Microarray Based Screening of Peptide Nano Probes for HER2

Jul 28, 2015 - Xiangqian Jia , Weizhi Wang , Qiuju Han , Zihua Wang , Yunhong Jia , and ... Kuick , Scott R. Owens , Henry Appelman , and Thomas D. Wa...
0 downloads 0 Views 3MB Size
Download PDF
Article pubs.acs.org/ac

Microarray Based Screening of Peptide Nano Probes for HER2 Positive Tumor Zihua Wang,†,# Weizhi Wang,*,†,# Xiangli Bu,† Zewen Wei,† Lingling Geng,† Yue Wu,‡ Chengyan Dong,‡ Liqiang Li,‡ Di Zhang,† Shu Yang,† Fan Wang,‡ Christopher Lausted,§ Leroy Hood,§ and Zhiyuan Hu*,†,§,∥ †

CAS Key Laboratory for Biomedical Effects of Nanomaterials & Nanosafety National Center for Nanoscience and Technology of China, Beijing, China, 100190 ‡ Medical Isotopes Research Center, Department of Radiation Medicine, School of Basic Medical Sciences, Peking University, Beijing China, 100191 § Institute for Systems Biology, 401 Terry Avenue North, Seattle, Washington 98109, United States ∥ Beijing Proteome Research Center, Beijing Institute of Radiation Medicine, Beijing China, 102206 S Supporting Information *

ABSTRACT: Peptides are excellent biointerface molecules and diagnostic probes with many advantages such as good penetration, short turnover time, and low cost. We report here an efficient peptide screening strategy based on in situ single bead sequencing on a microarray. Two novel peptides YLFFVFER (H6) and KLRLEWNR (H10) specifically binding to the tumor biomarker human epidermal growth factor receptor 2 (HER2) with aKD of 10−8 M were obtained from a 105 library. Conjugated to nanoparticles, both the H6 and H10 probes showed specific accumulation in HER2-positive tumor tissues in xenografted mice by in vivo imaging.

B

HER2 (human epidermal growth factor receptor 2, also known as neu or ERBB-2) is a 185 kDa transmembrane tyrosine kinase receptor encoded by the member of the epidermal growth factor receptor (EGFR) family of the HER2 gene.15 HER2 plays important roles in cell proliferation, differentiation, and survival. HER2 is scarcely expressed in normal tissue and overexpressed in many human cancers, especially in breast cancer.16,17 Overexpression of HER2 occurs in approximately 20%−30% of breast cancer in both primary and metastatic breast tumors. Its expressional level is strongly associated with tumor aggressiveness, recurrence risk, and poor prognosis.18 Therefore, HER2 is a good biomarker for breast cancer diagnosis and therapy.19−21 The accessibility of its extracellular domain makes HER2 an ideal target for drug delivery as well as molecular imaging.22,23 Various approaches have been used to develop drugs or diagnosis reagents targeting HER2 in the clinical field. Tyrosine kinase receptors, the monoclonal antibodies (trastuzumab and pertuzumab) have been approved by the FDA (U.S. Food and Drug Administration) for the treatment of HER2 positive breast cancer patients.24 Recently, HER2 based circulating tumor cells (CTCs) detection is recognized as a valuable prognostic and

reast cancer is the leading type of cancer in women. Early diagnosis as well as prognosis evaluation can improve the clinical survival of breast cancer. Recently, molecular imaging provides a noninvasive method for early tumor diagnosis and real time therapeutic monitoring.1 Hereinto, molecular probes toward the oncotarget is the essential component in molecular imaging.2 Current biochemical probes for cancer diagnosis mostly rely on the recognition of antibodies toward specific biomarkers on the membrane of cancer cells. However, strong immunogenicity, poor tissue penetration, and slow blood clearance impede the application of antibodies in cancer imaging.3 These limitations have accelerated the search for small-molecule alternatives as cancer probes and diagnosis reagent. As small molecular probes, peptides own several advantages such as fast blood clearance, low immunogenicity, good penetration, good biocompatibility, and ease for chemical modification.4−6 Affinity peptides are also excellent interface molecules between nano materials and biosystems, which mediate specific molecular recognition, imaging, and drug targeting delivery.7−9 A number of peptides identified from peptide libraries are in clinical trials and are anticipated to have broad clinical applications with high efficiency while reducing the side effects.10−12 Also, a number of peptide modified nano materials showed good biocompatibility and targeting effects.13,14 © XXXX American Chemical Society

Received: April 27, 2015 Accepted: July 28, 2015

A

DOI: 10.1021/acs.analchem.5b01588 Anal. Chem. XXXX, XXX, XXX−XXX

Article

Analytical Chemistry

purchased from GL Biochem (China). Tentagel Resin was from Rapp Polymere (Germany, loading 0.53 mmol/g). Trifluoroacetic acid (TFA), fluorescein 5-isothiocyanate (FITC) hoechst 33342, 1-ethyl-3-(3-(dimethylamino)propyl) carbodiimide (EDC), CHCA (α-cyano-4-hydroxycinnamic acid), and streptavidin coated magnetic beads (1 μm) were from Sigma-Aldrich. Silicon wafer (N/1-0-0, 500 μm) was from KYKY Tech. (China). HER2 protein was from Proteintech. Biotin labeling kit was from SoluLink. DMEM/high glucose medium, RPMI 1640 medium, and trypsin were purchased from GE Healthcare Life Sciences. The human breast cancer cell line SKBR-3 (HTB-30) and human embryonic kidney cell lines 293A (HEK) were purchased from Chinese Academy of Medical Sciences (China). All cell lines were supplemented with 10% fetal bovine serum (FBS), (Gibco) and 100 U/mL penicillin and 100 U/mL streptomycin (Gibco). BALB/c nude mice were purchased from Vitalriver (China). The plasmid pRNAi-HER2 for HER2 siRNA expressing was constructed by gene clone and restriction digest recombination in our lab. OBOC Peptide Library Construction and Magnetic Beads Assisted Screening. Fmoc SPPS (solid phase peptide synthesis) strategy was employed to synthesize the photo labile peptide library. Tentagel resin was used as the solid phase support. The pool and split method was employed to construct the OBOC combinatorial chemistry library.40,41 Scheme S1 shows the synthesis process, and the synthesis detail was described in the Supporting Information. HER2 protein was biotinylated using a biotin labeling kit at the ratio of 4 biotin/protein. Then biotinHER2 was purified by the Zeba spin column.42 The peptide beads in the library were incubated with biotin-HER2 and streptavidin coated magnetic beads successively. Then all the peptide beads were introduced into a Teflon tube (diameter 1 mm, flow rate 600 μL/min) with a magnet closely next to the outer wall of the tube. Finally, the magnet was removed. Then the trapped beads were flushed out and collected. The mixture of positive peptide beads and streptavidin coated magnetic beads were suspended in a tube and centrifuged for a very short time to collect the beads at the bottom. After sequencing, positive peptides H6 and H10 were resynthesized using Fmoc SPPS protocols on Wang resin. After the peptide prolongation, Fmoc-ε-aminocaproic acid (Fmoc-ε-Acp) was attached to the N terminal using HBTU coupling strategy. Then FITC coupling was also carried out through solid phase synthesis in the dark in pyridine/DMF/DCM (12/7/5) solution overnight.43 Then, FITC-labeled peptides were cleaved from the resins by TFA treatment. The crude peptides were purified by high-performance liquid chromatography (HPLC) and determined by MALDI-TOF mass spectrometry. In Situ MALDI-TOF Sequencing of the Peptide on the Single Bead. MALDI-TOF-MS analysis was performed on a Bruker ULTRAFLEXTREME mass spectrometer (Bruker Daltonics, Germany) equipped with a nitrogen laser (wavelength 337 nm, laser pulse duration 3 ns) with reflectron and positive-ion modes. CHCA was dissolved in solvent TA30 (30:70 (v/v) acetonitrile/TFA 0.1% in water) to prepare a matrix solution. Peptide beads were suspended in matrix solution and loaded into the wells of the microarray chip. The laser power energy was adjusted between 0% and 100% to provide laser pulse energy between 0 and 100 μJ per pulse. The mass spectra were typically recorded at an accelerating voltage of 19 kV, a reflection voltage of 20 kV, and with laser pulse energy of 60 μJ. Each mass spectrum was acquired as an average of 500 laser shots. For in situ “one well one bead” analysis, the laser beam

predictive marker with real-time information to guide individualized therapeutics for breast cancers.25 However, the abovementioned HER2 recognition was mainly based on antigen− antibody interaction, and small ligands for HER2 are highly desirable. Affinity peptides could be usually screened out of the highthroughput peptide libraries of which the one-bead onecompound (OBOC) combinatorial library approach is the most popular one.26,27 Nowadays, new generations of OBOC screening have been developed to accelerate the peptide screening processes.28−31 With magnetic conjugation assay, OBOC peptide beads could be trapped by the magnetic field instead of being picked out manually.32 High-throughput peptides could also be coded by quantum dots, and fluorescent identification was achieved.33 The continuous flow beads sorting method is developed to make the screening process very efficient.34,35 Our previous work is concentrated on the screening of high affinity peptide ligands with the integrated microscale fabricated devices.36−39 On the basis of the works of others and ours, we are aiming to discover novel peptide ligands toward HER2. We report here an integrated lab-on-chip system to perform the whole peptide screening process: positive peptide isolation, single bead trapping, and in situ sequencing. A one-bead-one-well microarray which is compatible with in situ matrix assisted laser desorption ionization timeof-flight mass spectrometry (MALDI-TOF-MS) sequencing was realized (Figure 1). Two novel octapeptides, H6 and

Figure 1. Screening system and alignment of novel HER2-binding peptides: (a) microscopic image of the positive peptide beads interacting with the magnetic beads, (b) microscopic image of the negative peptide beads interacting with the magnetic beads, (c) whole structure of the microfluidic chip, (d) one well one bead trapping, (e) single positive peptide bead detection, and (f) alignment of novel HER2-binding peptides.

H10, have been identified from the 7 × 105 candidates with nanomolar affinity property. In vivo and ex vivo experiments have further proved that both of the novel peptide ligands have high affinity and high specificity toward HER2, which are prospective diagnostic probes for HER2 positive breast cancer.

■

EXPERIMENTAL SECTION Materials. 9-Fluorenylmethoxycarbonyl (Fmoc)-protected amino acids, Wang resin, and 2-(1H-benzotriazole-1-yl)-1,1,3, 3-tetramethyluronium hexafluoro-phosphate (HBTU) were B

DOI: 10.1021/acs.analchem.5b01588 Anal. Chem. XXXX, XXX, XXX−XXX

Article

Analytical Chemistry

control tumor-bearing nude mice were intravenously injected with QDs. The mice were anesthetized by chloral hydrate (4%, 500 μL) and placed into the small animal in vivo imaging system (CRI Maestro 2). Near-infrared fluoresce (NIRF) imaging was achieved (ex, 450 nm ±10 nm; em, 705 nm ±17.5 nm; 20 s exposure). Then the nude mice were sacrificed. Tumors and the main organs were dissected and then the NIFR images were taken.

was directed to the bottom of the microwell (stepping well by well) with the light spot area of 0.02 mm2. Cell Culture and Confocal Fluorescence Imaging of Living Cancer Cells. The human breast cancer cell line SKBR-3 and human embryonic kidney cell lines 293A were cultured in RPMI 1640 medium and Hyclone DMEM/high glucose with 10% FBS, penicillin and streptomycin at 37 °C containing 5% CO2. The recombinant plasmid pRNAi-HER2 was constructed by combination of double-strand DNA of HER2 with vector pRNAi that underwent a restriction digest. The pRNAi-HER2 plasmid was transfected by liposome into SKBR-3 cells according to lipofectamine2000 transfection reagent protocol (Invitrogen). For SKBR-3 and 293A cells, approximately 1 × 105 mL−1 cells were seeded into culture dishes and cultured overnight for cell adherence. FITC-labeled peptide (H6 or H10) was dissolved in cold PBS at a concentration of 5.0 × 10−5 M. The cells were incubated with FITC-labeled peptide solution (200 μL, with hoechst 33342 (1 mM) in the dark for 30 min at 4 °C. Finally, the cells were washed three times with cold PBS. Confocal fluorescence imaging was performed on an Olympus FV1000-IX81 confocal-laser scanning microscope. A FV5-LAMAR 488 nm laser was the excitation source for FITC throughout the experiment, and emission was collected between 520 and 620 nm. Hoechst 33342 was excited at 50 mW; Ex, 405 nm; Em, 472 nm. The objective lens used for imaging was a UPLSAPO 100× oil-immersion objective (Olympus). Surface Plasmon Resonance Imaging (SPRi) for Detection of the Affinity Peptides toward HER2. SPRi analysis was performed on a Plexera PlexArray HT system (Plexera LLC, Bothell, WA) using a bare gold SPRi chip (Nanocapture gold chips, with a gold layer of 47.5 nm thickness). All the purified peptides were printed onto the gold chip surface by the thiol group of the cystein residue. The printed chip was then incubated in 4 °C overnight in a humid box. The SPRi chip was washed and blocked using 5% (m/v) nonfat milk in PBS overnight before use. The SPRi analysis procedure follows the following cycle of injections: running buffer (PBST, baseline stabilization); sample (five concentrations of the protein, binding); running buffer (PBST, washing); and 0.5% (v/v) H3PO4 in deionized water (regeneration). HER2 protein was diluted with PBST to concentrations of 10 μg/mL, 5 μg/mL, 2.5 μg/mL, 1.25 μg/mL, and 0.625 μg/mL. Real-time binding signals were recorded and analyzed by PlexArray HT software. In Vivo Fluorescence Imaging. All the animal experiments were conducted in compliance with the guide for the care and use of laboratory animals of Beijing University Animal Study Committee’s requirements. The xenografted tumors were established by subcutaneously injecting 1 × 107 SKBR-3 cells into the right hind leg of the 8 week-old female BALB/c nude mice. Tumor growth was measured periodically until the tumors reached to ≥8 mm in diameter. Qdot705 ITK carboxyl quantum dots (QDs) with a maximum emission wavelength at 705 nm were purchased from Invitrogen. The conjugation between the peptides (H6 and H10) and the QDs was carried out by the following method: 8 μM of QDs solution (25 μL) in 1× PBS was activated by 10 mg/mL (5 μL) EDC (30 min) and then incubated with 25 μL of 0.1 mg/mL peptides solution overnight. There are 20−40 peptides per QD based on the coupling efficiency of 40−50%. Fluorescence images of nude mice bearing subcutaneous tumor were acquired after tail vein injection of H6-QDs and H10-QDs (2 μM, 200 μL), while the

■

RESULTS AND DISCUSSION Design and Construction of HER2 Targeting OBOC Peptide Library. A peptide library with random amino acid residues at specific positions was constructed to create a 10-mer OBOC combinatorial library.44 A photo cleavable linker, ANP (3-amino-3-(2-nitrophenyl) propionic acid) was used for in situ UV (ultraviolet) cleavage. The general sequence of the library is X1X2X3X4X5X6X7X8CG-ANP. X1 represents any of V, N, Y, A, and K residues at N-terminal to form hydrophobic or hydrophilic interactions with HER2 as reported.45,46 X2 represents H, S, W, L, or D. X3 represents R, Q, F, L, or D. X4 represents K, S, F, L, or D. X5 represents T, S, V, or E. X6 represents P, F, Y, or W. X7 represents L, N, or E. From X2 to X7, amino acids with different properties were chosen to improve the diversity of the peptide.47 X8 represents H, R, or K residues because basic amino acid residues at the C-terminal were reported to improve ligands interaction with HER2 protein.48 In the library, the sequence on each bead was randomly distributed so that the complexity of the peptide library was 5 × 5 × 5 × 5 × 4 × 4 × 3 × 3 = 90 000 and the redundancy of the library was eight. As a result, peptide screening was carried out from 7 × 105 candidate beads. After the incubation of the library with biotinylated HER2, peptide beads with high affinity toward HER2 will be covered with biotinylated HER2. The positive peptide beads would be recognized by strepavidin coated magnetic beads in an affinity-dependent manner. Because of the size difference between the peptide beads (160 μm in diameter) and magnetic beads (1 μm in diameter), positive peptide beads would be wrapped up by magnetic beads (Figure 1a), whereas negative peptide beads have fewer or no magnetic beads bound (Figure 1b). In Situ Screening and Identification Based on the Microarray. A microarray chip was fabricated for in situ trapping and characterization of the positive peptides. The chip was fabricated with silicon. The microarray chip consisted of a 2 × 10 microarray of which each microarray contains 10 × 10 microwells (Figure 1c). The microwell array was prepared by a conventional soft lithography procedure as we reported before. Each microwell has a regular prismoid shape of a suitable size to trap individual peptide beads in a one-well-one-bead manner. (Figure 1d,e). The microwell array was utilized for in situ MALDI-TOF-MS sequencing.39 The wavelength of the laser is 365 nm in the UV region. The photo labile ANP linker could be in situ cleaved from the beads under the laser shooting. The TOF-TOF MS spectra were characterized by searching the MASCOT server after uploading the sequences list into the Swiss-Prot database. We obtained 84 positive sequences and aligned them as shown in Figure 1f. Using the software ClustalX2,49 conserved sequences were determined and the motif X-Leu-XXX-Trp(Phe)-X-Arg was revealed to hit the highest probability. The consensus sequences that emerged are YLFFVFER (named H6) and KLRLEWNR (named H10) with the most frequent matchings among all eight alterable residues. The representative MS-MS spectra were shown in Figure S1. It is notable that this in situ sequencing microarray provides C

DOI: 10.1021/acs.analchem.5b01588 Anal. Chem. XXXX, XXX, XXX−XXX

Article

Analytical Chemistry

Specificity of the Peptide Probes toward HER2. Further cell experiments were carried out to confirm that HER2 is the specific target of peptides on the complicated membrane structure. SKBR-3 cell lines were again used as the model cells. Recombinant plasmids were transfected by liposome into SKBR-3 cells to construct a RNA interference (RNAi) model cell. Self-made recombinant plasmid pRNAi-HER2 could significantly downregulate HER2 expression of SKBR-3 (Figure S3). As expected, FITC-H6 and FITC-H10 preserve the binding ability toward SKBR-3 cells (Figure 3a−h). For RNAi SKBR-3 cells, an

a high-throughput and high-efficiency strategy for affinity peptide identification. Affinity of the Peptide Probes toward Living Cancer Cells. To test the binding behavior of H6 and H10 toward HER2 protein in living cancer cells, the HER2 high-expressing human breast cancer cell line SKBR-3 (HTB-30) was chosen as an in vitro positive model. FITC labeled peptide H6 (FITC-H6) and FITC-H10 were incubated with SKBR-3 cells. It was clear that both H6 and H10 showed high signal on the membranes (Figure 2a,e). For quantitative comparison,

Figure 2. Confocal images of the FITC labeled peptides binding toward SKBR-3 cell lines (a, e, i). Confocal image of FITC labeled peptides binding toward 293A cells (b, f, j). The fluorescent intensity profiles along the red arrows through the SKBR-3 cell membrane of H6, H10, and P7, respectively (c, g, k). SPRi detection of the binding affinity of H10, H6, and P7 toward HER2, respectively (d, h, l).

fluorescence intensity profiles along through the red arrows were determined and H6 showed higher fluorescent signal than H10 (Figure 2c,g). Additionally, a control peptide with scramble sequence KDFLSWLR (P7) has little binding to the HER2 positive cells (Figure 2i,k). To check the specificity of the peptides toward HER2, human embryonic kidney cell lines 293A (HEK) with low expression of HER2 served as the negative cells. As illustrated in Figure 2b, f ,and j, none of the peptides showed binding to the cell surface. It was revealed that both the peptides H6 and H10 bind to HER2-positive cells in a specific manner. The binding rates of FITC-H6 and FITC-H10 toward SKBR-3 were measured by flow cytometer and the results were calculated as 69.59% and 53.32%, respectively (Figure S2). Furthermore, dissociation constants (KD) between the peptide ligands and HER2 protein were determined by SPRi. The dissociation constants were calculated from kinetic constants obtained by curve-fitting of the association and dissociation rates to real-time binding and washing data (Figure 2d, h, and l). The KDs of H6 and H10 were calculated as 6.70 × 10−8 mol/L and 3.04 × 10−8 mol/L, respectively. Meanwhile, the SPRi curve of P7 was near to the “square wave” of which the signal was only increased while the concentration increasing. The dissociation constant of P7 was calculated as 1.35 × 10−4 mol/L, which indicated very low binding affinity. The SPRi results agreed nicely with the fluorescence results. In genaral, 67 nM affinity of H6 and 30 nM affinity of H10 performed a satisfactory peptide−protein recognition behavior.

Figure 3. Specificity confirmation of the peptides toward HER2: (a−d) interaction of FITC-H6 with SKBR-3 cells and RNAi SKBR-3 cells, (e−h) interaction of FITC-H10 with SKBR-3 cells and RNAi SKBR-3 cells, and (i−l) fluorescent profiles along the red arrows through membranes.

obvious decrease in green fluorescence could be observed for both peptides due to the low expression of HER2 protein (Figure 3i−l). These results further confirmed that HER2 protein is the specific target of the peptide ligands. In addition, specifity identification has been done at the molecular level by SPRi detection. Another two proteins from the human epidermal growth factor receptor (HER) family, EGFR (epidermal growth factor receptor, HER1) and HER3 were chosen to verify the specifity of the peptide toward HER2. Human serum albumin (HSA) was chosen to test the nonspecific absorption. As shown in Figure S4, the SPRi curves of the peptides toward the three proteins were nearly to the nonebinding “square wave”. It was suggested that H6 and H10 show good specificity toward HER2. It was indicated that there was low nonspecific bindings of the peptides toward the three control proteins. Beside the high affinity and good specificity, low cytotoxicity was also an important criterion for a good molecular probe. The MTT (3-(4,5-cimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay had indicated that the survival rates of cells were close to 100% when they were exposed to H6 and H10 at concentrations ranging from 0.1 μM to 100 μM (Figure S5). As a result, D

DOI: 10.1021/acs.analchem.5b01588 Anal. Chem. XXXX, XXX, XXX−XXX

Article

Analytical Chemistry

factors caused by a combination of EPR (enhanced permeability and retention) effect of the nano particle QDs. It was shown that the specificity of H10 toward HER2 was better than H6. Furthermore, different performances between the two peptides were determined by pharmacokinetic experiments. The results were shown in the Figure S6 in the Supporting Information. The plasma half-lives of the peptides were determined as 17 min of H6 and 13 min of H10. Since it has a shorter half-life, peptide H10 could be cleared more rapidly than H6 in the blood flow. It was demonstrated that the combination between peptide ligands and nano materials could be developed into useful probes for in vivo imaging. Additionally, low cytotoxicity of both the nano probes was confirmed by MTT assay. After 48 h, the cell viability is near 70% for cancer cells (Figure S7) and near 80% for normal cells, endothelial cells, and fibroblast cells (Figure S8). Also, peptide-QDs showed lower toxicity than QDs only, which indicated that the nano probes show not only good target effects but also good biocompatibilities.

both the peptides could recognize the cells safely without stimulation of growth and inhibition of cell viability. In Vivo and ex Vivo Imaging of the Tumor Homing Peptide Nano Probes. Peptide-mediated molecular diagnosis toward the tumor usually resulted in the increased diagnostic efficacy and lower systemic toxicity.50 QDs are popular fluorescent nanocomposites for in vivo imaging. QDs have unique optical properties and high levels of fluorescence quantum yield.51 Therefore, peptides modified QDs were estimated as wonderful molecular probes. To further evaluate the HER2 tumor imaging of H6 and H10, both the peptides were labeled with CdSe QDs and were injected into SKBR-3 xenograft mice. The biodistribution of peptide probes in tumor-bearing mice were monitored by the small animal imaging system. The mice that were treated with intravenous injection of QDs only were used as a negative control. As shown in Figure 4a,b, after half an

■

CONCLUSION The microarray screening strategy could be universally and practicably applied to almost any membrane proteins. Using the efficient in situ single bead sequencing system, novel peptide probes toward HER2 have been successfully obtained and validated not only on living cancer cells but also on in vivo explant mice models. It is expected that the peptide-assisted nano probes could be used for breast cancer imaging and diagnosis. We are now developing multivalence nano probes by the modification of the nano materials with multiple peptide ligands. Our goal is to enhance the affinity and specificity to achieve good biodistribution in living systems.

■

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.analchem.5b01588. MALDI-TOF-MS spectra, flow cytometer results, pharmacokinetic experiments, and MTT assay (PDF)

■

Figure 4. In vivo and ex vivo imaging of tumor-bearing mice using H6-QDs and H10-QDs probes: (a, b), in vivo fluorescence imaging of peptide probes toward tumors, (c, e) ex vivo fluorescence imaging and biodistribution of dissected organs of tumor-bearing nude mice, and (d, f) quantification of the fluorescence signals in ex vivo experiments. All the fluorescent intensities were measured in counts/energy/area and were presented as an average (n = 3).

AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. Author Contributions #

Z. Wang and W. Wang contribute equally to the work.

hour, the fluorescent signal appeared at the tumor sites with H6-QDs displaying a higher signal than H10-QDs, which is consistent with the cell experiments. After 4 h, the mice were sacrificed and dissected. Compared with QDs only, the peptideQDs uptakes were observed in tumor in a specific manner. As shown in Figure 4c,d, H6-QDs showed the highest level of tumor accumulation. Peptide-QDs probes were also gathered in the livers and spleens, which may due to the metabolism of nanomaterials by the blood and lymph circulation. However, it was interesting that the accumulation of H10 in spleen and liver was relatively low while remained high in tumor. It was revealed that although the dissociation constants of both the peptides were close to each other, the specificity of the two peptides might be different. So the specificities of the nano probes were attributed by the recognition of the peptide ligands. It is also indicated that high accumulation at 4 h is attributable to kinetic

Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS We acknowledge funding from the Beijing Natural Science Foundation (Grant 2144058), National Natural Science Foundation of China (Grants 21305023, 31270875, 31470049, 31400702), and Project of Chinese Academy of Science (Grant YZ201217).

■

REFERENCES

(1) Alford, R.; Ogawa, M.; Choyke, P. L.; Kobayashi, H. Mol. BioSyst. 2009, 5, 1279−1291. (2) Yuan, L.; Lin, W.; Zheng, K.; He, L.; Huang, W. Chem. Soc. Rev. 2013, 42, 622−661. (3) Scott, A. M.; Wolchok, J. D.; Old, L. J. Nat. Rev. Cancer 2012, 12, 278−287. E

DOI: 10.1021/acs.analchem.5b01588 Anal. Chem. XXXX, XXX, XXX−XXX

Article

Analytical Chemistry (4) McGuire, M. J.; Gray, B. P.; Li, S.; Cupka, D.; Byers, L. A.; Wu, L.; Rezaie, S.; Liu, Y. H.; Pattisapu, N.; Issac, J.; Oyama, T.; Diao, L.; Heymach, J. V.; Xie, X. J.; Minna, J. D.; Brown, K. C. Sci. Rep. 2014, 4, 4480. (5) Xiong, N.; Dong, X.-Y.; Zheng, J.; Liu, F.-F.; Sun, Y. ACS Appl. Mater. Interfaces 2015, 7, 5650−5662. (6) Yu, Y.; Li, H.; Zhang, B.; Pan, X.; Zhu, X.; Ding, Y.; Li, G. ACS Appl. Mater. Interfaces 2015, 7, 4401−4405. (7) Aina, O. H.; Sroka, T. C.; Chen, M. L.; Lam, K. S. Biopolymers 2002, 66, 184−99. (8) Kim, Y. H.; Jeon, J.; Hong, S. H.; Rhim, W. K.; Lee, Y. S.; Youn, H.; Chung, J. K.; Lee, M. C.; Lee, D. S.; Kang, K. W. Small 2011, 7, 2052−2060. (9) Jin, S.; Wang, Y.; Zhu, H.; Wang, Y.; Zhao, S.; Zhao, M.; Liu, J.; Wu, J.; Gao, W.; Peng, S. ACS Nano 2013, 7, 7664−7673. (10) Steinmetz, N. F.; Ablack, A. L.; Hickey, J. L.; Ablack, J.; Manocha, B.; Mymryk, J. S.; Luyt, L. G.; Lewis, J. D. Small 2011, 7, 1664−1672. (11) Bartneck, M.; Ritz, T.; Keul, H. A.; Wambach, M.; Bornemann, J.; Gbureck, U.; Ehling, J.; Lammers, T.; Heymann, F.; Gassler, N.; Luedde, T.; Trautwein, C.; Groll, J.; Tacke, F. ACS Nano 2012, 6, 8767−8777. (12) Shokeen, M.; Pressly, E. D.; Hagooly, A.; Zheleznyak, A.; Ramos, N.; Fiamengo, A. L.; Welch, M. J.; Hawker, C. J.; Anderson, C. J. ACS Nano 2011, 5, 738−747. (13) Zhang, M. Z.; Yu, Y.; Yu, R. N.; Wan, M.; Zhang, R. Y.; Zhao, Y. D. Small 2013, 9, 4183−4193. (14) Gao, J.; Liang, G.; Zhang, B.; Kuang, Y.; Zhang, X.; Xu, B. J. Am. Chem. Soc. 2007, 129, 1428−1433. (15) Carney, W.; Leitzel, K.; Ali, S.; Neumann, R.; Lipton, A. Breast Cancer Res. 2007, 9, 207. (16) Montemurro, F.; Scaltriti, M. J. Pathol. 2014, 232, 219−229. (17) Tai, W.; Mahato, R.; Cheng, K. J. Controlled Release 2010, 146, 264−75. (18) Gutierrez, C.; Schiff, R. Arch. Pathol. Lab. Med. 2011, 135, 55− 62. (19) Arya, S. K.; Lim, B.; Rahman, A. R. Lab Chip 2013, 13, 1995− 2027. (20) Bai, L.; Du, Y.; Peng, J.; Liu, Y.; Wang, Y.; Yang, Y.; Wang, C. J. Mater. Chem. B 2014, 2, 4080−4088. (21) Cristofanilli, M.; Budd, G.; Ellis, M.; Stopeck, A.; Matera, J.; Miller, M.; Reuben, J.; Doyle, G.; Allard, W.; Terstappen, L.; Hayes, D. N. Engl. J. Med. 2004, 351, 781−791. (22) Nielsen, D. L.; Andersson, M.; Kamby, C. Cancer Treat. Rev. 2009, 35, 121−36. (23) Larimer, B. M.; Thomas, W. D.; Smith, G. P.; Deutscher, S. L. Mol. Imaging Biol. 2014, 16, 449−58. (24) Sliwkowski, M. X.; Mellman, I. Science 2013, 341, 1192−1198. (25) Budd, G.; Cristofanilli, M.; Ellis, M.; Stopeck, A.; Borden, E.; Miller, M.; Matera, J.; Repollet, M.; Doyle, G.; Terstappen, L.; Hayes, D. Clin. Cancer Res. 2006, 12, 6403−6409. (26) Cho, C. F.; Behnam Azad, B.; Luyt, L. G.; Lewis, J. D. ACS Comb. Sci. 2013, 15, 393−400. (27) Cho, C. F.; Amadei, G. A.; Breadner, D.; Luyt, L. G.; Lewis, J. D. Nano Lett. 2012, 12, 5957−5965. (28) Doran, T. M.; Gao, Y.; Mendes, K.; Dean, S.; Simanski, S.; Kodadek, T. ACS Comb. Sci. 2014, 16, 259−270. (29) Lian, W.; Upadhyaya, P.; Rhodes, C. A.; Liu, Y.; Pei, D. J. Am. Chem. Soc. 2013, 135, 11990−11995. (30) Trinh, T. B.; Xiao, Q.; Pei, D. Biochemistry 2013, 52, 5645− 5655. (31) Gao, Y.; Amar, S.; Pahwa, S.; Fields, G. B.; Kodadek, T. ACS Comb. Sci. 2015, 17, 49−59. (32) Astle, J. M.; Simpson, L. S.; Huang, Y.; Reddy, M. M.; Wilson, R.; Connell, S.; Wilson, J.; Kodadek, T. Chem. Biol. 2010, 17, 38−45. (33) Choi, Y.; Lee, J.; Kim, K.; Kim, H.; Sommer, P.; Song, R. Chem. Commun. 2010, 46, 9146−9148.

(34) Burns, A. R.; Kwok, T. C. Y.; Howard, A.; Houston, E.; Johanson, K.; Chan, A.; Cutler, S. R.; McCourt, P.; Roy, P. J. Nat. Prot. 2006, 1, 1906−1914. (35) Price, A. K.; MacConnell, A. B.; Paegel, B. M. Anal. Chem. 2014, 86, 5039−5044. (36) Wang, W.; Wei, Z.; Wang, Z.; Ma, H.; Bu, X.; Hu, Z. RSC Adv. 2014, 4, 61767−61770. (37) Wang, W.; Wei, Z.; Zhang, D.; Ma, H.; Wang, Z.; Bu, X.; Li, M.; Geng, L.; Lausted, C.; Hood, L.; Fang, Q.; Wang, H.; Hu, Z. Anal. Chem. 2014, 86, 11854−11859. (38) Wang, W.; Zhang, D.; Wei, Z.; Wang, Z.; Bu, X.; Yang, S.; Fang, Q.; Hu, Z. Talanta 2015, 134, 705−711. (39) Wang, W.; Li, M.; Wei, Z.; Wang, Z.; Bu, X.; Lai, W.; Yang, S.; Gong, H.; Zheng, H.; Wang, Y.; Liu, Y.; Li, Q.; Fang, Q.; Hu, Z. Anal. Chem. 2014, 86, 3703−3707. (40) Hintersteiner, M.; Kimmerlin, T.; Kalthoff, F.; Stoeckli, M.; Garavel, G.; Seifert, J.-M.; Meisner, N.-C.; Uhl, V.; Buehler, C.; Weidemann, T.; Auer, M. Chem. Biol. 2009, 16, 724−735. (41) Pei, D. Chem. Biol. 2010, 17, 3−4. (42) Reitinger, S.; Petriv, O. I.; Mehr, K.; Hansen, C. L.; Withers, S. G. J. Virol. Methods 2012, 185, 171−174. (43) Jullian, M.; Hernandez, A.; Maurras, A.; Puget, K.; Amblard, M.; Martinez, J.; Subra, G. Tetrahedron Lett. 2009, 50, 260−263. (44) Banappagari, S.; McCall, A.; Fontenot, K.; Vicente, M. G.; Gujar, A.; Satyanarayanajois, S. Eur. J. Med. Chem. 2013, 65, 60−69. (45) Lemmon, M. A. Exp. Cell Res. 2009, 315, 638−648. (46) Franklin, M. C.; Carey, K. D.; Vajdos, F. F.; Leahy, D. J.; de Vos, A. M.; Sliwkowski, M. X. Cancer Cell 2004, 5, 317−328. (47) Bostrom, J.; Yu, S. F.; Kan, D.; Appleton, B. A.; Lee, C. V.; Billeci, K.; Man, W.; Peale, F.; Ross, S.; Wiesmann, C.; Fuh, G. Science 2009, 323, 1610−1614. (48) Garrett, T. P. J.; McKern, N. M.; Lou, M. Z.; Elleman, T. C.; Adams, T. E.; Lovrecz, G. O.; Zhu, H. J.; Walker, F.; Frenkel, M. J.; Hoyne, P. A.; Jorissen, R. N.; Nice, E. C.; Burgess, A. W.; Ward, C. W. Cell 2002, 110, 763−773. (49) Chenna, R.; Sugawara, H.; Koike, T.; Lopez, R.; Gibson, T. J.; Higgins, D. G.; Thompson, J. D. Nucleic. Acids. Res. 2003, 31, 3497− 3500. (50) Chang, D. K.; Li, P. C.; Lu, R. M.; Jane, W. N.; Wu, H. C. PLoS One 2013, 8, e83239. (51) Gao, X.; Cui, Y.; Levenson, R. M.; Chung, L. W.; Nie, S. Nat. Biotechnol. 2004, 22, 969−976.

F

DOI: 10.1021/acs.analchem.5b01588 Anal. Chem. XXXX, XXX, XXX−XXX