Design and Proof of Programmed 5-Aminolevulinic Acid Prodrug

Research Article www.acsami.org

Design and Proof of Programmed 5‑Aminolevulinic Acid Prodrug Nanocarriers for Targeted Photodynamic Cancer Therapy Jina Wu, Haijie Han, Qiao Jin,* Zuhong Li, Huan Li, and Jian Ji* MOE Key Laboratory of Macromolecule Synthesis and Functionalization, Department of Polymer Science and Engineering, Zhejiang University, Hangzhou 310027, China S Supporting Information *

ABSTRACT: 5-Aminolevulinic acid (ALA), the precursor of photosensitizer protoporphyrin IX (PpIX), is a U.S. FDA-approved photodynamic therapeutic agent. However, realizing efficient delivery of ALA is still a big challenge as it is hydrophilic and cannot be recognized and selectively accumulated in tumor cells. In this study, matrix metalloproteinase-2 (MMP-2) and pH dual-sensitive ALA prodrug nanocarriers were constructed as a programmed delivery strategy for the targeted delivery of ALA. The nanocarriers were prepared by the co-modification of gold nanoparticles (AuNPs) with hydrazone-linked ALA and MMP-2-activatable cell-penetrating peptides (CPPs). Cationic CPP RRRRRRRR (R8) was shielded by zwitterionic stealth peptide EKEKEKEKEKEKEKEKEKEK (EK10) via MMP-2 substrate peptide PLGLAG. The zwitterionic stealth peptide EK10 is designed to endow ALA prodrug nanocarriers with strong antifouling ability and prolonged circulation time. Upon arriving at the tumor tissue, the shielded cationic CPP R8 can be activated by tumor-microenvironment-overexpressed MMP-2, which enabled enhanced cellular uptake of ALA. The results of drug loading and release, cellular uptake, PpIX generation and accumulation, photodynamic cytotoxicity, and photodynamic tumor inhibition demonstrated that such tumor-microenvironment-sensitive ALA prodrug nanocarriers could be considered as potential candidates for targeted photodynamic cancer therapy. KEYWORDS: 5-aminolevulinic acid, prodrug nanocarriers, programmed targeting, enzyme, photodynamic cancer therapy

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INTRODUCTION Photodynamic therapy (PDT), as a promising approach for the noninvasive treatment of tumors and other diseases, has attracted increasing attention.1−7 During the PDT process, the injected photosensitizer (PS) can be activated by specific light irradiation to produce reactive oxygen species (ROS), which is quite toxic to tumor cells.8 Therefore, because of the noninvasive property and minimal side effects, PDT has attracted more and more attention for tumor treatment. However, among all of the presented PSs, only the following three drugs and their derivatives are approved by U.S. FDA: Photofrin, Visudyne, and Levulan (5-aminolevulinic acid, ALA). Different from the former two PSs, ALA, which is an endogenous metabolic precursor of PS protoporphyrin IX (PpIX), has attracted increasing attention because of its fast clearance and reduced concomitant photosensitivity.9,10 When internalized by tumor cells, ALA will be converted to PpIX by heme biosynthetic pathway. The in situ produced PpIX will accumulate intracellularly, and ROS can be produced after light irradiation. However, ALA is hydrophilic and lacks targeting ability. Therefore, realizing efficient internalization across the lipophilic cell membrane is still a challenge, which greatly hampers its practical applications for ALA-based PDT.9,11 To achieve maximum therapeutic efficacy of ALA-based PDT, © 2017 American Chemical Society

efficient delivery vehicles that help ALA to be selectively accumulated in tumor tissue and then bypass the lipophilic barrier of cell membrane would be highly expected. Nanocarriers were hence developed by encapsulation of ALA into polymeric micelles or inorganic nanoparticles.12−14 However, in most of the present studies, ALA was encapsulated into nanocarriers by electrostatic interaction, which may result in undesirable leakage in blood circulation and unwanted distribution in normal tissues.11,15−17 The prodrug strategy has been proved as an effective way to avoid undesirable leakage during circulation.18 However, ALA prodrug nanocarriers are still rarely explored to date. Thus, efficient ALA prodrug nanocarriers, which can respond to endogenous biological stimulus, are highly expected. For ideal drug-delivery systems, the nanocarriers should be “stealthy” during circulation and have a long blood circulation time. Zwitterions are considered as an attractive alternative to stealth coatings of nanoparticles, which show strong resistance to nonspecific adsorption.19 Zwitterionic nanoparticles even exhibited longer blood circulation time than poly(ethylene Received: December 19, 2016 Accepted: April 11, 2017 Published: April 11, 2017 14596

DOI: 10.1021/acsami.6b15853 ACS Appl. Mater. Interfaces 2017, 9, 14596−14605

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ACS Applied Materials & Interfaces

Scheme 1. Schematic Illustration of Enzyme and pH Dual-Sensitive ALA-Conjugated AuNPs for Targeted PDT

glycol) (PEG)-modified nanoparticles.20,21 Meanwhile, after being accumulated in tumor tissue by enhanced permeability and retention effect, the nanocarriers should be selectively uptaken by cancer cells. Realizing a long circulation time as well as enhancing cancer cell uptake is still a big challenge. For example, cell-penetrating peptides (CPPs) can promote the internalization of various cargos by cancer cells. Nevertheless, nanocarriers modified with CPPs can be easily cleared by reticuloendothelial system (RES) and therefore cannot be effectively accumulated in tumor tissue.22,23 To overcome this dilemma, it is critical to construct drug nanocarriers that can “turn off” and “turn on” the internalization ability during circulation and in tumor tissue, respectively. One strategy is to protect or bury the targeting group with bioinert molecules to suppress the nonspecific interactions during circulation. Moreover, after accumulation in tumor tissues, the shield can be easily removed, and the targeting ligand will be activated upon exposure to specific tumor microenvironment.24−27 For instance, tumor-acidity-sensitive drug nanocarriers were well developed as the pH (6.2−6.9) in the tumor part is lower than that in normal tissue (pH 7.2−7.4).27−31 However, it is not very specific because mild acidic environment does not only exist in tumor tissue but also in an inflammation region. On the contrary, the dysregulation of some enzymes is a more specific character for many kinds of tumors.32,33 For example, the overexpression of matrix metalloproteinase-2 (MMP-2) is always observed in various cancers and associated with tumor invasiveness, metastasis, and angiogenesis.34,35 Therefore, the dysregulated enzymes can be used as specific tumor localization signals to design tumor-microenvironment-sensitive drug nanocarriers.36−41 In this study, taking advantage of the programmed delivery strategy, we aim to fabricate novel ALA prodrug nanocarriers that exhibit prolonged blood circulation time, tumor-microenvironment-triggered enhanced cellular uptake, and intracellular smart drug release. To prove this concept, AuNPs were adopted in this study because of their excellent stability,

biocompatibility, and easy functionalization. As illustrated in Scheme 1, ALA was conjugated to AuNPs by a pH-responsive hydrazone bond that could be cleaved at endosomal/lysosomal pH (5.5). Meanwhile, the AuNPs were co-modified with thiolated peptide CGGGRRRRRRRRPLGLAGEKEKEKEKEKEKEKEKEKEK (HS-R8-PLGLAG-EK10). PLGLAG, which can be selectively cleaved by MMP-2, was utilized to link CPP sequence RRRRRRRR (R8) and zwitterionic shield stealth peptide EKEKEKEKEKEKEKEKEKEK (EK10). The outer zwitterionic shield EK10 ensures stability and “antifouling” of the nanocarriers during circulation.42,43 On reaching the tumor site, the shield peptide EK10 would be removed because of the cleavage of PLGLAG by MMP-2, leading to the exposure of CPP sequence R8. Benefiting from this unique design, the internalization of ALA prodrug nanocarriers can be greatly enhanced by cancer cells. Moreover, with the release of PDT precursor ALA at endosomal/lysosomal pH, enhanced PDT is expected. Such enzyme and pH dual-responsive drug nanocarriers might provide a promising way for ALA-based PDT for the treatment of different tumors.

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EXPERIMENTAL SECTION

Materials. 5-Aminolevulinic acid (ALA) was obtained from Dalian Meilun Biological Technology Co., Ltd. Peptides CGGGRRRRRRRRPLGLAGEKEKEKEKEKEKEKEKEKEK (HSR8-PLGLAG-EK10), CGGGRRRRRRRRPGGGAGEKEKEKEKEKEKEKEKEKEK (HS-R8-PGGGAG-EK10), and CGGGRRRRRRRR (HS-R8) were purchased from Toppeptide Co., Ltd. Hydrogen tetrachloroaurate hydrate (HAuCl4·4H2O) and trisodium citrate dehydrate (C6H5Na3O7·2H2O) were supplied by Sinopharm Chemical Reagent Co., Ltd. Milli-Q water was prepared from Millipore Milli-Q Academic Water Purification System. All other reagents were used without further purification. SCC-7 cells were supplied by Prof. XianZheng Zhang in Wuhan University. Healthy female Institute of Cancer Research (ICR) mice and nude mice were purchase from the animal center of Zhejiang Academy of Medical Sciences. Synthesis of 2-Mercapto Acetohydrazide-Conjugated ALA (HS-hyd-ALA). HS-hyd-ALA was synthesized as shown in Scheme S1. 14597


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For fluorescent measurement, SCC-7 cells were seeded in 96-well plates (0.8 × 104 cells per well) with DMEM and incubated for 36 h. The ALA-conjugated AuNPs with or without MMP-2 sensitivity were then added. The medium was removed after 4 or 8 h, and the cells were washed with fresh PBS. Finally, 40 μL of PBS was added to each well. The fluorescent images were observed by fluorescence microscopy. As a control, the MMP-2 low-expressed A549 cells were incubated with ALA-conjugated AuNPs using the same method for fluorescent imaging. Investigation of the Internalization Mechanism. To study the cellular uptake pathway of ALA-conjugated AuNPs, cell culture was carried out at 4 °C or they were pretreated with NaN3 under regular incubation conditions. Low-Temperature Incubation at 4 °C. SCC-7 cells were seeded in a confocal dish at 1 × 105 cells per well and incubated for 36 h at 37 °C. The medium was replaced with ALA-conjugated AuNPs (10 μg/ mL equivalent concentration of ALA) in fresh culture medium. The SCC-7 cells were then incubated at 4 °C instead of the regular 37 °C condition. Incubation with Cells under ATP Depletion. SCC-7 cells were seeded in a confocal dish at 1 × 105 cells per well and incubated at 37 °C for 36 h. The cells were then pretreated with 10 mM NaN3 and 50 mM 2-deoxy-D-glucose at 37 °C for 30 min. After that, the medium was replaced with ALA-conjugated AuNPs (10 μg/mL equivalent concentration of ALA) in fresh culture medium. After incubation for 3 h, the medium was removed, and the cells were washed with PBS for three times. The fluorescent images were recorded by fluorescence microscopy. Measurement of ROS. SCC-7 cells were seeded in 96-well plates (0.8 × 104 cells per well) with DMEM and incubated for 36 h. The ALA-conjugated AuNPs with or without MMP-2 sensitivity were then added. After 4 h, the medium was replaced with 40 μL of a serum-free medium containing 25 μM 2′,7′-dichlorofluorescein diacetate (DCFDA). The cells were then washed with the serum-free medium after another 0.5 h. After the cells were irradiated for 30 s, fluorescent images were obtained by fluorescence microscopy. Cytotoxicity Assay. SCC-7 cells were seeded in 96-well plates (1 × 104 cells per well) with DMEM and incubated for 36 h. Then, the MMP-2 sensitive ALA-conjugated AuNPs were added at a 10 μg/mL equivalent concentration of ALA. After 4 h, the cells were washed by PBS three times. Finally, 60 μL of DMEM without fetal bovine serum was added into each well. Then, the samples were irradiated by a laser of wavelength 635 nm (730 mW/cm2) for different times, ranging from 1 to 10 min. After that, the cells were incubated for another 12 h. For MTT measurements, 20 μL of 5 mg/mL MTT solution was added in every well, and the cells were incubated for another 4 h. The culture medium in every well was then replaced with 150 μL of dimethyl sulfoxide (DMSO). The absorbance at 490 nm was recorded. The cytotoxicities of free ALA and ALA-conjugated AuNPs with or without MMP-2 sensitivity were investigated by MTT using the same method. The concentration of ALA in each group ranged from 5 to 20 μg/mL, and the irradiation time remained 2 min for each sample. In Vivo Circulation Evaluation and PDT Treatment. All animal experiments were approved by the Institutional Animal Care and Use Committee of Zhejiang University and in full compliance with international ethics guidelines. Healthy female Institute of Cancer Research (ICR) mice were chosen as the model for in vivo circulation evaluation. The concentration of the remaining Au in blood at different time intervals was measured by ICP-MS. Blood samples were collected at 0.75, 1.5, 4, 6, 10, and 24 h. The total weight of blood was estimated to be about 7% of body weight. Female nude mice bearing SCC-7 tumors were chosen as the model for in vivo PDT treatment. All of the selected mice were divided into four groups randomly with five mice in each group. PBS, free ALA, and ALA-conjugated AuNPs with or without MMP-2 sensitivity were injected via tail vein every 2 days. The concentration of ALA in the latter three groups was 15 mg/ kg. After 4 h, the tumor tissues were irradiated for 6 min by a 2840 mW/cm2 laser with a wavelength of 635 nm. The therapeutic efficacy of the modified AuNPs was assessed by measuring the tumor volume. The side effects of the formulation were assessed by measuring the

Briefly, 12.02 g (0.1 mol) of ethyl thioglycolate was dissolved in 30 mL of ethanol, and 7.01 g (0.14 mol) of hydrazine monohydrate was then added. After stirring at reflux for 24 h, the reaction was stopped and the solution was concentrated in vacuum. The crude was purified through silica gel column to obtain 2-mercapto acetohydrazide; 1H NMR (500 MHz, DMSO-d6), δ 3.25 (s, 2H, CH2). To 20 mL of methanol, 0.53 g (0.005 mol) of 2-mercapto acetohydrazide and 0.84 g (0.005 mol) of 5-aminolevulinic acid hydrochloride were added. Then, trifluoroacetic acid (10 μL) was added to this mixture. After stirring at room temperature for 24 h, the crude was obtained by concentrating the mixture in vacuum. Finally, this crude was purified through silica gel column to obtain 2-mercapto acetohydrazide-conjugated ALA (HS-hyd-ALA). Synthesis of ALA-Conjugated AuNPs. ALA-conjugated AuNPs were prepared by ligand exchange. Citrate-capped AuNPs were synthesized according to a traditional route, as previously reported.44 Then, ALA-conjugated AuNPs with MMP-2 sensitivity were synthesized by exchanging citrate molecules with HS-hyd-ALA and HS-R8-PLGLAG-EK10. Briefly, 1 μmol of HS-hyd-ALA and 3 μmol of HS-R8-PLGLAG-EK10 were dissolved in water. The mixture was then added to the original 10 mL of citrate-coated AuNP solution, adjusting the pH to 8.0. The mixture was then stirred at room temperature for 24 h. The ALA-conjugated AuNPs were obtained by further ultrafiltration and then redispersed in 10 mM phosphate buffer solution (PBS) (pH 7.4). The MMP-2 nonsensitive AuNPs or AuNPs without EK10 shielding were synthesized by the same method unless HS-R8-PGGGAG-EK10 or HS-R8 was used instead of HS-R8PLGLAG-EK10. MMP-2-Triggered Removal of Stealth Peptide EK10. ALAconjugated AuNPs with or without MMP-2 sensitivity (10 μg/mL equivalent concentration of ALA) were incubated with MMP-2 (5 μg/ mL) in TCNB buffer solution (composed of 100 mM Tris, 5 mM calcium chloride, 200 mM NaCl, and 0.1% Brij). The zeta potential of the solution was recorded at given time intervals. To study the MMP-2-concentration-dependent zeta-potential change, MMP-2-sensitive AuNPs (10 μg/mL equivalent concentration of ALA) were incubated with MMP-2 in different concentrations in TCNB buffer solution. After 2 h, the zeta potential of the solution was recorded. In Vitro Release of ALA. A thorough release of ALA is a crucial parameter for the generation of PS PpIX. The release of ALA was performed under different pH media (pHs 5.5 and 7.4) at 37 °C. Typically, ALA-conjugated AuNPs (2 mL) were added to a dialysis bag (MWCO 3500). The dialysis bag was then immersed in 8 mL of PBS (pH 5.5 or 7.4) with constant shaking (100 rpm) at 37 °C. At fixed time intervals, 1 mL of the released solution was taken out and 1 mL fresh PBS medium was added. The release of ALA was investigated by a ultraviolet−visible (UV−vis) spectrometer using a trinitrobenzene sulfonic acid (TNBS) kit according to the manufacturer’s instructions.45 Cellular Uptake and Generation of PpIX. The cellular uptake of AuNPs was measured using inductively coupled plasma mass spectrometry (ICP-MS). The endogenous generation of PpIX from ALA was determined by flow cytometry and fluorescence microscopy. SCC-7 (squamous cell carcinoma) cell was used as a model cell line and incubated in 24-well plates (1 × 105 cells per well) with Dulbecco’s modified Eagle’s medium (DMEM) for 36 h. The ALAconjugated AuNPs with or without MMP-2 sensitivity were added. After 4 or 8 h, the cells were washed by PBS and then treated with aqua regia (HNO3/HCl = 1:3, v/v) for 2 h. The solution was then diluted 10 times, and the Au concentration internalized by SCC-7 cells was measured by ICP-MS. For flow cytometry assay, SCC-7 cells were seeded in 24-well plates (2 × 105 cells per well) with DMEM and incubated for 36 h. The ALA-conjugated AuNPs with or without MMP-2 sensitivity were then added. After incubation for 4 and 8 h, respectively, the cells were washed with PBS and then treated with Trypsin. Finally, 1 × 104 cells were collected by a FACSCalibur flow cytometer, and the results were analyzed using WinMDI 2.9 software. 14598


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Figure 1. (a) DLS plot and (b) TEM image of ALA-conjugated AuNPs, indicating their stability. (c) UV−vis spectrum of HS-R8-PLGLAG-EK10 and ALA co-modified AuNPs in PBS at different time intervals. (d) DLS results of the peptide HS-R8-PLGLAG-EK10 and HS-R8-coated ALAconjugated AuNPs incubated with PBS, human lysozyme, and BSA. body weight of mice in each group. The tumor volume can be calculated as (tumor length) × (tumor width)2/2. Histological Analysis. For the immunohistochemistry staining profile, H&E staining, Ki67 staining, and TUNEL assays were employed to further confirm the proliferation and apoptosis after treated once. PBS, free ALA, and ALA-conjugated AuNPs with or without MMP-2 sensitivity were injected via tail vein. The concentration of ALA in the latter three groups was 15 mg/kg. After 4 h, the tumor tissues were irradiated for 6 min by a 635 nm laser (2840 mW/cm2). The mice were sacrificed, and the tumor tissues were harvested after 24 h. The tissue samples were then fixed in 4% neutral buffered formalin, processed routinely into paraffin, sectioned into 4 μm, and stained with hematoxylin and eosin (H&E), Ki67 antibody, and TdT-mediated dUTP nick end labeling (TUNEL). The samples were measured using an optical microscope. Characterization. 1H NMR spectra were recorded on a Bruker DMX500 spectrometer operating at 500 MHz. Electrospray ionization mass spectrometry (ESI-MS) was performed on a Bruker Esquire 3000 plus ion trap mass spectrometer equipped with an ESI source. Dynamic light scattering (DLS) and zeta-potential measurements were performed with Zetasizer Nano-ZS from Malvern Instruments, equipped with a He−Ne laser at a wavelength of 633 nm at 25 °C. Transmission electron microscopy (TEM) measurements were conducted on a JEM-3010 instrument operating at 80 kV. UV−vis spectra analysis was carried out by a UV−vis Shimadzu UV-2505

spectrophotometer. ICP-MS (Thermo Elemental Corporation) was used to measure Au concentration. Cell viability was measured by a microplatereader (MODEL 550; Bio-Rad) with absorbance at 490 nm.

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RESULTS AND DISCUSSIONS Synthesis and Characterization of ALA-Conjugated AuNPs. Citrate-capped AuNPs were first prepared as previously described.44 The number-average hydrodynamic diameter of the citrate-capped AuNPs was 14.3 nm, with a plasmon band at 519 nm (data not shown). 2-Mercapto acetohydrazide-conjugated ALA (HS-hyd-ALA) was synthesized by the reaction between hydrazine groups and ketone groups (Scheme S1). The 1H NMR and ESI-MS results of the HS-hyd-ALA are shown in Figures S1 and S2 respectively. ALA and peptide co-modified AuNPs were formed by the incubation of 2-mercapto acetohydrazide-conjugated ALA (HS-hyd-ALA) and HS-R8-PLGLAG-EK10 with citrate-capped AuNPs over 24 h. The size and morphology of the ALA-conjugated AuNPs were examined by DLS and TEM measurements (Figure 1a,b). The number-average hydrodynamic diameter (Dh) of the ALAconjugated AuNPs was 17.0 ± 0.5 nm. The TEM images revealed that the AuNPs remained monodisperse with spherical 14599


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Figure 2. (a) Zeta-potential change of the ALA-conjugated AuNPs with or without MMP-2 sensitivity after incubation with MMP-2 for different time intervals. (b) In vitro cumulative release of ALA from the ALA-conjugated AuNPs with MMP-2 sensitivity under different pH conditions (7.4 and 5.5).

morphologies after surface modification. The drug-loading content of ALA was measured by the detection of the amino groups of ALA using the TNBS method.45 The concentration of the AuNPs can be estimated by the Beer−Lambert law.46 It can be calculated that ∼2300 ALA molecules were conjugated to the surface of each AuNP. The stability and the antifouling ability of AuNPs are the first criteria for biomedical applications. In this study, zwitterionic peptides of alternating glutamic acid (E) and lysine (K) were used as antifouling coating for surface tailoring of AuNPs. The stability of HS-R8PLGLAG-EK10 and ALA co-modified AuNPs was measured by UV−vis spectroscopy and DLS. HS-R8 and ALA co-modified AuNPs without stealth peptide EK10 were chosen as a control to further demonstrate the stealth ability of zwitterionic peptides EK10. At first, ALA-conjugated AuNPs coated with peptide HS-R8-PLGLAG-EK10 were incubated in PBS at pH 7.4. The UV−vis spectrum showed a characteristic peak at about 520 nm. In addition, no significant change was observed in the UV−vis spectrum even after 24 or 48 h, which suggested excellent stability in PBS (Figure 1c). As is known, nonspecific protein adsorption is always a big problem in blood circulation. Therefore, negatively charged bovine serum albumin (BSA) and positively charged human lysozyme were used as model proteins to evaluate the antifouling ability of the AuNPs. The hydrodynamic diameters of HS-R8-PLGLAG-EK10 and ALA co-modified AuNPs incubated in BSA, lysozyme, and PBS were almost the same as in water (Figure 1d). In contrast, the hydrodynamic diameter of HS-R8 and ALA co-modified AuNPs significantly changed (Figure 1d). The hydrodynamic diameter in PBS was 962.2 nm, indicating the aggregation of HS-R8-coated AuNPs. After incubation with human lysozyme and BSA, the hydrodynamic diameters of HS-R8 coated AuNPs increased to 25.0 and 28.1 nm, respectively. The change in zeta potential was also tested to further identify whether the increased hydrodynamic diameter of HS-R8-protected AuNPs was ascribed to the absorption of the incubated protein. The zeta potential of HS-R8-protected AuNPs in water was 20.5 mV, whereas the corresponding values in human lysozyme and BSA were 28.6 and −21.8 mV, respectively, suggesting the

nonspecific protein absorption. Therefore, owing to the zwitterionic EK10 coating, HS-R8-PLGLAG-EK10 and ALA co-modified AuNPs exhibited excellent stability and antifouling ability. Enzyme-Sensitive Removal of Shield Peptide EK10. ALA-conjugated AuNPs were coated with MMP-2-sensitive peptide HS-R8-PLGLAG-EK10. The CPP R8 was masked with stealth peptide EK10. In the presence of MMP-2, PLGLAG peptide can be cleaved between glycine (G) and leucine (L) to remove the shield peptide from CPP. To evaluate the MMP-2sensitive deshielding effect, the change in zeta potential was recorded after incubating AuNPs with MMP-2 (Figure 2a). The AuNPs coated with MMP-2-nonsensitive peptide CGGGRRRRRRRRPGGGAGEKEKEKEKEKEKEKEKEKEK (HS-R8-PGGGAG-EK10) was used as a control. The zeta potential of HS-R8-PLGLAG-EK10-coated AuNPs was −4.2 mV in PBS. After incubation with MMP-2, the value increased significantly to 10.3 mV (Figure 2a), which might be attributed to the cleavage of PLGLAG and the removal of shield peptide EK10. Therefore, the positively charged CPP, R8, was exposed, which resulted in the increase of zeta potential. However, if the AuNPs coated with MMP-2-nonsensitive peptide HS-R8PGGGAG-EK10 were incubated with MMP-2, no noticeable change was observed in zeta potential even after 6 h. Moreover, as shown in Figure S3, the zeta-potential change enhanced with increasing concentration of MMP-2, which suggested that the cleavage of MMP-2-sensitive peptide was MMP-2 dependent. Given that MMP-2 is always overexpressed in tumor microenvironment, the charge-conversion behavior of HS-R8PLGLAG-EK10-coated AuNPs is very beneficial for their internalization in tumor tissue. In Vitro Release of ALA. As ALA was conjugated to AuNPs by a pH-responsive hydrazone bond, the drug-release behavior was observed at pH 5.5 or 7.4. As shown in Figure 2b, when ALA-conjugated AuNPs were incubated at pH 7.4, only about 20% ALA was released even after 8 h. However, a much faster and complete release was achieved at pH 5.5; more than 80% ALA was released after 8 h probably because of the cleavage of hydrazone bond at acidic media. This result 14600


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Figure 3. (a) Cellular uptake of ALA-conjugated AuNPs with or without MMP-2 sensitivity at different time intervals. All error bars represent standard deviations (n = 3); * and ** indicate P < 0.05 and P < 0.01, respectively. (b) Fluorescence microscopy images of PpIX generated in SCC-7 cells after incubation with ALA-conjugated AuNPs with or without MMP-2 sensitivity at different time intervals. (c) Flow cytometric histogram profiles of SCC-7 cells incubated with ALA-conjugated AuNPs with or without MMP-2 sensitivity at different time intervals.

demonstrated that ALA remained stable in circulation but underwent a quick release at endosomal/lysosomal pH, which might overcome the dilemma between unwanted leakage during circulation and fast release after internalization. Cellular Uptake and Generation of PpIX. To examine the tumor-microenvironment-activatable targeting ability, cellular uptake and generation of PpIX assays were performed on SCC-7 cells that overexpress MMP-2.47 ALA-conjugated AuNPs coated with MMP-2-sensitive peptide HS-R8PLGLAG-EK10 were incubated with SCC-7 cells, and MMP2-nonsensitive HS-R8-PGGGAG-EK10-coated AuNPs were used as control. The cellular uptake of AuNPs by SCC-7 cells was measured after aqua regia digestion by ICP-MS. After incubation for 4 h, the cellular uptake of HS-R8-PLGLAGEK10-coated AuNPs was 1.48-fold higher that of MMP-2nonsensitive HS-R8-PGGGAG-EK10-coated AuNPs (Figure 3a). Over time, the cellular uptake increased in both groups. However, the uptake of HS-R8-PLGLAG-EK10-coated AuNPs was even higher (1.65-fold) than that of the control group after 8 h incubation. These results might be attributed to the MMP-2 removable shield peptide, EK10. Upon incubation of HS-R8PLGLAG-EK10-coated AuNPs with SCC-7 cells, the secreted MMP-2 would cleave the peptide PLGLAG, resulting in the

exposure of positively charged CPP R8. Therefore, the cellular internalization can be greatly enhanced. ALA is an endogenous precursor of PS PpIX. After internalization of ALA-conjugated AuNPs by cancer cells, ALA was expected to be released in endosome/lysosome. The production and accumulation of PpIX in cancer cells are very important for ALA-based PDT. As is known, PpIX is redfluorescent and can be used as a diagnostic agent. Flow cytometry and fluorescence microscopy were employed to investigate the accumulation of PpIX in SCC-7 cells. The SCC7 cells were incubated with MMP-2-sensitive ALA-conjugated AuNPs for 4 and 8 h. As shown in Figure 3b, a strong red fluorescence of PpIX could be observed in the SCC-7 cells, which demonstrated the successful intracellular production of PpIX. The fluorescence increased with time. Meanwhile, the red fluorescence in the SCC-7 cells that incubated with MMP2-sensitive AuNPs was much stronger than that of the control group. Furthermore, the quantification of the generated PpIX was studied by flow cytometry, the results of which showed that the fluorescence intensities of PpIX in the SCC-7 cells increased with incubation time (Figure 3c). In addition, the fluorescence intensity of SCC-7 cells incubated with MMP-2sensitive AuNPs was much higher than that of the MMP-214601


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ACS Applied Materials & Interfaces nonsensitive AuNPs. The flow cytometry and fluorescence microscopy results suggested that the MMP-2-triggered exposure of CPP R8 was helpful for the successful internalization by SCC-7 cells. Moreover, the MMP-2 low-expressed A549 cells were used as the negative control to demonstrate the activity of MMP-2sensitive or MMP-2-nonsensitive AuNPs. Fluorescent images (Figure S4) showed that the fluorescence of A549 cells incubated with MMP-2-sensitive AuNPs was almost the same as that with MMP-2-nonsensitive AuNPs, which further demonstrated that overexpressed MMP-2 is the key factor to trigger the exposure of CPP R8 and the enhancement of cellular internalization. Investigation of the Internalization Mechanism. As ALA was conjugated to the AuNPs via a pH-sensitive covalent bond, the internalization mechanism of ALA-conjugated AuNPs might be different from that of free ALA. Therefore, the internalization mechanism of ALA-conjugated AuNPs was further investigated. As is known, endocytosis is an energydependent process and can usually be inhibited by incubating cells at low temperature (4 °C instead of 37 °C) or in ATP (adenosine triphosphate)-depleted environments, such as pretreating with NaN3.48 As shown in Figure S5, compared to that in the cells incubated under normal condition, the intracellular fluorescence was much weaker in the cells incubated at 4 °C or pretreated with NaN3, which confirmed that ALA-conjugated AuNPs were internalized into cells by endocytosis. In Vitro Measurement of Reactive Oxygen Generation. The generation of ROS upon light irradiation plays a crucial role in the PDT process. Therefore, a widely used ROS detector, DCFDA, was employed in this study. DCFDA is nonfluorescent but can be oxidized to 2′,7′-dichlorofluorescin (DCF) by ROS, which is green fluorescent. After SCC-7 cells were incubated with ALA-conjugated AuNPs, the green fluorescence of DCF can be clearly observed after light irradiation, indicating the production of ROS. More interestingly, compared to that in SCC-7 cells incubated with MMP-2nonsensitive AuNPs, a stronger green fluorescence was observed in those incubated with MMP-2-sensitive AuNPs, which may attribute to the better internalization by the SCC-7 cells (Figure S6). This result was consistent with the production of PpIX in SCC-7 cells (Figure 3a−c). Taking advantage of the tumor-microenvironment-sensitive cellular uptake, MMP-2-sensitive ALA-conjugated AuNPs were very beneficial for photodynamic ablation of cancer cells. In Vitro Photodynamic Cytotoxicity Assay. Typical PDT drugs are expected to have low cytotoxicity when no light was applied, whereas an effective capability of killing tumor cells upon light irradiation. We first assessed the cytotoxicity of MMP-2-sensitive ALA-conjugated AuNPs upon different light irradiation times (Figure 4a). The results demonstrated that negligible cytotoxicity was observed before light irradiation. However, with prolonged irradiation time, the cell viability decreased dramatically, exhibiting strong photodynamic ablation of SCC-7 cells. The cytotoxicities of MMP-2-sensitive AuNPs, MMP-2-nonsensitive AuNPs, and free ALA at different concentrations were further investigated after light irradiation. As shown in Figure 4b, a dose-dependent cytotoxicity was observed upon irradiation. The half-maximal inhibitory concentrations (IC50) of SCC-7 cells incubated with MMP-2sensitive AuNPs and MMP-2-nonsensitive AuNPs were 12 and 20 μg/mL, respectively. Both these values were much lower

Figure 4. (a) SCC-7 cell viability exposed to MMP-2-sensitive AuNPs with an equivalent ALA concentration of 10 μg/mL and irradiated for different times. (b) SCC-7 cell viability exposed to different concentrations of free ALA, MMP-2-sensitive AuNPs, and MMP-2nonsensitive AuNPs and irradiated with CW 635 nm light (n = 4).

than that of free ALA. The ALA prodrug nanocarriers were more effective in PDT than in free ALA probably because of the more effective ALA delivery into cancer cells. It should be noted that MMP-2-sensitive ALA-conjugated AuNPs induced a higher amount of cell death than the MMP-2-nonsensitive ones, which again indicated that tumor-microenvironmentsensitive internalization is a key factor for enhanced PDT. In Vivo Evaluation of Circulation and PDT Treatment. To realize effective accumulation of the drugs in tumor tissues, it is necessary that the drug-delivery system should have long blood circulation time. In this study, ICR mice were chosen as a model for the in vivo evaluation of circulation. Blood circulation curves of the two kinds of AuNPs (HS-R8-coated AuNPs and HS-R8-PLGLGA-EK10-coated AuNPs) are shown in Figure 5a (given in % injected dose (% ID) and % ID/g blood). A quick clearance of AuNPs coated with HS-R8 was observed. Nearly no Au content in blood was detected by ICPMS even after 45 min of injection, which may be due to its fast clearance by RES. However, owing to the outstanding antifouling ability of the zwitterionic stealth peptide EK10, the circulation time of AuNPs coated with HS-R8-PLGLGAEK10 was remarkably prolonged. More than 20% ID/g Au 14602


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Figure 5. (a) Blood circulation curves of peptide HS-R8-PLGLGA-EK10- and HS-R8-coated AuNPs in female ICR mice. In vivo antitumor study via intravenous injection. (b) Relative tumor volume, with the arrow representing the injection of PBS, free ALA, MMP-2-sensitive AuNPs, and MMP-2nonsensitive AuNPs; *p < 0.05 compared to that of MMP-2-nonsensitive AuNPs. (c) H&E staining, Ki67 staining, and TUNEL assay of tumor tissue after one injection. The scale bar is 50 μm (n = 5).

AuNPs grew much slower than that of the mice treated with MMP-2-nonsensitive ALA-conjugated AuNPs and free ALA (Figure 5b). The mice were sacrificed after 12 days, and the tumor tissues were harvested. After that, the tumor inhibition rate (TIR) was calculated. The TIR of the mice treated with MMP-2-sensitive ALA-conjugated AuNPs was 64.0%, which was much higher than that of the mice injected with MMP-2nonsensitive ones (34.5%) and free ALA (16.2%), suggesting significantly improved therapeutic efficacy. In addition, although the mice had multiple injections, no significant difference in their body weights was observed in all four groups (Figure S7), indicating negligible systematic toxicity.

content in blood was detected even after 24 h, suggesting prolonged circulation time. In vivo photodynamic therapeutic effect is a crucial index for the clinical application of PDT. Nude mice bearing SCC-7 tumors were chosen as the xenograft tumor model for the in vivo PDT treatment. SCC-7 tumor-bearing nude mice were treated with MMP-2-sensitive ALA-conjugated AuNPs, MMP2-nonsensitive ones, and free ALA with a final ALA concentration of ∼15 mg/kg by tail vein injection. The tumor tissues were irradiated with 635 nm light 4 h after injection. PBS groups were used as the negative control. The tumor of mice treated with MMP-2-sensitive ALA-conjugated 14603



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Cell proliferation and apoptosis in the tumor region after the first irradiation were further examined by immunohistochemical staining, including H&E staining, Ki67 staining, and TUNEL assay (Figure 5c). The results demonstrated that compared to that in PBS, free ALA, and MMP-2-nonsensitive AuNP groups, the cell proliferation was greatly inhibited in MMP-2-sensitive AuNPs when the cell apoptosis was largely enhanced, which is consistent with the above results of tumor growth measurement.

CONCLUSIONS In summary, we have successfully fabricated an efficient ALA prodrug delivery system with programmed tumor targeting ability for enhanced PDT. The ALA prodrug nanocarriers were prepared by modifying AuNPs with HS-hyd-ALA and HS-R8PLGLAG-EK10. The zwitterionic shield peptide, EK10, can avoid fast clearance by RES and endow the nanoparticles with prolonged blood circulation time. Once the peptide PLGLAG was cleaved by tumor-microenvironment-overexpressed MMP2, the cationic CPP, R8, was exposed, thereby accelerating internalization by cancer cells. The PDT precursor, ALA, was then released at endosomal/lysosomal pH, which can further produce PpIX endogenously for PDT. The in vitro and in vivo results showed that MMP-2-sensitive ALA prodrug nanocarriers exhibited better photodynamic therapeutic efficacy than the MMP-2-nonsensitive ones and free ALA. Such MMP-2sensitive ALA prodrug nanocarriers can achieve minimal unwanted leakage during circulation and fast drug release after internalization. Moreover, such programmed delivery strategy could overcome the conflict between zwitterionic stealth surface tailoring for prolonged blood circulation and CPPs facilitating internalization in tumor tissues. The design of programmed enzyme and pH dual-sensitive ALA prodrug nanocarriers provides a new strategy for targeted photodynamic cancer therapy. ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.6b15853. Synthetic scheme and additional characterization data (PDF)

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REFERENCES

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected] (Q.J.). *E-mail: [email protected]. Tel: +86-571-87953729. Fax: +86571-87953729 (J.J.). ORCID

Jian Ji: 0000-0001-9870-4038 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Financial supports from the National Natural Science Foundation of China (51573160 and 21574114), the Key Science Technology Innovation Team of Zhejiang Province (2013TD02), and the Fundamental Research Funds for the Central Universities (2016QNA4033) are gratefully acknowledged. The authors appreciate Prof. Xian-Zheng Zhang in Wuhan University for supplying SCC-7 cells. 14604


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