Polyethylene glycol-modified dendrimer-entrapped gold nanoparticles enhance CT imaging of blood pool in atherosclerotic mice
- Kaichuang Ye†1,
- Jinbao Qin†1,
- Zhiyou Peng†1,
- Xinrui Yang1,
- Lijia Huang1,
- Fukang Yuan1,
- Chen Peng2Email author,
- Mier Jiang1, 3Email author and
- Xinwu Lu1, 3Email author
© Ye et al.; licensee Springer. 2014
Received: 18 July 2014
Accepted: 20 September 2014
Published: 26 September 2014
We report a new use of dendrimer-entrapped gold nanoparticles (Au DENPs) modified by polyethylene glycol (PEG) with good biocompatibility for in vitro and in vivo imaging of atherosclerotic mice by computed tomography (CT). In this study, Au DENPs were synthesized using poly(amidoamine) (PAMAM) dendrimers of generation 5 (G5.NH2) modified by PEG monomethyl ether (G5.NH2-m PEG20) as templates. In vitro cytotoxicity and flow cytometry assays show that the formed PEGylated Au DENPs have good biocompatibility and are non-cytotoxic at the Au concentration up to 300 μM. Silver staining and transmission electron microscopy (TEM) further confirm that the Au DENPs are able to be uptaken by macrophages and are located dominantly in the lysosomes of the cells. Importantly, the formed PEGylated Au DENPs are able to be used for CT imaging of murine macrophages in vitro and macrophages in atherosclerotic mice in vivo using apolipoprotein-E-gene-deficient mice as a model. These findings suggest that the formed PEGylated Au DENPs are a promising contrast agent for CT imaging of atherosclerosis.
Atherosclerosis and its complications are the leading causes of morbidity and mortality in modern societies[1, 2]. Atherosclerosis is a processed disease, characterized by endothelial injury, lipid deposition, inflammation, and plaque formation[3–6]. Among the complications, ruptures of vulnerable atherosclerotic plaques are the major cause of death in cardiovascular diseases[7, 8]. Thus, the enhancement of early detection and identification of patients who are prone to plaque rupture is highly important. Recent studies have emerged to show the importance of macrophages in the formation and progression of atherosclerosis[9, 10], and the high macrophage content is one of the principal factors associated with plaque instability. Thus, new platforms and technologies are crucial for noninvasive imaging and assessment of the macrophage burden in atherosclerotic plaques.
The emergence of molecular imaging (MI) has drastically changed medical science in many ways, including the diagnosis of diseases in the early stages, the assessment of risks, the treatment outcome, and the follow-up[12–14]. Computed tomography (CT), one of the most reliable MI modalities, is widely used in cardiovascular diseases because of its properties of high resolution, sharp contrast, three-dimensional (3D) capabilities, and low cost in comparison to other imaging modalities[15–17]. Numerous studies showed that the key element in CT imaging of cardiovascular diseases is to design applicable and safe contrast agents with long imaging time, sharp contrast sensitivity, and excellent stability in vitro and in vivo[18, 19]. Given that clinically used ionic or non-ionic CT contrast agents have several drawbacks such as allergic reaction, short imaging time, renal toxicity at high concentrations, and nonspecificity[20, 21], developing novel nanoparticle (NP)-based contrast agents with excellent biocompatibility and prolonged blood circulation time is crucial for CT imaging.
Gold nanoparticles (AuNPs), one of the most promising nanomaterials, are widely used as X-ray CT imaging contrast agents in the diagnosis and therapy of tumors because of their in vitro and in vivo stability, easy surface modification, good biocompatibility, and higher X-ray attenuation characteristics than those of clinically used ionic or non-ionic iodinated CT contrast agents[17, 22]. However, to develop AuNP-based CT contrast agents with desirable properties, such as good biocompatibility, easy and controllable surface modification, sharp contrast, and prolonged imaging time[23–25], multiple complicated steps are generally required. Recent studies have shown that proper modification of AuNPs can extend their circulation time in cardiovascular diseases by decreasing the rapid uptake and clearance by the reticuloendothelial system (RES)[19, 26–28]. One promising and efficient technique to achieve this goal is to modify AuNPs with polyethylene glycol (PEG) molecules[29, 30].
In our previous reports[31, 32], we have shown that acetylated dendrimer-entrapped AuNPs (Au DENPs) synthesized using generation 5 (G5) poly(amidoamine) (PAMAM) dendrimers as templates are able to be used as contrast agents for CT imaging of C57 mice after intravenous injection. X-ray absorption coefficient measurements verified that the X-ray attenuation of Au DENPs was significantly higher than that of clinically used iodinated contrast agents at the same concentration (Au versus iodine)[32, 33]. The inferior vena cava (IVC) and pulmonary veins of the mice were clearly identified after intravenous injection of the Au DENPs. In our another work, we have shown that Au DENPs formed using PEGylated G5 dendrimers as templates have improved biocompatibility and are able to be used as contrast agents for blood pool and tumor CT imaging. These prior successes lead us to hypothesize that PEGylated Au DENPs can also be used to image blood pool in atherosclerotic mice in vitro and in vivo.
In this study, PEGylated Au DENPs were generated using G5 PAMAM dendrimers modified with PEG as templates. The formed PEGylated Au DENPs were systematically assessed in terms of their stability, biocompatibility, X-ray attenuation characteristics, and feasibility as contrast agents for CT imaging of murine macrophages in atherosclerosis. The biocompatibility of PEGylated Au DENPs was evaluated by cell counting kit-8 (CCK-8) assay for cell viability and flow cytometry for cell apoptosis. The distribution of Au DENPs within macrophages associated with atherosclerosis was observed by silver staining and transmission electron microscopy (TEM). Then, the PEGylated Au DENPs were used to diagnose the atherosclerotic mice model successfully. To our knowledge, this is the first report related to the PEGylated Au DENPs used as contrast agents for CT imaging of blood pool in atherosclerotic mice in vitro and atherosclerotic mice model in vivo.
Ethylenediamine core amine-terminated G5 dendrimers (G5.NH2) with a polydispersity index less than 1.08 were purchased from Dendritech (Dendritech Inc., Midland, MI, USA). PEG monomethyl ether with one end of maleimide (mPEG-MAL) was from Shanghai Yanyi Biotechnology Corporation (Shanghai, China). All other chemicals were obtained from Aldrich (Sigma-Aldrich, St. Louis, MO, USA) and used as received. Ana-1 cell line (a mouse macrophage cell line) was purchased from Shanghai Cell Bank, the Chinese Academy of Sciences (Shanghai, China). Penicillin, streptomycin, and fetal bovine serum (FBS) were purchased from Sigma (Sigma-Aldrich, St. Louis, MO, USA). Trypsin-EDTA, Dulbecco's PBS, RPMI 1640 medium, and bovine serum albumin were obtained from GIBCO-BRL (Life Technologies, Gaithersburg, MD, USA). The water used in all the experiments was purified using a Milli-Q Plus 185 water purification system (Millipore, Bedford, MA, USA) with a resistivity higher than 18 mΩ cm. Regenerated cellulose dialysis membranes (molecular weight cutoff, MWCO = 10,000) were acquired from Fisher (Thermo Fisher Scientific, Waltham, MA, USA).
Synthesis of [(Au0)300-G5.NHAc-m PEG] DENPs
The procedure used to synthesize [(Au0)300-G5.NHAc-m PEG] DENPs was adopted from our previous report. The solution of G5.NH2-m PEG and HAuCl4 (molar ratio between G5.NH2-m PEG and HAuCl4 is 1 to 300) was mixed for 30 min, followed by an addition of NaBH4 (four times molar excess of the Au salt) solution (V(H2O):V(CH3OH) = 1:1) under vigorous stirring for 2 h to obtain a wine red solution (denoted as [(Au0)300-G5.NH2-m PEG] DENPs).
Then, triethylamine (five times molar excess of the total primary amines of G5.NH2) was added to the aqueous solution of [(Au0)300-G5.NH2-m PEG] DENPs while under magnetic stirring. After 30 min, acetic anhydride (four times molar excess of the total primary amines of G5.NH2) was added to the above mixture solution under stirring, and the mixture was allowed to react for 24 h. The final reaction mixture solution was extensively dialyzed against PBS buffer (three times, 4 L) and water (three times, 4 L) for 3 days to remove the excess reactants and by-products, followed by lyophilization to obtain the PEGylated Au DENPs ([(Au0)300-G5.NHAc-m PEG] DENPs).
UV-vis spectra were obtained using a Lambda 25 UV/Vis spectrophotometer (Perkin Elmer, Waltham, Massachusetts, USA). Samples were dissolved in water before the experiments. TEM was performed using a JEOL 2010 F analytical electron microscope (JEOL Ltd., Akishima-shi, Japan) operating at 200 kV. An aqueous solution of [(Au0)300-G5.NHAc-m PEG] DENPs (5 μL, 1 mg/mL) was dropped onto a carbon-coated copper grid and air-dried prior to measurements.
Cell culture and characterization
Murine macrophages Ana-1 cells were cultured in RPMI 1640 medium with 10% heat-inactivated FBS and 1% v/v penicillin/streptomycin at 37°C and 5% CO2. Immunofluorescent staining was performed to characterize the Ana-1 cells. Ana-1 cells were fixed with 4% paraformaldehyde for 20 min at 37°C. After permeabilization in 0.3% Triton-X100 (diluted in PBS), the cells were incubated with 10% goat serum albumin diluted in 0.5% bovine serum albumin (BSA) for 30 min at 37°C to block nonspecific antibody adhesion. The primary macrophage antibodies F4/80 (rat monoclonal, 1:500 diluted in 0.5% BSA; Abcam, Cambridge, UK) and MAC-3 (rat monoclonal, 1:500 diluted in 0.5% BSA; BD, Franklin Lakes, NJ, USA) were incubated at 4°C overnight. After rinsing with PBS, Alexa Fluor® 555 (goat anti-rat, 1:500 diluted in 0.5% BSA; Invitrogen, Grand Island, NY, USA) was applied and incubated for 1 h at 37°C in the dark. After further washing with PBS, the cell nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI) (1:500, diluted in 0.5% BSA, DAKO, Real Carpinteria, CA, USA) for 20 s. After a final washing with PBS, the cells were examined under fluorescence microscope (Nikon, Chiyoda-ku, Tokyo, Japan).
To characterize the phenotypes of Ana-1 cells, 1 × 106 cells were washed with PBS and incubated with phycoerythrin (PE)-conjugated anti-mouse antibodies against F4/80 for 30 min at 4°C in the dark. Isotype control antibodies were used as the control group (all from eBioscience, San Diego, CA, USA). After washing, the cells were analyzed by a flow cytometer (Beckman Coulter, Fullerton, CA, USA).
Cytotoxicity assay of PEGylated Au DENPs
The viability of macrophages treated with PEGylated Au DENPs was evaluated via CCK-8 (Dojindo Laboratories, Kumamoto, Japan) assay. The assays were carried out according to the procedure described in a previous report. In brief, Ana-1 cells were seeded in 96-well culture plates at a density of 1 × 105 cells per well in quadruplicate and allowed to grow for 24 h. Then, the medium was replaced by RPMI 1640 medium containing PEGylated Au DENPs with different concentrations (0 to 4 μM), and the cells were incubated for 24 h at 37°C. After treatment, the medium was aspirated and cells were washed with PBS thrice. Then, 10 μL of CCK-8 diluted in 100 μL of fresh RPMI 1640 medium was added to each well and the cells were incubated for additional 2 h at 37°C. The absorbance was measured at 450 nm. The assay was carried out according to the manufacturer's instructions. For each concentration of Au DENPs, mean and standard deviation of the quadruplicate wells were reported.
The cytotoxicity of PEGylated Au DENPs was further examined by flow cytometric detection of cell apoptosis. Ana-1 cells were seeded in 6-well plates at a density of 2 × 106 cells per well in quadruplicate and grown to confluence for 24 h. The medium was replaced with a fresh medium containing PEGylated Au DENPs with two different concentrations (1 and 3 μM), and the cells were incubated for 24 h. Then, the cells were harvested, washed with PBS, fixed in 0.5% paraformaldehyde citrate buffer for 2 h, centrifuged, and resuspended with PBS at a cell density of 1 × 106 cells/mL. The cell suspensions were stained with Annexin V for apoptosis tests and analyzed by Cell Quest software (FACS Calibur, BD, Franklin Lakes, NJ, USA).
In vitro micro-CT imaging of macrophages
Ana-1 cells were seeded in 6-well plates at a density of 2 × 106 cells per well in triplicate and allowed to grow to 80% to 90% confluence for 24 h. Then, Ana-1 cells were incubated with fresh medium containing PEGylated Au DENPs (0, 100, and 300 μM, respectively) for 4 h at 37°C. After washing with PBS, cells were trypsinized, centrifuged, and resuspended with 100 μL PBS in a 0.5 mL Eppendorf tube containing approximately 4.0 × 106 Ana-1 cells for each tube. The cell suspension in each tube was placed in a self-designed scanning holder and then scanned using a micro-CT imaging system (eXplore Locus, GE Healthcare, London, Ontario, Canada) with the following operating parameters: tube voltage, 80 kV; tube current, 450 μA; exposure time, 400 ms; slice thickness, 45 μm; and scan field of view, 45 × 80 mm. Images were reconstructed on a micro-CT imaging workstation (GEHC microView, GE Healthcare, London, Ontario, Canada) using the following parameters: voxel, 45 × 45 × 45 μm; display field of view, 10 × 25 mm. CT values were acquired on the same workstation using the software supplied by the manufacturer. Each experiment was carried out in triplicate.
Cellular uptake of PEGylated Au DENPs
The cellular uptake of PEGylated Au DENPs was verified by silver staining and TEM according to methods reported in our previous work[17, 29]. For silver staining experiments, Ana-1 cells were seeded at a density of 2 × 106 per well in 6-well plates inserted with polylysine-coated cover slips for 24 h and treated with fresh medium containing PEGylated Au DENPs (1 and 3 μM) for 24 h at 37°C. After washing with PBS, the cells were fixed in 4% paraformaldehyde solution for 2 h. Silver staining was performed according to the manufacturer's instruction. Cells were washed with PBS thrice, stained with 1% nuclear fast red to stain the cell nuclei, washed, air-dried, dehydrated, cleaned, and mounted for light microscope observation.
For TEM imaging of the cellular uptake of PEGylated Au DENPs, Ana-1 cells were seeded at a density of 2 × 106 per well in 6-well plates for 24 h to grow to 90% confluence and treated with a fresh medium containing PEGylated Au DENPs (3 μM) for 24 h at 37°C. After washing thrice with PBS, the cells were trypsinized, centrifuged, and fixed with 2.5% glutaraldehyde in 0.2 M phosphate buffer (pH 7.2) for 12 h at 4°C, and post-fixed with 1% OsO4 in 0.2 M phosphate buffer (pH 7.2) for 2 h at 4°C. After washing with the phosphate buffer, the cells were dehydrated in a series of ethanol solutions of 30%, 50%, 70%, 95%, and 100% for 50 min, and then embedded with Epon 812 (Shell Chemical, London, UK) for polymerization. The embedded cells were sectioned to achieve a thickness of 75 nm using a Reichert ultramicrotome, and the samples were mounted onto 200 mesh copper grids. After negative staining with uranyl acetate and lead citrate for 5 min, the grids were visualized using an H600 transmission electron microscope (Hitachi Ltd, Chiyoda-ku, Japan) with an operating voltage of 60 kV.
CT imaging of an atherosclerotic mice model
We used an apolipoprotein-E-gene-deficient (ApoE-/-) mice model of atherosclerosis in the experiment. This animal model is generally accepted in the field. This model can imitate pathological changes in atherosclerotic lesions with high consistency when fed with a Western diet. Animal experiments and animal care were carried out according to protocols approved by the Institutional Committee for Animal Care and in accordance with the policy of the National Ministry of Health. For CT imaging, ApoE-/- mice (30 to 45 g, 1 year old, n = 8) purchased from the Shanghai Research Center for Model Organisms (Shanghai, China) were anesthetized by intraperitoneal injection of pentobarbital sodium (40 mg/kg). PEGylated Au DENPs (2.0 μmol Au/g body weight) dispersed in PBS buffer were then intravenously injected into the mice through the tail vein. The mice were scanned by a micro-CT imaging system (eXplore Locus, GE Healthcare, London, Ontario, Canada) with a tube voltage of 80 kV, an electrical current of 450 μA, and a slice thickness of 45 μm. CT scanning was performed both before and after intravenous injection of PEGylated Au DENPs (100 μL, [Au] = 0.1 M) at time points of 20 min, 2, 4, and 6 h post-injection, respectively. Images were reconstructed on a micro-CT imaging workstation using the following parameters: voxel, 45 × 45 × 45 μm; display field of view, 10 to 25 mm. CT values were acquired on the same workstation using the software supplied by the manufacturer.
Histological characteristics and macrophage uptake of PEGylated Au DENPs
After micro-CT scanning, the animals were anesthetized and perfusion-fixation was performed. The heart, lung, liver, spleen, kidney, intestine, and vessel were harvested and immediately fixed in a 4% formalin solution. Then the organs were dehydrated and embedded with paraffin. The paraffin-embedded samples were cut into 5-μm sections using a conventional microtome. For silver staining, the sections were dewaxed, washed thoroughly in PBS, and stained with the silver enhancement kit according to the manufacturer's instructions. Sections were then washed with PBS and counterstained with 1% nuclear fast red. After washing thrice with PBS, the sections were then air-dried, dehydrated, and mounted onto cover slips for light microscope observation.
For HE staining, the pre-treated sample sections were dipped in a Coplin jar containing hematoxylin for 30 s. The samples were then rinsed with water for 1 min and stained with 1% eosin Y solution for 30 to 60 s. Finally, the cover slips were dehydrated and mounted onto glass slides before microscopic imaging.
In vivo biodistribution of PEGylated Au DENPs
After CT imaging of macrophages in ApoE-/- mice model of atherosclerosis at 6 h post-injection of PEGylated Au DENPs via intravenous injection, the mice were euthanized. Then, the heart, lung, stomach, spleen, liver, intestine, kidney, testicle, blood, and brain were extracted and weighed. The organs were cut into 1- to 2-mm2 pieces and incubated in aqua regia solution for 4 h. Au content was determined by a Leeman Prodigy inductively coupled plasma-atomic emission spectroscopy (ICP-AES) system (Teledyne Leeman Labs, Hudson, NH, USA).
The data were expressed as mean values ± standard deviation (SD). For quantitative comparison and analysis, the values were evaluated by Student's t-test and a one-way analysis of variance (ANOVA) method. A probability value 0.05 was considered to indicate statistical significance, and the data were labeled with (*) for p < 0.05 and (**) for p < 0.01.
Results and discussion
Synthesis of PEGylated Au DENPs
Our previous work has shown that G5.NH2 dendrimers modified with m PEG-MAL (G5.NH2-m PEG) can be used as templates to entrap AuNPs with higher Au loading content and good stability. The modification of m PEG-MAL effectively improved the cytocompatibility of the formed PEGylated Au DENPs.
Characterization of macrophages
Cytotoxicity of PEGylated Au DENPs
Cell apoptosis is regarded as one of the most important characteristics for cytotoxicity. To test the influence of PEGylated Au DENPs on macrophage apoptosis, the cells incubated with the particles were analyzed by flow cytometry (Additional file1: Figure S1). The apoptotic index of Ana-1 cells treated with PEGylated Au DENPs at Au concentrations of 100 and 300 μM were estimated to be 3.3% ± 0.29% (Additional file1: Figure S1b) and 3.57% ± 0.16% (Additional file1: Figure S1c), respectively, with no statistically significant difference from that of the control cells treated with PBS (2.6% ± 0.27%, p > 0.05). This result suggests that PEGylated Au DENPs have no apoptotic effect on macrophage. Thus, the combined results of CCK-8 assay for cell cytotoxicity and flow cytometry analysis for cell apoptosis indicate that the PEGylated Au DENPs are non-cytotoxic in the given concentration range, which is essential for their uses in CT imaging of macrophages in atherosclerosis.
In vitro micro-CT imaging
Cellular uptake of PEGylated Au DENPs
TEM imaging of macrophages treated with PEGylated Au DENPs was also performed to identify the particle distribution in subcellular structures. After incubation with PEGylated Au DENPs (300 μM) for 24 h, numerous high electron-staining particles were found in the cytoplasm of cells (Figure 5d,e), especially in the lysosomes or phagocytic vacuoles. By sharp contrast, no electron-staining particles were observed in the cytoplasm of the control Ana-1 cells treated with PBS (Additional file2: Figure S2). The TEM results show that PEGylated Au DENPs were phagocytized by macrophages instead of adhering to the surface of cells. The internalization of PEGylated Au DENPs likely occurs through two different mechanisms, such as phagocytosis and diffusion via cell walls, in agreement with previous literature reports[37, 38]. The TEM data also verify that the incubation of PEGylated Au DENPs with a high Au concentration (300 μM) do not significantly affect the morphology and subcellular structures of cells, corroborating the CCK-8 and cell apoptosis assay data.
CT imaging of an atherosclerotic mice model
In vivo macrophage uptake of PEGylated Au DENPs
Biodistribution of PEGylated Au DENPs
In summary, PEGylated Au DENPs were synthesized and used as contrast agents for CT imaging of macrophages associated with atherosclerosis in vitro and in vivo. The combined results of CCK-8 assay and flow cytometry analysis show that the PEGylated Au DENPs are non-cytotoxic at the Au concentration up to 300 μM. Silver staining and TEM data confirm that the PEGylated Au DENPs can be efficiently phagocytized and are mainly located in the lysosomes of macrophages. Further in vitro CT imaging of Ana-1 cells treated with the PEGylated Au DENPs and in vivo blood pool CT imaging of ApoE-/- mice intravenously injected with the particles indicates that the developed PEGylated Au DENPs are able to image macrophages in vitro and the atherosclerotic mice in vivo. Findings from our study suggest that the developed Au DENPs have a great potential to be used as contrast agents for CT imaging of blood pool in atherosclerotic mice, which is important for the understanding of the pathological physiology of cardiovascular disease.
This research is financially supported by the National Natural Science Foundation of China (81370423, 81371517, and 81401458), the Shanghai Jiao Tong University School of Medicine Doctoral Innovation foundation (BXJ201231, for JQ), and the Fund of the Science and Technology Commission of Shanghai Municipality (Grant nos. 134119a2100, 14ZR1432400, 20124Y132 and 124119a4900). The authors appreciate the technical supports from Wei-Liu, De-min Yin, Li-juan Zong, Juan-juan Wu, Jing-jun Chen, and Wan-yao Xia in the laboratory.
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