- Nano Express
- Open Access
AMP-Conjugated Quantum Dots: Low Immunotoxicity Both In Vitro and In Vivo
© Dai et al. 2015
- Received: 24 August 2015
- Accepted: 28 September 2015
- Published: 5 November 2015
Quantum dots (QDs) are engineered nanoparticles that possess special optical and electronic properties and have shown great promise for future biomedical applications. In this work, adenosine 5′-monophosphate (AMP), a small biocompatible molecular, was conjugated to organic QDs to produce hydrophilic AMP-QDs. Using macrophage J774A.1 as the cell model, AMP-QDs exhibited both prior imaging property and low toxicity, and more importantly, triggered limited innate immune responses in macrophage, indicating low immunotoxicity in vitro. Using BALB/c mice as the animal model, AMP-QDs were found to be detained in immune organs but did not evoke robust inflammation responses or obvious histopathological abnormalities, which reveals low immunotoxicity in vivo. This work suggests that AMP is an excellent surface ligand with low immunotoxicity, and potentially used in surface modification for more extensive nanoparticles.
- Quantum dots
- In vivo
Quantum dots (QDs) have garnered a great deal of attention because of their attractive photophysical properties, including a high photoluminescence quantum yield (PLQY), superior photostability and a narrow and symmetric emission spectrum coupled with a broad and continuous excitation spectrum [1–4]. CdSe nanocrystals are among the brightest, best-studied and most widely available quantum dots used for bioimaging , and they have been successfully prepared via the organometallic route with a PLQY as high as 85 % . However, organic synthesised QDs are typically hydrophobic in nature and thus cannot be used directly in biological applications. Post-treatment is therefore required to render QDs with aqueous dispersibility [7, 8]. The most common strategy for doing so is to engineer QDs via surface modifications [9–11]. However, surface coatings may lead to a significant size increase beyond the desired range [12, 13] and a decrease of the PLQY . More distinctly, different surface ligands have reportedly caused additional toxicity by interacting with host’s innate immune system and evoking significant inflammatory effects [15–17]. For example, the proinflammatory effects of poly(ethyleneglycol) (PEG)ylated CdSe/ZnS QDs were reported as being strongly associated with the functional groups (-COOH, -NH2, -OH and -OCH3) at the end of the PEG chain . It is no doubt that excessive immune responses will, to a certain degree, hamper QDs’ use in biological living systems.
Recently, Liu et al. reported the synthesis of adenosine 5′-monophosphate (AMP) modified CdSe/CdS/ZnS QDs (AMP-QDs) , which maintained a high PLQY (one nearly identical to that of the original oil-soluble QDs) and a small hydrodynamic size (~7.1 nm), and also showed excellent stability even under various extreme conditions (pH ranging from 3 to 13 and NaCl concentrations up to 5 M). All of these features make AMP-QDs potentially great tools for biological imaging. Because AMP is an universal molecule in biological systems, we hypothesised that AMP coatings could, to a certain degree, assist QDs in evading host’s innate immune system and thus render QDs with a low immunotoxicity.
In this work, AMP-modified quantum dots (AMP-QDs) were prepared as described by Liu et al. . To further explore the potential use of AMP-QDs in biological systems, we investigated their imaging behaviour in macrophage and evaluated their immunotoxicity both in vitro and in vivo.
Using J774A.1 as the macrophage cell model, we first investigated AMP-QDs’ imaging property by confocal laser scanning microscopy (CLSM) and cytotoxicity by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). Subsequently, acute inflammation responses in macrophage to AMP-QDs were assayed by real-time PCR (RT-PCR). Furthermore, using BALB/c mice as the animal model, blood circulation and biodistribution of AMP-QDs were studied by measuring Cd content, which was quantified with inductively coupled plasma-mass spectrometry (ICP-MS). Proinflammatory responses in immune organs to AMP-QDs were conducted by measuring key cytokines transcription levels including tumour necrosis factor-α (TNF-α) and interleukin (IL)-1β. Histopathological assay of immune organs was evaluated by haematoxylin and eosin (H&E) staining.
The water-soluble AMP or 3-mercaptopropionic acid (MPA) capped CdSe/CdS/ZnS QDs (AMP/MPA-QDs) were synthesized according to the procedure developed by Liu et al. [19, 20]. Oil phase CdSe/CdS/ZnS QDs with a fluorescence emission of 610 nm were used as the starting materials. All chemicals were obtained from Sigma (St. Louis, MO, USA) unless otherwise noted.
Characterisation of Quantum Dots
Absorption was measured on a Shimadzu UV-2450 spectrophotometer. Fluorescence emission spectra were obtained by a Cary Eclipse (Varian) fluorescence spectrophotometer. The morphology and size of quantum dots were analysed by transmission electron microscopy (TEM) obtained on a JEOL JEM-1400. The hydrodynamic size of quantum dots was investigated by dynamic light scattering (DLS) with Zetasizer NanoZS Instrument (Malvern Instrument Corporation).
Cell Lines and Cell Culture
The macrophage cell line J774A.1 was purchased from the China Center for Type Culture Collection (Wuhan, China). J774A.1 cells were cultured in DMEM media supplemented with 10 % foetal bovine serum and were cultured in a 5 % carbon dioxide atmosphere at 37 °C.
Imaging of QDs in J774A.1 Macrophage Cells
J774A.1 cells were seeded onto sterilised 17-mm-diameter glass coverslips in 12-well plates (1 × 105 cells per well) and incubated for 24 h at 37 °C. Cells were then washed with phosphate buffer saline (PBS) and incubated in a media in the presence of 50 nM AMP/MPA-QDs. After 12 h, they were then washed with PBS and prepared for staining using a fixative solution for 10 min at room temperature, and the nuclei were stained with 4', 6-diamidino-2-phenylindole (DAPI). The slides were imaged with a laser scanning confocal microscope.
Cell Uptake Efficiency of QDs Measured by ICP-MS
J774A.1 cells were seeded in 12-well plates (1 × 105 cells per well) and incubated for 24 h at 37 °C. Cells were then washed with PBS and incubated in a media in the presence of 50 nM AMP/MPA-QDs. After 12 h, they were then washed with PBS, and the cells were lysed in a 1-ml digest solution (HNO3: HCl ratio of 10:1). The intracellular Cd2+ content was quantified using inductively coupled plasma-mass spectrometry (ICP-MS) and compared with standards.
Twenty-four hours after cell seeding, J774A.1 cells were incubated with a range of concentrations of AMP/MPA-QDs for 24 or 48 h at 37 °C, and then 10 μl of MTT (5 mg/ml) was added to each well and allowed to incubate for 4 h. Next, 100 μl of 10 % sodium dodecyl sulfate (SDS) solution was added to dissolve the formazan crystals during an additional 4-h incubation. The absorbance of the MTT formazan was determined at A570 nm with a spectrophotometer (SpectraMax M5, Molecular Devices, USA) following noncellular background (i.e., a blank consisting of the complete media, yellow MTT and SDS solution) subtraction. Results are expressed as the percent of MTT conversion activity for the media-treated control cells and are composed of six biological replicates.
Quantitative RT-PCR Analysis of Gene in Macrophage
Primer sequences for the analysed genes in J774A.1 cells
Six-week-old female BALB/c mice were purchased from Shanghai SLAC Laboratory Animal Co., Ltd. The study was approved by the Experimental Animal Management and Ethics Committees of Shanghai Jiaotong University School of Pharmacy. For the AMP-QDs in vivo toxicity experiments, there were six mice per group at each time point for statistical analysis. The mice were intravenously injected with 100 μL of PBS solution as the control group and 100 μL of AMP-QDs solution containing 0.4 nmol QDs as the experimental group. We collected the mouse body weights at indicated time for 60 days. The female BALB/c mice were sacrificed and blood and organs were collected at several time points post-injection.
Blood Circulation and Biodistribution Experiments
After injection of the AMP-QDs and PBS solution, the mice were sacrificed by exsanguination at various time points. The immune organs and tissues, which included the liver, spleen, kidney and blood, were weighed and then dissolved in 5 ml of a digest solution (HNO3:HCl ratio of 10:1) overnight. A microwave digestion system was used to ensure continuous digestion. The mixed solution became clear following digestion, and it was then cooled down at room temperature. Each of the samples was diluted to 10 ml by Milli-Q water. ICP-MS was used to analyse the concentration of Cd2+ in each sample.
BALB/c mice were sacrificed. The major immune organs, including the liver, spleen and kidney, were collected and fixed with 10 % buffered formalin following a rinse with PBS. They were then embedded in paraffin, sectioned and finally stained with H&E staining to prepare them for examination by digital microscopy.
Transcription Analysis of Proinflammation Responses to AMP-QDs in Mice
Total RNAs from the liver, spleen and kidney were extracted by the TRIzol lysis reagent according to the manufacturer’s protocol. The expression of key inflammatory cytokines such as TNF-α and IL-1β was measured using RT-PCR as described in the above method.
The results were showed in mean ± standard deviation (s.d.). Statistical analysis was measured by two-tailed Student’s t test. A difference of P < 0.05 was considered to be statistically significant.
Characterization of Quantum Dots
Imaging of Macrophage with AMP-QDs
Cytotoxicity Analysis of AMP-QDs by MTT
Acute Inflammation Responses in Macrophage to AMP-QDs
In previous study, nanoparticles can induce acute inflammation in immune cells . In order to get more conclusive information about the immune response profile elicited by AMP-QDs in macrophages, the transcriptional levels of acute inflammation response genes at 4 h after adding AMP-QDs into J774A.1 cell cultures were determined by RT-PCR method.
Upon activation, TLRs recruit adaptor proteins such as myeloid differentiating factor 88 (MyD88) and trigger downstream signalling proteins such as NF-κB to regulate subsequent inflammation responses. NF-κB is a cytosolic transcription factor binding to nuclear DNA and activating transcription of target genes. In the classical activation pathway, activation of NF-κB is controlled by its inhibitory subunit, inhibitor of NF-κB (I-κB), which prevents NF-κB subunits from leaving the cytosol. As showed in Fig. 5b, slight upregulation of MyD88 (1.78-fold) combined with NF-κB (1.71-fold) and downregulation of I-κB (0.89-fold) were found in AMP-QDs-treated group, compared to the control group. This result suggest that AMP-QDs, followed by activating TLR2, further transduced the signals to MyD88 and NF-κB pathway.
Activated NF-κB pathway could induce proinflammatory cytokines including IL-1β and TNF-α , and eventually result in diverse cellular inflammatory responses including secretion of cytokines. Results are showed in Fig. 5c. In the cells treated by AMP-QDs, the mRNA expression of TNF-α and IL-1β are slightly increased by 1.62- and 1.60-fold, and the expression levels of TGF-β and MCP-1 are nearly not changed. These data revealed that AMP-QDs induced a low inflammation level in macrophage, while MPA-QDs could highly improve inflammation levels .
Together, we profiled the acute inflammation responses for AMP-QDs in macrophage, which involve the cascade activation from TLR2 to MyD88/NF-κB pathway then to proinflammatory cytokines. Our data proved that AMP-QDs orchestrated a mild inflammatory response in macrophage, which leads to a low level of immunotoxicity.
Blood Circulation and Biodistribution of AMP-QDs in Mice
Proinflammatory Responses in Mice to AMP-QDs
Body Weight Measurement
In this study, AMP, a small molecule universal to biological systems, was conjugated to oil QDs to synthesize hydrophilic AMP-QDs. AMP-conjugated QDs were shown to have prior imaging property, and more essentially a low immunotoxicity both in vitro and in vivo. Our results suggested that AMP-based surface conjugation might be applied as a general strategy to endow nanoparticles with more desirable biocompatibility.
The authors would like to thank Professor Xinhua Zhong for providing the oil phase CdSe/CdS/ZnS QDs. This work was supported by the National Special Fund for State Key Laboratory of Bioreactor Engineering, Grant No. 2060204.
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.
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