Efficacy and toxicity of the DPCPX nanoconjugate drug study for the treatment of spinal cord injury in rats

Abstract

Effects of the Adenosine A1 blockade using 8-cyclopentyl-1,3-diprophyxanthine (DPCPX) nanoconjugate on inducing recovery of the hemidiaphragm paralyzed by hemisection have been thoroughly examined previously; however, the toxicology of DPCPX nanoconjugate remains unknown. This research study investigates the therapeutic efficacy and toxicology of the nanoconjugate DPCPX in the cervical spinal cord injury (SCI) rat model. We hypothesized that a single injection of nanoconjugate DPCPX in the paralyzed left hemidiaphragm (LDH) of hemisected rats at the 2nd cervical segment (C2Hx) would lead to the long-term recovery of LDH while showing minimal toxicity. Adult male rats underwent left C2Hx surgery and the diaphragms’ baseline electromyography (EMG). Subsequently, rats were randomized into a control group and four treated subgroups. Three subgroups received a single intradiaphragmatic dose of either 0.09, 0.15, or 0.27 µg/kg, and one subgroup received 0.1 mg/kg of native DPCPX two times per day intravenously (i.v.) for 3 days (total 0.6 mg/kg). Rats were monitored for a total of 56 days. Compared with control, the treatment with nanoconjugate DPCPX at 0.09 µg/kg, 0.15 µg/kg, and 0.27 µg/kg doses elicited significant recovery of paralyzed LDH (i.e., 67% recovery at 8 wk) (P < 0.05). DPCPX nanoconjugate-treated rats had significant weight loss for first 2 wk but recovered significantly by day 56 (P < 0.05). The levels of gold in the blood and body tissues were below the recommended levels. No sign of weakness, histology of tissue damage, or organ abnormality was observed. A dose of DPCPX nanoconjugate can induce long-term diaphragm recovery after SCI without observed toxicity.

NEW & NOTEWORTHY The intradiaphragmatic administration of nanoconjugate is safe and has the promise to significantly reduce the therapeutic dosage for the treatment and achieve long-term and possibly permanent recovery in respiratory muscle dysfunction after SCI. No toxicity of nanoconjugate was found in any of the experimental animals.

INTRODUCTION

Respiratory complications are the leading cause of death in humans after spinal cord injury (SCI) (1). There are ∼17,700 new SCI cases every year in the United States alone, and more than half of such injuries occur at the cervical level (2). Currently, there are no known cures for muscle paralysis after SCI. In cases where patients cannot maintain adequate ventilation, long-term mechanical ventilator support is necessary, but dependence on such support can lead to complications such as infection, pneumonia, atelectasis, and even death (3, 4). The other potential treatment is through drug administration. Systemic administration (either by mouth or intravenously) of methylxanthines or theophylline phosphodiesterase (PDE) inhibitors is frequently accompanied by undesirable side effects (5); however, there has been limited preclinical and clinical therapeutic development in this disease area. It was determined that while theophylline similarly worked in humans as in rats, most patients with SCI could not tolerate theophylline when the drug was delivered systemically at standard therapeutic dose levels due to undesirable side effects. This has prevented this class of drugs in their clinical use. Thus, the problem is not with the choice of the drug but rather with administrating the drug. To directly address the problem of the side effects following systemic drug therapy in patients with cervical SCI being treated for respiratory muscle weakness, we developed a novel approach to intradiaphragmatic injection that combines nanotechnology with proven neurobiological principles to selectively target only the lower motor phrenic motoneurons (PMs) and premotor rostral ventral respiratory group (rVRG) neurons responsible for diaphragm function using 8-cyclopentyl-1,3-diprophyxanthine (DPCPX) nanoconjugate; a nanoconjugate-bound A1 adenosine receptor antagonist contains DPCPX (8-cyclopentyl-1,3-diprophyxanthine). The left phrenic nerve recovery was 2.2% and 72.4% after administering the theophylline nanoconjugate at a concentration of 0.12 mg/kg and the DPCPX nanoconjugate at 0.00015 mg/kg (or 0.15 μg/kg) (6) consequently. Our recent report showed that the administration of the DPCPX nanoconjugate facilitated respiratory-related recovery following cervical SCI (7). We established quality control in nanoconjugate synthesis, storage, and administration that further paved the way for this therapeutic avenue to be translated clinically to patients with SCI suffering debilitating respiratory insufficiency. However, the optimal dose and tolerance of the DPCPX nanoconjugate are not known. We, therefore, focus our current and near-term efforts on optimizing the DPCPX nanoconjugate chemistry and its further therapeutic development. The present study sought to 1) test the therapeutic effect of nanoconjugate DPCPX needed to be injected in the paralyzed left hemidiaphragm (LDH) following cervical SCI that will lead to long-term recovery and 2) determine the safety and toxicity of nanoconjugate DPCPX. We hypothesized that a single injection of nanoconjugate DPCPX in paralyzed left hemidiaphragm (LDH) leads to long-term recovery after SCI induced via left hemisection (C2Hx). We further hypothesized that the novel nanoconjugate DPCPX will be well tolerated by the treated animals and result in low toxicity levels.

MATERIALS AND METHODS

The treatment of rats, all experimental protocols, and surgical procedures used in this study were reviewed and approved by the Wayne State University Institutional Animal Care and Use Committee and were performed in accordance with the Guide for the Care and Use of Laboratory Animals published by the National Research Council (8).

Experimental Design

Adult male Sprague-Dawley (SD) rats (weight: 325 to 350 g) were obtained from Charles River Laboratories (Wilmington, MA). Animals were housed in a temperature (20°C–23°C) and humidity (50% to 60%)-controlled room on a 12:12-h light: dark schedule. On arrival, in the first 7 days of the acclimation period, the rats were started immediately into their respective experimental conditions by providing an enriched diet and tap water provided ad libitum similar to what was given acutely postoperative before experimental use. Body weight-matched animals were used within any given experiment. All rats underwent left hemisection (C2Hx) surgery on day 0 and were randomized into four treated subgroups. Each group received its specified treatment of DPCPX nanoconjugate as a single intradiaphragmatic administration dose of either 0.09, 0.15, 0.27 µg/kg, or systemic administration 0.1 mg/kg of native DPCPX, two times per day intravenously (i.v.) for 3 days (total DPCPX amount = 0.6 mg/kg). An extra group of rats C2Hx was used to serve as a normal control group, without intradiaphragmatic injections, to test for the presence of spontaneous recovery. All injection volume was adjusted based on the animal weight. First, the quantitative respiratory function measured by the electromyography (EMG) was recorded on days 0 (immediately after hemisection before the intradiaphragm), 14, and 56 following the SCI. Next, the amount of gold (either from the nanoconjugate or from native DPCPX) was measured from the target organs (spinal cord and brain) and nontarget organs, such as reticuloendothelial system (RES) organ tissues (from heart, kidney, and liver) in addition to samples from the blood, using inductively coupled plasma mass spectrometry (ICP-MS) and assessed at days 3 (blood) and 56 (blood and tissues) after injection. Then, animals were weighed individually daily for 7 days initially, then once a week for 8 wk (56 days, end point). After that, the signs of illness were monitored daily. Finally, at the end of this study of 56 days, target and nontarget tissues were collected for organ morphology and tissue histology.

Perioperative Care

Animals were prepared for aseptic survival surgery according to the Handbook for Laboratory Animal Care and Use. Animals were anesthetized with an isoflurane vaporizer-gas anesthetic machine (Vaporizer Sales & Services Inc. Rockmart, GA). Periodically, the animal’s level of anesthesia was assessed by pedal withdrawal reflex. The observed pattern and rate of respiration and body temperature were monitored rectally and kept constant at ∼36.5°C using a homeothermic blanket (Harvard Apparatus, Holliston, MA) throughout the surgery.

Left C2 Hemisection

After induction of anesthesia, on day 0, animals underwent a left hemisection at the second cervical spinal segment (C2Hx) to induce SCI as previously described in the laboratory (9). Briefly, once the animals had reached a surgical plane of anesthesia, the dorsal aspect of the neck and the head was shaved and cleansed with betadine and 70% ethyl alcohol three times. Carprofen (5 mg/kg) was administered subcutaneously (s.c.) and bupivacaine hydrochloride (1.5 mg/kg) intramuscularly (i.m.) along with the incision site. Body temperature was maintained through the surgical procedure at ∼36°C. A dorsal midline incision was made through the skin and paravertebral muscles to the first four cervical vertebrae to expose the second cervical vertebra (C2). A C2 laminectomy and durotomy were performed to expose the spinal cord. The left half of the spinal cord was cut just caudal to the C2 roots and extended from the midline to the most lateral aspects of the left side of the spinal cord using a micro scissor (Zeiss, Oberkochen, West Germany). The wound was sutured in two layers: the paravertebral muscles and skin.

Electromyography

To verify the completeness of the hemisection, electromyography was performed similarly to how it was done previously in our laboratory (10). Immediately after hemisection, the abdominal region was shaved and disinfected. Two 5-cm incisions on each side of the abdomen were performed 1.5 cm below and parallel to the rib cage and 1.5 cm below the xiphoid process (laparotomy). The abdominal surface of the diaphragm was exposed. Bipolar platinum electrodes (Natus Medical Incorporated, Middleton, WI) were placed in the anterior (sternal), lateral (costal), and posterior (crural) area of the left and right hemidiaphragm (LHD and RHD). The same animal had repeated abdominal surgeries two times to achieve different time point (14 and 56 days) recordings produced in this study. Raw signals were amplified (20,000×) and bandpass-filtered (30—3,000 Hz) using a Grass P511 amplifier (Grass Technologies). EMG data were acquired using a data acquisition system (CED1401) and Spike 2 program (Cambridge Electronic Design System). No animal was tested >3 times for respiratory recovery due to the stress of multiple surgeries. Most importantly, the EMG electrodes were placed in the equivalent sites of LHD and RHD during each recording for the same animal concerning the group of study.

Nanoconjugate Synthesis and Quality Control

The synthesis and quality control of procedures of the three-part nanoconjugate gold-drug-protein were described in our previous publication (11). Briefly, DPCPX was converted to pro-DPCPX containing a reactive hydroxyl group using the Mannich reaction (12). In a separate step, carboxyl-terminated 3.5–4-nm gold nanoparticles (AuNP) were synthesized by sodium borohydride reduction in the presence of mercaptosuccinic acid (MSA). Pro-DPCPX was chemically conjugated to AuNP, followed by chemical conjugation with wheat germ agglutinin-horseradish peroxidase (WGA-HRP) using standard bioconjugation methods as described in our previous work (13).

We determined in previous work that 1) the batch-to-batch particle size variation is <10%; 2) the drug dosage variation at an optimized DPCPX dosage of 0.15 μg/kg is <10%; 3) the nanoconjugate solution can be stored at 2°C–8°C away from light for up to 4 mo without significant precipitation; and 4) the nanoconjugate is capable of slow drug release lasting for days at physiological pH.

The Nanoconjugate Administration

The DPCPX nanoconjugate was prepared in different dosages (0.09, 0.15, or 0.27 µg/kg), and each of these doses was injected into the paralyzed left hemidiaphragm by making five separate 10-μL injections along the entire length of the left hemidiaphragm using a 28-gauge Hamilton syringe (Hamilton, Reno, NV). The injections were equally spread over the posterior, lateral, and anterior areas of the diaphragm. A single injection did not exceed 10 µL. The needle was inserted parallel to the muscle fibers to put the bevel up and visualize the needle tip to the naked eye before administration. A small bubble was created in the diaphragm where the solution was deposited. The needle was held for 5–10 s before it was withdrawn from the muscle to prevent nanoconjugate leakage outside the diaphragm. After administering the injections, the animals were cleaned, and the wound was sutured.

Postoperative Care

Animals were monitored closely with respect to their food and water intake. Weight and incision areas were monitored daily for the initial 10 days after surgery. The animals were given Buprenorphine-SR (slow release) (0.5–1.0 mg/kg, sc) once before surgery to minimize the pain and sterile saline (10 mL sc) twice a day for 3 days to avoid dehydration. Also, Carprofen (5 mg/kg sc) was given once a day for 3 days to reduce inflammation. Animals usually regain bladder function 24 h after the surgery. The bladder was carefully monitored and expressed as needed as advised by the Division of Laboratory Animal Resources (DLAR). Animals received food supplements such as cereal, diet gel, liquid diet, Ensure, and peanut butter during the acclimation period before surgery and until they returned to presurgical weight. E-collar was applied immediately after the laparotomy surgery to prevent chewing the incision and scratching their eyes and skin. Following SCI, on occasion, animals demonstrated autophagia of their hind limbs. To prevent that, animals were monitored closely daily and inspected for scratches and smaller wounds on their legs. If there was evidence of excessive grooming of the paws, the hind legs were painted with new skin/metronidazole powder.

Transcardial Perfusion

The rats were anesthetized with an isoflurane vaporizer and monitored to ensure a decreased respiratory rate; the blood was collected via intracardiac puncture before transcardial perfusion. A 4% paraformaldehyde (PAF) (Santa Cruz Biotechnology, Inc. Dallas, TX) was used with a mini pump (Grainger, Macedonia, OH) to complete the perfusion of animal bodies and harvest the organs’ tissues. Following the transcardial perfusion, the spleen, liver, diaphragm, kidneys, heart, brain (medulla), and C3–C6 cervical spinal cord segments were dissected and stored at −80°C.

Inductively Coupled Plasma Mass Spectrometry

To detect the gold concentration in tissue and blood samples, ICP-MS was used. Tissue samples (weight: 0.1–0.5 g) were dried before microwave digestion using 9:1 HNO₃:HCl (Optima grade) and then were diluted to analyze gold concentrations. Measurements were performed using a Thermo Scientific iCAP Q ICP-MS. The lowest point in the standard calibration curve was 0.1 ppb for Au and samples were ∼0.02 ppb. A saline blank solution was used as the negative control. External calibration using a series of standards (High Purity Standards) was performed before the measurements. The instrument was rinsed with 0.1 M HCl (Optima grade) between measurements to prevent gold precipitation on the internal sampling surface.

Blood Serum Collection

Blood was collected from the animals via tail vein on day 3 and via intracardiac puncture at the end of the study, i.e., 56 days postoperative under surgical stage level of anesthesia with isoflurane vaporizer, allowed to clot for 30 min to 1 h at room temperature, and centrifuged at 4,000 rpm for 10 min. The serum was collected in aliquots, analyzed, and stored at −80°C for further analysis.

Data Analysis

Electromyography recording.

The EMG recording from both hemidiaphragm occurred at baseline (day 0) after hemisection and was repeated on days 14 and 56. The purpose of baseline recording was to confirm that all animals had successfully hemisected and established a diaphragm activity baseline. All recordings were obtained once the animals had reached a surgical stage of anesthesia, and a 1-min time window was recorded over each area of the diaphragm (posterior, lateral, and anterior). The incidence of “functional recovery” was defined as recovery of the paralyzed LHD side and was considered positive in each animal only if the following conditions were met: 1) the EMG activity was detected in at least two of the three diaphragm areas (posterior, lateral, and anterior) and 2) if the activity persists for the duration of the study. The percentage of respiratory recovery was calculated by dividing the number of animals that showed EMG activity over the total number of animals in the group.

ICP-MS.

The amount of gold to the target (brain and spinal cord) and nontarget-tissues (spleen, liver, diaphragm, kidneys, and heart), including blood, was examined by ICP-MS. At the end of the study, various tissues as described above were measured and compared. The blood samples were collected on days 3 and 56 postoperatively. In this study, significant accumulation was defined as 100 µg (Au)/kg (sample) or higher in the brain and others in any single specimen (rats) from the dosing group.

Bodyweight.

Initially, animals were weighed daily for 7 days and then at weekly intervals for 8 wk (56 days) to ascertain the effect of nanoconjugate on weight gain/loss. Each group’s bodyweight percentage change was calculated on days 0, 14, and 56 postoperatively.

Statistical Analysis

Results were analyzed via two-sample t statistic (bodyweight) and z statistic (EMG respiratory recovery, body weight, and gold concentration) with the minimal expected power of 0.8, using Statistical Analysis System Software (SAS Institute Inc., Cary, NC) with two-sided type 1 error 0.05. Data were considered statistically significant if P < 0.05 and were reported as means ± standard error, with n denoting the sample size.

RESULTS

Respiratory Functional Recovery after Treatment following the Hemisection

Twenty-nine SD rats were included in control (n = 5) and four treated subgroups [0.09 (n = 6), 0.15 (n = 6), 0.27 µg/kg (n = 6), DPCPX nanoconjugates or DPCPX i.v. (n = 6)]. As shown in Fig. 1, no EMG activity was noted in LHD at baseline after C2Hx in all tested areas of the hemidiaphragm (later, anterior, and posterior), whereas the EMG recording in the right hemidiaphragm (RHD) remained intact in all groups. Figure 2 depicts the LHD recovery in different treatment groups in representative examples of EMG recording on day 14. The treatment with nanoconjugate DPCPX at all doses (0.09 µg/kg, 0.15 µg/kg, and 0.27 µg/kg) elicited paralyzed LDH recovery after a single injection; however, the treated group with 0.27 µg/kg of DPCPX nanoconjugate elicited diaphragm activity recovery in two out of three areas, from anterior to lateral, but not in the posterior area and persists for the duration of the experiment up to and including 56 days postinjection (Fig. 3). Neither control nor native DPCPX administered intravenously could elicit any measurable EMG in LHD.

Figure 1.

Representative examples of diaphragmatic EMG recordings of the anterior, lateral, and posterior areas of the LHD and RHD were taken on a 0-day baseline from the left hemidiaphragm (LHD) and right hemidiaphragm (RHD) following a single intradiaphragmatic administration of DPCPX nanoconjugate immediately after SCI at a dose (µg/kg). Control had no injection (left) or 0.09 (second left), 0.15 (middle), 0.27 (second right), and 0.1 mg/kg of the native DPCPX, 2 times/day for 3 days (total = 0.6 mg/kg) intravenous injection into the tail vein (right). Note the absence of all LHD muscle activity in all groups on day 0, whereas all RHD were unaffected. Data in blue color indicated the lack and in dark color demonstrated the normal uninjured diaphragm muscle activity. The amplitude of the EMG recordings, y-axis, was expressed in mV. x-axis represents time, measured in seconds. Calibration: 2 s. DPCPX, 8-cyclopentyl-1,3-diprophyxanthine; EMG, electromyography; SCI, spinal cord injury.

Figure 2.

Fourteen days after treatment (as described previously in Fig. 1), the left LHD muscle activity recovery in all 3 of 3 areas in 0.09 and 0.15 groups and 2 of 3 areas in 0.27 µg/kg of the DPCPX nanoconjugate group. Data in red color indicated the recovery of diaphragm muscle activity. The absence of the left LHD muscle activity on day 14 in control and intravenous groups was the same as produced on day 0. DPCPX, 8-cyclopentyl-1,3-diprophyxanthine; LHD, left hemidiaphragm.

Figure 3.

Fifty-six days after treatment (as described previously in Fig. 1), the recovery of LHD muscle activity was the same as on day 14 and persisted for 56 days. The absence of the left LHD muscle activity on day 56 in the control and intravenous groups was the same as produced on day 0. LHD, left hemidiaphragm.

The incidence of “functional recovery” 14 and 56 days after treatment was 50% (3 of 6), and 60% (3 of 5) of rats that received a low dose of 0.09 µg/kg of DPCPX nanoconjugate displayed functional recovery (P = 0.0143 and 0.006, respectively). In animals treated with 0.15 µg/kg of DPCPX nanoconjugate, the recovery was detected in 66.7% (4 of 6) of rats on both days 14 and 56 (P < 0.0005). Finally, at the high dose of 0.27 µg/kg of the DPCPX nanoconjugate, 33.3% (2 of 6) and 50% (2 of 4) of induced functional recoveries were detected on days 14 and 56 (P = 0.0825 and 0.045, respectively). The recovery was only 16.7% (1 of 6) in animals treated intravenously with a total of 0.6 mg/kg native DPCPX on days 14 and 56 (P = 0.273). All control animals had absent functional recovery in all follow-up EMG recordings (Fig. 4).

Figure 4.

Vertical bar graphs represent the percentage of animals that illustrated recovery of LHD function after treatment with the DPCPX nanoconjugate, either 0.09, 0.15, 0.27 µg/kg, or total 0.6 mg/kg of native DPCPX on days 14 (A) and 56 (B). The values in the graph represent the “functional recovery” percentage. Data in white indicate no recovery portion and in gray indicate recovery portion. The sample size was N = 5, 6, 6, 6, 6 rats and n = 5, 5, 6, 4, 6 rats at control, 0.09, 0.15, 0.27 µg/kg, and 0.6 mg/kg on days 14 and 56, respectively. *P < 0.05 vs. Control by two-sample t test. DPCPX, 8-cyclopentyl-1,3-diprophyxanthine; EMG, electromyography.

Signs of Illness

Rats were regularly monitored for signs of illness, such as lethargy, ruffled fur, food withdrawal, listlessness, infection or pain, and seizure or tremor. No animals showed signs of disease over the study period (56 days), including signs of infection or pain, abnormal histopathological examination of the brain or spinal cord tissues, or observation of seizure or tremor.

Gold Concentration in Tissues and Blood Serum

Figure 5 presents the DPCPX nanoconjugate gold (Au) biodistribution in C2 hemisected rats at different time points compared with control and native DPCPX. In this study, the concentration of gold (Au) after 56 days of DPCPX nanoconjugate intradiaphragmatic administration at a dose of 0.09 µg/kg was 5.15 µg/kg in the brain (n = 5, P = 0.3777). At a dose of 0.15 µg/kg, Au concentration was 3.39 µg/kg in the brain (n = 8, P = 0.0889). At a dose of 0.27 µg/kg, Au concentration was 2.47 µg/kg in the brain (n = 5, P = 0.118).

Figure 5.

The concentrations of gold were measured by ICP-MS in target (brain and spinal cord) and nontarget (spleen, kidney, liver, and heart) tissues on day 56 (A) and in the blood serum on days 3 and 56 (B). Line of Au (gold) significant accumulation as illustrated at 100 µg/kg is indicated in red color. The values in the graph represent means ± SE. ICP-MS, inductively coupled plasma mass spectrometry.

Body Weight

Figure 6 illustrates the summary results of body weight percentage change of control group (n = 6), DPCPX nanoconjugate 0.09 µg/kg dose (n = 6), DPCPX nanoconjugate 0.15 µg/kg (n = 7), 0.27 µg/kg (n = 6), or i.v. group at 0.6 mg/kg of native DPCPX (n = 7). On day 14 of this study, body weight decreased significantly compared with baseline in 0.09 µg/kg and 0.27 µg/kg groups, 7% (P = 0.04) and 9% (P = 0.009), respectively. No significant change in body weight was noted in rats that received 0.15 µg/kg and native DPCPX. On day 56 of this study, body weight increased significantly compared with baseline in all groups except in the 0.27 µg/kg group (P = 0.053).

Figure 6.

Body weight was monitored from the time of hemisection day 0 to the end point 56 days postoperative. Data showed body weight change percentage (%) on days 0, 14, 56 postoperative among each experimental group. Animal size in each group on day 14: control (n = 6 rats, P = 0.771), 0.09 (n = 6 rats, P = 0.04), 0.15 (n = 7 rats, P = 0.287), or 0.27 (n = 6 rats, P = 0.009) µg/kg of DPCPX nanoconjugate, and 0.6 mg/kg (n = 7 rats, P = 0.055) of native DPCPX. On day 56, sample size was the same as on day 14; however, the P values were as follows: in control (P = 0.0006), 0.09 (P < 0.0005), 0.15 (P < 0.0005), 0.27 (P = 0.053) µg/kg of DPCPX nanoconjugate, and 0.6 mg/kg (P = 0.021) of native DPCPX. *P < 0.05 indicates the body weight was significantly decreased on day 14 compared with control baseline; **P < 0.05 indicates the body weight was significantly increased at end of study day 56 compared with control baseline. Each group is compared within three time points. Each time point is compared with “0-day” by two-sample t test. DPCPX, 8-cyclopentyl-1,3-diprophyxanthine.

DISCUSSION

Summary of Findings

These experiments demonstrated a single intradiaphragmatic administration of DPCPX nanoconjugate at three doses (0.09, 0.15, and 0.27 µg/kg) after SCI in rats restores respiratory function and persists for 2 mo. The treatment with DPCPX nanoconjugate resulted in significant weight loss but recovered quickly after that. No evidence of gold accumulation in the vital organs was observed throughout this study. Together, these findings suggest that the adenosine A1 receptor antagonist DPCPX nanoconjugate is effective in enhancing respiratory recovery after acute cervical SCI was well-tolerated without significant toxicity,

Effect of DPCPX Nanoconjugate on Respiratory Recovery

We previously showed that DPCPX nanoconjugate induced recovery in the paralyzed hemidiaphragm following the same model of SCI (C2Hx) for up to 28 days following the SCI. This study extended the recovery findings to 56 days, confirming the drug’s long-term effect due to the slow release of active drugs from the formulation of nanomaterials. We selected gold nanoparticles as our nanomaterial platform because they are biocompatible, nontoxic, and suitable for attaching drugs and proteins (14). A recent study showed that AuNPs have been used extensively as a label for tracking protein distribution in vivo with no apparent toxicity (15). Currently, most nanotherapeutics are focused on systemic drug delivery to treat various cancers, and none of them (except our work) have been applied to treat respiratory problems associated with SCI. Furthermore, a thorough pharmacokinetics study of DPCPX nanoconjugate was performed recently by our group and found stability of the drug preparation for months and the ability to sustain active drug (DPCPX) release lasting for days at physiological pH slowly.

The results of DPCPX nanoconjugate efficacy were quantified by electromyography techniques and demonstrated that all three nanoconjugate doses could stimulate recovery of LHD activity after SCI. Somewhat surprisingly, there was a pronounced trend for animals. First, the EMG recovery following DPCPX nanoconjugate treatment did not follow a dose-depended trend. Second, if the induced recovery of paralyzed left hemidiaphragm was detected 56 days postinjection, no matter the tested dosage or administration method (intradiaphragm or iv), the respiratory function recovery persisted in the same area(s) of the diaphragm.

This study showed that after treatment, the diaphragm recovers in the pattern beginning from anterior to lateral areas and then posterior regions. The methods of injection sites did not change this observation. Still, the innervation and blood supply leading to the anterior injection sites may be greater than in posterior areas. The difference noted on day 14 of diaphragm functional recovery was detected significantly in 0.09 and 0.15 µg/kg of DPCPX nanoconjugate groups compared with the control group. However, the respiratory recovery in the 0.27 µg/kg DPCPX nanoconjugate group was observed in some animals but was not statistically significant compared with 0.09 and 0.15 µg/kg of DPCPX nanoconjugate groups. On day 56, the functional diaphragm recovery was significantly higher among all nanoconjugate groups than in the control group. Once the recovery of the paralyzed diaphragm was detected by EMG in the first 3 days in the groups of animals that received DPCPX nanoconjugate at any doses, the diaphragm EMG recovery persisted until the end of this study (56 days); likewise, if no recovery occurred within first 3 days, such as in the native DPCPX systemic administration, no recovery was detected throughout the monitoring period (56 days). This finding of no significant effect from administering the drug systematically could be due to the low dosage when administered intravenously or the active drug (DPCPX) may not cross brain-blood barriers. However, in a recent study from our laboratory, we found a significant effect on respiratory function recovery (tidal volume and minute ventilation) when the drug (DPCPX) was administered orally (16).

Our results suggest that the dose of 0.15 µg/kg of DPCPX nanoconjugate might be the optimal dose. It produced the highest respiratory function recovery compared with 0.09 and 0.27 µg/kg of DPCPX nanoconjugate at any given time point. In addition, this dose was associated with the lowest body weight loss and highest body weight gain on days 14 and 56. These results agree with a previous study from our research group, which showed that respiratory recovery on day 3 persists for up to 4 wk. The optimal dose of DPCPX delivered via nanoconjugate was 0.15 µg/kg. However, this new study emphasizes that the steady recovery pattern induced by DPCPX nanoconjugate can restore respiratory function recovery in the paralyzed diaphragm as early as on day 3 but has long-term effects and possibly permanent respiratory recovery after only one-time administration. Therefore, the ability of the novel drug delivery approach represents a major advancement in the field, and it provides a potential new treatment for patients with respiratory dysfunction after SCI with drugs previously not well-tolerated by other methods.

Our results differ from the previous studies due to the difference in particle size, surface chemistry, and injection methods (17, 18). These studies focused on unmodified simple AuNPs. The concentration of gold in brain regions diminished dramatically, indicating that most of the gold was in venous blood and not in the brain tissues (19). This study was carried out on 4-nm Au nanoconjugate intradiaphragmatic injection in SCI rats concerning biodistribution. The size of AuNP (4 nm) was chosen to be below the kidney filtration threshold (6–8 nm) to be cleared through renal excretion to avoid long-term retention in reticuloendothelial system organs. Gold reached significant amounts in targeted tissues, including the spinal cord and medulla in the brain by our method according to our previous study.

In comparison, 10-nm unmodified AuNP intravenously injected in the tail vein of rats showed little accumulation in the brain and wide distribution in blood and other organs 24 h postinjection (20). Overall, the concentration range of gold in tissues and blood was low to approach the instrument’s detection limit, which we attribute to the low dose administered and the manner of administrating the drug in our case. The resultant concentrations of gold detected in tissues and blood serum were far below the recommended level of gold in animal models (21, 22).

To evaluate nanoconjugate safety, the next step toward clinical translation is to carry out P.K. and biodistribution studies to assess the targeted drug delivery efficiency and nanoparticle safety (23), and the studies on tissue distribution of nanoparticles did not show accumulations in target and nontarget tissue, which can provide adequate safety preclinical data for future clinical trials to avoid unexpected toxicities in human subjects. Our effort is part of accelerating trends that translate basic nanoscience discoveries into nanotherapeutic products (24). This result provided significant evidence that DPCPX nanoconjugate injected intradiaphragmatic at all three doses of 0.09, 0.15, or 0.27 µg/kg delivered and released sufficient pharmacologically active (free) drug at targeted sites (spinal cord and brain) with all animals well tolerated, and no adverse effects were observed.

Although the bioavailability of DPCPX nanoconjugate was low, however, there was still a concern about whether in vivo effective doses of DPCPX nanoconjugate can be aggregated and toxic. The 0.09, 0.15, and 0.27 µg/kg of DPCPX nanoconjugate was considered achievable in vivo dose, as shown above. Quite surprisingly, as was shown recently from our laboratory, there is no visually observed AuNP aggregation in the injection site at the three tested doses after intradiaphragmatic injection, where most of the absorption occurs (a significant advantage of AuNPs compared with other colorless nanomaterials). Our recent study also carried out a transmission electron microscopy (TEM) study to examine the tissue after 48 h at the injection site by using the maximum dosage of 0.27 µg/kg for signs of nanoconjugate aggregation. We neither observed AuNP aggregation in any tissue sections nor signs of inflammation or fibrosis in any of the studied rats. We, therefore, conclude that the nanoconjugate particles moved away from the injection site 48 h after injection, and the particles do not induce cellular toxicity (cell injury, necrosis, and apoptosis) at the injection site. These data are consistent with our previously established data that there is no significant aggregation of DPCPX nanoconjugate since there is no such aggregation or adverse effect after injecting 0.09, 0.15, or 0.27 µg/kg of the nanoconjugate in rats (n = 35). It is possible because very low concentrations of AuNPs were used.

Regarding body weight, the interpretation of body weight loss in the context of severity assessment also constitutes a challenge that needs to take into account that appetite, food intake, and body weight development are regulated in a very complex manner (25). Weight loss can reflect decreased appetite due to distress, fear, and pain (26). In our study, even though the routine-effective analgesic dosage was administered during and after the surgery to minimize the pain and the pain-related disruption to body weight gain, there are still some parameters that need to be considered regarding the findings of this study. First of all, the hemisection procedure involves manipulating and removing the lamina (bone), the back part of a vertebra that covers the spinal canal, and is therefore considered a highly invasive surgical procedure and severely painful to the animals. Second, the laparotomy EMG recording and intradiaphragmatic administration procedures performed simultaneously with hemisection make the surgical procedure last 2.5–3.0 h to be completed for each animal under deep anesthesia. Lastly, one of the known complications of bladder dysfunction after SCI is weight loss. However, after 14 days postoperatively, animals gradually return to presurgical weight.

Mechanistic considerations.

The mechanisms of action for this novel nanoconjugate drug compound in the respiratory recovery in this animal model following SCI are multiple. First, the nanoconjugate delivers the active drug DPCPX (an adenosine receptor antagonist) that is capable of inducing long-term recovery through the elevation of intracellular cAMP levels and activation of the cAMP-PKA cascade, which would change the latent pathway to an effective pathway (27); second, the study used nanoconjugates that have been shown to travel trasnsynaptically (28) and result in targeted drug delivery to the site of action including the respiratory motoneurons (phrenic nucleus and rVRG) in the paralyzed side of the C2Hx model; third, the drug release as was shown previously starts quickly (within 48–72 h) and persists for a period for up to 4 wk leading to long-term recovery; finally, adenosine A1 receptor antagonists have been shown to enhance respiratory plasticity through an interplay between adenosine and serotonin systems following SCI (29). In contrast, Adenosine A2a receptors had an inhibitory action on serotonin-mediated respiratory plasticity. Therefore, A2a receptors may block the actions of the adenosine A1 receptor subtype on ventilation in the first 2 wk following SCI.

Therapeutic considerations.

There are no known cures for muscle paralysis after SCI. However, there has been limited preclinical and clinical therapeutic development in this disease area. One drug being studied is theophylline, which is FDA-approved, and both an adenosine receptor antagonist and a PDE inhibitor. The clinical studies of theophylline as described above showed significant side effects on patients. In other studies, multiple administrations of rolipram and pentoxifylline can also induce persistent functional recovery of the paralyzed hemidiaphragm in the SCI rat model for as long as 10 days. Although rolipram and pentoxifylline have been approved for clinical use, it is well known that these first-generation PDE inhibitors can induce the same side effects as theophylline, i.e., nausea and vomiting (30).

Interestingly, second-generation PDE inhibitors such as roflumilast (31) and cilomilast (32) have been developed and tested in phase III clinical trials in Europe for their effectiveness as anti-inflammatory agents in treating chronic obstructive pulmonary disease (COPD). Although early reports have suggested reduced side effects compared with the first-generation inhibitors, phase III efficacy studies in COPD involving cilomilast and roflumilast have been hampered by a low therapeutic ratio. As the dose is increased to reach therapeutic levels, side effects also increase, including nausea, diarrhea, abdominal pain, vomiting, and dyspepsia. Many of the patients withdrew from the phase III study because of adverse side effects, and the percentage of patients withdrawing from the trial increased with increasing doses [GlaxoSmithKline, Cilomilast (Ariflo, SB-207499), New Drug Application 21–573]. Currently, third-generation inhibitors are being pursued by several drug companies to maximize therapeutic efficacy and further decrease adverse effects (33). However, these new drugs are still in the preclinical animal phase of testing, and it is uncertain when, if ever, the drugs will be adopted for clinical use.

To directly address the problem of the side effects following systemic drug therapy in patients with cervical SCI being treated for respiratory muscle weakness, we developed a novel approach that combines nanotechnology with proven neurobiological principles to selectively target only the lower motor and premotor neurons (rVRG) responsible for diaphragm function. Our nanotherapeutic design targets a transporter protein, wheat germ agglutinin conjugated to horseradish peroxidase (WGA-HRP), chemically conjugated to a gold nanoparticle (AuNP), which in turn is chemically conjugated to a pro-drug, pro-theophylline, or pro-DPCPX. WGA-HRP is taken up by the terminals of phrenic axons when injected into diaphragm muscle and retrogradely transported to phrenic motoneurons; importantly, WGA-HRP is further transported transsynaptically across physiologically active synapses to neurons in the rVRG and does not transport to any other neuron centers (34). WGA-HRP has been used as a tracer to establish the ross phrenic pathway (CPP).

Our targeted drug administration can induce recovery of the hemidiaphragm in SCI rats by using a fraction of the systemic dose necessary to induce the same recovery. For comparison, the systemic dose of theophylline in rats is 15 mg/kg (10), whereas the theophylline content in the nanoconjugate is only 0.12 mg/kg. The systemic dose of DPCPX in rats is 0.1 mg/kg (35), whereas the DPCPX content in the nanoconjugate is 0.15 µg/kg, ∼0.1% of the systemic dose. In addition, the nanoconjugate can induce persistent recovery after only one injection. Hence, this work is part of an accelerating trend to translate basic nanoscience discoveries into nanotherapeutic products (36).

Nanotherapeutics promise to improve: 1) formulation for low-solubility drugs, 2) in vitro and in vivo drug stability, 3) pharmacokinetic (PK) and biodistribution, and 4) efficacy, all of which can potentially reduce toxicity and side effects (37). We selected gold nanoparticles (AuNPs) as our nanomaterial platform because they are biocompatible, nontoxic, and suitable for attaching drugs and proteins (21). AuNPs have been used extensively as a label for tracking protein distribution in vivo with no apparent toxicity. Currently, most nanotherapeutics are focused on systemic drug delivery to treat various cancers. None of them (except for our work) have been applied to treat respiratory problems associated with SCI.

Methodological Considerations

Our laboratory has used the left C2Hx procedure in rats for years to assess the respiratory recovery after SCI (16). Nevertheless, few methodological considerations may affect the interpretation of the findings of this study. First, we studied a small number of animals in five groups. However, there was a significant difference between the treatment groups and the control group. Therefore, studying more animals is not justified for the welfare of the animals. Second, we studied only male rats; however, it is less likely that sex played a role in this experiment. Animal strain than sex was shown to affect respiratory function and chemoreflex sensitivity (38). In addition, it is well known that in SCI, the vast majority of patients are male. However, the effect of sex in respiratory recovery following SCI is not well studied and needs further investigation. Third, although the treatment with DPCPX nanoconjugate had a U-shape response (as observed in Figs. 2 and 3), there was no significant statistical difference between 0.15 and lower or higher doses. However, the sample size is small, limiting this observation’s generalizability. Fourth, we used a previously well-established animal model of C2 hemisection (as described earlier) (10, 16, 39) as it is the most-studied model of SCI in the context of respiratory function. In addition, it allows for a neurologically complete injury to one-half of the spinal cord and a clear assessment of the hemidiaphragm activity before and after treatment. The protocol includes animals that only have complete ipsilateral diaphragm paralysis to assess for recovery. Other models, such as contusion-induced injury, are associated with a different and, often variable, degree of tissue sparing, which can confound the data. Fifth, we used a negative control to decrease the surgical burden on the animal with large laparotomy, which may limit the comparability between groups; however, we have shown previously that water vehicles did not have the effect (similar to the negative control in this study). Finally, the study monitored the animals for up to 2 mo, the longest period of in vivo monitoring after nanotherapeutic intervention in the SCI model. Nevertheless, a more extended period of monitoring and assessing other animals is needed to allow more comprehensive safety studies in preparation for future pilot phase 1 clinical trials in human subjects.

Overall, this study suggests that through a C2-hemisected animal model, the nanoconjugate approach promises to reduce the therapeutic dosage for SCI treatment significantly. It is possible to reduce and even eliminate the side effects by targeted drug delivery using nanotechnology and achieve long-term and possibly permanent recovery in respiratory muscle function with only one-time administration.

GRANTS

The research reported in this publication was supported by the National Institute of Neurological Disorders and Stroke of the National Institutes of Health under Award Number R61NS112443 (MPI: A.S. and G.M.).

DISCLAIMERS

The content authors' responsibility of the authors and does not necessarily represent the official views of the Wayne State University Office of Research and development.

DISCLOSURES

The nano device has a U.S. Patent #US9649381 entitled “transporter protein-coupled nanodevices for targeted drug delivery” (date May 16, 2017). The authors have no other conflicts of interest, financial or otherwise, to disclose.

AUTHOR CONTRIBUTIONS

G.M. and A.S. conceived and designed research; X.G., M.M.H., and G.M. performed experiments; X.G., M.M.H., and S.G. analyzed data; X.G., M.M.H., G.M., and A.S. interpreted results of experiments; X.G. and A.S. prepared figures; X.G. and A.S. drafted manuscript; X.G., M.M.H., S.G., G.M., and A.S. edited and revised manuscript; X.G., M.M.H., S.G., G.M., and A.S. approved final version of manuscript.