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Bioinspired, Manganese-Chelated Alginate-Poly(Dopamine) Nanomaterials for Efficient In Vivo T1#Weighted Magnetic Resonance Imaging (MRI) Kefyalew Dagnew Addisu, Balkew Zewge Hailemeskel, Shewaye Lakew Mekuria, Abegaz Tizazu Andrgie, Yu-Chun Lin, and Hsieh-Chih Tsai ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b13396 • Publication Date (Web): 26 Dec 2017 Downloaded from http://pubs.acs.org on December 26, 2017

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ACS Applied Materials & Interfaces

Bioinspired, Manganese Chelated Alginate-Poly (Dopamine) Nanomaterials for Efficient in Vivo T1_Weighted Magnetic Resonance Imaging (MRI)

Kefyalew Dagnew Addisu1, Balkew Zewge Hailemeskel1, Shewaye Lakew Mekuria1, Abegaz Tizazu Andrgie1, Yu-Chun Lin2, Hsieh-Chih Tsai1*

1

Graduate Institute of Applied Science and Technology, National Taiwan University of Science

and Technology, Taipei 106, Taiwan, ROC 2

Tri-Service General Hospital, Department of Pathology, National Defense Medical Center,

Taipei Taiwan, ROC [*] To whom correspondence and reprint requests should be addressed. Prof. Hsieh-Chih Tsai E-mail: [email protected] Tel: +886-2-27303625 Fax: +886-2-27303733

1

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ABSTRACT Manganese-based nanomaterials are an emerging new class of magnetic resonance imaging (MRI) contrast agents (CAs) that provide impressive contrast abilities. MRI CAs that can respond to pathophysiological parameters like pH or redox potential are also highly demanded for MRI guided tumor diagnosis. Until now, synthesizing nanomaterials with good biocompatibility, physio-chemical stability, and good contrast effects remain challenging. This study investigated two new systems of calcium/manganese cations complexed with either alginate-polydopamine or alginate-dopamine nanogels (AlgPDA(Ca/Mn) NG or AlgDA (Ca/Mn) NG). Under such systems, Ca cations form ionic interaction via carboxylic acids of the Alg backbone to enhance the stability of the synthetic nanogels (NG). Likewise, complexation of Mn cations also increased the colloidal stability of the synthetic nanogel. Magnetic property of prepared CAs was confirmed with superconducting quantum interference device (SQUID) measurements, proving potential paramagnetic property. Hence, T1 relaxivity measurement showed that PDA-complexed synthetic NG reveal a strong positive contrast enhancement with r1=12.54 mM-1·s-1 in 7.0 T MRI images while DA-complexed synthetic NG showed relatively lower T1 relaxivity effect with r1 =10.13 mM-1·s-1 value. In addition, both synthetic NGs exhibits negligible cytotoxicity with >92% cell viability up to 0.25 mM concentration when incubated with mouse macrophage (RAW 264.7) and HeLa cells, and high biocompatibility under in vivo analysis. An in vivo MRI test indicate that the synthetic NG exhibits high signal to noise ratio for longer hours, which provides a longer image acquisition time for tumor and anatomical imaging. Furthermore, T1-weighted MRI results revealed that PEGylated AlgPDA(Ca/Mn) NGs significantly enhanced the signals from liver and tumor tissues. Therefore, owing to enhanced permeability and retention (EPR) effect, significantly enhanced both in vitro and in vivo imaging, low cost and one-pot synthesis method, the Mn-based biomimetic approach used in the study provides a promising and competitive alternative for non-invasive tumor detection and comprehensive anatomical diagnosis. KEYWORDS: Alginate, Longitudinal relaxation, T1-Magnetic Resonance Imaging (MRI), Polydopamine, Tumor diagnosis 2


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1. INTRODUCTION Magnetic Resonance Imaging (MRI) is a non-invasive clinical imaging technique with excellent spatial and temporal resolutions.1-5 One of the major applications of MRI is to observe the threedimensional (3D) anatomy of body and to amplify soft tissue contrast using non-ionizing radiofrequency.3, 5 During MRI scanning, contrast agents (CAs) are employed to further increase the inherent low sensitivity signal intensities of the body by accelerating proton relaxations of water. CAs are also extremely useful for locating diseases and early cancer detection.5-7 Paramagnetic nanomaterials have been used for medical imaging as CAs due to their ability to carry large payload of magnetic centers, helping to shorten water proton relaxation time either longitudinally (positive, T1) or transversely (negative, T2).3 Such CAs are able to enhance contrasts between organs of interest and generate a brighter image for T1.4 Current practices of MRI scanning are using progressively more and more CAs, but the fabrication of nanomaterials with high sensitivity, high specificity, and low toxicity remains a challenge.4 For instance, gadolinium (Gd3+), a non-biological rare earth metal, has been extensively applied as a clinical CA owing to its high paramagnetic nature and its long electronic relaxation time. However, Gdbased CAs have low tissue sensitivity and low efficacy. Low levels of Gd3+ accumulation can also result in serious toxic side effects when Gd3+ ions are released from the nanoparticles, leading to conditions such as nephrogenic systemic fibrosis(NSF),8-9 a rare, idiopathic systemic fibrosing disorder that can be serious to patients with acute or chronic kidney disease and cause severe renal dysfunction.10 Moreover, recent studies reported accumulation of intravenously 3



injected Gd3+ in the brains of patients with normal renal function.11 Organic ligands were used to reduce Gd3+ toxicity, but free Gd3+ ions can still leach out and enter the physiological environment of body.12 To effectively address the toxicity of Gd3+, Gd-based CAs were confined within stimuliresponsive multidentate ligands, such as hydrogels13-14 and micelles. However, such CAs have multiple disadvantages such a slow efficiency and inability of instantly responding to external stimuli. Furthermore, growing concerns for patient safety, limited diagnostic information, other concerns from the USA Food and Drug Administration (FDA), and problems caused by bioaccumulation led to widespread concerns and the need for alternatives. 9 A new approach based on non-lanthanide metals, such as manganese (Mn) cations, received more and more attention in MRI application. 9, 15 Mn cations exhibit some characteristics of Gd3+, including high spin quantum numbers (S=7/2 for Gd, S=5/2 for Mn), long longitudinal electronic relaxation times, and fast water exchange property. While Mn cations are biogenic and can be intravenously injected,1, 16-18 Mnbased nanoparticles (NPs) usually show low relaxation rate and small MR imaging performance efficiency. Specifically, Mn relaxation rate was reduced when functionalized with materials such as mesoporous silica and gold NPs. These issues pose a significant challenge for the synthesis of a biocompatible Mn-based MRICA.9, 15 Recently, numerous approaches have been proposed to increase the relaxation rate of Mnbased NPs. Such approaches include modifying the morphology of the Mn-based NPs to provide large surface areas and easy water contact. For example, MnO NPs with a hollow structure demonstrate

longitudinal relaxivity (r1) values of 1.417 mM−1·s−1 using 1.5T MRI scanner,

which is more than 6-fold as compared to that of sphere-shaped equivalents. 19-21 A recent report 4


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also mentioned using Mn coordinated with polydopamine (Mn-PDA) as a new Mn-based MRICA,

22

compared

which provided a relaxivity coeffcient of 6.55 mM-1·s-1, an improvement as to

clinical

T1 used

CAs,

Gd-DTPA

and

Gd-DOPA-(3,4-dihydroxy-

L-

phenylalanine).22 Nevertheless, more attention should be directed towards the design of novel CAs with excellent biocompatible and biodegradable polymers. Polydopamine (PDA) is a natural melanin (eumelanin) and a mussel-inspired material prepared by dopamine (DA) self-polymerization,23-24 and PDA has many functional groups (such as hydroxyl, carboxyl, and amino groups).

The inherent free structure, stable π-electron free

radical species, and high hydrophilicity of PDA make it an appropriate material for capturing large amounts of water molecules around the NGs, allowing more effective water exchange capacity. 25-26 Moreover, although PDA is produced in a facile and simple polymerization route, PDA has long been the topic of scientific debate, which lasts up to now, because of its multifaceted polymerization and reaction mechanism. 26 PDA has been extensively used in biomedical fields because of its excellent biocompatibility and biogenic nature.25 Essentially, DA and PDA can form strong coordination complexes with metals via two hydroxyl groups adjacent to each other.24, 27 Therefore, Mn based NPs coordinated with organic ligands like PDA, DA and Alg have been promising alternatives due to its good biodegradability and zero long-term toxicity.8, 15 More attractively, Mn-chelation was also helpful for nano-platforms to be well functionalized with polyethylene glycol (PEG), providing the resulting NPs with better blood pool stability and making in vivo applications possible.28 The current study demonstrated Ca/Mn-chelated alginate-polydopamine/dopamine (AlgPDA/DA (Ca/Mn)) NGs inspired by adhesive proteins in mussels and their functions as magnetic 5



resonance imaging contrasting agents (MRI CAs). Thus, melanin-like properties make the prepared AlgPDA/DA (Ca/Mn) NGs highly biogenic while enhancing positive T1 relaxivity as compared to clinically used Gd-based CAs. Moreover, the AlgPDA/DA(Ca/Mn) NGs have impressive in vitro and in vivo imaging potentials that offer the following advantages: simple synthesis, zero toxicity, and not requiring high temperature processing. Moreover, the presence of high negative surface charge and PEG as the capping agent (PEG-AlgPDA(Ca/Mn), that is used to improve the surface hydrophilicity of the CA and to increase blood circulation halflife time by reducing NGs interactions with blood proteins and mononuclear phagocyte system (MPS).28-29 Therefore, tumor uptake can be improved on the basis of enhanced permeability and retention (EPR) effect.30 Thus, PEG-AlgPDA(Ca/Mn NGs could be more competitive than other Mn-based MRI CAs.

EXPERIMENTAL SECTION 1.1.Materials Dopamine hydrochloride (98%), manganese (II) chloride tetrahydrate (MnCl2· 4H2O), O-(2amino ethyl) polyethylene glycol (with a molecular weight of 10000 Da), [4,5-dimethyl-2thiazolyl]-2,5- diphenyl-2H-tetrazolium bromide (MTT), 2-(N-morpholino) ethanesulfonic acid hydrate (MES)and D2O were purchased from Sigma-Aldrich. Ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS), calcium sulfate (anhydrous powder) were purchased from Acros Organics (Geel, Belgium). Alginic acid in sodium salt was purchased from Tianjin Jiang Tian Reagent Chemicals. Dulbecco’s modified eagle medium (DMEM), penicillin, sodium pyruvate, trypsin, sterilized fetal bovine serum (FBS), and L-glutamine were purchased from Gibco (Carlsbad, CA). RAW 264.7 cells (mouse 6


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monocyte macrophages) and human cervical carcinoma (HeLa) cells were obtained from the bioresource collection and research center (Hsinchu, Taiwan). Cellulose dialysis membrane (Molecular weight cut-off (MWCO), 6−8 kDa and 14kDa) was purchased from Orange Scientific. Deionized water in the experiments was obtained by using a Millipore water purification system. The other chemical reagents and buffer solution components were analytical grade preparations. 1.2. Synthesis of Alginate-Dopamine (AlgDA) conjugates The alginate-dopamine (AlgDA) conjugate was synthesized by chemically reacting 1-ethyl-3-(3dimethylaminopropyl carbodiimide (EDC) with N-hydroxysulfosuccinimide(NHS). Purified sodium alginate (100 mg,0.46 mmole in terms of repeating unit) was dissolved in 20 mL of a buffer consisting of 0.1M2-(N-morpholino) ethanesulfonic acid (MES) at pH5.9. After alginate was dissolved, in equal molar amounts of NHS (64.45 mg, 0.56 mmole) and EDC (109.26 mg, 0.56 mmol) were first dissolved in 1 mL MES buffer and then added to the solution. The reaction mixture was stirred at room temperature for up to 4 hours to fully activate the carboxyl groups of the alginate molecules. Then, prescribed amount of dopamine (106.19 mg, 0.56 mmole) was dissolved in 1 mL of MES buffer and added to the reaction mixture. The reaction mixture was then stirred for 24hours at room temperature under N2 gas protection to prevent dopamine selfpolymerization. AlgDA was purified by dialyzing the reaction mixture against water by means of a cellular membrane with a cutoff of 6-8 kDa against distilled water followed by lyophilization. 1.3. Preparation of AlgDA(Ca/Mn) and AlgPDA(Ca/Mn) NGs AlgDA(Ca/Mn) NGs were prepared using a reported method with minor modification.31 In a typical preparation, 50mg of AlgDA was first dissolved in 15mL of distilled water, followed by 7



adding 10 mL of acetone, drop wise, into the dissolved solution with continuous stirring. After that, 1mL of MnCl2·4H2O solution (0.12M) was added, through an injection needle and with vigorous stirring, to the 15mL colloidal solution of AlgDA.22 After 3 hours of incubation, the sample was filtered with filter paper to remove any large aggregate, and dialyzed against acetone solution (40% v/v) containing 10 mg CaSO4 for 4 hours using a dialysis bag (MWCO, 6-8 kDa) to perform the cross-linking reaction of AlgDA(Ca/Mn)NGs. Finally, the resulting product was extensively purified by dialysis membrane against distilled water using a dialysis bag (MWCO= 6-8kDa), followed by lyophilization to obtain the final product of AlgDA(Ca/Mn) NGs.31-32 AlgPDA(Ca/Mn) NGs were prepared using a method similar to that of AlgDA(Ca/Mn) except for a minor difference. 50 mg of AlgDA was dissolved in 15mL of alkaline Tris-HCl solution (50 mM, pH 8.0) for 24 hours to complete polymerization at room temperature. Then, 10 mL acetone was added drop wise into the dissolved sample with vigorous stirring to form colloidal particles.32 1mL of MnCl2·4H2O solution (0.12M) was added through an injection needle with stirring. After 3 hours of incubation, the sample suspension was filtered with filter paper to remove large aggregates. Finally, the filtered product underwent dialysis against acetone solution (40% v/v) comprising 10mg CaSO4 for 4hours using a dialysis bag (MWCO, 6-8KDa) to form AlgPDA(Ca/Mn) NGs. 1.4.Characterization The conjugation of alginate with dopamine (AlgDA) was confirmed by UV-vis spectra measured using JASCO-V-650 spectrophotometer, attenuated total reflectance (ATR) spectroscopy (JASCO, ATR-FTIR-6700) and nuclear magnetic resonance (1H-NMR) (Bruker Avance 500.163 MHz) using D2O as the solvent. Hydrodynamic diameter and zeta potential of the synthesized MRI samples were measured by Horiba Zeta sizer-100 system (Malvern Instruments, UK). 8


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Transmission electron microscopy (TEM, Technai F20 FEI-TEM system) and field-emission scanning electron microscopy (FESEM, JSM 6500F, JEOL) were used to observe the morphology and to estimate the size of the AlgDA conjugates using carbon-copper grid and silicon wafer sample holders, respectively. An energy dispersive X-ray spectroscopy (EDS) detector (Bruker Co.) equipped on SEM wasused for composition analysis. The crystalline structures of AlgDA(Ca/Mn) and AlgPDA(Ca/Mn) were characterized by x-ray diffraction (XRD) using CuKα radiation source in the range of 10-80° (2θ). The manganese content of both prepared MRI CA samples were measured by inductively coupled-plasma mass spectrometry (ICP-MS). Surface compositions of the samples were measured using x-ray photoelectron spectroscopy (XPS) with British VG Scientific ESCALAB 250 spectrometer fitted with an XR5 Monochromatic X-ray gun. Magnetic properties of obtained materials were analyzed using superconducting quantum interference device (SQUID) measurements, where prepared MRI CA samples in aqueous phase were lyophilized and then quantified with the range of -5 kOe to +5kOe. 1.5. In vitro T1 and T2 Relaxivity MRI studies The relaxation rates of r1 and r2 of AlgPDA(Ca/Mn) and AlgDA(Ca/Mn) NGs were determined using 7 Tesla (T) MRI analyzer using bruker paravision version 4.0 scanning software (Bruker Biospec 70/30 US) which offers dramatically enhanced sensitivity, contrast and spectral dispersion. Both T1 and T2 relaxation times and R1 and R2 map images were measured by spin echo (RARE) and Multi-Slice Multi-Echo Sequence (MSME) sequence imaging methods. A series of five aqueous solutions of various concentrations (0.015, 0.031, 0.0625, 0.125 and 0.25 mM) were prepared by diluting the MRI CAs solution with distilled water. The r1 and r2 relaxivities of each sample solution were then respectively determined from the slopes of curves of 1/T1 and 1/T2 against Mn concentration obtained by ICP-MS using the following parameters: 9



repetition time (TR) = 800ms, echo time (TE) = 11ms; flip angle (FA) = 180°; slice thickness = 1.0 mm; and field of view (FOV) =6.0cm. 1.6.Cytotoxicity test (MTT assay) Cytotoxicity of AlgPDA(Ca/Mn) and AlgDA(Ca/Mn) nanogels (NGs) was evaluated using HeLa and RAW 264.7 cells as a model. The following provides a summary description of the cytotoxicity test: growing HeLa and RAW 264.7 cells overnight in a DMEM ( medium supplement with 10% FBS, 1% penicillin, 1% glutamine, and 1% sodium pyruvate at 37 °C and 5% CO2 in 96-well plates at a density of 3.0 ×105 cells per well, followed by another 24 hours of culturing in fresh medium containing NGs with various concentrations (0, 0.015, 0.031, 0.0625, 0.125 and 0.25mM); mixing 5 mg/mL of MTT into each well to achieve a final concentration, and incubating the plate for 4 hours at 37 °C for the formation of formazan dye; solubilizing the formazan crystals using 100 µL of dimethyl sulfoxide (DMSO); and finally analyzing absorbance using an enzyme-linked immunosorbent assay (ELISA) reader (Multiskan FC, Skanlt, Software 3.0.0.64 RE) at 570 and 450 wavelengths (nm). Results were quantified as the percentage of viable cells after treatment compared to a control of untreated cells. Cell viability was expressed using the following formula.33 Cell viability (%) = absorbance of test cells / absorbance of controlled cells ×100 1.7. In vivo MR imaging 7-week old female BALB/c nude mice weighing 19-20 gram were purchased from BioLASCO (Taiwan Co., LTD). All animal care and handling procedures were performed according to the guidelines of the institutional animal care and research committee at Taiwan National University (NTU). The method comprises culturing human cervical carcinoma (HeLa) cells in DMEM (supplement with 10% FBS, 1% penicillin, 1% glutamine, and 1% sodium pyruvate) culture medium 10


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at 37 °C, atmosphere of 5% CO2 and 95% air. Once reached 80 % confluence, HeLa cells were washed twice with phosphate buffered saline (PBS) and treated with 1 ml of trypsin to detach from culture dishes. For cell implantation, the skin of the mouse was pinched between index finger and thumb and the skin was away from the body of the mouse. Then 200 µL of HeLa cells suspended in DMEM medium with 5.3 × 106 cell density were injected subcutaneously in to flank of 7 to 8-week old female BALB/c nude mice (n= 6 to 10) using 3 cc/ml syringes with a 24 ( 0.55 × 25 mm)

gauge needle to prepare tumor models.34 In order to prevent variable

tumor development and subcutaneous tumor spread growth, HeLa cells were inoculated evenly into the pouch formed by the fingers. After the nodule develop at the inoculation site, the tumor size was estimated by external measurement of its length and width of the tumors in two dimensions via a caliper when tumors reached measurable extent.34 To scan the mice using a 7T MRI System, the mice were anaesthetized by 2% isoflurane under an oxygen-enriched environment before administering the contrasting agent (CA). Positive contrast imaging was performed before and after the injection of PEG-AlgPDA(Ca/Mn) MRI sample using the following parameters: TR=1300s; TE=9.0; FA = 15; slice thickness = 0.7 mm; and field of view (FOV) = 90×40 mm before. The following provides a brief description of the imaging method: administering, through a tail vein of a mouse, PEG-AlgPDA(Ca/Mn) MRI sample dissolved in PBS was injected at a volume of 0.2ml (0.1mmol) of Mn per kg body weight of the mouse (measured by ICP-MS). Then measuring signal intensities (SI) of various organs and tissues like liver, kidney, spleen and tumor at different time intervals of 5, 30, 45, 60, 90,120, and 150 minutes using RADiANT DICOM viewer 4.0.3; and acquiring SI from the defined regions of interest (ROIs) at comparable positions within organs and tumor sites. The standard deviation of SIs were calculated from a group of ROIs (n = 3). 11



1.8. Histological and bio-distribution study To study histological effects, 200 µL of PEG-AlgPDA(Ca/Mn) CAs solution (0.1 mmol Mn/kg body weight, n =3) was administered into mice (experimental group, n =3), with the control group being mice administered with PBS (n=3). Heart, liver, lungs, kidneys and spleen tissues were then collected, fixed in 10% formalin, and stained with Hematoxylin and Eosin (H&E) for histopathology examination after 16 days. To quantitatively estimate the bio distribution of PEGAlgPDA(Ca/Mn) NGs (similar dosage as that in the histological administration) in the viscera and tumor tissues, the organs (liver, kidney and spleen) and tumor tissue were grounded, homogenized and treated with 1N HNO3 for 12 hours for dissolution after a post-injection period of 4 hours, 24hours,5days, and 16 days. The amount of manganese in these organs was then analyzed with ICP-MS.

2. RESULTS AND DISCUSSION 2.1.Synthesis and characterization of Alginate-polydopamine (AlgPDA) and Alginate dopamine (AlgDA) conjugates Alginate conjugate with dopamine (AlgDA) was synthesized by coupling alginate (Alg) with dopamine (DA) through a standard carbodiimide process via EDC/NHS coupling reaction, as illustrated in scheme-1.35-36The following provides a summary of the process: purging nitrogen (N2) gas into the reaction mixture to prevent DA self-polymerization; using MES buffer (pH 5.9) to inhibit DA oxidation while reacting Alg with DA at 1: 1.21 molar ratio; dialyzing the reaction solution against distilled water, and performing characterization of the AlgDA conjugate using UV-vis spectrophotometer. As shown in Fig. 1A, DA was successfully grafted onto the Alg backbone as indicated by the absorption peak around 280 nm, an observation consistent with previous reports.35-36 No absorption peak was observed in Alg solution from 240 to 300 nm 12


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wavelength range (Fig. 1A).37 To evaluate the amount of DA coupled to the carboxyl groups of the alginate, a DA standard curve was constructed with UV-vis spectrophotometer at 280 nm. The concentration of DA was determined as 0.263 mM using the standard curve as shown in Fig. S1A.Therefore, around 12.7 ± 0.16% of DA was coupled with Alg polymer, as indicted in Fig. 2B, a result highly consistent with previous reports.31 DA is known to oxidize readily and will undergo self-polymerization in mildly basic environments (pH 8.0). 40 Interestingly, the progress of DA polymerization could be observed through a gradual color change from pale to dark brown, as clearly illustrated in Fig.1C. Moreover, the UV-vis spectra (Fig.1D) demonstrated the gradual oxidative behavior of DA in solution, in which the absorbance values increase with time. Hence, an absorbance peak around 300 nm indicates the polymerization of AlgDA. 35 Measurements taken from the ATR- FTIR spectrum confirmed the conjugation of DA into Alg matrix with the presence of distinctive infrared (IR) bands and vibration shifts related to Alg and DA interaction, as shown in Fig. 2A. A broad peak and increase intensities are observed around the range 3,700-3,100 cm−1, attributed to stretching vibrations modes of N-H/O-H after DA conjugation with Alg. Additionally, the broad peak at 3,273.5 cm−1 in the Alg spectrum was derived from the -OH groups and the displayed Alg peak. The peak at 3,273.5 cm−1 shifted towards 3,300 cm−1 in AlgDA and 3,314 cm−1 in AlgPDA. The peak shift from Alg appears as a result of stretching band overlapping among -OH and –NH groups from AlgDA.35 Similarly, the broad peak centered at 1,711 cm−1 for AlgDA and at 1,738 cm−1for AlgPDA, correspond to the carbonyl group of -COOH while confirming the occurrence of quinine groups.

35

Additionally,

the peak at 1,422 cm−1corresponds to presence of catechol motif together with the aryl bands, while 1,217 cm−1corresponds to phenolic alcohol bands and 1,365cm-1corresponds to indole features, supporting presence of melanin-like polymers. Hence, these synthesized ATR-FTIR 13



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bands measurements demonstrate that Alg was successfully conjugated with DA, and result is supported with previous reports. 35 The conjugation of Alg with DA was also confirmed by 1H-NMR spectroscopy as revealed in Fig. 2B and Fig. S1B. When Alg is coupled with DA, the peak region from 6.5 to 7.0 ppm corresponds to the aromatic protons in DA. Likewise, the peak at 3.2 ppm corresponds to the two protons of CH2 adjacent to the N(C=O) group, which had no clear chemical shift when compared to the corresponding peaks of DA coupled with alginate (Fig. S1b). These identified 1H-NMR peaks further indicate the successful conjugation of DA with Alg polymer.37

Therefore,

according to the results of UV-vis, ATR-FTIR and1H-NMR spectrum, DA was successfully conjugated on to Alg matrix.

14


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Scheme 1. Schematic illustration of the synthesis of mussel-inspired alginate-dopamine (AlgDA) conjugates by coupling alginate (Alg) with dopamine (DA) under the catalysis of EDC and NHS

15



FIGURE 1.(A) UV-vis spectrum, DA, Alg AlgDA and AlgPDA (B) dopamine calibration curve at 280 nm (C) AlgDA time-dependent images during oxidation (D) AlgDA time dependent UVvis spectra during oxidation

16


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FIGURE 2. Characterization of AlgDA conjugate synthesis. (A) Shows the ATR-FTIR spectra of DA, Alg, AlgDA and AlgPDA, while (B) shows the 1H-NMR spectra analysis. Both confirmed AlgDA conjugation. The solvent used is D2O 2.2.Preparation and characterization of AlgPDA(Ca/Mn) and AlgDA(Ca/Mn) NGs Two kinds of mussel-inspired alginate nanogels (NG), AlgPDA(Ca/Mn) and AlgDA(Ca/Mn), were prepared through calcium and manganese ions based complexation, as revealed in Scheme2.

17



Scheme-2. Schematic illustration showing the preparation of efficient MRI contrasting agents (CAs) using Ca and Mn cations: (A) (AlgPDA(Ca/Mn) (B) AlgDA(Ca/Mn) NGs

As clearly illustrated in Scheme-2A, the process involves the following steps: (1) mixing AlgDA conjugate with acetone in tris-HCl buffer; (2) cross-linked assembly polymerization between DA moieties in AlgDA solution in a mildly alkaline medium (pH 8.0) at room temperature; and (3) adsorbing metal ions using the strong affinity of the polydopamine (PDA) and the carboxyl group of alginate to prepare the AlgPDA(Ca/Mn)nanogel (NG). In brief, DA undergoes oxidation and forms 5, 6-indolequinone (DHI) though 1, 4-Micheal addition reactions.38-39 Multiple isomers, like dimers and oligomers, with various degrees of polymerization can be formed using DHI and its oxides. Hence, dimers and oligomers can undergo several cross18


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linked assembly via the reverse dimutation reaction.23-24 Metal ions such as Mn2+, Cu2+, and Fe2+ can coordinate with the PDA moieties in AlgPDA such as o-quinine, amino, and phenolic groups to increase the local concentration by assembling the nearby dimers and oligomers as presented in Scheme-2A.35, 40 Ca cations undergo ionic interaction via carboxyl groups of the Alg backbone to form the alginate egg-box structure that is consistent with previously reported observations. Likewise, Mn cations complexation increase the structural

stability of the prepared the

nanogel.41 Finally, a stable AlgPDA(Ca/Mn) NG was prepared as depicted in Scheme-2A, while AlgDA(Ca/Mn) NG was prepared using the process of Scheme-2B, which is similar to AlgPDA(Ca/Mn) NG except minor modification. Under Scheme-2B, distilled water at neutral pH was applied to prepare AlgDA(Ca/Mn) NGs. The strong affinity of the positive charge of Mn cation leads the formation of Mn-catechol complexation, in which the high electro negativity of catechol and carboxyl groups in AlgDA conjugate act as a cross-linking agent between AlgDA polymers, as clearly illustrated in Scheme-2B. Similar results consistent with the current work, Mn-catechol complexation, were noted in previous reports.35, 41 The particle size and charge distribution of AlgPDA(Ca/Mn) NGs and AlgDA(Ca/Mn) NGs were confirmed using dynamic light scattering (DLS). As shown in Table S1, the average hydrodynamic diameters of both nanogels were reduced when adding Mn cation; while final mean zeta potential was respectively changed -24.26± 2.8mV for AlgPDA(Ca/Mn) NG and 27.7 ± 3.5mV for AlgDA(Ca/Mn) as shown in Fig.3 A-B. Moreover, the DLS results from Fig.3 C-D and Table S1 showed that the mean hydrodynamic sizes were 66.30 ± 6.5 nm for AlgPDA(Ca/Mn) NGs and 135.24 ± 4.5 nm for AlgDA(Ca/Mn) NGs, the result showed that the sizes of the NGs were highly dependent on the interaction between Mn and AlgPDA/DA conjugates. Moreover, AlgPDA(Ca/Mn) NGs revealed

less polydispersity index (PDI) with

19



mean PDI value of 0.13 ± 0.21 unlike AlgDA(Ca/Mn) (PDI= 0.64 ±0.14) NGs (Table S1). Reduced particle size could be attributed to the high affinity between AlgPDA/DA NG catechol ligands and metal cations. Chelation between AlgPDA/DA(Ca/Mn) NGs conjugates and Mn cations was also confirmed by a decrease in zeta potential charge once Mn cations were added, as indicated in Table S1. The morphology and size of the prepared NGs were compared using SEM and TEM. Fig.S2A-B and Fig.3E-F show that both the prepared NGs assumed a spherical morphology. In addition, AlgPDA(Ca/Mn) NGs reveal a size distribution with moderate uniformity relative to AlgDA(Ca/Mn) NGs as illustrated in Table S1and Fig.3E-F. Moreover, SEM and TEM images of AlgPDA/DA (Ca/Mn) NGs showed there was little decrease in NG size distribution than that of DLS measurements. This could be due to the fact that TEM and SEM images were acquired under dry and high vacuum environment while DLS measurements include the core particle size as well as the surrounding water molecules.22 Successful Mn and Ca cations chelation was also confirmed by energy dispersive spectroscopy (EDS) mapping. In EDS mapping, in AlgPDA/DA(Ca/Mn) NGs contain carbon, oxygen, Ca and Mn as shown in Fig.3G-H. In addition, the quantity of Mn loaded in the NGs were measured using ICP-MS, with approximately 0.16 % in AlgPDA(Ca/Mn) NG and 0.027% of

Mn in AlgDA(Ca/Mn) NG were complexed. Higher Mn loading was occurred in

AlgPDA(Ca/Mn) NG’s, this may be because of AlgPDA(Ca/Mn) NG’s: rich functional groups, a high surface area, large number of active sites for binding metal ions, coordination or chelation, π-π stacking interactions or due to its high surface area.42 Powdered XRD measurement were applied to confirm the crystalline structure of the NGs. Complexed Mn and Ca cations in AlgPDA/DA(Ca/Mn) NGs maybe either in amorphous structures or in crystalline form. As 20


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shown in Fig.S3, three diffraction peaks at 2θ values of 13.3°, 22°, and 39° were observed due to plane reflection of the poly guluronate and poly mannuronate units. The amorphous character, on the other hand, is caused by sodium alginate. When compared to peaks in alginate, AlgPDA(Ca/Mn) and AlgDA(Ca/Mn) NG peaks exhibit a broad XRD pattern, ensuring that alginate crystallization was greatly affected due to Ca/Mn cations interaction.43 Moreover, Ca and Mn cations complexing with various ligands of the nanogel were studied using XPS spectroscopy. XPS measurements were clearly shown in Fig.4A-D and demonstrated possible Mn-chelation with various ligands in the nanogel. Fig. S4A-B also shows the survey spectrum of AlgPDA(Ca/Mn) NGs that demonstrated various forms of Ca and Mn cation complexation forms. Mn comprises various binding energies with different spin orbitals. For example, Mn 2p spectra of AlgPDA(Ca/Mn) NGs can split into two major peaks, with Fig. 4A shows that binding energy is at 653 eV when Mn binds with O (Mn-O) and at 641 eV when Mn binds with N (Mn-N).44 Similarly, Fig. 4B shows that the binding energy of the Mn 2p spectra in AlgDA(Ca/Mn) NG is at 649.5 eV for Mn-O and 638 eV for Mn-N.45-46 The corresponding peaks in both the AlgPDA(Ca/Mn) and AlgDA(Ca/Mn) NGs show that Mn cations can coordinate with amino, hydroxyl and carboxyl of the nanogels.25 Ca cations can also interact in the same fashion with the carboxyl groups of the NGs, with corresponding peaks at 348.5 eV and 345.4 eV for AlgPDA(Ca/Mn) and 345.5 eV, 346.4 eV and 349.6 eV for AlgDA(Ca/Mn) (Fig. 4C-D).39 Hence, stable chelating could be mainly attributed to coordination effect between Ca or Mn cations and catechol or carboxyl groups inAlgPDA(Ca/Mn)and AlgDA(Ca/Mn)NGs.22

21



FIGURE-3. Zeta potential of (A) AlgPDA(Ca/Mn) and (B)AlgDA(Ca/Mn) in water; hydrodynamic particle size of (C) AlgPDA(Ca/Mn) and (D) AlgDA(Ca/Mn) in water; TEM image of (E) AlgPDA(Ca/Mn) and (F) AlgDA(Ca/Mn); and EDS elemental mapping (G) AlgPDA(Ca/Mn) and (H)AlgDA(Ca/Mn)

22


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FIGURE 4. X-ray photoelectron spectrum (XPS) of AlgPDA(Ca/Mn) NGs:(A) Mn (2p) and (C) Ca(2p) and AlgDA(Ca/Mn) NG: (B) Mn (2p) and (D) Ca (2p)

2.3.Magnetic properties The magnetic property of AlgPDA/DA(Ca/Mn) NGs was measured using superconducting quantum interference device (SQUID). Fig. 5A-B show magnetization versus applied field (MH) curves (-5 Tesla ≤H≤ 5 Tesla) at a temperature(T) of 5K and 300K, while Fig. 5C-D show Zero-field-cooled (ZFC) magnetization versus temperature (M–T) curves (5 ≤ T≤ 300K) at H=300 Oersted(Oe). The coercivities and remanences of both samples were zero. In other words, there is no hysteresis in the M–H curves at temperatures of 5Kand 300K. The absence of both 23



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hysteresis and magnetic transition at temperatures 5K or less in the M–T curves indicate that the NGs have paramagnetic property below 5K, which is consistent with previously reported observations5, 47-48.The M–H magnetization curves shown in Fig. 5A-B of AlgPDA/DA (Ca/Mn) NGs at H=5 Tesla at T=5K and 300 K were used to estimate M as shown in Table 1. M values of the NGs at a temperature of 5K were estimated to be 8.0 emu/g for AlgDA(Ca/Mn) NGs and 42.0 emu/g for AlgPDA(Ca/Mn) NGs. Thus, Mn-containing NGs are able to efficiently induce larger water proton relaxivities at room temperature. Table-1.Magnetic properties of AlgDA(Ca/Mn) and AlgPDA(Ca/Mn) NGs Magnetization (emu·g-1) Sample

Magnetism

5K

300K

AlgDA(Ca/Mn)

Para magnetism

8.0

0

AlgPDA (Ca/Mn)

Para magnetism

42.0

1

24


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FIGURE-5. Field dependent magnetization (M-H) curve of (A) AlgPDA(Ca/Mn) and (B) AlgDA(Ca/Mn) NGs at temperatures of 5k and 300k; and mass-corrected temperature-dependent magnetization curve (M-T) of (C) AlgPDA(Ca/Mn) and (D) AlgDA(Ca/Mn) NGs at H of 300 Oe. 2.4. In vitro MRI measurements Longitudinal (1/T1) and transverse relaxation (1/T2) times of AlgPDA/DA(Ca/Mn) NGs were plotted against Mn cation concentrations with the curves shown in Fig. 6. A-B. r1 and r2 values were compared with the respective slopes as illustrated in Table-2. To quantitatively estimate MRI contrast enhancements, r1 and r2 relaxivity values were calculated using the curve fitting relaxation time versus Mn concentrations from 0.015 mM to 0.25 mM as shown in Fig. 6A-B. 25



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Relaxivity values are: r1=12.54 mM-1s-1 and r2=141.38 mM-1s-1 for AlgPDA (Ca/Mn) NG; and r1 =10.13 mM-1s-1 and r2 =165.60 mM-1s-1 for AlgDA(Ca/Mn)NG.

The relaxivity provided

AlgPDA(Ca/Mn) was the highest value and 3 times higher when compared to clinically used GdDTPA (r1= 4.11 mM-1s-1 and r2=4.2 mM-1s-1).49 AlgPDA(Ca/Mn) NGs

A small size, approximately 66.3 nm, of

distribution could contribute for higher r1 relaxivity enhancement to

make a short water proton relaxation time.50 Compared to larger AlgDA(Ca/Mn) NGs (around 135.24nm), smaller nanoparticles with larger surface to volume ratio (s/v) may have greater possibility to access to water molecules for inducing relaxation, which is in agreement with the previous report.50 Furthermore, the nano-particulate character of melanin can further improve relaxivity. Melanin or its derivatives confined within the nanocarrier can enhance r1 relaxivity that can be attributed to the restricted rotational mobility of the complex.4,

51

Hence,

polydopamine (or melanin)-based CAs such as AlgPDA(Ca/Mn)NG tend to provide more chelation and relaxivity when compared to non-polymerized AlgDA(Ca/Mn) NGfor in vivo MRI. On the other hand, the inverse relationship of particle size with their relaxivity, smaller NPs possessing larger r1 relaxivity, revealed an insignificant r1 relaxivity variation when the particles size lowered to ultra-small level.54 For example, relaxivity studies of Gd2O3 has confirmed that particles less than 10 nm have much greater r1 value than on the order of 30 nm diameter; whereas, greater similarity in r1 values were found, when the particles sizes ranged around 71 and 145 nm with their r1 relaxivity 2.56 and 2.34 mM-1s-1,respectively, which is in line with our study.50, 52 53 Results depicted in Fig.6C-D show that both NGs indicate a dose-dependent contrast progression within their R1 and R2 map images. Mn-containing NG was able to achieve the highest contrast enhancement ability due to the presence of catechol or carboxyl groups that 26


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make water molecules bond to oxygen atoms.4, 51 Mn can coordinate with various groups such as catechol and carboxyl in the NGs, providing a second-sphere hydrogen bonding mechanism that enhances r1 relaxivity.22,

54

r1 relaxivity is also related to water hydration number (q) for

coordinating with unpaired electrons of the contrasting agents (CAs). Free sites in the five unpaired electrons of the Mn cation can be used for water ligation, thereby providing a higher r1 value.1, 55The results were consistent with our hypothesis that the prepared NGs offer higher water holding and exchanging capacity, and allows water to easily diffuse into the prepared NGs and contact the manganese core, there by achieving efficient relaxation of water.1 Additionally, compared with those of Mn@CQDs and MnCO3@PDA NPs, the r1 values

of the

AlgPDA(Ca/Mn) NGs were higher. These results demonstrate that AlgPDA(Ca/Mn) NGs are effective in T1 contrast agent , and the AlgPDA coating can further induce a higher r1 relaxivity of manganese as shown in in Table-2. Table 2. Water proton relaxivities (r1 and r2) of various nanoparticles Nanoparticle (Material)

Field (T)

r1 (mM-1 s-1)

r2(mM-1 s-1)

Ref

MnCO3@PDA NPs

7.0T

8.3

-

25

Mn@CQDs

7.0 T

7.43

140.7

56

AlgPDA(Ca/Mn) NGs

7.0T

12.54

141.38

This study

AlgPDA(Ca/Mn) NGs

7.0T

10.13

165.60

This study

27



FIGURE 6. (A) 1/T1 inverse relaxation times of AlgPDA(Ca/Mn) and AlgDA(Ca/Mn) NGs; (B) 1/T2 inverse relaxation times of AlgPDA(Ca/Mn) and AlgDA(Ca/Mn) NGs; (C) T1-weighted MR images of AlgPDA(Ca/Mn) and AlgDA(Ca/Mn) NGs at different Mn concentrations (mM); and (D) T2-weighted MR images of AlgPDA(Ca/Mn) and AlgDA(Ca/Mn) NGs at different Mn concentrations (mM) 2.5.Manganese ion release and cytotoxicity assessment The human cervical cancer HeLa cell line and the mouse monocyte macrophage Raw 264.7 cell line were used to evaluate the cytotoxicity of AlgPDA/DA(Ca/Mn). The standard MTT assay demonstrated that both NGs have no obvious toxic effect after a 24-hour incubation period at a high concentration of 0.25 mM (Fig. 7A). Results show that about 92.97% of normal (Raw 264.7) and 94.56% of cancer (HeLa) cells remained viable after incubation with 0.25mM AlgDA(Ca/Mn), while better cell viability was observed in AlgPDA(Ca/Mn) NGs with 93.14% of Raw 264.7 normal cells and 97.33% of HeLa cancer cells remaining viable after incubation. Low Mn ion release in physiological environments (pH 7.4) was observed in AlgPDA(Ca/Mn), and this could be the reason for improved cell growth and viability. To imitate 28


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Mn-release actions of AlgPDA(Ca/Mn) in the microenvironments inside and outside cells, Mnrelease experiments were carried out using PBS solutions at different pH conditions for 4 hours at 37 °C, with results shown in Fig. 7B. Microenvironments inside and outside cells were mimicked by providing a pH of 7.4, 6.5, and 5.0 that respectively represent physiological, tumor extra cellular, and endosome environments.25, 57 The xylenol orange test was used to investigate Mn ion leakage from AlgPDA(Ca/Mn) NGs under various pH (5.0, 6.5, and 7.4) conditions.58 Xylenol orange is highly sensitive to Mn ions and is able to detect Mn at a low concentration of 1mM due to the high ion charge density difference of the Mn cation.59 Fig. 7B and Table S2 shows that after 4 hours, pH 5.0 condition resulted in the highest release of Mn ions followed by pH 6.5 (Mn ion release: pH 5.0>6.5), while pH 7.4 has the lowest release rates. That is, approximately 0.37, 0.094 and 0.02 mM of Mn ion was release from pH 5.0, 6.5 and 7.4, respectively as shown in Fig S5 (calibration curve) and Table S2. The results indicate that 52.53% of Mn ions were released from pH 5.0, while insignificant level of Mn was leaked from the blood physiological environment (pH 7.4). This phenomenon was probably due to the pH-dependent reversible coordination between Mn cations and catechol (PDA) ligands, in which increasing pH strengthens PDA affinity towards Mn cations and induces the formation of stable bis- and tris-complexes.40 In contrast, complexation is reduced at low pH environments and results in the release of free Mn ions.40 Accelerated leaching of Mn ions from AlgPDA NGs at acidic pH environments may be also due to reduced interactions between AlgPDA and Mn upon protonation of the catechol and carboxyl groups of the NGs.60 Such pH-responsive characteristics of the contrasting agents (CAs) are very useful for tumor tissue diagnosis. Tumor extracellular environments has a pH of about 6.5, while

29



cellular endosomes and lysosomes are more acidic (pH5.0-5.5).53 MTT and xylenol orange tests further confirm that the AlgPDA(Ca/Mn) NGs are safe for subsequent in vivo studies.

FIGURE 7. (A) RAW 264.7 and HeLa cells incubated with different concentrations of AlgPDA(Ca/Mn) andAlgDA(Ca/Mn) NGs for 24 hours, and estimates of cell viabilities using the standard MTT assay; (B) Mn-release from AlgPDA(Ca/Mn) in the presence of xylenol orange at different pH conditions. 2.6. In vivo mice T1- MR images After receiving encouraging results from in vitro MRI, T1-weighted MR imaging was performed on mice with tumors. As clearly indicated in Scheme-3, the level of MRI contrast was evaluated using BALB/c nude female mice bearing subcutaneous tumor from human cervical carcinoma with polyethylene glycol-modified AlgPDA(Ca/Mn) (PEG-AlgPDA(Ca/Mn)) (Scheme 3A and Fig. S6).

30


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Scheme-3. Schematic illustrations for: (A) the process of preparing PEG-AlgPDA(Ca/Mn) NG; and (B) a passive tumor targeting of PEG-AlgPDA(Ca/Mn) NG through the defective tumor microvasculature pH is used to trigger local Mn ion release from the MRI CAs in the tumor.

PEG surface modification was used to enhance blood circulation time and biocompatibility of an MRI sample solution.22,

30

The method comprises administering 0.2mL of contrasting agent

nanogel (CA-NG) into a mouse tail vein, then taking successive in vivo T1 images with the passage of time. Fig.8 shows the T1-weighted images of mice before and after the injection of CA-NG at a dose of 0.1mmol of Mn per kg body weight. In the axial T1-weighted MRI, signal 31



enhancement in tumors was rapidly achieved after 5 minutes of administration as shown in Fig.8A. The morphology of tumor was highly contrasted (the bright areas) after administering T1-MRI CAs, which may be attributed to the distribution of PEG-AlgPDA(Ca/Mn) NGs and Mn release in tumor tissues (Scheme-3B, Fig.8) as well as less contrast enhancements in the surrounding normal tissues. Tumor progression at cancer tissue acidifies the interstitial pH of adjacent tumor tissues and cancer intracellular regions (Scheme-3B), stimulating the release of coordinated Mn cations from the MRI sample solution. Moreover, impairment of the blood tissue barrier due to tumor development allows PEG-AlgPDA(Ca/Mn)NGs to readily access the tumor and achieve long-term tumor retention, improving the contrast between tumor and normal tissues.4, 61 Manganese localization in the tumor tissue was confirmed by ICP-MS measurements. Significant T1 contrast effects were also observed from coronal T1-weighted MR images in different organs and organ positions after injection, as shown in Fig.8B. Signal-to-noise ratio (SNR) of the regions of interest (ROIs) at different body parts of mice were measured, with results shown in Fig. 9. A strong positive contrast enhancement in liver can be easily observed 5 minutes after PEG-AlgPDA(Ca/Mn) NG

injection. The spleen and kidneys also show

considerable positive contrast enhancements 5 minutes after injection, though the level of contrast was less than that of the liver. Significant hyper intensity change in the liver ROI area demonstrates that NGs may be taken up by the reticuloendothelial system (RES) of the liver organ,62-63 especially since the NGs have large hydrodynamic sizes (approximately 66.3nm). Positive signal enhancement in the kidneys shows that part of the NGs may be excreted through the kidneys, an important feature for clinical applications. Induced toxicity of PEG-AlgPDA(Ca/Mn) CAs were evaluated using Haemotoxylin and Eosin (H&E) staining. Fig.10 shows neither morphological change, appreciable sign of organ 32


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damage, inflammatory lesion, nor other forms of abnormality in all major organs such as the heart, liver, spleen, kidneys and lungs of the mice. The evaluation did not identify heart swelling and vacuolization nor inflammatory reaction in the lung and liver samples, and the glomerular and tubular structures in the kidney samples were clearly displayed. All these results confirmed that PEG-AlgPDA(Ca/Mn) CAs did not induce pathological changes, and thus can be regarded as a safe potential candidate as an MRI CAs.

33



FIGURE8. In-vivo T1-weighted MR imaging of a BALB/c nude female mice bearing subcutaneous tumor from human cervical carcinoma after: (A) tail injection of PEGAlgPDA(Ca/Mn), wherein the yellow circles indicate tumor tissues; and (B) intravenous (i.v) injection, wherein the organs identified by i, ii, iii, iv, and v represent liver, kidneys, spleen, bladder and tumor, respectively, and the yellow circles indicate the organ positions.

FIGURE 9. Signal to noise ratio (S/N) of the regions of interest (ROI) of different organs with respect to PEG-AlgPDA(Ca/Mn) NG injection at different time intervals. Error bars were derived from triplicate measurements.

FIGURE 10. Haemotoxylin and Eosin (H&E) staining images of major organs (Liver, Kidney, Spleen, Heart, and Lung) of human cervical carcinoma bearing nude mice injected phosphate buffered saline (PBS) as a control group and PEG-AlgPDA(Ca/Mn) NGs for 16 days as post injection (the scale bar is 100 µm).

34


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To investigate tumor targeting efficiency and excretion, the bio-distribution of PEGAlgPDA(Ca/Mn) NGs were

studied. Human cervical carcinoma tumor-bearing mice were

sacrificed at 4 hours, 24 hours, 5 days, and 16 days after injected with PEG-AlgPDA(Ca/Mn) NGs, while nude mice administrated with

PBS were

serving as the control group. Bio-

distribution profiles of PEG-AlgPDA(Ca/Mn) NGs were compared between vital organs and tumor tissue by investigating the remaining Mn using ICP-MS. The method therefore estimates Mn contents in PEG-AlgPDA(Ca/Mn) NGs at various times points and various organs, namely the kidneys, liver, spleen and tumor. Results are shown in Fig.11 and Table-S3,

significant

quantity of Mn was found in the liver, with accumulation reaching up to a mean of 222.3 ng/ml of Mn after one day. Early Mn buildup by the liver and spleen is predictable and is linked with the removal of NPs from the blood by cells of the MPS.62, 64 However, rapid drop of Mn-based NGs in the liver and spleen was observed after 1 day, possibly attributed to the PEG coating that may facilitate easy escape from the liver and spleen RES. After 16 days, lower uptake by the liver and spleen was observed, indicates a clearance of NGs through hepatobiliary transport.

62,

Typically, the hepatobiliary system serves as the main excretory pathway for nanoparticles bigger than 5-6 nm not excreted through the kidneys.65-66 In addition, to alleviate any toxicity caused by excessive accumulation, PEG surface coating is usually used to offer stealth properties to various nanoparticles for biological applications.63, 67 Mn rapidly accumulated in the tumor, reaching 32.29 ng/mL at 4 hours and rising to 140.9 ng/mL after 24 hours as illustrated in Fig.11 and Table S3. The leaky tumor vasculature and poor lymphatic drainage of tumors allow NGs

to accumulate in the tumor much more than that in

normal tissues, thereby increasing tumor uptake on the basis of enhanced EPR effect.39,

62

Alternatively, since PDA exists naturally in the body, AlgPDA(Ca/Mn)NGs have good 35



biocompatibility. The negative charge, proper size, and stealth properties of NGs may hinder NG-cell interactions during the blood circulation.39 These features make PEG-AlgPDA(Ca/Mn) NGs to extend blood circulation half-life time. Hence, improving tumor uptake because of EPR effect.

39, 62

Unlike most polymeric nanoparticles which severely accumulate in the RES

organs65-66, 68, the highest levels of Mn were noticed in the kidneys, demonstrating that PEGAlgPDA(Ca/Mn) NGs undergo renal clearance in vivo. Considering the kidney filtration threshold (KFT, 5.5 nm)68, PEG-AlgPDA(Ca/Mn) NGs should be disassembled into smaller molecules to be excreted from the kidneys. The Mn bio-distribution assessment results and the extend of Mn presence in vital organs and tissues as shown in Fig. 11 and Table S3a re consistent with the results of in vivo MRI experiments.

FIGURE 11. In vivo bio-distribution of Mn at various time points in major organs (4 hours, 24 hours,5 days, and 16 days) after injection AlgPDA(Ca/Mn) NGs and control without injection of the contrast agent . Error bars were based on triplicate measurements

3. CONCLUSIONS

36


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Manganese-based nanogels (Mn-based NGs) with high biocompatibility and contrasting abilities have been successfully prepared as a novel MRI agent using a very simple and non-toxic procedure that introduces calcium (Ca) and manganese (Mn) cations. Both AlgPDA(Ca/Mn) NGs and AlgDA(Ca/Mn) NGs showed significantly brighter MRI signals due to inherent Mnchelating properties of the NG. The longitudinal relaxation rate, measured using a 7.0 T MRI measurement system, was as high as 12.54 mM-1·s-1 in AlgPDA(Ca/Mn) and 10.13 mM-1·s-1for AlgDA(Ca/Mn) NG. The use of Mn ions as contrasting agents (CAs) and biogenic polymers as chelating agents provide a prepared CA with low cytotoxicity, demonstrating the superiority of the prepared CA over other MRI CAs in non-invasive tumor tissue recognition and comprehensive anatomical diagnosis. Therefore, the current bio-mimetic approach of coordinating Ca and Mn cations form AlgPDA/DA(Ca/Mn) NGs provides a promising potential MRI CAs for T1-weighted MR imaging. ASSOCIATED CONTENT Supporting Information Free manganese ions test using Xylenol orange dye, PEG surface coating, Mn ion released from AlgPDA(Ca/Mn), In vivo bio-distribution of Mn at various time points in major organs, DLS and zeta potential, standard curve showing Dopamine (DA) conjugation with (Alg),

1

alginate

H-NMR of DA, Alg and AlgDA, AlgPDA and AlgPDA (Ca/Mn)-PEG; SEM image of

AlgPDA(Ca/Mn) and AlgDA(Ca/Mn); XRD patterns of AlgDA(Ca/Mn), AlgPDA(Ca/Mn) and Alginate(Alg); XPS Surveys spectra of (A) AlgPDA(Ca/Mn) (B) AlgPDA(C/Mn) NGs;

PEG

surface coating to form AlgPDA(Ca/Mn)-PEG preparation; FTIR spectra of PEG, AlgPDA(Ca/Mn)-PEG, AlgDA and AlgPDA

AUTHOR INFORMATION Corresponding Author 37



Prof. Hsieh-Chih Tsai, E-mail: [email protected] Tel: +886-2-27303625, Fax: +886-227303733 Competing Interests The authors have declared that no competing interest exists.

ACKNOWLEDGMENTS The authors would like to thank the Ministry of Science and Technology, Taiwan, (MOST 1052221-E-011-133-MY3 and 105-E-2221-011-151-MY3) for providing the financial support.

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solid

lipid

nanoparticles

for

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resonance

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Nanomedicine:

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Bioinspired, Manganese-Chelated Alginate ... - ACS Publications

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