Employing antagonistic C-X-C motif chemokine receptor 4 antagonistic peptide functionalized NaGdF4 nanodots for magnetic resonance imaging-guided biotherapy of breast cancer

C-X-C motif chemokine receptor 4 (CXCR4) is a promising therapeutic target of breast cancer because it is overexpressed on cell surface of all molecular subtypes of breast cancer including triplenegative breast cancer (TNBC). Herein, CXCR4 antagonistic peptide-NaGdF4 nanodot conjugates (termed as anti-CXCR4-NaGdF4 NDs) have been constructed for magnetic resonance imaging (MRI)-guided biotherapy of TNBC through conjugation of the C-X-C Motif Chemokine 12 (CXCL12)-derived cyclic peptide with tryptone coated NaGdF4 nanodots (5 ± 0.5 nm in diameter, termed as Try-NaGdF4 NDs). The as-prepared anti-CXCR4-NaGdF4 NDs exhibits high longitudinal relaxivity (r1) value (21.87 mM−1S−1), reasonable biocompatibility and good tumor accumulation ability. The features of anti-CXCR4-NaGdF4 NDs improve the tumor-MRI sensitivity and facilitate tumor biotherapy after injection in mouse-bearing MDA-MB-231 tumor model in vivo. MRI-guided biotherapy using anti-CXCR4-NaGdF4 NDs enables to suppress 46% tumor growth. In addition, about 47% injection dose of anti-CXCR4-NaGdF4 NDs is found in the mouse urine at 24 h post-injection. These findings demonstrate that anti-CXCR4-NaGdF4 NDs enable to be used as renal clearable nanomedicine for biotherapy and MRI of breast cancer.

Due to its high soft-tissue contrast, the magnetic resonance imaging (MRI) has been recognized as a powerful non-invasive tool for diagnosis of various diseases including cancers.In order to improve diagnosis sensitivity of MRI, the contrast agents are widely employed to enhance the intensity of MR signal.Gd-chelates are a well-known kind of MRI contrast agents with high T 1 -weighted MRI contrast enhancement capability, and have achieved great clinical and commercial success because trivalent gadolinium ion (Gd 3+ ) has seven unpaired electrons with a large magnetic moment [28][29][30][31] .Recently, Gd 3+ -contained nanodots (Gd NDs) with small size (< 10 nm in diameter) have emerged as promising competitors to molecular Gd 3+ compound because Gd NDs can be not only accumulated into tumor through enhanced permeability and retention (EPR) effect, but also excreted from body like Gd-chelates through renal clearance [32][33][34][35][36][37][38][39][40][41][42][43] .In addition, the Gd NDs can be further functionalized for achieving high tumor-targeting ability and/or MRI-guided therapy of tumor through conjugation of molecules with specific functionalities (e.g., phosphorylated peptides and hydrophilic block copolymer) 41,42 .
C-X-C chemokine receptor 4 (CXCR4) is an important member of CXC chemokine receptor family, which is identified as a cell surface biomarker associated with the multiple malignant tumors, such as breast cancer and glioblastoma [44][45][46][47][48] .Previous studies indicate that antagonists against CXCR4 have shown enormous potential as cancer diagnostic and therapeutic agents [49][50][51][52][53] .For instance, gold nanoparticle (GNPs)-anti-CXCR4 antibody conjugates (termed as cGNPs) can enhance the efficiency of TNBC radiotherapy by increasing oxidative stress and DNA damage because the cellular internalization amount of cGNPs can be increased significantly through the interactions of overexpressed CXCR4 on TNBC cell membrane and conjugated anti-CXCR4 antibodies on GNP surface 52 .

Reagents and materials
The CXC ligand 12 (CXCL12)-derived cyclic peptide (sequence motif cyclo[K(1,5-pentanedioic acid)R-(2-Nal)-GY], termed as anti-CXCR4 peptide) was purchased from Shanghai Qiangyao biology.The molecular structure of CXC ligand 12 (CXCL12)-derived cyclic peptide was shown in Fig. S1.The details of reagents used were shown in Supporting Information, which were of analytical grade except specific indication.All of reagents were used without further purification.Milli-Q water (18.2MΩ cm) was used in all experiments.

Instrumentation
The details of instruments used were shown in Supporting Information.

Cell uptake
The MDA-MB-231 cells (1 × 10 4 cells per well) were seeded into 96-well plates with 100 µL RPMI 1640 culture medium supplemented with 10% (wt/v) FBS and 100 U/mL chlorostreptomycin.After the cells were cultured for 24 h, the culture medium was replaced by 100 μL of fresh culture medium containing various concentrations of anti-CXCR4-NaGdF 4 NDs and Try-NaGdF 4 NDs, respectively.After incubated for another 24 h, the NDs contained culture medium was discharged.The NDs stained cells were washed by 100 µL fresh culture medium (3 times), and 100 µL PBS, detached by 100 μL trypsin, counted by cell counter, and centrifuged at 2000 r/min

In vivo measurements
BALB/c nude mice with average bodyweight of 20 g were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd., (Beijing, China).All small animal experiments were in agreement with the guidelines of the Regional Ethics Committee for Animal Experiments established by Changchun Institute of Applied Chemistry Institutional Animal Care and Use (Ref.No. 20240007).The MDA-MB-231 tumor models were achieved by inoculating subcutaneously 5 × 10 6 MDA-MB-231 cells suspended in PBS (100 μL) to the right flanks of BALB/c nude mice.
For in vivo tumor accumulation study, MDA-MB-231 tumor-bearing BALB/c nude mice were anesthetized by using chloral hydrate (10 wt%), pre-injected intravenously with desired amounts (10 mg [Gd] kg −1 body weight) of anti-CXCR4-NaGdF 4 NDs and Try-NaGdF 4 NDs in PBS (100 μL) through tail vein, respectively, and acquired the T 1 -weighted MR images at desired time points after injection by a Siemens 1.5 T MRI scanner (see Supporting Information for details).
For biodistribution analysis, the MDA-MB-231 tumor-bearing BALB/c nude mice were sacrificed at 2 and 24 h post injection of the anti-CXCR4-NaGdF 4 NDs and Try-NaGdF 4 NDs.The tissues (tumor, heart, liver, spleen, lung and kidneys) were collected and digested in aqua regia at 80 °C for 2 h.The amounts of Gd element in the as-obtained liquids were detemined by inductively coupled plasma mass spectrometer (ICP-MS).
For biotherapy, the BALB/c nude mice with tumor sizes of 3-4 mm in diameter were divided into four randomized groups, which were treated by PBS, anti-CXCR4-peptide, Try-NaGdF 4 NDs and anti-CXCR4-NaGdF 4 NDs, respectively.The tumor sizes (tumor length and tumor width) were measured by a caliper for 21 days.The tumor volumes were calculated by (tumor length) × (tumor width) 2 /2.The relative tumor volumes were calculated as V/V 0 (V 0 was the tumor volume when the treatment was initiated).The tumors were collected, and recorded by a digital camera at 21th day post-injection.In addition, the MDA-MB-231 tumor-bearing mice were treated as descripted above, and the Kaplan-Meier survival curves were recorded within 40 days after treatment for evaluation of survival rate.
For toxicology analysis, healthy mice were treated by the anti-CXCR4-NaGdF 4 NDs and Try-NaGdF 4 NDs in PBS (100 μL) at a dose of 10 mg [Gd] kg −1 body weight through intravenous administration, respectively.Mice treated with PBS were used as the control group.The mice were sacrificed at 30th day post-injection, and the tissues including heart, spleen, liver, lung and kidneys were fixed in 10% neutral buffered formalin for the histological analysis.

Synthesis and characterization of anti-CXCR4-NaGdF 4 NDs
The synthetic procedure and biomedical application of anti-CXCR4-NaGdF 4 NDs are shown in Scheme 1.Briefly, the hydrophobic OA-NaGdF 4 NDs were synthesized by reported solvothermal methods 34,41,54 .The hydrophilic Try-NaGdF 4 NDs were easily prepared through mixing hydrophobic OA-NaGdF 4 NDs with tryptone at room temperature under vigorous stirring.Since tryptone contains large amounts of the casein phosphopeptides (CPPs) with motif -Ser(P)-Ser(P)-Ser(P)-Glu-Glu-, the hydrophobic OA molecules on the surface of NaGdF 4 NDs were replaced by the hydrophilic tryptone via formation of robust Gd 3+ -phosphate coordination bonds 34 .Subsequently, the carboxyl groups of tryptone on Try-NaGdF 4 NDs were activated by EDC and sulfo-NHS.Finally, the anti-CXCR4-NaGdF 4 NDs were prepared by the amidation reaction between activated carboxyl group of Try-NaGdF 4 NDs and the amine group of anti-CXCR4 peptide.There are negligible change of the morphology, size and crystalline nature of NaGdF 4 NDs after ligand exchange and anti-CXCR4 functionality (as shown in Fig. 1).The average size of as-prepared NaGdF 4 NDs is 5 ± 0.5 nm in diameter.The Zeta potentials of Try-NaGdF 4 NDs and anti-CXCR4-NaGdF 4 NDs are −5.1 ± 0.6 mV and 5.8 ± 0.8 mV, respectively (as shown in Fig. S2).The result is consistent with the negatively charged nature of phosphopeptide outlayer of Try-NaGdF 4 NDs and the positively charged nature of anti-CXCR4 peptide (PI = 10.5)outlayer of anti-CXCR4-NaGdF 4 Scheme 1.The schematic presentation of the synthsis procedure of anti-CXCR4-NaGdF 4 NDs.
NDs.In addition, the longitudinal relaxivity (r 1 ) value of anti-CXCR4-NaGdF 4 NDs (21.87 mM −1 S −1 ) is much higher than that of Try-NaGdF 4 NDs (10.75 mM −1 S −1 , as shown in Fig. 2).This result indicates that anti-CXCR4 enhances the interactions of H 2 O molecules and NaGdF 4 NDs, which may be due to the strong hydrogen bonding of polar amino acids (lysine, tyrosine and arginine) in the monocyclic peptide and H 2 O (as shown in Fig. S3, the hydrogen bond energies between the side chain of lysine, tyrosine, arginine and H 2 O molecules were 21.16, 20.65, 22.14 kJ/mol, respectively).The phenomenon suggests that anti-CXCR4-NaGdF 4 NDs have strong T1-weighted MRI contrast enhancement capability.

In vitro studies
MDA-MB-231 cell line was selected for evaluating the interaction of anti-CXCR4-NaGdF 4 NDs with cancer cells because the human TNBC is a typical refractory tumor.The MDA-MB-231 cells exhibit less than 70% viability after incubated with 400 ng mL −1 anti-CXCR4-NaGdF 4 NDs for 24 h, while the cells exhibit more than 95% viability after incubated with 400 ng mL −1 Try-NaGdF 4 NDs 24 h (as shown in Fig. 3a  www.nature.com/scientificreports/shown in Fig. 3b).The cytotoxicity and MRI results indicate that anti-CXCR4-NaGdF 4 NDs could be used as efficient agents for T 1 -weighted MRI-guided biotherapy of cancer.

In vivo tumor-targeting capability of anti-CXCR4-NaGdF 4 NDs
The

In vivo biodistribution and biotoxicity of anti-CXCR4-NaGdF 4 NDs
For further studying their biodistribution and clearance pathway, the Try-NaGdF 4 NDs and anti-CXCR4-NaGdF 4 NDs treated mice were sacrificed at 2 and 24 h post-injection, respectively.The amounts of Gd element in tumors, main organs, blood and urine were measured by ICP-MS.As shown in Fig. 5, the amounts of Gd in kidneys are higher than those in other organs during the whole period.More than 47% ID of NDs were found in the urine at 24 h post-injection.In addition, the amounts of Gd in blood of anti-CXCR4-NaGdF 4 NDs treated mice are higher than those in Try-NaGdF 4 NDs treated mice.The result is consistent with that of MRI experiment, which confirms the renal clearance of Try-NaGdF 4 NDs and anti-CXCR4-NaGdF 4 NDs.The efficient renal clearance of anti-CXCR4-NaGdF 4 NDs enables to eliminate potential long-term in vivo toxicity.Furthermore, the healthy BALB/c mice were treated with a single dose of anti-CXCR4-NaGdF 4 NDs, and sacrificed for histology analysis at 30-day post-injection.In comparison with the control group, negligible lesions and/or abnormalities were observed, indicating low in vivo toxicity of anti-CXCR4-NaGdF 4 NDs (as shown in Fig. S7).

Biotherapy of anti-CXCR4-NaGdF 4 NDs
The  reached 100%, which is much higher than those of other groups (PBS only, anti-CXCR4-peptide and Try-NaGdF 4 NDs).The results indicate that the anti-CXCR4-NaGdF 4 NDs have certain biotherapeutic effects, which could significantly prolong the survival time of MDA-MB-231 tumor-bearing mice.

Conclusion
In summary, bifunctional anti-CXCR4-NaGdF 4 NDs have been successfully fabricated for MRI-guided biotherapy of cancer by a two-step procedure through ligand exchange reaction between oleate and CPPs in tryptone, and the amidation reaction between activated carboxyl group of tryptone and amine group of anti-CXCR4.Both in vitro and in vivo experimental results indicate that the as-prepared anti-CXCR4-NaGdF 4 NDs exhibit high MRI enhancing performance, good MDA-MB-231 tumor suppression capacity and low toxicity in vivo.Furthermore, the anti-CXCR4-NaGdF 4 NDs can be efficiently excreted from body through renal clearance.Therefore, the anti-CXCR4-NaGdF 4 NDs could be employed as a targeting nanomedicine for detection and biotherapy of CXCR4-overexpression tumor.However, the biotherapeutic mechanism of anti-CXCR4-NaGdF 4 NDs has not been clearly addressed, which should be clarified in future.