Bioluminescent magnetic nanoparticles as potential imaging agents for mammalian spermatozoa.
Background
Nanoparticle-based biomedical applications include nanomedicine, bioimaging and theranostics [1]. Recently, nanoparticle-based bioimaging technologies have focused on bioluminescent resonance energy transfer-conjugated quantum dots (BRET-QDs), such as PbS, CdSe/ZnS, and CdTe/CdS QDs [2–6]. However, BRET-QDs are under scrutiny due to their cadmium content, since cadmium has known toxicity [7, 8].
An alternative noninvasive bioimaging system could explore luciferase enzymes, which are found in nature and has inherent light emission characteristics, for bioluminescence imaging in whole animal and cellular systems. For example, luciferase obtained from Renilla reniformis has been coupled with CdSe/ZnS QDs to create self-illuminating nanoparticles for dual imaging purposes. In this complex, the chemical energy generated by the reaction of luciferase with its substrate (coelenterazine or luciferin) produced light (bioluminescence) which excited the QD for a bright fluorescence emission [9, 10]. Similarly, firefly Photinus pyralis luciferase has been combined with core–shell quantum rods (CdSe/CdS or CdSe/CdS/ZnS), producing a significant increase and optimization in BRET ratios [11].
In biomedical applications, core–shell nanostructures comprised of a magnetic core present a unique opportunity for multi-functionality, incorporating optical imaging with tracking, sorting and/or cellular manipulation [12, 13]. Superparamagnetic iron oxide nanoparticles are clinically approved by the European Medicines Agency (EMA) and U. S. Food and Drug Administration (FDA), and have been used to label and track cells via MRI techniques [14].
Magnetic nanoparticles have shown viability in labeling and tracking applications; however, we are interested in using magnetic nanoparticles to enable cell detection, labeling and sorting without further perturbation of their viability—which would be the case when using fluorescent agents requiring additional excitation. In this study, coupling firefly luciferase (Photinus pyralis) with a magnetic nanoparticle carrier is expected to provide a multifunctional nanocomposite with both magnetic manipulation and bioimaging properties.
One objective of this work is to describe the synthesis and in situ characterization of core–shell nanocomposites comprised of a citric acid-stabilized magnetic nanoparticle core surrounded by a spherical shell of the bioluminescent firefly luciferase (Photinus pyralis) enzyme. A second objective is to analyze preliminary bioluminescence data from boar spermatozoa incubated with the newly synthesized luciferase-modified magnetic nanoparticles (Luc + MNP).
This analysis is presented using chemical and morphological characterization of the luciferase-magnetic nanoparticle composite and bioluminescence imaging, along with comparative data from a commercial BRET-QD that served as bioluminescence control.
Results
Iron oxide magnetic nanoparticles (MNPs) were synthesized using a co-precipitation technique that is described in detail in the methods section. After synthesis, MNPs were stabilized with a citric acid coating to form the citric acid-magnetic nanoparticle (CA-MNP) conjugate. A second reaction added the firefly luciferase enzyme onto the periphery of the CA-MNP to form the luciferase-CA-MNP (Luc + MNP) complex.
With the goal of performing cell sorting through nanotechnology tools, one advantage of using luciferase enzymes as imaging probes resides on the avoidance of additional light excitation that may damage cells when using fluorescent probes [18].
In situ ATR-FTIR spectroscopy was used to confirm the chemical changes in the MNPs for each reaction step (Fig. 1). For CA-MNPs, a strong peak at ~1645 cm−1 was observed corresponding to the symmetric carbonyl (C = O) vibrations of the carboxylic acid groups (–COOH) in citric acid when bound to iron oxide [19]. FTIR spectra of neat firefly luciferase showed distinctive peaks at 1550 cm−1 and 1515 cm−1, which are amide-II vibrations characteristic of luciferase [20].
In addition, the peak near 1650 cm−1 corresponds to an amide-I band commonly observed when multiple α- and β-functional groups are present, and has been previously observed for luciferase [21]. After addition of luciferase to the CA-MNPs complex, distinctive peaks were observed for the Luc + MNP samples that match the spectral signature for neat luciferase and indicate strong binding between luciferase and the CA-MNPs. Amide-II peaks observed in the neat luciferase spectra were also observed in the luciferase-CA-MNP (Luc + MNP) spectra.
In addition, the strong peaks at ~1400 and ~1350 cm−1 and the broadening and slight shift of the Amide-I peak is indicative of strong binding interactions between the amide/amine groups of luciferase and the carboxylic acid groups on the surface of the citric-acid modified iron oxide nanoparticles [19].
Transmission electron microscopy (TEM) was used to examine the structure and uniformity of the synthesized nanocomposites. Figure 2a shows a high-resolution TEM image of the as-synthesized CA-MNPs; homogeneous particles, approximately 17 nm in diameter, were observed. Cryo-TEM was performed on CA-MNPs to confirm the primary particle size and gain information on the in situ nanoparticle dispersion (Fig. 2b).
Samples imaged under cryogenic conditions showed a more dispersed particle phase that is expected to be more representative of the actual dispersion in solution [22]. Cryo-TEM images of Luc + MNPs (Fig. 2c) show the nanocomposite diameter ranging from 40–50 nm were observed, supporting the addition of firefly luciferase to the nanoparticles. Luc + MNP showed as distinct core–shell morphology with a lighter- colored luciferase shell (~5 nm) surrounding the darker CA-MNP core.
(Note that the lighter-colored strands between and near some of the Luc + MNP structures are likely luciferase strands which have extended/unfolded during the cryogenic sample preparation and are partially coordinated with the Luc + MNPs as an effect of rapid temperature changes as discussed elsewhere [23]). These TEM images not only validate the formation of the Luc + MNP complexes, but also demonstrate the formation of core-shell structures, as has been reported elsewhere for luciferase complexation with silver nanoparticles [24].
Dynamic light scattering (DLS) was used to evaluate the diameter of CA-MNP both before (31.5 ± 1.5 nm) and after (119.7 ± 23.9 nm) firefly luciferase adsorption. The increase in particle diameter and particle size distribution (Additional file 1: Figures S1, S2) is attributed to the addition of a firefly luciferase shell. While the trends are the same, the DLS particle sizes are larger than those measured using TEM.
However, the DLS data is expected to be more representative of the in situ particle sizes since the DLS data is collected in the luciferase enzyme hydrated state and the DLS experiment gives statistically significant data for the average particle sizes and particle size distributions.
Electrostatic charge on nanoparticle surfaces can be used to confirm the surface modification of nanoparticles, including binding with enzymes and proteins [24]. Here, phase-angle light scattering (PALS) measurements were used to study the surface charge of CA-MNPs at neutral pH before and after the addition of firefly luciferase on the CA-MNP periphery.
For CA-MNPs, PALS data shows a negative ζ-potential (−21.5 ± 2.0 mV). After addition of luciferase, the ζ-potential of nanoparticles increased to 4.5 ± 0.5 mV. The shift from negative to positive ζ-potential values confirms that luciferase is present as a shell on the exterior in the Luc + MNPs complexes.
Spermatozoa Labeling And Bioluminescence Imaging
After confirming the chemical and morphological characteristics of Luc + MNPs using a variety of in situ analyses, the newly synthesized Luc + MNP nanoparticles were evaluated for sperm labeling and imaging with the aid of in situ bioluminescence imaging (BLI) experiments to demonstrate the bioluminescent properties. Labeling procedures and imaging were followed as previously described [2, 15]. In this study, purified motile boar spermatozoa were prepared and labeled without or with the CA-MNP and Luc + MNP nanoparticles.
Unlabeled sperm samples and samples labeled with BRET-QD were used as negative and positive controls, respectively. Briefly, PBS-suspended spermatozoa were labeled and then washed three times by centrifugation to remove excess nanoparticles. All sperm pellets and their corresponding supernatants were mixed with luciferase substrate (coelenterazine) and immediately imaged (In Vivo Imaging System, IVIS 100; Xenogen; Fig. 3).
Compared to CA-MNP and supernatant, BLI signal intensity was higher in spermatozoa samples incubated with Luc + MNPs. For the Luc + MNP sample, relative BLI intensities for the sperm pellet and supernatant indicate that while excess unbound MNPs are present in the supernatant, there is a higher signal from the sperm pellet and indicates positive interactions between the spermatozoa and Luc + MNP. Significantly lower BLI intensities were observed for the CA-MNP sample and supernatant as compared with neat luciferase and Luc + MNP samples (Fig. 3).
Therefore, the strong BLI signal from the Luc + MNP coupled with spermatozoa, before and after centrifugation, confirms the presence and viability of the MNP- bound luciferase (Luc + MNP) as an image agent. The BLI for neat luciferase (with coelenterazine added) is presented as a positive control, and as expected shows the highest BLI signal (Luc, Fig. 3a), at least in part because the luciferase is not split between sample and supernatant aliquots. Spermatozoa in the Luc + MNP sample showed slightly lower BLI levels as compared to the luciferase alone, which is certainly due to substantial levels of Luc + MNP in the supernatant.
After in situ bioluminescence imaging, the sperm Luc + MNP labeled samples were smeared on microscope slides and light emissions imaged using two microscopic techniques, UV–visible-NIR and laser confocal. Using a series of in situ experiments, it was observed that the Luc + MNP and CA-MNP nanoparticles were in a non-aggregated state. It should be noted that the presence of luciferase, in particular while in the presence of biological media such as PBS (which was the as- received solution for boar spermatozoa specimens), could cause aggregation in situ and during drying [25–28].
To examine these issues, we collected an additional TEM image under non-cryo conditions (i.e., evaporative drying) that revealed possible aggregation of Luc + MNPs which could be a result of the PBS solution and/or the drying process (Additional file 1: Figure S3). Despite this observation, we can still investigate one of the primary objectives of this study—a preliminary evaluation of binding interactions between Luc + MNPs and boar spermatozoa.
After Luc + MNPs are added to boar sperm, round nanoparticle structures were observed on the surface of the sperm cells using the UV–vis-NIR microscope (see arrow heads, Fig. 4). Control experiments for QDs (Additional file 1: Figure S4) and neat spermatozoa cells (Additional file 1: Figure S5) were conducted, which reveal that the round nanoparticle structure are the MNP.
While these preliminary finding confirm that the nanoparticles interact with the cell surface (Fig. 4), the MNP density per cell, cell and MNP agglomeration, and time-dependency of the cell-MNP binding will be examined in future work.
Conclusions
Magnetic nanoparticles were synthesized and complexed with firefly (Photinus pyralis) luciferase enzyme to produce a multifunctional nanocomposite, Luc + MNP. Inherent bioluminescence in the presence of mammalian spermatozoa was examined, showing Luc + MNP as a promising candidate to enhance or replace some current bioimaging technologies. By utilizing FDA-approved iron oxide magnetic nanoparticles and a natural enzyme, such as firefly luciferase, this nanocomposite has potential for a lower toxicity than quantum dots, as well as the ability to magnetically manipulate cells and track them in vivo.
The results presented here demonstrate the possibility of using this luciferase-modified magnetic nanoparticle for cellular binding and imaging. Additional studies on the optimum concentrations and solution composition for cell binding and viability, cell tracking and magnetic manipulation, and time-dependence of the labeling and bioluminescence will allow for a better understanding of the parameters for implementing Luc + MNPs for assisted reproductive technologies.
Other activation mechanisms (beyond coelenterazine addition), such as ATP/Mg2+ sources, are also interesting avenues for future studies.
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