Targeted-delivery of nanomedicine-enabled methylprednisolone to injured spinal cord promotes neuroprotection and functional recovery after acute spinal cord injury in rats

Abstract

To date, no therapy has been proven to be efficacious in fully restoring neurological functions after spinal cord injury (SCI). Systemic high-dose methylprednisolone (MP) improves neurological recovery after acute SCI in both animal and human. MP therapy remains controversial due to its modest effect on functional recovery and significant adverse effects. To overcome the limitation of MP therapy, we have developed a N-(2-hydroxypropyl) methacrylamide copolymer-based MP prodrug nanomedicine (Nano-MP) that can selectively deliver MP to the SCI lesion when administered systemically in a rat model of acute SCI. Our in vivo data reveal that Nano-MP is significantly more effective than free MP in attenuating secondary injuries and neuronal apoptosis. Nano-MP is superior to free MP in improving functional recovery after acute SCI in rats. These data support Nano-MP as a promising neurotherapeutic candidate, which may provide potent neuroprotection and accelerate functional recovery with improved safety for patients with acute SCI.

Introduction

Spinal cord injury (SCI) affects up to 300,000 people in the United States (US) with about 17,700 new SCI cases each year.¹ SCI often results in catastrophic neurological impairments and life-long disability which present significant health, economic and social challenges for the patients, their families and the society.^2,3 Total health care costs associated with SCI exceed $10 billion annually in the US, and lifetime per person direct and indirect costs can exceed $3 million.^1,4 Multiple promising therapies for SCI have been investigated, including surgical, non-pharmacological interventions (e.g., hypothermia), pharmacological agents (e.g., targeting myelin-associated inhibitors of regeneration), and cellular transplantation therapies (e.g., Schwann cells and human embryonic stem cells).⁵ Unfortunately, none of these attempted strategies demonstrated sufficiently robust efficacy to be widely accepted by the clinical community.⁵

To date, methylprednisolone (MP) is the only FDA approved therapy for acute SCI treatment, although it is no longer widely used clinically.⁶ Damage to the spinal cord results both from the initial trauma and then from secondary processes that occur several hours to days following the initial injury. The secondary injury processes induce oxidative stress, calcium-mediated damage, excitotoxicity, immune reactions that culminate in neuronal apoptosis.⁷ While the extent of primary injury is determined by the initial trauma, which is not amenable to therapeutic intervention, the relatively slow progression of secondary neurological injury response (hours to days after the initial injury) provides a crucial post-acute SCI window of opportunity to improve functional outcomes. Should spinal cord tissue be spared due to dramatic reductions in secondary injury, one may anticipate significantly improved neurofunctional recovery. It is with this premise, systemic MP therapy was developed as an option to treat acute traumatic SCI.^6,8

In 1990, the second National Spinal Cord Injury Study (NASCIS II) trial reported its comparison of MP, naloxone and placebo showed no difference between the groups for functional recovery in patients with acute SCI. However, a subgroup of patients identified on post hoc analysis who were treated within 8 h of injury with MP (30 mg/kg bolus followed by 5.4 mg/kg over 23 h) had better motor scores than the placebo group.⁹ Based on this tenuous result, MP quickly became established as a standard of care. In 1997 and 1998, based on findings of the NASCIS III trial, it was recommended to initiate a 24-h course of MP if treatment was started within 3 h of injury and a 48-h course if treatment was started within 3–8 h of injury^6,10 with both adhering to NASCIS II protocol. The use of systemic high-dose MP in patients with acute SCI remains controversial because of the modest neuroprotective benefit, which should be weighed against its potential serious adverse side effects, including susceptibility to infection and wound complications, gastric bleeding, sepsis, diabetic complications, pneumonia, and acute corticosteroid myopathy.^8,9,11–13 Because of these concerns, the Guidelines for the Management of Acute Cervical Spine and Spinal Cord Injuries list MP administration merely as a treatment option to be considered for the treatment of acute SCI.¹⁴ Current AOSpine guidelines recommends that its administration be considered within 8 h after acute SCI and administered for 24 h in those without medical contraindications.^15,16

While the overarching objective of the current systemic MP therapy is to dampen the secondary injury pathology after SCI, the regimen exposes the entire body to high doses of MP in order to achieve effective local MP concentrations at the injury site, which results in the serious adverse side effects that in turn hinder the utility of higher and more efficacious MP levels at the lesion. Therefore, we hypothesize that targeted MP delivery to the injury site, once achieved, will significantly mitigate the systemic exposure of MP therapy and allow the medication to be delivered at far higher concentrations to the SCI site. As the direct outcome of such an approach, we anticipate markedly improved functional neurological outcome and safety profile. As such, we envision that improved MP therapy may be integrated as part of the standard post SCI care protocol to address the unmet urgent need among SCI patients.

Polymeric glucocorticoid (GC) prodrug nanomedicines have been developed and tested extensively as novel therapeutic agents for inflammatory and autoimmune diseases. When administered systemically, their passive targeting to inflammatory tissues in pathologies such as rheumatoid arthritis (RA),^17–19 lupus nephritis (LN),^20–22 traumatic brain injury,²³ or orthopedic implant loosening^24,25 is governed by Extravasation through Leaky Vasculature and Inflammatory cell-mediated Sequestration (ELVIS) effect.²⁶ Upon delivery to the sites of pathology, the prodrugs are activated by pathological environmental cues (e.g., acidosis and/or reduced lysosomal pH), which provides an additional level of specificity of drug for the pathological site. This unique pathophysiology-driven prodrug design has been proven to be very effective to potentiate the anti-inflammatory and immunoregulatory efficacy of GC and drastically reduce their toxicities. To ensure streamlined clinical translation, well-established biocompatible water-soluble polymers such as N-(2-hydroxypropyl) methacrylamide (HPMA) copolymers and polyethylene glycol have been used as the carrier systems in prodrug nanomedicine design. Given the extensive inflammation associated with acute SCI,²⁷ we posit that the polymeric GC prodrug nanomedicine design that has been successfully used in other inflammatory pathologies in AA and LN may also be applied to improve the treatment of acute SCI. Despite many other HPMA-conjugated glucocorticoids, no group has synthesized HPMA-based MP.

Several studies have demonstrated that, local MP nanoparticle formulations significantly reduced lesion volume and improved behavioral outcomes in animals. However, these reported approaches utilized local delivery via surgical procedures, which is not practical for most patients after acute SCI.^28–31 The main objective of the present study is to develop a HPMA copolymer-based MP prodrug nanomedicine (Nano-MP) via intravenous injection that selectively delivers MP to the spinal cord lesion. We first performed the biodistribution study to examine the selective uptake of the polymeric prodrug at the injured spinal cord (Study 1) after SCI. Additional acute analysis (Study 2 Part I) was performed at 2 days post injury to quantify specific protein expression indicative of secondary injury. Subacute analysis (Study 3) was performed at 7 days post injury to quantify the degree of the injury-related cellular reactivity responsible for wound-healing as well as neural apoptotic indices. The functional recovery of the animals was assessed pre-operatively and post-operatively over 2 months (Study 4 Part I). In parallel, Study 2 Part II, Study 3 and Study 4 Part II were conducted to assess reductions in side effects with the use of Nano-MP compared to systemic parent MP molecule; the findings of this work will be reported as a separate report in conjunction with this report in this issue of the journal. Because approximately 80 % of persons with SCI are male, these studies were performed using male rats. The design and major endpoints of Study 1–4 are outlined in Fig. 1.

Fig. 1.

The experimental design and a summary on major endpoints of each study.

Materials and methods

Materials, instruments and synthesis of Nano-MP

Please refer to “Supplemental Materials” for this part in details.

Surgery and in vivo experimental design

All animals were maintained on a 12:12-h light/dark cycle with lights on at 07:00 am in a temperature-controlled (20 ± 2 °C) vivarium, with food and water ad libitum. All procedures were approved by the IACUC at James J. Peters Veterans Affairs Medical Center and conformed to all guidelines and regulations for the protection of the welfare of animals. Complete spinal cord transection model was selected to be used in Study 1, 2 and 3, and was conducted as previously described.^32–38 For the function recovery assessment in Study 4, a moderate contusion SCI was produced at the interspace between the 8th and 10th thoracic vertebra using by an Infinite Horizontal (IH) Impactor.^39,40 Post SCI induction, urine was manually expressed 3 times/day until automaticity develops, and then as needed. Baytril was administered postoperatively for the first 3–5 days then as needed to manage cloudy/bloody urine or wound infection. Sham-transected/ contusion animals received a laminectomy-only surgery. See Supplemental Materials for details of SCI models’ justification, induction procedures and post-surgical care. In Study 1, three groups of animals were investigated: Healthy control (n = 10), laminectomy-only (Sham, n = 10), and SCI animal (SCI, n = 10). In Studies 2, 3 and 4, four groups of animals were planned: laminectomy-only (Sham, n = 12), SCI animals with vehicle (SCI + veh, n = 12), SCI animals treated with MP (SCI + MP, n = 12), and SCI animals treated with Nano-MP (SCI + Nano-MP, n = 12).

Treatments

An Alzet pump was implanted in a subcutaneous pocket during surgery. Immediately after SCI, a single dose of Nano-MP (60 mg/kg containing ~6 mg/kg of free MP) was administered to the SCI rats via intravenous (i.v.) administration. Unconjugated MP (30 mg/kg) and vehicle (saline) were also subjected to a bolus i.v. administration and followed by 24-h infusion of MP via the Alzet pump at 5.4 mg/kg/h in SCI + MP group, or vehicle in SCI + veh and SCI + Nano-MP groups, respectively. On an mg/kg basis, the dosing protocol for intravenous MP administration is identical to that prescribed by the Bracken protocol.^10,41 The dose-equivalent free MP level in the systemic Nano-MP treatment is equal to 3.75 % of the systemic MP dose clinically used. Sustained low-dose local delivery of MP (about 5 % of the systemically administered dose) could provide neuroprotection.²⁸ Sham-transected/contusion animals underwent laminectomy and implantation of the same Alzet pumps, with vehicle infusion instead, as baseline controls.

Assessment of Nano-MP biodistribution

In Study 1, two fluorescently tagged MP prodrugs (Nano-MP–IRDye, 1 mg/rat; Nano-MP-Alexa, 2 mg/rat) were co-administered via tail vein immediately after T₄ spinal cord transection. Prior to injection or at 1, 24 and 48 h post administration, the animals were anesthetized through inhalation of isoflurane and were optically imaged to evaluate the distribution of the Nano-MP using LI-COR Odessey Small Animal Imaging System. The animals were euthanized on day 3. At euthanasia, the spinal cords were isolated for ex vivo optical imaging with the same imager. The spinal cords were then sectioned and stained with fluorescence-labeled cell markers for confocal microscopic analyses to assess cell-specific localization of Nano-MP.⁴²

Tissue preparation and histological analysis

At 2 and 7 days after injury, animals (n = 5–6/condition) were perfused intracardially with 4 % paraformaldehyde. The spinal cords were removed and fixed overnight in the same fixative. Longitudinal 10 μm thick tissue sections were cryosectioned. Serial sections were blocked and followed by incubation of primary antibodies including: TNF-α, GFAP (astrocyte marker), nitrotyrosine (oxidative stress marker), μ-Calpain (pro-apoptotic protein), ED1 (macrophages/reactive microglia marker), CSPG (impaired axon regeneration marker), Bax (pro-apoptotic marker), activated Caspase-3 (apoptotic marker), and SM32 (motor neuron marker). After washing and incubation with Alexa Fluor 647-conjugated secondary antibodies, slides were mounted and subjected to fluorescent microscopy. For animals that received Nano-MP-Alexa 647 injection, slides were either immunostained with GFAP followed by Alexa 647-conjugated secondary antibody or directly stained with Cresyl violet. Images were taken using the EVOS FL Auto system. Fluorescence intensity quantifications were performed in the ImageJ software (NIH).

Lipid peroxidation

At 2-day post injury, the animals were sacrificed and sections of spinal cord centered around the site of lesion or laminectomy were dissected out and stored at −80 °C. The injured spinal cords (~ 25 mg) lysed in RIPA buffer and lipid peroxidation was measured using the TBARS Assay Kit following the manufacturer’s instructions. Malondialdehyde (MDA) concentration was determined using the colorimetric method.

Locomotor function testing

Use of the hindlimbs was scored using the Basso, Beattie and Bresnahan (BBB) locomotor score. A score of 0 indicates no hind-limb movement whereas a score of 21 indicates unimpaired locomotion as observed in normal uninjured rats. The test was performed pre-operatively, day 1, 3, and 7 post injury, and then once weekly postoperatively, as described previously⁴³ and in “Supplemental Materials”. Briefly, animals were placed at the center of a square box and observed as they move toward a wall/corner, which was recorded by a video camera. The final BBB scores for each time point were determined by combining the BBB paper record with the results of the video record review by two accessors.

Statistics

Standard power analyses were used to determine the requisite minimum number of animals to ensure sufficient statistical power, as described previously.⁴⁴ Data are expressed as mean ± SEM; the number of independent samples (n) is provided in the legend of each figure. The statistical significance of differences among means was tested using one-way analysis of variance and Bonferroni’s post hoc test to determine the significance of differences between individual pairs of means using a P value of 0.05 as the cutoff for significance. Statistical calculations were performed using Prism 4.0c (GraphPad Software, CA).

Results

Synthesis of Nano-MP and labeling with fluorescent dyes

Nano-MP was synthesized according to the route shown in Fig. 2. Different from the previous synthesis of HPMA copolymer-based dexamethasone prodrug (P-Dex), in which C3 carbonyl of A-ring was the preferred hydrazone linker formation site, MP preferentially conjugated to the polymeric carrier via C20 carbonyl. Given that HPMA copolymer is not biodegradable, the molecular weight of Nano-MP was maintained at 30 kDa to facilitate its clearance through the kidney glomerular filtration with threshold of 45 kDa for HPMA copolymer.⁴⁵ The dispersity is narrow with Ð = 1.03. To determine the amount of MP in Nano-MP, it was hydrolyzed in 0.1 N HCl and analyzed with HPLC. The MP content in Nano-MP was determined to be ~10 wt%. Nano-MP–IRDye and Nano-MP-Alexa were synthesized via polymer analogous reaction between primary amine-containing Nano-MP and IRDye 800CW-NHS or Alexa Fluor 647-NHS. The IRDye and Alexa Fluor 647 contents in Nano-MP were determined to be 4.92 × 10⁻⁶ mol/g and 5.23 × 10⁻⁶ mol/g, respectively.

Fig. 2.

Synthesis of HPMA copolymer-based methylprednisolone prodrug nanomedicine (Nano-MP).

Selective uptake of Nano-MP in the injured spinal cord

To visualize Nano-MP biodistribution in vivo in Study 1, Nano-MP–IRDye and Nano-MP-Alexa were co-administered i.v. immediately after SCI. Blood vessels in the rat ear were visible within 1 h after injection, indicating Nano-MP’s presence in circulation. At day 2 and 3, no fluorescent signal is detectable in the rat ear, suggesting clearance of Nano-MP from the circulation (Fig. 3A). Importantly, the presence of Nano-MP-IRDye in the spinal cord was only detectable in rats with SCI, not in normal or Sham rats with laminectomy (Fig. 3B), confirming the selective targeting and local retention of Nano-MP at the spinal cord injured site. The targeted delivery of Nano-MP to injured spinal cord is further confirmed by the conventional fluorescence microscopy analysis, which demonstrated the presence of Nano-MP-Alexa in the spinal cord section of SCI rats, but in not in the Healthy or Sham controls (Fig. 3C).

Fig. 3.

Selective uptake of Nano-MP in the injured spinal cord. (A) Male rats underwent complete T₄ spinal cord transection or laminectomy only. Nano-MP-IRDye (1 mg/rat) was injected via tail veins immediately after SCI. The biodistribution of the Nano-MP in rat ear was examined at different time points post-injection. (B–C) Nano-MP can be detected in the injured spinal cord (B) or spinal cord section (C) at day 3, but not that in normal or laminectomy-only rats. Images were taken using a LI-COR imaging system (X4) for (A) and (B) and fluorescent microscopy for (C) (scale bar = 400 μm), respectively. n = 10/group.

Internalization of Nano-MP by microglia and astrocytes

To further evaluate the subcellular-localization of Nano-MP-Alexa, longitudinal spinal cord sections were examined by fluorescent microscopy. No colocalization between Nano-MP-Alexa 647-fluorescence and Nissl staining (Fig. 4A, B) or SM32 staining (Supplemental Fig. 1) were detectable, suggesting weak internalization by spinal cord motor neurons. Conversely, Nano-MP-Alexa colocalized with CD11⁺ microglia (Fig. 4C) and GFAP⁺ astrocytes (Fig. 4D) respectively, suggesting strong sequestrations by these cell phenotypes.

Fig. 4.

Internalization of Nano-MP by microglia and astrocytes. Nano-MP-Alexa 647 (2 mg/rat) was injected via tail veins immediately after SCI and detected in injured spinal cord at about 1 cm caudal to the lesion epicenter by confocal microscopy. (A and B) Nano-MP (green color) did not accumulate in motor neurons (stained by Nissl Staining; purple). Scale bar = 100 μm. Accumulation of Nano-MP in CD11⁺ microglia (C) and GFAP⁺ astrocytes (D) (n = 10/group). Motor neurons were stained by SM32 antibody (red). Nuclei were stained by DAPI (blue). White arrows show double-labeling in intact cells. Scale bar = 20 μm.

A single dose Nano-MP significantly inhibited oxidative stress in the spinal cord

Oxidative stress is considered a hallmark of SCI or damage to other sites of the central nervous system. MP was reported to have significant antioxidant activities and improve the recovery of patients with SCI in clinical trials, primarily through the inhibition of reactive oxygen induced by lipid peroxidation and inflammation.⁸ The level of nitrotyrosine, an indirect chemical indicator of toxic nitric oxide and peroxynitric-induced cellular damage, was examined in the spinal cord. SCI-Nano-MP group had significantly lower SCI-induced accumulation of nitrotyrosine at 2 days (−33.9 % vs. SCI + veh; Fig. 5) and 7 days post injury (−34.0 % vs. SCI + veh; Supplemental Fig. 2) whereas the SCI-MP group did not. The TBARS assay was used to assess the levels of MDA as an indication of lipid peroxidation.⁴⁶ Acute SCI significantly increased the MDA level in the spinal cord as compared to Sham control animals at 2 days post injury (9.44 ± 0.49 μM vs. 5.00 ± 0.62 μM). Notably, while systemic MP treatment had no effect, Nano-MP treatment reduced the MDA level (5.47 ± 0.85 μM), thus protecting the animals from SCI-mediated MDA accumulation and lipid peroxidation in the spinal cord (Fig. 5B).

Fig. 5.

Nano-MP attenuated SCI-induced oxidative stress and lipid peroxidation in the spinal cord. A-a. Representative images showing the nitrotyrosine level in the spinal cord (longitudinally sectioned) and fluorescent intensity quantification (A-b). B. Malondialdehyde (MDA) level was measured using TBARS assay. Data are expressed as Mean ± SEM. n = 5–6/group. *p < 0.05, **p < 0.01, ***p < 0.001 by one-way ANOVA. Scale bar = 40 μm.

A single dose of the Nano-MP reduced inflammation and injury-related cellular markers in the spinal cord

Inflammation responses are a major element of secondary injury and play a pivotal role in regulating the pathogenesis of acute and chronic SCI. From 3 to 24 h post SCI, upregulation of TNF-α could be detected around the injured area.^47,48 As expected, acute SCI led to increased TNF-α reactivity in the spinal cord. Interestingly, similar to oxidative stress markers, TNF-α also showed a significant decrease at 2 days after injury (−51.3 % vs. SCI + veh) upon treatment with Nano-MP while MP treatment had no effect (Fig. 6A). At 7 days post injury, both Nano-MP and free MP treatments significantly reduced SCI-induced TNF-α expression to similar levels (−33.9 % and −42.5 % vs. SCI + veh, respectively) (Supplemental Fig. 3). Acute SCI also resulted in increased reactivity of GFAP⁺ astrocytes (Fig. 7A), ED-1⁺ macrophages/reactive microglia (Fig. 7B) and CSPG deposition (Supplemental Fig. 4) at 7 days post injury. Both Nano-MP and free MP treatment reduced the GFAP reactivity and ED-1 reactivity (Fig. 7A and B) whereas Nano-MP, but not free MP, inhibited CSPG deposition (indicating greater axon regeneration potential) at 7 days after acute SCI.

Fig. 6.

Effects of Nano-MP on inflammation and pro-apoptotic markers after acute SCI. Representative images showing the TNF-α (A), Bax (B) and Calpain (C) in the spinal cord (longitudinally sectioned) and fluorescent intensity quantification (n = 5–6/group). *p < 0.05, **p < 0.01, ***p < 0.001 by one way ANOVA. Scale bar = 40 μm.

Fig. 7.

Effect of Nano-MP on the expressions of injury-related cellular marker and caspase-3 after acute SCI. Representative images showing the GFAP (A), ED-1 (B) and caspase-3 (C) in the spinal cord (longitudinally sectioned) and fluorescent intensity quantification (n = 5–6/group). *p < 0.05, **p < 0.01, ***p < 0.001 by one way ANOVA. Scale bar = 40 μm.

A single dose of the Nano-MP administration significantly enhanced neuroprotection after acute SCI

Apoptosis, as demonstrated by nuclear DNA fragmentation and caspase activation, was a prominent feature in the spinal cord post SCI. After SCI, some cells at the lesion site die by post-traumatic necrosis, whereas others die by apoptosis.⁴⁹ Pro-apoptotic proteins such as Bax, Caplain (a Ca²⁺-dependent protease)^28,50 and caspase-3 activation play central roles in cell apoptosis. As expected, acute SCI led to upregulation of Bax and Caplain at 2 days post injury. Nano-MP treatment significantly reduced the reactivity to Bax and Caplain at the site of injury when compared to control (veh) treatment (Fig. 6B and C). Nano-MP, but not MP significantly reduced the expression of Bax as compared to SCI control animals at 2 days (−27.0 % and −8.3 % vs. SCI + veh, respectively) post injury (Fig. 6B). Caspase-3 activation was also evaluated. Both Nano-MP and free MP significantly reduced SCI-induced Caspase-3 activation at 7 days post injury (−49.8 % and −30.0 % vs. SCI + veh, respectively), with Nano-MP treatment showing more robust effect than free MP (Fig. 7C).

Further studies were conducted to examine cellular location of caspase-3 and Bax in injured spinal cord tissue sections. As expected, acute SCI led to increased caspase-3 activation and Bax expression. Interestingly, the caspase-3 and Bax immunoreactive signals selectively co-localized with motor neuron-specific immunoreactivity (SM32) as determined by overlapping caspase-3/Bax and SM32 staining (Fig. 8A–b, c and 8B–b,c). Of note, animals in the Nano-MP group displayed significantly reduced immunoreactive signal for caspase-3 activation (Fig. 8A–d) and Bax expression in motor neurons (Fig. 8B–d), suggesting that Nano-MP treatment would lead to a significantly greater reduction in neuronal damage, indicative of enhanced neuroprotection of the spinal cord after acute SCI when compared to MP treatment (Fig. 8A–c and 8B–c). Collectively, these findings support Nano-MP’s potential in providing more significant protection against SCI-induced apoptosis in animals than free MP treatment.

Fig. 8.

Nano-MP decreased SCI-induced motor neuron apoptosis in the spinal cord and promoted functional recovery after acute SCI. Representative images show the caspase-3 (A) or Bax (B) immunofluorescence staining in the spinal cord (longitudinally sectioned, n = 5–6/group). Caspase-3 or Bax stain in green; SMI32 (red) and DAPI (purple) stain motor neuron and nucleus, respectively. Scale bar = 20 μm. Arrow, co-localization. (C) Functional recovery evaluated by the BBB scale after SCI. Vehicle, free MP or Nano-MP was administered immediately after contusion SCI. n = 12/group; *p < 0.05, **p < 0.01 by two-way ANOVA and Bonferroni post tests.

Effects of a single dose of the Nano-MP on functional recovery after acute SCI

The potential impacts of Nano-MP on improving animal functional outcome after acute SCI were assessed (Study 4 Part I). Immediately after a moderate contusion SCI at T₉ induced by IH impactor device, rats were administered free MP, Nano-MP, or vehicle. Hindlimb motor function was then assessed pre-operatively, Day 1, 3 and 7 post injury, and then once weekly postoperatively. At Day 1 or 3 after SCI, BBB scores were similar among all groups. At other time points, Nano-MP-treated rats exhibited a more rapid recovery, yielding higher improvement on BBB scores than that of animals given free MP or those in the control groups (Fig. 8C). At 56 days post SCI, Nano-MP-treated rats had an average of ~12 BBB scores and regained the ability to take weight-supported steps without consistent forelimbs and hindlimbs coordination whereas MP-treated rats had an average of ~9 BBB scores and demonstrated weight support in stance only without consistent stepping. This observation demonstrated that Nano-MP is superior to free MP in its ability to improve functional motor recovery after acute SCI in the rodent model employed in this study.

Discussion

SCI is a catastrophic event that causes loss of sensory, motor, and autonomic function due to central nervous system damage. To date, there are no therapeutic approaches that provide neurological functional recovery after acute SCI. MP is an FDA-approved SCI management option to improve neurological functions after acute injury, but it is not routinely used clinically. The use of this drug remains controversial as the modest protective functions on nerve cells are overshadowed by the unfavorable side effects to the rest of the body. To overcome this challenge, we have developed a macromolecular prodrug nanomedicine Nano-MP that can selectively deliver MP to sites of injured and/or inflamed spinal cord in a rat model of acute SCI. Our overarching hypothesis is that such targeted delivery strategy would revitalize MP therapy with improved therapeutic efficacy and safety. In the present study, we demonstrated that a single intravenous injection of Nano-MP is able to preferentially deliver MP to the site of the injured spinal cord where the polymeric prodrug is sequestered and activated predominantly by CD11⁺ infiltrating inflammatory cells (extrapolated from the action of the parent MP molecule), thereby exerting its therapeutic effects. Nano-MP treatment significantly inhibited lipid peroxidation and inflammation of the injured spinal cord, resulting in reduced neuronal damage and enhanced neuroprotection after acute SCI. Importantly, the administration of Nano-MP provided better functional improvement after acute SCI in rodents compared to the systemic delivery of parent MP. In our companion publication in this issue of the journal, we report that compared to conventional intravenous delivery of the regular MP molecule, Nano-MP that was selectively delivered to the injury site, showed to have far fewer systemic adverse side effects—that is, reduced muscle atrophy, bone loss, and less carbohydrate intolerance. Collectively, this novel targeted-delivery system of MP may be significantly safer than standard MP therapy and can be translated as a highly feasible treatment approach in patients with acute SCI to improve functional recovery.

The optical imaging results confirmed our hypothesis that Nano-MP can selectively target to the SCI lesion with sustained presence for >3 days, which may be attributed to the compromised blood-spinal cord barrier (BSCB) and the neuroinflammation-induced neovascularization and vascular fenestration. In addition to localizing in the transected spinal cord (Fig. 3), the NIR fluorescent signal associated with Nano-MP was also seen in liver and kidney (data not shown), the two clearance organs, as often observed in other systemic delivery of nanomedicines. Overt extravasation of macromolecules has been reported within the first hour after SCI,⁵¹ a period that coincided with marked tissue disruption within the epicenter of the lesion. Abnormal BSCB permeability remains significantly elevated at 24 h after injury. A second peak of abnormal barrier permeability has been observed at 3–7 days postinjury, which coincides with prominent revascularization of the epicenter. BSCB integrity is restored at 21 days post SCI.⁵¹ Given this chronological sequence of the peri-spinal cord vasculature remodeling/healing process, Nano-MP was i.v. administered immediately after SCI surgery in our study to take advantage of accessibility to the injured spinal cord. Given this unique pathology-driven passive-targeting of Nano-MP to the injury site, the nanomedicine treatment must be administered as soon as possible post injury, similar to the MP treatment protocol.

Interestingly, as we further investigate the cells that were responsible for Nano-MP’s sustained local retention at SCI pathology, microglia and astrocytes were identified as the main cell phenotypes that sequestered the nanomedicine at sites of SCI pathology. It has been well-established that these two types of cells play pivotal roles in SCI pathogenesis, repair, and healing.^1,52–55 As part of the spontaneous repair process, these cells have adopted highly phagocytic phenotypes,^56,57 which were responsible for the swift internalization of Nano-MP. Furthermore, activation of microglia and astrocytes results in further acidification of lysosomes,⁵⁸ where Nano-MP likely resides. The reduced lysosomal pH value would in turn accelerate the cleavage of the hydrazone bond linking MP to the HPMA copolymer carrier and the release of free MP in order to dampen excessive local inflammation and induce resolution and neuroregenerative repair processes. Distinctively different from other attempts at MP formulation improvement,^{28–31,52,59} the macromolecular prodrug design of Nano-MP is very unique because its simple structure affords a MP therapy with high specificities to SCI pathology on subcellular, cellular and tissue levels. The evidence presented in this report regarding Nano-MP’s capacity to reduce oxidative stress, dampen inflammation, facilitate neuroprotection and regeneration, and most importantly to improve neurofunctional outcome post SCI, validated the principles we adopted in designing Nano-MP. Due to the aforementioned unique features and characteristics of macromolecular prodrugs such as Nano-MP, we may consider the extrapolation of the same design principle to several other drug candidates in order to improve their efficacy and safety. The pharmacological agents for SCI that may be considered include CSPG inhibitors which reduce glia scar, PTEN, Nogo-A and Rho inhibitors, or other neurotrophic factors which promote axon regeneration.

Given the strong evidence presented regarding Nano-MP’s efficacy and safety, we will perform a temporal and/or dose-response curve of Nano-MP and/or other adjunctive therapies on biologic, pathologic and functional outcome measures in the future to facilitate clinical translation of Nano-MP therapy. We will also perform additional experiments to develop in-depth understanding of Nano-MP’s mechanism of action (e.g., inflammatory cascades, glia scar formation, axon regeneration, etc.). Collectively, these studies will guide the design of future treatment regimens by identifying the appropriate time window and the minimum effective dose of Nano-MP or other combinatorial approaches capable of maintaining therapeutic efficacy. As a limitation of the present study, only male animals were investigated. Therefore, it remains of interest and relevance to investigate if the observed beneficial effects of Nano-MP can be reproduced in female animals as well.

In summary, the present work presents the development of a novel macromolecular prodrug nanomedicine Nano-MP, which enabled selective targeting of MP to the injured spinal cord via i.v. systemic administration. The Nano-MP was sequestered in and retained by activated microglia and astrocytes within an SCI lesion via the ELVIS mechanism where it remained bioactive in vivo at least for a few days [verse the intravenous parent MP with a short half-life for 3 h⁶⁰]. Nano-MP mediated substantially decreases in secondary injury-related inflammation, oxidation, lipid peroxidation, and neuronal death, resulting in considerably enhanced neuroprotection and functional recovery after acute SCI as compared to conventional intravenous delivery of the parent MP molecule. These extraordinary findings and the significantly improved safety profile presented in the companion paper strongly support the potential of Nano-MP for continued efforts toward clinical translation as a highly promising drug candidate for improved clinical management of SCI and its complications.

Abbreviations:

AA

adjuvant-induced arthritis

Bax

Bcl-2-associated X protein

BBB

Basso, Beattie and Bresnahan locomotor scale method

BSCB

blood-spinal cord barrier

CMCht/PAMAM

carboxymethyl-chitosan polyamidoamine

CSPG

chondroitin sulfate proteoglycan

Dex

dexamethasone

ELVIS

Extravasation through Leaky Vasculature and subsequent Inflammatory cell-mediated Sequestration

GC

glucocorticoid

GFAP

Glial Fibrillary Acidic Protein

H&E

hematoxylin-eosin

HPMA

N-(2-hydroxypropyl) methacrylamide

IH

Infinite Horizons

LN

lupus nephritis

MDA

malondialdehyde

MP

methylprednisolone

M_w

molecular weight

Nano-MP

N-(2-hydroxypropyl) methacrylamide copolymer-based methylprednisolone prodrug nanomedicine

NASCIS

National Acute SCI Study

NIR

near infrared

NO

nitric oxide

NT

nitrotyrosine

PDI

polydispersity

RAFT

reversible addition-fragmentation chain transfer

TBARS

thiobarbituric acid reactive substance assay

TNFα

tumor necrosis factor alpha

veh

vehicle

Data availability statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.