Preclinical evaluation of triiodothyronine nanoparticles as a novel therapeutic intervention for resuscitation from cardiac arrest

Abstract

Background: Given emerging evidence of rapid non-genomic cytoprotective effects of triiodothyronine (T3), we evaluated the resuscitative efficacy of two nanoparticle formulations of T3 (T3np) designed to prolong cell membrane receptor-mediated signaling.

Methods: Swine (n = 40) were randomized to intravenous vehicle (empty np), EPI (0.015 mg/kg), T3np (0.125 mg/kg), or T3np loaded with phosphocreatine (T3np + PCr; 0.125 mg/kg) during CPR following 7-min cardiac arrest (n = 10/group). Hemodynamics and biomarkers of heart (cardiac troponin I; cTnI) and brain (neuron-specific enolase; NSE) injury were assessed for up to 4-hours post-ROSC, at which time the heart and brain were collected for post-mortem analysis.

Results: Compared with vehicle (4/10), the rate of ROSC was higher in swine receiving T3np (10/10; p < 0.01), T3np + PCr (8/10; p = 0.08) or EPI (10/10; p < 0.01) during CPR. Although time to ROSC and survival duration were comparable between groups, EPI was associated with a ∼2-fold higher post-ROSC concentration of cTnI vs T3np and T3np + PCr and the early post-ROSC rise in NSE and neuronal injury were attenuated in T3np-treated vs EPI-treated animals. Analysis of hippocampal ultrastructure revealed deterioration of mitochondrial integrity, reduced active zone length, and increased axonal vacuolization in EPI-treated animals vs controls. However, the frequency of these abnormalities was diminished in animals resuscitated with T3np.

Conclusions: T3np achieved a ROSC rate and post-ROSC survival that was superior to vehicle and comparable to EPI. The attenuation of selected biomarkers of cardiac and neurologic injury at individual early post-ROSC timepoints in T3np-treated vs EPI-treated animals suggests that T3np administration during CPR may lead to more favorable outcomes in cardiac arrest.

Keywords: Cardiac Arrest; Ischemic Injury; Nanoparticles; Phosphocreatine; Thyroid Hormone; Triiodothyronine.

Figure 1:. Experimental Protocol for Blinded, Randomized, and Vehicle-Controlled Preclinical Trial of T3np, T3np+PCr, and EPI in Swine Subjected to Cardiac Arrest.

(A) Swine were subjected to electrical induction of ventricular fibrillation and 7-minutes of untreated cardiac arrest, followed by initiation of CPR with manual chest compressions and mechanical ventilation. Defibrillation was attempted 1-minute after the onset of CPR and every 2-minutes thereafter as needed. Animals that were not successfully resuscitated by CPR and defibrillation alone were randomized to receive empty NP (vehicle), EPI, T3np, or T3np+PCr at the 2-minute CPR timepoint via intravenous infusion in a blinded fashion. Resuscitation efforts continued for up to 20 minutes or until the ROSC was achieved (defined as unassisted SBP ≥ 80 mmHg for at least 1 minute). (B) Following ROSC, animals were followed for up to 4-hours, during which time an additional dose of the test drug was given if hypotension developed and only 1 dose of the drug had been given during the resuscitation period. Serial echocardiography and blood sampling were performed throughout the 4-hour post-resuscitation period, after which animals were humanely euthanized and tissue was collected for post-mortem analysis. Please see text for additional details. CPR = cardiopulmonary resuscitation; EPI = epinephrine; ROSC = return of spontaneous circulation; NP = nanoparticles; SBP = systolic blood pressure; T3np = triiodothyronine nanoparticles; T3np+PCr = triiodothyronine nanoparticles with encapsulated phosphocreatine.

Figure 2:. Rate of ROSC and Post-ROSC Survival in Swine Resuscitated with T3np, T3np+PCR, and EPI After Cardiac Arrest.

(A) Compared with vehicle treatment, EPI, T3np, and T3np+PCr administration during CPR resulted in a significant improvement in the rate of ROSC that was not different between treatment groups. The time needed to achieve ROSC was also similar among treatment groups. (B) The timecourse of post-ROSC survival among each group (left panel) reveals a similar pattern among EPI-, T3np-, and T3np+PCR-treated animals such that a majority of animals died prior to the 4-hour post-ROSC timepoint. Overall, average post-ROSC survival duration did not differ among treatment groups (right panel). Values are mean±SEM. *p<0.05 vs. empty NP; †p=0.08 vs. empty NP. ROSC = return of spontaneous circulation; NP = nanoparticles; T3np = triiodothyronine nanoparticles; T3np+PCr = triiodothyronine nanoparticles with encapsulated phosphocreatine.

Figure 3:. Post-ROSC Hemodynamics and Left Ventricular Function in Swine Resuscitated with T3np, T3np+PCR, and EPI After Cardiac Arrest.

Hemodynamic and echocardiographic parameters were similar among treatment groups prior to cardiac arrest and were generally similar throughout the post-resuscitation period. Notable differences among groups were observed only at the 15-minute post-ROSC timepoint, as EPI-treated animals exhibited an elevated heart rate, LV dP/dt_max, and LV ejection fraction relative to T3np-treated animals. Values are mean±SEM. *p<0.05: T3np vs. EPI; †p<0.05: T3np+PCr vs. EPI. LV = left ventricular; ROSC = return of spontaneous circulation; T3np = triiodothyronine nanoparticles; T3np+PCr = triiodothyronine nanoparticles with encapsulated phosphocreatine.

Figure 4:. Circulating Cardiac Troponin I Concentrations in Swine Resuscitated with T3np, T3np+PCR, and EPI Before and After Cardiac Arrest.

Circulating cTnI concentrations were low, but detectable, at baseline and did not exceed the 99^th percentile of normal animals (0.04 ng/mL). Within the first hour after ROSC, circulating cTnI levels rose in all animals, but the magnitude of cTnI elevation was greater in EPI-treated animals compared with animals treated with either T3np formulation. Values are mean±SEM. *p<0.05 vs. EPI; †p=0.06 vs. EPI. cTnI = cardiac troponin I; ROSC = return of spontaneous circulation; T3np = triiodothyronine nanoparticles; T3np+PCr = triiodothyronine nanoparticles with encapsulated phosphocreatine

Figure 5:. Post-ROSC Hippocampal Ultrastructure in Swine Resuscitated with T3np, T3np+PCR, and EPI After Cardiac Arrest.

(A) Hippocampal mitochondrial integrity was assessed by examining cristae organization and fragmentation (left panel; blue arrows), with assignment of a score on a scale from 1–5 (5 = mitochondria with preserved ultrastructural integrity). Based on this scoring system, mitochondrial integrity was significantly reduced in EPI-treated animals (left panel; red arrows) compared with sham controls but was largely preserved in T3np-treated animals. (B) Active zone length (left panel; outlined in blue) at neuronal synapses was significantly reduced in EPI-treated animals vs. sham controls, suggestive of impaired neurotransmitter release from synaptic vesicles in these animals. However, T3np-treated animals did not exhibit significant changes in active zone length compared with sham controls. (C) Ultrastructural changes to hippocampal axons were assessed as shown in the left panel, with a normal axon labeled with a blue asterisk, dysfunctional axons labeled with red asterisks, and vacuoles outlined in red. Dysfunctional axons were more prevalent in EPI-treated animals and this group also exhibited a significant increase in axonal vacuolization, an early sign of axonal injury. However, this was attenuated in animals treated with either formulation of T3np, indicating better preservation of axonal structure in these groups. Values are mean±SEM. *p<0.05 vs. sham control; †p=0.07 vs. CA+EPI. CA = cardiac arrest; EPI = epinephrine; T3np = triiodothyronine nanoparticles; T3np+PCr = triiodothyronine nanoparticles with encapsulated phosphocreatine.