负载R848疏水性介孔二氧化硅纳米粒的制备及复极化巨噬细胞的研究

doi:10.3877/cma.j.issn.2095-1221.2025.06.003

目的

制备苯基修饰的介孔二氧化硅（PMSN）负载免疫调节剂雷西莫特（R848），探讨PMSN-R848是否能将M2型巨噬细胞复极化为M1型。

方法

通过煅烧法制备介孔二氧化硅（MSN），加入苯基三乙氧基硅烷（PhTES）将MSN进行苯基修饰后负载R848得到PMSN-R848纳米粒，拟合R848标准曲线用酶标仪测量计算出PMSN负载R848的载药率，透射电镜观察PMSN和PMSN-R848结构，通过CCK-8测量PMSN-R848的细胞毒性，荧光电子显微镜检测PMSN-R848与巨噬细胞溶酶体共定位情况，流式细胞术检测PMSN-R848是否能复极化巨噬细胞。多组间比较采用单因素方差分析，组间两两比较采用LSD-t检验。

结果

PMSN负载R848载药率为（36.97 ± 5.80）%，PMSN透射电镜呈有孔道的球形结构，PMSN-R848在球形结构表面可见吸附的颗粒，PMSN-R848无细胞毒性且能很好的定位于巨噬细胞溶酶体，M2型巨噬细胞与PMSN-R848共孵育后复极化为M1型。

结论

通过PMSN负载R848后能有效的将M2型巨噬细胞复极化为M1型。

关键词: 介孔二氧化硅, 巨噬细胞, 雷西莫特, 免疫调节

Objective

Phenyl-modified mesoporous silica nanoparticles (PMSN) -loaded immunomodulator Resiquimod (R848) was prepared to investigate whether PMSN-R848 could repolarize M2-type macrophages to M1-type.

Methods

Mesoporous silica (MSN) was prepared by calcination, Phenyltriethoxysilane (PhTES) was added to modify MSN with phenyl and loaded with R848 to obtain PMSN-R848 nanoparticles. The drug loading efficiency of R848 on PMSN was calculated by measuring the R848 standard curve using an enzyme-linked immunosorbent assay (ELISA) reader. The structure of PMSN and PMSN-R848 was observed by transmission electron microscopy. The cytotoxicity of PMSN-R848 was assessed via the CCK-8 assay. Fluorescence electron microscopy (FEM) was employed to detect the colocalization of PMSN-R848 with macrophage lysosomes. Flow cytometry was performed to determine whether PMSN-R848 could repolarize macrophages. One-way analysis of variance was used for comparison between multiple groups, and LSD-t test was used for pairwise comparison between groups.

Results

The drug loading rate of PMSN loaded with R848 was (36.97 ± 5.80) %, the PMSN transmission electron microscope showed a spherical structure with pores, adsorbed particles could be seen on the surface of the spherical structure of PMSN-R848, PMSN-R848 was non-cytotoxic and could be well localized in macrophage lysosomes, and M2 macrophages were repolarized to the M1 phenotype after co-incubation with PMSN-R848.

Conclusion

The PMSN loaded with R848 could effectively repolarize M2 macrophages to M1.

Key words: Mesoporous silica, Macrophages, Resiquimod, Immunomodulation

图1 R848在无水乙醇中的光谱扫描和标准曲线检测注：a图为光谱扫描；b图为标准曲线

图2 透射电镜下观察PMSN和PMSN-R848的状态注：a图为PMSN；b图为PMSN-R848；×20 000；比例尺100 nm；PMSN为苯基修饰的介孔二氧化硅

图3 不同浓度的PMSN-R848细胞毒性检测（n = 3）注：PMSN为苯基修饰的介孔二氧化硅

图4 共聚焦显微镜下观察PMSN-R848与Raw264.7溶酶体共定位（DAPI-Cy5.5-溶酶体探针染色，×40）注：PMSN为苯基修饰的介孔二氧化硅

图5 显微镜下巨噬细胞的形态（×20，比例尺200 μm）注：IL为白细胞介素；PMSN为苯基修饰的介孔二氧化硅；LPS为脂多糖

图6 PMSN-R848对巨噬细胞复极化的结果注：IL为白细胞介素；PMSN为苯基修饰的介孔二氧化硅；LPS为脂多糖；a图为经过不同处理的巨噬细胞表达CD86分子的百分比（n = 3）；b图为经过不同处理的巨噬细胞表达CD206分子的百分比（n = 3）；^*P < 0.05

表1 各组巨噬细胞表达CD86及CD206分子的百分比（

± s，n = 3）

分组	CD86表达的百分比（%）	CD206表达的百分比（%）
IL-4	29.66 ± 0.20	49.30 ± 0.47
IL-4+PMSN	38.18 ± 2.55^a	51.47 ± 2.93
IL-4+R848	45.00 ± 2.25^ab	23.44 ± 2.40^ab
IL-4+PMSN-R848	46.92 ± 1.15^ab	22.09 ± 1.32^ab
LPS	52.67 ± 3.34^abc	8.91 ± 1.40^abcd
F值	48.740	283.900
P值	< 0.0 001	< 0.0 001

表1 各组巨噬细胞表达CD86及CD206分子的百分比（

± s，n = 3）

分组	CD86表达的百分比（%）	CD206表达的百分比（%）
IL-4	29.66 ± 0.20	49.30 ± 0.47
IL-4+PMSN	38.18 ± 2.55^a	51.47 ± 2.93
IL-4+R848	45.00 ± 2.25^ab	23.44 ± 2.40^ab
IL-4+PMSN-R848	46.92 ± 1.15^ab	22.09 ± 1.32^ab
LPS	52.67 ± 3.34^abc	8.91 ± 1.40^abcd
F值	48.740	283.900
P值	< 0.0 001	< 0.0 001

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Preparation of R848-loaded hydrophobic mesoporous silica nanoparticles and study on its effects on the repolarization of macrophages

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Preparation of R848-loaded hydrophobic mesoporous silica nanoparticles and study on its effects on the repolarization of macrophages