Engineered liposomes targeting the gut–CNS Axis for comprehensive therapy of spinal cord injury
Graphical abstract
Introduction
The oral route is the primary and most convenient drug administration route that allows for self-administration and is associated with good patient compliance as compared with various other administration routes [1,2]. Therefore, oral dosage forms of drug candidates are a major focus in research, clinics and marketing [3]. However, despite the advancements in targeted delivery systems and smart nanomedicines, progress in the state-of-the-art design and construction of oral delivery systems, especially targeted oral delivery systems, is far from satisfactory [4,5]. On the one hand, the harsh environment in the gastrointestinal (GI) tract has severe impacts on the functionalities of nanomedicines. On the other hand, compared with other administration routes, e.g., the intravenous route, via which delivery systems can rapidly distribute to the target sites, the GI tract acts as an extra barrier, hampering timely accumulation of delivery systems in target sites [6,7]. With the improved knowledge of gut pathology and physiology, it has become clear that gut function is not limited to digestion. In fact, direct connections exist between the gut and various important organs [[8], [9], [10], [11]], such as the gut–liver axis, gut–lung axis, and gut–brain axis. Accumulating evidence suggests that many diseases and their complications are directly or indirectly related to the gut, and the existence of relevant targets in the GI tract provides a substantial basis for the development of relevant treatments [12,13]. With the support of rapid-developing biochemistry and material sciences for targeted drug delivery system design, it is anticipated that, besides being an administration route, the gut will also serve as a “shortcut” for therapeutic agents in the treatment and intervention of various diseases.
Spinal cord injury (SCI) is a pathological damage to the spinal cord induced by trauma, inflammation, and various other causes, with severe symptoms, difficult recovery, and often a life-long course. SCI occurs mostly in young men, resulting in heavy financial and psychological burdens for patients, families, and society. It is estimated that approximately 1,000,000 people suffer from SCI each year [14], however, safe and effective therapeutic agents with simple administration are lacking. Methylprednisolone is the major clinical drug used to treat SCI, but its therapeutic effect is limited, and it causes significant side effects [15].
Recently, astrocytes have emerged as an important treatment target for SCI [16,17]. In normal conditions, astrocytes support the maintenance of the physiological functions of the central nervous system (CNS) [18]. In the pathological process of SCI, however, astrogliosis occurs, in which reactive astrocytes form dense glial scar tissue at the lesions as physical barriers for nerve regeneration, and secrete a series of inhibitors for nerve regeneration [19]. The regulation of astrogliosis and astrocyte characteristics has been suggested as an important strategy for the treatment of SCI [20]. Agents such as curcumin and triptolide reportedly inhibit the activation of astrocytes, thus reducing the formation of glial scar tissues and promoting the regeneration and functional recovery of spinal nerves [[21], [22], [23]]. Efficient delivery of potential therapeutic agents towards the lesion sites, concerning functional moieties and relevant targeted delivery systems with specific recognition of astrocytes and effective penetration through the blood-spinal cord barrier, are areas of focus for such an approach to become effective.
Complications, especially, intestinal complications and relevant dysfunctions, add to the burden of SCI sufferers, severely affecting their quality of life [24,25]. There clearly is an urgent need for relevant and effective therapeutic agents. Enteric glial cells (EGCs), an important component of the gut–CNS axis which resembles astrocytes in morphology, function, and biomarker expression, are a promising target [13]. EGCs are distributed throughout the intestinal system, including the mucous, muscle layers, and the enteric nervous system [26]. Similar to the function of astrocytes in the CNS, EGCs regulate intestinal homeostasis [27], and especially, motor functions [28]. EGCs play an important role in various intestinal dysfunctions, and the toll-like receptor (TLR) expressed on EGCs can be activated by pathological stimuli, leading to activation of the TLR-NFκB pathway and NO production, finally resulting in motor dysfunction [12,29,30]. EGCs may be even directly involved in pathological changes in the CNS [31]. Therefore, we assumed that EGCs may be a potential target for the treatment of SCI and related enteric complications. As EGCs and astrocytes express similar biomarkers, relevant ligands of such specific receptors could be adopted in the construction of dual-targeted delivery systems. It can be expected that, after oral administration, such systems could deliver therapeutic agents to both EGCs after crossing the epithelial layer and astrocytes in the spinal cord after entering the systemic circulation, thus achieving a comprehensive therapeutic effect against SCI and related enteric complications.
Based on the above reasonings, the neuropeptide apamin [32], a ligand of the small conductance calcium-activated potassium (SK) channel that can effectively penetrate the blood-spinal cord barrier and specifically recognize astrocytes and EGCs, was adopted as a targeting moiety, and the disulfide bond in the apamin molecule was replaced with a diselenide bond for enhanced stability in vivo [33]. The conventional liposome was selected as a carrier, and the natural compound curcumin (CUR) was encapsulated to regulate glial cell function [34]. To improve liposome functionality further, the liposome surface was covered with a temporary protective layer of non-covalent cross-linked chitosan oligosaccharide lactate (COL) that facilitates penetration of the intestinal mucosa and dissociates in situ [35]. The delivery system (Fig. 1) was systematically evaluated in molecular and animal studies.
Section snippets
Materials
Curcumin (CUR) was purchased from Rongsheng Biotechnology (Xi'an, China). HSPC and NHS-PEG2000-DSPE were purchased from A.V.T. Pharmaceutical (Shanghai, China). DiIC18(3) (DiI) and DiOC18(3) (DiO) were purchased from US Everbright (Suzhou, China). Chitosan oligosaccharide lactate (COL) and IR-780 were purchased from Sigma-Aldrich (USA). Rabbit anti-S100β antibody was obtained from Abcam (Cambridge, UK). Rabbit anti-claudin 4, anti-MUC5B, anti-Sox10, anti-glial fibrillary acidic protein (GFAP),
Targeting compound synthesis and characterization
We first replaced the disulfide bonds in apamin with diselenide bonds to obtain diseleno bond-stabilized apamin (Se-apamin), which maintained the helical structure of apamin (Fig. 2a). This result was consistent with a three-dimensional (3D) structural model of Se-apamin constructed based on energy minimization in the solvent model, which confirmed that the two molecules have largely coincident structures (Fig. 2a). Analysis of the stability of Se-apamin under simulated physiological conditions
Conclusions
We achieved effective intervention in SCI and related enteric complications through oral drug delivery based on the following two principles: 1) simultaneous recognition of a specific receptor shared by astrocytes and EGCs by a relevant peptide ligand with improved stability, and 2) significant improvement in penetration through oral absorption barriers by coating the liposome with a water-soluble chitosan derivative, which also had an obvious advantage in the preparation process as compared
Author contributions
‡X.W. and J.W. contributed to this work equally. All authors have prepared and approved the final version of the manuscript.
Declaration of Competing Interest
The authors declare no competing financial interest.
Acknowledgement
The research was financially supported by Natural Science Foundation of Chongqing (cstc2015jcyjBX0100), the National Natural Science Foundation of China (NSFC No. 81673376, 81803472), and Fundamental Research Funds for the Central Universities (XDJK2017D150).
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These authors contributed equally to this work.