Zwitterionic Gadolinium(III)-Complexed Dendrimer-Entrapped Gold

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Biological and Medical Applications of Materials and Interfaces

Zwitterionic Gadolinium (III)-Complexed Dendrimer-Entrapped Gold Nanoparticles for Enhanced CT/MR Imaging of Lung Cancer Metastasis Jinyuan Liu, Zhijuan Xiong, Jiulong Zhang, Chen Peng, Barbara Klajnert-Maculewicz, Mingwu Shen, and Xiangyang Shi ACS Appl. Mater. Interfaces, Just Accepted Manuscript • Publication Date (Web): 09 Apr 2019 Downloaded from http://pubs.acs.org on April 9, 2019

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ACS Applied Materials & Interfaces

Zwitterionic Gadolinium (III)-Complexed Dendrimer-Entrapped Gold Nanoparticles for Enhanced CT/MR Imaging of Lung Cancer Metastasis

Jinyuan Liua, Zhijuan Xionga, Jiulong Zhangb, Chen Pengb,c*, Barbara Klajnert-Maculewiczd*, Mingwu Shena*, Xiangyang Shia,e*

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, P. R. China b Department

of Radiology, Shanghai Public Health Clinical Center, Fudan University, Shanghai

201508, P. R. China c Cancer

Center, Shanghai Tenth People’s Hospital, Tongji University School of Medicine, Shanghai

200072, P. R. China d Department

of General Biophysics, Faculty of Biology and Environmental Protection, University of

Lodz, 141/143 Pomorska St., 90-236 Lodz, Poland e

CQM - Centro de Química da Madeira, Universidade da Madeira, Campus da Penteada, 9020-105

Funchal, Portugal

Keywords: dendrimers; gold nanoparticles; RGD peptide; zwitterions; dual mode CT/MR imaging; lung cancer metastasis

________________________________ *

Corresponding

author.

Email

addresses:

[email protected]

(C.

Peng),

[email protected] (B. Klajnert-Maculewicz), [email protected] (M. Shen), and [email protected] (X. Shi)

1

ACS Paragon Plus Environment

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Abstract Design of dual mode or multimode contrast agents or nanoplatforms with antifouling properties is crucial for improved cancer diagnosis since the antifouling materials are able to escape the clearance of reticuloendothelial system with improved pharmacokinetics. Herein, we present the creation of zwitterionic gadolinium (III) (Gd(III))-complexed dendrimer-entrapped gold nanoparticles (Au DEN) for enhanced dual mode CT/MR imaging of lung cancer metastasis. In this present work, poly(amidoamine) (PAMAM) dendrimers of generation 5 were partially decorated with carboxybetanie acrylamide (CBAA), 2-methacryloyloxyethyl phosphorylcholine (MPC), and 1,3propane sultone (1,3-PS), respectively at different degrees, then used to entrap Au NPs within their interiors, and finally acetylated to cover their remaining amine termini. Through protein resistance, macrophage cellular uptake and pharmacokinetics assays, we show that zwitterionic Au DEN modified with 1,3-PS exhibit the best antifouling property with the longest half-decay time (37.07 h) when compared to the CBAA- and MPC-modified Au DEN. Furthermore, with the optimized zwitterion type, we then prepared zwitterionic Gd(III)-loaded Au DEN modified with arginineglycine-aspartic acid peptide for targeted dual mode CT/MR imaging of a lung cancer metastasis model. We disclose that the designed multifunctional Au DEN having an Au core size of 2.7 nm and a surface potential of 7.6 ± 0.9 mV display a good X-ray attenuation property, relatively high r1 relaxivity (13.17 mM s-1), acceptable cytocompatibility and targeting specificity to αvβ3 integrinexpressing cancer cells, and enable effective dual mode CT/MR imaging of a lung cancer metastasis model in vivo. The developed multifunctional zwitterion-functionalized Au DEN may be potentially adopted as an effective nanoprobe for enhanced dual-modal CT/MR imaging of other cancer types.

2


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Introduction Molecular imaging technologies have been increasingly employed for clinical diagnosis.1-2 The clinic imaging modalities include magnetic resonance (MR) imaging, computed tomography (CT), single photon emission computed tomography (SPECT), and positron emission tomography.3-6 Each imaging mode owns its inherent advantages and weaknesses, which makes it difficult to get accurate and reliable diagnosis results using single mode imaging techniques.7 To solve this problem, the combination of different imaging modes has been developed, such as CT/MR,8-13 SPECT/CT,14 SPECT/MR.15-16 For enhanced imaging purposes, these dual mode imaging practices are unable to be achieved due to the fact that the conventional clinical contrast agents are just for single mode imaging applications and their inherent drawbacks such as short blood circulation time, non-specificity to the target tissue and high concentration-induced renal toxicity. Rapid advances of nanotechnology have stimulated the development of a wide range of organic/inorganic nanomaterials as imaging agents to overcome the weaknesses of clinically used contrast agents.17-20 The major advantages of multifunctional nanoparticles for CT/MR imaging are 1) to improve the imaging precision; 2) to target tumor lesion with improved specificity; and 3) to have improved contrast enhancement when compared to conventional small molecular CT or MR contrast agents. In particular, polyethylenimine (PEI)-entrapped gold nanoparticles (Au NPs) can be coordinated with Gd(III) for dual modal tumor CT/MR imaging,21 and the imaging performance can be further improved by loading these particles within alginate nanogels.22 Similarly, Au NPs hybrid with iron oxide NPs can also be prepared via a self-assembly method,23 one-step hydrothermal approach,16 or thermal decomposition strategy24 for dual mode CT/T2-weighted MR imaging applications. In our previous work, we have shown that poly(amidoamine) (PAMAM) dendrimers of generation 5 (G5) are able to be entrapped with Au NPs and surface decorated with 2,2’,2’’-(10-(2(2,5-dioxopyrrolidin-1-yloxy)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl) acid/Gd(III) (DOTA/ Gd(III)) complexes for CT/MR imaging of different biosystems.23,

triacetic 25-26

The

distinct merits of the dendritic nanotechnology lie in the fact that the dendrimer surface can be 3



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decorated with different molecules through covalent bonding to have multifunctionalities, while their internal hydrophobic cavity can be used to entrap metal or inorganic NPs or hydrophobic drugs for different biomedical applications.27-28 For tumor diagnosis, it is essential to design a nanoparticulate system that can be efficiently taken up within the tumor site through not only enhanced permeability and retention (EPR)-based passive targeting, but also active targeting after the particles were linked with specific ligand such as folic acid (FA)29 or arginine-glycine-aspartic acid (RGD) peptide.30 Even with these properties owned, the NPs still have very limited accumulation in the tumor site after intravenous injection,31 mainly due to the huge clearance of NPs by the reticuloendothelial system (RES) organs (e.g., liver, spleen and lung). In order to overcome this issue, one has to modify the NPs with antifouling property, allowing for the escape of NPs from the RES organs to have improved pharmacokinetics. Traditional way to render the NPs with antifouling properties is to modify the particle surface with a hydrophilic polymer, polyethylene glycol (PEG).32-33 However, PEG possesses a temperaturedependent antifouling property, immunogenicity34-35 and is susceptible to oxidation in the presence of oxygen and transition metal ions,36-37 thereby limiting its practical applications. Recently, zwitterion modifications have received increasing attention to generate antifouling NPs,38-43 due to the unique structure of zwitterions with an equiv. molar ratio of positive and negative charges that can form a thick hydration layer onto the particle surface to render them with improved colloidal stability,44 corrosion resistance elimination of immunity,45 and prolonged blood circulation time.46 For instance, Mn3O4 NPs47 or ultrasmall Fe3O4 NPs48 can be coated with zwitterions of cysteine to possess extended blood circulation time for improved T1-weighted MR imaging of tumors. Dendrimer-entrapped Au NPs (Au DEN) modified with carboxybetaine acrylamide (CBAA) can be rendered with desired antifouling properties, thus enabling enhanced CT imaging of blood pool, lymph node, and tumors.49 In addition to carboxybetaine, other zwitterion-inducing agents, including phosphobetaine and sulfobetaine have also been widely studied.50-52 Unfortunately, currently there is no report specifically comparing the antifouling properties of NPs possessing different kinds of zwitterion modifications and further development of optimized zwitterionic NPs for dual mode CT/MR imaging of tumors. 4


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In the present work, inspired by the zwitterion-rendered antifouling property and our previous success in the development of Gd(III)-complexed Au DEN for dual mode CT/MR imaging,53 we report the optimization of three kinds of zwitterionic Au DEN with different modification degrees in terms of their antifouling properties, then the zwitterionic Au DEN with the optimal antifouling property were further developed to have targeting agent RGD and DOTA/Gd(III) complexes modified onto their surface. The materials were thoroughly characterized and their potential to be utilized for enhanced dual mode CT/MR imaging of a lung cancer metastasis model was explored. According to our thorough literature investigation, this is the original example relating to the optimization of zwitterion modification of Au DEN and the development of multifunctional zwitterionic Au DEN for enhanced CT/MR imaging applications.

Results and Discussion Synthesis and Characterization of CBAA-, MPC-, and 1,3-PS-Modified Au DEN The protocols we used to synthesize the three types of zwitterionic Au DEN are shown in Figure 1a. First, G5 PAMAM dendrimers with amine termini were partially functionalized with CBAA, MPC, and 1,3-PS, respectively, entrapped with Au NPs inside their internal cavities, and then acetylated to shield their remaining terminal amines. 1H NMR was performed to characterize the structure of the synthesized Au DEN before final acetylation step (Figure S1). For {(Au0)100-G5.NH2-CBAA20} and {(Au0)100-G5.NH2-CBAA80}, by comparison of the CBAA -CH2- proton peak at 1.86 ppm with the G5 methylene protons,49 the number of CBAA coupled to each G5 dendrimer was calculated to be 19 and 78, respectively (Figure S1a-b). Similarly, by comparison of the methyl proton of MPC at 0.97 ppm54 with dendrimer methylene protons, the {(Au0)100-G5.NH2-MPC20} and {(Au0)100-G5.NH2MPC80} were calculated to have 20 and 79 MPC moieties linked onto each dendrimer, respectively. Further, the comparison of the characteristic methylene protons of 1,3-PS at 1.88 ppm55 with those of G5 dendrimers led us to conclude that the number of 1,3-PS attached to each G5 dendrimer was 20 and 80, respectively for the {(Au0)100-G5.NH2-PS20} and {(Au0)100-G5.NH2-PS80}. Since the number 5



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of zwitterionic molecules attached onto each G5 dendrimer for each type of Au DEN are more or less similar, the further comparison of the antifouling property of these three kinds of zwitterionic Au DEN is reasonable. Acetylation was carried out to shield the residue dendrimer terminal amines according to the literature protocols.32 NMR was employed to validate the generation of dendrimer acetamides (Figure S2). Clearly, after the acetylation, the peaks at 1.8 ppm associated to the acetyl groups appear. Although there is somehow an overlap between the acetyl protons and the CBAA and 1,3-PS -CH2protons, the sharp peaks of acetyl protons are prominent.

Figure. 1 (a) Schematic representation of the preparation of CBAA-, MPC- or 1,3-PS-modified Au DEN. Et3N and Ac2O denote triethylamine and acetic anhydride, respectively. (b) The synthesis of RGD-Gd-Au DEN-PS. 6


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We further measured the surface potentials and hydrodynamic sizes of different zwitterionic Au DEN (Table S3). All of these Au DEN exhibit a surface potential in a range of 5-8 mV, suggesting that both zwitterion and acetylation modifications are effective to reduce the surface potential of amine-terminated dendrimers.49 Furthermore, the hydrodynamic sizes of all zwitterionic Au DEN are within a range of 130-176 nm with a low polydispersity index (0.2-0.5). It seems that with 1,3-PS modification, the hydrodynamic size of the Au DEN is slight larger than that modified with MPC or CBAA. UV-vis spectrometry was adopted to validate the creation of Au DEN (Figure S3). For all zwitterionic Au DEN, the peak at around 520 nm can be attributed to the typical surface plasmon resonance (SPR) peak of Au core NPs. In contrast, fully acetylated G5 dendrimers without entrapment of Au NPs (G5.NHAc) do not show such kind of absorption feature. The size and morphology of the Au core NPs were visualized by TEM (Figure S4). For all cases, the Au core NPs are within a range of 2.3-2.7 nm, suggesting that regardless of the different surface modifications of CBAA, 1,3-PS, and MPC with different modification degrees, the dendritic effect to entrap Au NPs using G5 dendrimer templates does not seem to be significantly impacted. It should be noted that the small sized Au core particles do not seem to correspond well with their SPR peak at 520 nm. We could attribute this phenomenon to the contribution of the largest gold core NPs in the sample. The gold content of each material was quantitatively measured by ICP-OES. Our data show that the added Au salt is able to be fully reduced to form the Au NPs. Synthesis and Characterization of Zwitterionic Dendrimeric CT/MR Contrast Agents According to our optimization of the antifouling property of zwitterionic Au DEN (see below), we selected final dendrimer amine coverage with 1,3-PS to generate multifunctional Au DEN for dual mode CT/MR imaging applications (Figure 1b). First, RGD-PEG-COOH was synthesized and confirmed by 1H NMR,56 and each PEG was found to be linked with 0.7 RGD moieties (Figure S5ab). G5.NH2 dendrimers were linked with DOTA-NHS and RGD-PEG-COOH, entrapped with Au NPs, chelated with Gd(III) ions via DOTA ligands, and finally modified with 1,3-PS through a ring opening 7



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reaction to generate zwitterionic {(Au0)100-G5-DOTA(Gd)-(PEG-RGD)-PS} NPs (for short, RGDGd-Au DEN-PS). To compare the targeting specificity, non-targeted counterpart materials of {(Au0)100-G5-DOTA(Gd)-mPEG-PS} NPs (for short, Gd-Au DEN-PS) were also prepared using mPEG-COOH, instead of RGD-PEG-COOH under the same experimental conditions. The modification degrees of DOTA and COOH-PEG-RGD (or mPEG-COOH) onto the surface of G5 dendrimers were quantified by 1H NMR. According to the integration data, the numbers of RGD-COOH-PEG and mPEG-COOH coupled to each dendrimer were found to be 10.1 and 8.9, respectively (Figure S5c-d). What’s more, according to the results of ICP-OES, for the RGD-Gd-Au DEN-PS and Gd-Au DEN-PS, the molar ratios of Au atom/dendrimer were found to be 98:1 and 96:1, respectively, further validating the complete transformation of Au(III) to Au(0). In addition, the Gd(III)/dendrimer molar ratios were measured to be 9.7:1 and 9.4:1 for RGD-Gd-Au DEN-PS and Gd-Au DEN-PS, respectively. These molecular compositions of the particles ensure the reasonable comparison of their x-ray attenuation property and r1 relaxivity for imaging applications (see below). The entrapment of Au NPs within the internal cavity of G5 dendrimers were verified by UV-Vis spectroscopy (Figure S6) and TEM imaging to have an average Au core size of 2.7 nm and 2.8 nm, respectively for Gd-Au DEN-PS and RGD-Gd-Au DEN-PS (Figure S7), similar to those of CBAA-, MPC-, and 1,3-PS-modified Au DEN (Figure S4). This suggests that the decoration of RGD-PEG and DOTA(Gd) onto the dendrimer surface does not seem to impact the size of Au core NPs. We then measured the hydrodynamic sizes of the Gd-Au DEN-PS and RGD-Gd-Au DEN-PS NPs in water to be 168.4 nm and 173.7 nm, which are significantly larger than those of Au core NPs determined by TEM. This is likely because TEM only images the single Au core particles, while dynamic light scattering (DLS) evaluates the whole particles composed of both dendrimers and Au cores in aqueous solution that may form a certain degree of aggregation. Hence, the result reflects the size of clusters that consist of many single Au DEN, in consistence with the literature.32 Furthermore, the surface potentials of the Gd-Au DEN-PS and RGD-Gd-Au DEN-PS were measured to be 6.3 mV and 7.6 mV, respectively. Owing to the 1,3-PS modification, the surface potentials of both particles were close to be neutral, much lower than those before modification (22.8 and 24.6 mV for {(Au0)1008


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G5-DOTA(Gd)-(PEG-RGD)} and {(Au0)100-G5-DOTA(Gd)-mPEG}, respectively). The stability of the developed RGD-Gd-Au DEN-PS and Gd-Au DEN-PS was assessed by DLS and ICP-OES, respectively (Figure S8). Clearly, the hydrodynamic sizes of both functional Au DEN dispersed in water do not exhibit obvious change, confirming their good colloidal stability. To examine the possible Gd(III) leakage, the relative Gd contents (% of origin) in the outer phase PBS solution after both particles were dialyzed for 1, 3 and 7 days at 37 oC are all less than 0.5% (Figure S9), indicating that the RGD-Gd-Au DEN-PS and Gd-Au DEN-PS NPs possess good stability without significant Gd(III) leakage.

Figure 2. Protein resistance assay of BSA (1 mg mL-1) incubated with {(Au0)100-G5.NH2-CBAA20}, {(Au0)100-G5.NH2-MPC20} and {(Au0)100-G5.NH2-PS20} (a), and {(Au0)100-G5.NH2-CBAA80}, {(Au0)100-G5.NH2-MPC80} and {(Au0)100-G5.NH2-PS80} (b) at different particle concentrations for 2 h. The absorbance at 278 nm was measured before and after centrifugation.

Protein Resistance Assay of CBAA-, MPC-, and 1,3-PS-Modified Au DEN Protein resistance assay was used to assess the antifouling property of the CBAA-, MPC-, and 1,3-PS-modified Au DEN according to the literature.49 Bovine serum albumin (BSA) exhibits a characteristic absorption peak at 278 nm. After 4 h incubation, the change of absorbance ( Δ absorbance) was calculated with a lower value meaning the better antifouling property (Figure 2). In all groups, the Δabsorbance increases with the dendrimer concentration, andΔabsorbance follows 9



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the order of {(Au0)100-G5.NHAc-PS20} < {(Au0)100-G5.NHAc-CBAA20} < {(Au0)100-G5.NHAcMPC20} at the same concentrations (Figure 2a). For the same type of zwitterion modification, the higher modification degree leads to the lowerΔabsorbance (Figure 2b). Overall, the mixture of BSA and {(Au0)100-G5.NHAc-PS80} shows the minimal value of Δabsorbance. Our results suggest that 1,3-PS modification may render the Au DEN with the optimal antifouling property. Cytotoxicity and Cellular Uptake Assays of CBAA-, MPC-, and 1,3-PS-modified Au DEN Cell counting kit-8 (CCK-8) assay was executed to analyze the cytotoxicity of zwitterionic Au DEN to macrophage cells (Figure 3a-b). For all materials, even at the maximum Au concentration (400 μM), the viabilities of cells are above 90%, implying that CBAA-, MPC-, and 1,3-PS-modified Au DEN exhibit negligible cytotoxicity in the range of studied concentrations.

Figure 3. CCK-8 assay of the viability of RAW264.7 cells treated with Au DEN with low (a) and high (b) zwitterion modification degrees at different Au concentrations for 24 h. Au uptake in RAW264.7 cells treated with Au DEN with low (c) and high (d) zwitterion modification degrees for 4 h at different 10


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dendrimer concentrations.

To further compare the antifouling properties of CBAA-, MPC-, and 1,3-PS-modified Au DEN, macrophage cellular uptake assay was performed. As shown in Figure 3c-d, cellular Au uptake increases with the concentration of Au DEN. At the same dendrimer concentration, macrophage cells always exhibit the lowest Au uptake for 1,3-PS-modified Au DEN when compared with the other two types of zwitterionic Au DEN. Higher modification degree of zwitterions, regardless their types, leads to much less Au uptake for the same type of zwitterion modification. Among these Au DEN, {(Au0)100G5.NHAc-PS80} shows the minimum macrophage cellular uptake of Au. Pharmacokinetics All animal experiments were performed complying with the protocols approved by the ethical committee of Shanghai Public Clinical Health Center and also in accordance with the policy of the National Ministry of Health. The pharmacokinetics of the Au DEN was investigated to validate the antifouling properties of the three types of zwitterionic Au DEN with a high modification degree (Figure 4 and Figure S10). The half-delay lives of the {(Au0)100-G5.NHAc-CBAA80}, {(Au0)100G5.NHAc-MPC80} and {(Au0)100-G5.NHAc-PS80} were measured to be 29.9, 28.9 and 37.7 h, respectively. The {(Au0)100-G5.NHAc-PS80} has the longest half-delay time, hence the best antifouling property, corroborating the protein resistance and macrophage cellular uptake assay results. 0

{(Au )100-G5.NHAc-CBAA80}

Au concentration (μg/g)

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Figure 4. Blood circulation and pharmacokinetics data of different zwitterionic Au DEN after intravenous injection of the corresponding Au DEN ([Au] = 0.1 M, in 100 μL saline for each mouse). The data represent the mean ± SD (n = 3).

Cytotoxicity and Cellular Uptake Assays of RGD-Gd-Au DEN-PS Based on the optimized type of zwitterions, we prepared RGD-targeted Au DEN chelated with Gd(III) with the remaining dendrimer terminal amines being covered with 1,3-PS. The prepared multifunctional RGD-Gd-Au DEN-PS and the non-targeted Gd-Au DEN-PS were subjected to cytotoxicity assay (Figure 5a). Apparently, the prepared RGD-Gd-Au DEN-PS and Gd-Au DEN-PS are non-toxic to B16 cells, and the cell viability is in all cases more than 90% in the investigated range of Au concentration.

Figure 5. (a) CCK-8 assay of the viability of B16 cells after they were treated with the Gd-Au DENPS or RGD-Gd-Au DEN-PS at varying Au concentrations for 24 h. (b) Au uptake in B16 cells that were treated with the RGD-Gd-Au DEN-PS or Gd-Au DEN-PS for 4 h at different dendrimer concentrations. RGD blocking + RGD-Gd-Au DEN-PS group represents the prior free RGD (5 μM) treatment overnight, followed by 4 h incubation with the RGD-Gd-Au DEN-PS.

To investigate the active targeting specificity of the RGD-Gd-Au DEN-PS, the Au uptake in B16 cells was tested (Figure 5b). Clearly, B16 cells treated with the RGD-Gd-Au DEN-PS display a 12


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significantly higher Au uptake than those treated with the non-targeted Gd-Au DEN-PS, and those pretreated with free RGD, followed by treatment of the RGD-Gd-Au DEN-PS at the same Au concentrations (p < 0.001). At the dendrimer concentration of 4 M, the cellular Au uptake of the GdAu DEN-PS, RGD blocking + RGD-Gd-Au DEN-PS and RGD-Gd-Au DEN-PS groups was 19.34, 19.47 and 27.96 pg/cell, respectively. These results suggest that RGD modification renders the prepared RGD-Gd-Au DEN-PS with specificity to target cancer cells expressing αvβ3 integrin. X-Ray Attenuation Property and T1 MR Relaxometry To check the property of the RGD-Gd-Au DEN-PS for imaging applications, CT and MR phantom studies were performed (Figure 6). Clearly, Au DEN and Omnipaque exhibit increased brightness and CT values with the Au or I concentration (Figure 6a). The CT values of RGD-Gd-Au DEN-PS and Gd-Au DEN-PS are much larger than Omnipaque at the same radiodense element concentrations (Figure 6b), suggesting their better X-ray attenuation effect than Omnipaque.

Figure 6. CT images (a) and CT values (b) of the RGD-Gd-Au DEN-PS (1), Gd-Au DEN-PS (2) and 13



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Omnipaque (3) at different Au or iodine concentrations. (c) T1-weighted MR images of the RGD-GdAu DEN-PS (1), Gd-Au DEN-PS (2) and RGD-Gd-Au DEN (3) at different Gd concentrations. (d) Linear fitting of 1/T1 versus Gd concentration of the above three types of particles.

To investigate the use of the RGD-Gd-Au DEN-PS for MR imaging, the r1 relaxivity of the particles was measured. We also included the {(Au0)100-G5-DOTA-(PEG-RGD)} (RGD-Gd-Au DEN) without 1,3-PS modification for comparison. All NPs are able to positively enhance the MR signal intensity in a dose-dependent manner (Figure 6c). The RGD-Gd-Au DEN-PS and Gd-Au DEN-PS display a similar r1 relaxivity (13.17 and 12.29 mM-1 s-1, respectively), which are seven times higher than the RGD-Gd-Au DEN (1.76 mM-1 s-1) without zwitterion modification (Figure 6d). The higher r1 relaxivity is likely due to the zwitterion coating that affords the particles with a significantly better water accessibility. Meanwhile, due to the hydration layer, the increased molecular volume of the particles render the complexed Gd(III) with a much longer rotational correlation time than that of particles without zwitterion modification. Hence, it is safe to conclude that 1,3-PS modification of the Gd-Au DEN can significantly improve the r1 relaxivity for sensitive MR imaging applications. It should be noted that the low r1 relaxivity of RGD-Gd-Au DEN without dendrimer surface zwitterion modification may be because the strong interaction of a portion of Gd(III) ions with the entrapped Au NP cores limits the water accessibility of the Gd(III) ions, in accordance with the literature.25 For both RGD-targeted and non-targeted Gd-Au DEN, their X-ray attenuation properties and r1 relaxivities are quite similar, ensuring the reasonable comparison of the RGD-mediated targeting specificity for imaging applications. Targeted Dual Mode CT/MR Imaging of Lung Cancer Metastasis Model in Vivo Next, we validated the feasibility to apply the RGD-Gd-Au DEN-PS for dual mode CT/MR imaging of a B16 lung cancer metastasis model (Figure 7). The CT images (Figure 7a) and CT value measurements (Figure 7c) obviously show that the tumor area exhibits a significant CT contrast enhancement with a higher CT value than with that before injection and after a peak time point of 60 min, the CT values start to gradually decline. The tumor CT values for the RGD-Gd-Au DEN-PS 14


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group are much higher than those for the non-targeted Gd-Au DEN-PS group at the same time points (Figure 7c). At 60 min postinjection, the CT value of tumor treated with the RGD-Gd-Au DEN-PS is 22.8 ± 1.2 HU, significantly higher than that treated with the Gd-Au DEN-PS (15.9 ± 1.1, p < 0.001). The results imply that both targeted and non-targeted Gd-Au DEN can reach the tumor site for CT imaging via passive EPR effect, while the RGD modification endows the particles with targeting specificity to αvβ3 integrin-expressing tumor cells, hence having enhanced tumor CT imaging.

Figure 7. In vivo CT images (a), T1-weighted MR images (b), CT values (c), and MR SNR (d) of B16 lung cancer metastasis model at different time points post intravenous injection of the RGD-Gd-Au DEN-PS (1) or Gd-Au DEN-PS (2). The red circles indicate the tumor area.

Similarly, the MR imaging results show that the tumor site has the highest MR imaging intensity at 60 min and then the MR signal to noise ratio (SNR) starts to decrease gradually, which is possibly owing to the metabolism of the Au DEN (Figure 7b and Figure 7d). Meanwhile, the tumor MR imaging intensity for the targeted group is obviously higher than that for the non-targeted group. At 60 min 15



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postinjection, the tumor MR SNR for the RGD-Gd-Au DEN-PS group is 61.7 ± 1.1, much higher than that for the Gd-Au DEN-PS group (43.8 ± 1.2, Figure 7d). Taken together with the CT imaging data, it is reasonable to claim that the prepared zwitterionic Gd-Au DEN modified with RGD enable targeted dual mode CT/MR imaging of a B16 lung cancer metastasis model. It is interesting to note that the CT/MR imaging signals reach the maximum at 1 h postinjection while the circulation half life time of the particles is around 37.7 h. This can be explained as follows: At 1 h postinjection of the hybrid particles, the injected particles are able to be quickly accumulated and remain saturated in the tumor region via a passive EPR-based targeting mechanism. With the time postinjection, part of the particles in the tumor lesion may intravasate back to the blood circulation, which may contribute to the prolonged half-decay time of the materials. Further in vivo biodistribution (Figure S11) and histological examination (Figure S12) studies reveal that the developed functional Au DEN are able to be metabolized and finally cleared out of the body and do not generate systemic toxicity to the major organs after intravenous injection. It is interesting to note that we have not confirmed that the Au DEN containing a non-biodegradable gold core are removed from body by renal clearance as the literature suggests.57 This is definitely an interesting point deserving our further study. With the RGD mediation, the RGD-Gd-Au DEN-PS display a higher Au uptake in lung than the Gd-Au DEN-PS at the earlier time points of 1 and 4 h, thereby enabled specific dual mode CT/MR imaging of the metastasis cancer model.

Conclusions In summary, we decorated Au DEN with three types of zwitterions at different modification degrees and reveal that 1,3-PS modification (with each G5 dendrimer reacted with 80 molar equiv. of 1,3-PS) renders the Au DEN with the best antifouling properties as verified through protein resistance, macrophage cellular uptake, and pharmacokinetics assays. Based on the optimization, we further developed stable RGD-targeted Gd-Au DEN-PS with an Au core size of 2.7 nm. With the 1,3-PS and RGD modifications and the presence of Au NPs, the multifunctional Gd-Au DEN display a good X16


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ray attenuation property, enhanced r1 relaxivity, and targeting specificity to cancer cells expressing αvβ3 integrin. With these properties owned along with the good cytocompatibility and biosafety, the developed RGD-Gd-Au DEN-PS enabled targeted dual mode CT/MR imaging of a B16 lung cancer metastasis model. Such design of zwitterionic Au DEN may be applied to the preparation of other functional NPs with good antifouling properties for different theranostic applications.

Experimental Section Synthesis of CBAA-, MPC-, and PS-Modified Au DEN. CBAA was first synthesized following previous literature protocols.49 G5 PAMAM dendrimers were partially modified by CBAA via Michael addition reaction according to the literature49 with slight modification to get the product of G5.NH2-CBAA20 or G5.NH2-CBAA80 dendrimers. G5.NH2 dendrimers were also partially modified with MPC via Michael addition. MPC (26.5 mg or 83.5 mg, in 5 mL water) was dropped into an aqueous solution of G5.NH2 dendrimers (10 mg, 5 mL) under stirring at room temperature. Three days later, the reaction mixture was dialyzed and lyophilized to get the G5.NH2-MPC20 or G5.NH2-MPC80 dendrimers according to protocols described above. Similarly, G5.NH2 dendrimers were also modified with 1,3-PS via a one-step ring-opening reaction. To be brief, 1, 3-PS (1.2 mg or 3.8 mg, in 5 mL water) was dropped into the G5.NH2 water solution (10 mg, 5 mL) under stirring at room temperature. After 3 days’ reaction, the mixture was subjected to the same dialysis and lyophilization processes to generate the G5.NH2-PS20 and G5.NH2PS80 products (see details in Table S1). Next, we prepared Au DEN using the above dendrimer derivatives as templates according to the literature.32 In all cases, the dendrimer/Au salt molar ratio was kept at 1:100. Under similar reaction and purification conditions, products of {(Au0)100-G5.NHAc-CBAA80}, {(Au0)100-G5.NHAc-MPC20}, {(Au0)100-G5.NHAc-MPC80}, {(Au0)100-G5.NHAc-PS20}, and {(Au0)100-G5.NHAc-PS80} DENPs were produced (Table S2). 17



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Synthesis of Dendrimer-Based CT/MR Contrast Agents. After optimization of the antifouling property of the zwitterion-modified Au DEN, we selected 1,3-PS-modified Au DEN for further preparation of multifunctional dendrimeric nanodevices for dual-mode CT/MR imaging application. To be brief, G5.NH2 dendrimer (20 mg, in 10 mL DMSO) was reacted with 10 molar equiv. of DOTANHS (5.86 mg, in 5 mL DMSO) under stirring at room temperature for 24 h, followed by dialysis and lyophilization according to the above procedures to get the G5.NH2-DOTA product. RGD peptide was linked with a dual functional PEG (COOH-PEG-NH2) through EDC chemistry to generate RGD-PEG-COOH according to the literature.58 Then RGD-PEG-COOH (20.68 mg, 5.0 mL MDSO) with 10 molar equiv. of G5 dendrimer was activated by EDC (14.74 mg, 3 mL DMSO) under stirring for 3 h, and reacted with G5.NH2-DOTA dendrimer (25.86 mg, 5 mL DMSO) under vigorous stirring for 3 days. Followed by dialysis and lyophilization, G5-DOTA-(PEG-RGD) dendrimers were obtained. The synthesized G5-DOTA-(PEG-RGD) dendrimers were used as templates to entrap Au NPs according to the literature.32 Briefly, the G5-DOTA-(PEG-RGD) dendrimer (20 mg) was dissolved in 20 mL of water, added with HAuCl4 solution (30 mg/mL, 0.44 mL water) under stirring for 30 min, and the mixture was reacted with an icy cold NaBH4 solution (4.23 mg, 1 mL water) with 3 molar equiv. of Au salt under stirring. After 3 h, an aqueous Gd(NO3)3 solution (1.45 mg, 1 mL water) was added to the mixture under stirring for 3 h to enable chelation of Gd(III) ions with dendrimers through the DOTA ligands. Lastly, the raw product of {(Au0)100-G5.NH2DOTA-(PEG-RGD)} dendrimers were reacted with 1, 3-PS (0.99 mg, in 10 mL water) to cover the remaining dendrimer terminal amines. The reaction mixture was stirred for 12 h, dialyzed, and lyophilized to get the final product of {(Au0)100-G5.NHPS-DOTA(Gd)-(PEG-RGD)} (for short, RGDGd-Au DEN-PS). For comparison, we also prepared the non-targeted {(Au0)100-G5.NHPSDOTA(Gd)-mPEG} DENPs (for short, Gd-Au DEN-PS) under similar experimental conditions using mPEG-COOH to modify the G5 dendrimers. See full description of experimental procedures in Supporting Information. ASSOCIATED CONTENT 18


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Supporting Information Full experimental section and additional data including zeta potentials, hydrodynamic size, 1H NMR spectra, UV-vis spectra, TEM images, stability assessment, biodistribution, and H&E staining of the main organ slices. AUTHOR INFORMATION Corresponding Author *E-mail address: [email protected] (C. Peng), [email protected] (B. Klajnert-Maculewicz), [email protected] (M. Shen), and [email protected] (X. Shi). Notes The authors declare no competing financial interest.

ACKNOWLEDGMENTS This study was financially supported by the National Natural Science Foundation of China (21773026 and 81761148028) and the Science and Technology Commission of Shanghai Municipality (17540712000 and 18520750400).

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Zwitterionic Gadolinium(III)-Complexed Dendrimer-Entrapped Gold

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