细胞内氧化还原响应型肝靶向嵌段聚合物的合成及纳米自组装性能

doi:10.7503/cjcu20170714

摘要/Abstract

摘要：

通过两步可逆加成-断裂链转移自由基聚合(RAFT)和缩醛脱保护反应合成了一种亲水段含半乳糖侧基和疏水段含吡啶环二硫键侧基的两亲性嵌段聚合物PMAIgGP-b-PPDSMA(PMgPP), 用核磁共振氢谱(¹H NMR)和凝胶渗透色谱(GPC)分析验证了目标产物的化学结构. 利用纳米沉淀技术和巯基氧化自交联反应制备内核二硫键交联的PMgPP纳米粒(PMgPP-CC NPs). 动态光散射(DLS)和透射电子显微镜(TEM)测定结果表明, PMgPP-CC NPs粒径较小(<30 nm), 且粒径分布较窄. 在谷胱甘肽(GSH)还原环境下, PMgPP-CC NPs粒径不断增大, 发生了解组装. 以阿霉素(DOX)为模型药物制备了PMgPP-CC/DOX NPs, 载药量可达12.5%, 对应包封率为83.3%, 其粒径与空白PMgPP-CC NPs粒径大小相近, 且粒径分布均匀. 体外药物释放实验表明, PMgPP-CC/DOX NPs在体液条件下46 h释放了4.47%的DOX, 而在10 mmol/L GSH条件下累积释放量达到了50.6%. 细胞胞吞实验进一步验证了PMgPP-CC/DOX NPs可高效入胞并在细胞内快速释放DOX. 体外细胞毒性(MTT)实验表明, PMgPP-CC/DOX NPs对肝癌HepG-2细胞表现出良好的增殖抑制活性. 因此, 多功能PMgPP-CC NPs在实现肝靶向纳米精准给药上呈现出良好的应用前景.

关键词: 半乳糖, 氧化还原敏感, 核交联嵌段聚合物, 可逆加成-断裂链转移自由基聚合(RAFT), 药物递送

Abstract:

The amphiphilic block copolymers PMAIgGP-b-PPDSMA(PMgPP) with a hydrophilic block containing galactose side groups and a hydrophobic segment containing pyridine ring disulfide pendant groups were prepared by two-step reversible addition-fragmentation chain transfer(RAFT) polymerization and acetal deprotection reaction. Their well-defined chemical structures were confirmed by proton nuclear magnetic resonance(¹H NMR) and gel permeation chromatography(GPC). Core-crosslinked PMgPP nanoparticles(PMgPP-CC NPs) were prepared by nano-precipitation technology and thiol oxidation self-crosslinking reaction. Dynamic light scattering(DLS) and transmission electron microscopy(TEM) were used to determine the size of PMgPP-CC NPs(<30 nm) and the particle size distribution indexes were narrow. Under the reduction environment of GSH, the particle sizes of PMgPP-CC NPs were increasing with incubation time, indicating the disassembly of PMgPP-CC NPs. PMgPP-CC/DOX NPs were prepared by DOX as the model drug, and drug loading content up to 12.5% and entrapment efficiency of 83.3%. The particle sizes were similar to that of the corresponding blank CC NPs, and the particle size distribution was uniform. Within 46 h in vitro, 4.47% of DOX was released from the PMgPP-CC/DOX NPs in normal physiological conditions, whereas 50.6% was released in the presence of 10 mmol/L GSH condition analogous to the reductive microenvironment in cytoplasm. The cell uptake tests further confirmed that PMgPP-CC/DOX NPs could be efficiently released in the HepG-2 cells. 3-(4,5-dimethyethiazol-2-yl)-2,5-diphemptetrazolium bromide(MTT) assays show that PMgPP-CC/DOX NPs has good proliferation inhibitory activity against HepG-2 cells. Therefore, multifunctional PMgPP-CC NPs have a good prospect in the field of precise hepatoma-targeting drug delivery.

Key words: Galactose, Redox-sensitive, Core-crosslinked block copolymer, Reversible addition-fragmentation chaintransfer(RAFT) polymerization, Drug delivery

中图分类号:

O631
TQ463

TrendMD:

赵军强, 闫彩霞, 陈泽, 杨宁, 冯霞, 赵义平, 陈莉. 细胞内氧化还原响应型肝靶向嵌段聚合物的合成及纳米自组装性能. 高等学校化学学报, 2018, 39(7): 1592.

ZHAO Junqiang, YAN Caixia, CHEN Ze, YANG Ning, FENG Xia, ZHAO Yiping, CHEN Li. Synthesis and Self-assembly Properties of Intracellular Redox Bioresponsive Block Copolymers with Hepatoma-targeting Groups^†. Chem. J. Chinese Universities, 2018, 39(7): 1592.

图/表 14

Scheme 1 Schematic illustration of PMgPP-CC/DOX NPs with intracellular redox-responsive properties for GAL-mediated targeted delivery of DOX

Scheme 2 ynthesis routes of PMgPP

Fig.1 1H NMR spectra of MAIpGP(A) and PDSMA(B) monomers

Fig.2 1H NMR spectra of PMAIpGP-CTA(A), PMpPP(I)(B) and PMgPP(Ⅱ)(C) and GPC elution chromatograms of PMAIpGP-CTA and PMpPP(D)

Table 1 Chemical structure and compositions of PMpPP(Ⅰ) and PMgPP(Ⅱ)

Sample	Composition(/unit)(¹H NMR)		M_n (¹H NMR)	M_w/M_n(GPC)	M_n(PMAIpGP)/M_n(PPDSMA) (¹H NMR)
Sample	MAIpGP	PDSMA	M_n (¹H NMR)	M_w/M_n(GPC)	M_n(PMAIpGP)/M_n(PPDSMA) (¹H NMR)
PMpPP(Ⅰ)	25.0	29.0	16013	1.09	0.63
PMpPP(Ⅱ)	25.0	39.0	18566	1.32	0.50

Fig.3 DLS measurements of 1.0 mg/mL PMgPP(Ⅱ)-NC NPs(a), 1.0 mg/mL PMgPP(Ⅱ)-CC NPs(b), 1.0 μg/mL PMgPP(Ⅱ)-NC NPs(c) and 1.0 μg/mL PMgPP(Ⅱ)-CC NPs(d)

Table 2 Characteristics of blank PMgPP-NC NPs and PMgPP-CC NPs

Sample	NC NPs		CC NPs
Sample	Size/nm	PDI	Size/nm	PDI
PMgPPI	23.9±0.8	0.14	22.7±1.5	0.16
PMgPPII	26.0±1.6	0.15	23.7±0.2	0.18

Fig.4 Reduction-induced size changes of PMgPP(Ⅱ)-CC NPs^Concentration of GSH/(mmol·L-1) and time/h: a. 0, 0; b. 10, 5; c. 10, 10; d. 10, 24.

Table 3 Characteristics of PMgPP-NC/DOX NPs and PMgPP-CC/DOX NPs*

Sample	NC/DOX NPs				CC/DOX NPs
Sample	Size/nm	PDI	DLC(%)	DLE(%)	Size/nm	PDI	DLC(%)	DLE(%)
PMgPP(Ⅰ)	21.5 ± 4.8	0.16	11.9	79.3	20.1 ± 4.8	0.19	11.7	78.0
PMgPP(Ⅱ)	25.0 ± 3.2	0.19	12.0	80.0	24.8 ± 3.9	0.18	12.5	83.3

Fig.5 TEM images of PMgPP(Ⅱ)-NC/DOX NPs(A) and PMgPP(Ⅱ)-CC/DOX NPs(B)

Fig.6 GSH-triggered DOX release profiles from PMgPP(Ⅱ)-NC/DOX NPs(a, b) and PMgPP(Ⅱ)-CC/DOX NPs(c, d)^Concentration of GSH(mmol·L-1): a, c. 0; b, d. 10.

Fig.7 CLSM images of HepG-2 cells after incubated with PMgPP(Ⅱ)-NC/DOX NPs(A—C), PMgPP(Ⅱ)-CC/DOX NPs(D—F) and free DOX(G—I) at equivalent DOX concentration of 10.0 μg/mL for 8 h^Images from left to right show DOX fluorescence in cells(red) and cell nuclei stained by DAPI(blue) and overlays of two images.

Fig.8 In vitro cytotoxicity of blank PMgPP(Ⅱ)-NC NPs and PMgPP(Ⅱ)-CC NPs towards HepG-2 cells

Fig.9 Cell growth inhibition activity of free DOX, PMgPP(Ⅱ)-NC/DOX NPs and PMgPP(Ⅱ)-CC/DOX NPs as a function of DOX dosages towards HepG-2 cells

参考文献 23

[1]	Dai Y. L., Xu C., Sun X. L., Chen X. Y., Chem. Soc. Rev., 2017, 46(12), 3830—3852
[2]	Li R., Xie Y., J. Controlled Release, 2017, 251, 49—67
[3]	Tibbitt M. W., Dahlman J. E., Langer R., J. Am. Chem. Soc., 2016, 138(3), 704—717
[4]	Shi J. J., Kantoff P. W., Wooster R., Farokhzad O. C., Nature Reviews Cancer, 2017, 17, 20—37
[5]	Blum A. P., Kammeyer J. K., Rush A. M., Callmann C. E., Hahn M. E., Gianneschi N. C., J. Am. Chem. Soc., 2015, 137(6), 2140—2154
[6]	Karimi M., Ghasemi A., Zangabad P. S., Rahighi R., Moosavi Basri S. M., Mirshekari H., Amiri M., Chem. Soc. Rev., 2016, 45(5), 1457—1501
[7]	Zhang X. J., Dong H., Fu S. L., Zhong Z. L., Zhuo R., Macromol. Rapid Commun., 2016, 37, 993—997
[8]	Li D. W., Bu Y. Z., Zhang L. N., Wang X., Yang Y. Y., Zhuang Y. P., Yang F., Shen H., Wu D. C., Biomacromolecules, 2016, 17(1), 291—300
[9]	Talelli M., Barz M., Rijcken C. J., Kiessling F., Hennink W. E., Lammers T., Nano Today, 2015, 10(1), 93—117
[10]	Xiao W. W., Suby N., Xiao K., Lin T. Y., Awwad N. A., Lam K. S., Li Y. P., J. Controlled Release, 2017, 264, 169—179
[11]	Huang M. M., Zhao K. J., Wang L., Lin S. Q., Li J. J., Chen J. B., Zhao C. G., Ge Z. S., ACS Appl. Mater. Interfaces, 2016, 8, 11226—11236
[12]	Zhang Q. Q., He J. L., Zhang M. Z., Ni P. H., J. Mater. Chem. B, 2015, 3(24), 4922—4932
[13]	Kong F. P., Liang Z. Y., Luan D. G., Liu X. J., Xu K. H., Tang B., Anal. Chem., 2016, 88(12), 6450—6456
[14]	Zhang P., Zhang H. Y., He W. X., Zhao D. J., Song A. X., Luan Y. X., Biomacromolecules, 2016, 17, 1621—1632
[15]	Deng B., Ma P., Xie Y., Nanoscale, 2015, 7(30), 12773—12795
[16]	Han H. S., Choi K. Y., Ko H., Jeon J., Saravanakumar G., Suh Y. D., Lee D. S., Park J. H., J. Controlled Release, 2015, 200, 158—166
[17]	Bertrand N., Wu J., Xu X. Y., Kamaly N., Farokhzad O. C., Adv. Drug Delivery Rev., 2014, 66, 2—25
[18]	Hu X. L., Liu G. H., Li Y., Wang X. R., Liu S. Y., J. Am. Chem. Soc., 2015, 137(1), 362—368
[19]	Fu L. Y., Sun C. Y., Yan L. F., ACS Appl. Mater. Interfaces, 2015, 7(3), 2104—2115
[20]	Wang Z., Luo T., Sheng R. L., Li H., Sun J. J., Cao A., Biomacromolecules, 2016, 17(1), 98—110
[21]	Wang Y., Hong C. Y., Pan C. Y., Biomacromolecules, 2013, 14(5), 1444—1451
[22]	Moad G., Chong Y. K., Postma A., Rizzardo E., Thang S. H., Polymer, 2005, 46(19), 8458—8468
[23]	Chen W., Meng F. H., Cheng R., Deng C., Feijen J., Zhong Z. Y., J. Controlled Release, 2015, 210, 125—133