Intravascular infusion of PEGylated Au nanoparticles affects cardiovascular function in healthy mice

Abstract

Recent advances of nanotechnology in clinical settings have spurred the development of various complex engineered nanoparticles (NPs). NPs share characteristics with ultrafine particles (UFPs; <1 μm) that can cross the pulmonary epithelium and disturb cardiovascular functions. Since these particles are injected directly into the blood stream, it is imperative to clarify whether NPs disrupt cardiovascular functions similar to UFPs. Therefore, we investigated whether engineered polyethylene glycol (PEG)-coated aluminum NPs for biomedical uses disturb cardiovascular functions in healthy mice. Mean arterial blood pressure (MAP) was measured in mice chronically instrumented with telemetric blood pressure transducers, and NPs were administered intravenously (10 mg kg⁻¹). The NPs caused a prolonged lowering of MAP 7 days after injection (119.3 ± 3.3 vs. 97.4 ± 7.5 min⁻¹), with no effect on the endothelial function as revealed by normal endothelial function of small vessels mounted in a myograph.

Introduction

Complex engineered nanoparticles (NPs) with distinct chemical, physical and biological properties are currently being developed for therapeutic usage and as contrast agents. Because these NPs are injected directly into the blood stream, ^1,2 it is imperative to study whether they interfere with the endothelial cells and affect blood pressure. However, while NPs share characteristics with ultra fine particles (UFPs), which are known to elicit cardiorespiratory malfunctions ³ and increase mortality and morbidity from pulmonary arrhythmias, ^{4 –6} very little is known about their specific cardiovascular effects, ⁶ precluding successful mitigation of their negative impact.

Here we apply telemetric probes for continuous long-term measurements of blood pressure, heart rate and locomotor activity in healthy mice receiving intravenously injected engineered aluminum (Au) NP. We recently showed that intravenous injections of polyacrylic acid (PAA)-coated γ-Fe₂O₃ NPs (10 mg kg⁻¹) decreased blood pressure 12–24 h after injection in mice in conjunction with reduced vessel contractility induced by endothelial impairment. ⁷ Besides γ-Fe₂O₃ NPs, some of the most promising NP candidates are Au NP, which are explored for photodiagnostics, photothermal cancer therapy and drug vectorization. ⁸ Given the promising biomedical use of Au NPs and our recent findings of γ-Fe₂O₃ NPs, we evaluate the effects of polyethylene glycerol (PEG)ylated Au NP on normal cardiovascular functions at the proposed clinically relevant dose of 10 mg kg⁻¹. Since UFP may damage systemic endothelial cells, we further study endothelial function in vitro using myography by determining contractile performance in small resistance vessels from mice at 24 h and 7 days after intravenously injected PEGylated Au NPs. We demonstrate that intravenously injected Au NP caused prolonged lowering of blood pressure without interfering with the endothelial contractility.

Materials and methods

Animals and housing

Experiments were performed on 60 BLAB/cJ mice of both sexes aged 8–12 weeks. A total of 18 mice (20.8 ± 2.6 g) were used for telemetric measurements of blood pressure, heart rate and locomotor activity, while 32 mice (21.3 ± 0.8 g) were used to study isolated blood vessels in a myograph. All mice were purchased from Taconic (Ry, Denmark) and transported to the animal facility at the Department of Biosciences (Aarhus University), where they were maintained under standard light (12:12 dark–light) and temperature conditions with free access to rodent chow and tap water. Animal care followed the guidelines of the Danish National Institute of Health.

Telemetric probe implantation procedure

Surgical anaesthesia was induced by an intraperitoneal injection of ketamine (75 mg kg⁻¹) and xylazine (10 mg kg⁻¹). A small thermistor was inserted into the rectum to maintain body temperature at 36–37.5°C throughout surgery, and the eyes were protected against dehydration with an eye ointment (Ophtha A/S, Copenhagen, Denmark). After the neck had been shaved and disinfected, the mouse was placed in a supine position and a ventral midline skin incision was made from the lower mandible posteriorly to the sternum (3 cm). The submaxillary glands were gently separated and the left common carotid artery was isolated using fine forceps, without disrupting the vagus nerve parallel to the artery. Two sterile silk sutures (6-0, Agnthos, Sweden) were passed under the vessel for temporary occlusion of the artery during catherization and to secure the catheter. The catheter from the telemetric probe (TA11PA-C10: 1.1 cm³, 1.4 g; Data Sciences International (DSI), St Paul, Minnesota, USA) was inserted into the artery, and a subcutaneous pouch was formed for placement of the transmitter body along the animal’s right flank. The neck incision was closed using 5-0 silk. Mice were allowed to recover from surgery in a climate chamber at 28°C for the first 24 h after surgery. Twice within this re-covalence period, the mice received subcutaneous injections of temgesic for analgesia (buprenorphinum, 0.3 mg ml⁻¹, 3.75 mg kg⁻¹).

Telemetry protocol

Following recovery from surgery and anesthesia, mice were returned to their cages (placed on top of the telemetry receivers) and monitored daily throughout the study. Radio telemeters (DSI, St Paul, Minnesota, USA) were magnetically activated 7 days after surgery, and blood pressure (pulsatile waveforms and mean arterial pressure (MAP)), heart rate and locomotor activity were recorded continuously as 10-s averages each minute for 7 days. After recordings of cardiovascular parameters for 24 h, NP or saline were injected in the tail vein of experimental and control mice. The telemetered pressure signals (absolute pressure) were corrected automatically for changes in atmospheric pressure measured by an ambient pressure monitor.

Isometric force measurements in mesenteric small arteries

A total of 32 mice of both sexes were infused with either NPs or saline in the tail vein and mesenteric arteries were removed at different time points for in vitro studies. Mice that received Au NP (100 µl; 10 mg kg⁻¹) were killed after 24 h and 7 days (n = 8 in each groups,), respectively, while control animals that received saline (n = 8 in the two control groups) were killed at the same time point as the treated mice. These time points were chosen due to the previously demonstrated decrease in MAP in mice that received γ-Fe₂O₃ NP and where the measured cardiovascular parameters were restored after 7 days. ⁷

In all the mice, the mesenteric bed was carefully pinned out in a Petric dish and four 2-mm-long segments of third-order branches were dissected free from fat and connective tissue. ⁹ The isolated arteries were threaded onto two stainless steel wires (40 µm in diameter) and mounted as ring preparations in a quadruple wire myograph (Model 610M, Danish Myo Technology, Aarhus University) for isometric force measurements. Data were sampled at 100 Hz (16 bit) using a Biopack MP100 data acquisitions system (Biopac system Inc. Santa Barbara, California, USA). Upon mounting, the arteries were allowed to equilibrate for 20 min before they were stretched stepwise to characterize the passive elastic properties. ¹⁰ The mesenteric arteries were stretched to 90% of L ₁₀₀, where L ₁₀₀ is defined as the circumference of the relaxed artery transmural pressure of 100 mmHg. Prior to the experimental protocol, the arteries were stimulated three times with 10 µM noradrenaline (NA) dissolved in 125 mM K⁺-containing physiological saltwater solution (PSSS (KPSS)) for 3 min to verify viability. Subsequently, NA (3 × 10⁻³) was added and at the maximal response, a single dose of acetylcholine (ACh; 1 µM) was given to ensure relaxation of the vessel. This was repeated with an increased dose of ACh to 3 µM. Vessels that failed to contract more than 125% of the forced developed at L ₁₀₀ were discarded. Both NA and ACh were added directly to the bath. To evaluate the contractile properties, we constructed cumulative NA concentration–response curves (0.1–0.3 µM NA), increasing the concentration every 2 min to allow the force to reach a steady state.

Solutions and nanoparticles

The vessels were dissected, mounted and, if not indicated otherwise, held relaxed in physiological salt solution (PSS) of the following composition in millimoles: NaCl, 119; NaHCO₃, 25; KC1, 4.9; CaC1₂, 2.5; H₂PO₄, 1.18; MgSO₄, 1.17; EDTA, 0.026 and glucose, 11. KPSS was PSS in which Na⁺ was substituted by an equimolar concentration of K⁺ to reach 15 mM K⁺. All the solutions were bubbled with 5% CO₂ in O₂ at 37°C and the pH was adjusted to 7.4 immediately before use.

The gold NPs were capped with PEGylated thiol with carboxyl functional end group (IMEC, Kapeldreef, Leuven, Belgium). They were concentrated with an optical density of 2. The particles were given at a dose of 100 µl of Au (0.2 mg ml⁻¹). UV–Vis absorption spectrum revealed an localized surface plasmon resonance (LSPR) peak at 523.5 nm and the dynamic light scattering (DLS) spectrum with an average size of 22.5 nm.

Data and statistical analyses

The telemetry data were collected continuously by sampling for 10 s every minute and stored using the Dataquest ART data acquisition system (DSI, St Paul, Minnesota, USA). After the experimental session, the obtained data were uploaded into the ACQ analysis program (DSI, St Paul, Minnesota, USA) and exported into Excel. The data from the myograph were collected at 50 Hz and transferred directly into Excel. All further data analyses from both experimental sessions were analyzed in Excel. Data were tested for homogeneity of variance and normal distribution prior to parametric tests. Statistical significance of differences between treatments on blood pressure, heart rate or locomotor activity obtained in mice receiving PEG-coated Au NPs or saline was evaluated by a two-factor (time and treatment) analysis of variance (ANOVA) for repeated measures followed by a Tukey’s multiple comparison. In the myograph studies, the effect of treatment, that is  PEG-coated Au NPs or saline and the cumulative dose–response curve of noradrenalin on tension and pressure was also evaluated by a two-factor (noradrenalin and treatment) ANOVA for repeated measures followed by Tukey’s multiple comparison. Differences between mean values were considered statistically significant when p ≤ 0.05 and all results are presented as mean values ± SEM.

Results and discussion

Intravenously injected Au NPs, at a clinically relevant dose for hyperthermia cancer treatment, caused a prolonged decrease in MAP. However, our myograph studies on isolated vessels show that this hypotension was not associated with impaired endothelial function. This is in contrast to the intravenously injected PAA-coated γ-Fe₂O₃ particles, which caused an acute decrease in MAP associated with a failure of obtaining maximal force development in small mesenteric arteries in response to NA. ⁷ Together with the Au NP data obtained in the present study, this highlights the importance of evaluating the cardiovascular effects of NPs before its use in clinical trials and further how different NPs affect the cardiovascular system differently.

Telemetry allows for MAP to be measured accurately under relatively stress-free conditions in freely moving, conscious animals, alleviating the effects of stress from recent anesthesia and surgery. ¹¹ The example of continuous measurements of MAP, heart rate (f _H) and physical activity obtained 8 days after surgery (Figure 1) reveals typical murine circadian rhythms with elevated and more variable MAP, f _H and activity during dark periods. The rise in MAP during activity appears to be sympathetically mediated ¹² and the locomotor activity is considered to be a major determinant of the variation in MAP among individuals.

Figure 1.

Representative 36 h data trace from one healthy BALB/c mouse 10 days after instrumentation. (a) MAP, (b) heart rate (f _H) and (c) locomotor activity. The dotted lines indicate the shift from light to dark phases, respectively. MAP: mean arterial blood pressure.

To account for these effects, the present obtained telemetric data were analyzed as proposed by Van Vliet et al. ¹³ as follows:

$\begin{array}{r}{\mathrm{M}\mathrm{A}\mathrm{P}}_{24\mathrm{h}}={\mathrm{M}\mathrm{A}\mathrm{P}}_{\mathrm{i}\mathrm{n}\mathrm{a}\mathrm{c}\mathrm{t}\mathrm{i}\mathrm{v}\mathrm{e}}\\ +T_{\mathrm{a}\mathrm{c}\mathrm{t}\mathrm{i}\mathrm{v}\mathrm{e}}({\mathrm{M}\mathrm{A}\mathrm{P}}_{\mathrm{a}\mathrm{c}\mathrm{t}\mathrm{i}\mathrm{v}\mathrm{e}}-{\mathrm{M}\mathrm{A}\mathrm{P}}_{\mathrm{i}\mathrm{n}\mathrm{a}\mathrm{c}\mathrm{t}\mathrm{i}\mathrm{v}\mathrm{e}})\end{array}$

where MAP_24h represents mean MAP over the entire 24 h period, MAP_inactive and MAP_active are the MAP during inactivity (no activity signal) and activity, respectively. T _active represents the proportion of time spent in activity. By emphasizing the impact of activity, the calculated MAP depends on the extent of the change in T _active and the magnitude of the effect of activity on MAP (MAP_active − MAP_inactive). ¹³ Figure 2 shows a significant reduction in MAP on the seventh day after injecting Au NPs, while no effect was observed after injecting saline.

Figure 2.

MAP in the three experimental groups of mice that received PEG-coated Au NP (□) and saline (•). (a) Calculated from Van Vliet et al. ¹³ and (b) when data were normalized to the control value in each group. Data are presented as mean ± SE. * indicates significant difference from control values. Au: aluminum; MAP: mean arterial blood pressure; NP: nanoparticles; PEG: polyethylene glycol; SE: standard error.

The cardiovascular effects caused by the Au NPs are consistent with previous reports on inhalation or injected NPs and UFPs ^{14 –18} and may have been caused by the formation of oxidative stress that impairs normal endothelial function. ¹⁹ We recently showed that γ-Fe₂O₃ NPs are taken up by endothelial cells, particularly those in liver and kidney, and that small mesenteric resistance vessels ²⁰ exposed short term to these γ-Fe₂O₃ NPs failed to obtain the maximal force development when stimulated with NA, although the major endothelial-dependent relaxing pathways seemed intact. ⁷ All mesenteric arteries in the present study exhibited a concentration-dependent contraction in response to NA but showed no effect of treatment with the PEGylated Au NPs of either 24 h or 7 days (Figure 3). In contrast, Mo et al. ²¹ showed that UFPs, at a nontoxic dose, induced reactive oxygen species generation in mouse pulmonary microvascular endothelial cells. Thus, endothelial cells that line the inner surface of blood vessels are in direct contact with these particles, emphasizing the relevance of particle–endothelial interactions. The difference in the present data when compared with the previous published data on γ-Fe₂O₃ NPs may be explained by the different interference with the normal endothelial function, although this cannot explain the prolonged decrease of the MAP for Au NPs. The specific mechanism underlying this is, therefore, unknown. If a pro-inflammatory stimulation of endothelial cells by nano-scaled particles occurs in vivo, a chronic inflammation could be a possible consequence. Importantly, the present results underlines the need for future studies in addressing the cause of the difference in the effect and mechanism on the cardiovascular system and whether this can be ascribed to the effect of size, coating or core type of the NPs.

Figure 3.

Cumulative dose–response curves of tension and pressure of small mesenteric arteries from healthy BALB/c mice after receiving PEG-coated Au NP (□) and saline (•) to increasing concentrations of NA. (a, c) Intravenously injected PEG-coated Au NP (□) and saline (•) after 24 h and  (b, d)  after 7 days. Data are presented as mean ± SEM. Au: aluminum; MAP: mean arterial blood pressure; NA: noradrenaline; NP: nanoparticles; PEG: polyethylene glycol; SEM: standard error of mean.

In conclusion, intravenous injection of Au NP causes prolonged reduction in MAP, whereas PAA-coated γ-Fe₂O₃ NPs causes much shorter hypotensive effects. The difference of these two types of NPs may be due to the core type of the particle, size or the different coatings. Furthermore, it has been shown that hypertensive rats exposed to instilled UFPs increased f _H and MAP, while the normal rats did not respond. ¹⁸ The authors therefore hypothesized that inhaled UFPs causes systemic effects by evoking a stress that may be compensated in healthy individuals but that severely disrupts homeostatic processes in individuals with cardiovascular or pulmonary diseases. ¹⁸ This underpins the importance of future studies on the cardiovascular effects of NPs during disease states, because most patients who receive NP are likely to suffer from various diseases, including hypertension.