And an equivalent amount of mixture of arginine-rich peptide dendron and DOX also had extremely poor interactions with BMs (Kd value was 1388.9 μM, Figure 2E), due to lack of amphipathic structures. However, no obvious binding energy was detected between APDCs (Compound 6) within unactivated NAs (Figure 2F) at the physiological pH 7.4 condition, indicating that chemical inactivation of APDCs



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successfully hided the membrane-perturbing effects to avoid interference on the normal cells. The activated APDCs within NAs could recover robust interactions with BMs (Figure 2G), having a similar Kd value (2.7 μM) as previous Compound 1 of N As-1. The results suggested that i) our design on amphipathic structure, arginine-rich cationic dendron and lipophilic drug moiety synergistically played an important role for generating strong membrane-perturbing ability, and ii) chemical inactivation is able to avoid the detrimental effects to normal cells, while tumor specific activation would completely recover the strong membrane-perturbing ability of APDCs. Taken together, we could presume that APDCs from NAs were able to disturb structures and functions of natural membrane system, as well as bring about antitumor activity for drug resistance reversal. 3.2. APDC-Induced Disturbance on Tumor Plasma/Organelle Membranes. Afterwards, we turned to explore membrane-perturbing ability of APDCs within NAs on the natural membrane systems of SKOV3/R cells. It is well known that plasma membrane (PM) is the primary obstacle of drug-resistant tumor cells for lowering drug intake and increasing drug efflux. We used a commercial amphipathic dye (CellMask) to stain tumor PM, observing by CLSM. The CLSM images (Figure 3A) showed that after culture with activated NAs containing 2.0 μM APDCs at pH 6.5 for 0.5 h, abundant APDCs (red fluorescence) interacted with the SKOV3/R tumor cells, and some of APDCs evidently associated with tumor PM (green). The observations agreed with the line scanning profile of fluorescent colocalization, suggesting strong intercalation of APDCs from NAs into bilayer membrane as the commercial amphipathic dyes. However, the fluorescently-labeled (FITC) arginine-rich dendrons were hindered by the drug-resistant PM with low cell internalization and poor interactions towards subcellular membrane (Figure S14). And incubation of the SKOV3/R tumor cells with DOX or DOX.HCl alone showed weak


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fluorescence signal overlapping with the tumor PM (Figure S15 and Figure S16). Additionally, at the normal physiological pH 7.4 condition, unactivated NAs composing of APDCs were rarely internalized into the tumor cells and colocalized with the drug-resistant PM (Figure S17). More importantly, the APDC-induced membrane perturbation caused an evident leakage of LDH (Figure 3A), which is an over-expressed metabolic enzyme in the tumor cytoplasm and has a close relationship with drug resistance.56 Within 0.5 h, a drastic loss of LDH (about 42%) from the tumor cytoplasm clearly stated the APDC-induced PM dysfunction. This damage suggested the great potentials of activated APDCs from NAs for multidrug resistance reversal, because the decrease of LDH in tumor cytoplasm would affect mitochondrial membrane potential, arouse ROS generation and accelerate the cell death.57 Thus, it can be seen that the activated APDCs from NAs indeed squeezed into PM and perturbed their structures and functions. Once the activated APDCs from NAs entered into the drug-resistant tumor cells, the structures and functions of organelle membranes also could be disturbed by supramolecular interactions. Endoplasmic reticulum (ER) having the maximum membrane structures in cells was labeled with a commercial ER-Tracker. The CLSM analysis indicated that the activated APDCs highly colocalized with ER membrane and showed a high Pearson's coefficient value of 0.95 with the ER membrane (Figure 3B and Figure S18). Moreover, the vital ER function of Ca2+ storage was damaged by the APDC-induced disturbance, hence the cytoplasmic Ca2+ level of APDC-treated cells rose to 2.4-fold as compared with the untreated cells (*p < 0.001). The Ca2+ efflux indicated the ER membrane perturbation and homeostasis alteration, thus helping to induce mitochondriadependent apoptosis and increase ROS production for multidrug resistance reversal.58 Likewise, the activated APDCs with red fluorescence were also observably located with the LysoTrackerlabeled acidic compartments, indicating the activated APDCs from NAs could perturb the



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subcellular lysosome membranes (Figure 3C). The release of lysosome-located cathepsins into the cytosol is an important indicator of lysosomal rupture and dysfunction, which was considered as an attractive strategy to intensively induce drug-resistant cancer cell death. As shown in the Figure 3C, cathepsins which was marked by Magic Red (RR)2 with red fluorescence, widely distributed in the cytoplasm after treatment with activated APDCs from NAs. The leakage of over-expressed cathepsins in the drug-resistant tumor cells would bring about the mitochondrial injury and cell apoptosis.59 Nevertheless, single incubation with DOX, DOX.HCl and the unactivated APDCs failed to alter the cathepsin distribution in SKOV3/R tumor cells. The intense interactions between activated APDCs from NAs and the acidic compartments would trigger the cleavage of hydrazone bond and the degradation of dendritic peptides to release the native drug (DOX) and exert the anticancer activity. Of course, the activated APDCs from NAs could powerfully attack the high-electronegative tumor mitochondria (Mito), which are central to tumor apoptosis.60, 61 CLSM images and the line scanning profile displayed that the activated APDCs observably interacted with mitochondria (stained by MitoView with green fluorescence, Figure 3D), bringing about mitochondrial membrane permeabilization and a 68% loss of Cyt C (Figure 3D). The devastation on mitochondria was urgently pursued for overcoming the multidrug resistance, since mitochondria control both pro-apoptotic signaling pathways and antiapoptosis mechanisms. In general, DOX, DOX.HCl and the unactivated APDCs from NAs hardly influenced on the structures and functions of the tumor plasma/organelle membrane system. Accumulating evidences suggested that PM-, ER-, lysosome-, and Mito-disturbed responses would streamline into a common death pathway.8, 11 As a result, APDCs would concurrently provoke multiple


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apoptotic stimulations against drug-resistant tumor cells, which is expected to arouse ultra-strong apoptotic induction and reverse multidrug resistance. 3.3. APDC-Related Biological Mechanisms for Multidrug Resistance Reversal. We turn to investigate whether such heavy disturbance on the tumor plasma/organelle membrane system would promote apoptosis and inhibit anti-apoptosis in drug-resistant tumor cell line. Flow cytometry results showed that treatment with the activated APDCs (2.0 μM, 24 h) led to a sharp decrement of ΔΨm, while other formulations (such as DOX.HCl and unactivated APDCs) had no obvious influence on the tumor ΔΨm (Figure 4A). 65% loss of ΔΨm should be attributed to intense mitochondrial membrane permeabilization, which was induced by damage of tumor membrane system and activation of apoptotic signaling (such as leakage of LDH, Ca2+, cathepsin). 8, 11 8, 11 8, 11 As envisioned, the above-mentioned homeostasis disbalance of mitochondria gave rise to a great deal of ROS generation (Figure 4B), which would denature some proteins, lipids and nucleic acids. After incubation with APDCs from activated NAs for 24 h, ROS content in SKOV3/R tumor cells was about three times of the untreated tumor cells. Undoubtedly, ΔΨm decrease and ROS increase aggravated mitochondrial-dependent apoptosis. For another, we quantified the intracellular distribution of activated APDCs based on DOX fluorescence, after treatment with the APDCs at pH 6.5 medium for 12 h (Figure S25 and Figure S26). Nearly all of the activated APDCs from NAs were internalized into the SKOV3/R tumor cells, whereas other formulations (even the positive control of DOX.HCl) demonstrated an extremely low drug bioavailability due to solid biological barrier of drug-resistant tumor membrane systems. CLSM images indicated that some of DOX was disassociated from the Cyanine5.5 (Cy5.5)-labelled dendrons and passed into nuclei to exert antitumor activity on nuclear apoptosis (Figure S27).



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At molecular biology level, we monitored the intracellular content of critical apoptosis-related proteins using western blot assay. As showed in the Figure 4C, only activated APDCs were capable of down-regulating anti-apoptotic Bcl-2 protein level in the drug-resistant cells. The proapoptotic caspase-3 protein was usually scarce in drug-resistant tumor cells, but the activated APDCs also could markedly up-regulate the caspase-3 protein level. 19 19 19 Conspicuously, wildtype p53 protein level was also elevated after incubation with the activated APDCs, which could impede DNA repair, cause cycle arrest and accelerate apoptosis in the tumor cells. In addition, the pro-apoptotic caspase-3 protein activity was also largely promoted by our activated APDCs from NAs (Figure S28). In the meantime, the mRNA levels of apoptosis-related proteins were quantified using qPCR analysis. The caspase-3 and p53 mRNA levels were raised up 67.5% and 187.2% after treatment with the activated APDCs, respectively (Figure 4D). And the activated APDCs from NAs also led to 64.5% down-regulation of Bcl-2 mRNA level in the drug-resistant tumor cells. In contrast, in vitro treatment with DOX, DOX.HCl and the unactivated APDCs failed to arouse the apoptosis-regulated protein/mRNA (e.g., caspase-3 and p53) and lower antiapoptosis protein/mRNA level. These results suggested that APDC-induced damage and dysfunction of tumor membrane system converge on the central death induction in drug-resistant tumor cell, including apparent apoptotic activation and anti-apoptotic inhibition. Therefore, according to previous overwhelming researches, our tailor-made amphipathic dendronized compounds must be able to activate the multiple apoptosis pathway for killing the drug-resistant tumor cells. 3.4. Antitumor Activity of APDCs against Drug-Resistant Tumor Cell Lines. Finally, we carried out a series of quantitative studies to evaluate the antitumor pharmacological activity of activated APDCs against the drug-resistant tumor cells. A CCK-8 array was adopted to determine


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the cytotoxicity of each compound towards the SKOV3/R tumor cells. The IC50 value (the concentration causing 50% growth inhibition) of the activated NAs composing of APDCs against SKOV3/R tumor cells was 3.3 μM (Figure 5A), which was similar to the IC50 value of Compound 1 from NAs-1 (about 3.3 μΜ, Figure S31) but much more efficient than the positive control of DOX.HCl. However, the unactivated APDCs showed no obvious cytotoxicity to SKOV3/R tumor cells even at relatively high concentration. Expectedly, the unactivated APDCs within NAs hid their membrane-perturbing potentials, while tumor microenvironment acidity activation fully awakened membrane-perturbing ability of APDCs within NAs to SKOV3/R tumor cells for combating multidrug resistance. Additionally, the Compound 2 within NAs-2 with lysine-rich dendron displayed poor antitumor effects (IC50 = 10.5 μM, Figure S32), and the Compound 3 from NAs-3 with oleylamine segment revealed very poor cytotoxicity towards SKOV3/R tumor cells (Figure S33). And the mixture of Compound 4 and Compound 5 also showed weak antitumor ability to SKOV3/R tumor cells (Figure S35). As expected, the antitumor activity of these compounds or mixture highly depended on their membrane-perturbing ability, verifying the importance of amphipathic peptide-dendronized molecular design with arginine-rich dendron and doxorubicin for defeating drug-resistant cells. Compared with other compounds, the high antitumor activity of activated APDCs against the SKOV3/R tumor cells confirmed the multiple apoptotic pathways were successfully provoked in multidrug resistance. Furthermore, the activated APDCs also provided distinct antitumor activity to human drug-resistant colorectal carcinoma (LoVo/R) cell line and human drug-resistant breast cancer (MCF-7/R) cell line (Figure 5A), implying APDCs had versatile antitumor activity for drug-resistant reversal. Likewise, the antitumor activity of other compounds or mixture was quite limited against LoVo/R and MCF-7/R tumor cell lines.



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Cell cycle distribution analysis and apoptotic assay were used to further disclose the fate of the SKOV3/R tumor cells after incubation with the activated APDCs. In line with the many previous reports, DOX.HCl arrested the cell cycle of the SKOV3/R cells at the G2 phase to exert the antitumor activity (Figure 5B).50 Nevertheless, the activated APDCs within NAs mainly restrained the cell cycle of SKOV3/R cells at the S phase (49.89%), showing the unique pharmacological activity of membrane-perturbing APDCs from NAs. Due to serious drug tolerance, DOX, DOX.HCl and unactivated APDCs failed to induce the late apoptosis of drug-resistant tumor cells (Figure 5C). Attractively, more than 90% SKOV3/R cells were guided to the late apoptosis after incubation with 2.0 μM APDCs, confirming the high-efficiency antitumor activity to drug-resistant tumor cells. It was concluded that APDCs from NAs would be an excellent candidate compound for coping with multidrug resistance. 4. CONCLUSIONS In conclusion, we have successfully demonstrated molecular engineering of amphipathic peptide-dendronized compound for perturbing tumor plasma/organelle membrane systems and surmounting multidrug resistance. The precise amphipathic peptide-dendronized compounds have dendritic arginine-rich segments for generating multivalent interactions with tumor membrane and lyophobic doxorubicin for intensifying membrane-inserting ability. Chemical inactivation is capable of concealing membrane-perturbing potentials of APDCs at the normal physiological condition, while the tumor-specific activation on the unactivated APDCs could completely arouse membrane-perturbing ability to attack the drug-resistant tumor cells. These APDCs could assemble into NAs through effects in aqueous solutions. Once the NAs composing of APDCs met membrane systems, the activated APDCs would strongly interact with tumor plasma/organelle membrane and cause severe dysfunctions of membrane system (Figure 6),


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including plasma membrane breakage with LDH leakage, endoplasmic reticulum membrane damage with Ca2+ loss, lysosome membrane rupture with cathepsin release, and mitochondria membrane destruction with Cyt C leakage. The strong disturbances on plasma/organelle membrane would converge together to arouse multiple apoptotic pathways, inhibit anti-apoptotic pathways and arrest cell cycle at the S phase for multidrug resistance reversal, accompanying with a sharp decrease of mitochondrial membrane potentials, a high increase of ROS generation, dramatic up-regulation of caspase-3/p53 protein/mRNA levels, and down-regulation of Bcl-2 protein/mRNA level. Owing to their highly-efficient perturbation on drug-resistant tumor membrane systems, the NAs composing of APDCs displayed outstanding antitumor activity towards some drug-resistant tumor cell lines. We believe that this work will initiate a new concept to design membrane-perturbing materials for resolving the difficulty on chemotherapy resistance. ASSOCIATED CONTENT Supporting Information. Materials, experimental details, additional data, Schemes S1-S6, Figures S1-S35 and Table S1. This material is available free of charge via the Internet at http://pubs.acs.org. AUTHOR INFORMATION Corresponding Author *E-mail: [email protected] (X. Xu), *E-mail: [email protected] (Z. Gu). ACKNOWLEDGMENT



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This work was supported by the National Natural Science Foundation of China (NSFC, 21674067, 81361140343, 51503128, and 31771067), the Scientific Research Foundation for Talent Introduction of Nanjing Tech University (39803130, 39803134), the National Key Research and Development Program of China (2017YFC1103501), Miaozi Project in Science and Technology Innovation Program of Sichuan Province (2018067), and Scientific Research Foundation for Outstanding Young Scholars in Sichuan University (2016SCU04A19). REFERENCES 1.

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Figure 1. Schematic illustration of anticancer amphipathic peptide-dendronized compounds with membrane-perturbing ability. (A) Chemical structure of Compound 1 and other control compounds (from Compound 2 to Compound 5), (B) chemical inactivation and tumor-acidity activation of amphipathic peptide-dendronized compounds, and supramolecular formation of NAs, (C) tumor-activated membrane-perturbing processes of APDCs from NAs (i. tumor-acidity



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activation, ii. strong attachment and interaction, iii. insertion and membrane disturbance), membrane dysfunctions and molecular leakage.


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Figure 2. Supramolecular aggregations of APDCs into NAs. (A) CD spectra and the secondary structure compositions of activated APDCs within NAs (20 μM) in the absence and presence of BMs at various molar ratios (BMs/APDCs) in PBS (pH 6.5, ionic strength = 0.01 M) at 25 °C. The ITC profiles for the titration of BMs (6.0 mM) with (B) Compound 1 within NAs-1, (C) Compound 2 within NAs-2, (D) Compound 3 within NAs-3, (E) mixture of Compound 4 and Compound 5, (F) unactivated APDCs within NAs and (G) activated APDCs within NAs (150 μM) in PBS (ionic strength = 0.01 M) at 25 °C over 120 min.



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Table 1. Kinetic and Thermodynamic Parameters for Various Compounds Binding to BMs at 25 °C. Groups

Kda

∆Hb

T∆Sc

∆Gd

Compound 1

1.4

-3.5

4.5

-8.0

Compound 2

1063.8

-1.3

2.8

-4.1

Compound 3

66.7

2.1

8.7

-6.6

Compound 4 and 5

1388.9

-3.3

0.6

-3.9

Unactivated APDCs

n.d.e

n.d.e

n.d.e

n.d.e

Activated APDCs

2.7

-3.5

4.1

-7.6

a

Kd, dissociation constant, μM; b∆H, change in enthalpy, kcal mol-1; cT∆S, change in entropy,

kcal mol-1; d∆G, change in Gibbs free energy, kcal mol-1; en.d., not determined.


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Figure 3. APDC-induced disturbance on the structures and functions of tumor plasma/organelle membranes. (A1) CLSM images for PM (green, CellMask)/APDCs (red) colocalization after incubation with 2.0 μM activated APDCs for 0.5 h, the scale bar represents 25 μm; (A2) Cytoplasmic LDH levels of SKOV3/R cells after treatment with DOX, unactivated APDCs, activated APDCs and DOX.HCl (2.0 μM) with the different incubating time (n = 3). (B1) CLSM images for ER (green, ER-Tracker Red)/APDCs (red) colocalization after incubation with 2.0 μM activated APDCs for 0.5 h; (B2) Cytoplasmic Ca2+ levels of SKOV3/R cells after treatment with DOX, unactivated APDCs, activated APDCs and DOX.HCl (2.0 μM) for 24 h as measured by Fluo 4-AM assay (n = 3, *p < 0.001). (C1) CLSM images for lysosomes (green,



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LysoTracker)/APDCs (red) colocalization after incubation with 2.0 μM activated APDCs for 0.5 h; (C2) Intracellular cathepsin B distribution of SKOV3/R cells after treatment with DOX, unactivated APDCs, activated APDCs and DOX.HCl (2.0 μM) for 24 h identified by Magic Red assay. (D1) CLSM images for Mito (green, MitoView 633)/APDCs (red) colocalization after incubation with 2.0 μM activated APDCs for 0.5 h; (D2) Intracellular Cyt C distribution in Mito and cytosol after treatment with DOX, unactivated APDCs, activated APDCs and DOX.HCl (2.0 μM) for 24 h as detected by Cyt C Elisa Kit.


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Figure 4. APDC-induced disturbance on tumor membrane system for multidrug resistance reversal. (A) Flow analysis (left) and CLSM images (right) for determining mitochondrial membrane potentials of SKOV3/R cells after incubation with DOX, unactivated APDCs, activated APDCs and DOX.HCl for 24 h at a same concentration of 2.0 μM using MitoView 633 staining. The scale bar represents 100 μm. (B) Relative ROS level of SKOV3/R cells after incubation with DOX, unactivated APDCs, activated APDCs and DOX.HCl at the same molar concentration of 2.0 μM with different incubating time (n = 3). (C) Western blot assay for protein levels and (D) qPCR analysis for mRNA levels (n = 3) of Bcl-2, caspase-3 and p53 in the SKOV3/R tumor cells after incubation with DOX, unactivated APDCs, activated APDCs and DOX.HCl (2.0 μM) for 48 h.



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Figure 5. Antitumor activity of APDCs against drug-resistant tumor cell lines. (A) Cell viability of SKOV3/R, LoVo/R and MCF-7/R tumor cells vs molar concentrations of DOX (orange), unactivated APDCs from NAs (blue), activated APDCs (red) and DOX.HCl (green) with 48 h incubation (n = 6). (B) Cell cycle distribution of SKOV3/R tumor cells after incubation with DOX, unactivated APDCs, activated APDCs and DOX.HCl (2.0 μM) for 24 h detected by flow cytometry. (C) Apoptosis analysis of the SKOV3/R tumor cells after incubation with the different formulations (2.0 μM) for 48 h using an Annexin V-APC/7-AAD assay and flow cytometry detection.


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Figure 6. Mechanism of activated APDCs from NAs for multidrug resistance reversal, including dysfunctions of plasma/organelle membrane, leakage of critical biomolecules (LDH, Ca2+, cathepsin, Cyt C), a common death pathway evocation with ROS generation, up-regulation of caspase-3/p53 protein and down-regulation of Bcl-2 protein, and the cell cycle arrest.



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Table of Contents Graphic


Engineering Anticancer Amphipathic Peptide-Dendronized

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