Folate Receptor-Targeted and Cathepsin B-Activatable Nanoprobe for

Mar 4, 2015 - A Graphene Oxide-Based Fluorescent Method for the Detection of Human ... Aptamer loaded MoS2 nanoplates as nanoprobes for detection of ...
0 downloads 0 Views 5MB Size
Download PDF
Article pubs.acs.org/ac

Folate Receptor-Targeted and Cathepsin B‑Activatable Nanoprobe for In Situ Therapeutic Monitoring of Photosensitive Cell Death Jiangwei Tian,†,‡,§ Lin Ding,†,§ Quanbo Wang,† Yaoping Hu,† Li Jia,† Jun-Sheng Yu,† and Huangxian Ju*,† †

State Key Laboratory of Analytical Chemistry for Life Science, School of Chemistry and Chemical Engineering, Nanjing University, Nanjing, Jiangsu 210093, P.R. China ‡ Jiangsu Key Laboratory of TCM Evaluation and Translational Research, Department of Complex Prescription of TCM, China Pharmaceutical University, Nanjing, Jiangsu 211198, P. R. China S Supporting Information *

ABSTRACT: The integration of diagnostic and therapeutic functions in a single system holds great promise to enhance the theranostic efficacy and prevent the under- or overtreatment. Herein, a folate receptor-targeted and cathepsin B-activatable nanoprobe is designed for background-free cancer imaging and selective therapy. The nanoprobe is prepared by noncovalently assembling phospholipid-poly(ethylene oxide) modified folate and photosensitizer-labeled peptide on the surface of graphene oxide. After selective uptake of the nanoprobe into lysosome of cancer cells via folate receptor-mediated endocytosis, the peptide can be cleaved to release the photosensitizer in the presence of cancerassociated cathepsin B, which leads to 18-fold fluorescence enhancement for cancer discrimination and specific detection of intracellular cathepsin B. Under irradiation, the released photosensitizer induces the formation of cytotoxic singlet oxygen for triggering photosensitive lysosomal cell death. After lysosomal destruction, the lighted photosensitizer diffuses from lysosome into cytoplasm, which provides a visible method for in situ monitoring of therapeutic efficacy. The nanoprobe exhibits negligible dark toxicity and high phototoxicity with the cell mortality rate of 0.06% and 72.1%, respectively, and the latter is specific to folate receptor-positive cancer cells. Therefore, this work provides a simple but powerful protocol with great potential in precise cancer imaging, therapy, and therapeutic monitoring. way to overcome these shortcomings is to design an “Off-On” nanoprobe that can be activated with a cancer-associated stimulus. Our previous work designed an acidic pH-activatable nanoparticle, achieving highly selective photodynamic therapy against cancer by specially damaging the lysosome of cancer cells.17 Unfortunately, these nanoparticles failed to in situ evaluate the therapeutic efficacy. Cathepsin B (CaB, EC 3.4.22.1) is a lysosomal cysteine protease which participates in the degradation of phagotrophic material18 with specific peptide cleaving capability.19 This protease plays crucial roles in cancer progression and has been suggested to be a target enzyme for the diagnosis of cancer.20 Thus, it offers an attractive choice for triggering cancer theranostics. Since lysosome is the main organelle involved in the FR-mediated endocytosis, CaB activation can be smoothly connected with FR-based targeting. Particularly, lysosome has been regarded to be involved in the lysosomal cell-death pathway through releasing lysosomal hydrolases into the cytosol after lysosomal destruction,21 and this can be induced by cytotoxic singlet oxygen (1O2) generated under irradiation in the presence of

O

ptical nanoplatforms can integrate multiple functionalities such as imaging, drug delivery, and photodynamic or photothermal therapy in a single system, thus offering excellent prospects for noninvasive diagnosis and treatment of cancer.1 Various functional nanomaterials including polymeric nanoparticles,2 dendrimers,3 quantum dots,4 gold nanovesicles,5 upconversion nanoparticles,6 and graphene oxide nanosheets7 have shown promising potential in cancer imaging and therapy. The major challenges for their applications are the selectivity of imaging and therapy against cancer cells8−11 and the in situ monitoring of therapeutic efficacy for preventing under- or overtreatment.12,13 The “Always-On” property of some functional agents also leads to high background and therapeutic side effects.9 These challenges and limitations have driven the further development of this field. Folate receptor subtype alpha (FR-α), with high affinity for folate (FA), is aberrantly up-regulated on the surface of many cancer cells.14 Although FR subtype beta (FR-β) has been found in several normal cells, the receptor lacks affinity for folate.14 Thus, FR-α has been extensively used as target by conjugation of FA to the nanomaterials for selective delivery into cancer cells via the endosome-lysosome endocytosis pathway.15,16 However, this uptake pathway cannot completely eliminate the disadvantages of “Always-On” agents. An efficient © XXXX American Chemical Society

Received: December 8, 2014 Accepted: March 4, 2015

A

DOI: 10.1021/acs.analchem.5b00429 Anal. Chem. XXXX, XXX, XXX−XXX

Article

Analytical Chemistry photosensitizer.17,22 Thus, it is beneficial to design a FRtargeted and CaB-activatable nanoprobe for realizing background-free imaging and selective therapy against cancer. This work reports a nanoprobe for triggering photosensitive lysosomal cell death and in situ therapeutic monitoring. The nanoprobe uses 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[folate(poly ethylene glycol)-2000] (DSPE-PEG2000FA) to achieve FR-mediated endocytosis and chlorin e6-labeled peptide substrate (Ce6-GRRGKGGFFFF, Ce6-Pep) to perform a cancer-specific stimulus via CaB activation, which leads to the release of Ce6 from the graphene oxide (GO) sheet as a delivery carrier and quencher of Ce6 fluorescence (Scheme 1).

cell death (Scheme 1b). The diffusion of the lighted Ce6 into cytoplasm upon lysosomal destruction provides self-feedback information on therapeutic efficacy, thus contributing to the improvement of theranostic efficiency.

■

EXPERIMENTAL SECTION Synthesis of Nanoprobe. A 1.0 mg mL−1 suspension of GO in ultrapure water was sonicated for 24 h to break the GO sheets into much smaller GO pieces. The mixture of Ce6-Pep and DSPE-PEG2000-FA with a weight ratio of 4:1 was added into the GO suspension and incubated at room temperature in the dark overnight to form the nanocomposite of Ce6-Pep, DSPE-PEG2000-FA, and GO, named as the nanoprobe. The unbound excess Ce6-Pep and DSPE-PEG2000-FA were removed by filtration through a 30 kDa MWCO centrifugal filter (Amicon Ultra) at 4000g and washed with ultrapure water until the filtrate became free of green color. 0.5 mg of the obtained nanoprobe was then resuspended in 1.0 mL of phosphate buffer (pH 5.0, 50 mM) and stored at 4 °C. The amount of Ce6-Pep loaded on the nanoprobe was determined by the Ce6 characteristic absorption peak at 404 nm with a molar absorption coefficient of 1.1 × 105 M−1 cm−1 after subtracting the absorbance of the GO at this wavelength, which was obtained from the GO suspension with the same concentration.26 Similar procedures were used to prepare the nanocomposite of Ce6-Pep/GO as FA free nanoprobe with Ce6-Pep, DSPE-MPEG2000, and GO and nonspecific peptide nanoprobe assembled by Ce6-GRGRKGGFFFF, DSPEPEG2000-FA, and GO. Fluorescence Assay. CaB (2.5 U mL−1) was incubated with Ce6-Pep/GO (2.6 μM Ce6 equiv) in 1 mL of 50 mM phosphate buffer (pH 5.0) containing 100 mM NaCl, 2 mM EDTA, and 2 mM L-cysteine. The similar experimental conditions were applied to the mixture of CaB (2.5 U mL−1), Ce6-Pep/GO (2.6 μM Ce6 equiv), and antipain dihydrochloride as an inhibitor (50 μg mL−1). In addition, Ce6-Pep/ GO (2.6 μM Ce6 equiv) was also incubated with cathepsin D (CaD, 2.5 U mL−1) in 100 mM sodium acetate buffer (pH 3.5) containing 200 mM NaCl or with cathepsin L (CaL, 0.01 U mL−1) in 50 mM MES buffer (pH 6.5) containing 5 mM dithiothreitol (DTT). After a 60 min incubation at 37 °C, the fluorescence intensity of Ce6 was measured using a spectrofluorometer with the excitation at 400 nm and emission from 600 to 750 nm. The excitation and emission band widths were set to 5.0 and 5.0 nm, respectively. Determination of Singlet Oxygen Generation. CaB (2.5 U mL−1) was incubated with Ce6-Pep/GO (2.6 μM Ce6 equiv) in 1 mL of 50 mM phosphate buffer (pH 5.0) containing 100 mM NaCl, 2 mM EDTA, 2 mM L-cysteine, and 1.0 μM singlet oxygen sensor green (SOSG, S-36002). SOSG was used as a singlet oxygen fluorescent probe27 to evaluate the 1O2 generation. The solution was gently stirred at 37 °C for 60 min and irradiated with a 660 nm laser at a power of 250 mW cm−2 for 200 s. The fluorescence intensity of SOSG was measured by a spectrofluorometer with excitation at 480 nm and emission at 525 nm. The excitation and emission band widths were set to 3.0 and 3.0 nm, respectively. Cell Culture. Human nasopharyngeal epidermal carcinoma KB cell line, human lung carcinoma A549 cell line, human cervical carcinoma HeLa cell line, and immortalized human epidermal HaCaT cell line were obtained from KeyGEN Biotech (Nanjing, China). KB and A549 cells were, respectively, maintained in minimum essential medium

Scheme 1. Schematic Illustration of (a) Nanoprobe and (b) FR-Targeted Delivery and Cathepsin B Activation for Photosensitive Lysosomal Cell Death and In Situ Therapeutic Monitoring

It can be conveniently prepared by simple noncovalent assembly of DSPE-PEG2000-FA and Ce6-Pep on the surface of the GO sheet (Scheme 1a). Ce6 is both a fluorescence dye with a fluorescence quantum yield (ΦF) of 0.18 and a highly efficient photosensitizer with a singlet oxygen quantum yield (ΦΔ) of 0.75.23−25 Close proximity of Ce6-Pep to the GO sheet results in strong inhibition of both fluorescence and 1O2 generation. After the nanoprobe is specifically internalized into lysosome of cancer cells, the peptide can be cleaved by CaB to release the Ce6 from the surface of the GO sheet. Under 660 nm irradiation, the free Ce6 can efficiently induce the formation of cytotoxic 1O2 to damage the lysosome, leading to lysosomal B

DOI: 10.1021/acs.analchem.5b00429 Anal. Chem. XXXX, XXX, XXX−XXX

Article

Analytical Chemistry

After rinsing with PBS, HeLa cells were incubated with 200 μL of culture media containing serial concentrations of the nanoprobe for 4 h. One plate was kept in the dark for studying the dark toxicity, and another plate was irradiated using a 660 nm laser at a power of 250 mW cm−2 for 200 s. The cell death induced by the nanoprobe in the presence or absence of irradiation was evaluated by the MTT assay according to the manufacturer’s instruction. The cell viability was calculated with the equation: cell viability (%) = (mean OD value of treatment group/mean OD value of control) × 100%. For the flow cytometric assay, HeLa cells were subjected to 4 groups: group 1, untreated; group 2, laser exposure; group 3, incubation with nanoprobe; group 4, incubation with nanoprobe and then laser exposure. The incubation was performed with fresh serum-free culture medium containing nanoprobe (2.0 μM Ce6 equiv) at 37 °C for 4 h. The laser exposure was performed with a 660 nm laser at a power of 250 mW cm−2 for 200 s. Afterward, these cells were stained with a LIVE/DEAD viability assay kit to perform the flow cytometric assay. For propidium iodide (PI) or TUNEL staining, HeLa or HaCaT cells were incubated with the nanoprobe (2.0 μM Ce6 equiv) and irradiated with a 660 nm laser at a power of 250 mW cm−2 for 200 s. Afterward, the cells were stained with PI or TUNEL according to the manufacturer’s instruction. The apoptotic fluorescence was visualized with a confocal laser scanning microscope at an excitation wavelength of 543 nm and collected from 580 to 620 nm. Lysosomal Integrity Assays. Confocal fluorescence images of HeLa cells after treatment with PBS (control), laser exposure, nanoprobe incubation, or nanoprobe incubation and then laser exposure were stained with 5.0 μM AO for 15 min to perform confocal fluorescence imaging. The images are collected from 515 to 545 nm (green) and 610 to 640 nm (red) at an excitation wavelength of 488 nm. The lysosomal CaB release for different treatment groups of HeLa cells was examined with the CaB activity assay kit. In Vivo Tumor Imaging Assay. Specific pathogen-free female BALB/c nude mice, 5−6 weeks of age, were purchased from Shanghai Laboratory Animal Center, Chinese Academy of Sciences (SLACCAS), and bred in an axenic environment. All animal operations were in accord with institutional animal use and care regulations approved by the Model Animal Research Center of Nanjing University (MARC). The HeLa tumor model was established by subcutaneous injection of HeLa cells (1 × 106) into the selected position of nude mice. Tumors were then allowed to grow to 8−10 mm in diameter. For tumor imaging, 200 μL of nanoprobe (2.0 μM Ce6 equiv) was injected via a tail vein. In vivo imaging was then performed using a Maestro EX in vivo imaging system (CRI, Inc.; excitation, 631 nm; emission, 645 nm long-pass) at 24 h postinjection. The mice were then euthanized to obtain the organs and tumor tissues for ex vivo imaging. In Vivo Phototoxicity Assay. The HeLa-tumor bearing mice were subjected to 4 different treatments: group 1, untreated; group 2, laser exposure only; group 3, intravenous injection of nanoprobe only; group 4, intravenous injection of nanoprobe and then laser exposure. Each group contained 6 mice. 200 μL of nanoprobe (2.0 μM Ce6 equiv) in PBS was injected into the mice via a tail vein of groups 3 and 4, respectively. Twenty-four hours later, laser exposure was performed on groups 2 and 4 by irradiating the tumor region with a 660 nm laser at the power of 250 mW cm−2 for 200 s. The mice from different treatment groups were monitored by

(MEM) with 10% fetal bovine serum (FBS) and F12K media with 10% FBS. HeLa and HaCaT cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% FBS, 100 μg mL−1 streptomycin, and 100 U mL−1 penicillin. The cells were cultured at 37 °C in a humidified incubator containing 5% CO2 and 95% air. The medium was replenished every other day, and the cells were subcultured after reaching confluence. Cell Uptake, Colocalization, and Competition Assays. HeLa cells were seeded into 35 mm confocal dishes (Glass Bottom Dish) at a density of 1 × 104 per dish and incubated for 24 h at 37 °C. The medium was then replaced with fresh serum-free culture medium containing nanoprobe (2.0 μM Ce6 equiv) and incubated at 37 °C for different times. Before imaging, the cells were rinsed three times with PBS (pH 7.4) and kept in phenol red-free culture medium. The fluorescence of cells was visualized with a confocal laser scanning microscope at stationary parameters including the laser intensity, exposure time, and objective lens. The cells were excited at 633 nm with a helium−neon laser, and the emission was collected from 645 to 700 nm. Compared with the excitation at 400 nm, it could reduce cellular damage and autofluorescence from biological samples. All images were digitized and analyzed by Leica Application Suite Advanced Fluorescence (LAS-AF) software package. For the colocalization assay of the nanoprobe-loaded HeLa cells, the cells were washed with PBS and further incubated with 1.0 μM LysoTracker Green and 1.0 μM Hoechst 33342 for 15 min. LysoTracker Green was excited at 488 nm with an argon ion laser, and the emission was collected from 505 to 535 nm. Hoechst 33342 was excited with a violet 405 nm laser diode, and the emission was collected from 420 to 480 nm. HeLa cells were seeded into 35 mm confocal dishes (1 × 104 per dish) or 6-well plates (1 × 105 per well) and incubated in complete medium for 24 h at 37 °C. Then, HeLa cells were randomly divided into five groups for the following treatments: group 1, incubation with FA free nanoprobe; group 2, adding 10 μM FA for 30 min and then incubation with nanoprobe; group 3, adding 100 μg mL−1 antipain for 30 min and then incubation with nanoprobe; group 4, incubation with nonspecific peptide nanoprobe; group 5, incubation with nanoprobe. The concentration of nanoprobe was 2.0 μM Ce6 equiv. After a 4 h incubation, confocal fluorescence imaging was performed to visualize the intracellular Ce6 fluorescence. The different treatment groups of HeLa cells were also trypsinized, harvested, rinsed with PBS and resuspended, and subjected to the flow cytometric assay using Cytomics FC500 Flow Cytometry. All experiments detected at least 10 000 cells, and the data were analyzed with FCS Express V3. Imaging Specificity Assays. HeLa or HaCaT cells were incubated with nanoprobe (2.0 μM Ce6 equiv) at 37 °C for 4 h and stained with 1.0 μM Hoechst 33342 for 15 min. Then, the cells were rinsed three times with PBS (pH 7.4) and kept in phenol red-free culture medium, and confocal fluorescence imaging was performed. Flow cytometry was also used to evaluate the specificity of the nanoprobe to cancer cells. After incubation with the nanoprobe (2.0 μM Ce6 equiv) at 37 °C for 4 h, HeLa or HaCaT cells were trypsinized, harvested, rinsed with PBS and resuspended, and subjected to the flow cytometric assay. Determination of Cell Viability. For MTT assay, HeLa cells (1 × 104 per well) were seeded into two 96-well plates in 200 μL of complete medium and incubated at 37 °C for 24 h. C

DOI: 10.1021/acs.analchem.5b00429 Anal. Chem. XXXX, XXX, XXX−XXX

Article

Analytical Chemistry measuring the tumor size using a vernier caliper for 12 d after the treatment. The tumor volume was calculated using length × width2 × 0.5, where the length is the greatest longitudinal diameter and the width is the greatest transverse diameter of each tumor. The relative tumor volume for each mouse was calculated as V/V0, where V0 is the tumor volume before the treatment. Statistical Analysis. Data were expressed as means ± SD from at least three experiments. One-way ANOVA was used to compare the treatment effects. P < 0.05 was considered to be statistically significant.

■

RESULTS AND DISCUSSION Characterization of Ce6-Pep/GO. In light of the large surface area, good biocompatibility, easy surface modification, and ultrahigh fluorescence quenching efficiency,28−31 GO sheets were chosen as the vector for preparation of the FRtargeted and CaB-activatable nanoprobe. After sonication treatment, most GO sheets showed single-layered dispersion with a dimension less than 100 nm and thickness of 0.4 nm (Figure 1a). The zeta potential of −40.9 mV indicated good

Figure 2. Absorption spectra of 11.4 μM Ce6-Pep, 0.5 mg mL−1 GO, and 0.5 mg mL−1 Ce6-Pep/GO, respectively.

surface of GO. The loading amount of Ce6-Pep on GO could be determined by measuring the absorption at 404 nm after subtracting the absorbance of GO at this wavelength.26,35 The loading amount of Ce6-Pep was determined to be 5.2 nmol mg−1 with 46% loading efficiency. Fluorescence Response and Specificity to CaB. The quenching and CaB-activatable recovery of Ce6 fluorescence were examined with absorption and fluorescence spectra. Under 400 nm excitation, the Ce6-Pep displayed a fluorescence peak at 645 nm (Figure S1 in the Supporting Information). Because of the broad absorption from the UV to NIR region (Figure S1 in the Supporting Information), GO could be used as a highly efficient quencher. After Ce6-Pep was adsorbed on the GO surface, the fluorescence intensity of Ce6-Pep was dramatically reduced (i.e., 4.6% of free Ce6-Pep) (Figure 3a) due to the energy transfer from the excited Ce6 to the GO. The high quenching efficiency manifested the low background signal. Furthermore, no fluorescence recovery of the nanoprobe was observed at different times, pHs, and temperatures (Figures S2−S4 in the Supporting Information), indicating the stability of Ce6-peptide assembled on the surface of the GO sheet. Upon addition of CaB to Ce6-Pep/GO solution, the fluorescence intensity of Ce6 at 645 nm increased rapidly (Figure S2 in the Supporting Information) and reached 85% of the intensity of free Ce6-Pep at 1 h (Figure 3a). The fluorescence recovery was attributed to the cleavage of peptide sequence by CaB to release Ce6 from the GO surface. The fluorescence intensity further increased and trended to the maximum value after reaction for 1 h (Figure S2 in the Supporting Information), and the time corresponding to the plateau value was chosen as the reaction time for in vitro experiments. The cleavage reaction also depended on the solution pH (Figure S3 in the Supporting Information) and temperature (Figure S4 in the Supporting Information), which showed the optimal pH range of 4.5−6.0 and optimal temperature of 37 °C. Thus, pH 5.0 was used for the following in vitro experiments considering the microenvironment of lysosome. The cleavage of the designed peptide showed good selectivity to CaB. In the presence of CaD or CaL, Ce6-Pep/GO did not show the fluorescence recovery (Figure 3b). Furthermore, the addition of antipain as a CaB inhibitor into Ce6-Pep/GO solution could inhibit the recovery, indicating the specific activation of Ce6-Pep/GO by CaB. 1 O2 Generation Assays. The controllable release of 1O2 is a major challenge for photodynamic therapy against cancer.17 The 1O2 production of Ce6-Pep/GO under irradiation before

Figure 1. (a) Atomic force microscopy and (b) zeta potential characterization of GO and Ce6-Pep/GO, respectively.

stability32 of GO sheets (Figure 1b). Ce6 was covalently linked with CaB specific substrate peptide. The peptide sequence was GRRGKGGFFFF with RR as the recognition sequence.33,34 The FFFF sequence ensured the close proximity between Ce6Pep and aromatic regions of the GO by the hydrophobic interaction and π−π stacking.28 After assembling Ce6-Pep on GO, the height became 0.7 nm (Figure 1a), and the zeta potential also changed to −22.1 mV (Figure 1b) owing to the positive charge of the peptide. These characterizations demonstrated the successful assembly of Ce6-Pep/GO. The absorption spectrum of Ce6-Pep displayed a strong peak at 404 with a molar absorption coefficient ε of 1.1 × 105 M−1 cm−1 and two weak peaks at 500 nm (1.0 × 104 M−1 cm−1) and 660 nm (2.5 × 104 M−1 cm−1) (Figure 2). The absorption of GO spanned over a broad range from the UV to NIR region. The appearance of an absorption peak at 404 nm of Ce6-Pep/ GO indicated that Ce6-Pep was successfully loaded onto the D

DOI: 10.1021/acs.analchem.5b00429 Anal. Chem. XXXX, XXX, XXX−XXX

Article

Analytical Chemistry

dispersed with an increased thickness of 1.2 nm (Figure S5 in the Supporting Information) compared with 0.7 nm of Ce6Pep/GO (Figure 1a), which suggested the successful assembly of DSPE-PEG2000-FA to GO. The zeta potential was measured to be −31.6 mV (Figure S6 in the Supporting Information), indicating good stability of the nanoprobe. By measuring the absorbance of nanoprobe at 404 nm (Figure S7 in the Supporting Information), the loading amount of Ce6-Pep was determined to be 4.0 nmol mg−1 with 35% loading efficiency. The nanoprobe displayed a low fluorescence background and specific fluorescence activation by CaB (Figure S8 in the Supporting Information). After a 1 h incubation of human cervical carcinoma HeLa cells with the nanoprobe, some fluorescent spots of Ce6 were observed in the cells (Figure 4a), indicating the fast uptake and subsequent activation of the nanoprobe by CaB. The amount and fluorescence intensity of fluorescent spots increased gradually with the increasing incubation time and reached a maximum at 4 h. The amplified confocal fluorescence images (Figure 4b) clearly revealed that the intracellular fluorescence intensity was strongly position dependent. After the nanoprobe-incubated HeLa cells were costained with a lysosome probe, LysoTracker Green, and a nucleus dye, Hoechst 33342, the overlap of red Ce6 fluorescence with LysoTracker green fluorescence could be observed (Figure 4c), which demonstrated that the fluorescence activation of the nanoprobe happened in lysosome. HeLa cells were also incubated with FA free nanoprobe, Ce6Pep/DSPE-MPEG2000/GO (Figure 5a). The incubated HeLa cells did not show any Ce6 fluorescence signal due to the repulsive interaction between the negatively charged cell membrane and the negatively charged FA-free nanoprobe. As an additional control, a blocking dose of 10 μM FA was added for 30 min before the nanoprobe incubation (FA + nanoprobe); the intracellular fluorescence was obviously reduced, which indicated FA played important roles in the targeted delivery of the nanoprobe into cancer cells. The FR-mediated delivery was further confirmed by comparing the uptake of nanoprobe in FR-positive KB cells and FR-negative A549 cells (Figure S9 in the Supporting Information). To validate the specificity of CaB activation, HeLa cells were incubated with the nanoprobe in the presence of antipain (CaB inhibitor + nanoprobe) or the nonspecific peptide nanoprobe. In these cases, the intracellular fluorescence was much weaker than that treated with the nanoprobe. These results confirmed the FRtargeted and CaB-activatable fluorescence imaging of cancer cells with the designed nanoprobe, which was further verified by the flow cytometric assay (Figure 5b). Imaging Specificity Assay. The imaging specificity to cancer cells was evaluated by incubating FR-positive HeLa cells and FR-negative immortalized human epidermal HaCaT cells with the nanoprobe for 4 h at 37 °C. Unlike HeLa cells, the confocal image of the nanoprobe treated HaCaT cells did not show observable fluorescence (Figure 6a) due to the low-level expression of FA in normal cells. The imaging selectivity of the nanoprobe for HeLa cells was also verified by flow cytometric analysis (Figure 6b). The significant difference of fluorescence signal between cancer and normal cells provided a tool for cancer discrimination. The occurrence of Ce6 fluorescence in the nanoprobe treated HeLa and HaCaT cells could be used for in situ specific detection of intracellular CaB activity. Phototoxicity and Therapeutic Selectivity of Nanoprobe. The cytotoxicity of nanoprobe to HeLa cells was

Figure 3. CaB-activatable fluorescence and 1O2 generation of Ce6Pep/GO. (a) Fluorescence emission spectra of different solutions and (b) fluorescence intensity of Ce6-Pep/GO (2.6 μM Ce6 equiv) at 645 nm after treatment with CaB, CaD, CaL, and the mixture of CaB and inhibitor for 1 h along with corresponding imaging sections of the 96well plate. (c) 1O2 generation determined by SOSG fluorescence intensity at 525 nm.

and after the addition of CaB was demonstrated with Singlet Oxygen Sensor Green (SOSG) as a 1O2 indicator. This indicator could emit strong fluorescence at 525 nm upon reaction with 1O2.27 After exposing Ce6-Pep/GO solution to 660 nm irradiation for 200 s, a minor fluorescence increase of SOSG was observed compared with the solution without irradiation. However, the fluorescence intensity increased greatly after CaB was added in the Ce6-Pep/GO solution under irradiation (Figure 3c). The enhanced fluorescence emission showed the intensity similar to that of the Ce6-Pep solution after irradiation. These results indicated the high 1O2 inhibition ability of GO and the efficient cleavage of peptide by CaB for release of Ce6 from the GO surface to induce the generation of 1O2. In the presence of CaB inhibitor, the SOSG fluorescence intensity displayed an obvious reduction, implying the specific activation of 1O2 generation by CaB. Cell Imaging, Colocalization, and Competition Assays. FA has been extensively used as a targeting ligand to achieve selective recognition of nanomaterials to FR-positive cancer cells.15−17 Here, the functionalization of nanomaterials with FA was achieved by noncovalent assembly of DSPEPEG2000-FA and Ce6-Pep on GO. DSPE-PEG2000-FA was an amphiphilic polymer with DSPE as the hydrophobic chain for nonconvalent binding to the surface of GO, and FA terminated PEG as the hydrophilic portion for stability and targeting. AFM measurement showed that the obtained nanoprobe was well E

DOI: 10.1021/acs.analchem.5b00429 Anal. Chem. XXXX, XXX, XXX−XXX

Article

Analytical Chemistry

Figure 4. Confocal fluorescence imaging of HeLa cells incubated with the nanoprobe. (a) Time course fluorescence and DIC images of HeLa cells upon incubation with the nanoprobe (2.0 μM Ce6 equiv). (b) Images of HeLa cells incubated with the nanoprobe for 4 h with green frames showing the amplified region. (c) Costaining of nanoprobe-loaded HeLa cells with a Lysotracker green and Hoechst 33342.

Figure 6. (a) Confocal fluorescence images and DIC images and (b) flow cytometric assay of HeLa and HaCaT cells incubated with the nanoprobe (2.0 μM Ce6 equiv) for 4 h.

Figure 5. (a) Confocal fluorescence images and (b) flow cytometric assay of HeLa cells incubated with FA free nanoprobe, nanoprobe in the presence of excess FA, nanoprobe in the presence of antipain as an CaB inhibitor, nonspecific peptide nanoprobe, and nanoprobe for 4 h.

Moreover, the phototoxicity increased along with the concentration of the nanoprobe with a half lethal dose (IC50) of 1.4 μM (Ce6 equiv) at a 4 h incubation (Figure S10a in the Supporting Information). HeLa cells were also incubated with FA free nanoprobe, FA + nanoprobe, CaB inhibitor + nanoprobe, or nonspecific peptide nanoprobe and irradiated with a 660 nm laser at 250 mW cm−2 for 200 s, respectively. The cell viability of these groups could be maintained by more than 87%, while for the nanoprobe-treated HeLa cells, the cell viability was reduced to 24% (Figure S10b in the Supporting Information), which confirmed the FR-targeted and CaB-activatable phototoxicity to cancer cells.

examined using MTT and flow cytometric assays. In the absence of 660 nm irradiation, the nanoprobe was basically noncytotoxic (Figure S10a in the Supporting Information), and the 4 h incubation with the nanoprobe could maintain 99.4% cell viability, which was close to 99.9% for the cells without any treatment (Figure S11 in the Supporting Information). Under the 200 s irradiation with 50 J cm−2 dose (250 mW cm−2), the nanoprobe exhibited high phototoxicity, which led to the cell mortality rate of 72.1%, much higher than 0.04% in the absence of the nanoprobe (Figure S11 in the Supporting Information). F

DOI: 10.1021/acs.analchem.5b00429 Anal. Chem. XXXX, XXX, XXX−XXX

Analytical Chemistry

Article

■

CONCLUSIONS A FR-targeted and CaB-activatable nanoprobe has been presented for triggering photosensitive lysosomal cell death and in situ therapeutic monitoring by integrating the DSPEPEG2000-FA and Ce6-conjugated substrate peptide on the surface of the GO sheet. The fluorescence of Ce6 and the 1O2 generation induced by Ce6 are greatly inhibited by the GO vector. After FR-mediated delivery into the lysosome of cancer cells, the Ce6 is released from the GO surface via the specific cleavage of peptide by CaB. Thus, the nanoprobe can be specifically activated by CaB for fluorescent discrimination of cancer and irradiated triggering of lysosomal cell death, respectively. The diffusion of lighted Ce6 from lysosome to cytoplasm demonstrates the lysosomal destruction and provides a visual feedback for in situ therapeutic monitoring. We believe that this work provides a successful theranostic paradigm to develop the cancer-selective and activatable nanoprobe for lysosome-aimed cancer imaging, therapy, and therapeutic monitoring.

After HeLa and HaCaT cells were incubated with the nanoprobe and irradiated with a 660 nm laser, only HeLa cells showed strong apoptotic fluorescence during the TUNEL assay (Figure S12 in the Supporting Information), while the apoptotic fluorescence from HaCaT cells was negligible. In addition, after the HeLa and HaCaT cells were stained with PI, a membrane-impermeable dye for indicating dead cells, bright PI fluorescence was observed in the nucleus of HeLa cells, while no PI fluorescence was observed in HaCaT cells (Figure S13 in the Supporting Information), further verifying the selective phototoxicity of the nanoprobe against cancer cells due to the FR-targeting. In Situ Therapeutic Efficacy Evaluation. Under irradiation, the nanoprobe-loaded HeLa cells showed obvious morphological change, and the bright fluorescence gradually diffused from lysosomes to cytoplasm (Figure S14a in the Supporting Information). The swelling of HeLa cells and the ruptured outer membrane as shown in the differential interference contrast (DIC) image indicated obvious cell death. The fluorescence diffusion suggested the release of lighted Ce6 to cytoplasm due to the lysosomal destruction, thus providing a convenient method for in situ therapeutic effect evaluation. To confirm the lysosomal destruction, AO was employed as an integrity indicator of acidic organelle, which emits red fluorescence in acidic lysosome and green fluorescence in the cytosol and nuclei.36 The acidic organelles in HeLa cells displayed bright red fluorescence in the absence of nanoprobe or/and irradiation (Figure S14b in the Supporting Information), suggesting that the lysosomal compartments were maintained well. After the nanoprobe-loaded cells were irradiated, the red AO fluorescence disappeared (Figure S14b in the Supporting Information). Thus, the irradiation led to the destruction of the lysosomal membrane in the presence of nanoprobe. Lysosome has been demonstrated to be an effective target for cancer therapy by releasing cathepsins to cytosol after lysosomal destruction.22 Therefore, the CaB activity assay kit was used to examine the CaB release.37 The labeled RR was detectable in cytosol for the group of nanoprobe + irradiation (Figure S14c in the Supporting Information), indicating the CaB release from lysosome to cytosol. These results suggested that the nanoprobe specifically damaged the lysosome after irradiation to trigger the lysosomal cell-death pathway. In Vivo Tumor Imaging and Phototoxicity Evaluation. The feasibility of nanoprobe for in vivo tumor imaging was investigated in subcutaneous HeLa tumor-bearing mice. After intravenous injection of the nanoprobe for 24 h, the tumor region displayed strong fluorescence and could be distinguished from the normal tissues (Figure S15a in the Supporting Information). The ex vivo imaging of excised tissues showed the strongest fluorescence in the tumor tissue, while other organs including heart, liver, spleen, lung, kidneys, intestine, and muscle showed weak fluorescence. The high contrast for tumor imaging was attributed to FR-targeting and CaB activation. The in vivo phototoxicity of nanoprobe to tumor was assessed by monitoring the change of relative tumor volume. After the tumor bearing mice were treated with nanoprobe and irradiation, tumor growth was significantly suppressed, while no therapeutic effect was observed without treatment with nanoprobe or/and irradiation (Figure S15b in the Supporting Information), revealing the strong phototoxicity of the nanoprobe. These results indicated the significance of the designed nanoprobe for in vivo theranostic application.

■

ASSOCIATED CONTENT

S Supporting Information *

Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org.

■

AUTHOR INFORMATION

Corresponding Author

*Phone/Fax: +86-25-83593593. E-mail: [email protected]. Author Contributions §

J.T. and L.D. contributed equally to this work.

Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS This study was supported by the National Basic Research Program (2010CB732400, 2014CB744501), National Science Fund for Creative Research Groups (21121091), and National Natural Science Foundation of China (21322506, 21135002, 91213301).

■

REFERENCES

(1) Barreto, J. A.; O’Malley, W.; Kubeil, M.; Graham, B.; Stephan, H.; Spiccia, L. Adv. Mater. 2011, 23, H18−H40. (2) Wang, S.; Kim, G.; Lee, Y. E.; Hah, H. J.; Ethirajan, M.; Pandey, R. K.; Kopelman, R. ACS Nano 2012, 6, 6843−6851. (3) Menjoge, A. R.; Kannan, R. M.; Tomalia, D. A. Drug Discovery Today 2010, 15, 171−185. (4) Bagalkot, V.; Zhang, L.; Levy-Nissenbaum, E.; Jon, S.; Kantoff, P. W.; Langer, R.; Farokhzad, O. C. Nano Lett. 2007, 7, 3065−3070. (5) Park, K.; Lee, S.; Huang, E. P.; Lin, J.; Li, W.; Rong, P.; Wang, Z.; Wang, S.; Wang, X.; Sun, X.; Aronova, M.; Niu, G.; Leapman, R. D.; Nie, Z.; Chen, X. Angew. Chem., Int. Ed. 2013, 52, 13958−13964. (6) Cui, S.; Yin, D.; Chen, Y.; Di, Y.; Chen, H.; Ma, Y.; Achilefu, S.; Gu, Y. ACS Nano 2013, 7, 676−688. (7) Lia, J.-L.; Tanga, B.; Yuanb, B.; Suna, L.; Wang, X.-G. Biomaterials 2013, 34, 9519−9534. (8) Urano, Y.; Asanuma, D.; Hama, Y.; Koyama, Y.; Barrett, T.; Kamiya, M.; Nagano, T.; Watanabe, T.; Hasegawa, A.; Choyke, P. L.; Kobayashi, H. Nat. Med. 2009, 15, 104−109. (9) Kobayashi, H.; Choyke, P. L. Acc. Chem. Res. 2011, 44, 83−90. (10) Mitsunaga, M.; Ogawa, M.; Kosaka, N.; Rosenblum, L. T.; Choyke, P. L.; Kobayashi, H. Nat. Med. 2011, 17, 1685−1691. (11) Tian, X.; Baeka, K.-H.; Shin, I. Chem. Sci. 2013, 4, 947−956.

G

DOI: 10.1021/acs.analchem.5b00429 Anal. Chem. XXXX, XXX, XXX−XXX

Article

Analytical Chemistry (12) Kelkar, S. S.; Reineke, T. M. Bioconjugate Chem. 2011, 22, 1879−1903. (13) Kievit, F. M.; Zhang, M. Adv. Mater. 2011, 23, H217−H247. (14) Sudimack, J.; Lee, R. J. Adv. Drug Delivery Rev. 2000, 41, 147− 162. (15) Nayak, S.; Lee, H.; Chmielewski, J.; Lyon, L. A. J. Am. Chem. Soc. 2004, 126, 10258−10259. (16) Huang, P.; Xu, C.; Lin, J.; Wang, C.; Wang, X.; Zhang, C.; Zhou, X.; Guo, S.; Cui, D. Theranostics 2011, 1, 240−250. (17) Tian, J.; Ding, L.; Xu, H. J.; Shen, Z.; Ju, H.; Jia, L.; Bao, L.; Yu, J. S. J. Am. Chem. Soc. 2013, 135, 18850−18858. (18) Mohamed, M. M.; Sloane, B. F. Nat. Rev. Cancer 2006, 6, 764− 775. (19) Ryu, J. H.; Na, J. H.; Ko, H. K.; You, D. G.; Park, S.; Jun, E.; Yeom, H. J.; Seo, D. H.; Park, J. H.; Jeong, S. Y.; Kim, I. S.; Kim, B. S.; Kwon, I. C.; Choi, K.; Kim, K. Biomaterials 2014, 35, 2302−2311. (20) Kim, C. J.; Lee, D. I.; Kim, C.; Lee, K.; Lee, C. H.; Ahn, I. S. Anal. Chem. 2014, 86, 3825−3833. (21) Aits, S.; Jäaẗ telä, M. J. Cell Sci. 2013, 126, 1905−1912. (22) Tian, J.; Ding, L.; Ju, H.; Yang, Y.; Li, X.; Shen, Z.; Zhu, Z.; Yu, J. S.; Yang, C. J. Angew. Chem., Int. Ed. 2014, 53, 9544−9549. (23) Isakau, H. A.; Parkhats, M. V.; Knyukshto, V. N.; Dzhagarov, B. M.; Petrov, E. P.; Petrov, P. T. J. Photochem. Photobiol., B 2008, 92, 165−174. (24) Huang, P.; Lin, J.; Wang, S.; Zhou, Z.; Li, Z.; Wang, Z.; Zhang, C.; Yue, X.; Niu, G.; Yang, M.; Cui, D.; Chen, X. Biomaterials 2013, 34, 4643−4654. (25) Huang, P.; Lin, J.; Wang, X.; Wang, Z.; Zhang, C.; He, M.; Wang, K.; Chen, F.; Li, Z.; Shen, G.; Cui, D.; Chen, X. Adv. Mater. 2012, 24, 5104−5110. (26) Tian, B.; Wang, C.; Zhang, S.; Feng, L.; Liu, Z. ACS Nano 2011, 5, 7000−7009. (27) Kim, S.; Fujitsuka, M.; Majima, T. J. Phys. Chem. B 2013, 117, 13985−13992. (28) Liu, Z.; Robinson, J. T.; Sun, X.; Dai, H. J. Am. Chem. Soc. 2008, 130, 10876−10877. (29) Wang, H.; Zhang, Q.; Chu, X.; Chen, T.; Ge, J.; Yu, R. Angew. Chem., Int. Ed. 2011, 50, 7065−7069. (30) Dong, H.; Gao, W.; Yan, F.; Ji, H.; Ju, H. Anal. Chem. 2010, 82, 5511−5517. (31) Cho, Y.; Kim, H.; Choi, Y. Chem. Commun. 2013, 49, 1202− 1204. (32) White, B.; Banerjee, S.; O’Brien, S.; Turro, N. J.; Herman, I. P. J. Phys. Chem. C 2007, 111, 13684−13690. (33) Gao, W.; Cao, W.; Zhang, H.; Li, P.; Xu, K.; Tang, B. Chem. Commun. 2014, 50, 8117−8120. (34) Ryu, J. H.; Kim, S. A.; Koo, H.; Yhee, J. Y.; Lee, A.; Na, J. H.; Youn, I.; Choi, K.; Kwon, I. C.; Kim, B.-S.; Kim, K. J. Mater. Chem. 2011, 21, 17631−17634. (35) Liu, Z.; Fan, A. C.; Rakhra, K.; Sherlock, S.; Goodwin, A.; Chen, X.; Yang, Q.; Felsher, D. W.; Dai, H. Angew. Chem., Int. Ed. 2009, 48, 7668−7672. (36) Chen, H.; Xiao, L.; Anraku, Y.; Mi, P.; Liu, X.; Cabral, H.; Inoue, A.; Nomoto, T.; Kishimura, A.; Nishiyama, N.; Kataoka, K. J. Am. Chem. Soc. 2014, 136, 157−163. (37) Kindy, M. S.; Yu, J.; Zhu, H.; El-Amouri, S. S.; Hook, V.; Hook, G. R. J. Alzheimer’s Dis. 2012, 29, 827−840.

H

DOI: 10.1021/acs.analchem.5b00429 Anal. Chem. XXXX, XXX, XXX−XXX