Zwitterionic Polydopamine-Coated Manganese Oxide Nanoparticles

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Interface Components: Nanoparticles, Colloids, Emulsions, Surfactants, Proteins, Polymers

Zwitterionic Polydopamine-Coated Manganese Oxide Nanoparticles with Ultrahigh Longitudinal Relaxivity for Tumor-Targeted MR Imaging Peng Wang, Xiaoying Xu, Yue Wang, Benqing Zhou, Jiao Qu, Jin Li, Mingwu Shen, Jindong Xia, and Xiangyang Shi Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.9b00013 • Publication Date (Web): 28 Feb 2019 Downloaded from http://pubs.acs.org on March 4, 2019

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Zwitterionic Polydopamine-Coated Manganese Oxide Nanoparticles with Ultrahigh Longitudinal Relaxivity for Tumor-Targeted MR Imaging

Peng Wang,a,1 Xiaoying Xu,a,1 Yue Wang,b, 1 Benqing Zhou,a Jiao Qu,b Jin Li,a Mingwu Shen,a Jindong Xia,*b Xiangyang Shi*a

a

State Key Laboratory for Modification of Chemical Fibers and Polymer Materials, International

Joint Laboratory for Advanced Fiber and Low-dimension Materials, College of Chemistry, Chemical Engineering and Biotechnology, Donghua University, Shanghai 201620, People’s Republic of China b

Department of Radiology, Shanghai Songjiang District Central Hospital, Shanghai 201600, People's Republic of China

Keywords: zwitterion; manganese oxide nanoparticles; antifouling property; polydopamine; targeted tumor MR imaging

_________________________________________________________________ * To whom correspondence should be addressed. E-mail: [email protected] (J. Xia) and [email protected] (X. Shi) 1

Authors equally contributed to this paper.

1

ACS Paragon Plus Environment

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ABSTRACT We present the design of antifouling zwitterion-functionalized manganese oxide (Mn3O4) nanoparticles (NPs) modified with folic acid (FA) for targeted tumor magnetic resonance (MR) imaging. In the current work, diethylene glycol-stabilized Mn3O4 NPs were initially prepared via a solvothermal approach, coated with polydopamine (PDA), fluorescently labeled with rhodamine B, conjugated with FA via amide bond formation, and finally covered with zwitterions of L-lysine (Lys). The thus generated multifunctional Mn3O4 NPs display excellent water-dispersibility and colloidal stability, good protein-resistance ability, and desirable cytocompatibility. With the PDA and Lys modification, the multifunctional Mn3O4 NPs own an ultrahigh r1 relaxivity (89.30 mM-1s-1), and enable targeted tumor MR imaging owing to the linked FA ligands. The designed antifouling zwitterion-functionalized Mn3O4 NPs may be employed as an excellent MR contrast agent for targeted MR imaging of other biological systems.

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INTRODUCTION Recent developments of nanotechnology have afforded the creation of a variety of nanoparticulate systems for theranostics of cancer.1-2 In general, most of the nanosystems are subjected to rapid clearance by the reticuloendothelial system (RES) organs such as liver, spleen, and lung after intravenous injection and the accumulation of the nanoscale therapeutic agents is usually less than 2% of the whole injected dose.3 To overcome such an obstacle, it is important to modify nanoparticle (NP)-based contrast agents (CAs) or drug payload with antifouling properties, allowing for extended blood circulation time and reasonable RES escape.4 The conventional adopted strategy to render the NPs with antifouling properties is to decorate polyethylene glycol (PEG), a hydrophilic and biocompatible polymer onto the particle surface.5 However, PEGylated NPs are easy to aggregate under a high ionic strength,6-7 and the PEG polymer possesses immunogenicity8 and is easy to be oxidized in the presence of oxygen and transition metal ions,9 hence the application of PEGylated NPs for nanomedicinal applications is quite limited for practical applications. Zwitterionic molecules contain equal amounts of positive and negative charges, resulting in a super-hydrophilic surface after the NPs are modified with them.10 Due to these properties, zwitterionic functionalization can be used to afford NPs with antifouling properties to have protein resistance ability, reduced uptake in macrophage cells, and prolonged blood half life.11-12 For instance, dendrimer-entrapped gold NPs modified with zwitterions of carboxybetaine acrylamide displayed good protein resistance and extended blood circulation time, allowing for effective blood pool, lymph node, and tumor computed tomography imaging.13 Ultrasmall iron oxide14 or manganese oxide15 NPs can be surface decorated with L-cysteine to have antifouling properties for improved tumor magnetic resonance (MR) imaging. L-lysine (Lys), as a kind of zwitterion containing an α-amino group and an α-carboxylic acid group, can be protonated and deprotonated 3


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under biological conditions, respectively.16 Besides, the remaining -amino group is easy to be modified with other active groups (e. g., targeting ligands or fluorescent dyes). Therefore, Lys with low fouling property has attracted great interest in resisting non-specific protein adsorption and cell adhesion.17 For instance, Lys has been covalently decorated onto the surfaces of gold18 and silica NPs,19 silica-titania,20 titanium oxide,21 niobium oxide,22 poly(ethylene terephthalate) sheet,23 and polyvinylidene difluoride membrane24 to attain a superior antifouling performance. To achieve accurate cancer diagnosis, it is of vital importance to select an appropriate imaging technique to distinguish tumors from normal tissues and organs. MR imaging, as a noninvasive medical diagnostic tool, can offer high spatial resolution and superb soft tissue contrast details as well as real-time monitoring feature. Various CAs, particularly T1-weighted positive CAs, are commonly used to improve the MR imaging sensitivity.25-26 Owing to the risk of nephrogenic systemic fibrosis induced by Gd(III)-based CAs, Mn-based CAs with low side effects have been regarded as one of the favorable alternatives for T1-weighted MR imaging,27-28 although their r1 relaxivity is usually lower than that of commercial Gd(III)-based CAs.15, 29-31 Recently, polydopamine (PDA) coating has been found to effectively increase the r1 relaxivity of Mn-based CAs. For example, PDA-coated Mn3O4 NPs loaded with quantum dots displayed an increased r1 relaxivity of 3.46 mM-1s-1, which was much larger than that of Mn3O4 NPs (0.33 mM-1s-1) before PDA coating.32 The r1 relaxivity of poly(acrylic acid)-modified MnCO3 NPs (6.90 mM-1s-1) could be increased to 8.30 mM-1s-1 under pH 6.0 after PDA coating.33 The PDA coating-rendered improved r1 relaxivity of Mn-based CAs is likely due to the fact that the PDA coating increases the overall molecular volume of the CAs to have prolonged rotational correlation time, and the hydrophilic nature of the polymer does not seem to impact the water accessibility of the core particles.32-34 Furthermore, PDA coating was demonstrated to significantly improve the 4


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water dispersibility and colloidal stability of the particles.35 Based on the antifouling characteristics of Lys zwitterions and the advantages of PDA coating, we report here a novel antifouling Mn3O4 NP-based probe with an ultrahigh r1 relaxivity for targeted tumor MR imaging. We first synthesized Mn3O4 NPs via a solvothermal method, coated them with PDA, fluorescently labeled them with rhodamine B (RB), conjugated folic acid (FA) ligands onto their surface, and finally covered the particle surface with zwitterions of Lys (Figure 1). The as-prepared multifunctional Mn3O4-PDA-RB-FA-Lys NPs were well characterized to delineate their structure, composition, morphology, stability, protein resistance ability, r1 relaxivity, and cytocompatibility. Their targeting specificity to FA receptor-expressing cancer cells was investigated by flow cytometry. Finally, the developed Mn3O4-PDA-RB-FA-Lys NPs were employed as a nanoprobe for targeted MR imaging of a tumor model. According to our literature investigation, this study is an innovative report concerning the preparation of Lys-modified Mn3O4 NPs with ultrahigh r1 relaxivity for targeted MR imaging of tumors.

Figure 1. Schematic presentation of the preparation of Mn3O4-PDA-RB-FA-Lys NPs.

EXPERIMENTAL SECTION Synthesis of the Mn3O4-PDA-RB-FA-Lys NPs. Mn(acac)3 (481.2 mg) was dissolved in 25 mL of DEG and continuously stirred for 3 h at 70 oC to form a homogeneous solution. After that, the 5


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solution was placed in a Teflon-lined stainless-steel autoclave (50 mL) and sealed under air atmosphere. Then the autoclave was heated at 180 °C for 24 h in an oven, and cooled down to room temperature. The reaction solution was dialyzed against water to get the water/DEG interface. Then upper phase of DEG was removed, and the remaining solution was dialyzed against water to obtain an aqueous solution containing manganese oxide (Mn3O4) NPs. The Mn3O4 NPs (50 mg, 9 mL water) was mixed with ammonia aqueous solution (NH4OH, 75 μL, 20%) and ethanol (4 mL) under mild stirring at room temperature for 30 min. Dopamine hydrochloride (50 mg) dissolved in water (1 mL) was injected into the above mixture solution, and the reaction was allowed to proceed for 30 h according to the literature.36 The PDA-coated Mn3O4 (Mn3O4-PDA) NPs were obtained by centrifugation in MicroSep™ Advance Centrifugal Device (Millipore, Billerica, MA) and rinsing with water. After that, RB (2 mg, 1 mL methanol) was mixed with the Mn3O4-PDA NPs (50 mg, 5 mL water) under vigorous stirring at room temperature for 24 h. The solution was dialyzed against water to obtain a water solution of Mn3O4-PDA-RB NPs. Afterwards, FA (5 mg, 1 mL DMSO) was activated with EDC (10.9 mg, 0.5 mL DMSO) and NHS (6.5 mg, 0.5 mL DMSO), followed by addition of Mn3O4-PDA-RB NPs (40 mg, 8 mL water) under stirring at room temperature for 3 days. The reaction mixture was dialyzed against water to obtain a water solution of the Mn3O4-PDA-RB-FA NPs. Subsequently, Lys solution (2 mg, 100 μL water) was added into the mixture of Mn3O4-PDA-RB-FA aqueous solution (30 mg, 10 mL water) and Tris buffer (pH = 9.0, 20 mM, 10 mL) under continuous stirring at room temperature for 24 h. The final Mn3O4-PDA-RB-FA-Lys NPs were acquired by centrifugation in MicroSep™ Advance Centrifugal Device and rinsing with water for three times. The final product was kept at 4 oC before further use. The non-targeted Mn3O4-PDA-RB-Lys NPs were also prepared under the same conditions. See full experimental details in Supporting Information.

RESULTS AND DISCUSSION 6


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Synthesis and Characterization of Mn3O4-PDA-RB-FA-Lys NPs. One-step solvothermal method was utilized to synthesize pristine Mn3O4 NPs through thermal decomposition of manganese salt Mn(acac)3 in diethylene glycol (DEG). Self-polymerization of dopamine was utilized to coat the Mn3O4 NPs to improve their r1 relaxivity, water dispersibility and colloidal stability. Subsequently, the Mn3O4-PDA NPs were modified with RB and FA and finally covered with zwitterion Lys via Michael addition (Figure 1). Fourier transform infrared spectroscopy (FTIR) spectra were used to confirm the PDA coating onto the particle surface (Figure S1). The conjugation of RB and FA onto the surface of PDA-coated Mn3O4 NPs was validated by UV-vis spectroscopy. As shown in Figure S2, after RB modification, the PDA-coated Mn3O4 NPs displayed an apparent absorption peak at 563 nm, which is the typical peak of RB. The modification of FA led to an obvious absorption peak at 282 nm, which can be ascribed to the conjugated FA ligands. These results demonstrated the successful grafting of RB and FA molecules onto the surface of PDA-coated Mn3O4 NPs. Next, thermogravimetric analysis (TGA) was performed to quantitatively assess the coating and decoration of PDA, RB, FA and Lys onto the surface of Mn3O4 NPs (Figure 2a). The pristine Mn3O4 NPs have a weight loss of 10.42% at 750 oC owing to the presence of DEG on the particle surface.37-38 After sequential reaction with the RB, FA and Lys, the weight loss of Mn3O4-PDA, Mn3O4-PDA-RB, Mn3O4-PDA-RB-FA and Mn3O4-PDA-RB-FA-Lys NPs are 34.36%, 34.92%, 38.11% and 41.92%, respectively at 750 oC. As a result, the loading percentages of PDA, RB, FA and Lys on the surface of the Mn3O4 NPs were quantified to be 23.94%, 0.56%, 3.19% and 3.81%, respectively. Meanwhile, the weight loss of Mn3O4-PDA-RB-Lys (as a control) was estimated to be 37.26%. Therefore, the loading percentages of Lys on the surface of the Mn3O4 NPs was quantified to be 2.34%.

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Figure 2. (a) TGA curves of Mn3O4, Mn3O4-PDA, Mn3O4-PDA-RB, Mn3O4-PDA-RB-Lys, Mn3O4-PDA-RB-FA and Mn3O4-PDA-RB-FA-Lys NPs, respectively. (b, c) TEM images and diameter distribution histogram of the Mn3O4-PDA-RB-FA-Lys NPs. (d) Protein resistance assay of the Mn3O4-PDA-RB-Lys, Mn3O4-PDA-RB-FA and Mn3O4-PDA-RB-FA-Lys NPs at different Mn concentrations incubated with BSA (1 mg mL-1) for 2 h. The mixture was centrifuged (8000 rpm, 10 min), and the absorbance at 278 nm before and after centrifugation were recorded. The reduced absorbance was used to reflect the protein resistance capability.

DLS and zeta-potential measurements were executed to further verify the surface conjugation of the Mn3O4 NPs (Table S1). Owing to the existence of DEG on the surface, the pristine Mn3O4 NPs exhibit a negative surface potential (-24.90 mV). Subsequent PDA coating obviously renders the particles to have an increased hydrodynamic size of 174.60 nm when compared to the pristine 8


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particles (118.20 nm) and does not seem to appreciably change their surface potential. Further modification of RB, FA, and Lys slightly affects the surface potential of the particles, while each step of modification appears to enlarge the hydrodynamic size of the particles from 180.3 nm (for RB) to 205.45 nm (for FA) and 198.7 nm (for Lys). It seems that the final Lys coverage of the particles slightly shrinks the particles likely due to the presence of strong hydration shell that can reduce

aggregation

and

improve

monodispersity

of

the

particles.

The

non-targeted

Mn3O4-PDA-RB-Lys NPs display the comparable surface potential of the targeted ones, and slightly smaller hydrodynamic size (170.8 nm) than the targeted ones (198.7 nm) due to the lack of FA modification. Besides, Mn3O4-PDA-RB-Lys, Mn3O4-PDA-RB-FA and Mn3O4-PDA-RB-FA-Lys NPs possessed good colloidal stability in water, PBS solution and cell culture medium, and no precipitates could be seen during one week’s storage at room temperature (Figure S3a). Moreover, there are no obvious changes in the hydrodynamic sizes of the Mn3O4-PDA-RB-Lys, Mn3O4-PDA-RB-FA and Mn3O4-PDA-RB-FA-Lys NPs after 4 days’ storage in water at 4 oC, indicating their desired colloidal stability (Figure S3b). The morphology and size of the Mn3O4-PDA-RB-FA-Lys NPs were checked by transmission electron microscopy (TEM, Figure 2b-c). Clearly, the Mn3O4 core particles display an elongated grain shape with a mean size of 3.89 ± 0.99 nm, and are crystalline in nature. It should be noted that the diffused TEM image of the Mn3O4-PDA-RB-FA-Lys NPs may stem from the thick PDA shell modification onto the particle surface. Protein Resistance Assay. To assess the antifouling property of the Mn3O4-PDA-RB-FA-Lys NPs, protein resistance assay was carried out through UV-vis spectroscopy monitoring. The changes of absorbance of the mixture containing bovine serum albumin (BSA) and Mn3O4 NPs with different types of surface modification at 278 nm were measured after incubation for 2 h, followed by 9


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centrifugation. As shown in Figure 2d, the protein adsorption of Mn3O4-PDA-RB-Lys, Mn3O4-PDA-RB-FA and Mn3O4-PDA-RB-FA-Lys NPs is Mn concentration-dependent with a higher Mn concentration resulting in a higher BSA adsorption. At the same Mn concentration of 50 M or above, the zwitterionic Mn3O4-PDA-RB-Lys and Mn3O4-PDA-RB-FA-Lys NPs display much less BSA adsorption than the Mn3O4-PDA-RB-FA NPs without Lys modification (p < 0.001). At the highest Mn concentration (200 μM) tested, the Mn3O4-PDA-RB-FA NPs absorb almost all BSA protein, which is approximately 2 and 3 times more than the Mn3O4-PDA-RB-Lys and Mn3O4-PDA-RB-FA-Lys NPs, respectively. It seems that at the highest Mn concentration tested, the antifouling property of the Mn3O4-PDA-RB-FA-Lys NPs is better than that of the Mn3O4-PDA-RB-Lys NPs. These results imply that the surface decoration of zwitterion Lys endows the PDA-coated Mn3O4 NPs with good protein resistance performance. MR Relaxometry. T1-weighted MR phantom studies of the Mn3O4-PDA-RB-FA-Lys NPs were carried

out.

The

aqueous

solutions

of

Mn3O4-PDA-RB-Lys,

Mn3O4-PDA-RB-FA

and

Mn3O4-PDA-RB-FA-Lys NPs showed enhanced MR contrast with the Mn concentration (Figure 3a). By plotting the relaxation rate (1/T1) versus Mn concentration, the r1 relaxivities of the Mn3O4-PDA-RB-Lys, Mn3O4-PDA-RB-FA and Mn3O4-PDA-RB-FA-Lys NPs were calculated to be 78.49, 65.87 and 89.30 mM-1s-1, respectively (Figure 3b), which are not only much higher than the Mn3O4 NPs described in our previous work15, 39-40 and commercial Magnevist, but also significantly higher than the PDA-coated Mn3O4 NPs reported in the literature.32-34 The ultrahigh r1 relaxivity might be attributed to the surface coating of PDA and modification of zwitterion Lys. In general, PDA coating renders the Mn3O4 NPs with enlarged molecular volume (accordingly extended rotational correlation time) and non-compromised water accessibility, thus having a large r1 value (65.87 mM-1s-1). Further zwitterion Lys modification increases the overall molecular volume of the 10


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particles without affecting their water accessibility. Hence, the Mn3O4-PDA-RB-FA-Lys NPs display a larger r1 value than Mn3O4-PDA-RB-FA NPs without Lys. The T1-weighted MR phantom studies and the T1 relaxometry data imply that the PDA-coated Mn3O4 NPs with or without FA or Lys modification can be employed as promising T1-weighted CAs for MR imaging applications. Besides,

the

r2

relaxivities

of

the

Mn3O4-PDA-RB-Lys,

Mn3O4-PDA-RB-FA

and

Mn3O4-PDA-RB-FA-Lys NPs were also measured to be 34.65, 53.21 and 25.99 mM-1s-1, respectively (Figure S4). The low r2/r1 ratios (0.29- 0.81) for the above three materials do not merit their applications for T2-weighted MR imaging.

Figure 3. (a) T1-weighted MR imaging of the Mn3O4-PDA-RB-Lys (I), Mn3O4-PDA-RB-FA (II) or Mn3O4-PDA-RB-FA-Lys (III) NPs at different Mn concentrations. (b) Linear fitting of 1/T1 of the corresponding particles versus Mn concentration.

Cytotoxicity and Cellular Uptake Assays. CCK-8 cell viability assay of KB cells was 11


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performed to evaluate the cytocompatibility of the Mn3O4-PDA-RB-FA-Lys NPs (Figure S5). Both Mn3O4-PDA-RB-Lys and Mn3O4-PDA-RB-FA-Lys NPs exhibit good cytocompatibility in the Mn concentration range of 0-400 μM. At the studied highest Mn concentration, the cells treated with both non-targeted Mn3O4-PDA-RB-Lys NPs and targeted Mn3O4-PDA-RB-FA-Lys NPs still display a viability of 92%, whereas the cells treated with Mn3O4-PDA-RB-FA NPs without Lys modification just have a viability of 65%. These data imply that the zwitterion Lys conjugation is beneficial to endow the particles with improved cytocompatibility. This is likely due to the fact that the zwitterionic modification of particles renders their lower affinity to and uptake by cells than the counterpart non-zwitterionic materials. The cytocompatibility of the Mn3O4-PDA-RB-FA-Lys NPs was further validated by cell morphology observation (Figure S6). To check the FA-mediated targeting specificity, cells treated with both Mn3O4-PDA-RB-Lys and Mn3O4-PDA-RB-FA-Lys NPs were subjected to flow cytometry assay. The FA-targeted Mn3O4-PDA-RB-FA NPs without Lys were also evaluated for comparison. As can be seen in Figure S7, at all Mn concentrations studied, non-targeted Mn3O4 NPs with Lys display much lower cellular Mn uptake than FA-targeted Mn3O4 NPs with or without Lys. These results imply that the linked FA affords the Mn3O4 NPs with targeting specificity to FA receptor-expressing cancer cells. At the Mn concentration of 100 μM, it seems that the Mn3O4-PDA-RB-FA-Lys NPs have a better cellular uptake than the Mn3O4-PDA-RB-FA NPs without Lys (p < 0.001). This means that with the zwitterion coverage the targeting specificity of the particles can be further improved at a relatively high Mn concentration, likely due to the good antifouling property of the particles rendered by Lys modification. In Vivo MR Imaging of a Xenografted Tumor Model. Next, we assessed the potential to use the designed Mn3O4-PDA-RB-FA-Lys NPs for targeted tumor MR imaging in vivo (Figure 4). All 12


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animal experiments were carried out following the protocols approved by the Ethical Committee of Shanghai Songjiang District Central Hospital and the policy of the National Ministry of Health. The MR signal intensity of the tumor treated with the targeted zwitterionic Mn3O4-PDA-RB-FA-Lys NPs significantly increases at 20 min postinjection, then levels off and gradually decreases (Figure 4a). In contrast, the MR signal intensity of the tumor treated with non-targeted zwitterionic Mn3O4-PDA-RB-Lys NPs slightly increases after injection and reaches the peak value at 130 min postinjection, which is more or less similar to the group of targeted Mn3O4-PDA-RB-FA NPs without Lys. This can be validated by quantitative tumor MR SNR data (Figure 4b). At the same time point postinjection, the tumor MR SNR follows the order of Mn3O4-PDA-RB-FA-Lys > Mn3O4-PDA-RB-Lys > Mn3O4-PDA-RB-FA. This means that with both zwitterion Lys and FA modification, the Mn3O4 NPs are able to be significantly delivered to the tumor site after intravenous injection, thereby affording improved targeted MR imaging of tumors. Without Lys decoration, the Mn3O4-PDA-RB-FA NPs might be largely cleared by RES-rich organs, hence having much less tumor uptake. The zwitterionic Mn3O4-PDA-RB-Lys NPs without FA just display EPR-based passive targeting to tumor site, hence having much less particle uptake in tumor region than the targeted ones. To sum up, our data suggest that the prepared Mn3O4-PDA-RB-FA-Lys NPs could be employed as a powerful nanoprobe for T1-weighted MR imaging of tumors. Biodistribution. In order to delineate the metabolism of the Mn3O4-PDA-RB-FA-Lys NPs in vivo, ICP-OES was executed to assess the biodistribution of the particles (Figure S8). At 1 h postinjection, the particles were predominantly taken up by liver, kidney and spleen, then were metabolized and gradually cleared out of body with the time postinjection. This indicates that the particles have a great biocompatibility and biological safety in vivo. Noting that, because maximal accumulation in tumor of the particles was at 20 min postinjection, the content of the particles in 13


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tumor was very low in the given studied time points.

Figure 4. (a) In vivo T1-weighted MR images of the KB tumor before and after intravenous injection of the Mn3O4-PDA-RB-FA, Mn3O4-PDA-RB-Lys or Mn3O4-PDA-RB-FA-Lys NPs ([Mn] = 5 μg/μL, in 100 μL PBS for each mouse, n = 1). The yellow arrow points to the tumor site. (b) MR SNR of the KB tumor before and after intravenous injection of the Mn3O4-PDA-RB-FA, Mn3O4-PDA-RB-Lys or Mn3O4-PDA-RB-FA-Lys NPs ([Mn] = 5 μg/μL, in 100 μL PBS for each mouse, n = 3, meaning that three different round locations in the same mouse tumor were chosen to calculate the MR signal intensity and background intensity, each circular position had a diameter of 4 mm).

CONCLUSION We have developed a convenient approach to prepare Mn3O4-PDA-RB-FA-Lys NPs with an ultrahigh r1 relaxivity and great antifouling property for targeted tumor MR imaging applications. Pristine DEG-stabilized Mn3O4 NPs are able to be coated with PDA, followed by sequential modification with RB, FA, and Lys. The last step of zwitterion Lys coverage is important to afford the particles with excellent antifouling property and to contribute to their ultrahigh r1 relaxivity (89.30 mM-1s-1) along with the PDA coating. The designed Mn3O4-PDA-RB-FA-Lys NPs display 14


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good water dispersibility and desired cytocompatibility, can target FA receptor-expressing cancer cells, and enable targeted and enhanced T1-weighted MR imaging of a xenografted tumor model. The fabricated Mn3O4-PDA-RB-FA-Lys NPs may be developed as an excellent nanoprobe for T1-weighted MR imaging of different types of FA receptor-expressing cancer.

ASSOCIATED CONTENT

Supporting Information Full experimental details, and data of DLS, zeta potential, FTIR spectra, UV-vis spectra, r2 relaxivity measurements, cytotoxicity assay, cell morphology observation, flow cytometry assay of KB cells, and in vivo biodistribution. AUTHOR INFORMATION Corresponding Author *E-mail address: [email protected] (J. Xia) and [email protected] (X. Shi) Notes The authors declare no competing financial interest.

ACKNOWLEDGMENTS This research is financially supported by the National Natural Science Foundation of China (81761148028 and 21773026), the Science and Technology Commission of Shanghai Municipality (17540712000 and 18520750400), and the Fundamental Research Funds for the Central Universities.

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Zwitterionic Polydopamine-Coated Manganese Oxide Nanoparticles

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