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Polysialic acid-based micelles promote neural regeneration in spinal cord injury therapy Xiao-Juan Wang, Chen-Han Peng, Shuo Zhang, Xiao-Ling Xu, Gao-Feng Shu, Jing Qi, Ya-Fang Zhu, De-Min Xu, Xu-Qi Kang, Kong-Jun Lu, Fei-Yang Jin, Risheng Yu, Xiao-Ying Ying, Jian You, Yong-Zhong Du, and Jiansong Ji Nano Lett., Just Accepted Manuscript • Publication Date (Web): 03 Jan 2019 Downloaded from http://pubs.acs.org on January 3, 2019
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Nano Letters
Polysialic acid-based micelles promote neural regeneration in spinal cord injury therapy Xiao-Juan Wang 1, Chen-Han Peng 1, Shuo Zhang 1, Xiao-Ling Xu 1, Gao-Feng Shu1, 2
, Jing Qi 1, Ya-Fang Zhu 1, De-Min Xu 3, Xu-Qi Kang 1, Kong-Jun Lu 1, Fei-Yang
Jin 1, Ri-Sheng Yu 3, Xiao-Ying Ying 1, Jian You 1, Yong-Zhong Du 1*, Jian-Song Ji 2*
Affiliations: 1
Institute of Pharmaceutics, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou 310058, PR China; 2
Key Laboratory of Imaging Diagnosis and Minimally Invasive Intervention
Research, Lishui Hospital of Zhejiang University, Lishui 323000, China 3
Department of Radiology, Second Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou 310009, PR China;
* Correspondence to: Dr. Y. Z. Du (Institute of Pharmaceutics, College of Pharmaceutical Sciences, Zhejiang University), 866 Yu-Hang-Tang Road, Hangzhou, 310058, China. Tel: +86-571-88208435; Fax: +86-571-88208439. E-mail:
[email protected]; Dr. J. S. Ji (Key Laboratory of Imaging Diagnosis and Minimally Invasive Intervention
Research,
Lishui
Hospital
of
Zhejiang
University),
Tel:
+86-578-2285011; Fax: +86-578-2285011; E-mail:
[email protected]. 1
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KEY WORDS: Spinal cord injury, polysialic acid, minocycline, neural regeneration, synergistic therapy ABSTRACT Spinal cord injury (SCI) routinely causes the immediate loss and disruption of neurons, followed by complicated secondary injuries, including inflammation, oxidative stress and dense glial scar formation. Inhibitory factors in the lesion scar and poor intrinsic neural regeneration capacity restrict functional recovery after injury. Minocycline, which has neuroprotective activity, can alleviate secondary injury, but the long-term administration of this drug may cause toxicity. Polysialic acid (PSA) is a large cell-surface carbohydrate that is critical for central nervous system development and is capable of promoting precursor cell migration, axon path-finding and synaptic remodeling; thus, PSA plays a vital role in tissue repair and regeneration. Here, we developed a PSA-based minocycline-loaded nanodrug delivery system (PSM) for the synergistic therapy of spinal cord injury. The prepared PSM exerted marked anti-inflammatory and neuroprotective activities both in vitro and in vivo. The administration of PSM could significantly protect neurons and myelin sheaths from damage, reduce the formation of glial scar, recruit endogenous neural stem cells to the lesion site, and promote the regeneration of neurons and the extension of long axons throughout the glial scar, thereby largely improving the locomotor function of SCI rats and exerting a superior therapeutic effect. The findings might provide a novel strategy for SCI synergistic therapy and the utilization of PSA in other central nervous system diseases. 2
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Nano Letters
Graphic abstract Lesion site
Polysialic acid Octadecylamine Minocycline Microglia Neurons
Neural regeneration
Inhibition of inflammation
3
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1
Spinal cord injury (SCI) commonly begins with a primary injury, such as
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contusion and compression, and it results in the irreversible loss of neurons and the
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disruption of axons in spinal cord tissue.
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subsequently occurs,3 which can cause further damage to the normal tissue around the
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lesion site, leading to the spread and exacerbation of tissue injury and ultimately
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causing motor dysfunction below the level of the lesion.4, 5 Current strategies for the
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treatment of SCI mainly include neuroregeneration and neuroprotection.
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Neuroregeneration, e.g., tissue engineering
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rewiring the injured neuronal connections, promoting the regeneration of axons and
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neurons, and reinstating the neural loss via recellularization in the injured tissue. 6 In
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contrast, neuroprotection, e.g., treatment with methylprednisolone,9 dexamethasone,10
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riluzole
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degeneration induced by secondary injury, thereby limiting the damage to the spinal
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cord. Although most therapies have shown efficacy in animal studies, they have been
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shown to be largely unsuccessful in clinical trials, which is possibly due to targeting a
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relatively limited number of processes. A therapeutic strategy combining
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neuroprotection with neuroregeneration might be promising for SCI therapy.
18
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, erythropoietin
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1, 2
Secondary injury, such as inflammation
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and cell transplantation,
and epothilone B,
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8
6
focuses on
aims to prevent the extensive
Minocycline (MC), which is a clinically available antibiotic, could target various 14, 15
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secondary injury mechanisms
, including those previously described, and could
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exert therapeutic effects in spinal cord contusion
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anti-inflammatory, antioxidant and anti-apoptotic capacities.
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long-term and systemic administration of a high dose of MC might cause toxicity and
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and compression injury via its 17
However, the
4
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even death.18 Thus, there has been a growing demand for drug delivery systems that
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have sustained drug release behaviors and abilities to target the lesion site.
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Polysialic acid (PSA) is an endogenous carbohydrate polymer composed of
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alpha-2,8-linked-N-acetyl neuraminic acid units, and it is frequently attached to neural
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cell adhesion molecule (NCAM). 19 Polysialylated NCAM (PSA-NCAM), known as a
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marker of neural stem cells,
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Additionally, PSA plays a vital role in the migration and differentiation of neural
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precursors, neuronal guidance, synapse formation and axon path-finding.21 The roles
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of PSA and its mimetics in contributing to nerve repair and regeneration have also
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been extensively studied.22, 23, 24 Previous studies have shown that PSA has advantages
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in the design of drug delivery systems (DDS), showing great potential in prolonging
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circulation lifetime and reducing the rate of drug elimination in vivo. 25-28
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is a critical factor for improving neural regeneration.
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Nano-DDS (NDDS) is an effective approach for altering the in vivo biological
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distribution of drugs, and it has recently been used for SCI therapy.4, 5 Furthermore,
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the impaired blood-spinal cord barrier and the enhanced permeability of vessels after
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SCI 29 could endow NDDS with the ability to passively target the lesion site. Herein, a
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novel functional PSA-based carrier for delivering MC was developed for SCI therapy.
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We hypothesize that the use of PSA could improve neural regeneration and
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remyelination after delivering minocycline to the lesion site, thereby representing an
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effective treatment for SCI through the combined therapies of neuroprotection and
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neuroregeneration.
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First, PSA was hydrophobically modified by an acylation reaction with 5
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octadecylamine (ODA). The illustration showing the synthesis of the amphiphilic
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polymer (PSO) is shown in Figure 1a. The 1H-NMR spectrum of the PSO conjugate
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exhibited the characteristic peaks of both PSA (∼2.0 ppm) and ODA (∼0.9 ppm),
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indicating successful modification (Figure 1b). The degree of substitution (DS) of
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PSO was 4.33%, 7.15% and 16.11% when the ODA feeding ratio was 5%, 10% and
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20%, respectively (Table S1). The PSO conjugate could self-assemble into micelles,
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and its critical micelle concentration (CMC) gradually decreased with increasing
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ODA feeding ratios (Figure 1c and Table S1). In addition, the particle size and zeta
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potential of the micelles also decreased with increasing DS (Table S1).
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Then, minocycline-loaded PSO micelles (PSM) were prepared by dialysis using
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their free base form, and the drug loading (DL) capacity and encapsulation efficiency
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(EE) were calculated by UV spectrophotometry. At a fixed amount of MC addition
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(20%), PSO micelle with 10% and 20% ODA feeding ratio could effectively
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encapsulate MC with DL and EE being of 12.48%, 12.94% and 71.16%, 72.24%,
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respectively. PSM displayed an initial rapid drug release during the first 12 h,
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followed by a prolonged sustained release through 72 h (Figure 1d). Notably, the
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PSM with a 20% ODA feeding ratio presented slower drug release than the PSM with
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a 10% ODA feeding ratio. Considering that the acute inflammatory response occurs
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immediately after injury,30 the PSM with a 10% ODA feeding ratio was used for
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further studies because of the relatively faster drug release. To further investigate the
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loading capacity of PSO micelles, PSMs with MC feeding ratios ranging from 5% to
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20% were prepared, and the corresponding physicochemical properties are shown in 6
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Table S2. Both the PSO micelles and PSM with a 20% MC feeding ratio showed
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narrow and monomodal size distributions, with average sizes of 118.0 ± 4.58 and
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60.89 ± 5.98 nm, respectively (Figure 1e). The transmission electron microscope
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(TEM) images (Figure 1e) showed that both micelles had uniform and round
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morphologies.
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Figure 1 Preparation and characterization of PSM. (a) Synthetic scheme of the PSO polymer. (b) 1H-NMR spectra of PSA, ODA and PSO from top to bottom, respectively. (c) The variations in fluorescence intensity ratios (I1/I3) presented as the change in logarithmic concentrations of PSO conjugate with different ODA feeding ratios (5%, 10% and 20%) as measured by a fluorescence spectrophotometer. (d) Drug release behaviors of the prepared PSM with ODA feeding ratios of 10% and 20%. (e) Size distributions of PSO and PSM micelles, and 7
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the corresponding TEM images.
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The PSO conjugate exhibited good biocompatibility in BV2 and SH-SY5Y cells
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(Figure S1). In addition, PSO micelles had a good cellular internalization capacity in
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microglial cells and BV2 cells (Figures S2a, b). Both of these cells are immune cells
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in the central nervous system that can release pro-inflammatory cytokines after
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activation, and then amplify inflammation in the injured tissue.31 Then, BV2 cells
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were used to investigate the cellular uptake mechanism. Both lipopolysaccharide
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(LPS)-activated and normal BV2 cells showed a remarkable decrease in fluorescence
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after incubation with PSO micelles at 4 ℃ (Figure S2c), indicating that the cellular
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uptake process was energy dependent. To further determine the possible pathway,
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different inhibitors were added in advance. In the case of normal BV2 cells, the
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fluorescence was reduced by 30.8% and 19.2% when the cells were pretreated with
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chlorpromazine (CPZ) and 5-(N-ethyl -N-isopropyl) amiloride (EIPA), respectively
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(Figures S2c, d and f), suggesting that normal BV2 cells took up PSO micelles
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mainly through the clathrin-mediated pathway and phagocytosis. For LPS-activated
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BV2 cells, the fluorescence was reduced by 26.5% and 51.7% after the cells were
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preincubated with CPZ and EIPA, respectively (Figures S2c, e and f), illustrating that
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phagocytosis played a more important role in the cellular uptake process.
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To evaluate the anti-inflammatory capacity of PSM, BV2 cells and microglial
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cells were activated by LPS, and then incubated with PSM. The concentrations of
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nitric oxide (NO), TNF-α and IL-6 in the culture medium were detected. The results
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demonstrated
that
LPS-activated
cells
released
much
higher
levels
of 8
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pro-inflammatory cytokines than normal cells (Figures 2a, b and c). After
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LPS-activated cells were incubated with minocycline hydrochloride (MC.HCl) and
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PSM, the concentrations of pro-inflammatory cytokines were significantly reduced
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(p
