Renal-clearable hyaluronic acid functionalized NaGdF4 nanodots with enhanced tumor accumulation

Integration of high tumor-targeting capacity, controlling in vivo transport and low normal tissue retention into one engineered nanoparticle is a critical issue for future clinically translatable anti-cancer nanomedicines. Herein, hyaluronic acid functionalized 3.8 nm NaGdF4 nanodots (named NaGdF4 ND@HAs) have been prepared through conjugation of tryptone capped NaGdF4 nanodots (NaGdF4 ND@tryptone) with hyaluronic acid (HA, a naturally occurring glycosaminoglycan), which can recognize the overexpressed CD44 on cancer cell membranes. The as-prepared NaGdF4 ND@HAs have good paramagnetic properties (longitudinal relaxivity (r1) = 7.57 × 10−3 M S−1) and low cytotoxicity. The in vivo experimental results demonstrate that the NaGdF4 ND@HAs can not only efficiently accumulate in mouse-bearing MDA-MB-231 tumors (ca. 5.3% injection dosage (ID) g−1 at 2 h post-injection), but also have an excellent renal clearance efficiency (ca. 75% injection dosage (ID) at 24 h post-injection). The as-prepared NaGdF4 ND@HAs have good paramagnetic properties with enhanced tumor-targeting capacity, which provides a useful strategy for the preparation of renal clearable magnetic resonance imaging (MRI) contrast agents for tumors.


Introduction
As one of the prospective directions of contemporary medical sciences, nanomaterial-based theranostics (known as nanomedicines) have been intensively studied because of their potential applications in various domains including diagnosis of and therapies for diseases (e.g., tumor), tracking the location of biomarkers/signal molecules in vivo, and evaluating their therapeutic effect. [1][2][3][4][5][6][7][8][9][10] To date, more than one hundred nanomedicines have been approved by the Food and Drug Administration (FDA) in the USA, or are in the FDA clinical trial stage. 4 However, it is a great challenge to translate nanomedicines from pre-clinical proof-of-concept to clinical applications because there is an increasing consideration of the biosafety of nanomedicines. [11][12][13][14][15][16][17][18] Currently, many promising nanomedicines normally eliminated through the hepatobiliary route are constructed from relatively large sized (>10 nm in diameter) and nonbiodegradable inorganic nanoparticles. [16][17][18][19] However, the interactions of nanomedicines with mononuclear phagocyte system (MPS) cells (e.g., Kupffer cells) in spleen and liver signicantly increase their retention time in vivo (even more than months), which causes potential toxicity. [11][12][13][14][15][16][17][18][19] The biosafety concern can be addressed by the development of nanomedicines that undergo renal clearance. [20][21][22] During the last few years, several ultra-small sized inorganic nanoparticles (<5.5 nm) have been employed to generate renal clearable nanomedicines for the diagnoses and therapies of various diseases including cancers. [23][24][25][26][27][28][29][30][31][32][33] For instance, Zheng's group has been developed a series of gold nanoparticles/nanoclusters for delivering anticancer drugs, 30 evaluating kidney function, 31 and imaging tumors. 32,33 As a powerful tool, magnetic resonance imaging (MRI) has been extensively applied for non-invasive diagnoses of various diseases through producing excellent so-tissue contrast. Compared with other modalities of medical imaging, the sensitivity of MRI is poor. Because trivalent gadolinium ion (Gd 3+ ) has seven unpaired electrons with a large magnetic moment, Gd-chelates and Gd nanoparticles have been employed as contrast agents for enhancing the sensitivity of T 1weighted MRI. [34][35][36][37][38] Among Gd-based T 1 -weighted MRI contrast agents, ultrasmall Gd nanoparticles (also known as Gd nanodots (Gd NDs)) not only have high contrast enhancement capability, but also can be eliminated from body through renal clearance. [39][40][41][42][43][44][45][46][47] The Gd NDs are normally coated hydrophilic or amphiphilic ligands for improving their colloidal stability and biocompatibility, then further modied with specic biomolecules for generating high tumor-targeting ability. As a major component of extracellular matrices, hyaluronic acid (hyaluronan, HA) has high binding affinity with cell surface receptor, CD44. [48][49][50] Therefore, HA can be used as an active tumortargeting ligand for constructing drug delivery systems since tumor cells normally express high level of CD44. [50][51][52][53][54] For example, Parayath and coauthors have fabricated hyaluronic acid-poly(ethylenimine) (HA-PEI)-based nanoparticles encapsulating miR-125b for anticancer immunotherapy through the interactions of HA with CD44. 53 In addition, Guo et al. have found that Gd 3+ -labeled peptide dendron-HA conjugate-based hybrid (dendronized-HA-DOTA-Gd) has better biocompatibility and higher accumulation in tumors than those of Gd-DTPA. 51 The results suggest that HA might be employed as an active tumor-targeting ligand for generating renal clearable Gd NDs.
In this study, a highly renal-clearable HA functionalized NaGdF 4 nanodots (NaGdF 4 ND@HAs) were synthesized by a two-step reaction, and evaluated as an active tumor-targeting MRI contrast agent. The tryptone was employed as phase transfer agent for transferring hydrophobic oleic acid coated NaGdF 4 nanodots (NaGdF 4 ND@OAs, 3.8 nm in diameter) through the Gd-phosphate coordination reaction. HA was then conjugated with the tryptone coated NaGdF 4 NDs (NaGdF 4 ND@tryptone) by the use of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) as coupling agent. Both in vitro cellular studies and in vivo small animal experiments of mouse-bearing MDA-MB-231 breast cancer model demonstrated that NaGdF 4 ND@HAs exhibit low toxicity and can be used for specic detection of tumors with abundant HA receptor. Characterizations TEM micrographs were recorded by TECNAI G2 high-resolution TEM (FEI Co., USA). Dynamic light scattering and zeta potential of the as-prepared samples were carried out on a Zetasizer Nano ZS (Malvern Instruments Ltd, UK). The analysis of elements was conducted with an ELAN 9000/DRC ICP-MS system (Perki-nElmer, USA). The relaxation times of the samples were carried out on a Siemens Prisma 3.0 T MR scanner (Erlangen, Germany). Energy-dispersive X-ray spectra (EDS) were inspected on an energy dispersive spectroscopy (FEI Co., USA). X-ray diffraction analysis were carried out on a D8 ADVANCE diffractometer (Bruker Co., Germany) using Cu Ka (0.15406 nm) radiation. The infrared spectra were conducted with a Vertex 70 Fourier transform infrared (FTIR) spectrometer (Bruker, Germany). XPS measurements were conducted with a VG ESCALAB MKII X-ray photoelectron spectrometer (VG Scientic Ltd, UK) spectroscopy (XPS). Siemens Prisma 3.0 T MR scanner (Erlangen, Germany) was employed to acquire T 1 -weighted MR images.

In vivo MRI study
Female Balb/c nude mice and Balb/c mice with average body weight of 20 g were purchased from Liaoning Changsheng Biotechnology Ltd (Liaoning, China). The mice having free access to food and water were kept for 12 h in light/12 h in dawn daily at 20 C. All animal procedures were approved by the Local Ethics Committee for Institutional Animal Care and Use of Jilin University. MDA-MB-231 cells (1 Â 10 6 cells in 100 mL PBS) were subcutaneously injected into the hind ank of female Balb/c nude mouse. The volume of tumor (V) was evaluated by the following formula: V ¼ length Â (width) 2 /2. When V reached about 60 mm 3 , the MDA-MB-231 tumor-bearing nude mice were injected intravenously with 100 mL 0.9 wt% NaCl solution containing 2 mg mL À1 (Gd 3+ content) NaGdF 4 ND@tryptone or NaGdF 4 ND@HAs through tail veins. The in vivo MR images of tumors were taken at 0, 0.5, 2, 4, 8, 12, and 24 h post-injection by Siemens 3.0 T MRI scanner with the following scanning parameters: 1.2 mm slice thickness, 3000 ms TR, 9.1 ms TE and 120 mm Â 72 mm eld of view. In addition, the mice were sacriced at 2 h post-injection, and the main organs as well as tumors were collected for ICP-MS measurement.

Biocompatibility analysis
The healthy female Balb/c mice were randomly divided into 2 groups: control group and NaGdF 4 ND@HAs treated group. The mice in treated group were injected intravenously with 100 mL 0.9 wt% NaCl solution containing 10 mg kg À1 (Gd 3+ content) NaGdF 4 ND@HAs, respectively. The mice in control group were only injected intravenously with 100 mL 0.9 wt% NaCl solution respectively. The body weights of mice were measured every 2 days until 30 days aer injection. The mice were sacriced at the 1 st and the 30 th day post-injection, and main organs including heart, liver, spleen, lung and kidneys were xed in 4% (w/v) paraformaldehyde solution, embedded in paraffin, sectioned, and nally stained with hematoxylin-eosin (H&E). Meanwhile, the tumors were xed in 4% (w/v) paraformaldehyde solution, embedded in paraffin, sectioned, and nally stained by hematoxylin-eosin (H&E) and anti-CD44v6 immunohistochemistry. The blood samples of mice were collected at the 1 st and the 30 th day post-injection, also analysed by the blood routine assay.

Synthesis and characterization of NaGdF 4 ND@HAs
The synthetic route and application of NaGdF 4 ND@HAs is shown in Scheme 1. The hydrophobic NaGdF 4 ND@OAs (3.8 AE 0.4 nm in diameter) were prepared by previously reported procedure with a slight modication (as shown in Fig. 1a). 40,43,47 As the digestion product of casein, tryptone contains ca. 10-20% casein phosphopeptide (CPP) with the sequence -Ser(P)-Ser(P)-Ser(P)-Glu-Glu-, which can form robust Gd 3+ -phosphate coordination bonds under mild conditions through the reaction of phosphoseryl serine residue (Ser(P)) and trivalent Gd ions. 43,47 Aer mixing NaGdF 4 ND@OAs with tryptone, hydrophilic NaGdF 4 ND@tryptone were generated through replacing the original OA ligand of NaGdF 4 ND@OAs by tryptone. Subsequently, HA was activated by EDC and sulfo-NHS, also conjugated on NaGdF 4 ND@tryptone surface through the amidation reaction between carboxy group of HA and amine group of tryptone. HA and tryptone, which have been extensively used for producing drugs and health care products, are raw materials approved by the US Food and Drug Administration (FDA). Therefore, the toxicity of NaGdF 4 ND can be reduced by HA and tryptone coating. Aer ligand exchange and HA functionalization, the morphology, size and crystalline nature of NaGdF 4 NDs exhibit negligible changes (as shown in Fig. 1b and c). The hydrodynamic diameter and zeta potential of NaGdF 4 ND@tryptone were 11.99 nm and À5.86 mV, respectively. The result was consistent with the structure of NaGdF 4 ND@tryptone which contains individual solid NaGdF 4 ND core and a exible phosphopeptide outlayer. The phenomenon Scheme 1 Schematic diagram of NaGdF4 ND@HAs synthesis, and the application in MRI of tumor through recognizing the overexpressed CD44 on cancer cell membrane.
suggested that NaGdF 4 ND@tryptone exhibits good monodispersity and negative surface charge. Because HA is negatively charged biomacromolecule, the hydrodynamic diameter of NaGdF 4 ND@HAs was increased to 29.05 nm, while the zeta potential of NaGdF 4 ND@HAs was deceased to À12.16 mV. The negative surface charge helps to reduce the nonspecic interactions of NaGdF 4 NDs with cells. The successful preparation of NaGdF 4 ND@tryptone and NaGdF 4 ND@HAs were also investigated by XPS, EDS and FTIR. Aer incubation with tryptone, the phosphorus and nitrogen peaks were clearly observed in the XPS (P 2p (133 eV) and N 1s (400 eV)) and EDS (P (2.01 keV) and N (0.39 keV)) spectra of NaGdF 4 NDs (as shown in Fig. S1 and S2 †). 43,47,55 In addition, the XPS spectrum of NaGdF 4 ND@HAs exhibited relatively high intensity of C 1s (284 eV). Compared to NaGdF 4 ND@OAs, two additional IR bands at 683 cm À1 and 1080 cm À1 are observed in FTIR spectrum of NaGdF 4 ND@tryptone (as shown in Fig. S3 †), which are corresponded to the out-of-plane bending vibration of C-H bond on benzene ring and antisymmetric bending mode of PO 4 3À , respectively. 56 A new IR band at 1010 cm À1 is observed in FTIR spectrum of NaGdF 4 ND@HAs (as shown in Fig. S3 †), which is corresponded to stretching vibration of C-O band of primary alcohol of HA. 57 As shown in Fig. 2, the longitudinal relaxivity (r 1 ) value (7.57 mM À1 S À1 ) of NaGdF 4 ND@HAs is higher than those of NaGdF 4 ND@tryptone (6.03 mM À1 S À1 ) as well as commercial Gd 3+ chelates (e.g., (4.3 mM À1 S À1 )). The high r 1 value of NaGdF 4 ND@HAs may stem from the strong polarity of HA molecule which may cause a spatial agglomeration of water around the Gd 3+ , leading to boost the relaxivity to high value. The result indicates that NaGdF 4 ND@HAs can be used as efficient T 1 -weighted MRI contrast agent.

The interactions of NaGdF 4 ND with living cells
Human triple-negative breast carcinoma MDA-MB-231 cell line was selected as a typical model because it expresses high level of CD44. 58 As shown in Fig. S4, † the MDA-MB-231, MCF-7 and 293 cells exhibit higher than 90% viability aer incubated with up to 200 mg mL À1 NaGdF 4 ND@HAs or NaGdF 4 ND@tryptone for 24 h. The result indicates that both of NaGdF 4 ND@HAs and NaGdF 4 ND@tryptone have low cytotoxicity. The T 1 -weighted MR signal intensity of NaGdF 4 ND@HAs stained MDA-MB-231 cells was stronger than which of NaGdF 4 ND@tryptone stained MDA-MB-231 cells or NaGdF 4 ND@HAs stained MCF-7 (as shown in Fig. 3a-c). The T 1 -weighted MR signal intensity  This journal is © The Royal Society of Chemistry 2020 RSC Adv., 2020, 10, 13872-13878 | 13875 (Fig. 3, group 2) of NaGdF4 ND@HAs stained HA treated MDA-MB-231 cells is much lower than which of NaGdF4 ND@HAs stained untreated MDA-MB-231 cells (Fig. 3, group 4). In addition, the cellular internalization amount of NaGdF 4 ND@HAs is much higher than which of NaGdF 4 ND@tryptone (as shown in Fig. 3d). The results demonstrate that the NaGdF 4 ND@HAs have high affinity with MDA-MB-231 cells, and can be used to recognize CD44-overexpression cells with high specicity.

In vivo MRI of MDA-MB-231 tumor
A Balb/c nude mouse bearing MDA-MB-231 tumor model was established for evaluating the active tumor-targeting capacity of NaGdF 4 ND@HAs. Both of NaGdF 4 ND@HAs and NaGdF 4 ND@tryptone (10 mg Gd kg À1 body weight in 0.9 wt% NaCl) were intravenously injected into the mouse bearing MDA-MB-231 tumor through tail vein and the MR signals of tumor sites were recorded at desired timed intervals within 24 h postinjection. The NaGdF 4 ND@tryptone can be accumulated in tumor site through enhanced permeability and retention (EPR) effect (i.e., passive tumor-targeting), while NaGdF 4 ND@HAs can be accumulated in tumor site by EPR effect and binding of HA with CD44 (i.e., active tumor-targeting). As expected, the MR signals of the tumor sites gradually increased by numbers in between 0 and 2 h post-injection (as shown in Fig. 4). Maximum MR contrast enhancement is obtained at 2 h post-injection of NaGdF 4 NDs. In particular, the MR signal intensity in tumor site of NaGdF 4 ND@HAs treated mouse is higher than which of NaGdF 4 ND@tryptone treated mouse at the same post-injection time point. The maximum MR contrast enhancement in tumor site of NaGdF 4 ND@HAs treated mouse is 1.6 times higher than which of the NaGdF 4 ND@tryptone treated mouse. The phenomenon may due to high binding affinity of HA with over expressed CD44 on MDA-MB-231 cells. The result demonstrated that the as-prepared NaGdF 4 ND@HAs can be severed as an excellent T 1 -weighted MRI contrast agent for detection of CD44overexpression tumors (e.g., triple-negative breast cancer). The active tumor-targeting capacity of NaGdF 4 ND@HAs was also conrmed by ICP-MS measurement. As shown in Fig. 4d, the amounts of Gd in kidneys and tumors were relatively higher than in other organs, indicating that both NaGdF 4 ND@tryptone and NaGdF 4 ND@HAs can be efficiently accumulated in tumor sites and excreted by renal clearance. In particular, the Gd content in tumor of NaGdF 4 ND@HAs treated mouse treated mouse was 1.87 times higher than which of NaGdF 4 ND@tryptone treated mouse. The result indicated that the accumulation amount of NaGdF 4 ND@HAs in tumor site is clearly improved through EPR effect as well as recognition of HA receptors of tumor cells.

In vivo biodistribution and toxicology of NaGdF 4 ND@HAs
For evaluating its biodistribution and clearance pathway, NaGdF 4 ND@HAs (10 mg Gd kg À1 body weight in 0.9 wt% NaCl) were injected into healthy Balb/c mice through tail vein. The MR signals in the liver, kidneys and bladder were recorded at different timed intervals of post-injection (as shown in Fig. 5a and S5 †). The MR signal of liver exhibited little change during the whole period, indicating low accumulation of NaGdF 4 ND@HAs in liver. MR signals in the kidneys and bladder were clearly enhanced within 24 h post-injection, and recovered to pre-injection levels aer 24 h post-injection. The result demonstrated that NaGdF 4 ND@HAs are excreted from the body by renal clearance. Aer intravenous injection of NaGdF 4 ND@HAs, the Gd content in urine of mouse was measured for addressing the pharmacokinetics behavior of NaGdF 4 ND@HAs  (as shown in Fig. 5b). The total of Gd element in urine of NaGdF 4 ND@HAs treated mouse increased signicantly from 0 to 12 h post-injection. About 75% Gd was found in urine aer 24 h administration, conrming that NaGdF 4 ND@HAs were efficiently excreted with the urine. In addition, the NaGdF 4 ND@HAs showed a negligible morphology change aer in vivo circulation, which indicated that the NaGdF 4 ND@HAs have good colloidal stability in vivo (as shown in Fig. S6 †). The efficient renal clearance of NaGdF 4 ND@HAs helped to eliminate potential hazards of long-term in vivo toxicity.
For further evaluating the biocompatibility, the healthy Balb/ c mice were intravenously injected at a single dose of NaGdF 4 ND@HAs (10 mg Gd kg À1 body weight in 0.9 wt% NaCl). The bodyweights of NaGdF 4 ND@HAs treated mice increased steadily as the time prolonged, which was consistent with those of control group (as shown in Fig. S7 †). The result suggested that NaGdF 4 ND@HAs have little effect on the growth and development of mice. The main organs (heart, liver, spleen, lung and kidneys) were collected for histology analysis at the 1 st day and the 30 th day post-injection. Comparing with the control group, the main organs of NaGdF 4 ND@HAs treated mice showed negligible lesions or abnormalities (as shown in Fig. 6). Tumor tissue from the mice were collected for H&E staining and anti-CD44v6 staining, respectively. The experimental result indicated that cell membrane surface receptor CD44 was overexpressed on the tumor tissue (as shown in Fig. S8 †). Hematology analysis was carried out for monitoring acute and long-term toxicity of NaGdF 4 ND@HAs at the 1 st day and the 30 th day post-injection, respectively. There was little difference between NaGdF 4 ND@HAs treated group and control group (as shown in Table S1 †). These results demonstrated that NaGdF 4 ND@HAs have good biocompatibilities.

Conclusions
In summary, the renal clearable NaGdF 4 ND@HAs have been prepared by a two-step reaction through strong interaction of Gd 3+ with phosphonate groups in tryptone and amidation reaction between carboxy group of HA and amine group of tryptone. In vitro and in vivo experimental results demonstrate that the as-prepared NaGdF 4 ND@HAs display high MDA-MB-231 tumor-targeting capacity. The NaGdF 4 ND@HAs have held great potential as an excellent MR contrast agent for detection CD44-overexpression tumor since advantages of NaGdF 4 ND@HAs including high tumor targeting ability, efficient renal clearance capacity and excellent biocompatibility satised the basic standards of clinical applications.

Conflicts of interest
There are no conicts to declare.  This journal is © The Royal Society of Chemistry 2020 RSC Adv., 2020, 10, 13872-13878 | 13877