Hybrid Prodrug Nanoparticles with Tumor ... - ACS Publications

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Hybrid Prodrug Nanoparticles with Tumor Penetration and Programmed Drug Activation for Enhanced Chemoresistant Cancer Therapy Caiyan Zhao,†,‡ Leihou Shao,†,‡ Jianqing Lu,† Xiongwei Deng,† Yujia Tong,† and Yan Wu*,†,‡ †

CAS Key Laboratory for Biomedical Effects of Nanomaterials and Nanosafety, CAS Center for Excellence in Nanoscience, National Center for Nanoscience and Technology, Beijing 100190, China ‡ University of Chinese Academy of Sciences, Beijing 100049, China S Supporting Information *

ABSTRACT: Despite nanomedicine having shown great potential for reversing cancer cell resistance, the suboptimal transport across multiple biological obstacles seriously impedes its reaching targets at an efficacious level, which remains a challenging hurdle for clinical success in resistant cancer therapy. Here, a lipid-based hybrid nanoparticle was designed to efficiently deliver the therapeutics to resistant cells and treat resistant cancer in vivo. The hybrid nanoparticles (D-NPs/ tetrandrine (TET)) are composed of a pH-responsive prodrug 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE)doxorubicin (DOX), an efflux inhibitor TET, and a surfactant DSPE-[methoxy (poly(ethylene glycol))-2000] (DSPE-mPEG2000), which hierarchically combatted the sequential physiological and pathological barriers of drug resistance and exhibited prolonged blood circulation, high tumor accumulation, and deep tumor parenchyma penetration. In the meantime, the programmed stepwise activation of encapsulated TET and DOX suppressed the function of resistance-related P-glycoprotein in a timely manner and facilitated the DOX sustained accommodation in tumor cells. Through systematic studies, the results show that such a nanosystem dramatically enhances drug potency and significantly overcomes the DOX resistance of breast cancer with negligible systemic toxicities. These findings provide new strategies to systemically combat chemoresistant cancers. KEYWORDS: nanomedicine, cancer resistance, biological barriers, tumor tissue penetration, programmed activation

1. INTRODUCTION Under evolutionary pressure from chemotherapy, cancer cells frequently develop drug resistance by overexpressing the Pglycoprotein (P-gp) and upregulating its activity.1−3 P-gp, encoded by the multidrug resistance gene (MDR-1) and functioning as a typical ATP-binding cassette membrane transporter, can actively effuse the anticancer drugs and prevent intracellular accumulation of the drug, hence decreasing the chemosensitivity of cancer cells against anticancer drugs.4−6 Recently, nanoparticle-based delivery systems have offered great promise for combatting drug resistance.7,8 Several attempts have indicated that nanoparticles could enter cells via an endocytosis pathway independent of the P-gp pathway.9−11 More importantly, it is increasingly becoming evident that the use of combinatorial nanomedicines provides superior efficacy for circumventing drug resistance.12−14 However, few nanosystems have entered clinical trials. A possible reason behind such a disappointing outcome is that they fail to address the intricate mechanism of drug resistance, including sequential physiological obstacles.15,16 Most conventional methods have rarely examined whether nanosystems are capable of simultaneously overcoming intracellular drug efflux and crossing a © 2017 American Chemical Society

series of extracellular biological barriers imposed by the body. These systematic biological obstacles could considerably prevent efficacious drug distribution within the tumor tissue and powerfully limit various countermeasures to fight against cancer cells resistance, hence resulting in treatment failure.17,18 Aiming at optimal therapy efficiency of nanosystems to tumor resistance, it is of great importance to understand and overcome the various extra/intracellular barriers. After intravenous injection, the reticuloendothelial system (RES) recognizes and clears the circulating nanoparticles, leading to a major loss of the injected dose.19,20 Only the particles not rapidly cleared by the RES are able to reach the leaky tumor vasculature, through which the nanoparticles accumulate in the tumor tissues by the enhanced permeability and retention (EPR) effect.21,22 However, the drugs are often restricted at the tumor periphery after extravasation from the tumor vessels. Efficiently crossing the tumor mass and permeating deep into the tumor parenchyma still remain intractable problems due to Received: February 8, 2017 Accepted: May 17, 2017 Published: May 17, 2017 18450

DOI: 10.1021/acsami.7b01908 ACS Appl. Mater. Interfaces 2017, 9, 18450−18461

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ACS Applied Materials & Interfaces

Scheme 1. Schematic Illustration of the D-NPs/TET Nanoplatform Construction and the in Vivo Drug Delivery and Cancer Therapy Strategya

a

The PEG protected D-NPs/TET was favorable for prolonged blood circulation and targeted tumor tissue through the EPR effect. Once extravasated from leaky tumor vasculature, D-NPs/TET with weak diffusional hindrance penetrated into both the tumor periphery and intratumor parenchyma. After cellular uptake, acidic endo/lysosome induced the decomposition of nanoparticles. The fast-released TET inhibited P-gp expression first and facilitated the intracellular DOX sustained accumulation, leading to tumor cell apoptosis.

superior tumor penetration capacity due to their weak diffusional hindrance in the tumor matrix.29−31 So, lipid-based nanomicelles have inherent advantages in cancer therapy by virtue of their small size.32−34 DSPE-PEG2000 was used to form a PEGylated micelle to impart the particle with high stability and prolong blood circulation because the PEG referred to as “stealth” layer could bypass the surveillance of the RES.35,36 Tetrandrine (TET), a third-generation P-gp inhibitor, was encapsulated in the hydrophobic lipid “core” to inhibit the drug efflux of tumor cells.37,38 The distinct encapsulated properties of TET and DOX endowed the nanosystem with an ondemand release property and hence efficiently overcame the DOX resistance of tumor cells. Compared with the conventional nanoparticle, this system is supposed to systematically target and effectively kill the resistant tumor cells after intravenous injection in the following steps: (i) prolonging the blood circulation for beneficial tumor accumulation, (ii) penetrating deep into the intratumor parenchyma to reach more cancer cells, (iii) motivating TET and DOX sequentially to inhibit the DOX efflux in a timely manner and continuously

the dysfunctional structure of the tumor vessels, the high interstitial fluid pressure, and dense extracellular matrix of tumor tissue.16,23−25 In addition, controlled drug release is another bottleneck for successful cancer therapy.26,27 A programmed drug release approach that can sequentially motivate active agents in an on-demand manner will offer vast promise for the treatment of resistant cancer.28 Therefore, developing the sophisticated nanomedicines that are able to systematically cross the sequential biological barriers to accumulate at tumor cells and are also capable of reversal of the tumor cell resistance is a pressing task in the systemic therapy of resistant tumors. Herein, we developed a long-circulating, tumor-penetrating, and programmed activated prodrug nanoparticle for systematically treating drug-resistant breast cancer. The nanoparticles were constructed through the coassembly of pH-cleavable bond-linked prodrug conjugates 1,2-distearoyl-sn-glycero-3phosphoethanolamine (DSPE)-doxorubicin (DOX, a first-inclass chemotherapeutic drug) and DSPE-[methoxy (poly(ethylene glycol))-2000] (DSPE-mPEG2000). It has been demonstrated that the smaller nanoparticles exhibited a 18451


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Trypsin-EDTA (0.25%) and antibiotic solution (penicillin and streptomycin) were from Invitrogen (Invitrogen, Carlsbad, CA). MCF-7S and MCF-7R cells were maintained in Dulbecco’s modified Eagle’s medium containing 10% FBS and 1% antibiotic solution in a humidified atmosphere with 5% CO2 at 37 °C. 2.8. Cellular Uptake. Cellular uptake was investigated by confocal imaging and flow cytometry analysis. The cells were seeded into specified wells, incubated at 37 °C for 24 h, and then treated with different drug formulations at a DOX concentration of 15 μg/mL for 4 h. For confocal microscopy imaging, the lysosomes were stained with LysoTracker Deep red (green); then, the cells were observed using confocal laser scanning microscopy (CLSM; Carl Zeiss Inc.). For flow cytometry, the cells were collected and analyzed using an Attune acoustic focusing cytometer (Applied Biosystems, Life Technologies). 2.9. Drug Efflux. MCF-7 R cells were seeded into six-well plates and incubated at 37 °C for 24 h. Then, the culture medium was replaced with fresh complete medium containing naked DOX, DOXTET, D-NPs, or D-NPs/TET at a DOX concentration of 15 μg/mL. After 5 h, the medium was removed and the cells were rinsed twice with PBS and subsequently incubated with fresh complete medium for different time intervals (0, 1, 2, or 4 h). At set time intervals, the cells were collected and analyzed by flow cytometry.39 2.10. In Vitro Cytotoxicity. The cells were seeded into a 96-well plate and incubated at 37 °C for 24 h. Then, the various drug formulations at DOX concentrations ranging from 0.625 to 20 μg/mL were added. The cells were incubated for another 24 h. Afterward, the medium was replaced by 100 μL of MTT solution (5 mg/mL). After 4 h, the MTT solution was removed and 150 μL of DMSO was added. The absorbance was measured with a microplate reader (Tecan) at 570 nm. 2.11. Western Blot Analysis. MCF-7R cells were seeded in sixwell plates at a density of 200 000 cells per well at 37 °C for 24 h. Then, they were treated with naked DOX, DOX-TET, D-NPs, or DNPs/TET at a DOX concentration of 5 μg/mL. After 15 h, the cells were washed twice with PBS, scraped off the dishes, and then centrifuged at 2000 rpm for 5 min. The collected cells were lysed in cold RIPA buffer (Beyotime, China) for 30 min, and the lysates were clarified by centrifugation at 12 000g for 15 min at 4 °C. The obtained protein was separated on gel, transferred onto a poly(vinylidene difluoride) (PVDF) membrane, and blocked with 5% milk. The PVDF membrane was incubated with primary antibody (anti-P-gp, diluted 1:1000; Sigma; anti-caspase-3: diluted 1:1000, Cst; β-actin: diluted 1:1000; Beyotime, China) at 4 °C overnight and then immunoblotted with secondary antibody (diluted 1:5000; zsBio, China). 2.12. Cell Apoptosis. MCF-7R cells were seeded into six-well plates at a density of 200 000 cells per well and incubated at 37 °C for 24 h. Then, the culture medium was replaced with a fresh complete medium containing different drug formulations at a DOX concentration of 5 μg/mL. After 15 h, the cells were harvested and rinsed twice with sodium azide-free buffers. Then, the cells were resuspended with 100 μL sodium azide- and protein-free Dulbecco’s phosphatebuffered saline (1× DPBS). The Fixable Viability Stain 450 (FVS450) stock solution (0.1 μL) was added into the above cell suspension and incubated for 15 min protected from light. Subsequently, the cells were rinsed twice and resuspended with 100 μL 1× annexin-binding buffer. APC Annexin V solution (annexin V-FITC) (5.0 μL) was added and incubated for another 15 min in dark. Finally, the cells were rinsed and analyzed by flow cytometry. 2.13. Penetration Multicellular Spheroids (MCSs). MCSs were produced with MCF-7R cells as follows. Agarose solution (1%, w/v) was prepared by heating at 90 °C for 15 min; then, 50 μL of the agarose solution was coated on the bottom of the 96-well plates. The agarose-coated 96-well plates were exposed to UV irradiation for 30 min. Next, the 100 μL of MCF-7R cell suspensions were seeded into the agarose-coated 96-well plates at 500 cells per well. The medium was replaced every other day for 1 week to form the MCF-7R MCSs. To determine the drug penetration ability, the MCF-7R MCSs were incubated with free DOX, DOX-TET, D-NPs, or D-NPs/TET at a DOX concentration of 15 μg/mL. After 5 h incubation, the medium was removed and the MCSs were washed with PBS for three times;

enhance intracellular DOX accumulation in a timely manner, and (iv) inducing resistant cell apoptosis (Scheme 1).

2. EXPERIMENTAL SECTION 2.1. Materials. DSPE and DSPE-mPEG2000 were purchased from Avanti Polar Lipids (Alabaster, AL). 1,4-Phthalaldehyde was obtained from TCI Development Co. Ltd. (Shanghai, China); TET was obtained from Selleckchem (Huston, TX); DOX hydrochloride was purchased from Beijing Huafeng United Technology Co., Ltd. (Beijing, China); cy5 was purchased from Beijing Fanbo Biochemicals Co,. Ltd. (Beijing, China); 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) was obtained from Sigma-Aldrich (Milwaukee, WI); and LysoTracker Deep Red was purchased from Molecular Probes Inc. (Eugene, OR). Dichloromethane (DCM) and dimethyl sulfoxide (DMSO) were purified by distilling or refluxing before use. Other solvents were of analytical grade and used as received. 2.2. Activation of DSPE. DSPE (100 mg, 0.135 mmol) in dried DCM was stirred and refluxed at 45 °C until the solution was transparent. Then, 1,4-phthalaldehyde (362 mg, 2.7 mmol) was added dropwise to the above solution using a syringe. The reaction was stirred at 40 °C for another 24 h. Then, the resulting solution was concentrated by vacuum rotary evaporation and precipitated in cold diethyl ether. The product was filtered and washed three times, then dried under vacuum. Yield: 71%. 2.3. Syntheses of DSPE-DOX. The activated DSPE (170 mg, 0.2 mmol) was dissolved in dried DMSO. DOX (130 mg, 0.24 mmol) was added into the above solution. The mixture was reacted for 24 h at 40 °C. Subsequently, the crude product was dialyzed (MWCO = 1000 Da) against methanol for 12 h then against distilled water at pH 7.4 for another 24 h and lyophilized. Yield: 78%. 2.4. Fabrication of Hybrid Nanoparticles. The nanoparticles DNPs/TET were prepared by the film dispersion method. DSPE-DOX, DSPE-mPEG2000, and TET with a specified ratio were dissolved in chloroform/2,2,2-trifluoroethyl alcohol (2:1, v/v) and dried by vacuum rotary evaporation followed by hydration with PBS for 30 min. The TET noncontaining nanoparticles D-NPs were prepared using the identical procedure. The cy5-labeled fluorescent nanoparticles D-NPs/TET&cy5 were prepared as above with the mixture of TET and cy5 instead of TET. 2.5. Characterization. The structure of each compound was determined by NMR (AVANCE, Bruker) and Fourier transform infrared spectroscopy (FT-IR, Perkin-Elmer). Size distribution and ζ potential of nanoparticles were measured using dynamic light scattering (Zetasizer Nano ZS, Malvern), and the morphology of the nanoparticles was characterized by transmission electron microscopy (TEM, Tecnai G2 20 STWIN; Philips). DOX concentrations were estimated using a Perkin-Elmer LS 55 luminescence spectrometer (F4500; Hitachi) with 480 nm excitation and 590 nm emission. The TET was analyzed by high-performance liquid chromatography (HPLC; Waters 2796 with a Waters 2996 PDA detector) on a reverse-phase C18 column (4.6 mm × 250 mm; Agilent). The flow rate was maintained at 1.0 mL/min, and the chromatograms were collected at 280 nm using methanol/methanoic acid/water (72:8:20, v/v/v) as the mobile phase. 2.6. In Vitro Drug Release. In vitro release of DOX and TET from D-NPs/TET was performed at 37 °C under acetate buffer (10 mM, pH 5.0) or phosphate buffer (PB, 10 mM, pH 7.4). D-NPs/TET solution (1 mL) was added into a dialysis tube (MWCO 3000 Da) and the samples were dialyzed against 20 mL of buffer containing 1% Tween 80. The incubation medium (1.0 mL) was taken out at predetermined time intervals then replaced with 1.0 mL of fresh medium. The DOX and TET in the outside medium were quantified by luminescence spectrometer or HPLC. 2.7. Cell Culture. DOX-resistant MCF-7 (MCF-7R) cells and DOX-sensitive MCF-7 (MCF-7S) cells were purchased from the American Type Culture Collection (ATCC; Manassas, VA). The fetal bovine serum (FBS) and cell culture medium obtained were from Wisent Inc. (Multicell, Wisent Inc., St Bruno, Quebec, Canada). 18452


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Figure 1. Construction and physicochemical characterizations of D-NPs/TET. (a) Size distribution of D-NPs/TET determined by DLS; (b) TEM image of the D-NPs/TET; pH triggered release of DOX (c) and TET (d) from D-NPs/TET at 37 °C. then, the spheroids were transferred to confocal dishes and observed by CLSM. 2.14. In Vivo Anticancer Activity Assay. Female BALB/c nude mice (20−22 g) were purchased from the Vital River Laboratory Animal Center (Beijing, China). The animal experiments were performed in accordance with the protocols approved by the ethics committee of Peking University. Briefly, 1.0 × 107 MCF-7R cells were suspended in 100 μL of PBS buffer/Matrigel (BD Pharmingen) mixed solution (1:1, v/v) and subcutaneously inoculated into the armpit of the female BALB/c mice. When the tumor volume was about ∼100 mm3, the mice were injected by tail vein with naked DOX, DOX-TET, D-NPs, or D-NPs/TET at the DOX dose of 5 mg/kg every 3 days 5 times (n = 6 per group). The tumor sizes were estimated using the caliper measurements and calculated by the following formula: volume = 1/2 × LW2 (L represented the maximum diameter and W represented the minimum diameter of the tumor). The body weights of the mice were recorded simultaneously. Finally, the mice were sacrificed. The sera were collected for biochemical studies. The organs and tumors were excised, embedded in paraffin, and stained by hematoxylin-eosin-safran (H&E); they were then analyzed by 40× magnification. 2.15. In Vivo Biodistribution. The female BALB/c nude mice bearing MCF-7R tumors were treated with 200 μL of naked cy5 or DNPs/TET&cy5 at a cy5 dose of 150 ng/μL by tail vein injection. The realtime distributions of naked cy5 and D-NPs/TET&cy5 were carried out at 3, 5, 8, 12, and 24 h postinjection using an in vivo imaging system (IVIS, Cambridge Research & Instrumentation, Inc.). Finally, the mice were sacrificed and the tumor, heart, liver, spleen, lung, and kidney were excised for fluorescence intensity measurement. 2.16. In Vivo Tumor Penetration. The female BALB/c nude mice bearing MCF-7R tumors were injected by tail vein with 100 μL of naked DOX, DOX-TET, D-NPs, or D-NPs/TET at the DOX dose of 5 mg/kg. After 12 h, the mice were sacrificed and the tumors were

excised and embedded in an optimal cutting temperature compound. The samples were cut into 10 μm slides for histological analysis. The tumor vessels were stained with anti-CD31 antibody. Briefly, the sections were fixed in 4% (w/v) formaldehyde solution for 20 min; washed with PBS three times, followed by incubation with 10% (w/v) BSA for 1 h at 37 °C; and then exposed to rat anti-CD31 (diluted 1:200, BD Pharmingen) antibody at 4 °C overnight. The sections were then washed in PBS three times and stained with a goat anti-rat IgG H&L (Alexa Rluor 647) secondary antibody (diluted 1:100, Santa Cruz Biotechnology, Inc.) in the dark for 1 h. Then, the nuclei were labeled with 4′,6-diamidino-2-phenylindole (DAPI). The sections were washed with PBS three times, covered with a coverslip, and observed with an inverted fluorescence microscope (Olympus IX 70; Olympus).

3. RESULTS AND DISCUSSION 3.1. Construction and Characterization of Prodrug Nanoparticle D-NPs/TET. The prodrug DSPE-DOX was synthesized by conjugating DSPE with DOX by a benzoic imine bond (Figures S1−S6). The resultant benzoic imine bond-linked DSPE-DOX was acid labile and could be cleaved at the acidic endo/lysosome. The TET-containing pH-sensitive DOX prodrug nanoparticle (D-NPs/TET) was prepared from the coassembly of DSPE-DOX, DSPE-PEG2000, and TET by the film dispersion method. The mass ratio of DOX and TET was selected as 4:1 according to previous reports.40 The molar ratio of DSPE-DOX and DSPE-PEG was optimized as 1:1 on the basis of the drug encapsulation efficiency, loading content, particle size distribution, and ζ potential (Table S1). The hydrodynamic diameter and ζ potential of the D-NPs/TET was examined by DLS. As shown in Figure 1a, the size distribution of the particles was approximately 28 nm, and the ζ potential 18453


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Figure 2. Effect of various drug formulations on intracellular drug accumulation and drug efflux. (a) CLSM imaging of the MCF-7R cells after incubation with naked DOX, DOX-TET, D-NPs, or D-NPs/TET for 4 h. Lysosomes were stained with LysoTracker Deep Red (green). Scale bars are 20 μm; (b) cellular uptake quantitative analysis in MCF-7R using flow cytometry after treatment with the above different drug formulations; (c, d) inhibition of DOX efflux in MCF-7R cells determined by flow cytometry. (e) P-gp expression of MCF-7R cells detected using western blotting after being treated with naked DOX, DOX-TET, D-NPs, or D-NPs/TET for 15 h.

was about −13 mV. The morphology of D-NPs/TET was recorded by TEM imaging (Figure 1b), which indicated that it was well dispersed as a nanosized droplet with a typical spherical shape. Stability is very important for the drug delivery systems to be applied in vivo. So, the stability of D-NPs/TET was determined under conditions similar to those in blood plasma. As shown in Figure S7, the D-NPs/TET was incubated with distilled water, PBS of pH 7.4 or 10% FBS. No significant size change was observed when dissolved in distilled water or PBS for up to 3 days. Although a slight size decrease was observed when dissolved in 10% FBS, the diameter of D-NPs/TET could be consistently maintained over a range of 23−25 nm for 3 days. These results indicated that the D-NPs/TET had good stability and were suitable for in vivo studies.

Next, the release kinetics of DOX and TET from D-NPs/ TET was monitored at pH 7.4 or 5.0 (Figure 1c,d). Unexpectedly, at pH 5.0, the physically encapsulated TET was released fast and in bursts over the initial 12 h, whereas slower and continuous release was observed with covalent conjugated DOX for up to 48 h. The fast-released TET primarily inhibited the P-gp expression in the tumor cells, followed by sustained activation and retention of DOX. Such a programmed release profile contributed to the intracellular stepwise sustained accumulation of DOX for the activation of the intrinsic apoptosis pathway. Besides, at pH 7.4, only 31.7% DOX and 32.8% TET were released from the particles even after 48 h incubation. The low drug release of DOX-NPs/TET at physiological pH will definitely be beneficial to reduce undesired side effects. 18454


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Figure 3. In vitro therapeutic efficacy of various drug formulations. (a, b) The in vitro cytotoxicity of naked DOX, DOX-TET, D-NPs, or D-NPs/ TET in MCF-7R (a) and MCF-7S (b) cells; (c) flow cytometry analysis of MCF-7R cell apoptosis induced by the above different drug formulations using the Annexin V-FITC/FVS450 staining.

3.2. Drug Resistance Reversal in MCF-7R Cells. We inspected the cellular uptake of D-NPs/TET in DOX-resistant MCF-7R cells. As shown in Figure 2a, indeed, the D-NPs/TET and DOX-TET (representative of the mixture of naked DOX and naked TET)-treated groups significantly presented strong fluorescence signals, whereas nearly no fluorescence was observed in TET noncontaining groups (naked DOX or DNPs), suggesting that the TET promoted the internalization and accumulation of DOX in resistant MCF-7R cells effectively. Moreover, the DOX fluorescence of the cells treated with DOX-TET was located in the nucleus, but it was also colocalized in the lysosomes and cytoplasm for the cells treated with D-NPs/TET, suggesting that the D-NPs/TET were

internalized and entered into the endo/lysosomes by endocytosis. The enhanced cellular uptake of D-NPs/TET in MCF-7R cells was further validated by flow cytometry (Figure 2b). The uptake of naked DOX in MCF-7R cells was obviously null, whereas it was practically effective for D-NPs/TET or DOX-TET. Collectively, these results suggested that the codelivery of DOX and TET increased the sensitivity of MCF-7R cells to DOX, making the DOX successfully enter and efficiently accumulate at cancer cells. Drug resistance of tumor cells is often associated with the overexpressed drug efflux pumps on the cell membrane.41 We therefore assessed the drug efflux in MCF-7R cells by flow cytometry (Figure 2c,d). The results showed that after being 18455


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Figure 4. Penetration ability evaluation of various drug formulations. (a) CLSM Z-stack scanning images of MCF-7R MCSs after being treated with naked DOX, DOX-TET, D-NPs, or D-NPs/TET for 5 h; (b) immunofluorescence staining images of the intratumor distribution of naked DOX, DOX-TET, D-NPs, or D-NPs/TET. The tumor vessels were stained with Alexa Rluor 647-tagged CD31 antibody (red), cell nuclei were stained with DAPI (blue), and DOX were represented by green.

3.3. Tumor Cell Killing of Various Drug Formulations. To reconcile the above exciting findings with the improved cell killing rate, we evaluated the antiproliferation efficiency of different drug formulations in MCF-7R cells. As shown in Figure 3a, the cell death rate was less than 20% after being treated with naked DOX for MCF-7R cells, whereas both DNPs/TET and DOX-TET exhibited strong cytotoxic effects to MCF-7R cells with IC50 values of 3.29 and 2.09 μg/mL. In contrast, the cytotoxicity was further evaluated in DOXsensitive MCF-7S cells (Figure 3b). Differently, the antiproliferation effect of naked DOX was higher than that of all nanodrugs, which might be attributed to the incomplete drug release of nanodrugs over a short period. In addition, for only TET-treated groups, the cell viability reached more than 74% in both MCF-7R and MCF-7S cells (Figure S9), indicating that TET with a low dosage nearly had no effect on cell viability. Furthermore, the death pathway of MCF-7R cells was examined by exploring annexin V-FITC and FVS450 staining methods. As we know, at the initial stage of apoptosis, the phosphatidylserine was exposed outside the cell membrane, and the annexin V-FITC bound to phosphatidylserine without permeabilization. When the late apoptosis stage induces the

treated with naked DOX, most of the drug was eliminated from MCF-7R cells within 1 h, whereas a less amount of the drug was expelled in D-NPs/TET or DOX-TET-treated groups even up to 4 h. These results highlighted that TET-containing DNPs/TET could make the DOX effectively escape the drug efflux action and lead to substantial intracellular drug accumulation in MCF-7R cells. It is of note that P-gp is the major efflux pump overexpressed on drug-resistant MCF-7R cells for opposing the cellular uptake of DOX.42,43 To investigate whether the D-NPs/TET could inhibit the expression of P-gp on the cell membrane, we examined the effect of different drug formulations on P-gp expression in MCF-7R cells using western blot analysis (Figure 2e). It was shown that P-gp had a very high expression level in MCF-7R cells after being treated with naked DOX. However, the treatment with TET-contained samples remarkably decreased the expression of P-gp in comparison with naked DOX or D-NPs. These phenomena indicated that the codelivery of TET and DOX by D-NPs/TET protected DOX from being extruded from the drug-resistant MCF-7R cells by inhibition of P-gp expression and hence facilitated the DOX to accumulate in the resistant cells sufficiently. 18456


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Figure 5. In vivo tumor accumulation. (a) In vivo fluorescence images of tumor-bearing nude mice at 3, 5, 8, 12, and 24 h post intravenous injection of D-NPs/TET&cy5. The tumors were visually indicated by the white circles. (b) Average fluorescence signals of tumor and normal organs 24 h post intravenous injection. (c) Ex vivo images of tumor and normal organs at 24 h after administration.

dropped sharply. By contrast, noticeable red fluorescence signals in the internal area of MCSs were detected from both D-NPs/TET and D-NPs even at a depth of 75 μm, demonstrating that the small size nanoparticles had deep tumor-penetrating characteristics due to their weak diffusional hindrance. In addition, it could be seen that the fluorescence signal in DOX-TET or D-NPs/TET-treated MCSs was much stronger than that in the DOX or D-NP treated groups, consistent with the above-mentioned high cellular accumulation of TET-containing groups. The enhanced tumor penetration was further validated by immunofluorescence staining. The tumors were excised from mice and sectioned, and the tumor vessels were labeled with fluorescent-tagged antibody against the endothelial marker CD31 (red). As shown in Figure 4b, for naked DOX or DOXTET treatment groups, only a little green fluorescence (DOX) could be seen, and they were colocalized with blood vessels. For the D-NP treatment group, the green fluorescence intensity in the tumor space was slightly weak. In contrast, highly concentrated D-NPs/TET with bright green fluorescence could be observed in the tumor space. Remarkably, a majority of DNPs/TET could efficiently extravasate from tumor vessels and penetrate into the tumor parenchyma, strongly suggesting the better tumor penetration of D-NPs/TET in vivo. 3.5. Tumor Accumulation of D-NPs/TET in Vivo. Importantly, it is well known that specific accumulation at the tumor sites is the prerequisite for tumor tissue penetration and intracellular drug accommodation. To evaluate the nanoparticle-mediated drug targeting, the in vivo distribution of D-NPs/TET was progressively measured in the tumorbearing mice model. The D-NPs/TET was labeled with the near-infrared fluorescence probe cy5 by physical encapsulation for determination. The naked cy5 dye alone was used as a

disruption of the cell membrane upon the onset of necrosis, the FVS450 entered into cells and interacted with intracellular amines. As shown in Figure 3c, neither annexin V-FITC nor FVS450-stained cells were detected for the untreated group. For the naked DOX or D-NP treated groups, the percentage of cells at the early apoptosis stage and late apoptosis stage were less than 10%. By contrast, a much higher percentage of apoptotic cells were clearly observed after treatment with DNPs/TET, indicating that the TET restored the drug sensitivity of MCF-7R cells to DOX and the codelivery of TET and DOX by D-NPs/TET induced resistant MCF-7R cell apoptosis. We further confirmed the flow cytometry data through employing another apoptosis assay by examination of the activity of the apoptosis factor caspase-3, which showed an identical trend. As shown in Figure S10, the increased expression of activated caspase-3 was detected clearly, when MCF-7R cells were treated with D-NPs/TET or DOX-TET. These results demonstrated that D-NPs/TET were considerably more effective than naked DOX or D-NPs in inhibiting cell proliferation in drug-resistant MCF-7R cells and ultimately led to apoptosis-induced anticancer activity. 3.4. MCS Penetration and Intratumor Spatial Distribution. Crossing the high tumor interstitial pressure and penetration into intratumors is a significant bottleneck for drug delivery to achieve an efficient therapy response.44 Here, the 3D-cultured MCSs derived from MCF-7R cells were chosen to mimic the solid tumor in vitro to evaluate the penetration efficacy of the D-NPs/TET. As shown in Figure 4a, the MCSs were incubated with DOX, DOX-TET, D-NPs, or D-NPs/TET for 5 h, then, the Z-stack image was observed by CLSM. Notably, for naked DOX or DOX-TET treatment, the red fluorescence attached to the periphery of the MCSs. At a depth of 45 μm, the fluorescence in the interior area of the MCSs 18457


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Figure 6. In vivo tumor growth inhibition studies. (a) Tumor growth curves of various drug formulations over the course of the treatments; (b) mice body weight changes of various drug formulations over the course of the treatments; (c) representative H&E sections of tumor tissue of tumorbearing nude mice after treatment with various drug formulations.

bearing mice is moderately better than that with naked drug treatment, which possibly attributed to the good drug availability of nanodrugs. Significantly, the treatment with DNPs/TET almost completely inhibited tumor growth. The average tumor size was nearly 4-fold smaller than that treated with naked DOX on the 19th day, confirming that the D-NPs/ TET possessed optimal efficiency in the suppression of drugresistant tumors. During the treatment period, the body weights of the mice were measured simultaneously, which did not show an obvious body weight drop (Figure 6b), indicating the reliability of the above assays and the negligible toxic side effects of D-NPs/TET. The antitumor effect of D-NPs/TET was further evaluated by a postmortem histopathology assay (Figure 6c). Consistent with the in vivo antitumor study, the DNPs/TET effectively promoted apoptosis of tumor cells, revealing the best therapeutic efficiency. To evaluate the potential damage of different drug formulations to major organs in tumor-bearing mice, we studied the postmortem histopathology of the heart, liver, spleen, lung, and kidney. As shown in Figure S12, the myocardial fiber breakage and liver injury were observed in the groups treated with naked DOX or DOX-TET, suggesting the possible cardiotoxicity and liver toxicity associated with the naked anticancer drug, whereas no obvious morphological change and organic damage were observed among the drugloaded NP treatment groups. To further examine the efficacy of these drug treatments, we measured the serum biochemical indices of all groups (Figure 7). Remarkably, significant differences were detected between the normal nude mice (control) and the tumor-bearing mice, which may be induced by the tumor xenografts. After treatment with D-NPs/TET, the liver, heart, and kidney functions of tumor-bearing mice tended to be at the normal level. However, for the mice treated with the naked drug, the organ functions showed serious abnormalities. These findings indicated that the D-NPs/TET

negative control. The realtime biodistribution and tumor accumulation were recorded at 3.0, 5.0, 8.0, 12.0, and 24.0 h post injection. As shown in Figure 5a, the fluorescence signal in the tumor sites was observed in D-NPs/TET&cy5 treated groups after 3 h, which implied its effective delivery to the tumor. As time elapsed, a notably increased D-NPs/TET&cy5 signal was observed in the tumor, whereas the fluorescence intensity in the normal sites continued to lower, indicating the superb targeting efficiency of D-NPs/TET&cy5. By contrast, an obviously decreased fluorescence intensity throughout the body was found for naked cy5 treated, even at 3 h post, and little fluorescence was located in the tumor regions (Figure S11a), which also confirmed the prolonged persistence of the nanodrug in bloodstream. Then, the mice were autopsied after 24 h injection and the ex vivo fluorescence of the major organs was further observed under IVIS (Figures 5b,c and S11b,c). It was found that the fluorescence from D-NPs/ TET&cy5 mainly localized in the tumor. The calculated results demonstrated that the relative fluorescence intensity from DNPs/TET&cy5 in the tumor to the total organs was about 36%. These exciting results further suggested the high accumulation of D-NPs/TET&cy5 in tumor tissue. 3.6. Tumor Therapeutic Effect and Potential Toxic Effects of D-NPs/TET in Vivo. Finally, we evaluated the in vivo tumor growth inhibition efficiency of different drug formulations. The tumor volumes were measured every 2 days after being intravenously injected with the following samples: saline, DOX, DOX-TET, D-NPs, and D-NPs/TET. As shown in Figure 6a, a fast tumor growth of the saline-treated group was observed within 19 days. The naked DOX and DOX-TETtreated mice showed little tumor growing inhibition compared to that of the saline-treated group, indicating that the naked drug barely had physiological activity against the resistant tumor due to poor biodistribution in the tumor. After being treated with D-NPs, the therapeutic effect on MCF-7R cell 18458


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Figure 7. Serum biochemical examination of tumor-bearing nude mice after treatment with various drug formulations. Normal BALB/c nude mice served as the control group. ALP, alkaline phosphatase; ALT, alanine aminotransferase; ALB, albumin; AST, aspartate transaminase; TBIL, total bilirubin; CK, creatine kinase; LDH, lactate dehydrogenase; BUN, blood urea nitrogen; CRE, creatinine.

did not have an obvious toxic effect on the major organs and demonstrated that such a nanodrug had good in vivo safety and biocompatibility.

response to the intracellular acidic endo/lysosomes for tumorselective and stepwise activation of the P-gp inhibitor TET and the anticancer drug DOX. The fast burst release of TET effectively inhibited the P-gp expression and subsequently the DOX accumulated in tumor cells in a sustained manner. Based on the advantage of successively overcoming these interconnected barriers, the D-NPs/TET achieved an efficient therapeutic outcome against drug resistance systematically. Concomitantly, the prodrug D-NPs/TET reduced the potential side effects of DOX in vivo due to the low risk of drug leakage. Therefore, this versatile nanosystem may open doors for the systemic treatment of chemoresistance.

4. CONCLUSIONS In summary, a nanoparticle-based therapeutic D-NPs/TET with the capability to adaptively cross a series of sequential biological barriers was successfully developed for combatting cancer resistance. Our results demonstrated that D-NPs/TET had the potential to prolong blood circulation, specifically accumulate in tumor tissue, and penetrate into the tumor deep parenchyma. Moreover, D-NPs/TET displayed an intelligent 18459


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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.7b01908. Detailed synthetic route and supporting results, 1H NMR and FT-IR spectra, nanoparticle stability, hydrodynamic diameter, cell viability evaluation, western blot analysis, in vivo fluorescence images, and representative H&E sections of various organs (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel: +86 10 82545614. Fax: +86 10 62656765. ORCID

Yan Wu: 0000-0001-8508-7077 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This study was supported by the National Natural Science Foundation of China (81472850) and supported by the Strategic Priority Research Program of the Chinese Academy of Sciences (Grant No. XDA09030301).

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