Alginate/chitosan multilayer films coated on IL-4-loaded TiO2 nanotubes for modulation of macrophage phenotype

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Abstract

Macrophage phenotype conversion is crucial for improving post-traumatic angiogenesis and tissue repair. Biomaterials with the ability of skewing macrophage phenotype have attracted widespread attention in the field of tissue engineering. The aim of this study was to transform macrophage phenotype by a three-step process; anodizing, drug loading and coating with polyelectrolyte multilayer (PEM) films. Interleukin (IL)-4, an anti-inflammatory cytokine, was loaded into titania nanotubes (TNTs) on the titanium surface. Subsequently, sodium alginate (ALG) and chitosan (CS) were alternately assembled onto IL-4-loaded TNTs and cross-linked with genipin/calcium chloride, finally forming cross-linked PEM films. The IL-4 release profile and cellular immune response of the modified surface was investigated. In the simulated biological solution, only 20% of IL-4 were detected in the first 3 days, with a sustained release of approximately 5 ng over 10 days. The results of gene expression and protein secretion in macrophages indicated that IL-4-loaded PEM films significantly attenuated the inflammatory activity of macrophages at the later stage through down-regulating the mRNA and protein levels of inflammatory markers. In summary, IL-4 was controlled released from the cross-linked PEM films deposited on the nanotubes, leading to the temporal conversion of macrophage phenotype.

Graphic abstract

A functionalized material regulates macrophage phenotype via controlled release of IL-4 from multilayer films on titania nanotubes.

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Introduction

Inflammation, an inevitable early immune response in natural wound healing, has been shown to play a crucial role in tissue regeneration. Macrophage is one of the most important effector cells in the material-induced inflammatory response. The function of macrophages is initially thought to be merely pro-inflammatory due to their destructive nature of the phagocytic process. More recently, macrophages are found to contribute to tissue repair based on their polarization states [1,2].

During the early stage of wound healing (1–3 days), cytokines, such as interferon gamma (IFN-γ), stimulate macrophages into a classically activated (M1) phenotype [3]. Such phenotype not only demonstrate abilities of pro-inflammation and antigen presentation, but also secrete other cytokines, such as vascular endothelial growth factor (VEGF), to enhance neovascularization [4,5]. In the later stage (4–10 days), anti-inflammatory mediators like interleukin (IL)-4 or IL-10 induce macrophages into an alternatively activated (M2) phenotype [2,5], which release platelet-derived growth factor (PDGF)-BB to promote vessel maturation and tissue repair [6]. Thus, certain types of inflammatory response are beneficial for angiogenesis and tissue remodeling. However, the pro-inflammatory secretion of M1 macrophages must be controlled to prevent excessive inflammation from damaging normal tissue and delaying wound healing. Therefore, it is essential to temporally manipulate the M1-to-M2 transition of macrophages.

Considering the critical role of angiogenesis in successful implantation, biomaterials that bind to angiogenic growth factors have been evaluated for the induction of vascularization [7,8]. However, angiogenesis is a complex process that involves a coordinated network of multiple cytokines. The stimulated release of various cytokines by regulating macrophage phenotype can improve vascularization and wound repair [4,9]. Previous studies have demonstrated that macrophages could convert to anti-inflammatory phenotype upon the stimulation of IL-4 released from biomaterials [[10], [11], [12]]. Yet, a limited number of studies have focused on bone grafting for the modulation of macrophage phenotype to attenuate implantation failures triggered by inflammation. For instance, Spiller et al. [5] designed modified decellularized bone scaffolds with two inflammatory factors to enhance vascularization. Yuan et al. [13] examined the immunomodulatory effects of calcium and strontium co-doped titanium oxides on osteogenesis.

Titanium (Ti) and its alloys have been widely used as bone replacement materials in clinical practice. Fabrication of titania nanotubes (TNTs) on Ti surface has been proposed to improve the bioactivity [14,15] and anti-infection ability [16] of Ti material. In addition, TNTs have been reported to exert anti-inflammatory effect in vitro [[17], [18], [19]] and serve as nanoreservoirs for local drug delivery, but the release profile often exhibit an initial burst release phase [20,21]. To achieve a sustained release of drugs, biodegradable coatings are commonly used to seal the open end of TNTs [[21], [22], [23]]. Polyelectrolyte multilayer (PEM) films prepared by a layer-by-layer (LbL) technique have been widely applied in surface modification [24,25] and controlled release of biomolecules [[26], [27], [28], [29]]. Many studies reported that PEM films can improve the activity, proliferation and differentiation of osteoblasts [30,31], and enhance the adhesion and proliferation of endothelial cells and mesenchymal stem cells [32,33]. Sodium alginate (ALG) and chitosan (CS), two natural polyelectrolytes, are commonly used in drug delivery due to their optimal biocompatibility and osteogenic potential [34,35]. Specifically, CS exhibits anti-inflammatory effect and wound healing properties in inflammatory macrophages by enhancing arginase activity [36,37].

In the present study, ALG/CS PEM films were fabricated on IL-4-loaded TNTs via LbL technique. The PEM films were expected to control the release of IL-4 from TNTs and maintain its bioactivity for the modulation of macrophage phenotype. Lipopolysaccharide (LPS) and IFN-γ were firstly added into a growth medium to induce M1 macrophages, and then IL-4 was released from PEM films at the later stage to activate M2 macrophages. After that, the functions of macrophages were evaluated by cell viability, surface marker expression, mRNA expression and protein secretion, in order to determine the immune response of RAW264.7 cells to IL-4-loaded PEM films.

Section snippets

Materials and reagents

Chitosan (CS, Mw~75 kDa, 75–85% deacetylated) was obtained from Sigma-Aldrich (USA). Sodium alginate (ALG), genipin and calcium chloride (CaCl2) were supplied by Aladdin (China). Recombinant murine interleukin-4 (IL-4, 13.5 kDa) and interferon-γ (IFN-γ, 15.6 kDa) were purchased from Peprotech (USA). Lipopolysaccharide (LPS) was also obtained from Sigma-Aldrich (USA). For cell culture, Dulbecco's modified Eagle Medium-High Glucose(DMEM-H), penicillin-streptomycin and phenol red-free medium were

Surface morphology

The surface morphologies of different samples detected by SEM are illustrated in Fig. 2. As shown in Fig. 2A, the diameter of TNTs was approximately 100 nm. The enlarged image of T4 (Fig. 2B) clearly indicated that the white nanoparticles were uniformly distributed around the open end of TNTs. EDX analysis of T4 sample revealed the existence of Ti, C, N and O elements (Fig. S2). Obviously, C and N elements were primarily attributed to precipitated protein from IL-4 solution, mainly containing

Discussion

Bone defect repair remains a major challenge in hard tissue engineering. Novel therapeutic strategies that utilize implanted biomaterials to restore the body's natural ability to heal itself are of great concern over recent decades. The inflammatory response to injury or to implanted biomaterials plays a pivotal role in angiogenesis and tissue repair. Macrophages are key cellular components involved in the host immune response. Previous studies have reported that the sequential activation of M1

Conclusions

In this study, TNTs were employed as drug nanoreservoirs for the storage and delivery of IL-4. The IL-4-loaded TNTs were then covered with ALG/CS PEM films through LbL technique. In addition, a cross-linking reaction was applied to regulate the release profile of IL-4 in PEM films. In the simulated biological environment, IL-4-loaded PEM films exhibited a sustained release of IL-4 at earlier stage and massive release in the later stage. Gene expression and protein secretion analyses of

Notes

The authors declare no competing financial interest.

Acknowledgment

This work was supported by the National Key Research and Development Program of China (2017YFB0702602) and the National Natural Science Foundation of China (31570955).

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