Peptide-functionalized NaGdF4 nanoparticles for tumor-targeted magnetic resonance imaging and effective therapy

Metallic nanoparticles showed potent efficacy for diagnosis and therapy of cancer, but their clinical applications are limited by their poor tumor-targeting ability. Herein, peptide-functionalized 9 nm NaGdF4 nanoparticles (termed as, NaGdF4@bp-peptide NPs) have been synthesized through the Gd–phosphate coordination reaction of the spherical NaGdF4 nanoparticles with phosphopeptides (sequence: KLAKLAKKLAKLAKG(p-S)GAKRGARSTA, p-S means phosphorylated serine) including a p32 protein binding motif incorporating a cell-penetrating function, and a proapoptotic domain. The NaGdF4@bp-peptide NPs are ready to be efficiently internalized by cancer cells; they show a much higher cytotoxicity in MCF-7 breast cancer cells than the casein phosphopeptide (CPP) modified NaGdF4 nanoparticles (termed as, NaGdF4@CPP NPs). Using mouse-bearing MCF-7 breast cancer as a model system, the in vivo experimental results demonstrate that NaGdF4@bp-peptide NPs have integration of T1-weighted magnetic resonance imaging (MRI) contrast and tumor-targeting functionalities, and are able to suppress tumor growth without causing systemic toxicity.


Introduction
Nanotheranostics (also known as nanomedicines), integrating diagnostic and therapeutic functions into one nanosized material, have many advantages over the conventional molecular medicines, such as prolonged blood circulation time, controlled clearance pathways, and tunable physical properties for multimodal imaging. [1][2][3][4][5][6][7] To date, numerous nanomaterials, including inorganic nanoparticles, organic nanoparticles, nanohydrogels, and organic-inorganic hybrid nanomaterials have been developed as promising nanotheranostics for precise treatment of cancer (as well as many other diseases). Although a few nanotheranostics have been approved by the U. S. Food and Drug Administration (FDA) for drug delivery (e.g., Doxil and Abraxane) or contrast agents (e.g., Feridex IV) for magnetic resonance imaging (MRI), clinical translation efforts of have been hampered by their low tumor delivery efficiency. [8][9][10] For instance, Chan and co-authors found that only 0.59% injected dose of trastuzumab-coated nanoparticles reached the mousebearing SKOV-3 xenogra tumor through intravenous administration. 10 The tumor delivery efficiency of nanomaterials is strongly determined by their own physicochemical properties including size, surface modication, and stimuliresponsiveness to tumor microenvironment. [11][12][13][14][15][16] Therefore, there is a strong desire for the development of new modication strategies for generating nanomaterials with improved diagnostic and therapeutic capabilities through increasing their tumor accumulating amounts.
Because of its low ionizing radiation, magnetic resonance imaging (MRI) enables noninvasive detecting and assessing disease progression at relatively frequent time intervals. [17][18][19] However, it has been clinically found that the sensitivity of MRI is poor, resulting in difficulty in accurate diagnosis of tumor at early stage. In order to overcome detection limitation of MRI, contrast agents (CAs) are usually used for improvement of the signal-to-noise ratios (SNRs) in the imaging process through altering the relaxation time of local water molecules in lesion regions. 20,21 Gadolinium (Gd) chelates (i.e., Gd-DTPA and Gd-DOTA) are currently clinical used CAs, which suffer low tumor accumulation efficiency and short circulation time. Beneting from their unique physicochemical properties, the application of nanoparticles in bioimaging eld has been demonstrated as a very promising strategy for circumventing the inherent defects of traditional small molecular CAs in terms of bioavailability and targeting ability. [21][22][23][24][25][26][27][28][29][30] Versatile nanoparticle-based CAs have been constructed for MRI, computed tomography (CT), photoacoustic imaging (PAI) and uorescence imaging (FI) to enhance the spatial resolution and detection sensitivity of these bioimaging modes. For example, nanoparticles containing Gd such as gadolinium oxide (Gd 2 O 3 ) nanoparticles and sodium tetrauoro gadolinium (NaGdF 4 ) nanoparticles are appropriate CAs of MRI for visualization of tumor details and/or MRI-guided therapy of tumors. 31,32 Peptides are one kind of important biological materials, which normally present structure-dependent functions. Peptides and peptide derivatives have been extensively employed for constructing multifunctional nanotheranostics since they exhibit chemical versatility and enable to specically recognize other biomacromolecules. [33][34][35][36][37][38][39][40][41][42][43][44][45][46][47][48][49][50][51] In particular, conjugation of nanoparticles with tumor-homing peptides and/or therapeutic peptides can generate novel nanotheranostics for highly sensitive tumor imaging and effective tumor-targeted therapy. For example, Ruoslahti and coauthors have been developed a theranostic nanosystem, which consists of iron oxide nanoworms conjugated with a composite peptide with proapoptotic domain (i.e., therapy motif) and cell surface p32 protein binding domain (i.e., tumor-homing motif). 52,53 The in vivo experimental results indicate that the asprepared theranostic nanosystem has excellent homing and penetrating activity in mouse-bearing breast tumor models, and enables to effectively retard tumor growth. Very recently, we also synthesized a kind of peptide mixture-modied NaGdF 4 nanodot with active tumor targeting ability, which can be used as high efficient T 1 -weighted MRI CA for tracking small drug induced orthotopic colorectal tumor (c.a., 195 mm 3 in volume) in mouse. 54 Herein, a tumor-specic multifunctional nanotheranostic composed of NaGdF 4 nanoparticle together with phosphopeptides has been developed for T 1 -weighted MRI guided cancer therapy. Strategically, the p32 protein binding peptide (sequence: AKRGARSTA) and the pro-apoptotic peptide (sequence: KLAKLAKKLAKLAK) were linearly linked together to form a new peptide (sequence: KLAKLAKKLAKLAKG(p-S) GAKRGARSTA, named as bp-peptide) through the phosphorylated tripeptide (-G(p-S)G-: -glycine-phosphorylated serineglycine-). Beneting from the strong coordination reaction between Gd(III) ion and phosphate, the hydrophilic bp-peptide conjugated NaGdF 4 nanoparticles (NaGdF 4 @bp-peptide NPs) can be easily achieved through replacing initial hydrophobic oleate ligand by the bp-peptide molecules due to the phosphopeptides can be selectively enriched by the rare-earth materials through the coordination reactions of rare-earth ions with phosphate moieties in the peptides. 54,55 Both of in vivo and in vitro experimental results demonstrated statistically signicant improvement in tumor-targeted uptake and tumor suppression of NaGdF 4 @bp-peptide NPs over these of passive tumor-targeting the casein phosphopeptide (CPP) modied NaGdF 4 nanoparticles (NaGdF 4 @CPP NPs). The mice had free access to food and water and were raised on a 12 h light/12 h dark cycle at 20 C.

Synthesis of NaGdF 4 nanoparticles
NaGdF 4 nanoparticles were synthesized by literature reported procedure with slight modication. 54 Briey, 1 mL GdCl 3 stocking solution was dried at 100 C. Subsequently, 6 mL OA and 22.5 mL ODE were added and well mixed at 160 C in Ar atmosphere. Aer cooled to 60 C, 15 mL CH 3 OH solution containing 6 mmol NH 4 F and 3.75 mmol NaOH were added dropwise, and vigorously mixed for 12 h. Aer evaporating CH 3 OH completely at 90 C, the temperature of mixture was slowly (10 C per minute) increased to 250 C and maintained for 10 minutes in Ar atmosphere. Aer cooling to room temperature, the as-prepared NaGdF 4 nanoparticles were puri-ed by repeated centrifugation (10 000 rpm, three times) and redispersed in 10 mL cyclohexane.

Cytotoxicity evaluation
Human breast cancer cells (MCF-7) and normal kidney tissue cells (293) were purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences. The MCF-7 cells and 293 cells were co-cultured with various concentrations of NaGdF 4 @bppeptide NPs and NaGdF 4 @CPP NPs in DMEM (containing 10% (v/v) FBS and 100 U mL À1 penicillium-streptomycin) for 24 h, respectively. Then, the cells were washed and the MTT assays were used to detect cell viabilities. The normally cultured cells were used as control groups.

Biocompatibility analysis
Kunming mice with average body weight of 35 g were ordered from Liaoning Changsheng Biotechnology Ltd. (Liaoning, China). The mice were randomly divided into three groups: control group, NaGdF 4 @bp-peptide NPs treated group and NaGdF 4 @CPP NPs treated group. The mice in treated groups were injected intravenously with 100 mL NaGdF 4 @bp-peptide NPs or NaGdF 4 @CPP NPs (10 mg Gd per kg) in PBS through tail vein, respectively, while the mice in control group were only injected intravenously with 100 mL PBS. The bloods were taken for routine blood tests at 24 h post-injection. The body weights of mice were measured every 2 days until 31 days aer injection. The mice were sacriced, and main organs including heart, liver, spleen, lung and kidneys were collected for hematoxylineosin (H&E) staining analysis.

In vivo study
Balb/c nude mice with average body weight of 20 g were purchased from Beijing Vital River Laboratory Animal Technology Ltd. (Beijing, China). The tumor model was constructed by subcutaneous injection of MCF-7 cells (1 Â 10 6 cells in 100 mL PBS). The tumor size (V) was evaluated by the following formula: V ¼ length Â (width) 2 /2; the inhibition rate of tumor growth (IRT) was calculated as follows: (1 À V NPs /V Ctrl ) Â 100% (V NPs : tumor volume of NaGdF 4 @bp-peptide NPs treated mouse; V Ctrl : tumor volume of PBS treated mouse). The MCF-7 tumor-bearing Balb/c nude mice were anesthetized with 100 mL chloral hydrate (10 v/ v%) while the size of tumor reached about 400 mm 3 in volume. Subsequently, the mice were treated with 100 mL NaGdF 4 @bppeptide NPs or NaGdF 4 @CPP NPs (10 mg Gd per kg) in PBS through intravenous injection via the tail vein, respectively. MRI of nude mice were performed at pre-injection (0 h), 1 h, 2 h, 4 h, 8 h and 24 h post-injection by Siemens 3.0 T MRI scanner with the following parameters: TR, 358 ms; TE, 15 ms; eld of view, 120 mm Â 72 mm and slice thickness, 2.0 mm. In addition, three mice were sacriced at 24 h post-injection, and main organs and tumors were collected for ICP-MS measurement.

Treatment efficiency evaluation
9 MCF-7 tumor-bearing nude mice were randomly divided into 3 groups which were treated by 100 mL PBS only (control group), 100 mL PBS containing NaGdF 4 @bp-peptide NPs (10 mg Gd per kg, NaGdF 4 @bp-peptide NPs treated group) and 100 mL PBS containing NaGdF 4 @CPP NPs (10 mg Gd per kg, NaGdF 4 @CPP NPs treated group) through tail vein, respectively. The tumor sites of mice were measured every 2 days until 31 days aer injection.

Synthesis and characterization of nanoparticles
The hydrophobic OA-coated NaGdF 4 nanoparticles (9.0 AE 1.0 nm in diameter) were synthesized by literature reported strategy with slight modication. [54][55][56] In this case, the phosphopeptides (sequence: KLAKLAKKLAKLAKG(p-S) GAKRGARSTA, named as bp-peptide) were employed as a bifunctionalized ligand and phase transfer agent for preparing hydrophilic NaGdF 4 nanoparticles (NaGdF 4 @bppeptide NPs), and through formation of Gd 3+ -phosphate coordination bond in the ligand exchange reaction under mild experimental conditions because phosphopeptides have the ability to react with multivalent cations and form robust metalphosphate coordination bonds (as shown in Fig. 1a). 54,55,57 Strategically, the p32 protein binding peptide (AKRGARSTA) is linearly linked to the pro-apoptotic peptide (KLAKLAKKLA-KLAK) through the phosphorylated tripeptide (glycine-phosphorylated serine-glycine). The p32 protein (HAPB1, gC1qR or C1qbp) is a mitochondrial protein which highly expresses on the cell surfaces of activated endothelial cells and various tumor cells. 58,59 Therefore, the p32 protein binding peptide can improve the tumor accumulation ability of nanoparticles. For comparison, the NaGdF 4 @CPP NPs were also prepared by same strategy. Aer ligand exchange, there are negligible changes on Fig. 1 The schematic representation of transfer and functionalization of OA-coated NaGdF 4 nanoparticles by bp-peptide (a), and TEM and HRTEM of NaGdF 4 nanoparticles (b and e), NaGdF 4 @bp-peptide NPs (c and f) and NaGdF 4 @bp-peptide NPs (d and g), respectively. morphology, size and crystalline nature of NaGdF 4 nanoparticles (as shown in Fig. 1b-g). The successful exchange of OA with phosphopeptides (bp-peptide and/or CPP) was demonstrated FTIR and EDS. Aer conjugation with phosphopeptides, the nitrogen and phosphorus peaks are clearly observed at 0.39 keV and 1.99 keV in the EDS spectra of NaGdF 4 @bp-peptide NPs and NaGdF 4 @CPP NPs (as shown in Fig. S1 in ESI †). The antisymmetric bending mode of PO 4 3À (1083 cm À1 ) and synthetic spectrum band of stretching vibration and bending vibration of PO 4 3À (1657 cm À1 ) are clearly observed in FTIR spectra of NaGdF 4 @bp-peptide NPs and NaGdF 4 @CPP NPs (as shown in Fig. S2 †). The results indicate that the phosphopeptides are successfully immobilized on the NaGdF 4 nanoparticle surface. The NaGdF 4 @bp-peptide NPs exhibit slight positive surface charges (0.63 mV) in H 2 O because the bp-peptide has relatively high isoelectric points (PI). The slightly positive charged surface may enhance the cellular uptake of NaGdF 4 @bp-peptide NPs. The hydrodynamic diameter (HD) and zeta potential of NaGdF 4 @bp-peptide NPs are 269.5 nm and À0.31 mV in culture medium, while the HD and zeta potential of NaGdF 4 @CPP NPs are 110.1 nm and À5.23 mV in culture medium. The phenomenon suggests that the interactions of NaGdF 4 @bp-peptide NPs with components of culture medium are stronger than those of NaGdF 4 @CPP NPs with components of culture medium. However, the longitudinal relaxivity (r 1 ) value (5.8 mM À1 s À1 ) of NaGdF 4 @bp-peptide NPs is lower than that (7.9 mM À1 s À1 ) of NaGdF 4 @CPP NPs (as shown in Fig. 2). The relative low r 1 value of NaGdF 4 @bp-peptide NPs may due to the branch structure and rigidity of bp-peptide which prolongs the exchange rate of the intra-spherical water molecules around the Gd 3+ . 60,61 Interactions of NaGdF 4 @bp-peptide NPs with cells In order to test the cytotoxicity of as-prepared NaGdF 4 @bppeptide NPs, the breast cancer cells (MCF-7) and human normal kidney tissue cells (293) were co-cultured the asprepared NaGdF 4 @bp-peptide NPs. Aer incubated with up to 200 mg mL À1 NaGdF 4 @bp-peptide NPs for 24 h, the viability of MCF-7 cells is less than 60%, while viability of 293 cells is higher than 80% (as shown in Fig. 3a and S3 †). The result suggests that NaGdF 4 @bp-peptide NPs have relatively low cytotoxicity to normal cells and could be used as antitumor agents. In addition, the NaGdF 4 @CPP NPs have low cytotoxicities to both of 293 cells and MCF-7 cells (as shown in Fig. 3a and S3 †). The result indicates that the high cytotoxicity of NaGdF 4 @bppeptide NPs towards MCF-7 cells originate from their ligands. The T 1 -weighted MR signal intensity of nanoparticle stained MCF-7 cell pellet is increased by increasing the concentration of nanoparticles in culture medium (as shown in Fig. 3b). Under same experimental condition, the MR signal intensity of NaGdF 4 @bp-peptide NPs stained MCF-7 cells is much stronger ($2.9 times) that of NaGdF 4 @CPP NPs stained MCF-7 cells. In addition, the cellular internalization amount of NaGdF 4 @bppeptide NPs is higher ($6.7 times) than that of NaGdF 4 @CPP NPs (as shown in Fig. 3c). These results demonstrate that the binding affinity of NaGdF 4 @bp-peptide NPs with MCF-7 cells is much higher than that of NaGdF 4 @CPP NPs with MCF-7 cells.

In vivo toxicity investigation
The healthy Kunming mice were intravenously injected a single dose (10 mg kg À1 of Gd) of NaGdF 4 @bp-peptide NPs or NaGdF 4 @CPP NPs, respectively. The blood routine analysis was used to test acute toxicities of nanoparticles. At 1 day postinjection, blood platelet count of NaGdF 4 @bp-peptide NPs treated mice is higher (2.44 times) than those of NaGdF 4 @CPP NPs treated mice and untreated mice, while other blood routine indicators of NaGdF 4 @bp-peptide NPs treated mice are comparable to those of NaGdF 4 @CPP NPs treated mice and untreated mice (as shown in Table S1 †). The blood platelet count of NaGdF 4 @bp-peptide NPs treated mice is decreased with increasing the post-injection time. There is little difference in blood components among treated groups and control group at 7 day of post-injection. The long-term in vivo toxicities of nanoparticles were evaluated by monitoring the body weight changes of mice, histology analysis of major organs, and blood biochemical assays at 31 day of post-injection. As shown in Fig. S4, † the body weights of mice in all tested groups were increased steadily as the time prolonged. Comparing with the untreated mice, the main organs (e.g., heart, liver, spleen, lung, kidneys) of NaGdF 4 @bp-peptide NPs or NaGdF 4 @CPP NPs treated mice exhibit little abnormalities or lesions (as shown in Fig. 4). For blood biochemical assays, there is negligible difference among untreated mice and NaGdF 4 @bp-peptide NPs (10 mg Gd per kg body weight) treated mice at 31 day of postinjection (as shown in Table S1 †). The results further conrm the good biocompatibility of NaGdF 4 @bp-peptide NPs. The results of in vivo acute and chronic toxicity analysis suggest that the NaGdF 4 @bp-peptide NPs have reasonable biocompatibility.
In vivo tumor-targeting capacity of NaGdF 4 @bp-peptide NPs The MCF-7 tumor-bearing Balb/c nude mice were used to investigate the tumor-targeting capacities of NaGdF 4 @bppeptide NPs and NaGdF 4 @CPP NPs. The nanoparticles were  Fig. 5a and c). The NaGdF 4 @CPP NPs show relatively poor MR contrast enhancement in the tumor site (as shown in Fig. 5b and c). The result of in vivo MRI indicates that NaGdF 4 @bp-peptide NPs have good tumor-targeting capacity. The relative strong MCF-7 tumor-targeting capacity of NaGdF 4 @bp-peptide NPs may due to high binding affinity of bp-peptide with MCF-7 cells. For further conrming tumor-targeting capacity of NaGdF 4 @bp-peptide NPs, the MCF-7 tumor-bearing Balb/c nude mice were sacriced at 1 h or 24 h post-injection, respectively. The main organs and tumors of mice were collected, and the total amounts of Gd element in these tissues were measured by ICP-MS. As shown in Fig. 6, large amounts of Gd element were found in liver, spleen, kidneys and tumor. The Gd amounts in these organs at 1 h post-injection are higher than the Gd amounts in these organs at 24 h post-injection. The result indicates that both of NaGdF 4 @bp-peptide NPs and NaGdF 4 @CPP NPs are mainly accumulated in the liver, spleen, kidneys and tumor, and gradually excreted from body by hepatic and renal clearance pathways. Notably, the Gd amount in tumor of NaGdF 4 @bp-peptide NPs treated mouse is higher (1.82 times at 1 h post-injection and 1.78 times at 24 postinjection) than the Gd amount in tumor of NaGdF 4 @CPP NPs   treated mouse. The result demonstrates that NaGdF 4 @bppeptide NPs have excellent tumor-targeting capacity.

In vivo antitumor efficacy of NaGdF 4 @bp-peptide NPs
The MCF-7 tumor-bearing Balb/c nude mice were divided randomly into three groups (n ¼ 3), control group, NaGdF 4 @bppeptide NPs group, and NaGdF 4 @CPP NPs, which were treated by 100 mL PBS, and single dose (10 mg Gd per kg body weight) of NaGdF 4 @bp-peptide NPs and NaGdF 4 @CPP NPs through tail vein. Aer the different treatments were administered, the tumor sizes were measured with calipers every 2 days. Comparison with the PBS treated and NaGdF 4 @CPP NPs treated mice, the NaGdF 4 @bp-peptide NPs treated mice exhibit a delay in the tumor growth (as shown in Fig. 7 and S5 †). The average tumor volume (1375 AE 885 mm 3 ) of NaGdF 4 @bp-peptide NPs treated mice is much smaller than those of PBS treated (5229 AE 1296 mm 3 ) and NaGdF 4 @CPP NPs treated (5160 AE 1501 mm 3 ) mice. This result suggested that the NaGdF 4 @bp-peptide NPs have good antitumor bioactivity with the IRT as high as 73.7%.

Conclusions
In summary, taking the advantage of strong interaction of phosphonate group with Gd 3+ , a new theranostic nanosystem (NaGdF 4 @bp-peptide NPs) has been constructed consisting of a NaGdF 4 NPs core as the T 1 -weighted MR contrast agent and a peptide as the active-tumor targeting ligand as well as antitumor agent. In vitro and in vivo experiments demonstrate that the as-prepared NaGdF 4 @bp-peptide NPs have reasonable biocompatibility and excellent tumor targeting capacity. Furthermore, NaGdF 4 @bp-peptide NPs exhibit good anticancer efficacy with the IRT as high as 73.7% in subcutaneous MCF-7 breast tumor Balb/c nude mouse models. Owing to the diverse biological functionalities of peptides, this work provides us a facile preparation method to fabricate theranostic nanoparticles which have potential as anticancer agents for molecular imaging guided therapy.

Conflicts of interest
There are no conicts to declare. Fig. 6 Total amounts of Gd element in the main organs and tumors of mice at 1 h (a) and 24 h (b) post-injection of NaGdF 4 @bp-peptide NPs or NaGdF 4 @CPP NPs, respectively. Error bars mean standard deviations (n ¼ 3, **P < 0.01 or ***P < 0.001 from an analysis of variance with Tukey's post-test). Fig. 7 Tumor growth curves of Balb/c nude mice after different intravenous treatments. Inset digital photographs excised tumors, (a) control, (b) NaGdF 4 @CPP NPs and (c) NaGdF 4 @bp-peptide NPs (10 mg Gd per kg body weight), respectively. Error bar mean standard deviations (n ¼ 3, *P < 0.05 from an analysis of variance with Tukey's posttest).