Cepharanthine loaded nanoparticles coated with macrophage membranes for lung inflammation therapy

Abstract

Acute lung injury (ALI) is a disease associated with suffering and high lethality, but to date without any effective pharmacological management in the clinic. In the pathological mechanisms of ALI, a strong inflammatory response plays an important role. Herein, based on macrophage 'homing' into inflammation sites and cell membrane coating nanotechnology, we developed a biomimetic anti-inflammation nanosystem (MM-CEP/NLCs) for the treatment of ALI. MM-CEP/NLCs were made with nanostructured lipid carriers (NLCs) coated with natural macrophage membranes (MMs) to achieve effective accumulation of cepharanthine (CEP) in lung inflammation to achieve the effect of treating ALI. With the advantage of suitable physicochemical properties of NLCs and unique biological functions of the macrophage membrane, MM-CEP/NLCs were stabilized and enabled sustained drug release, providing improved biocompatibility and long-term circulation. In vivo, the macrophage membranes enabled NLCs to be targeted and accumulated in the inflammation sites. Further, MM-CEP/NLCs significantly attenuated the severity of ALI, including lung water content, histopathology, bronchioalveolar lavage cellularity, protein concentration, and inflammation cytokines. Our results provide a bionic strategy via the biological properties of macrophages, which may have greater value and application prospects in the treatment of inflammation.

Keywords: Macrophage membrane; acute lung injury; bionic nanocarrier; cepharanthine; inflammation.

Figure 1.

Schematic synthesis of MM-CEP/NLCs and their inflammatory targeting procedure to the ALI mouse. CEP/NLCs are prepared by solvent evaporation-ultrasonic dispersion method. MMs were coated on these CEP/NLCs to fabricate MM-CEP/NLCs. After injected into ALI mouse, these MM-CEP/NLCs can target to the inflammatory sites just like the accumulation of macrophage in lungs when pneumonia burst.

Figure 2.

Characterization of the MM-coated biomimetic nanoparticles. (A) The size and (B) zeta potentials of MMs, NLCs and MM-NLCs (n = 3, mean ± SD). (C) Western blot results of CD44 and CD11b in MMs, NLCs and MM-NLCs. (D) TEM images of NLCs, MMs, and MM-NLCs (scale bar = 100 nm). (E) CLSM images of the colocalization of the nucleus (blue), MMs ‘shell’ (green) and NLCs ‘core’ (red) (scale bar = 10 μm). (F) Size stability of NLCs and MM-NLCs in solution with 10% FBS. (G) In vitro CEP release from CEP/NLCs or MM-CEP/NLCs in PBS (pH 7.4) at 37 °C.

Figure 3.

Preliminary safety evaluation of the biomimetic nanosystem. (A) Cell viability of HUVEC cells incubated for 24 h with different concentration of empty NLCs and MM-NLCs (n = 3, mean ± SD). (B) Hemolysis assay of empty formulations. 0.9% (w/v) sodium chloride solution and double distilled water were served as the negative and positive control, respectively (n = 3, mean ± SD, ***p < .001). (C) Histological staining of organs from healthy mice treated with different CEP-load formulations (scale bar = 100 μm).

Figure 4.

Long circulation feature. (A) CLSM images of RAW 264.7 cells after incubation with Cou6-tagged NLCs and MM-NLCs (scale bar = 10 μm). (B) FACS results and quantification of cellular uptake of NLCs and MM-NLCs in RAW264.7 cells (n = 3, mean ± SD, **p < .01). (C) Relative fluorescence intensity of NLCs and MM-NLCs in blood (n = 3, mean ± SD).

Figure 5.

In vivo targeting evaluation. (A) Representative ex vivo fluorescence images and (B) quantitative data of DiR fluorescent signals accumulated in lung at 24 h (n = 3, mean ± SD, ***p < .001). (C) CLSM images of accumulated MM-NLCs in lung at 12 h (scale bar = 50 μm). (D) ImageJ was utilized to quantify the distribution of NLCs and MM-NLCs in lung (n = 3, mean ± SD, *p < .05).

Figure 6.

MMs-coated biomimetic nanoparticles can improve the therapeutic efficacy of ALI in a mouse model. (A) Experimental protocol for evaluating the therapeutic effect of free CEP, CEP/NLCs and MM-CEP/NLCs. (B) Total cell counts in BALF. (C) Lung water content was assessed by the measurement of wet-to-dry ratio. (D) Protein contents in BALF were measured using a BCA kit. (E) TNF-α and (F) IL-6 in BALF were measured by ELISA kit. (G) Hematoxylin and eosin (H&E)-stained lung tissue sections were imaged (scale bar = 100 μm). (n = 4, mean ± SD, *p < .05, **p < .01, ***p < .001 and ns, no significance).