Chelator-Free and Biocompatible Melanin Nanoplatform with Facile

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A Chelator-free and Biocompatible Melanin Nanoplatform with Facile Loading Gadolinium and Copper-64 for Bioimaging Su Hyun Hong, Yao Sun, Chu Tang, Kai Cheng, Ruiping Zhang, Quli Fan, Liying Xu, Daijuan Huang, Anthony Zhao, and Zhen Cheng Bioconjugate Chem., Just Accepted Manuscript • Publication Date (Web): 08 Jun 2017 Downloaded from http://pubs.acs.org on June 9, 2017

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Bioconjugate Chemistry

A Chelator-free and Biocompatible Melanin Nanoplatform with Facile Loading Gadolinium and Copper-64 for Bioimaging Su Hyun Hong1, 2†, Yao Sun1†, Chu Tang3†, Kai Cheng1, Ruiping Zhang1, Quli Fan1, Liying Xu1, Daijuan Huang1, Anthony Zhao1, Zhen Cheng1* 1. Molecular Imaging Program at Stanford (MIPS), Department of Radiology and Bio-X Program, Canary Center at Stanford for Cancer Early Detection, Stanford University, 1201 Welch Road, Lucas Center, P095, Stanford, CA 94305, USA. 2. Department of Chemistry, Stanford University, William Keck Science Building Rm 125, Stanford, CA 94305, USA. 3. School of Life Sciences and Technology, Xidian University, Xipei Road Xinglong Section, Xi'an, Shaanxi 710126, China ‡

These authors contributed equally to this work

Corresponding

Author:

Zhen

Cheng,

PhD;

Phone:

[email protected]

1

ACS Paragon Plus Environment

650-210-6001,

Email:


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Abstract Development of a chelator-free and biocompatible platform for facileconstruction of gadolinium3+ (Gd3+) loaded nanoparticle-based probe for in vivo magentic resonance imaging (MRI) is still challenging. Herein, a biocompatible Gd3+ loading melanin dots (Gd-M-dots) has been easily preppared and exhibitedgood loading efficiency for Gd3+, high stability, and higher T1 relaxivity compared with the commercial Gd-DOTA agent. Furthermore, Gd-M-dots showed unique photoacoustic (PA) property, and high PA imaging signal could be observed in vivo after 1h injection. Compared to the traditional Gd3+ loaded nanoparticles for single modal MRI, Gd-M-dots can also be radiolabeled with

64

Cu2+ for positron emission tomography.

Overall, these attractive properteis of Gd-M-dots render it a promising imaging agent for various biomedical applications.

2


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INTRODUCTION Molecular imaging has evolved into a fast-growing research field and plays a crucial role in early and accurate diagnosis and therapy of diseases since it can provide us important information such as anatomic, physiologic, molecular information of diseases in living organisms.1-2 Tremendous efforts have been spent on the development of highly specific and sensitive molecular probes especially multimodal imaging probes to achieve these aims in recent years. Various types of platforms such as small molecules,3-6 polymers7and nanomaterials8-9 have been actively explored for the fabrication of molecular probes. Among them, nanoparticles-based systems have received significant attention because of their ease of preparation and straightforward integration of multiple functional moieties into one entity.10 Magnetic resonance imaging (MRI) is a noninvasive imaging technique that can provide images of intact, opaque organisms in three dimensions.11 Gd3+-based MRI contrast agents increase tissue contrast by increasing water proton relaxation, and they are widely used in clinical diagnostics.12 Among the various of Gd3+-based MRI contrast agents, Gd3+ loaded nanoparticles with high sensitivity and positive signal intensity on T1-weighted images have been developed for MRI in vivo.13-14 Despite these significant progress, some issues including complexity of preparation, instability in vivo and biocompatibility still limit their further applications in clinical research.15-16 For example, the additional coupling of metal chelators on nanoparticles makes the preparation 3



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strategy complex. Furthermore, some Gd-loaded nanoparticles show poor stability and release Gd ions, which causes latent cytotoxicity.16 Therefore, developing promising Gd-nanoparticle systems based MRI agents with chelator-free strategy, good biocompatibility, high stability and sensitivity are highly demanded. Melanin, a natural pigment that is widely distributed in many living organisms, is particularly attractive owing to its excellent physical and chemical properties, especially strong metal ions trapping ability.17-18 Melanin has traditionally been used as a tumor biomarker for melanoma imaging and therapy.19 Recently, by mimicking natural melanin, our group has prepared artificial ultrasmall size PEG modified melanin nanoparticles [named as MNPs or melanin dots (M-dots)] and further used it as a nanoplatform for chemical catalysis and biomedical imaging applications.20-22 These results demonstrated that the M-dots platform not only exhibits good biocompatibility and water-solubility but also actively chelates various metal ions via its intrinsic chelating functional groups with good in vivo stability. Considering the merits of M-dots, we hypothesized that loading of Gd3+ into M-dots can obtain a T1-MRI agent with attractive properties such as chelator-free, water-soluble, high biocompatible and stability. More importantly, combined with its intrinsic photoacoustic properties, Gd3+ loaded M-dots can be also easily loaded with radioactive metal ions such as

64

Cu2+ for multimodality imaging including

positron emission tomography (PET), MRI, and photoacoustic imaging (PAI). 4


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In this study, for proof of concept, we prepared a novel Gd3+ and

64

Cu2+ loaded

M-dots and demonstrated its excellent properties for T1-MRI, PET and PAI multimodal imaging ability in living subjects (Figure 1).

Figure 1. Synthetic scheme of M-dots preparation and application in multi-modality imaging.

RESULTS Synthesis and Characterization of Gd-M-dots Figure 1 schematically illustrates the procedure for preparing the M-dots. The M-dots were synthesized from commercial melanin granules according to our previous report.8 The average sizes of M-dots were ~13 nm as measured by dynamic laser scattering (DLS), and the zeta potential of M-dots was determined to be 2.02 mV (Table 1). In our previous study, PEG encapsulation was demonstrated to enhance biocompatibility, metal loading and water-solubility.8 5



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Materials

D, nm (DLS)

Z, meV

M-dots

13.66 ± 2.211

2.02 ± 7.30

Gd-M-dots

14.58 ± 2.725

6.93 ± 4.56

Table 1. DLS and Z potential of M-dots and Gd-M-dots.

Figure 2. Characterization of properties of Gd-M-dots. (a) TEM of Gd-M-dots; (b) The plot of the relationship between the number of Gd3+ ions attached on one M-dot with feed ration (WGd(III): WM-dot); (c) Stability study of Gd3+-chelated M-dots in buffers with different pH values (pH=7.4, 5.5, and 4.5); (d) Cellular toxicity of Gd-M-dots in NIH-3T3 and SKOV3 cell lines.

6


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The M-dots were then loaded with Gd3+ without chelators by simple addition of GdCl3 in buffer solution of pH = 5.5 followed by 1h incubation at 40oC. The Gd-M-dots were purified using a centrifugal filter (MWCO = 30 kDa). After purification, the fresh Gd-M-dots maintained good water and phosphate buffer (PBS)-solubility without any observed precipitation. The TEM image indicates that Gd-M-dots were monodispersed and homogeneous in water (Figure 2a). The Gd-M-dots exhibited good Gd3+ loading capacities. The maximum number of Gd3+ that one M-dot chelates is about 50 according to the inductively coupled plasma-mass spectrometry (ICP-MS) measurement results (Figure 2b). After Gd3+-chelation, the size of the Gd-M-dots slightly increased to ~15 nm and its zeta-potential went from 2.0 mV to 6.9 mV due to the introduction of positive Gd3+on the nanoparticle surface (Table 1).

Stability and Cell Viability of Gd-M-dots A stability assay of Gd-M-dots in buffers with different pH values showed that no Gd3+ release from nanoparticles over 48 h, indicating the high stability of the chelator-free nanoplatform (Figure 2c). Moreover, the high viability of NIH3T3 and SKOV3 cells after 24 h of incubation with Gd-M-dots demonstrated high biocompatibility of Gd-M-dots even at high concentration of nanoparticles (Figure 2d). Furthermore, Gd-M-dots were incubated with excess metal chelator DOTA (2 eq to Gd3+ ions containing in Gd-M-dots) in the PBS for 0 h, 12 h and 24 h. At different time points, Gd-M-dots was separated from the solvent by 30 kDa centrifugal-filter 7



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and washed with water for three times. The sample was analyzed by ICP-MS. The result showed no release of Gd from the M-dots under DOTA competition, suggesting high stability of Gd-M-dots even in the presence of metal chelator (Table 2).

Table 2. The stability study of Gd-M-dots in the presence of DOTA.

In Vitro Study of MRI of Gd-M-dots To investigate whether Gd3+ retains MR signal-enhancing property after being loaded on M-dots, T1 weighted MR images of various concentrations of Gd-M-dots in agarose gel were studied (250, 125, 62.5, 31.2, 0 µM). With the increase of the Gd-M-dots concentration, MR signal was significantly enhanced, which means that Gd-M-dots generate a high magnetic gradient on their surface (Figure 3a). Furthermore, T1 relaxivity signals of Gd-M-dots were much higher and brighter than a commercially available contrast agent, Gd-DOTA. The regional of interest (ROI) analysis also indicated the value of T1 relaxivity for Gd-M-dots was two times more than that of Gd-DOTA standard (Figure 3b, 6.8±1.1vs. 2.8± 0.9 for 0.25 mM).

8


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Figure 3. In vitro study of Gd-M-dots. (a) MR images of Gd3+ loaded melanin nanoparticles of different concentrations, and comparison with corresponding concentration of Gd-DOTA. (b) ROI values comparison between Gd-M-dots and Gd-DOTA .

In Vivo Study of MRI of Gd-M-dots A group of mice (n=5 per group) were first imaged with a 1T MRI scanner and then injected with Gd-M-dots via tail vain injection (100 µl of 8 mM). After 5 h, MRI was performed using the same scanner. Axial and Coronal T1-weighted MR images (TR 250 ms, TE 6.1 ms) of mice liver before (pre-scan) and after intravenous injection of Gd-M-dots was shown in Figure 4, and the relative MR signal intensity of liver at 5 h significantly increased compared with at 0 h, demonstrated that M-dots can be used as a platform for MRI. Furthermore, Gd-M-dots mainly accumulated on liver, suggesting the clearance routes of Gd-M-dots are predominantly through hepatobiliary systems.

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Figure 4. In vivo study of MRI of Gd-M-dots: Axial and Coronal T1-weighted MR images (TR 250 ms, TE 6.1 ms) of mouse liver before (pre-scan) and after intravenous injection of MNPs. Contrast agent accumulation is noted as a positive (bright) signal enhancement. White arrows indicate the liver.

In Vivo PAI and PET of Gd/Cu-M-dots To further investigate in vivo PAI properties, mice were injected with M-dots via tail vein injection. The M-dots accumulated in mice livers and showed obvious photoacoustic signals after 5 h post-injection than that of pre-scan (Figure 5a). To test the PET imaging properties of M-dots, the radionuclide,

64

Cu2+was used for

radiolabeling of M-dots for PET studies because of its easy loading to melanin nanoparticles and moderate half-life (12.7 h). A simple mixing of M-dots with 64Cu2+ 10


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allowed successfully labeling the M-dots in the radiolabeling yield of 85%. The resulting 64Cu-M-dots displayed excellent stability in PBS solution. The in vivo PET of 64Cu-M-dotswas performed on a Siemens Inveon microPET-CT. At different times after injection (1, 2, 4 and 24h), the mice were scanned by 3 min static scans (Figure 5b). The result showed that M-dots can be used as a platform for multimodality imaging. Therefore, together with its native photoacoustic properties, it can be used for multimodality imaging.

Figure 5. (a) In vivo study of PAI of M-dots: the overlay of ultrasonic (grey) and photoacoustic (red) imaging of mouse liver before and after tail vein injection of M-dots; (b) In vivo study of PET of

64

Cu-M-dots: Representative small animal PET

images of mice liver acquired at 2 h and 24 h post intravenous injection of 64

Cu-M-dots. White arrows indicate the location of liver.

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DISCUSSION MRI is commonly used for in vivo molecular imaging. It is non-invasive and enables high spatial and temporal resolution, high contrast and high sensitivity.10 Gd3+ can produce bright signals with positive contrast in T1-weighted MRI, and several types of Gd-loaded nanoparticles has been well designed for in vivo applications.13-14 However, most of Gd-loaded nanoparticles systems such as upconversion nanoparticles (UCNPs) and quantum dots (QDs) required additional chelators for maintaining the stability of Gd3+. Furthermore, the biocompatibility and in vivo stability still limit these systems for further applications. In this study, Gd3+ successfully loaded on M-dots using a simple strategy without any chelator ligands. Moreover, the obtained Gd-M-dots exhibited good in vitro and in vivo stability and biocompatibility, and produced brighter T1 signal than commercial Gd-DOTA. Melanin is widely distributed in many living organisms and has various biological functions, such as actively chelate metal ions. Encouraged by this promising property, we prepared novel Gd-M-dots for molecular imaging, which presents the following advantages: (1) the natural ligand of M-dots can directly coordinate Gd3+ without additional chelator and Gd-M-dots were easily and straightforward prepared; (2) the high stability and biocompatibility of Gd-M-dots greatly reduced the potential toxicity of Gd3+ ions, which is one major challenges of current Gd-loaded nanoparticle based MRI probe for in vivo imaging. Therefore, these excellent advantages could greatly expand the biomedical applications of Gd-loaded nanopariticle in molecular imaging. 12


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The unique ability of M-dots platform to combine the advantages of MRI/PAI/PET imaging together to get precise and complementary information for objects. For example, MRI provides high spatial resolution image and anatomical information on disease. PAI provides functional and molecular information on the tumor. PET efficiently provides the in vivo pharmacokinetics and biodistribution of the probe23. Therefore, MRI/PAI/PET allows us to image objects at different aspects with molecular and anatomical information. Multimodal also showed that M-dots based probes mainly accumulated on liver, suggesting the imaging clearance routes are predominantly through hepatobiliary systems, which is similar to many nanoparticle-based

probes.

Furthermore,

recent

developments

showed

that

combination of PET/MRI/PAI imaging together is anticipated to help for guiding both superficial and deep tumor surgery. Accordingly, our triple-modality nano-platform can be first used for PET and MRI to obtain detailed information of tumor for surgical planning in presurgery. PAI can then be used to guide the tumor resection in real time. The M-dots nanoplatform significantly simplifies the preparation of multimodal probes with a chelator-free procedure. However, there are also some disadvantages for this nano-platform such as the moderate PAI sensitivity of Gd-M-dots and concerns on the possible long term toxicity of Gd-M-dots.

CONCLUSION In short, a new type of Gd-M-dots probe were synthesized and characterized for in vivo MRI. Gd-M-dots is proven to be a high sensitive, biocompatible, stable and 13



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multimodal imaging probe without noticeable Gd3+ toxicity. It is expected that M-dots systems can serve as a metal loading nanoplatform for further bioimaging applications.

EXPERIMENTAL SECTION MATERIALS and METHODS.

The following materials were used: melanin

(Sigma-Aldrich, St. Louis, MO, USA), sodium hydroxide (Sigma-Aldrich), hydrochloric

acid

(37

wt%,

Sigma-Aldrich),

NH4OH

solution

(28

wt%,

Sigma-Aldrich), amine-PEG5000-amine (NH2-PEG5000-NH2; Laysan Bio, Arab, AL, USA),

3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium

(MTT;

Biotium,

Hayward, CA, USA), phosphate-buffered saline (PBS; Gibco, Waltham, MA, USA), and agarose (Invitrogen, Carlsbad, CA, USA). Millipore water (18 MOhm; Billerica, MA, USA) was also used. The NIH3T3 and SKOV3 cell lines were obtained from the American Type Culture Collection. Nude mice were purchased from Charles River Laboratory. Synthesis of Gd-M-dots. M-dots were obtained as described previously (see supplement information).8 Fresh GdCl3 (2.5 mL, 10 mg/mL) was added and the solution was stirred using a magnetic stirrer for1 h at 40°C to chelate Gd3+. The resulting chelation products were then filtered using a PD-10 Column (GE Healthcare, Chicago, IL, USA)to remove free Gd3+. Finally, the concentration of Gd-M-dots aqueous solution was adjusted to 10 mg/mL. ICP-MS analysis. The detected sample (100 µL) was firstly heated to evaporate the 14


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water solvent and then digested with 0.5 mL of concentrated nitric acid (70% w/w) under heating. After the solvent was evaporated, the residue was then dissolved in 7 mL of dilute nitric acid (2% w/w) for final ICP-MS analysis. Characterization of Gd-M-dots. The hydrodynamic sizes of M-dots and Gd-M-dots were measured by dynamic light scattering (DLS) instrument using a 90 Plus particle size analyzer (Malvern, Zetasizer Nano ZS90). Briefly, M-dots (200 µL) based samples were firstly passed through a 0.22 µm filter and then were filled with a 200 µL clean microcuvette for DLS analysis. We made 6 separate DLS measurements with each measurement consisting of 5 sub-runs with 10 second duration. Zeta potential was measured using a zeta potential analyzer (Malvern, Zetasizer Nano ZS90). Zeta cells should be rinsed thoroughly and dried using nitrogen before use. The zeta cell was filled with 200 µL of samples by gently depressing the syringe plunger. In order to establish measurement repeatability, each sample was performed for three runs. Transmission electron microscopy (TEM) images were recorded on a JEOL 2010 transmission electron microscope at accelerating voltage of 100 kV. The TEM specimens were made by placing a drop of the Gd-M-dots aqueous solution on a carbon-coated copper grid, followed by plasma cleaning. Cell viability. In vitro cytotoxicity of Gd-M-dots was determined in NIH3T3 and SKOV3 cells by the MTT assay. Cells were incubated on 96-well plate in DMEM medium containing 10% FBS and 1% penicillin/streptomycin at 37 °C in 5% CO2 humidified atmosphere for 24 h and 0.5×104 cells were seeded per well. Cells were then cultured in the medium supplemented with indicated doses of different 15



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Gd-M-dots for 24 h. The final concentrations of Gd-M-dots in the culture medium were fixed at 0, 6.25, 12.5, 25 and 50 µg/mL in the experiment. Addition of 10 µL of MTT (0.5 mg/mL) solution to each well and incubation for 3 h at 37 °C was followed to produce formazan crystals. Then, the supernatant was removed and the products were lysed with 200 µL of DMSO. The absorbance value was recorded at 590 nm using a microplate reader. The absorbance of the untreated cells was used as a control and its absorbance was as the reference value for calculating 100% cellular viability. 64

Cu Radiolabeling. The M-dots with Gd3+ was further radiolabeled with

addition of 1.5 mCi of

64

Cu by

64

CuCl2 in 0.1 N NaOAc (pH=5.5) buffer followed by 1 h

incubation at 40 °C. The radiolabeled MNPs were then purified by a PD-10 column. The product was washed out by PBS and passed through a 0.22 µm Millipore filter into a sterile vial for in vitro and animal experiments. Small-Animal MRI and PAI Imaging. MRI experiments were performed at 25°C in a magnetic resonance (MR) scanner (Siemens 1.0 T). Imaging analysis was performed using the ImageJ software. The contrast was adjusted. PAI was carried out using the Vevo LAZR PAI System. Similarly, image analysis was carried out using ImageJ. Small-Animal PET Imaging. The animal procedures were performed according to a protocol approved by the Stanford University Institutional Animal Care and Use Committee. The nude mice were injected with approximately ~100 µCi 64Cu-M-dots via the tail vein (n = 5 for each group). At the indicated times after injection, the mice were anesthetized with isoflurane (5% for induction and 2% for maintenance in 100% O2). All the small animal PET images were reconstructed using Irw4.0 software. The 16


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radioactivity uptake was calculated using a region of interest drawn over the whole organ region and expressed as a percentage of the injected radioactive dose per gram of tissue (% ID/g).

Corresponding Author Information Zhen Cheng, Tel 86 27 68759734, [email protected] Author Contributions Su Hyun Hong, Yao Sun and Chu Tang contributed equally to this work. Notes The authors declare no competing financial interest. Author ORCID information Zhen Cheng: 0000-0001-8177-9463

ACKNOWLEDGEMENTS This work was partially supported by the Office of Science (BER), U.S. Department of Energy (DE-SC0008397), NIH In vivo Cellular Molecular Imaging Center (ICMIC) grant P50 CA114747, NSFC (81571747, 81371628), Overseas students science and technology Projects, Shanxi Scholarship Council of China (No.2015057).

ABBREVIATIONS Magentic resonance imaging: MRI; Melanin dots: M-dots; Positron emission tomography: PET; Photoacoustic imaging: PAI; Transmission electron microscopy: 17



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TEM; Inductively coupled plasma-mass spectrometry: ICP-MS; Upconversion nanoparticles: UCNPs; Quantum dots: QDs; Dynamic laser scattering: DLS.

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Chelator-Free and Biocompatible Melanin Nanoplatform with Facile

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