A Peptide-Guided System with Programmable Subcellular

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A Peptide-Guided System with Programmable Subcellular Translocation for Targeted Therapy and Bypassing Multidrug Resistance Yuanyuan Zhu, Yanyan Huang, Yulong Jin, Shilang Gui, and Rui Zhao Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b03598 • Publication Date (Web): 10 Dec 2018 Downloaded from http://pubs.acs.org on December 11, 2018

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Analytical Chemistry

A Peptide-Guided System with Programmable Subcellular Translocation for Targeted Therapy and Bypassing Multidrug Resistance Yuanyuan Zhu,†,‡ Yanyan Huang,*,†,‡ Yulong Jin,†,‡ Shilang Gui,†,‡ and Rui Zhao*,†,‡ †Beijing

National Laboratory for Molecular Sciences, CAS Key Laboratory of Analytical Chemistry for Living Biosystems, Institute of Chemistry, Chinese Academy of Sciences, Beijing, 100190, China ‡University

of Chinese Academy of Sciences, Beijing, 100049, China

ABSTRACT: Non-selectivity and drug resistance are two major obstacles for cancer treatment. Although great advances have been made towards cell targeting or discovering novel delivery pathways, it is still desirable to simultaneously overcome the two hurdles to the success of cancer theranostics. Herein, a peptide-guided system was tailored by modular integration of a cancer biomarkerspecific peptide, a mitochondria-targeting motif and a cell toxin. Cell imaging analysis revealed that the dual-targeting peptide-drug conjugate (PDC) features in cancer cell-specific uptake, strong drug retention and programmable intracellular translocation. Facilitated by in-situ bond cleavage, PDC successfully diverted toxic effect of nucleus-localized drug to mitochondria. Mechanism investigation demonstrated that the cell damage pathway of the drug was also transformed, which is beneficial to reverse drug resistance in cancer cells. The effectiveness of PDC for cancer therapy was further demonstrated by in vivo imaging and tumor inhibition assay. With intravenous injection, targeted accumulation in tumor site and tumor suppressing efficacy without side effect exhibited its perspective for cancer treatment. The dual-targeting peptide-drug conjugate featuring tailored transportation route highlights a promising and generally applicable way to enhance overall therapeutic index of conventional anticancer drugs.

Highly selective and efficient recognition and localization are critical for both detection and cure of cancers.1-4 Due to the lack of specificity and cancer affinity, traditional drugs are usually accompanied with severe side effect, low absorption in tumor sites and drug resistance, leading to the failure in the treatment.57 To minimize systemic toxicity, drugs are given at suboptimal dosages, which however results in limited clinical efficacy.3 Multidrug resistance (MDR), as another obstacle for chemotherapeutics, can render drugs ineffective via different mechanisms, including reducing cellular uptake of drugs.1, 8-12 Discovery of novel approaches for simultaneously reducing side effect and bypassing drug resistance are highly desired for effectively probing cancers, however still remain a challenging task. Targeted drug delivery emerges as an effective way for cancer therapy benefiting from the advances in chemical biology and nanotechnology.13-16 Molecular-targeted approach directed towards molecular signatures of cancer such as proteins, enzymes and nucleic acids shows high specificity and affinity.17-20 With the guidance of targeting vehicles, drugs can be localized to cancer cells selectively and efficiently, thus minimizing side effect and improving the therapeutic index. However, drug resistance is still found for molecular specific chemotherapeutics.21 In comparison, targeting a subcellular organelle represents an attractive route to circumvent drug resistance.22-25 For example, mitochondrion as the powerhouse of cells and central regulator of programmed cell death, has been demonstrated to be a promising target for combating MDR cancer cells.22, 26, 27 Diverse damaging pathways, including inhibition of ATP synthesis, activation of cell apoptosis

machinery and damaging mitochondria DNA, or their combinations can be adopted to enhance anticancer activity.28, 29 Nevertheless, there are also hurdles includes low accumulation of drugs in mitochondria and nonselective uptake by both cancer and normal cells.26 Hence, it is highly demanded to develop new targeting approaches to simultaneously resolve problems concerning systemic toxicity and drug resistance. To address this, combination of different targeting approaches shows great potential.21 In our previous work, molecular-targeting approach was employed to develop a peptide-based prodrug. Mediated by peptide-receptor interaction, the prodrug effectively ablated cancer cells with minimal side cytotoxicity. However, the intracellular destination and cell damage pathway of the drug is not changed, which may be still problematic for drug resistant cells. Herein, a dual-targeting peptide-drug conjugate (PDC) was developed to achieve cancer cell-specific drug delivery and controlled release at subcellular scale. The modular design of PDC consists of three units, a cancer biomarker-specific peptide, a mitochondria-targeting moiety triphenylphosphonium (TPP)23 and cell toxin doxorubicin (DOX). DOX takes effect by accumulating in cell nucleus and exerts cytotoxicity via inhibition of topoisomerase II, DNA damage and reactive oxygen species (ROS) generation.26, 29 Despite its activity against a broad range of cancers, DOX is also well-known for its off-target toxicity and drug resistance.11, 30 The integration of molecular-targeting, organelle-targeting and a pH-responsive bond was expected to program the cell recognition, subcellular translocation processes of PDC and cancer cell damage mechanism. The specific

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Scheme 1. Design and synthesis of the peptide-drug conjugate.

binding, cellular distribution and cytotoxicity of PDC were monitored in different cells including DOX resistant cancer cells. In vivo application of PDC for tumor targeting and suppression was also investigated, which demonstrated the potential of this dual-targeting complex for effective cancer therapy.

EXPERIMENTAL SECTION Synthesis of AP2H-hydrazide. AP2H-hydrazide was synthesized manually by solid phase FMOC strategy. FMOCGly-Wang resin was used as the starting material. 20% piperidine in DMF was used for removal of the FMOC protecting group. 4-methylmorpholine (NMM) and HBTU were used as the activating reagents. The peptide synthesis was carried out as described in our previous work.31 After the peptide sequence was assembled, the FMOC group was removed and 3-fold of succine anhydride (SA) was introduced. After coupling and washing, 3-fold excess of FMOC-NH-NH2, HBTU and HOBt were added and reacted overnight. Cleavage of AP2H-hydrazide from the resin and global side-chain deprotection were carried out with a freshly prepared TFA cocktail (95%TFA, 2.5%H2O and 2.5% TIS). The product was purified on a preparative HPLC system. The purified AP2Hhydrazide was characterized with HPLC and high resolution MALDI-FT-ICR MS (Figure S-1).

Synthesis of TPP-DOX. TPP (45 mg, 0.10 mmol) were placed in a 25-mL flask with anhydrous DMF (10 mL) and stired until completely dissolved. DCC (25 mg, 0.12 mmol) and NHS (14 mg, 0.12 mmol) were then added in the flask. After stirring for 3 h, the mixture was filtered to remove dicyclohexylurea (DCU), and the supernatant was collected. DOX (60 mg, 0.10 mmol) was dissolved with anhydrous DMF (15 mL) in a 50-mL flask. Triethylamine (15 μL, 0.11 mmol) was then added dropwise in the flask while stirring. Afterwards, the activated TPP supernatant was added dropwise to the flask while stirring. The mixture was subjected for reacting in dark overnight at room temperature.26 The crude product was purified on the preparative HPLC system. The purified product was characterized with HPLC, high resolution MALDI-FT-ICR MS and NMR (Figure S-2, 4). Synthesis of PDC. AP2H-hydrazide (1.18 mg, 1 μmol), TPPDOX (1.05 mg, 1.2 μmol) and anhydrous DMSO (400 μL) were added to a 5-mL sealed vial. The reaction was carried out in dark overnight at room temperature. The resultant solution was then purified on the preparative HPLC system. The purified product was obtained as red powder after lyophilization. The obtained PDC was characterized with HPLC, high resolution ESI-FT-ICR MS, ESI-IT-MS/MS and 1H NMR (Figure S-3, 5). Cell imaging. In 35 mm glass-bottomed dishes, the cells (approximately 1.0×105/mL) were seeded and cultured overnight for adhesion. After removal of culture medium, the


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cells were treated with DOX, TPP-DOX and PDC solution (500 μL, 20 μM), respectively. After incubation, the cells were carefully washed with PBS for three times. Fluorescence imaging were performed on a FV 1000-IX81 CLSM (Olympus) with a UPLSAPO 100×oil-immersion objective (Olympus). Images were processed and analyzed with the Olympus FV10ASW software. For co-staining assay, the PDC loaded cells were subjected for the incubation with MitoTracker, LysoTracker, or Hoechst 33342 solutions respectively. After carefully washed with PBS for three times, the cells were observed with CLSM. PDC, DOX and TPP-DOX were excited by a FV5-LAMAR 488 nm laser and collected with a band-pass filter within the range of 570-670 nm. MitoTracker and LysoTracker were excited by a FV5-LAMAR 488 nm laser and collected with a band-pass filter within the range of 500-550 nm. Hoechst 33342 was excited by a 50 mW, 405 nm Laser Head FV5-LD405-2 and collected with a band-pass filter within the range of 425-475 nm. In vivo antitumor study. All animal studies were performed with the approval of the Chinese Academy of Sciences Institutional Animal Care and Use Committee. For in vivo antitumor study, 5-6 weeks old female BALB/c nude mice (Vital River Laboratories, Beijing, China) were subcutaneously implanted with HepG2 cells (3×107 /mL, 200 μL) at the right upper flank. When the volume of tumors reached about 50 mm3, 20 mice were randomly divided into four groups. In each group, mice were intravenously injected with 200 μL PBS, PDC, DOX and TPP-DOX solutions (20 μM) respectively every other day. Tumor volume were measured and calculated according to the formula: Tumor volume = (length × width2)/2. The mice were euthanized after 18 days and the tumor tissue, heart, liver, spleen, lung and kidney of each mice were collected and fixed in formalin for post-mortem histopathology analysis. In vivo optical imaging. Female BALB/c nude mice bearing HepG2 tumors of 50-60 mm3 were intravenously injected with PBS, PDC and DOX respectively. After 90 min, images of mice were taken on a small animal in vivo imaging system (PerkinElmer, Spectrum CT, Boston, USA). Afterwards, the mice were euthanized and tumor, heart, liver, spleen, lung and kidney were collected and subjected for fluorescence imaging. The results were from three individual experiments.

RESULTS AND DISCUSSION Design and synthesis. The modular structure and synthesis route of PDC are illustrated in Scheme 1. A decapeptide AP2H (IHGHHIISVG) targeting the extracellular fragment of a cancer-related protein LAPTM4B was employed to introduce cancer cell selectivity.32, 33 Mitochondria having their own DNA but lack of repair machinery were then chosen as the subcellular organelle to exert damage effect of DOX, and triphenylphosphonium (TPP), a well-established mitochondriatargeting moiety was incorporated (Scheme 1). Although DOX is known for nucleus accumulation, the conjugation with TPP is expected to redirect it to mitochondria and trigger different drug action mechanisms to bypass drug resistance. For the conjugation process, TPP was covalently linked to DOX via an amide bond. To introduce the peptide AP2H, a pHsensitive hydrazone bond was designed to achieve controllable release of the drug by responding to acidic environment. The synthesis was accomplished by modification of AP2H with a hydrazine group during solid phase synthesis. After cleavage, the hydrazone bond was facilely formed between the hydrazine group and the keto group in TPP-DOX (Scheme 1). After purification, PDC was obtained with a yield of 21%. The products were characterized with HPLC, high resolution MS, tandem MS, NMR, fluorescence spectrometry and dynamic light scattering (DLS) (Figure S-1-7). Monitoring the pH-responsive drug release in vitro. To study the pH triggered drug release behavior, PDC was treated with phosphate buffers of different pH at 37 oC. HPLC was used to monitor the drug release profiles (Figure S-8). Under pH 7.4 simulating general pH condition in body, negligible release of TPP-DOX was detected (Figure 1a), suggesting the high stability of PDC in neutral physiological environment. At pH 5.0 which mimics the low pH environment of endosomes and lysosomes, the content of PDC significantly lowered while the signal of TPP-DOX increased (Figure S-8), clearly demonstrating the cleavage of PDC by acidic condition and release of free TPP-DOX. With 48-h incubation, the release efficiency can reach 68% (Figure 1a). To mimic the low pH microenvironment of cancer,34, 35 buffer solution of pH 6.0 was used for the treatment of PDC. Due to the slight elevated pH, the release kinetics was slowed down compared

Figure 1. (a) Release profiles of TPP-DOX from PDC at pH 7.4, pH 6.0 and pH 5.0. (b) CLSM imaging of cancer cells (HepG2, MCF-7/WT and MCF-7/ADR) and normal cells (HEK293) after the treatment of DOX, PDC and TPP-DOX respectively. Scale bar: 20 m.



Figure 2. (a) Co-staining assays of HepG2 cells with nucleus indicator Hoechst (blue fluorescence). Cells were incubated with DOX and Hoechst or PDC and Hoechst respectively. Scale bar: 20 m. (b) Cytotoxicity assays of PDC, DOX and TPPDOX towards cancer cells (HepG2, MCF-7/WT and MCF7/ADR) and normal cells (HEK293). with the data from pH 5.0 (Figure S-8). After 48-h treatment, the drug release ratio was 26% (Figure 1a). The high stability and pH-sensitive drug release of PDC can avoid unexpected burst release in non-tumor parts and promote local drug concentration in cancer cells. Cancer cell-specific uptake. The fluorescence emission of DOX enables monitoring the cellular uptake with confocal laser scanning microscopy (CLSM). The fluorescence stability of DOX under different pH environments further facilitates the observation in cells and tumor sites (Figure S-9). Cancer cells HepG2 (human liver hepatocellular carcinoma cells) and normal cells HEK293 (human embryonic kidney cells) were firstly used for the incubation with PDC, TPP-DOX and DOX respectively. As shown in Figure 1b, free DOX emitted bright red fluorescence in both HepG2 and HEK293 cells, demonstrating its nonselective uptake by cancer and normal cells. The fluorescence signal was localized to cell nucleus by co-staining with nucleus indicator Hoechst (Figure 2a and Figure S-10). In contrast, PDC shows significantly difference in both cellular uptake and subcellular distribution. Red fluorescence only can be detected in cancerous HepG2 cells while HEK293 cells remained dark after the same treatment (Figure 1b), suggesting the high specificity of PDC for cancer cell recognition and internalization. The saturable cellular uptake behavior of PDC in cancer cells is well consistent with receptor-mediated internalization process, which reaches an equilibrium upon receptor binding (Figure S-11, 12). For the distribution pattern, the fluorescence signal in HepG2 cells indicates the absence of PDC from cell nucleus and an altered localization in cytoplasm (Figure 1b and 2a), which is

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inverse to that of DOX. As a control, TPP-DOX was also used to treat cells. Without the guidance of AP2H peptide, TPP-DOX non-selectively scattered in both HepG2 and HEK293 cells (Figure 1b). Moreover, the fluorescence intensity from HepG2 cells was weaker than that from PDC-loaded cells. These results manifest that targeted binding of AP2H not only facilitated the cancer cell-specific internalization, but also enhanced uptake efficiency of PDC into HepG2 cells. The cellular uptake by DOX resistant cancer cells was investigated using a multidrug-resistant breast cancer cell model MCF-7/ADR cells (Figure 1b). For free DOX, negligible fluorescence can be seen inside MCF-7/ADR cells after treatment (Figure 1b and Figure S-10). Instead, red fluorescence only can be detected in the peripheral of the cells, suggesting DOX was repelled by MCF-7/ADR cells and only nonspecifically retarded on cell membranes. This can be attributed to the presence of permeability glycoprotein (P-gp) in drug resistant cells, which pumps DOX out. For PDC treatment, bright dotted fluorescence can be detected in the cytoplasm of both MCF-7/ADR cells and MCF-7/WT cells. No sacrifice in signal intensity is detected for MCF-7/ADR cells. These results demonstrate the ability of PDC to enter and retain in drug resistant cancer cells, which offers the prerequisite for efficient killing of cancer cells with MDR. Selective cytotoxicity of PDC. The cytotoxicity of the compounds was assayed with the standard MTT (3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) method (Figure 2b). Free DOX and TPP-DOX killed both HepG2 and HEK293 cells to the viability below 20%. This is in accordance with their non-selective entrance into cancer and normal cells. The high toxicity of DOX and TPP-DOX towards normal cells would bring severe side effect. In contrast, PDC killed most of HepG2 cells with a high efficiency of 83% and left HEK293 cells unaffected. The cell viability of HEK293 cells was above 97% after 48-h incubation. The peptide-guided internalization of PDC allowed the drug to act only in cancer cells and protected normal cells. The cytotoxic effects towards drug resistant cancer cells was also examined (Figure 2b). After treated with DOX and TPP-DOX, only 13% and 22% of MCF7/WT cells remained alive respectively, while the viability of MCF-7/ADR cells rose to 66% and 53%. Their toxicity dramatically decreased towards drug resistant cancer cells. In comparison, PDC maintained almost the same cytotoxicity against MCF-7/ADR cells and MCF-7/WT cells (Figure 2b). No discrimination in killing drug resistant-cancer cells was detected. The cytotoxicity results were in good consistent with the different cellular uptake behavior of these compounds (Figure 2b). Benefiting from its targeting character, PDC changed delivery pathway and accumulated inside MCF7/ADR cells, which contributed to the ability to overcome drug resistance. IC50 values were calculated to evaluate the antiproliferative abilities of the compounds (Figure S-13, Table S-1). DOX killed HepG2 and MCF-7/WT cells with IC50 of 0.76 M and 1.1 M respectively. A significant decrease in its ability of ablating drug resistant MCF-7/ADR cells was revealed with an


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Figure 3. (a, b) Colocalization of PDC with LysoTracker (a) or MitoTracker (b) after different incubation periods of PDC with HepG2 cells. Scale bar: 20 m. (c) Changes in the mitochondrial membrane potential in different cells after the treatment of PDC for 18 h. (d) Illustration of the possible pathway of PDC for targeting and damaging mitochondria in cancer cells. IC50 of 40 M. For PDC, its IC50 values against MCF-7/WT (15 M) and MCF-7/ADR (18 M) are almost the same, confirming its effectiveness in bypass drug resistance. It is noted that PDC shows slightly lower toxicity than DOX towards non-drug resistant cells. This is most probably due to their different cell uptake manners (Figure S-14) and the release kinetics of PDC. Lysosomal escape and mitochondria targeting. To trace the subcellular translocation of PDC, a time-course imaging assay was carried out to examine the internalization process (Figure S-15). Initial binding of PDC to the target protein on cell membrane was observed with 1-h incubation. As the incubation time prolonged to 4 h, the fluorescence signal on the membrane diminished and bright fluorescence emerged inside the cells, conferring uptake and accumulation of PDC by the cells. The travelling directions of PDC inside cancer cells were monitored based on colocalization assays. After 4-h treatment with PDC, red fluorescence from PDC showed good overlap with the green signal from lysosomal tracker (correlation coefficient (R): 0.91, R=1 represents the perfect overlap) (Figure 3a), suggesting the transportation of PDC to the lysosomes. With longer incubation time of 8 h, the merge ratio between the fluorescence signal from PDC and LysoTracker decreased (R = 0.41). Since strong red fluorescence still emitted inside the cells, such drop in the colocalization ratio reveals the escape of PDC from lysosomes and its translocation. According to the design, with the acid-sensitive hydrazone bond, PDC is expected to be cleaved in lysosomes and released to mitochondria. To examine this, a mitochondrial indicator (MitoTracker) was used to co-stain with PDC. In the cells treated with PDC for 8 h, green fluorescence from MitoTracker shows partial overlap with the red fluorescence from TPP-DOX (Figure 3b), suggesting the successful delivery of TPP-DOX to mitochondria. The moderate R value of 0.66 can be attribute to the partial cleavage of PDC. As the incubation time increased to 17 h, a good merged image between the fluorescence signal from TPP-DOX and MitoTracker was obtained. At this stage almost all TPP-DOX has escaped from lysosome and travelled to mitochondria. Investigation of the cell death mechanism. DOX is known for its damage roles of DNA binding/cross-linking, ROS induction and topoisomerase II inhibition. To investigate the cytotoxic mechanism of PDC, its ability to bind DNA was firstly

examined because DNA binding is the prerequisite for DNA damaging and topoisomerase II inhibition. The DNA binding affinity was assayed with fluorescence titration with calfthymus DNA (ct-DNA) (Figure S-16). With a binding constant (Kb) of 6.41.8  106 mol-1, DOX shows the strongest binding affinity with DNA, which is consistent with its DNA damaging and topoisomerase inhibition effect. Loss in DNA binding ability was found for TPP-DOX (Kb=3.00.5106 mol-1) and PDC (Kb=1.80.7105 mol-1). The weak binding of TPP-DOX and PDC towards DNA limits their roles in DNA damage and topoisomerase II inhibition. Mitochondria are the major source of cellular ROS, meanwhile prone to oxidative damage. The abilities of the compouds to induce ROS was studied with a commercial ROS detection dye (Figure S-17). Strong ROS signal was detected from PDC and TPP-DOX treated cancer cells. Although DOX also induced ROS in cells, its intensity was about 50% lower than that generated by PDC. These results suggest the high ability of PDC and TPP-DOX in inducing ROS in cancer cells. Because mitochondrial metabolism plays important roles in the survival of drug resistant cells, the cell death mechanism was then studied in terms of mitochondria functions. Mitochondrial membrane potential (m), an indicator of the health status of mitochondria, was measured with a commercial fluorescence dye (Figure S-18). Without PDC treatment, NIR fluorescence from the m indicator was observed in cancer cells (HepG2, MCF-7/WT and MCF-7/ADR), indicating healthy polarized mitochondria (Figure S-19). After treated with PDC, the fluorescence intensity in HepG2, MCF-7/WT and MCF-7/ADR fell down to 13%, 32% and 35% of the original signals respectively, indicating critical drop in m in these cells (Figure 3c and Figure S-19). The ATP contents in different cells were measured using an ATP bioluminescent assay kit (Figure S-20). 18-h treatment with PDC led to 71%, 80% and 78% decrease in ATP contents in HepG2, MCF-7/WT and MCF7/ADR cells respectively, suggesting the inhibition of ATP synthesis in mitochondria. In contrast, for HEK293 cells, no influence to both mitochondrial membrane potential and ATP content was observed after PDC treatment (Figure 3c and Figure S-20). These results reveal the dramatic damage effect of TPP-DOX to the electron transport chain and oxidative phosphorylation in mitochondria regardless of drug resistance effect.



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Figure 4. In vivo imaging and therapeutic effect of PDC via intravenous injection. (a,b) Body imaging and tissue imaging of the distribution of PDC. Yellow circles indicate tumor sites. (c) Tumor volume up to day 18 after treatment with PDC (means.d., n = 5) (**P < 0.01; Student's t-test). (d) Change in the body weight of tumor-bearing mice up to day 18 after treatment. (e-h) H&E staining of tumor tissues from mice injected with PBS (e), PDC (f), DOX (g) and TPP-DOX (h) after 18 days, respectively. Scale bar: 100 m. P-gp, also known as ATP-binding cassette sub-family B member 1, acts as a transmembrane efflux pump and excludes drugs out of the cells.7, 8 The expression level of P-gp can be used a marker for the drug resistant ability of the cells. The influence of PDC to P-gp expression level was investigated with Western blot (Figure S-21). The treatment with PDC leads to 32% decrease in P-gp expression in MCF-7/ADR cells. Down regulation of P-gp expression was also detected in a drug resistant liver cancer cell line. These results confirm the capability of PDC to bypass and suppress the drug resistance effect. Based on the above analysis, a road map which PDC adopted for targeted drug delivery and cell damage can be summarized (Figure 3d). Initiated by the interaction between AP2H and membrane-anchored LAPTM4B protein, PDC specifically recognized and bound cancer cells. The receptor-mediated endocytic pathway then led to the delivery of PDC to lysosomes. The low pH environment in lysosomes efficiently cut the hydrazone bond and released TPP-DOX. The successful escape from lysosomes enabled further transportation of TPPDOX to mitochondria directed by targeting group TPP. Because TPP-DOX is highly active in producing ROS and mitochondria are prone to oxidative damage, the disruption in the electron transport chain and ATP synthesis finally lead to mitochondrial dysfunction and cell death. With dual-targeting ability, PDC can effectively bypass the interaction with P-gp. Meanwhile, due to the energy-dependent expression feature of P-gp, the damage to mitochondria inhibit P-gp expression, thus inhibit the efflux.9 The targeted and mitochondria interrupted cell damaging pathway allow PDC to exert toxicity both wild type and drug resistant cancer cells. In vivo imaging and targeted antitumor activity. The in vivo therapeutic performance of PDC was examined using HepG2 tumor xenograft model. After the tumor volume reached 50 mm3, the mice were randomly divided into four groups and the treatment was administrated by intravenous injection every other day. The delivery and accumulation in tumor site is the prerequisite to obtain antitumor activity. Hence, body imaging was performed after intravenous injection of saline, PDC and

DOX, respectively. As shown in Figure 4a and Figure S-22, strong fluorescence emitted in the tumor site of PDC-injected mice, demonstrating the targeted delivery and retention of PDC in tumor. For the DOX-treated mice, the fluorescence signal from tumor is weak, indicating low affinity and uptake of free DOX by tumor. After sacrifice, bright emission from tumor tissue of PDC-treated mice was clearly observed, while no fluorescence was detected in the organs including heart, liver, spleen, lung and kidney (Figure 4b and Figure S-22). These results further confirm the in vivo targeting ability of PDC. The antitumor activity was studied by monitoring tumor volume. Over the period of 18 days, the tumors in the control group grew rapidly from 50 mm3 to 535 mm3 (Figure 4c). With the injection of PDC, the rate of tumor growth significantly slowed down and the tumor size was obviously reduced. The average tumor volume shrunk by 55% at day 18 compared with that of the control group (Figure 4c). In contrast, DOX only showed a moderate inhibition of tumor growth by 41% (Figure 4c). This may be most probably caused by the non-specific biodistribution and low uptake in tumor site. With cancer cellspecific targeting ability, PDC shows high affinity and penetrability towards tumor. The damage of mitochondria leads to the high efficiency for tumor suppression. The systemic toxicity of PDC was evaluated by recording the body weight of mice (Figure 4d). After PDC treatment, the body weights were almost unchanged, suggesting the minimal systemic cytotoxicity. The good biocompatibility of PDC is further confirmed by hematoxylin and eosin (H&E) histopathological analysis. Significant cell damaging can be identified in H&E stained tumor section (Figure 4f). For the major organs including heart, liver, spleen, lung and kidney, no obvious pathological change or damage was observed after the treatment of PDC (Figure S-23). These results manifest the negligible side effect of PDC during blood circulation.

CONCLUSIONS In summary, the feasibility of simultaneously overcoming systemic toxicity and drug resistance of an established anticancer drug was demonstrated by changing its delivery


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pathway and subcellular distribution. Taking advantage of the fact that drug resistant cells are sensitive to mitochondria damage, PDC redirected the toxic effect to mitochondria. Its high activity in ROS generation leads to disruption in redox potential of mitochondria and inhibition in ATP supply in cancer cells. The potency of PDC for cancer therapy was further demonstrated by its targeted accumulation in tumor site and tumor inhibition efficacy without side effect in vivo. The dualtargeting peptide-drug conjugate featuring tailored transportation route highlights a promising and generally applicable way to enhance overall therapeutic index of conventional anticancer drugs.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website. Materials and reagents, equipment and apparatus. Experiment description of drug release kinetics in vitro, MTT, DNA binding assay, ROS detection, mitochondrial membrane potential assay, ATP assay and Western blot. Results of product characterization (HPLC, MS and NMR), DLS analysis, fluorescence stability, HPLC monitoring the pH-sensitive drug-releasing profiles, cell uptake process, intracellular distribution of free DOX, time-course assay, mitochondrial membrane potential assay, PDC-induced ATP change, P-gp expression levels, in vivo imaging, H&E analysis, and investigation of anticancer activity against drug resistant tumor model (PDF)

AUTHOR INFORMATION Corresponding Author *E-mail: [email protected], [email protected]. Phone: +8610-62557910. Fax: +86-10-62559373.

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

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT This work is supported by grants from National Natural Science Foundation of China (21475140, 21675161 and 21621062), Ministry of Science and Technology of China (2015CB856303), Chinese Academy of Sciences and Youth Innovation Promotion Association CAS (No. 2015027). We thank Dr. Junfeng Xiang from Institute of Chemistry, Chinese Academy of Sciences, for his valuable help in NMR analysis.

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Analytical Chemistry for TOC only


9


Scheme 1. Design and synthesis of the peptide-drug conjugate.


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Figure 1. (a) Release profiles of TPP-DOX from PDC at pH 7.4, pH 6.0 and pH 5.0. (b) CLSM imaging of cancer cells (HepG2, MCF-7/WT and MCF-7/ADR) and normal cells (HEK293) after the treatment of DOX, PDC and TPP-DOX respectively. Scale bar: 20 μm. 180x51mm (300 x 300 DPI)



Figure 2. (a) Co-staining assays of HepG2 cells with nucleus indicator Hoechst (blue fluorescence). Cells were incubated with DOX and Hoechst or PDC and Hoechst respectively. Scale bar: 20 μm. (b) Cytotoxicity assays of PDC, DOX and TPP-DOX towards cancer cells (HepG2, MCF-7/WT and MCF-7/ADR) and normal cells (HEK293). 75x72mm (300 x 300 DPI)


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Figure 3. (a, b) Colocalization of PDC with LysoTracker (a) or MitoTracker (b) after different incubation periods of PDC with HepG2 cells. Scale bar: 20 μm. (c) Changes in the mitochondrial membrane potential in different cells after the treatment of PDC for 18 h. (d) Illustration of the possible pathway of PDC for targeting and damaging mitochondria in cancer cells. 150x45mm (300 x 300 DPI)



Figure 4. In vivo imaging and therapeutic effect of PDC via intravenous injection. (a,b) Body imaging and tissue imaging of the distribution of PDC. Yellow circles indicate tumor sites. (c) Tumor volume up to day 18 after treatment with PDC (mean±s.d., n = 5) (**P < 0.01; Student's t-test). (d) Change in the body weight of tumor-bearing mice up to day 18 after treatment. (e-h) H&E staining of tumor tissues from mice injected with PBS (e), PDC (f), DOX (g) and TPP-DOX (h) after 18 days, respectively. Scale bar: 100 μm. 119x61mm (300 x 300 DPI)


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A Peptide-Guided System with Programmable Subcellular

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