Tumor-Specific Multiple Stimuli-Activated Dendrimeric

Tumor-Specific Multiple Stimuli-Activated Dendrimeric Nanoassemblies with Metabolic Blockade Surmount Chemotherapy Resistance Yachao Li,†,§ Xianghui Xu,*,†,§ Xiao Zhang,† Yunkun Li,† Zhijun Zhang,† and Zhongwei Gu*,†,‡ †

National Engineering Research Center for Biomaterials, Sichuan University, Chengdu, Sichuan 610064, P.R. China College of Materials Science and Engineering, Nanjing Tech University, Nanjing, Jiangsu 210009, P.R. China

‡

S Supporting Information *

ABSTRACT: Chemotherapy resistance remains a serious impediment to successful antitumor therapy around the world. However, existing chemotherapeutic approaches are difficult to cope with the notorious multidrug resistance in clinical treatment. Herein, we developed tumor-specific multiple stimuli-activated dendrimeric nanoassemblies with a metabolic blockade to completely combat both physiological barriers and cellular factors of multidrug resistance. With a sophisticated molecular and supramolecular engineering, this type of tumor-specific multiple stimuli-activated nanoassembly based on dendrimeric prodrugs can hierarchically break through the sequential physiological barriers of drug resistance, including stealthy dendritic PEGylated corona to optimize blood transportation, robust nanostructures for efficient tumor passive targeting and accumulation, enzyme-activated tumor microenvironment targeted to deepen tumor penetration and facilitate cellular uptake, cytoplasmic redox-sensitive disintegration for sufficient release of encapsulated agents, and lysosome acid-triggered nucleus delivery of antitumor drugs. In the meantime, we proposed a versatile tactic of a tumor-specific metabolism blockade for provoking several pathways (ATP restriction, apoptotic activation, and anti-apoptotic inhibition) to restrain multiple cellular factors of drug resistance. The highly efficient antitumor activity to drug-resistant MCF-7R tumor in vitro and in vivo supports this design and strongly defeats both physiological barriers and cellular factors of chemotherapy resistance. This work sets up an innovative dendrimeric nanosystem to surmount multidrug resistance, contributing to the development of a comprehensive nanoparticulate strategy for future clinical applications. KEYWORDS: multiresponsive nanoassemblies, dendrimeric prodrugs, metabolic blockade, multidrug resistance reversal, deep tumor penetration

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physiological barriers and cellular factors of chemotherapy resistance up to now.5,6 Very recently, supramolecular dendritic assemblies have attracted increasing attention for the development of sophisticated nanomaterials for nanomedicine,13−15 which have been exploited as a therapeutic entity for reversing drug resistance owing to their large dendritic void space for drug loading.16 However, the complicated MDR mechanisms are so difficult to cope with that more rational molecular and supramolecular engineering are urgently needed to invent a versatile supramolecular dendritic system for surmounting the physiological and pathological obstacles of drug resistance.17,18 Current advancements reveal that developing multiresponsive

linical cancer chemotherapy often fails due to lethal side effects and multidrug resistance (MDR).1,2 Encouragingly, numerous clinical evidence has proven that nanoparticulate formulations (e.g., Doxil, Genexol-PM, and Abraxane) largely decrease systemic toxicity of chemotherapeutics.3 However, drug resistance poses a serious threat to cancer patients for subsequent metastasis and recurrence, due to the intricate mechanism of chemotherapy resistance including sequential physiological barriers (e.g., poor blood circulation, undesirable drug penetration, and inadequate drug influx) and multiple cellular factors (e.g., high drug efflux capacity, reduced apoptosis, and elevated anti-apoptosis).4−6 Although massive efforts have been devoted to develop advanced nanovehicles based on inorganic,7,8 organic,9,10 and hybrid nanomaterials11,12 for retarding drug resistance, few appropriate strategies have been established to combat both © 2016 American Chemical Society

Received: September 12, 2016 Accepted: December 22, 2016 Published: December 22, 2016 416

DOI: 10.1021/acsnano.6b06161 ACS Nano 2017, 11, 416−429

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Scheme 1. Schematic Illustrations of (A) Molecular and Supramolecular Engineering on Tumor-Specific Multiple StimuliActivated Dendrimeric Nanoassemblies with Metabolic Blockade and (B) Their Synergistic Effects for Overcoming Physiological Barriers and Cellular Factors of Chemotherapy Resistance

specific burst drug release for fast killing cancer cells. For these reasons, customizing multiresponsive supramolecular dendritic systems with invincible systemic transportation, efficient tumor targeting, deep drug penetration, and site-specific sufficient delivery remains a valuable and challenging task for conquering sequential physiological barriers of drug resistance. On the other hand, we manage to put forward a versatile tactic to assist dendritic assemblies for reversing MDR cellular factors, directing against universal tumor pathophysiology. In contrast to normal cells, malignant tumor cells reprogram their metabolism and overly rely on aerobic glycolysis, which is termed as “Warburg effect”.32 This abnormal metabolism plays a crucial role in affording tumor anabolic and energetic demands (e.g., nucleic acids, proteins, lipids, and energy in the form of ATP) for uncontrolled tumor progression.33,34 With inspections of cellular drug-resistant mechanisms, aberrant metabolism is closely related to the drug-resistant generation, including drug efflux capacity by ATP-driven transporters, suppression on apoptotic signal transduction, and activation of anti-apoptotic pathway. Although some studies show that blocking tumor metabolic adaptations stands a chance for cancer therapy, rare practical protocols on metabolism regulation have been proposed for MDR reversal, owing to limited antitumor effects by metabolic blockade alone and poor bioavailability of metabolism blockers.33−36 Fortunately, a tailor-made supramolecular dendritic system not only is expected to defeat multiple physiological barriers but also serves as a versatile nanoplatform to resolve the aforementioned dilemma of tumor metabolic blockade for MDR reversal,

nanocarriers holds a great potential to readily adapt diversiform biological barriers,19−21 allowing to resolve the contradictions among long blood circulation, high tumor accumulation and efficient cellular internalization. Furthermore, tumor-specific responsiveness is also regarded as an emerging and efficient targeting strategy such as tumor microenvironment targeting,22 thereby highly selective and active tumor responsiveness, such as tumor-associated enzyme sensitivity,23−25 is imminently pursued for tumor-activated targeted delivery systems to address tough drug resistance barriers.26 Despite continuous advancements on smart dendritic systems response to a certain stimulus for molecular delivery, there have been rare reports on multiresponsive dendritic assemblies that meet the complicated demands on systemic therapeutic delivery.27 Noticeably, imperfect drug penetration in tumor frequently results in the rapid evolution of chemotherapy resistance,28,29 whereas most research on physiological barriers neglects the importance of drug distribution within the tumor tissue. It is encouraged that some smart nanoparticles with tunable components and nanostructures (e.g., sheddable PEGylation and shrinkable size) can largely enhance deep drug penetration of tumor tissue.30,31 Therefore, endowing dendritic assemblies with tunable tumor permeability is hopeful to facilitate drug penetration at the tumor site and to lower drug resistance. Last but not least, insufficient drug release is another main reason for the progression of drug resistance, because it badly restricts the intracellular drug concentration from reaching the therapeutic window.19 Constructing dendritic systems with sitespecific disintegration must be the best way to achieve site417


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Figure 1. Molecular multisensitivities of DPs. (A) RP-HPLC curves and (B) MALDI-TOF MS results of DPs (control) and DPs treated with MMP-2 (2 μg mL−1), DTT (10 mM), and acid conditions (pH 5.0) at 37 °C for 3 h, respectively.

Figure 2. Nanostructure and stimuli-responsive properties of dendrimeric nanoassemblies. (A) Size distribution, TEM and AFM images of DNs. (B) CD spectrum of supramolecular DNs. (C) Size variations (left) and TEM images (right) of DNs in the different biomimetic conditions. (D) DOX release profiles of the multiple-activated DNs in different biomimetic buffer solutions at 37 °C (means ± standard deviation (SD), n = 3).

between peptide dendrons, and subcellular lysosome acid-labile hydrazone bonds for dynamic conjugation of hydrophobic antitumor drugs (doxorubicin, DOX). Self-assembling these amphiphilic DPs into dendrimeric nanoassemblies (DNs) can generate enough void space for encapsulation of therapeutic agents as well as site-specific controlled release. The supramolecular nanoparticulate DPs are expected to hierarchically knock down the multiple physiological barriers of drug resistance with following advancements (Scheme 1B): (i) clinically proven PEGylated corona for optimal drug blood transportation; (ii) robust dendritic nanostructures to enhance passive targeting; (iii) tumor microenvironment targeting activated by drug-resistant tumor-overexpressed MMPs to promote drug accumulation; (iv) tumor-adaptive size and interface for deep tumor penetration; and (v) intracellular

thanks to the great potentials of supramolecular dendritic systems on multimodal drug transportation (e.g., multivalence for drug conjugation and abundant void space for drug encapsulation) and tumor-activated site-specific delivery. To confirm our concepts, we hope to build a tumor-specific multiple stimuli-activated supramolecular dendritic system with metabolic blockade to smoothly surmount both sequential physiological barriers and complicated cellular factors of multidrug resistance (Scheme 1A). Taking full advantage of controllable, highly branched and multivalent dendrimeric features,37−40 we devised tumor-specific multiple stimuliactivated dendrimeric prodrugs (DPs) with tumor microenvironment matrix metalloproteinase (MMP) sensitive peptides (GPLGLAG sequence) to decorate hydrophilic PEG segments, cytoplasmic redox-cleavable disulfide linkages 418


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Figure 3. Encapsulation of guest molecules and in vitro controlled release. (A) I372/I383 ratio in the emission spectra of pyrene vs DN concentrations in aqueous solution (means ± SD, n = 3). (B) Emission spectra of LND vs DN concentrations in aqueous solution. (C) Size distribution and TEM image of L-DNs in aqueous solution. (D) LND release profiles of L-DNs in different biomimetic TCNB buffer solutions at 37 °C (means ± SD, n = 3).

25.2 to 16.2 min following treatment with 2 μg mL−1 MMP-2, which is the most common MMP within drug-resistant tumors,42,43 suggesting that MMP-2 triggered the breakage of enzyme-sensitive peptides (Figure 1A). Mass spectrum verified that molecular fragments of mPEG-GAL-NH2 were removed from dendrimeric skeletons (Figure 1B). After incubation with 10 mM dithiothreitol (DTT) corresponding to intracellular redox condition, the changes in the RP-HPLC curve and the MS result should be attributed to the cleavage of disulfide linkage and disassociation of dendritic fragments. In addition, DPs could maintain stability under the condition of 10 μM DTT in analogy to extracellular environment (Figure S20). As speculated, hydrophobic fragments of two drugs would be discharged from dendrimers at pH 5.0 simulating lysosomal condition with a decreased molecular weight, but weak acid conditions (e.g., pH 6.8 at tumor microenvironment) did not influence the molecular structure of DPs (Figure S20). Altogether, we successfully synthesized tumor multiresponsive DPs, which are in urgent demand by nanomedicine and oncology. Supramolecular Fabrication and Multi-Responsive Features of DNs. In an aqueous medium, the amphiphilic DPs self-assembled into well-defined nanostructures with a hydrophilic PEGylated corona and a hydrophobic drug core, having an average size of 124.5 ± 3.0 nm by dynamic light scattering (DLS) detection (Figure 2A). Transmission electron microscopy (TEM) image and atomic force microscopy (AFM) image presented spherical nanostructures and three-dimensional (3D) architectures of DNs with a diameter of ∼130 nm. With supramolecular self-assembly of peptide dendritic components, DNs possessed highly ordered architectures with a typical secondary structure of 12.4% α-helix, 40.7% βsheet, 15.2% β-turn, and 31.7% random coil by circular dichroism (CD) spectroscopy (Figure 2B).44,45 Zeta potential

stimuli-disintegration for site-specific sufficient drug delivery. Also, we aim at targeting tumor hexokinase (HK) to arouse multiple pathways against complicated cellular factors of drug resistance, because HKs catalyze the first committed step of glycolysis metabolism.33−35 It is noted that HK2 is overexpressed in tumor cells, and inhibiting the activity of HK2 synchronously prevents substance and energy metabolism.41 In this regard, we attempt to interdict tumor metabolism by a HK2-targeting therapeutic agent for accelerating drug-resistant tumor cell death. As a result, it is reasonable to believe that tumor multiple stimuli-activated dendrimeric nanoassemblies with metabolic blockade can completely surmount the physiological barriers and cellular factors of multidrug resistance in cancer treatment.

RESULTS AND DISCUSSION Molecular Engineering and Multiple Sensitivities of DPs. The amphiphilic DPs were synthesized using a divergent−convergent approach, and the synthetic routes were illustrated in Supporting Information (Schemes S1−S6). The final matrix-assisted laser desorption ionization time-offlight mass spectroscopy (MALDI-TOF MS) result for DPs agreed well with the theoretical range of molecular weight distribution (Figure S19). The detailed characterizations of synthetic compounds can be found in Figures S1−S19. Additionally, this tumor multiple-activated molecular design can be extended to develop more DPs involving other dynamic bonds, such as tumor microenvironment acid-sensitive linkage,39 cytoplasmic caspase-3 responsive peptide, and lysosome cathepsin-B cleavable peptide.25 The multiresponsive behaviors of DPs were monitored by reversed-phase high-performance liquid chromatography (RPHPLC) analysis and MALDI-TOF MS measurement. As shown in the RP-HPLC curves, the elution time of DPs shifted from 419


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ACS Nano of DNs was −17.2 mV (Figure S21). The aggregation-caused quenching phenomena of DOX fluorescence also indicated the formation of compact DNs with the π−π stacking interactions in H2O (Figure S22). Moreover, DNs maintained nice dimensional stability and negative zeta potentials for a relatively long time, implying that our robust DNs would be beneficial to prolong blood circulation and minimize reticuloendothelial system clearance (Figures S23 and S24). Next, we investigated the stimuli-responsive size and structure transition of DNs under the various biomimetic environments. Incubation of DNs with MMP-2 (2 μg mL−1) caused a decrease in diameter to 70 nm, due to MMP-triggered deshielding of PEG corona and stronger hydrophobic interactions among DPs (Figure 2C). The MMP-activated PEG-sheddable and size-shrinkable DNs may promote tumor penetration and celluar internalization,30,31 thus helping to overcome drug resistance. After exposure to 10 mM DTT, redox-activated DNs converted into two types of supramolecular aggregations as shown in the DLS result and TEM image. It was inferred that redox cleavage of disulfide linkages induced rearrangement of the hydrophobic dendrons into larger aggregations (∼800 nm) and the relatively hydrophilic dendrons into loose nanoparticles (∼250 nm), and this phenomenon was good according to redox-responsive behaviors of linear polymeric assemblies.46 Thus, vast void pockets of DNs can be utilized as smart nanocontainers for site-specific molecular delivery. With the pH value adjusted to 5.0, DNs were dissipated into small single PEGylated dendrimers (∼18 nm), due to disassociation of the hydrophobic drug and protonation of dendrimers. Additionally, DNs were able to tolerate certain tumor microenvironment conditions (Figure S26). Furthermore, we determined in vitro DOX release profiles of DNs by dialysis tubes (MW cutoff of 1000 Da) in various biomimetic TCNB buffer solutions. In the line with the molecular and supramolecular stimuli-sensitivities, pH 5.0 would trigger the burst release of native DOX due to the acid-labile hydrazone bonds and sudden DN disassembly (Figure 2D), which was confirmed by electrospray ionization time-of-flight mass spectrometry (ESI-TOF MS, Figure S27). On the contrary, DNs were able to steadily hold drugs against several extracellular conditions, such as the biomimetic conditions of 2 μg mL−1 MMP-2, pH 6.8, and 10 μM DTT (Figures 2D and S28). Consequently, our DNs can prevent premature leakage of antitumor drugs and recognition by drugresistant transporters, but deliver drugs in tumor-specific conditions and to subcellular sites. Molecular Entrapment and Controlled Release. An increasing number of studies prove that supramolecular dendritic systems provide high molecular entrapment capacity, as compared to other organic nanocarriers such as liposomes and micelles. Since the intensity ratio between the first and the third peaks in the emission spectrum of pyrene (I1/I3) is sensitive to the polarity of local environment, pyrene was first used as a probe to investigate the encapsulation ability of DNs as guest molecules. As shown in Figure 3A, the curve of I1/I3 ratio (I372/I383) began to decrease after the DNs reached a fairly low concentration (∼2 μg mL−1), supporting that DNs can readily entrap guest molecules into their supramolecular cavities. Likewise, DNs also served as universal nanocarriers to load other hydrophilic and hydrophobic molecules (e.g., calcein and DiIC1(5), Figures S29 and S30). It is able to predict that the stimuli-responsive void spaces of DNs are advanta-

geous to integrate the metabolism-blocking capacity for reversing MDR. In this work, we selected lonidamine (LND) as a model molecule to inhibit overexpressed HK2 in drug-resistant cells, owing to its high potentials for clinical application (Phase III trials).33,34 With the assistance of tumor multiple-activated DNs, LND is expected to interdict tumor metabolism and combat cellular drug-resistant factors by minimizing drug efflux, apoptotic promotion, and anti-apoptotic inhibition. With the increasing DN concentration, the attenuation of LND fluorescence indicated the successful molecular encapsulation and construction of LND-loaded DNs (L-DNs, Figure 3B). DLS and TEM results demonstrated that L-DNs kept the welldefined nanostructures (about 130 nm in diameter, Figure 3C) and zeta potential (−17.8 mV, Figure S31) as original DNs. And maximum loading capacity of LND reached up to 21%. In vitro LND release profiles manifested that (i) intracellular reductive circumstance (10 mM DTT) could activate the sufficient release of LND in the cytoplasm (Figure 3D) and (ii) DNs steadily carried therapeutic agents against normal physiological conditions and tumor extracellular microenvironments to prevent premature leakage (Figure S32). In Vitro Antitumor Activity to MCF-7R Tumor Cell Line. Once we confirmed the fabrication of the tumor multipleactivated dendritic systems, antitumor efficiency was evaluated against DOX-resistant MCF-7 human breast cancer cells (MCF-7R) using CCK-8 arrays. As shown in Figure 4A, the

Figure 4. In vitro antitumor activity and MDR reversal. (A) Cell viability of MCF-7R tumor cells vs concentrations of various formulations with 48 h incubating, respectively (means ± SD, n = 6). (B) IC50 value of various formulations to MCF-7R tumor cells.

dendrimeric skeleton without drug conjugation was obviously nontoxic to MCF-7R tumor cells, but DNs showed much better antitumor effects to MCF-7R cells compared to DOX and DOX·HCl. The IC50 value (the concentration causing 50% growth inhibition) of DNs incubation with MCF-7R tumor cells was 17.8 μg mL−1 (DOX concentration, Figure 4B), which was obviously lower than that of native DOX (IC50 = 55.7 μg mL−1) and the positive control (DOX·HCl, 27.4 μg mL−1). These results indicated the tumor-activated DNs could circumvent cellular barriers to suppress drug resistance. Then, we began to investigate the utility of incorporation with metabolic blockade for combating drug resistance. In line with some previous reports on targeting tumor metabolism alone,35,36 single treatment with LND had very little impact on cell viability of MCF-7R cells even at the relatively high concentration of 10 μg mL−1. Nevertheless, encapsulating LND into DNs represented large improvement on cytotoxicity to 420


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Figure 5. Tumor multiple-activated DNs for combating physiological barriers at cellular level. (A) DOX positive cells and MFI for quantitative cellular uptake after incubation with DOX, DOX·HCl, and DNs for 2 h (means ± SD, n = 3, *p < 0.001). (B) Flow cytometric profiles of MCF-7R cells incubated with DNs in the presence of Phen and MMP-2 or without treatment. (C) CLSM images for untreated, BSOpretreated, and GSH-OEt-pretreated MCF-7R cells after incubation with DiIC1(5)-loaded DNs for 4 h, including Alexa Fluor 488 phalloidinstained cytoskeleton channel (green), DOX channel (red), and DiIC1(5) channel (blue). (D) CLSM images for tracking DOX delivery with LysoTracker-stained lysosome channel (green) or Hoechst 33342-stained nucleus channel (blue).

incubation, supplemental MMP-2 facilitated drug internalization and antitumor activity to a certain degree, whereas cellular uptake of drug markedly decreased in the existence of MMP-inhibitor due to the failure of PEG-deshielding. To track the intracellular-activated delivery, dendrimeric systems were monitored by CLSM, and DiIC1(5) was still used as a fluorescence guest molecule (blue). CLSM images showed that red fluorescence (DOX) and blue fluorescence totally overlapped in MCF-7R cells within 2 h (Figure S39), yet a portion of blue fluorescence disassociated with red fluorescence within 4 h (cell cytoskeleton was stained by Alexa Fluor 488 phalloidin with green, Figure 5C), suggesting intracellular reductive conditions triggered the disintegration of DNs and cytoplasmic delivery of encapsulated molecules. Furthermore, after pretreatment with glutathione monoester (GSH-OEt), upregulated intracellular reductive levels of MCF-7R cells accelerated the disassociation of blue fluorescence from red fluorescence as well as wide distribution in cytoplasm within 4 h. In contrast, downregulation of intracellular reductive condition by buthionine sulfoximine (BSO) distinctly lowered the intracellular fluorescence intensity and limited the fluorescence separation, because insufficient degradation of DNs hindered sufficient release of guest molecules. Meanwhile, L-DNs showed a lower cytotoxicity to BSO-pretreated MCF7R cells as compared to the untreated MCF-7R cells with 24 h incubation (Figure S40), indicating the importance of sufficient

MCF-7R cells, and IC50 value of L-DNs further reduced to 8.4 μg mL−1 (weight ratio of LND and DOX was fixed at 1:2, which is an optimal ratio for biological studies in consideration of loading capacity and antitumor efficiency, Figure S34), demonstrating apparent potentials of L-DNs for MDR reversal. Combating Cellular Biological Barriers. Encouraged by the satisfactory antitumor activity to MCF-7R cells, we started to disclose the processes of our design for defeating cell biological barriers. Quantitative drug intake from flow cytometry manifested that almost all MCF-7R cells (99.98%) took in DOX after exposure to DNs for 2 h, much more than other groups (Figure 5A). Moreover, the DOX mean fluorescence intensity (MFI) in the DN-treated group elevated to 10.7-fold as compared to the DOX·HCl group (*p < 0.001), indicating that cell membranes are the first key barriers broken down by the MMP-activated DNs. The corresponding phenomena could be observed by confocal laser scanning microscopy (CLSM) images (Figure S35). Of course, DNs also can largely increase the cellular uptake of encapsulated molecules such as DiIC1(5) (Figure S36). The prominent drug internalization mediated by DNs should be ascribed to the MMP-triggered PEG-deshielding and more compact dendrimeric nanostructures. To clarify tumor extracellular MMPtargeted drug delivery, we quantitatively evaluated the drug internalization in the presence of MMP-inhibitor (phenanthroline, Phen) and additional MMP-2 (Figure 5B). Within 2 h of 421


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Figure 6. Metabolism-blocked L-DNs for combating cellular factors of MDR. (A) Relative hexokinase activity and (B) ATP content of MCF7R cells after treatment with LND and L-DNs at different concentrations for 24 h (means ± SD, n = 3, *p < 0.01). (C) The drug efflux from MCF-7R cells. MCF-7R cells were incubated with various formulations for 2 h and then incubated with fresh media for detection (means ± SD, n = 3, *p < 0.05, **p < 0.005). (D) Flow cytometric profiles (left) and CLSM images (right) for mitochondrial membrane potentials in MCF-7R cells after treatment with DOX, DOX·HCl, LND, DNs, and L-DNs for 24 h with MitoView 633-stained mitochondria channel (red) and Hoechst 33342-stained nucleus channel (blue). (E) Western blot analysis for protein levels of caspase-3 and Bcl-2 expression in MCF-7R cells after incubation with various formulations for 48 h. (F) CLSM images (left) and quantitative results (right) for caspase-3 activity in MCF-7R tumor cells treated with various formulations for 36 h (means ± SD, n = 3, *p < 0.005). (G) Apoptosis analysis for MCF-7R tumor cells induced by various formulations for 36 h incubation using Annexin V-FITC/PI detection assay by flow cytometry. (H) Schematic illustration for synergetic mechanisms on combating cellular factors of chemotherapy resistance by L-DNs.

activated nucleus delivery heavily attacked cellular barriers to reverse MDR. Overcoming Cellular Factors of MDR. Following the analysis of intracellular fate, we turned to reveal the mechanism of L-DNs on combating cellular factors of drug resistance. After exposure to LND for 24 h alone, there was a modest decrease of HK activity of around 10% (Figure 6A). DN-mediated LND delivery for blocking metabolism caused a ∼50% decrease in HK activity, reflecting the high efficiency on sufficient sitespecific delivery of LND. As predicted, DNs dramatically promoted the LND capacity to terminate ATP production in MCF-7R cells (Figure 6B), thereby stopping biological functions of ATP-driven MDR transporters. The multiple-

LND delivery in cytoplasm for killing drug-resistant cells. When the incubation time was increased from 6 to 8 h, a large amount of DOX successfully escaped from the lysosomes and widely dispersed into the cytoplasm due to the acid-activated drug release (Figure 5D). Finally, DN-mediated DOX was delivered into the nuclei (Hoechst 33342 staining) to exert antitumor activity, but little DOX fluorescence was monitored in the DOX·HCl-treated cells (Figure S42). Nuclear delivery is an important index for MDR reversal, especially for many clinical antitumor drugs which played their therapeutic roles in the nuclei (e.g., DOX and cisplatin). Thus, it can be seen that the redox-activated disintegration for sufficient delivery and acid422


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Figure 7. In vitro and in vivo drug penetration of tumor-activated DNs. (A) CLSM images for 3D MCF-7R MTSs after treatment with DOX, DOX·HCl, and DNs for 3 h as well as coincubation with MMP-2 or Phen. Optical MTS sections imaged by CLSM from the top to bottom with 5 μm per section. (B) Percentage of DOX-positive cells and DOX MFI in MCF-7R MTSs quantified by flow cytometry (means ± SD, n = 3, *p < 0.001). (C) CLSM images for cryosections of MCF-7R tumor after intravenous administration of DOX·HCl and DNs for 12 h, including AF594-CD31 stained blood vessel channel (green), DOX channel (red), and DAPI-stained nucleus channel (blue).

Treatment with DOX and DOX·HCl had no significant influence on the caspase-3 mRNA level, while LND, DNs, and L-DNs caused 13.3%, 184.9%, and 297.4% upregulation of caspases-3 mRNA level, respectively. Unexpectedly, DOX, DOX·HCl, and LND upregulated the Bcl-2 mRNA level, but DNs and L-DNs led to 63.9% and 84.9% downregulation of Bcl-2 mRNA level. More importantly, we determined the caspase-3 activity using fluorescence-activatable NucView 405.48 The green fluorescence representing the active caspase-3 proteins was strikingly enhanced by L-DNs, whereas low fluorescence intensity was observed in the CLSM images for other groups (Figure 6F). As compared to the blank control, the relative caspase-3 activity was elevated more than 6 times in the L-DNs group (*p < 0.005). Overall, the metabolism-targeted L-DNs altered cell signaling pathways to hasten death of drug-resistant cells. After disclosure of their impact on signaling transduction, we carried out cell-cycle analysis and cell-apoptosis detection to investigate the decisive role on the drug-resistant cell fate. Due to tough drug tolerance, a single treatment with DOX failed to arrest the cell cycle at the G2 phase (∼18.6%, Figure S46), analogous to the untreated control group (∼18.7%). Compared with the G2 arrest levels induced by LND (∼17.5%) and DNs (∼41.9%), higher cell arrestment caused by L-DNs (∼54.1% in G2 phase) verified the tumor-activated DNs combined metabolic blockade greatly restored pharmaceutical sensitivity in MCF-7R cells, owing to synergetic efficacies. In the

activated DNs not only enhanced drug internalization but also decreased drug elimination (Figure 6C). ATP reduction by LDNs further restricted drug efflux (19.2% drug loss) after 6.0 h. The fluorescent images confirmed that the prominent drug intake and low drug efflux of L-DNs jointly contributed to the highest drug accumulation in the MCF-7R tumor cells in all treatment groups (Figure S43). Thereupon, we assessed the influence of L-DNs on cell signaling transduction. As shown in the Figure 6D, it was clearly found that blocking metabolism by L-DNs notably provoked a most significant disturbance on the mitochondrial membrane potentials (ΔΨm) of MCF-7R cells, which were performed using intracellular fluorescence intensity of MitoView 633 kit.47 By comparison with untreated MCF-7R cells, ΔΨm declined by 88.6% after incubation with L-DNs for 24 h, much more efficient than single LND treatment (Figure S44). The CLSM images visualized a notable dissipation of ΔΨm with the lowest red fluorescence in the L-DN treated MCF-7R cells. As expected, such severe mitochondrial membrane depolarization by L-DNs largely upregulated the caspase-3 protein level (Figure 6E), which is a crucial mediator of programmed apoptosis but often lowly expressed in drug-resistant tumor cells. In contrast to other control groups, L-DNs also revealed the highest ability to repress anti-apoptotic Bcl-2 protein expression in MCF-7R cells. Concurrently, mRNA expressions of both caspase-3 and Bcl-2 were identified by quantitative polymerase chain reaction (qPCR) analysis (Figure S45). 423


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Figure 8. In vivo tumor treatment and MDR reversal. (A) Tumor volume changes of nude mice bearing MCF-7R tumor administered with normal saline, DOX·HCl, DNs, and L-DNs by intravenous injection (2.5 mg LND kg−1 and 5.0 mg DOX kg−1, means ± SD, n = 6, *p < 0.001). (B) Tumor growth inhibition after treatment with DOX·HCl, DNs, and L-DNs over 24 d (means ± SD, n = 6, *p < 0.001). (C) Histological and immunohistochemical analysis of H&E, CD31, Ki-67, and TUNEL assays for MCF-7R tumors. CD31-positive vessels, Ki-67positive cells, and TUNEL-positive cells are stained brown. (D) In vivo pharmacokinetics (means ± SD, n = 3) and (E) drug distribution of DOX·HCl, DNs, and L-DNs (5.0 mg LND kg−1 and 10.0 mg DOX kg−1). (F) CLSM images for cryosections of MCF-7R tumors after 12 h intravenous administration, including DOX channel (red) and DAPI-stained nucleus channel (blue).

meantime, we detected the apoptosis of MCF-7R cells induced by the diverse formulations using the Annexin V-FITC/PI detection assay (Figure 6G). L-DNs resulted in the highest late apoptotic ratio of 55.2% in MCF-7R cells, yet other treatment with DOX, DOX·HCl, LND, and DNs just induced lower apoptosis of MCF-7R tumor cells by 3.6%, 5.1%, 0.5%, and 36.8%. In general, L-DNs caused the apoptosis of drug-resistant tumor cells and were much better than the sum of each formulation effects alone. To well illustrate the multiple cooperative mechanisms of dendrimeric nanoassemblies with metabolic blockade for surmounting chemotherapy resistance, we summarized the pivotal processes on the cellular level in the schematic diagram (Figure 6H). At first, MMP-activated PEG-deshielding and sizeshrinkable L-DNs can effectually circumvent membrane barriers and promote the cellular uptake of therapeutic agents. Besides, the nanoparticulate prodrug strategy could help drugs avoid recognition by MDR transporters. Once L-DNs were internalized into drug-resistant tumor cells, the intracellular high glutathione level led to the disintegration of L-DNs and exhaustive release of the metabolism-blocking agents. Soon after, lower pH conditions in lysosomes broke dynamic bonds to release the original antitumor drugs and facilitate nuclear delivery. As a matter of fact, the tumor-activated DNs already reduced drug resistance by optimized drug bioavailability. What’s more, metabolism-blocking strategy simultaneously evoked the multiple pathways to strengthen the drug-resistant

reversal including ATP depletion, apoptotic enhancement, and anti-apoptotic reduction. It is concluded that this design succeeded in defeating biological barriers and cellular factors to kill drug-resistant tumor cells. Tumor-Activated Deep Penetration in Vitro and in Vivo. To investigate whether our multiple stimuli-activated DNs resolved the difficulties of poor drug distribution in tumors, we evaluated the drug penetration using multicellular tumor spheroids (MTSs, about 200 μm in diameter) as 3D tumor models and tumor-bearing mouse models. After incubation for 3 h, low fluorescence signals were detected in the MCF-7R MTS across each equatorial section with DOX treatment, meanwhile a significant decrease of drug fluorescence intensity from the rim to the center was found in the DOX·HCl group (Figure 7A), because of inherent poor distribution of native drugs. Excitingly, high drug fluorescence was observed throughout the MTS sections after incubation with DNs, indicating enhanced drug penetration followed by the MMP-activated deshielding of PEGylated corona and sizeshrinkable nanostructure. To confirm MMP-activated penetration, we pretreated MTSs with a MMP inhibitor, thereby the drug fluorescence observably attenuated in the kernel area of MTS for limited penetration ability after the inhibition of MMP activity. And supplementary MMP-2 contributed to better drug penetration in MTSs. DNs also strongly assisted guest molecules to deeply penetrate in MTSs (Figure S48). Moreover, quantitative results verified that nearly all tumor 424


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L-DNs were more favorable for the drug accumulation at tumor sites with the discernible fluorescence signals, whereas little drug signals were captured in the DOX·HCl-treated group. Along with the extending of time, much stronger drug signals appeared at the solid tumor site, ascribing to the enhanced permeability and retention (EPR) effects. After the mice were sacrificed at the end of viewing time, the ex vivo fluorescence imaging for major organs and tumors also revealed that the antitumor drug was inclined to accumulate at the drug-resistant solid tumor (Figure S52). The in vivo drug distribution quantified by RP-HPLC also suggested that the DNs increased the drug distribution in tumor tissues, and DNs combined metabolism blockade further enhanced DOX accumulation at the tumor (Figure S53). Cryosections of tumor tissue manifested that DNs implemented nucleus delivery of antitumor drug in vivo, and integration with metabolism interdiction predominantly made more of the drug accumulate at the tumor site and drug-resistant cells (Figure 8F). These in vivo antitumor results suggested that the tumor-activated LDNs conquered physiological barriers during blood circulation, enhanced drug accumulation at tumor site, minimized the adverse effects of chemotherapy, and finally inhibited the growth of drug-resistant xenograft tumors.

cells in MTSs (98.2%) took in antitumor drugs, owing to the enhanced drug penetration by the tumor-activated DNs (Figure 7B), much more than the DOX group (31.4%) and DOX·HCl group (66.5%). In light of the excellent in vitro tumor penetration of DNs, in vivo drug distribution at tumor sites was observed by the cryosections using nude mice bearing MCF-7R xenograft tumors.49 CLSM images showed that a small amount of autofluorescent DOX·HCl was just distributed around the CD31-stained blood vessels (green), while DNs deeply penetrated into the tumor tissue much farther away from the tumor vessels (Figure 7C). The larger penetrating area and much stronger fluorescence signals should profit from MMPactivated DNs in tumor microenvironments. Taken together, we concluded that our design of tumor multiple activated DNs swimmingly overcame sequential biological obstacles of drug resistance including deep tumor penetration, enhanced drug internalization, sufficient site-specific release, and nucleartargeted delivery. In Vivo Chemotherapy Resistance Reversal. Motivated by the satisfactory performances of L-DNs on overcoming the tissue/cellular barriers and cellular factors of drug resistance, we evaluated in vivo therapeutic efficacy using nude mice bearing a MCF-7R tumor following systemic administration. As presented in Figure 8A, the rapid tumor growth of the DOX· HCl-treated mice must result from the serious resistance of MCF-7R xenograft tumors to antitumor drugs. However, average tumor volume of the DNs-treated mice was only half that of the DOX·HCl-treated mice. Relative to other groups, the metabolism-blocked L-DNs exhibited maximal antitumor activity against the MCF-7R tumors, without apparent growth in tumor size. At the end of the treatment course, tumor growth inhibition rates of DNs and L-DNs, respectively, reached to 69% and 84% (Figure 8B) as compared to the saline-treated group, which is much more efficient than DOX·HCl treatment (∼32%, *p < 0.001). Afterward, histological and immunohistochemical analyses showed that systemic administration of DNs and L-DNs caused a distinct drop in the number of tumor cells and more void spaces in hematoxylin and eosin (H&E) stained slices for the tumor tissue, unlike the compact tumor tissue of the saline-administered and DOX·HCl-administered groups (Figure 8C). Compared with other groups, L-DNs did provide the maximum effects to damage CD31-positive vessels, exterminate Ki-67-positive proliferating tumor cells, and induce apoptosis of MCF-7R cells (TUNEL-positive cells). At the end of the treatment course, obvious weight loss (approximately 18%) was observed in the DOX·HCl-treated group as compared to the saline-treated group, while the nanoparticulate formulations of DNs and L-DNs avoided the weight decrease after chemotherapy (Figure S50). And histological analysis indicated that DNs and L-DNs could prevent the side effects of DOX to heart tissue, which was distinguished from irregular and incompact heart slices of the DOX·HCl group (Figure S51). The plasma drug concentration vs injection time profiles (Figure 8D) and pharmacokinetic parameters (Table S2) revealed that area under the curve (AUC) of DNs (151.9 mg L−1 h) and L-DNs (152.2 mg L−1 h) was 3.2-fold higher than that of DOX·HCl (47.4 mg L−1 h), and the half-life period (t1/2) of DOX was prolonged about 10-fold by PEGylated DNs as compared to DOX·HCl. In the following, fluorescent imaging was used to perform an in vivo drug distribution (Figure 8E). In a short monitoring time of 1 h, both DNs and

CONCLUSIONS We have developed tumor-specific multiple stimuli-activated dendrimeric nanoassemblies with metabolic blockade for completely defeating multidrug resistance. This type of DN hierarchically broke through the sequential physiological barriers of chemotherapy resistance, owing to their multiresponsive features, including stealthy dendritic corona to prolong blood circulation, robust nanostructures for efficient passive targeting and tumor accumulation, enzyme-activated tumor microenvironment targeting to deepen tumor penetration and facilitate drug internalization, cytoplasmic redoxsensitive disintegration for sufficient LND release to rapidly reach a therapeutic window, and lysosome acid-triggered nucleus delivery of antitumor drugs. More importantly, the metabolism-blocked tactics well terminated the activity of overexpressed hexokinases in MCF-7R cells and further provoked multiple pathways to overcome cellular factors of drug resistance including ATP-restriction, apoptotic activation, and anti-apoptotic inhibition. The predominant curative effects to MCF-7R tumor in vitro and in vivo supporting our design strongly surmounted both physiological barriers and cellular factors of multidrug resistance. We believe that our strategy is highly valuable for developing advanced dendrimeric nanoassemblies and conquering chemotherapy resistance. MATERIALS AND METHODS Synthesis and Characterizations of DPs. The amphiphilic DPs were synthesized using a divergent−convergent approach according to our previous descriptions,37−40 and the detailed synthetic routes and procedures are illustrated in the Supporting Information. Briefly, methoxy polyethylene glycol (mPEG, Aladdin Reagents Company, China) as hydrophilic segments were grafted onto peripheral groups of dendritic skeleton via MMP-sensitive peptides (GPLGLAG sequence). The MALDI-TOF MS spectrum (Bruker Autoflex III, Germany) and 1 H NMR (Bruker Avance II NMR spectrometer, Germany) results indicated the hydrophilic dendrons were completely decorated with MMP-cleavable mPEG with a molecular weight distribution from 3000 to 5000 Da. DOX (Hisun Pharmaceutical, Zhejiang, China) as hydrophobic moiety was conjugated onto dendrons by a lysosome acid-labile hydrazone bond. Finally, a redox-responsive disulfide bond 425


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volume reached about 50 mm3, the in vivo experiments were conducted. All animal experiments were approved by the ethics committee of Sichuan University. In Vitro Antitumor Assay. MCF-7R tumor cells were seeded in 96-well plate (1 × 104 cells per well) and cultured for 24 h. Then the culture media was replaced with 100 μL of fresh media containing dendrimers, DOX, DOX·HCl, DNs, and L-DNs, respectively. After 48 h incubation, cell viability was determined by CCK-8 kit. The absorbance was measured at 450 nm by Varioskan Flash microplate reader (Thermo Fisher Scientific, USA). Cell viability of MCF-7R cells was calculated by the equation: (ODSample − ODBackground)/(ODControl − ODBackground) × 100%. Cellular Uptake and Intracellular Delivery. To quantify the internalization, MCF-7R cells were cultured in a 6-well plate (3 × 105 per well) for 24 h. After treatment with DOX, DOX·HCl, and DNs (10 μg mL−1 DOX) for 2 h, the cells were washed with PBS and harvested by centrifugation (1000 rpm for 5 min). Then MCF-7R cells were resuspended in PBS (300 μL) and analyzed by a fluorescenceactivated cell sorting (FACS, BD Biosciences, USA). Also, MCF-7R tumor cells were incubated with DNs in the presence of Phen (100 μM, Sigma, USA) and MMP-2 (2 μg mL−1) or without treatment to determine the MMP-2 dependent internalization. To track intracellular molecular delivery, MCF-7R tumor cells were cultured in glass-bottomed dishes (1 × 104 cells). Two of these samples were pretreated with BSO (0.1 mM, 12 h, Sigma, USA) and GSH-OEt (10 mM, 2 h, Sigma, USA), respectively. After exposure to DiIC1(5)-loaded DNs for 4 h, the cells were stained with Alexa Fluor 488 Phalloidin (Molecular Probes, USA) and imaged using CLSM (Leica TCS SP5, Germany). To further investigate subcellular delivery, MCF-7R cells were treated with DNs for the predetermined incubation time (6, 8, and 48 h). The MCF-7R cells were stained with LysoTracker (Molecular Probes, USA) or Hoechst 33342 (Dojindo, Japan) for CLSM observation. Hexokinase Activity and ATP Content Analysis. MCF-7R cells were seeded in 6-well plates at a density of 3 × 105 cells per well for 24 h culture time. Then the cells were incubated with LND (1, 5, and 10 μg mL−1) and corresponding L-DNs for 24 h (DOX concentration was fixed at 10 μg mL−1). HK activity was determined by Hexokinase Colorimetric Assay Kit (Sigma, USA), following the manufacturer’s instructions. Likewise, L-DN treated MCF-7R cells were used to determine the ATP content by an ATP Assay Kit (Beyotime Biotechnology, Jiangsu, China). The experiments were carried out in triplicate. Drug Efflux Assay. MCF-7R tumor cells were seeded in 12-well plate with 1 × 105 cells per well and cultured for 24 h. After treatment with DOX·HCl, DNs, and L-DNs (5 μg mL−1 LND and 10 μg mL−1 DOX) for 2 h, MCF-7R cells were incubated with fresh media for different monitoring times. At predesigned time points, the culture medium was collected, and the MCF-7R cells were lysed (n = 3 per group). The DOX amount was determined by fluorescence spectroscopy at an excitation wavelength at 480 nm and emission wavelength at 550 nm. Mitochondrial Membrane Potential Study. MCF-7R tumor cells were treated with DOX, DOX·HCl, LND, DNs, and L-DNs for 24 h (5 μg mL−1 LND and 10 μg mL DOX−1) in a 6-well plate. After staining with MitoView 633 (Biotum, USA) for 30 min,47 the cells were collected for flow cytometer analysis. On the other hand, MCF7R cells were seeded in glass-bottomed dishes (5 × 103 cells) with the above-mentioned treatment for CLSM imaging. The reduction of fluorescence intensity represents the depletion of mitochondrial transmembrane potential. Western Blot Analysis. MCF-7R cells were cultured in a 6-well plate for 24 h. Then MCF-7R cells were incubated with DOX, DOX· HCl, LND, DNs, and L-DNs (5 μg mL−1 LND and 10 μg mL−1 DOX) for 48 h, respectively. The harvested MCF-7R cells were lysed with RIPA buffer containing a protease inhibitor.37 After centrifugation at 12000 g for 20 min, supernatants were collected to determine the protein concentration using the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific, USA). The equal amount of total protein (40 μg) was separated on a sodium dodecyl sulfate polyacrylamide gel

was introduced to link hydrophilic dendrons and hydrophobic dendrons together. All amino acids and condensing agents were purchased from GL Biochem LTD (Shanghai, China). The characterizations of synthetic compounds can be found in the Supporting Information. To study the multiple sensitivities, DPs were incubated with different biomimetic buffer solutions at 37 °C for 3 h, including 2 μg mL−1 MMP-2 (R&D System, USA), 10 mM DTT (Aladdin Reagents Company, China), and pH 5.0, respectively. The cleaved properties of DPs were identified by RP-HPLC (Agilent HPLC System, USA) and MALDI-TOF MS. The RP-HPLC operated as following conditions: acetonitrile/water; 0−30 min (acetonitrile from 5% to 85%), with a flow rate at 1.0 mL/min; monitored by UV−vis detector at 480 nm. Preparation and Characterizations of DNs. First, DPs were fully dissolved in dimethyl sulfoxide (DMSO), and this solution was dropped into deionized water under ultrasonic condition. Then the solution containing DNs was dialyzed (MWCO, 2000 Da) and freezedried. The size and zeta potential of DNs were determined by DLS (Malvern NANO ZS, UK). The DN nanostructures were measured by TEM (FEI Tecnai GF20S-TWIN, USA) and AFM (MFP-3D-BIO, USA). CD spectrum of DNs was conducted on a Dichroism Spectropolarimeter Jasco (Japan). To investigate the stimuli-sensitivities, DNs were individually incubated in different biomimetic environments of pH 5.0, 10 mM DTT, and 2 μg mL−1 MMP-2 at 37 °C for 3 h. Next, the size and nanostructure transitions were measured by DLS and TEM. In vitro DOX release was carried out in a series of TCNB (50 mM Tris, 10 mM CaCl2, 150 mM NaCl, 0.05% Brij 35) buffers (pH 7.4, pH 5.0, 10 mM DTT, and 2 μg mL−1 MMP-2). The dialysis bags with 1 mL of DN solution were immersed in tubes containing 25 mL buffer solutions, continuously shaking at 37 °C (n = 3). At predetermined time points, 1 mL external medium in tube was collected as a sample, and the same amount of fresh medium was replenished. The DOX concentration was determined by a fluorescence spectrophotometer (Hitachi F-7000, Japan) with an excitation wavelength at 480 nm and emission wavelength at 550 nm. Molecular Entrapment and Controlled Release. The encapsulation properties were first examined by pyrene (Sigma, USA) as a polar probe. Different amounts of DNs were dissolved in the same pyrene solution to prepare a series of samples (n = 3 per concentration). The emission spectra of pyrene from 340 to 600 nm were performed with an excitation wavelength at 330 nm. The values of fluorescence intensity at 372 and 383 nm were recorded to calculate the I1/I3 ratio. Meanwhile, emission spectra of LND vs DN concentrations (1, 5, 25, 50, 60, and 80 μg mL−1) in aqueous solution were recorded with an excitation wavelength at 299 nm. For preparation of L-DNs, LND was dissolved along with DPs (DPs:LND 3:1 w/w) in DMSO under ultrasonic conditions. With the formation of DNs in aqueous solution, LND was encapsulated into void space of DNs. Then the L-DN solution was dialyzed (MWCO, 2000 Da) and freeze-dried to obtain L-DNs. The drug loading capacity was about 21.4% (n = 3). Drug loading capacity = (weight of LND in L-DNs/weight of L-DNs) × 100%. In vitro LND release was carried out according to the above-mentioned method of DOX release. The LND concentration was measured by RP-HPLC using acetonitrile/ water containing 0.1% TFA (50:50, v/v) at 1.0 mL/min and detected by UV absorbance at 260 nm. The experiments were carried out in triplicate. Cell Lines and Animals. DOX (adriamycin)-resistant human breast cancer cells (MCF-7R cells) were obtained from MEIXUAN Biological Science and Technology, LTD (Shanghai, China). MCF-7R cells were cultured in Dulbecco’s minimal essential medium (DMEM, Gibco, USA) with 10% fetal bovine serum (FBS, Gibco, USA) and 1% penicillin-streptomycin (Hyclone, USA) solution in a humidified atmosphere containing 5% CO2 at 37 °C. BALB/c mice and BALB/c nude mice (about 5-week-old) were purchased from Dashuo Experimental Animal Company (Chengdu, China). The MCF-7R xenograft tumor models were generated by injecting a 1 × 107 cell suspension (100 μL) containing 50 μL Matrigel (BD Biosciences, USA) in the right flank of the BALB/c nude mice. When the tumor 426


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ACS Nano and transferred onto a polyvinylidene fluoride (PVDF) membrane. The PVDF membrane was blocked by 5% (w/v) nonfat dry milk and incubated with primary antibody (Bcl-2, Caspase-3 and β-actin, Abcam, UK) in the dilution buffer overnight at 4 °C. Then the membranes were washed by Tris-buffered saline Tween (15 mL) for three times and incubated with polyclonal goat antirabbit IgG HPR (Abcam, UK). An enhanced ECL Western Blotting Detection kit (Thermo Fisher Scientific, USA) was used to detect the luminescence by a ChemiDoc XR+UV illuminator (Bio-Rad, USA). Caspase-3 Activity Analysis. NucView 405 kit (Biotum, USA) was used to analyze caspase-3 activity.48 MCF-7R cells were treated with DOX, DOX·HCl, LND, DNs, and L-DNs (5 μg mL−1 LND and 10 μg mL−1 DOX) for 36 h in confocal dishes. After incubation with NucView 405 for 30 min at 37 °C, MCF-7R cells were washed with PBS for CLSM imaging. The mean fluorescence intensity reflecting caspase activity was analyzed by the software of Leica Application Suite X 2.0. Apoptosis Assay. MCF-7R cells were cultured in a 6-well plate and cultured for 24 h. The cells were treated with DOX, DOX·HCl, LND, DNs, and L-DNs (5 μg mL−1 LND and 10 μg mL−1 DOX) for 36 h, respectively. After washing and centrifugation, the harvested cells were stained with the Annexin V-FITC/PI Apoptosis Detection Kit (Dojindo, Japan) for 15 min at room temperature. Then, the MCF-7R cells were analyzed on a flow cytometer with 488 nm excitation using a 515 nm bandpass filter for FITC detection and a 615 nm filter for propidium iodide (PI) detection. In Vitro Drug Penetration. According to our previous report, multicellular tumor spheroids based on MCF-7R tumor cells were obtained using a liquid overlay method.45 Cell suspension was seeded in pretreated 6-well plates, which were coated with 1 mL of a sterile 1% agar and cultured for approximately 10 days in 37 °C humidified incubator with 5% CO2. When the size of MTSs multicellular tumor spheroids reached to about 200 μm in diameter, MTSs were carefully transferred to confocal dishes and treated with DOX, DOX·HCl, DNs, and L-DNs (2.5 μg mL−1 LND and 5 μg mL−1 DOX) for 3 h. To disclose MMP-dependent penetration, we set the groups with coincubation of MMP-2 (2 μg mL−1) or Phen inhibitor (100 μM) in the culture media. Optical MTS sections imaged by CLSM from top to bottom with 5 μm per section. To quantify tumor penetration, MTSs were adequately disassociated into single cells by accutase reagent (Invitrogen, USA). DOX-positive cells and fluorescence intensity were measured by flow cytometer with three parallel samples. In Vivo Drug Penetration. The nude mice bearing MCF-7R tumors were intravenously injected with DOX·HCl, DNs, and L-DNs (5 mg LND kg−1and 10 mg DOX kg−1). The mice were sacrificed 12 h after injection, and solid tumors were excised for frozen sections. The fixed tumor samples were sectioned into 5 μm-thick slides and incubated with Alex Fluor 594 labeled anti-CD31 antibody (Abcam, UK) and DAPI (Dojindo, Japan). Confocal microscope was used to observe drug penetration in solid tumor. In Vivo Antitumor Treatment. The nude mice bearing MCF-7R tumor were randomly divided into four groups (n = 6). Normal saline, DOX·HCl, DNs, and L-DNs (2.5 mg LND kg−1 and 5 mg DOX kg−1) were intravenously injected into the mice via tail vein every 3 days for 4 times. Tumor volumes were measured by caliper every 3 days for 9 times and calculated using the following formula: V = L × W2/2, where L and W are the larger and smaller diameters, respectively. When mice were sacrificed at the end of the treatment course, tumors and major organs were excised for H&E staining and immunohistochemistry analysis.39,40 Pharmacokinetics Study. BALB/c mice were administered with DOX·HCl, DNs, and L-DNs at a dose of 10 mg DOX kg−1 via tail vein injection (n = 3). Blood was sampled from the mouse eyes at preset time points (1, 30, 60, 180, 360, and 720 min). These samples were centrifuged at 3000 g in 4 °C for 10 min to obtain the supernatant plasma. The drug was fully extracted from plasma by chloroform/ isopropanol (4:1, v/v), and the DOX concentration was quantified by RP-HPLC. Pharmacokinetic software of DAS 3.015 was used to analyze data.

In Vivo Drug Distribution. The tumor-bearing nude mice intravenously injected with 100 μL of DOX·HCl, DNs, and L-DNs (5 mg LND kg−1 and 10 mg DOX kg−1) via tail vein. Fluorescence imaging system (CRi Maestro EX, USA) for small animals was used to capture fluorescence signals of nude mice at 1, 3, 6, and 12 h postintravenous injection. At the end of monitoring time point (12 h), the mice were sacrificed, and the tumors were sectioned for DAPI staining (5 μm thickness). The DOX distribution in tumor tissue and tumor cells was imaged by CLSM.

ASSOCIATED CONTENT S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsnano.6b06161. Materials and methods, experimental details, additional data, Schemes S1−S6, Figures S1−S53, and Tables S1− S2 (PDF)

AUTHOR INFORMATION Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. ORCID

Yachao Li: 0000-0003-4792-0915 Xianghui Xu: 0000-0002-8885-0848 Zhongwei Gu: 0000-0003-1547-6880 Author Contributions §

These authors contributed equally to this work.

Notes

The authors declare no competing financial interest.

ACKNOWLEDGMENTS This work was supported by the National Natural Science Foundation of China (NSFC, 51133004, 81361140343, 51503128, and 21674067), Joint Sino-German Research Project (GZ905), and Scientific Research Foundation for Outstanding Young Scholars in Sichuan University (2016SCU04A19). We appreciate the help from Jiao Lu of Sichuan University with CLSM imaging. REFERENCES (1) Chabner, B. A.; Roberts, T. G. Timeline-Chemotherapy and the War on Cancer. Nat. Rev. Cancer 2005, 5, 65−72. (2) Holohan, C.; Van Schaeybroeck, S.; Longley, D. B.; Johnston, P. G. Cancer Drug Resistance: an Evolving Paradigm. Nat. Rev. Cancer 2013, 13, 714−726. (3) Davis, M. E.; Chen, Z. G.; Shin, D. M. Nanoparticle Therapeutics: an Emerging Treatment Modality for Cancer. Nat. Rev. Drug Discovery 2008, 7, 771−782. (4) Szakacs, G.; Paterson, J. K.; Ludwig, J. A.; Booth-Genthe, C.; Gottesman, M. M. Targeting Multidrug Resistance in Cancer. Nat. Rev. Drug Discovery 2006, 5, 219−234. (5) Patel, N. R.; Pattni, B. S.; Abouzeid, A. H.; Torchilin, V. P. Nanopreparations to Overcome Multidrug Resistance in Cancer. Adv. Drug Delivery Rev. 2013, 65, 1748−1762. (6) Szakacs, G.; Hall, M. D.; Gottesman, M. M.; Boumendjel, A.; Kachadourian, R.; Day, B. J.; Baubichon-Cortay, H.; Di Pietro, A. Targeting the Achilles Heel of Multidrug-Resistant Cancer by Exploiting the Fitness Cost of Resistance. Chem. Rev. 2014, 114, 5753−5774. (7) Meng, H.; Mai, W. X.; Zhang, H. Y.; Xue, M.; Xia, T.; Lin, S. J.; Wang, X.; Zhao, Y.; Ji, Z. X.; Zink, J. I.; Nel, A. E. Codelivery of an Optimal Drug/siRNA Combination Using Mesoporous Silica Nano427


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