Codelivery of minocycline hydrochloride and dextran sulfate via bionic liposomes for the treatment of spinal cord injury

Abstract

After primary injury to the spinal cord, a series of microenvironmental changes can lead to secondary injury. The use of nano-targeted drug delivery systems to improve the postinjury microenvironment, inhibit inflammation and reduce neuronal apoptosis can be of great help during spinal cord injury (SCI) recovery. In this study, we prepared primary macrophage membranes bionic modified nanoliposomes (MH-DS@M−Lips) loaded with minocycline hydrochloride (MH) and dextran sulfate (DS) to target their delivery to the site of injury to bind calcium ions in situ and form metal ion complexes. Complex formation reduced calcium ion concentrations and calcium-associated neuronal apoptosis, while MH was slowly released to produce better anti-inflammatory effects. The successful preparation of MH-DS@M−Lips was verified using transmission electron microscopy (TEM), confocal laser scanning microscopy (CLSM), western blotting and dynamic light scattering (DLS). The targeting capability of the MH-DS@M−Lips was demonstrated using a Transwell system and an in vivo imaging system. The therapeutic efficacy of MH-DS@M−Lips was examined in vitro and in vivo using flow cytometry, immunofluorescence, ELISA kits and western blotting. The results showed that SCI mice treated with MH-DS@M−Lips received high behavioral scores, which led to the conclusion that MH-DS@M−Lips have great potential for the treatment of SCI.

Introduction

SCI is a serious traumatic disorder of the central nervous system (CNS). More than one million new cases of SCI occur each year worldwide as a result of accidents (Charlifue et al., 2016, Li et al., 2019). These injuries result in motor and sensory dysfunction (da Silva et al., 2020), and current clinical treatment strategies are not very effective during recovery (Hachem et al., 2017, Toluse and Adeyemi, 2021), leading to patients incurring lifelong treatment costs (Nogueira et al., 2012).

The main clinical drug treatment for SCI is high-dose systemic administration of methylprednisolone, which has been reported to have the therapeutic effects of reducing inflammation and neuroprotection (Cheung et al., 2015). The blood-spinal cord barrier (BSCB) protects CNS homeostasis and prevents the entry of foreign substances (Jin et al., 2021). Although the BSCB opens briefly at the injury site after SCI, the BSCB still poses a great challenge for subsequent drug delivery (Whetstone et al., 2003). Because of the BSCB, the systemic administration of conservative doses of methylprednisolone is inefficient, difficult to enrich at the injury site, and therapeutically ineffective (Jin et al., 2021, Kim et al., 2009), while the toxicity that may be triggered by high doses can cause serious and unavoidable damage to the body, such as gastric bleeding, liver damage and reinfection (Ersayli et al., 2006). In practice, the BSCB makes it difficult to systemically administer most drugs at safe doses, so it is worth exploring nanodelivery systems for delivering drugs across the BSCB to target the damaged areas (Song et al., 2019). In recent years, nanodelivery systems such as liposomes (Gao et al., 2017), PLGA nanoparticles (Ren et al., 2014), silica nanoparticles (Zhang et al., 2021), metal nanoparticles (Kim et al., 2017), exosomes (Guo et al., 2019) and cell membrane vesicles (Liu et al., 2022) have been reported.

Among these, nanoliposomes as a drug delivery system are less toxic, simple to prepare and have a proven process for mass production (Li et al., 2018, Wang et al., 2021). Our previous studies have shown that macrophage-derived membrane nanovesicles have a good effect to target the site of SCI (Liu et al., 2022). Therefore, we performed primary macrophage membranes bionic modified nanoliposomes. The modification method was different from that of the previously reported membrane-modified bionanoparticles (Tang et al., 2021b, Zhuang et al., 2020), as it used a one-step method to dope a small amount of the primary macrophage membranes into lipids, which could reduce the number of membranes needed and solve the problem of the limited source of bionanomimetic materials. Additionally, as less bionanomimetic materials were used, the possible unknown side effects of these materials were reduced. It was found that the modified bionic nanoliposomes inherited primary macrophage membranes proteins, such as VLA4 (Integrins α4/β1) and Mac-1 (CD11b/CD18). These proteins could interact with the highly expressed vascular cell adhesion molecule 1 (VCAM-1) or intercellular adhesion molecules (ICAM) in endothelial cells of the inflammatory site (David and Kroner, 2011, Thawer et al., 2013), which could make the bionic nanoliposomes target and enrich in the damaged area.

Following primary injury to the spinal cord caused by external forces, the microenvironment around the injury point rapidly undergoes a series of changes leading to secondary injury, including M1 macrophage infiltration causing hyperinflammation, ROS stress, electrolyte disturbances and elevated intra- and extracellular calcium ion concentrations (Ahuja et al., 2017). Inflammation and elevated levels of calcium ions lead to an increase in peripheral neuron apoptosis (Beattie, 2004). Improving the microenvironment at this point (e.g., reducing calcium ion concentrations) can help tremendously in SCI recovery (Schanne et al., 1979, Wang et al., 2017). Inflammatory regulation and neuroprotection have a significant impact on subsequent motor recovery, which should be prominently considered (Ahuja et al., 2017, Beattie, 2004). MH, a broad-spectrum antibacterial tetracycline antibiotic, has been reported to be able to treat SCI and possesses a powerful anti-inflammatory effect (Kobayashi et al., 2013). According to previous reports, MH and DS can form complexes with divalent metal ions such as calcium ions and magnesium ions (Wang et al., 2017, Zhang et al., 2015). We attempted to load both MH and DS in macrophage membranes modified nanobionic liposomes (MH-DS@M−Lips). MH-DS@M−Lips were constructed to hopefully target the site of injury for drug enrichment, reduce calcium ion concentrations in situ, reduce neuronal apoptosis and produce anti-inflammatory effects. Experiments simulating the in vivo damage environment showed that MH and DS bind to calcium ions, producing metal ion complexes (Fig. S1) similar to those previously reported (Zhang et al., 2015). This system was studied both in vitro and in vivo, and it was found that MH combined with DS reduced the subsequent calcium-associated apoptotic cascade by binding large amounts of free calcium ions, resulting in neuroprotection while MH produced an anti-inflammatory effect. SCI mice treated with MH-DS@M−Lips also achieved higher behavioral scores. The design concept and mechanism of action are shown in Scheme 1, which will hopefully provide new ideas for the treatment of SCI.