Acidity-Triggered Ligand-Presenting Nanoparticles To Overcome

Jul 28, 2017 - Acidity-Triggered Ligand-Presenting Nanoparticles To Overcome Sequential Drug Delivery Barriers to Tumors ... In this study, we reporte...
17 downloads 8 Views 5MB Size
Download PDF
Letter pubs.acs.org/NanoLett

Acidity-Triggered Ligand-Presenting Nanoparticles To Overcome Sequential Drug Delivery Barriers to Tumors Tingting Wang,†,‡ Dangge Wang,†,‡ Jianping Liu,† Bing Feng,†,‡ Fangyuan Zhou,† Hanwu Zhang,† Lei Zhou,† Qi Yin,† Zhiwen Zhang,† Zhonglian Cao,§ Haijun Yu,*,† and Yaping Li*,† †

State Key Laboratory of Drug Research and Center of Pharmaceutics, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China ‡ University of Chinese Academy of Sciences, Beijing 100049, China § School of Pharmacy, Fudan University, Shanghai 201203, China S Supporting Information *

ABSTRACT: The success of cancer chemotherapy is impeded by poor drug delivery efficiency due to the existence of a series of pathophysiological barriers in the tumor. In this study, we reported a tumor acidity-triggered ligand-presenting (ATLP) nanoparticle for cancer therapy. The ATLP nanoparticles were composed of an acid-responsive diblock copolymer as a sheddable matrix and an iRGD-modified polymeric prodrug of doxorubicin (iPDOX) as an amphiphilic core. A PEG corona of the polymer matrix protected the iRGD ligand from serum degradation and nonspecific interactions with the normal tissues while circulating in the blood. The ATLP nanoparticles specifically accumulated at the tumor site through the enhanced permeability and retention (EPR) effect, followed by acid-triggered dissociation of the polymer matrix within the tumoral acidic microenvironment (pH ∼ 6.8) and subsequently exposing the iRGD ligand for facilitating tumor penetration and cellular uptake of the PDOX prodrug. Additionally, the acid-triggered dissociation of the polymer matrix induced a 4.5-fold increase of the fluorescent signal for monitoring nanoparticle activation in vivo. Upon near-infrared (NIR) laser irradiation, activation of Ce6-induced significant reactive oxygen species (ROS) generation, promoted drug diffusion inside the tumor mass and circumvented the acquired drug resistance by altering the gene expression profile of the tumor cells. The ATLP strategy might provide a novel insight for cancer nanomedicine. KEYWORDS: Drug delivery barriers, acid-responsive, ligand presentation, tumor microenvironment, cancer therapy

T

corona with the nanoparticles can extend their blood circulation time by suppressing protein absorption and reduces RES recognition.10 However, PEGylation dramatically shields the targeting effect and suppresses the internalization of nanoparticles.11−13 To address above paradoxes, stimuli-responsive nanocarriers had been tremendously exploited in the past few decades to improve the drug delivery efficacy by “normalizing” the tumor microenvironment14 or degrading the extracellular matrix (ECM) with recombinant human hyaluronidase.15−18 Shrinkable nanovectors were also designed to improve their tumor distribution.19,20 These nanoparticles can change their particle size to facilitate tumor penetration by responding to the biological or physical stimuli such as acidic pH,21,22 enzyme (e.g., esterase, matrix metalloproteinases (MMPs) or hyaluronidase),23−25 or UV−vis light (i.e., 350−500 nm).7,26 Despite

he principle goal of cancer nanomedicine is to improve the therapeutic efficacy while suppressing the adverse effects of anticancer drugs. However, the success of nanomedicine was significantly hampered with the sequential drug delivery barriers including blood circulation, extravasion from the blood vessels at the tumor site, deep tumor penetration, efficient internalization and drug release inside the tumor cells.1,2 Solid tumors are highly heterogeneous, surrounded by a dense extracellular matrix (ECM) and lack of lymphatic drainage. These three factors synergistically induce a reduced trans-capillary pressure gradient and elevated interstitial fluid pressure to impede tumor penetration with effective concentrations.3 The tumor accumulation and cellular uptake of drugloaded nanoparticles can be improved by surface modification of nanovectors with a targeting ligand (e.g., folic acid),4 or cellpenetrating peptide (e.g., TAT).5,6 Unfortunately, ligandmodified nanoparticles may have undesirable interactions with serum proteins and normal tissues, which results in nonspecific drug distribution and accelerated reticuloendothelial system (RES) clearance in the liver and spleen.7−9 Integrating a PEG © XXXX American Chemical Society

Received: May 15, 2017 Revised: July 12, 2017 Published: July 28, 2017 A

DOI: 10.1021/acs.nanolett.7b02031 Nano Lett. XXXX, XXX, XXX−XXX

Letter

Nano Letters

Figure 1. Schematic illustration of iPAPD nanoparticles with enhanced tumor penetration and cellular uptake for cancer therapy. (a) Fabrication of iPAPD nanoparticles. (b) Schematic illustration of tumor acidity-mediated activation of iPAPD nanoparticles with iRGD-enhanced tumor penetration and imaging-guided combination cancer therapy. (c) TEM images and hydrodynamic particle size distribution of iPAPD nanoparticles examined at pH 7.4 and 6.6. (d) Acid-triggered activation of fluorescence and photodynamic properties of iPAPD nanoparticles (fluorescence imaging was performed at Ex = 640 nm and Em = 680 nm for Ce6).

pKa value of 6.9.28,29 iRGD was employed to facilitate tumor penetration and cellular uptake of PDOX by binding with αvβ3/5 integrin and neuropilin-1 (Nrp-1) overexpressed on the membrane of tumor cells.30 iPAPD nanoparticles change their physiochemical properties in the tumoral acidic microenvironment. At physiological pH (i.e., 7.4), the PHMA matrix compresses the iPDOX prodrug into a compact nanostructure to shield the iRGD ligand from serum degradation and nonspecific interaction with normal organs. The iPAPD nanoparticles specifically accumulate at the tumor site through the enhanced permeability and retention (EPR) effect. Once deposited in the acidic tumor microenvironment, the PHMA matrix dissociates from iPAPD nanoparticles to disclose the targeting ligand iRGD and restore the fluorescence imaging of Ce6 for real-time monitoring the activation and deep tumor penetration of nanoparticles in vivo. Additionally, Ce6 induced significant ROS generation upon NIR laser irradiation to promote intratumoral drug diffusion and overcome the drug resistance by regulating gene expression profiles of the tumor cells (Figure 1b). To prepare iPAPD nanoparticles, acid-sensitive PHMA diblock copolymer bearing three Ce6 molecules on each

that the stimuli-responsive nanocarriers are promising for improved drug delivery efficacy, it remains a formidable challenge to develop more sophisticated nanomedicine with minimal interaction with the RES system while being specifically activated in the tumor microenvironment for a broad tumor spectrum. Moreover, there is also an increasing need to develop versatile nanoparticles with imaging function for real-time monitoring tumor penetration in vivo. It has been well-defined that solid tumors are of low extracellular pH (i.e., 6.5−6.8) due to anaerobic glycolysis, the so-called “Warburg effect”.27 Accordingly, we herein presented a tumor acidity-triggered ligand-presenting (ATLP) nanoparticle for tumor-specific penetration and cellular uptake by utilizing the acidic microenvironment existing in a broad range of solid tumors. The ATLP nanoparticles were fabricated by integrating a Ce6-modified acid-responsive poly(ethylene glycol)-b-poly(2-(hexamethyleneimino) ethyl methacrylate) (PEG-b-PHMA, termed as PHMA) diblock copolymer and an iRGD-modified polymeric prodrug of doxorubicin (iPDOX) into one single nanoplatform (termed as iPAPD) (Figure 1a). PHMA diblock copolymer undergoes amphiphilic to hydrophilic transition due to its ultra-acid-sensitivity with an apparent B

DOI: 10.1021/acs.nanolett.7b02031 Nano Lett. XXXX, XXX, XXX−XXX

Letter

Nano Letters

Figure 2. Acid-triggered tumor penetration and activation of iPAPD nanoparticles in vitro. (a) The 2.5D images and (b) CLSM images of iPAPD penetration into MCF-7/ADR MCSs (Ex = 488 nm and Em = 594 nm for DOX). (c) Flow cytometric analysis of intracellular fluorescence of DOX in the MCF-7/ADR MCSs as a function of medium pH (** p < 0.05, *** p < 0.01). (d) CLSM of 655 nm laser irradiation-induced ROS generation in MCF-7/ADR cells with a power density of 2.0 W/cm2 (Ex = 488 nm and Em = 529 nm for DCF; Ex = 633 nm and Em = 650 nm for Ce6) (scale bars = 50 μm). (e) Chemo- and phototoxicity of iPAPD nanoparticles in vitro upon 655 nm laser irradiation (** p < 0.05, *** p < 0.01). (f) Differential gene expression heat maps for Human Gene Expression Array of MCF-7/ADR cells treated with chemotherapy (DOX), PDT (PHMA + Laser) or chemotherapy + PDT (iPAPD + Laser), in comparison with nontreated groups.

polymer chain was synthesized using the atom transfer radical polymerization (ATRP) method by following a previously reported procedure (Figure S1).31 The chemical structure of PHMA was verified using 1H NMR spectra (Figure S2). Polymeric DOX prodrug iPDOX was synthesized by conjugating iRGD and DOX on the chain end of pluronic P85 copolymer via a MMP-2-labile PLGLAG spacer (Figure S3). The successful synthesis of iPDOX was confirmed using 1 H NMR spectra (Figures S4 and S5). The Ce6 and DOX grafting ratios in PHMA and iPDOX were determined to be ∼8.0 wt % using UV−vis and fluorescence spectrometric examinations, respectively (Figure S6). Pluronic copolymer P85 was selected for prodrug synthesis due to its intrinsic property to overcome acquired drug resistance by blocking the Pglycoprotein (P-gp) drug efflux pump.32 Over 90% of DOX was released from iPDOX within 3 h in the presence of 50 nM MMP-2, revealing the high sensitivity of the prodrug substrate to MMP-2 cleavage (Figure S7). iPAPD nanoparticles were then prepared by self-assembly of PHMA with iPDOX at a weight ratio of 1:1. An iRGD-free control of iPAPD (namely PAPD) was also prepared by coassembling PHMA with PDOX at the weight ratio of 1:1 as well. Meanwhile, an acid-insensitive analog of iPAPD was prepared by coassembly of iPDOX prodrug with Ce6-modified acid-insensitive poly(ethylene glycol)-block- poly(tertbutyl methacrylate-co-hydroxyl ethylene methacrylate) (PEG-b-PTBA) diblock polymer (PTBA) at 1:1 weight ratio, which was denoted as iPTPD (Figure S8). The resultant iPAPD and iPTPD nanoparticles both showed spherical morphology as determined using transmission electron microscopic (TEM) examination (Figure 1c and S9a). To test the acid-sensitivity of the ATLP nanoparticle, iPAPD nanoparticles were incubated in a series of phosphate buffer

(PB) solutions with different pH values, and their size change was monitored using DLS. At pH 7.4, iPAPD nanoparticles showed an average particle size of 34 ± 2.0 nm and surface charge of 1.1 ± 0.4 mV (Figure 1c). The nanoparticles swelled sharply to 155 ± 12 nm with a positive surface charge of 4.6 ± 0.2 mV at pH 6.6, a pH value within the range of the tumor acidic microenvironment, presumably due to protonationinduced dissociation of the PHMA matrix and formation of iPDOX aggregates with iRGD presented on the surface (Figure 1c). In contrast, iPTPD displayed a constant hydrodynamic particle size and neutral surface charge at pH 7.4 and 6.6 because it was acid insensitive (Figure S9b,c). The acid-triggered activation of fluorescence properties of iPAPD was evaluated using fluorescence spectrometer. iPAPD with Ce6 aggregated inside the hydrophobic core of the nanoparticles showed minimal fluorescence activation in neutral HEPES-buffered solution (20 mM, pH 7.4) and fresh mouse blood due to fluorescence resonance energy transfer (FRET)-induced fluorescence quenching between Ce6 molecules (Figure 1d and S9d). The fluorescence signal of iPAPD increased by 3.8-fold in HEPES-buffered acidic solutions (pH ≤ 6.8) compared to that observed at pH 7.4 due to acid-triggered dissociation of PHMA from iPAPD nanoparticles. Similar to the trend of fluorescence change, iPAPD displayed inert photoactivity at pH 7.4, which was dramatically activated by 3.4-fold at acidic pH (Figure 1d). In contrast, iPTPD nanoparticles showed minimal fluorescence and photoactivity from pH 7.4 to 6.4 because Ce6 was tightly encapsulated inside the nanoparticles (Figure S9e,f). Moreover, multispectral optoacoustic tomography (MSOT) examination showed that iPAPD nanoparticles induced a measurable photoacoustic (PA) C

DOI: 10.1021/acs.nanolett.7b02031 Nano Lett. XXXX, XXX, XXX−XXX

Letter

Nano Letters

Figure 3. Biodistribution and pharmacokinetics profiles of iPAPD nanoparticles in vivo and ex vivo. (a) Fluorescence imaging of nanoparticle distribution in MCF-7/ADR tumor-bearing nude mice at the desired time points post injection by examined Ce6 fluorescence (Ex = 640 nm and Em = 680 nm for Ce6). (b) Normalized Ce6 fluorescence intensity of nanoparticle-treated tumor-bearing nude mice. (c) Tumor distribution and penetration iPAPD nanoparticles examined using PA imaging in MCF-7/ADR tumor-bearing nude mice (examined = 680 nm). (d) PA intensity as a function of time duration post nanoparticle injection. (e) Plasma concentration of DOX as a function of injection time (*** p < 0.01). (f) Ex-vivo fluorescence imaging of DOX distribution in MCF-7/ADR tumor (Doxorubicin Filter for DOX). (g) Quantitative examination of DOX distribution in the tumor xenograft 2 or 24 h postinjection (*** p < 0.01).

signal as a function of Ce6 concentration, suggesting the potential use of iPAPD for PA imaging (Figure S9g). To demonstrate acid-triggered iRGD-presentation of iPAPD nanoparticles in vitro, we first evaluated the cellular uptake of iPAPD nanoparticles in monolayer cultured DOX-resistant MCF-7/ADR breast cancer cells, which overexpressing P-gp,33 Nrp-1, and MMP-2 as revealed using immunofluorescent staining, Western blot, and enzyme-linked immunosorbent assay (ELISA), respectively (Figures S10 and S11). The overactivation of Nrp-1 and MMP-2 in MCF-7/ADR cells may facilitate iRGD-mediated tumor penetration of iPAPD nanoparticles and cleavage of the PLGLAG spacer, respectively. The confocal laser scanning microscopic (CLSM) images showed negligible DOX uptake in MCF-7/ADR cells due to Pgp-mediated drug efflux (Figure S12a).33,34 The intracellular fluorescence of DOX increased dramatically in iPAPD or PAPD group by blocking the P-gp efflux pump with pluronic P85. The highest intracellular accumulation of DOX was observed in the PAPD + iRGD group due to iRGD-enhanced intracellular uptake of the nanoparticles. In contrast, iPAPD displayed similar cellular uptake efficiency as PAPD, suggesting iRGD was shielded within the PHMA matrix in the neutral extracellular microenvironment (Figure S12b). We next investigated the tumor penetration ability of iPAPD nanoparticles using MCF-7/ADR-derived multicellular spheroids (MCSs). MCS is a three-dimensional (3D) tumor model widely used for studying tumor biology and evaluating cancer therapeutics by mimicking the microenvironment of solid tumors, including the acidic and enzyme microenvironments.35

The pH value of the cell culture medium was adjusted to 6.6 before nanoparticle addition for mimicking the acidic tumoral microenvironment. The fluorescence of DOX distributed in the outer layer of MCSs in the DOX, DOX+iRGD, PAPD, and iPTPD groups disregarding the pH values when examined 8 h post nanoparticle addition (Figure 2a,b), which could be explained by the elevated interstitial fluid pressure in the tumor spheroid and drug efflux from the MCF-7/ADR cells. With the addition of free iRGD, PAPD nanoparticles diffused deep into the tumor spheroids at both pH 7.4 and 6.6 due to iRGDenhanced tumor penetration of the nanoparticles. The DOX fluorescence of iPAPD nanoparticles perfused throughout the tumor spheroid at pH 6.6, in sharp contrast with the highly restricted fluorescence distribution pattern at pH 7.4, verifying acid-triggered exposure of iRGD ligand dramatically promoted tumor penetration of iPAPD. The internalization of iPAPD in the MCSs was further quantitively analyzed using flow cytometric measurement 8 h post nanoparticle incubation. The acid-insensitive iPTPD nanoparticles were constant while low intracellular DOX fluorescence intensity was shown at both neutral and acidic conditions. In contrast, iPAPD displayed 2.3-fold higher intracellular uptake efficacy at pH 6.6 than that achieved at neutral pH (Figure 2c). Moreover, iPAPD-incubated MCSs at pH 6.6 showed 3.2-fold higher intracellular DOX fluorescence intensity than that of iPTPD group, which provided direct evidence for enhanced intracellular uptake of iPDOX via acidtriggered iRGD presentation. D

DOI: 10.1021/acs.nanolett.7b02031 Nano Lett. XXXX, XXX, XXX−XXX

Letter

Nano Letters

24 h postinjection whereas the signals from the PAPD group declined quickly, revealing highly improved tumor penetration and retention properties of iPAPD nanoparticles, which could be attributed to the tumor acidity-triggered presentation of iRGD (Figure 3a,b). As predicted, the PAPD + iRGD group showed lower tumoral fluorescence intensity than the iPAPD group due to the inconsistent pharmacokinetics profiles between iRGD and the PAPD nanoparticles.36−38 In contrast, iPTPD nanoparticles showed weak fluorescence signal at the tumor site and declined quickly by 12 h postinjection due to their always-off fluorescence property and rapid blood clearance from the tumor with iRGD shielded inside the nanoparticles (Figure S15). It has been widely exploited that the solid tumors generate an extracellular acidic microenvironment through aerobic glycolysis.39 To better understand the mechanisms underlying ATLP of iPAPD nanoparticles in vivo, a tumor glycolysis inhibitor 2deoxy-D-glucose (2-DG) was intratumorally injected (250 mg/ kg) to inhibit tumoral glucose uptake through cell surface glucose transporters and acidification of the tumoral microenvironment.40 Figure 3a showed that 2-DG noticeably suppressed the fluorescence signal of the iPAPD nanoparticles due to inhibited tumor acidification and subsequent Ce6 activation. iPAPD showed 2-fold higher tumoral signal to background fluorescence ratio than iPAPD + 2-DG when examined at 24 h postinjection (Figure 3b). iPTPD showed comparable intratumoral fluorescence intensity as iPAPD + 2DG, while 2.5-fold lower than iPAPD when examined 24 h postinjection (Figure S15a,b). This phenomenon confirmed tumor acidity-dependent activation of iPAPD fluorescence property in vivo. The iRGD-enhanced intratumoral accumulation and penetration of iPAPD nanoparticles was further investigated using PA imaging in vivo. The distribution area and signal intensity continuously increased in the first 8 h postinjection, indicating intratumoral accumulation and penetration of the nanoparticles into deep tumors (Figure 3c). The iPAPD group showed 1.5fold higher PA signal intensity than the PAPD group examined 8 h postinjection, indicating improved tumor penetration and accumulation of the iPAPD nanoparticles (Figure 3d). The PA imaging data further identified that the ATLP strategy robustly improved the tumor penetration of iPAPD nanoparticles. The pharmacokinetics profiles of iPAPD nanoparticles were investigated by measuring the plasma concentration of DOX after intravenous administration. The blood clearance half time (t1/2β) and bioavailability (area under the curve, AUC0‑t) of DOX in the iPAPD group was 4.7- and 64.8-fold higher than that of the DOX groups, respectively (Figure 3e and Table S1), suggesting that nanoparticle formulation dramatically elongated the blood circulation time of PDOX. Noticeably, iPTPD displayed comparable t1/2β and AUC0‑t as those of iPAPD, implying the ATLP property of iPAPD affected negligibly on the pharmacokinetic profiles of iPAPD nanoparticles. The organ (i.e., heart, liver, spleen, lung, and kidney) and tumor distribution of the prodrug-loaded nanoparticles was examined by fluorescence imaging ex vivo. iPAPD nanoparticles showed the highest tumoral ambulation when examined at 2 or 24 h post nanoparticles injection (Figure 3f). The organ distribution of the nanoparticles was then quantitively determined by measuring the organ concentration of DOX. The mixture of iRGD and PAPD showed moderately increased intratumoral DOX distribution. In contrast, iPAPD showed the highest intratumoral DOX accumulation, which was 3.0- and

We then examined the in vitro activation of iPAPD nanoparticles by measuring laser-triggered ROS generation. MCF-7/ADR cells were incubated with PDOX-free PHMA nanoparticles for 4 h at a Ce6 concentration of 5.0 μg/mL. The cells were then loaded with a fluorescent ROS probe 2′,7′dichlorfluorescein diacetate (DCFH-DA) and illuminated with 655 nm laser for 5 min at predetermined power density (i.e., 0.5, 1.0, 1.5, 2.0 W/cm2). Laser irradiation induced negligible ROS generation in PBS-treated cells (Figure S13). In contrast, the CLSM images and flow cytometric examination showed significant ROS generation in PHMA-treated cells in vitro as a function of power density (Figure 2d and S14a,b), implying noticeable photoactivity of PHMA nanoparticles in vitro. Laser irradiation caused significant cellular apoptosis of PHMA-treated cells in a photodensity-dependent manner (Figure S14c,d). The cumulative cytotoxicity of iPAPD nanoparticles between photodynamic therapy (PDT)-induced photoactivity and DOX-caused chemotoxicity was then evaluated in MCF-7/ADR cells using MTT assay. Figure 2e displayed that laser irradiation at photodensity of 1.0 W/cm2 dramatically reduced the viability of iPAPD-treated cells by 2.3fold, suggesting PDT noticeably sensitized MCF-7/ADR cells to DOX treatment. Compared to iPAPD, iPTPD showed negligible cytotoxicity without laser irradiation, implying iPTPD tightly compressed iPDOX inside the nanoparticles without intracellular release of free DOX. Upon laser irradiation at photodensity of 1.0 W/cm2, iPTPD nanoparticles displayed 1.9-fold lower phototoxicity than that of iPAPD, which could be attributed to the quenched photoactivity of iPTPD. Treatment-induced (i.e., chemotherapy, PDT or PDT + chemotherapy) gene expression alterations in MCF-7/ADR cells were investigated using an Agilent Human Gene Expression Array. Signature gene targets involved in cell death and proliferation were presented as gene expression heat maps by calculating the up- or down-regulation of gene expression based on the probes with the maximum signal intensity per gene. Figure 2f displayed a comparison of the potential gene targets after different treatments showed that PDT highly up-regulated genes related to metabolism (e.g., SCD5, ATP1A2, and ATP6 V0D2) and intracellular transports (e.g., HSPA5 and HSP90B1). In contrast, PDT downregulated genes involved in maintaining cell proliferation (i.e., marker of proliferation KI-67, MKI67), cell division (cell cycle regulator cyclin A1, CCNA1) as well as extracellular matrix proteins (e.g., COL18A1, COL23A1, and COL4A6), suggesting that iPAPD nanoparticle-mediated PDT indirectly contributed to the destruction the extracellular matrix (ECM). Therefore, PDT appears to not only strengthen the apoptosis-induced cell death mechanism from DOX (DOX sensitization) but also triggers ECM destruction-dependent necrosis of the tumor cells. To investigate the tumor accumulation and distribution profile of the ATLP nanoparticles in vivo, iPAPD nanoparticles were intravenously (iv) injected into nude mice bearing orthotopic MCF-7/ADR tumors (n = 3 in each group). PAPD nanoparticles without iRGD modification and iPTPD nanoparticles with always-OFF fluorescence property were used as the controls. The biodistribution of nanoparticles was examined using in vivo fluorescence imaging at the desired time points postinjection. Noticeable fluorescence signal was found in the first hour post PAPD injection, revealing significant tumor accumulation and fluorescence activation of the PAPD nanoparticles in vivo. The tumor to normal skin fluorescence signal ratio of the iPAPD group increased consistently over E

DOI: 10.1021/acs.nanolett.7b02031 Nano Lett. XXXX, XXX, XXX−XXX

Letter

Nano Letters

Figure 4. ROS generation and tumor penetration of iPAPD nanoparticles in vivo. (a) CLSM examination of laser-induced penetration of iPAPD nanoparticles in MCF-7/ADR tumors, the blood vessels were stained with green fluorescence-stained CD31 antibody (DAPI Ex = 405, Em = 460 nm; FITC Ex = 488 nm, Em = 518 nm; Ce6/DOX Ex = 590 nm, Em = 615 nm) (scale bar 100 μm). (b) CLSM images of MCF-7/ADR tumor sections 2 h post iPAPD administration, DCFH-DA was used as a ROS probe (DCF Ex = 488 nm, Em = 529 nm) (scale bar 100 μm). (c) Quantification scatter plots indicate mean fluorescent intensity of Ce6/DOX and (d) DCF from N > 30 individual fields (*** p < 0.01).

tumor bearing mice were iv injected with PAPD, iPAPD, and iPTPD nanoparticles at an identical DOX and Ce6 dose of 5.0 mg/kg. The mice were intratumorally injected with DCFH-DA and illuminated with 655 nm laser 2 h postinjection. The tumors were immediately collected following laser irradiation, frozen-sectioned, and examined using CLSM. Laser irradiation induced significant ROS generation in iPAPD group in vivo, and the intratumoral penetration and diffusion of iPAPD nanoparticles was further enhanced by laser irradiation as indicated by the increased diffusion of red fluorescence of Ce6/ DOX throughout the tumor (Figure 4b,c). Both the green fluorescence of ROS and red fluorescence of Ce6/DOX perfused into the tumor interstitium, confirming ROS-induced intratumoral penetration and diffusion of nanoparticles (Figure 4d). In contrast, laser irradiation negligibly promoted the intratumoral penetration of iPTPD nanoparticles (Figure S16). Therefore, laser-promoted tumor penetration of the ATLP nanoparticles could be attributed to ROS-induced degradation of tumoral ECM and increased extracellular transportation of PDOX. We next investigated the therapeutic effect of the iPAPD nanoparticles in vivo using MCF-7/ADR tumor bearing nude mouse model. When the tumor volume reached 200 mm3, the mice were randomly grouped (n = 6 in each group) and iv administrated with PBS, DOX, PHMA, PAPD + iRGD, or iPAPD nanoparticles at an identical DOX and Ce6 dose of 5.0 mg/kg. The PHMA and iPAPD groups were further illuminated with 655 nm laser 2 h postinjection at photodensity

3.5-fold more efficient than PAPD and free DOX groups, respectively, when examined 24 h postinjection (Figure 3g and S15c,d). The intratumoral distribution of the ATLP nanoparticles was further investigated using CLSM examination of anti-CD31 antibody-stained tumor sections. Figure 4a displayed that DOX/Ce6 in the PAPD group was primarily colocalized with CD31, a marker of the neovasculature, indicating PAPD nanoparticles were entrapped in the perivascular area of the tumors. As expected, iPAPD showed a more diffuse fluorescence pattern, indicating nanoparticle perfusion out of the blood vasculature. The fluorescence intensity of Ce6/DOX in iPAPD without laser group increased by 5.3-fold compared to PAPD, suggesting that the iRGD-modification significantly improved the tumor penetration of iPAPD nanoparticles. Laser irradiation further promoted the intratumoral penetration and diffusion of the iPAPD nanoparticles. In contrast, laser irradiation showed negligible influence on the intratumoral distribution of iPTPD nanoparticles, implying the photoactivity of iPTPD was quenched in vivo. Therefore, laser-promoted tumor penetration of iPAPD nanoparticles could be attributed to laser-induced ROS generation and degradation of tumoral ECM. PDT is reported to facilitate the tumor penetration of nanoparticles by triggering ROS generation.41,42 To investigate whether PDT can further enhance the intratumoral diffusion of the ATLP nanoparticles in vivo, we then examined the photoactivity of iPAPD nanoparticles in vivo. MCF-7/ADR F

DOI: 10.1021/acs.nanolett.7b02031 Nano Lett. XXXX, XXX, XXX−XXX

Letter

Nano Letters

Figure 5. In vivo antitumor performance of PDT and ATLP nanoparticles. (a) Therapy schedule for ATLP nanoparticles administration and PDT treatment. (b) Antitumor performance of iPAPD nanoparticles in combination with PDT (** p < 0.05, *** p < 0.01); (b) TUNEL staining of the tumor sections, and (c) relative apoptotic rate of different groups (** p < 0.05, *** p < 0.01). (d) H&E staining of tumor sections at the end of antitumor studies (scale bar 200 μm).

of 2.0 W/cm2 for 2 min. The treatments were repeated three times at a time interval of 4 days. The tumor volume and body mass were monitored during the duration of the study (Figure 5a). Free DOX slightly delayed the tumor growth due to the high DOX resistance of MCF-7/ADR cells (Figure 5b). PAPD + iRGD showed moderate therapeutic effect to inhibit ∼63% of tumor growth, whereas iPAPD nanoparticles dramatically suppressed ∼88% of the tumor growth. This highly efficacious antitumor effect is presumed to be due to the iRGD-mediated tumor accumulation, deep penetration, and cellular uptake of the PDOX prodrug in vivo. Noticeably, iPAPD + Laser showed the best antitumor effect to completely eliminate the tumor xenograft by the end of the study. The results demonstrated that iRGD and PDT collectively promoted the tumor penetration of anticancer drugs for improved cancer therapy. TUNEL staining of the tumor sections at the end of antitumor study showed that a combination of iPAPD treatment with laser irradiation significantly inhibited the proliferation and induced apoptosis of the tumor cells in vivo (apoptotic percentage >70%) (Figure 5c), which was 2-fold more efficient than iPAPD nanoparticles alone. These observations provided more evidence for the accumulative therapy effect between DOX-induced chemotherapy and PDT. Hematoxylin and eosin (H&E) staining of the tumor sections post treatment showed that the majority of tumor cells in the iPAPD group had lost their membrane integrity following laser irradiation (Figure 5d). These observations provided more evidence for the accumulative therapy effect between DOXinduced chemotherapy and PDT. The PHMA and iPAPD group showed comparable body weight change as that of the PBS group (Figure S17). H&E staining showed that no obvious toxicity in the major organs (i.e., heart, liver, spleen, lung, and kidney), indicating the ATLP nanoparticles were of good biosafety (Figure S18).

In summary, we have rationally designed an ATLP nanoparticle for improved cancer therapy. The ATLP nanoparticles possess several distinct advantages to combat the sequential drug delivery barriers. First, the ATLP nanoparticles displayed high colloidal stability, neutral surface charge and fully shielded targeting ligand (i.e., iRGD) in the blood circulation to minimize nonspecific interactions with the plasma proteins and RES. Second, once deposited in the tumor site through the passive accumulation, the targeting ligand of the ATLP nanoparticles was presented for improved tumor penetration and cellular uptake by responding to the tumor acidity microenvironment. Moreover, the P85 pluronic component of the nanoparticles can block the P-gp pump to prevent drug efflux from the tumor cells, thus reversing the acquired drug resistance. Furthermore, the ATLP nanoparticles are of acidity-activatable fluorescence property, which can be utilized to monitor the intratumoral activation of the ATLP nanoparticles, and perform image-guided PDT for promoting intracellular drug release and altering the gene expression profiles of the tumor cells, thereby sensitizing the drug-resistant cells to chemotherapy.

■

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.nanolett.7b02031. Materials, methods, and additional figures (PDF)

■

AUTHOR INFORMATION

Corresponding Authors

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

DOI: 10.1021/acs.nanolett.7b02031 Nano Lett. XXXX, XXX, XXX−XXX

Letter

Nano Letters ORCID

(26) Racker, E. Science 1983, 222, 232−232. (27) Webb, B.; Chimenti, M.; Jacobson, M.; Barber, D. Nat. Rev. Cancer 2011, 11, 671−677. (28) Li, H.; Du, J. Z.; Liu, J.; Du, X.; Shen, S.; Zhu, Y. H.; Wang, X.; Ye, X.; Nie, S.; Wang, J. ACS Nano 2016, 10, 6753−6761. (29) Wang, Y.; Zhou, K.; Huang, G.; Hensley, C.; Huang, X.; Ma, X.; Zhao, T.; Sumer, B.; DeBerardinis, R.; Gao, J. Nat. Mater. 2013, 13, 204−212. (30) Ruoslahti, E. Adv. Drug Delivery Rev. 2017, 110-111, 3−12. (31) Yu, H.; Xu, Z.; Wang, D.; Chen, X.; Zhang, Z.; Yin, Q.; Li, Y. Polym. Chem. 2013, 4, 5052−5055. (32) Yin, Q.; Shen, J.; Zhang, Z.; Yu, H.; Li, Y. Adv. Drug Delivery Rev. 2013, 65, 1699−1715. (33) Yu, P.; Yu, H.; Guo, C.; Cui, Z.; Chen, X.; Yin, Q.; Zhang, P.; Yang, X.; Cui, H.; Li, Y. Acta Biomater. 2015, 14, 115−124. (34) Duan, X.; Xiao, J.; Yin, Q.; Zhang, Z.; Yu, H.; Mao, S.; Li, Y. ACS Nano 2013, 7, 5858−5869. (35) Griffith, L. G.; Swartz, M. A. Nat. Rev. Mol. Cell Biol. 2006, 7, 211−224. (36) Sharma, S.; Kotamraju, V. R.; Molder, T.; Tobi, A.; Teesalu, T.; Ruoslahti, E. Nano Lett. 2017, 17, 1356−1364. (37) Sugahara, K.; Teesalu; Karmali, P.; Kotamraju, V.; Agemy, L.; Greenwald, D.; Ruoslahti, E. Science 2010, 328, 1031−1035. (38) Peng, Z.; Kopecek, J. J. Am. Chem. Soc. 2015, 137, 6726−6729. (39) Hanahan, D.; Weinberg, R. A. Cell 2011, 144, 646−674. (40) Sonveaux, P.; Vegran, F.; Schroeder, T.; Wergin, M. C.; Verrax, J.; Rabbani, Z. N.; De Saedeleer, C. J.; Kennedy, K. M.; Diepart, C.; Jordan, B. F.; Kelley, M. J.; Gallez, B.; Wahl, M. L.; Feron, O.; Dewhirst, M. W. J. Clin. Invest. 2008, 118, 3930−3942. (41) Zheng, M.; Yue, C.; Ma, Y.; Gong, P.; Zhao, P.; Zheng, C.; Sheng, Z.; Zhang, P.; Wang, Z.; Cai, L. ACS Nano 2013, 7, 2056− 2067. (42) Wang, D.; Wang, T.; Liu, J.; Yu, H.; Jiao, S.; Feng, B.; Zhou, F.; Fu, Y.; Yin, Q.; Zhang, P.; Zhang, Z.; Zhou, Z.; Li, Y. Nano Lett. 2016, 16, 5503−5513.

Haijun Yu: 0000-0002-3398-0880 Yaping Li: 0000-0002-0574-6966 Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS The authors thank the technician support from Merck Millipore for flow cytometric examination. Financial supports from the National Natural Science Foundation of China (31671024, 31622025, and 81521005) and the National Basic Research Program of China (2013CB932704) are gratefully acknowledged. All animal procedures were carried out under the guidelines approved by the Institutional Animal Care and Use Committee (IACUC) of the Shanghai Institute of Material Medica, Chinese Academy of Sciences.

■

REFERENCES

(1) Sun, Q.; Zhou, Z.; Qiu, N.; Shen, Y. Adv. Mater. 2017, 29, 1606628. (2) Sun, H.; Su, J.; Meng, Q.; Yin, Q.; Chen, L.; Gu, W.; Zhang, P.; Zhang, Z.; Yu, H.; Wang, S.; Li, Y. Adv. Mater. 2016, 28, 9581−9588. (3) Yu, H.; Cui, Z.; Yu, P.; Guo, C.; Feng, B.; Jiang, T.; Wang, S.; Yin, Q.; Zhong, D.; Yang, X.; Zhang, Z.; Li, Y. Adv. Funct. Mater. 2015, 25, 2489−2500. (4) Zhou, F.; Feng, B.; Yu, H.; Wang, D.; Wang, T.; Liu, J.; Meng, Q.; Wang, S.; Zhang, P.; Zhang, Z.; Li, Y. Theranostics 2016, 6, 679−687. (5) Yu, H.; Nie, Y.; Dohmen, C.; Li, Y.; Wagner, E. Biomacromolecules 2011, 12, 2039−2047. (6) Jiang, T.; Zhang, Z.; Zhang, Y.; Lv, H.; Zhou, J.; Li, C.; Hou, L.; Zhang, Q. Biomaterials 2012, 33, 9246−9258. (7) Wang, S.; Huang, P.; Chen, X. ACS Nano 2016, 10, 2991−2994. (8) Gullotti, E.; Yeo, Y. Mol. Pharmaceutics 2009, 6, 1041−1051. (9) Jin, E.; Zhang, B.; Sun, X.; Zhou, Z.; Ma, X.; Sun, Q.; Tang, J.; Shen, Y.; Van Kirk, E.; Murdoch, W. J.; Radosz, M. J. Am. Chem. Soc. 2013, 135, 933−940. (10) Yu, H.; Wagner, E. Curr. Opin. Mol. Ther. 2009, 11, 165−178. (11) Liu, J.; Wang, T.; Wang, D.; Dong, A.; Li, Y.; Yu, H. Acta Pharmacol. Sin. 2017, 38, 1−8. (12) Li, D.; Ma, Y.; Du, J.; Tao, W.; Du, X.; Yang, X.; Wang, J. Nano Lett. 2017, 17, 2871−2878. (13) Guan, X.; Guo, Z.; Lin, L.; Chen, J.; Tian, H.; Chen, X. Nano Lett. 2016, 16, 6823−6831. (14) Chauhan, V. P.; Jain, R. K. Nat. Mater. 2013, 12, 958−962. (15) Zhou, H.; Fan, Z.; Deng, J.; Lemons, P. K.; Arhontoulis, D. C.; Bowne, W. B.; Cheng, H. Nano Lett. 2016, 16, 3268−3277. (16) Gong, H.; Chao, Y.; Xiang, J.; Han, X.; Song, G.; Feng, L.; Liu, J.; Yang, G.; Chen, Q.; Liu, Z. Nano Lett. 2016, 16, 2512−2521. (17) Provenzano, P. P.; Cuevas, C.; Chang, A. E.; Goel, V. K.; Von Hoff, D. D.; Hingorani, S. R. Cancer Cell 2012, 21, 418−429. (18) Feig, C.; Gopinathan, A.; Neesse, A.; Chan, D. S.; Cook, N.; Tuveson, D. A. Clin. Cancer Res. 2012, 18, 4266−4276. (19) Zhu, L.; Wang, T.; Perche, F.; Taigind, A.; Torchilin, V. P. Proc. Natl. Acad. Sci. U. S. A. 2013, 110, 17047−17052. (20) Liu, X.; Xiang, J.; Zhu, D.; Jiang, L.; Zhou, Z.; Tang, J.; Huang, Y.; Shen, Y. Adv. Mater. 2016, 28, 1743−1752. (21) Li, H.; Du, J.; Du, X.; Xu, C. F.; Sun, C.; Wang, H.; Cao, Z.; Yang, X.; Zhu, Y.; Nie, S.; Wang, J. Proc. Natl. Acad. Sci. U. S. A. 2016, 113, 4164−4169. (22) Mo, R.; Sun, Q.; Xue, J.; Li, N.; Li, W.; Zhang, C.; Ping, Q. Adv. Mater. 2012, 24, 3659−3665. (23) Qiu, N.; Liu, X.; Zhong, Y.; Zhou, Z.; Piao, Y.; Miao, L.; Zhang, Q.; Tang, J.; Huang, L.; Shen, Y. Adv. Mater. 2016, 28, 10613−10622. (24) Zhu, L.; Kate, P.; Torchilin, V. P. ACS Nano 2012, 6, 3491− 3498. (25) Hu, Q.; Sun, W.; Lu, Y.; Bomba, H. N.; Ye, Y.; Jiang, T.; Isaacson, A. J.; Gu, Z. Nano Lett. 2016, 16, 1118−1126. H

DOI: 10.1021/acs.nanolett.7b02031 Nano Lett. XXXX, XXX, XXX−XXX