Editor’s note: The following is a report on research presented during a session on pain nanomedicine at the American Pain Society (APS) Annual Scientific Meeting. This year, the meeting was held in the form of a scientific summit that took place March 4-6, 2018, in sunny Anaheim, California, US—the home of Disneyland! See previous report from the meeting here.
Treatments that target only specific cell types may potentially relieve chronic pain from neuroinflammation while avoiding the side effects of systemic drugs. During “Exploiting Cellular and Molecular Mechanisms of Neuroinflammation for New Treatments of Chronic Pain,” a session at the 2018 annual meeting of the American Pain Society, several speakers highlighted pain treatments that target one specific cell type—macrophages—using a nanomedicine approach.
Jelena Janjic, Duquesne University School of Pharmacy, Pittsburgh, US, presented her work on targeted nanomedicine for chronic pain treatment. To start, Janjic said that nanomedicine promises to improve drug delivery for pain treatment by decreasing the amount of drug needed to alleviate pain; by enabling treatment adjustment as the patient recovers; by offering the potential to release drugs upon disease flare-ups; and by targeting drugs only to specific tissues.
Janjic has designed a new Theranostic Analgesic Regenerative Gel-Emulsion Technology (TARGET) platform for localized delivery of an anti-inflammatory drug to the site of injury. TARGET works by incorporating a nanoemulsion containing a drug into a gel, which helps promote extended drug release at the site of injury. The newly developed TARGET platform is being evaluated for safety, feasibility, and efficacy in experimental pain models at the research facilities of the 59th Medical Wing of the United States Air Force. (Speaker disclosure: This project is supported by an Air Force Medical Support Agency award granted to Duquesne University [Janjic, PI, Grant FA8650-17-2-6836] in collaboration with Col. Erik K. Weitzel, MD, FACS, deputy commander of the United States Army Institute for Surgical Research [USAISR] and Dr. Vijay Gorantla, MD, RESTOR Program, USAISR).
Macrophages associated with disease typically derive from blood monocytes and have two phenotypes: M1 macrophages are pro-inflammatory, while M2 macrophages are anti-inflammatory. Macrophages can switch between these phenotypes based on the cytokine signals they receive. Chronic inflammatory and neuropathic pain is perpetuated by M1 macrophages that express the enzyme cyclooxygenase-2 (COX-2). COX-2 signaling leads to elevated levels of prostaglandin E2 (PGE2), which is a physiologically active lipid linked to neuropathic pain through its effects on neuronal activity, cytokine production, and neuronal gene expression. Nonsteroidal anti-inflammatory drugs (NSAIDs) that inhibit COX-2 block production of PGE2. Thus, Janjic developed near infrared fluorescence-labeled (NIRF) theranostic nanoemulsions containing the COX-2-selective inhibitor celecoxib to target macrophages (Janjic et al., 2018). In this instance, NIRF is a fluorescence technique that allows for visualization of inflammation.
As part of this collaborative project, Janjic and John Pollock, Duquesne University, delivered the nanoemulsion via tail vein injection into rats eight days after they underwent chronic constriction injury (CCI), a model of neuropathic pain. These animals displayed significantly less mechanical hypersensitivity compared to rats that received a nanoemulsion containing no drug. Measurement of inflammation using NIRF labeling of macrophages that had engulfed the nanoemulsions without the drug revealed heightened fluorescence over the injured sciatic nerve, which was significantly decreased in animals that received the drug-containing nanoemulsion. This suggested that the macrophages containing celecoxib were most likely not of the inflammatory phenotype and did not migrate to the site of injury.
Next, the researchers studied the effects of the nanoemulsions on the number of infiltrating monocyte-derived macrophages, COX-2 expression, and PGE2 expression in the injured sciatic nerve using immunofluorescence. Treatment with celecoxib-containing nanoemulsions significantly reduced all three, compared to treatment with nanoemulsions without the drug. (Speaker disclosure: This work was funded in part by National Institute on Drug Abuse Award R21EB023104 [PI: J. Janjic]).
In sum, nanoemulsion therapy significantly reduced inflammation at the site of injury, which coincided with decreased pain hypersensitivity. This holds promise for future therapies to utilize nanomedicine, allowing for low-dose, targeted drug delivery that avoids damage to healthy tissues.
Theodore (Ted) Price, University of Texas at Dallas, US, discussed the role of AMP-activated protein kinase (AMPK) in chronic pain and the sex differences his group saw with macrophage-targeted nanomedicine. AMPK is an enzyme known to play a role in cell metabolism. Activation of AMPK decreases mTOR and ERK signaling in injured sensory neurons. mTOR and ERK are phosphorylation enzymes that regulate cell proliferation, translation, and cell growth.
mTOR and ERK signaling pathways are involved in sensitization of peripheral pain-sensing neurons, so activating AMPK should alleviate pain. Indeed, Price reported previously that AMPK activation with metformin, a drug typically used for treatment of type 2 diabetes, in the spared nerve injury (SNI) mouse model of neuropathic pain provided long-lasting reversal of mechanical hypersensitivity.
Since AMPK activation eased neuropathic pain, Price also tested the effect of this manipulation in a mouse model of postoperative pain. He and his colleagues used a modified Brennan incision model by incising the skin and muscle, and then retracting the muscle to produce a more severe and long-lasting injury. The researchers aimed to use resveratrol, a natural product and potent activator of AMPK. However, free resveratrol is poorly absorbed by the body. To overcome this obstacle, Price struck up a collaboration with Janjic, who had previously made resveratrol nanoemulsions (Herneisey et al., 2016). Tail vein injection of the nanoemulsions significantly reduced the mechanical hypersensitivity seen after incision, compared to vehicle nanoemulsions and free resveratrol injected at the site of injury.
Next up: Could the resveratrol nanoemulsions block hyperalgesic priming in mice? Hyperalgesic priming occurs when there is an insult, such as a surgical incision, that induces pain that resolves after a period of time. Then, a second insult occurring at the initial site of injury causes chronic hyperalgesia. Once the animals were no longer hypersensitive to mechanical stimuli following the priming injury (31 days after incision), Price and colleagues injected the inflammatory lipid PGE2 into the previously operated hindpaw to cause long-lasting hyperalgesia. Animals that had received the resveratrol nanoemulsion at the time of incision, and 24 and 48 hours following incision, did not develop mechanical hypersensitivity, whereas animals treated with the vehicle nanoemulsion did. Therefore, nanoemulsion delivery of resveratrol prevented hyperalgesic priming and subsequent chronic pain.
Sex differences
The previous experiments using the incision model were performed in male mice. But the nanoemulsions had no effect on incisional pain or hyperalgesic priming and the subsequent chronic pain in female mice. To address this sex discrepancy, the researchers gave daily injections, at the site of injury, of narciclasine, a potent AMPK activator that enters the circulation easily, to both female and male mice for seven days after incision. Narciclasine reversed mechanical hypersensitivity after injury and prevented hyperalgesic priming in both female and male mice. Thus, AMPK activation, and the ensuing decrease in mTOR and ERK signaling, was effective in treating surgical incision pain in both female and male mice. Price concluded that the sex differences that occurred with the nanoemulsion treatment were not due to differences in AMPK signaling but rather to differences in how the nanoemulsion reaches the site of injury.
Since the nanoemulsion is engulfed by macrophages that migrate to the site of injury, Price hypothesized that “a differential recruitment of macrophages occurs in males and females with surgery.” To test this, the researchers used the traditional Brennan incision model where the plantar hindpaw skin and plantaris muscle were incised, with no muscle retraction. The sciatic nerve was removed 24 or 48 hours after surgery for immunofluorescence analysis.
Immunostaining for IBA1, a marker for macrophages, along with measurement of inflammation using NIRF after nanoemulsion treatment, revealed that females had little, if any, macrophage infiltration into the sciatic nerve, whereas macrophages were robustly recruited in male mice. This suggested that the sex differences observed after resveratrol nanoemulsion treatment may result from sexual dimorphism of macrophage function.
Price concluded his talk by mentioning his group’s transcriptome analysis of dorsal root ganglia from humans with chronic pain. These analyses revealed that sexual dimorphism in macrophages exists in human chronic pain conditions as well. Currently, Price and colleagues are working to answer the question: "Can we identify neuroimmune or other interactions in human tibial nerve RNA sequencing data?"
Edgar Alfonso Romero-Sandoval, Wake Forest School of Medicine, Winston-Salem, US, spoke of the effect of macrophage gene modulation on chronic postoperative pain. Researchers know that chronic pain after surgery depends on the invasiveness of the surgery, genetic susceptibility, age, and sex. Major surgeries cause significant inflammation at the site of injury that lasts longer than less invasive surgeries. Romero-Sandoval’s goal is to reduce how long inflammation lasts after surgery; this could decrease the incidence of chronic pain following major surgeries. To achieve this goal, Romero-Sandoval and colleagues decided to target macrophages based on their previous work that focused on the role of macrophages in the initiation and resolution of inflammation (Bernal et al., 2017).
First, the researchers studied whether nanoparticles containing cDNA plasmids could be used in a safe and efficient way to cause gene overexpression in a human M1 (proinflammatory) macrophage cell line called THP-1. cDNA plasmids are used as carriers of a gene of interest that can replicate independently of the host’s DNA and cause expression of the gene. The group used polyethylenimine (PEI) nanoparticles attached to a mannose receptor ligand (Man-PEI). PEI nanoparticles are cationic (positively charged) protein polymers that prevent DNA degradation and have been successfully used in clinical trials for gene delivery. Meanwhile, the mannose receptor ligand addition allows for targeting of monocyte-derived macrophages that express the mannose receptor on their surface. The Man-PEI nanoparticles also contained a green fluorescent protein (GFP) plasmid to visualize how efficiently the macrophages engulfed the nanoparticles and to study effects on gene expression.
Results showed that the M1 macrophages efficiently took up the Man-PEI nanoparticles and expressed GFP by 24 hours, with significant increases in uptake seen by 48 hours, as measured by GFP fluorescence and mRNA levels. And, Man-PEI nanoparticles containing constitutively expressed genes (CD14 and CD68, two genes expressed by macrophages) resulted in overexpression of those genes in the human macrophages. Man-PEI nanoparticles did not kill the macrophages and had a small effect on cytokine production (slightly decreased IL-6 and IL-10). Overall, these results showed that Man-PEI nanoparticles could be used safely and effectively to cause gene expression in the cells.
Making the switch
Next, the researchers used the Man-PEI nanoparticles to switch M1 (proinflammatory) macrophages to the M2 (anti-inflammatory) phenotype during inflammation (Alvarado-Vazquez et al., 2017). To do so, they loaded the nanoparticles with a CD163 plasmid. CD163 is a scavenger receptor with high affinity for a complex consisting of hemoglobin and haptoglobin, and is exclusively expressed by M2 macrophages. Macrophage degradation of this complex via CD163 uptake causes the release of anti-inflammatory molecules. Also, CD163 expression on M2 macrophages is a marker of when inflammation has ceased.
Results showed that CD163 messenger RNA and protein were significantly upregulated in THP-1 (M1) macrophages challenged with lipopolysaccharide (LPS), a molecule found in bacteria that pushes macrophages toward the M1 phenotype. This meant that CD163 could now be expressed in M1 macrophages, and so the researchers could test if they could change the phenotype of these cells so that they were anti-inflammatory.
Measurement of anti-inflammatory cytokines released from CD163-overexpressing M1 macrophages revealed a significant increase in the anti-inflammatory IL-10 and IL-1 receptor antagonist (IL-1ra), compared to macrophages treated with empty plasmid Man-PEI nanoparticles. And, measurement of proinflammatory cytokines showed that overexpression of CD163 resulted in a decrease of monocyte chemoattractant protein 1 (MCP-1). These results indicated that overexpression of CD163 in M1 macrophages using Man-PEI nanoparticles led to an increase in anti-inflammatory cytokines and a decrease in a proinflammatory cytokine.
Using the same paradigm as above, the researchers overexpressed CD163 in primary human macrophages. This had different effects compared to the THP-1 cell line-derived macrophages. This time, only the anti-inflammatory IL-10 was significantly increased, and the proinflammatory cytokines TNF-α, IL-1β, and IL-6 were all significantly decreased; there was no change in MCP-1. Overall, overexpression of CD163 under inflammatory conditions (LPS treatment) altered macrophage cytokine expression toward an anti-inflammatory cytokine profile.
Romero-Sandoval and colleagues are now studying the effects of their macrophage-targeted nanomedicine approach in models of chronic pain, including the skin/muscle incision and retraction (SMIR) model. The group performed RNA sequencing studies in primary human macrophages transfected with an empty plasmid or a plasmid containing CD163. Then they performed a bioinformatic analysis to identify gene expression patterns. From this analysis, the investigators found that cytokine-cytokine receptor interaction pathways were strongly modulated by CD163. Based on those findings, they studied these pathways in the SMIR model. Romero-Sandoval presented preliminary findings showing that CD163 modulates specific cytokines in vivo at the surgical site in that model.
Ashley Cowie is a PhD candidate at the Medical College of Wisconsin, Milwaukee, US.
Image credit: jewhyte/123RF Stock Photo.
