Article pubs.acs.org/molecularpharmaceutics
Renal Clearable Peptide Functionalized NaGdF4 Nanodots for HighEfficiency Tracking Orthotopic Colorectal Tumor in Mouse Hongda Chen,†,§ Xiaodong Li,‡ Fuyao Liu,† Huimao Zhang,*,‡ and Zhenxin Wang*,† †
State Key Laboratory of Electroanalytical Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun 130022, P. R. China ‡ Department of Radiology, The First Hospital of Jilin University, Changchun 130021, P. R. China § University of Chinese Academy of Sciences, Beijing 100049, P. R. China S Supporting Information *
ABSTRACT: The effective delivery of bioimaging probes to a selected cancerous tissue has extensive significance for biological studies and clinical investigations. Herein, the peptide functionalized NaGdF4 nanodots (termed as, pPeptide-NaGdF4 nanodots) have been prepared for highly efficient magnetic resonance imaging (MRI) of tumor by formation of Gd-phosphonate coordinate bonds among hydrophobic NaGdF4 nanodots (4.2 nm in diameter) with mixed phosphorylated peptide ligands including a tumor targeting phosphopeptide and a cell penetrating phosphopeptide. The tumor targeting pPeptide-NaGdF4 nanodots have paramagnetic property with ultrasmall hydrodynamic diameter (HD, c.a., 7.3 nm) which greatly improves their MRI contrast ability of tumor and facilitates renal clearance. In detail, the capability of the pPeptide-NaGdF4 nanodots as high efficient contrast agent for in vivo MRI is evaluated successfully through tracking small drug induced orthotopic colorectal tumor (c.a., 195 mm3 in volume) in mouse. KEYWORDS: NaGdF4 nanodots, magnetic resonance imaging, tumor targeting peptide, cell penetrating peptide, orthotopic colorectal tumor, renal clearance
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fluoride nanoparticles have been extensively employed as T1weighted MRI contrast agents in the preclinical studies.18−27 In particular, Gd-based inorganic nanoparticles with small hydrodynamic diameter (HD) can steadily be eliminated by the kidneys which circumvent the likelihood of toxicity due to long residence time of nanoparticles.28−32 In order to improve their colloidal stability in biological medium, the ultrasmall gadolinium-based nanoparticles (also known as gadoliniumbased nanodots) are normally capped by hydrophilic polymers and/or encapsulated by amphiphilic polymers.31,32 For generating high in vivo targeting ability, the polymer stabilizer gadolinium-based nanodots are usually further modified with specific biomolecules. Unfortunately, the biomolecule conjugation leads to dramatically enlarge the HD of nanoparticles, which reduces removal efficiency of nanoparticles by kidney. As a consequence, it is necessary to develop a simple strategy to prepare functionalized gadolinium-based nanodots with small HD. To date, peptides with short amino acid sequences have attracted great attention since they have a diverse range of
INTRODUCTION As one of the most powerful noninvasive diagnostic molecular imaging methods, magnetic resonance imaging (MRI) can produce valuable medical images with functional information and submillimeter resolution anatomic details based on softtissue contrast.1−4 Compared with other bioimaging techniques, the major challenge for MRI is sensitivity.5,6 For enhancing the role of MRI in biomedical investigations, it is necessary to improve the sensitivity of involved contrast agents.7,8 Because of their inherent advantages (e.g., strong signal output and integration of multiple diverse functions in a single complex), nanomaterials have demonstrated extraordinary promise as a new generation of high performance MRI contrast agents for accurately diagnosing disease in early stage and/or monitoring treatment effect in a timely manner.9−15 Up to now, there are six clinically approved MRI contrast agents in the worldwide, four of them are gadolinium chelates. However, the low relaxivity of clinical Gd-chelates (c.a., 4 mM−1 s−1 at 3 T) strongly limits the application in sensitive detection. The T1weighted MRI contrast abilities of Gd-chelates can be improved significantly by confining these agents inside nanostructures including silicon nanoparticles with nanoporous structure and cross-linked hyaluronic acid nanoparticles.16,17 Due to the paramagnetic property induced by seven unpaired electrons of gadolinium ion (Gd3+), gadolinium oxide, and/or gadolinium © 2017 American Chemical Society
Received: Revised: Accepted: Published: 3134
May 2, 2017 July 13, 2017 July 20, 2017 July 20, 2017 DOI: 10.1021/acs.molpharmaceut.7b00361 Mol. Pharmaceutics 2017, 14, 3134−3141
Article
Molecular Pharmaceutics biological functionalities, including participation in signaling pathways, displaying membrane activity as drug carriers or lytic agents, and forming complex molecular assemblies with the lipid membrane.33−38 Based on their different functionalities, the peptides can be sorted into different families, such as cellpenetrating peptides (CPPs), tumor-specific targeting peptides, and therapeutic peptides. Conjugation of peptide with nanomaterial can generate novel theranostic nanosystems that are highly effective tumor-homing.37−39 For instance, Morshed and coauthors found that cell-penetrating peptide modified gold nanoparticle loading doxorubicin led to improved survival time of mouse bearing an xenograft intracranial breast cancer (MDA-MB-231-Br).39 In our previous study, we demonstrate that casein phosphopeptides, the major component of tryptone can easily conjugate with NaGdF4 nanodot through the formation of Gd-phosphate coordination bond.32 In vitro and in vivo experimental results show that the tryptone modified NaGdF4 nanodots have small HD, excellent colloidal and chemical stability, reasonable biocompatibility, strong T1weighted MRI enhancing capacity, and passive tumor targeting ability. The experimental results inspire us to fabricate phosphopeptide functionalized gadolinium nanoparticle-based theranostic nanosystems with highly active tumor targeting ability. Herein, we report a simple approach to synthesize active tumor targeting NaGdF4 nanodots (termed as, pPeptideNaGdF4 nanodots) through peptide-mediated phase transfer. In this case, the hydrophobic oleate ligand of NaGdF4 nanodots are replaced by the mixture of two hydrophilic phosphopeptides, including phosphorylated cytosol-localizing internalization peptide 6 (pCLIP6) and phosphorylated retro-inverso tumor-affinity peptide (pD-SP5), through the Gd-phosphate coordination reaction. The small animal experiments in drug induced orthotopic colorectal tumor mouse model demonstrate that the as-prepared pPeptide-NaGdF4 nanodots exhibit low toxicity, active tumor targeting ability, strong MR enhancement, and efficient renal clearable property.
purification. Ultrapure water (18.2 MΩ cm) was prepared by Milli-Q system, and used in all experiments. TECNAI G2 high-resolution transmission electron microscope (TEM, FEI Co., USA) and Zetasizer Nano ZS (Malvern Instruments Ltd., UK) were employed to record TEM micrographs, HD and Zeta (ζ) Potential of the as-prepared nanodots, respectively. Siemens Prisma 3.0 T MR scanner (Erlangen, Germany) was employed to study the relaxation times of samples. ELAN 9000/DRC inductively coupled plasma mass spectrometry (ICP-MS) system (PerkinElmer, USA) was used for analyzing the elements in samples. Vertex 70 Fourier transform infrared (FTIR) spectrometer (Bruker, Germany) was used to record infrared spectra. VG ESCALAB MKII X-ray photoelectron spectrometer (VG Scientific Ltd., UK) spectroscopy (XPS) was used to measure the XPS spectra of as-prepared materials. Energy-dispersive X-ray spectra (EDS) were inspected on an energy dispersive spectroscopy (FEI Co., USA). Thermogravimetric analysis (TGA) was inspected on a TGA-2 analyzer (PerkinElmer, USA) with temperature ranging from room temperature to 800 °C at a rate of 10 °C min−1. Philips Achieva 3.0 T MRI scanner (Magnetom Avanto, Philips, Netherlands) was employed to acquire T1-weighted MR images. Synthesis of pPeptide-NaGdF4 Nanodots. The oleatecapped NaGdF4 nanodots were synthesized and purified according to a previously reported procedure with slight modifications (see Supporting Information for details).31,32 Oleate-capped NaGdF4 nanodots solution (10 mL, 1.0 mg mL−1 in cyclohexane) was mixed with 10 mL water containing two phosphopeptides (10 mg pCLIP6 and 10 mg pD-SP5), and the two-phase mixture was agitated vigorously at 25 °C for 12 h. Then, aqueous phase was collected. The phosphopeptidefunctionalized NaGdF4 nanodots were purified by repeated centrifugation (10000 rpm, 10 min for three times) at 4 °C. The purified phosphopeptide-functionalized NaGdF4 nanodots (termed as pPeptided-NaGdF4 nanodots) were redispersed in water and stored at 4 °C. The pCLIP6, pD-SP5, or tryptone functionalized NaGdF4 nanodots (named as pCLIP6-NaGdF4 nanodots, pD-SP5-NaGdF4 nanodots, or tryptone-NaGdF4 nanodots) were prepared under same experimental conditions except using 1 mg mL−1 pCLIP6, pD-SP5, or tryptone aqueous solution, respectively. Decomposition Evaluation of Gd3+ from pPeptideNaGdF4 Nanodots. Ten milligrams (Gd content) pPeptideNaGdF4 nanodots in 10 mL 0.9 wt %/wt NaCl was sealed in a dialysis bag (MWcutoff = 8000) and submerged into PBS (50 mL, pH 7.4, containing 137 mM NaCl, 1.5 mM KH2PO4 and 8.1 mM Na2HPO4) plus 10% (w/w) FBS under mild stirring for 14 days. The total amount of Gd3+ in dialysis solution was determined by ICP-MS. Cytotoxicity and Cellular Internalization of pPeptided-NaGdF4 Nanodots. SW620 cells (106 cells mL−1) were cocultured with different amounts of pPeptide-NaGdF4 nanodots in 2 mL fresh L-15 under a humidified 5% CO2 atmosphere at 37 °C for 24 h, respectively. Then, pPeptideNaGdF4 nanodot-stained cells were washed with L-15 (2 mL, three times) and PBS (2 mL, pH 7.4, three times), respectively. For studying the cytotoxicity of pPeptided-NaGdF4 nanodots, the viabilities of pPeptide-NaGdF4 nanodot-stained SW620 cells and untreated SW620 cells (control sample) were determined by MTT assay. The relative cell viabilities (%) were calculated using the absorbance at 490 nm, assigning the relative viability of untreated cells as 100%. For evaluating their
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EXPERIMENTAL SECTION Materials and Characterizations. Gd2O3 (99.99% (w/ w)) were purchased from Alfa Aesar (Ward Hill, USA). Fetal bovine serum (FBS) and trypsin-EDTA cell detaching kit were purchased from Gibco Co. (New York, USA). 3-(4,5-Dimethyl2-thiazolyl)-2, 5-diphenyl-2-H-tetrazolium bromide (MTT), and Leibovitz’s L-15 culture medium (L-15) were purchased from Beijing Dingguo Biotechnology Ltd. (Beijing, China). Oleic acid (OA, 90% (v/v)) and 1-octadecene (ODE, 90% (v/ v)) were purchased from Sigma-Aldrich Co. (St. Louis, USA). Phosphorylated cytosol-localizing internalization peptide 6 (pCLIP6, sequence: KVRVRVRV(dP)P(p-T)RVRERVK, where p-T is a phosphoseryl threonine residue, and dP is Dproline, respectively. MW: 2.3 kDa) and phosphorylated retroinverso pD-SP5 peptide (sequence: D(PRPSPKMGV(p-S)VS), where p-S is a phosphoseryl serine residue, and others are retroinverso amino acids. MW: 1.4 kDa) were purchased from ChinaPeptides Co. Ltd. (Shanghai, China). Human colon carcinoma cell line, SW620 was purchased from Shanghai Cell Bank, CAS (Shanghai, China). BALB/C mice (c.a., 20 g body weight) were provided by Beijing Vital River Laboratory Animal Technology Co. Ltd., (Beijing, China). Other reagents used were of analytical grade (Beijing Chemical Reagents Co. Ltd., Beijing, China). All reagents were used without further 3135
DOI: 10.1021/acs.molpharmaceut.7b00361 Mol. Pharmaceutics 2017, 14, 3134−3141
Molecular Pharmaceutics
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cellular internalization efficiency, SW620 cells (106 cells mL−1) were incubated with 100 μg mL−1 pPeptided-NaGdF4 nanodots, pCLIP6-NaGdF4 nanodots, pD-SP5-NaGdF4 nanodots, or tryptone-NaGdF4 nanodots in 2 mL fresh L-15, respectively. Then, nanodot-stained cells were washed with fresh L-15 (2 mL, three times) and pH 7.4 PBS (2 mL, three times), detached from the culture plates by commercial trypsin-EDTA cell detaching kit, and centrifuged at 1000 rpm for 5 min, respectively. The amounts of Gd element in the nanodotstained cells were measured by ICP-MS. T1-Weighted MR Imaging. For in vitro T1-weighted MRI, 2 mL SW620 cells (106 cells mL−1 in fresh L-15) were cocultured with various amounts of pPeptide-NaGdF4 nanodots for 4 h, respectively. Subsequently, the pPeptide-NaGdF4 nanodot-stained cells were washed, detached, and centrifuged as previously described. The cell pellets were collected and immobilized by 1% agarose. MRI of nanodots-stained cells were performed using a Philips Achieva 3.0 T MRI scanner. The imaging parameters are as following: 15 ms (TE), 358 ms (TR), 0.5 mm (slice thickness), and 144 mm × 144 mm (field of view), respectively. All animal procedures were approved by and carried out under the guidelines of the Local Ethics Committee for Institutional Animal Care and Use of Jilin University. To model drug induced orthotopic colorectal tumors in mice, 5-week-old mice with average weight of 20 g were fed with 2% dextran sulfate solutions for daily drinking and intraperitoneally injected azoxymethane at the dose of 5 mg kg−1 body weight once per week for 7 weeks. The mice bearing drug induced orthotopic colorectal tumors were anesthetized by isoflurane. Desired amounts of pPeptides-NaGdF4 nanodots or tryptone-NaGdF4 nanodots in 100 μL NaCl solutions (0.9 wt%) were injected intravenously into the mice through tail vein. After injection, T1-weighted MR images were acquired at 1, 2, and 24 h by Philips Achieva 3.0 T MRI scanner with previously described imaging parameters. After MR imaging, the mice were sacrificed for pathological analysis of rectums. For biodistribution analysis, 100 μL of peptides-NaGdF4 nanodots (10 mg Gd kg−1 body) in 0.9 wt% NaCl solutions were injected intravenously into the mice bearing drug induced orthotopic colorectal tumors. The mice were sacrificed at 1, 2, and 24 h postinjection, respectively. The main organs, including heart, lung, spleen, liver, kidneys, and rectum, were surgically resected. The organs were pretreated by aqua regia at 80 °C for 2 h, respectively, and the obtained liquids were subjected to determine the amount of Gd by ICP-MS analysis. As a control, the amounts of Gd in the organs of health mice were also determined at 24 h postinjection. In Vivo Toxicology Analysis. Healthy mice were administrated intravenously with single dose of pPeptideNaGdF4 nanodots (10 mg Gd kg−1 body, 100 μL) in 0.9 wt% NaCl solutions by tail vein. The mice were sacrificed at 30 days postinjection. The tissues (heart, lung, spleen, liver, and kidneys) were collected and fixed in 10% paraformaldehyde in PBS. The histological slices were investigated and imaged by an optical microscope equipped with a CCD camera. For hematology analysis and blood biochemistry assay, the blood samples were obtained from pPeptide-NaGdF4 nanodot-treated mice at 30 days postinjection and normally raised mice, respectively.
Article
RESULTS AND DISCUSSION Synthesis and Characterization of pPeptide-NaGdF4. The oleate-capped NaGdF4 nanodots (4.2 ± 0.3 nm in diameter) were synthesized and purified by slight modification of a literature procedure.31,32 In this study, two kinds of phosphopeptides, pCLIP6 and pD-SP5, were arbitrarily selected to prepare the pPeptide-NaGdF4 nanodots. The pCLIP6 is derived from cytosol-localizing internalization peptide 6 (CLIP6) which can improve the cellular internalization ability of pPeptide-NaGdF4 nanodots.40 The pD-SP5 is phosphorylated D-SP5 peptide which has high affinity for human tumor cells.41 It was found that the cellular internalization amounts of pPeptide-NaGdF4 nanodots exhibited negligible change by changing the ratio of pD-SP5 with pCLIP6 in the range of 1 to 25 in the peptide mixture while keeping the total amount of peptides as constant. Therefore, the mixture of pCLIP6 and pD-SP5 (1:1 of mass ratio) was used to exchange original oleate ligand on the surface of hydrophobic NaGdF4 nanodots and generate hydrophilic pPeptide-NaGdF4 nanodots through the formation of Gd3+phosphate coordination bonds under mild conditions.42,43 As shown in Figure 1, after ligand exchange, the morphology, size,
Figure 1. TEM micrographs of hydrophobic oleate-NaGdF4 nanodots (a and b) and hydrophilic pPeptide-NaGdF4 nanodots (c and d).
and crystalline nature of NaGdF4 nanodots are essentially remained. The successful exchange of oleate with phosphopeptides was confirmed by XPS, FTIR, EDS, and TGA. After incubation with phosphopeptides, the phosphorus peaks are clearly observed in the XPS (133 eV, P 2p) and EDS (1.99 keV) spectra of nanodots (as shown in Figures S1 and S2).44 As shown in Figure S3, two IR bands at 544 and 1083 cm−1 are observed in FTIR spectrum of pPeptide-NaGdF4 nanodots, which are corresponded to the symmetric stretching vibration of PO43− and antisymmetric bending mode of PO43−, respectively.45 The results demonstrate that the phosphopeptides are conjugated successfully on the surface of NaGdF4 nanodot. Furthermore, TGA experimental result shows that the total weight loss of pPeptide-NaGdF4 nanodots and tryptoneNaGdF4 nanodots are 14 and 26%, respectively (as shown in 3136
DOI: 10.1021/acs.molpharmaceut.7b00361 Mol. Pharmaceutics 2017, 14, 3134−3141
Article
Molecular Pharmaceutics Figure S4). Tryptone is a peptide mixture containing 10−20% casein phosphopeptides with the molecular weights (MWs) in the range of 2.0 kDa to 3.2 kDa, which are derived from the casein digestion.46 The TGA result provides an additional evidence for that pPeptide-NaGdF4 nanodots are coated by the phosphopeptide mixture with high surface coverage. Combination of diversity of peptides and high peptide surface coverage of NaGdF4 nanodots, the pPeptide-NaGdF4 nanodots could have good loading capability and opportunity to carry therapeutic compounds through peptide-based acceptorreceptor interactions and/or the covalent reactions (e.g., amidation reaction of carboxylic acid with amine). The zeta potential and HD of pPeptide-NaGdF4 nanodots are 8.24 ± 0.87 mV and 7.3 ± 0.1 nm, respectively (as shown in Table S1). The experimental result is consistent with the composition of nanoparticles of pPeptide-NaGdF4 nanodot which contains individual solid NaGdF 4 nanodot core and a flexible phosphopeptide outlayer. The pPeptide-NaGdF4 nanodots have positive surface charges because both of CLIP6 peptide and D-SP5 peptide have high isoelectric points (PI). The slightly positive surface charge is favorable for the cellular internalization of pPeptide-NaGdF4 nanodots. The pPeptidesNaGdF4 nanodots exhibit good monodispersity in PBS of different pH values (as shown in Figure S5). Negligible amount of Gd3+ ions (
