Dual mode mass spectrometric imaging for determination of in vivo stability of nanoparticle monolayers

Abstract

Effective correlation of the in vitro and in vivo stability of nanoparticle-based platforms is a key challenge in their translation into the clinic. Here, we describe a novel dual imaging method that site-specifically reports the stability of monolayer-functionalized nanoparticles in vivo. This approach uses laser ablation inductively-coupled plasma mass spectrometry (LA-ICP-MS) imaging to monitor the distributions of the nanoparticle core material and laser desorption/ionization mass spectrometry (LDI-MS) imaging to report on the monolayers on the nanoparticles. Quantitative comparison of the images reveals nanoparticle stability at the organ and sub-organ level. The stability of particles observed in the spleen was location-dependent and qualitatively similar to in vitro studies. In contrast, in vivo stability of the nanoparticles in the liver differed dramatically from in vitro studies, demonstrating the importance of in vivo assessment of nanoparticle stability.

Graphical Abstract

Nanoparticles (NPs) offer key delivery^1,2 and imaging platforms^3,4 for biological applications.^5,6 NPs provide unique physico-chemical attributes with potential to overcome challenges including biological barriers,⁷ rapid clearance,^8,9 and ineffective delivery of the cargo to the target.^10,11 A wide range of therapeutic payloads ranging from small molecules to proteins and nucleic acids have been conjugated to NPs to provide delivery systems.^12,13 These systems have shown great promise in pre-clinical studies,^14,15,16,17 and in the clinic,^18,19 including AuroLase and Magnablate.^20–23

Effective biomedical application of nanomaterials requires effective understanding and correlation of in vitro and in vivo behavior.²⁴ In addition to physical parameters such as size and shape,^25,26 dynamic changes in the NP surface or structure in the blood stream or within organs will change their behavior.^27,28 This remodeling can dictate NP fate by altering cellular uptake,¹⁵ corona formation,^16–18 biodistribution,¹⁹ interactions with biomolecules,²⁰ pharmacokinetics,²⁹ and pharmacodynamics parameters.^30,31 Current strategies for tracking NPs generally focus on the quantification of the NP core^32,33 rather than considering surface stability.

Here, we describe a dual-mode mass spectrometric imaging approach that provides site-specific quantitative information on inorganic NP monolayer stability.^3,34 We use laser ablation inductively-coupled plasma mass spectrometry (LA-ICP-MS) imaging³⁵ to track the distributions of NP core and laser desorption/ionization (LDI) MS imaging^36,37 to track the distributions of ligands that are still bound to the NP core. Comparison of the two images indicates the extent to which the NPs monolayers retain their integrity. Using this strategy, we show how NP stability dramatically diverges in different organs and sub-organs and simultaneously establish that there can be varying stability profiles for NPs in vitro and in vivo.

Five different AuNPs (Figure 1(a), AuNPs 1–5) with the same core size (2 nm) but different surface monolayers were used to determine the ability of the dual mode imaging approach to report on monolayer stability. These ligands possess four functional segments: 1) a mono or dithiol group for anchoring ligands to the NP surface,³⁸ 2) a hydrophobic alkane group for core stability,³⁹ 3) a tetra(ethylene glycol) (TEG) group for biocompatibility,⁴⁰ and 4) a variable head group for chemical diversity.⁴¹ The chemical identity of these layers plays a role in the stability of the monolayer.^34,3 Dithiol groups offer a more stable anchor to the gold core than mono thiols; longer alkane chain lengths better protect the gold core than shorter chains; and the presence of the TEG group has been shown to impart greater NP stability.³⁴ When NPs like those shown in Figure 1 are injected into mice, their biodistribution typically feature high levels of accumulation in the liver and spleen, two important organs of reticulo-endothelial system (RES).^42,43 Due to the high accumulation of administered NPs in the RES system (30–99%),^44,45 we investigated the changes in AuNPs in the spleen and liver by tracking the monolayer stability using our combined imaging approach. The AuNPs (Figure 1) were injected into mice intravenously (see details in the SI), and after fixed time points the mice were sacrificed and their organs were collected.

Figure 1.

(a) Structure of the AuNPs used in this study. (b) Schematic representation of the experimental workflow.

We first investigated the stability of AuNPs in the spleen of mice injected with AuNP 1, with the expectation that the loss of monolayer in this organ would be slower than for the liver due to lower endogenous thiol levels.⁴⁶ Imaging of the spleen 24 h after injection (Figure 2a) provides preliminary evidence for retention of ligands of AuNP 1, as the signal associated with the monolayer ligand in the LDI-MS image (Figure 2b) arises from only ligands attached to the core.³⁷ As expected, this signal co-localizes with the signal from Au in the LA-ICP-MS image (Figure 2c), indicating the presence of AuNPs with intact monolayers.

Figure 2.

Comparison of the stability of AuNPs after 24 hours of injection in spleen slices from mice with AuNP 1. (a) Optical image of the spleen section. (b) LDI-MS image of AuNP 1 that reports on the monolayer signal for the spleen 24 h after injection, and (c) LA-ICP-MS image that reports on the Au signal for the spleen 24 h after injection. See the SI for instrument parameters and measurement details. (d) LDI-MS images of spleen slices at three different time points from mice injected with AuNP 1, AuNP 2, and AuNP 3. (e) Normalized LDI-MS intensities across the entire image for each AuNP, illustrating the loss of monolayer ligand signal over time. The approach for obtaining these normalized intensities, which accounts for differences in tissue slice sizes, can be found in the SI. Error bars represent variations in normalized intensities for images of multiple adjacent tissue slices (n=3).

We then investigated the spleen stability of AuNPs at different time points (4, 24, and 48 h) after their injection into the mice. AuNPs 1, 2, and 3 were simultaneously injected, and LDI-MS images for each NP’s monolayer signal were generated. We predicted that the ligand signal in the LDI-MS images would decrease after longer time points as the AuNPs are degraded over time. As expected, LDI-MS images of the spleen showed a decrease in the monolayer signal for each AuNP over time (Figure 2d). An evaluation of the ion intensities in these images (Figure 2e), after normalizing for the size of the tissue, confirms this observation and further indicates that the signal drops for each AuNP is similar. In contrast, the Au distributions in the spleen, as determined by LA-ICP-MS imaging, indicate that the Au amounts remain essentially constant when the data is normalized for mouse-to-mouse variations (e.g. Figure S1 and S2). Taken together, comparison of the LDI-MS and LA-ICP-MS imaging results reveal that ~50% of the monolayer stability is lost in the spleen over a 48 h period, with headgroup structure playing a rather small role in determining particle stability.

Studies using AuNPs 1–3 (Figure 2) show that these NPs have similar stability. The ability of the system to determine differential monolayer stability was probed using AuNPs 1 and 4; previous in vitro findings showed that incorporating TEG moieties in the monolayer provides enhanced stability in cells.³⁴ We first compared the LDI-MS behavior of these two ligand systems, finding that they had similar ionization efficiencies (Figure S3). To best compare the two sets of images, we imaged two sets of tissues that had similar total gold amounts based on ICP-MS measurements of tissue homogenates (Figure S4). The LDI-MS signals (shown in green) are qualitatively brighter for AuNP 1 (Figure 3a), indicating higher ligand stability for AuNP 1 relative to AuNP 4. A more quantitative comparison was made by summing the ion abundances in each pixel and dividing this value by the total area of the image, while also correcting for the slight differences in ionization efficiencies of the two monolayers (Figure 3b). From such an analysis, both AuNPs are found to have similar abundances per unit area in the LA-ICP-MS images but quite different ion abundances are observed per unit area in the LDI-MS images (Figure 3b). From these studies we can conclude that AuNP 1 is essentially unchanged in the spleen, whereas ~60% of the monolayer of AuNP4 was degraded. These outcomes correlate well with our previous in vitro results,³⁴ and provide a strong demonstration of the importance of monolayer structure on in vivo stability.

Figure 3.

Comparison of the stability of AuNP 1 and AuNP 4 in the spleen 24 h after injection. (a) LDI-MS and LA-ICP-MS images showing the NP-bound monolayer and Au distributions, respectively, in the spleens of mice IV-injected with either AuNP 1 or AuNP 4. (b) Summed relative ion abundances of the Au and ligand ions from the LA-ICP-MS and LDI-MS images in (a). The normalized pixel intensity calculations are described in the SI.

The use of imaging tools allows the monitoring of NP stability across individual organs. ⁴⁷ LDI-MS and LA-ICP-MS images of spleens from mice injected with AuNP 1 and 4 were compared to determine stability differences at the sub-organ level. H&E staining and Fe images from the LA-ICP-MS experiments were used to identify the three primary regions of the spleen: red pulp, white pulp, and marginal zone (Figure 4).⁴⁷ For the analysis, the average ligand and Au signals for each spleen region were determined separately, as described in the SI, and then a stability factor for each AuNP was calculated from the ratio of the ligand signal to Au signal (Figures S5, S6, S7, and Table S4). The calculated stability factor indicates that AuNP 1 and AuNP 4 have similar stability in the red pulp, while AuNP 1 is slightly more stable in the white pulp. Interestingly, AuNP 1 is significantly more stable than AuNP4 in the marginal zone (Table S4). This increased level of degradation suggests an increased level of biogenic thiols in the marginal zone relative to the rest of the spleen, a property that to our knowledge has been suggested,^48,49 but has not been experimentally observed.

Figure 4.

Example LA-ICP-MS and H&E staining images of a spleen tissue slice from a mouse injected with AuNP 4. The Fe image reflects the presence of the blood in the organ, distinguishing between the red pulp (higher Fe, red) and white pulp regions (lower Fe, black). The marginal zone is the interface between the red and white pulp regions and extends ~ 40 μm from the white pulp. In the H&E stains, the pale pink color indicates the red pulp region, while the dark purple regions indicate the white pulp regions.

Having established the ability of the dual imaging strategy to determine stability in the spleen, we next examined the stability of NPs in the liver. Mice were injected with AuNP 1, and after 24 h were sacrificed. Slices from the liver were then imaged by LDI-MS for the ligand and LA-ICP-MS for Au. As expected, LA-ICP-MS indicates high levels of Au in the liver (Figure 5a). In contrast, LDI-MS shows no ligand signal, indicating complete degradation of the AuNP monolayer. This outcome is surprising because previous studies showed that AuNPs with similar ligands were reasonably stable (~70% monolayer retention) for in vitro studies using liver (HepG2) cells.³⁴ In a control experiment, AuNPs that were added to liver tissue slices gave rise to significant ligand ion signal (Figure S8), eliminating the possibility that LDI-MS does not work in liver tissue.

Figure 5.

LDI-MS and LA-ICP-MS images of liver tissue slices from a mouse injected with (a) AuNP 1 and (b) AuNP 5 (24 h after injection).

Given the total degradation of the monolayer observed with even our most stable monothiol ligand, we performed a further 24 h trial using dithiol-functionalized AuNP 5 (Figure 1). Previous in vitro studies showed complete (100%) retention of the monolayer of AuNP 5 in HepG2 liver cells;³⁴ given this outcome we expected similar behavior in vivo. In stark contrast to the in vitro studies, however, no ligand is observed by LDI-MS (Figure 5b). These studies indicate a remarkable difference between in vitro and in vivo stability, a key observation that calls into question the utility of in vitro studies to predict in vivo behavior.^50,51 While the mechanism for enhanced degradation in vivo is unknown, the difference in behavior can be explained at least in part by increased levels of biogenic thiols in vivo arising from the complex network of inter-related cells including hepatocytes, Kupffer cells, macrophages, and immune cells (dendritic cells, natural killer cells, and lymphocytes) found in the liver.⁴⁴

In summary, we have developed a dual mode imaging approach that combines LA-ICP-MS and LDI-MS imaging to quantify AuNP monolayer stability in vivo. This technique provides a tool for quantifying particle stability in vivo, producing results dramatically different than those predicted from in vitro studies. Imaging studies of the liver revealed stark differences between in vitro and in vivo behavior. Even NPs that were completely stable in liver cells in vitro were completely degraded in vivo. The resolution of the techniques enabled determination of the sub-organ stability of the particles. These studies indicate marked differences in AuNP stability at different locations in the spleen, indicating that this dual-imaging strategy has the capability of providing unprecedented granularity of our understanding of the pharmacodynamic properties of nanomaterials. Taken together, these results indicate that in vitro systems have unexpected limitations in predicting in vivo pharmacodynamics, an issue that we will pursue in future imaging studies.