Introduction

Pyrroloquinoline quinone (PQQ) is a ubiquitous molecule that is found in locations that vary from interstellar dust to human tissues. It is water soluble, heat stable, and capable of undergoing continuous redox cycling at a level at least 25 times greater than that of many other redox agents. PQQ is a redox cofactor in bacteria and is found in plant and animal tissues [1–4]. As an electron donor or N-methyl d-aspartate receptor (NMDAR) redox reaction site, PQQ readily reacts with nucleophiles to form stable condensation products [5]. PQQ’s benefits as an antioxidant are so numerous that it has been considered for classification as a vitamin. PQQ’s activity inhibits β-amyloid (1–42) fibril formation, the cytotoxic effects of solubleβ-amyloid (1–42) and fibrillation of mouse prion protein. It also protects against oxidative damage to the brain and protects other organs from external sources such as poisons or drugs [6].

In the brain, NMDAR is considered to be among the most critical molecular regulators of cognition, memory, motor control and synaptic plasticity. These receptors contain a “redox modulatory site” that strongly regulates receptor function. NMDARs are a type of receptor for the neurotransmitter glutamate, which is the primary excitatory neurotransmitter in the brain. Glutamate is found in 100 % of pyramidal neurons, approximately 60 % of total brain neurons, and accounts for virtually all cortico-cortical neurotransmission. Disturbances in NMDAR-mediated glutamatergic neurotransmission in the brain, primarily related to excitotoxicity, are present in many neurological and psychiatric disorders including epilepsy, Alzheimer’s disease (AD) and schizophrenia [7–11]. It has been shown that PQQ interacts with the NMDAR redox site to inhibit glutamate-induced cytotoxicity and produce beneficial physiological and cognitive outcomes.

Understanding the function of NMDAR in brain continues to be limited by a lack of effective imaging methods in vivo. Currently, positron emission tomography (PET) and single photon emission computed tomography (SPECT) are favored methods of imaging the living brain, and it is believed that developing a broad battery of radioligands specific for NMDA receptors would contribute to our knowledge regarding the density, spacial distribution, and function of these receptors in brain [12]. Unfortunately, many radio-probes are not ideal and ultimately are limited by their low fat solubility and consequent high non-specific binding. There is an urgent need to develop improved probes that can show distinct changes in NMDA receptors over the course of neurological disorders.

Technetium-99m (99mTc) is the most popular SPECT radioisotope. It is used in up to 85 % of diagnostic nuclear medical procedures every year [13, 14].

99mTc-PQQ was previously prepared in our laboratory and found to potentially accumulate predominantly in the hippocampus and cortex, which have a high density of NMDAR [15]. However, 99mTc-PQQ is water-soluble and negatively charged and has chelating groups that further limit its ability to pass through the blood–brain barrier (BBB). Therefore, in our previous work, image reconstruction was restricted because hardly any 99mTc-PQQ entered the brain and even less bound with NMDARs. Our next research goal became to improve the ability of 99mTc-PQQ to pass through the BBB.

The BBB forms a physical blockade to exclude from the brain nearly all neurotherapeutics and small molecule drugs [16]. The end result is a bottleneck in brain drug development, and this is the single most important factor limiting the future growth of neurotherapeutics. Hyperosmotic opening of the BBB has been developed as a tool to reversibly disrupt the BBB during administration of chemotherapeutic drugs, allowing for access to hidden marginal tumor cells and better clinical outcomes [17]. Mannitol has been used clinically to open the BBB for the delivery of some agents into the brain tissue [18].

In this work, the specificity binding of 99mTc-PQQ to NMDAR was investigated by Radio-ligand receptor binding assay (RRA). Regional distribution of 99mTc-PQQ in brain was tested and the effect of the mannitol dose on BBB opening was studied. Moreover, whether BBB opening affect the specificity binding of 99mTc-PQQ to NMDA receptor were further studied. This provides a new method for improving the uptake of water-soluble imaging reagent in brain.

Experimental procedures

Materials and reagents

PQQ was obtained from ShanghaiMed Co. (China). SnF2 anhydrous, MK801, and Ebselen (EB) were purchased from Sigma-Aldrich (USA). All the other analytical chemical reagents employed were purchased from commercial sources and used without further purification. Na99mTcO4 pertechnetate obtained from locally produced fission based 99Mo/99mTc generator system (Chengdu Nuclear Isotope Qualcomm Inc., China) was used for radio labeling.

Animals

Sprague–Dawley (SD) rats were supplied by Jiangsu Institute of Nuclear Co., Ltd. (China). The NMDA receptor was prepared from SD rats. Mice (ICR) were purchased from Shanghai SLAC Laboratory Animal Center (Shanghai, China). ICR mice (20 ± 1 g) were divided randomly into six groups with three mice per group experiments for the opening BBB experiments by mannitol. The animal experiment in this study was approved by the Animal Care and Ethics Committee of Jiangsu Institute of Nuclear Medicine.

Preparation of 99mTc-PQQ

99mTc-PQQ was prepared as described previously [15]. Briefly, 0.05 mL EDTA-2Na (10 mg/mL) and 0.15 mL PQQ (6.6 mg/mL) were added to a 1.5-mL centrifuge tube, followed by 0.1 mL Na[99mTc]O4 (40 MBq) and then immediately by 0.03 mL freshly prepared SnF2 solution(1 mg/mL in 0.1 M HCl). The volume was adjusted to 1 mL using phosphate buffer (pH 6.0). The mixture was reacted at room temperature for 30 min. 99mTc-PQQ was purified by radio-HPLC to obtain high specific activity. Radiochemical purity (RCP) was tested by thin-layer chromatography (TLC) using acetone as the mobile phase.

RRA

Sources of NMDAR

NMDAR was extracted from SD rats as previously described [19]. Brains (removed midbrain, cerebellum, and spinal cord) were homogenized sufficiently by glass homogenizer in 10 volumes of buffer B (10 mM HEPES buffer which contented 1 mM EDTA). Then the homogenates were centrifuged at 2,400 r/min for 10 min at 4 °C to remove impurities. The remained supernatant was centrifuged for 20 min. The precipitants were resuspended in buffer B and centrifuged again. This procedure was repeated at least three times. Then the pellet was resuspended in buffer A (10 mM HEPES) in the original volume. The homogenates were then incubated at 37 °C for 30 min, followed by three additional centrifugation steps in buffer A. The entire operation was completed in an ice bath. The pellet was resuspended in buffer A in the original volume and stored in 1-mL aliquots at −20 °C until use. The protein content was determined by Coomassie Brilliant Blue Method.

The effects of NMDAR concentration and incubation time on specific binding

NMDAR was diluted to 0.025, 0.05, 0.1, 0.5, 1, 1.5, 2, and 2.5 mg/mL with buffer A. Samples were divided into three groups: total binding (TB), nonspecific binding (NSB) and blank (BLANK). TB was tested with 0.3 mL of each protein concentration. For each tube, 0.1 mL buffer C (5 % Tween-20 in buffer A) and 0.1 mL 99mTc-PQQ (1.0 μmol/L) were added, and buffer C was used to adjust the volume to 0.6 mL. NSB was tested using the same procedure, except that 0.1 mL PQQ (200 μmol/L) was added to the tube. The blank was assayed with only 0.1 mL 99mTc-PQQ and 0.5 mL buffer C. All the tubes were incubated at 37 °C for 30 min in a water bath and placed on ice for 5 min to stop the reaction. GF/B glass fiber filter paper was used for filtering with 5 % bovine serum albumin (BSA). The ZT2-type long-cell collector was used for filtration. The products were washed with 4 mL buffer C three times before removing the membranes to count the samples using γ-counter. The incubation time experiment was done in the same way, using 0.5 mg/mL protein in each tube and incubating the samples for 5, 10, 20, 30, 40, 50, or 60 min, followed by leaching and counting.

The effect of different socking mediums (including PEI, Tris-HCl, BSA, serum and saline), rinse time and amount on the impact of specific binding on NMDA receptor were studied as above described.

Determining equilibrium dissociation constant K d

The same steps described in the above section were used with 0.1 mL 99mTc-PQQ at 0.025, 0.5, 1, 2, 4, 8, or 16 μmol/L added. The samples were stirred, incubated at 37 °C for 30 min, leached, and counted. The equilibrium dissociation constant K d was estimated according to a saturation curve.

Competition binding assay

The three antagonists, EB (+)-MK-801, and halperidol were used for experiments following same steps described above with 0.1 mL 99mTc-PQQ (1 μmol/L) and the antagonists at 1 × 10−3, 1 × 10−4, 1 × 10−5, 1 × 10−6, 1 × 10−7, 1 × 10−8, 1 × 10−9, 1 × 10−10, 1 × 10−11, or 1 × 10−12 mol/L in each tube.

Opening the BBB

The dosage of mannitol

ICR mice were divided randomly into six groups with three animals weighing 20 ± 1 g per group. For BBB studies, different concentrations of 20 % mannitol (0.25, 2.5, 5, 7.5, 10, and 15 mL/kg) were injected 5 min prior to 99mTc-PQQ injection (7.4 MBq, 0.2 mL). The animals were sacrificed 5 min after injection of 99mTc-PQQ. The brain was rapidly removed, chilled and dissected. Samples from different brain regions (striatum [ST], hippocampus [HP], cerebellum [CB], thalamus [TH], brain stem [BS], frontal lobe [FT], parietal lobe [PT], temporal lobe [TR], and occipital lobe [OT]) were collected, weighed and counted. The percentage of injected dose per gram (%ID/g) was calculated by comparing its activating with appropriate standard of injected dose (ID).

Mannitol incubation time

ICR mice (20 ± 1 g) were divided into six groups with three mice per group experiments to determine the incubation time for opening the BBB. 99mTc-PQQ (RCP > 95 %,7.4 MBq, 0.2 mL) was injected via the tail vein after giving 20 % mannitol for 1, 15, 30, 60, 240, and 360 min, and the mice were sacrificed 5 min after injection of 99mTc-PQQ. Regional brain distribution was determined as described above.

Confirmation of optimized conditions

The brain biodistribution experiment in the presence of 20 % mannitol (7.5 mL/kg) with 0.9 % saline was as control group. Twenty minutes after the first injection of 20 % mannitol (control by 0.9 % saline), 99mTc-PQQ was injected (7.4 MBq, 0.2 mL). Mice in the two groups were sacrificed at 5, 15, 30, 60 180 or 360 min post-injection (n = 3). The average ratios and standard deviations of %ID/g tissue of the different brain regions relative to that in thalamus were calculated.

Reversible opening of the BBB

The effect of BBB opening on the specific binding of 99mTc-PQQ was studied using ICR mice by control (mannitol) and experiment (mannitol + EB) treatment. Twenty minutes after the first injection of 20 % mannitol (7.5 mL/kg), 99mTc-PQQ was injected (7.4 MBq, 0.2 mL) in the control group. EB (51.4 mg/kg) was also injected in the experiment group. Mice in the two groups were sacrificed 5, 30 or 60 min post-injection (n = 3). The ratios of the values in different brain regions to the value in thalamus were calculated.

Data analysis

The average count of the blank samples was subtracted from the average count of the TB and NSB samples, and SB (specific binding) = TB − NSB. B max and K d were calculated by using software written according to the Clarlc occupancy theory of zone B.

Results

NMDAR and radioligand preparation

For in vitro experiments, NMDAR was extracted from SD rats as described by Reynolds and Sharma [19]. Concentration was determined to be approximately 3.876 mg/mL. 99mTc-PQQ was prepared to a radiochemical purity of greater than 95 % according to the method of Kong et al. [15]. Its specific activity was 2.2 × 1013 Bq/g. The results of paper electrophoresis indicated that the complex has a negative charge. 99mTc-PQQ is water soluble due to its hydrophilic side group (lgP = −1.49).

Determination of specific binding

Our first goal was to establish and optimize the signal for specific binding between 99mTc-PQQ and NMDAR using RRA. A known concentration of radioligand (1 μmol/L) corresponding to approximately 625 counts per minute (cpm) was incubated with varied concentrations of NMDAR protein in order to determine total binding (TB). A parallel experiment to determine non-specific binding (NSB) was completed that included the addition of 200-fold excess of unlabeled PQQ to the binding reaction. This ensured that all the NMDAR sites would be occupied by unlabeled PQQ and the measured radio-activity would be the non-specific binding. Specific binding (SB) was calculated from the difference between TB and NSB. Data points were fit to a spline curve and the optimal NMDAR concentration was determined to be 0.8 mg/mL (C NMDAR = [C starting point + C highest point]/2) (Fig. 1).

Fig. 1
figure 1

Specific binding of 99mTc-PQQ to NMDAR at various concentrations

Full size image

99mTc-PQQ saturation curve

We determined maximum binding capacity B max and the equilibrium dissociation constant K d from the 99mTc-PQQ saturation curve (Fig. 2). NMDAR was incubated with varied concentrations of 99mTc-PQQ, and the data points were fit to a spline curve. B max was determined to be 6.648 μmol/L (n = 3) and K d 2.381 nmol/mg.

Fig. 2
figure 2

Saturation curve of 99mTc-PQQ to NMDA receptor

Full size image

Competition curves

To confirm that the binding in our reactions was legitimate and comparable to PQQ-NMDAR binding behavior in actual cells, we conducted competitive binding experiments using Ebselen (EB). Ebselen is demonstrated to have neuroprotective effects in clinical trials and has been shown to bind the NMDAR redox modulatory site. We first determined the % Bound for 99mTc-PQQ in the presence of varied amounts of EB. From plotting % Bound as a function of the log[EB], we obtained the half maximal inhibitory concentration (IC50) of EB, which was 1.968 nmol/L. We then were able to calculate the equilibrium dissociation constant for EB (K i), which was 1.919 nmol/L. This value indicates a slightly higher affinity for EB-NMDAR binding than for 99mTc-PQQ-NMDAR binding, and the data show a clear inhibitory effect by EB. Together, these results suggest that the radiolabeled ligand binds NMDAR in a manner that is comparable to binding behavior in live cells.

We also used an uncompetitive NMDAR antagonist, MK-801 in competition studies. MK-801 has been shown to bind NMDAR in the ion channel as a means of blocking receptor activity. The NMDAR redox modulatory site exerts effects on the activity of MK-801, and we investigated whether the reverse effects also applied to our system. Using the method just described, we found this to be the case in spite of the fact that MK-801 has an IC50 of 11.220 nmol/L and a K i of 10.940 nmol/L. High values of IC50 and K i suggest that MK-801 would not inhibit 99mTc-PQQ-NMDAR binding, but the inhibitory effects were seen in our results which suggested that some indirect mechanism maybe play a some role. As a control, we used haloperidol, a dopamine D 2 receptor and σ receptor antagonist. It had no effect on 99mTc-PQQ-NMDAR binding. As a control, we used haloperidol, a dopamine D 2 receptor and σ receptor antagonist. It had no effect on 99mTc-PQQ-NMDAR binding (Fig. 3).

Fig. 3
figure 3

Competition curves of EB and MK-801

Full size image

In vivo studies

The effect of mannitol concentration on opening the BBB

The studies of cerebral receptor imaging agents were generally from in vitro to in vivo, and include affinity, uptake in brain and other properties. In this work, opening BBB in vivo to improve the uptake in brain was performed using mannitol. Six parallel experiments were conducted using three ICR mice in each. Varied concentrations of 20 % mannitol were administered to the mice 5 min prior to 99mTc-PQQ injection. Saline was used as a control. Animals were sacrificed 5 min after injection of 99mTc-PQQ, and the brains were analyzed by comprehensive dissection. The effect of dose on the uptake of 99mTc-PQQ in brain is shown in Fig. 4, and the results for spacial distribution in several regions of the brain are summarized in Table 1. Thalamus (TH) has a lower distribution than other regions and is considered the “reference region.” Ratios of other brain regions compared to TH provide information on the relative abundance of NMDAR in different parts of the brain. To avoid damaging brain tissue with high doses of mannitol, 7.5 mL/kg 20 % mannitol was selected as the optimum dose in subsequent experiments. This concentration produced a percentage dose of approximately 0.4 %ID/g (Fig. 4).

Fig. 4
figure 4

The effect of mannitol dose on the brain uptake of 99mTc-PQQ

Full size image
Table 1 The effect of mannitol dose on the distribution of 99mTc-PQQ in brain
Full size table

The effect of mannitol incubation time on opening the BBB

Next, we determined the best mannitol incubation time. Six parallel experiments were conducted in which mice were treated with 7.5 mL/kg 20 % mannitol for different time durations. This was followed by 99mTc-PQQ treatment for 5 min, animal sacrifice, and comprehensive dissection. Maximum 99mTc-PQQ uptake occurred in the 30 min mannitol treatment group (See Table 2 and Fig. 5).

Table 2 The distribution of 99mTc-PQQ in brain at different time points after mannitol injection
Full size table
Fig. 5
figure 5

The effect of mannitol on the time to BBB opening

Full size image

Spacial distribution of 99mTc-PQQ uptake in brain

Using the optimized treatment parameters, we determined that the spacial distribution of 99mTc-PQQ in brain has the highest density in the HP and FT, as indicated by the HP/TH and FT/TH ratios in Table 3, particularly for the 5, 30 and 60 min time points. This result is in agreement with our previous findings. We conducted competition studies using EB and found that in vivo, as in our in vitro experiments, EB inhibited 99mTc-PQQ-NMDAR binding in the presence of mannitol.

Table 3 Uptake of 9mTc-PQQ in brain of mice with time after mannitol injection
Full size table

Reversible opening of the BBB on the specific binding of 99mTc-PQQ to NMDAR

To further confirm that mannitol does not affect the binding specificity of 99mTc-PQQ to NMDAR, we examined the distribution of 99mTc-PQQ upon closing the BBB with EB. As shown in Fig. 6, the HP/TH and FT/TH ratios in the presence of mannitol and EB were significantly lower than the group that received mannitol only. From our results, it is clear that mannitol does not affect the binding specificity of 99mTc-PQQ to NMDAR.

Fig. 6
figure 6

The effects of mannitol on the binding specificity of 99mTc-PQQ

Full size image

Discussion

Hyperosmolar mannitol has been used clinically to open the BBB for the administration of pharmacological agents to the brain tissue [18]. In addition, Brown also indicated that mannitol-induced BBB disruption may allow improved design and/or enhancement of the penetration of chemotherapeutics and other types of drugs to better treat a multitude of neurological disorders [17]. Previously, our laboratory produced 99mTc-labeled PQQ. Biodistribution studies in mice showed that the activity of 99mTc-PQQ in brain reached its maximum by 5 min. Here we characterized the binding between 99mTc-PQQ and NMDAR in vitro and in vivo. Using RRAs, we determined ligand-receptor binding parameters including specific binding, maximum binding capacity and equilibrium constant. We established a method for opening the BBB using mannitol to enable entry of 99mTc-PQQ into the brain and mapped the regional distribution of the radioligand in brain. Specifically, our study shows that using mannitol to open the BBB significantly increased the levels of 99mTc-PQQ in brain. Target tissues with abundant NMDAR, such as the hippocampus and cortex, also increased uptake and retention of 99mTc-PQQ. Using competitive inhibition and reversible BBB opening experiments with EB, we have validated the binding between receptor and radioligand to be comparable to unlabeled PQQ and not affected by mannitol. Furthermore, our results strongly suggest that the BBB can be opened multiple times without affecting 99mTc-PQQ-NMDAR binding. The use of mannitol with 99mTc-PQQ should contribute significantly to improving image quality by increasing the uptake of a water-soluble agent in brain.