Preparation, Structure and Pharmaceutical Analysis of Protamine-siRNA Complexes†

doi:10.7503/cjcu20180833

Abstract

Abstract:

Protamine-siRNA complexes of different morphologies were prepared by vortex mixing different mass ratios of protamine and siRNA at room temperature, which could be resolved by polyacrylamide gel electrophoresis with silver staining. The protamine-siRNA complexes were observed under atomic force and transmission electron microscopy, respectively. It was found that similarly shaped protamine-siRNA complexes could be formed by protamine with different siRNA molecules, indicated that the interaction between protamine and siRNA was independent on the base composition of siRNA. Moreover, the interactions between the protamine and the siRNA molecules were characterized by ionic probes, including Mg²⁺ etc. And the morphologies, the particle sizes as well as the structural properties of the protamine-siRNA complexes were further characterized using atomic force microscope, dynamic light scattering and transmission electron microscope, respectively. The results show that protamine formed toroid complexes and fibrous composites with siRNA molecules respectively in the light of the different mass ratios between protamine and siRNA. In the meantime, the phagocytosis and the pharmacodynamics of protamine-siRNA complexes of different morphologies were compared and measured using melanoma cells.

Key words: Protamine, siRNA, Melanoma, Drug delivery

CLC Number:

O629.74

TrendMD:

SHI Mai,JIANG Rui,CUI Xinxia,ZHANG Xin,SHEN Shigang,DING Liang,PAN Xuefeng. Preparation, Structure and Pharmaceutical Analysis of Protamine-siRNA Complexes^†[J]. Chem. J. Chinese Universities, 2019, 40(6): 1164.

Figures/Tables 10

Table 1 Preparation of protamine-siRNA-1 complex with a mass ratio of 20∶1

V(Total)/μL	V(siRNA)/μL	V(Protamine)/μL	V(HEPES)/μL
50.0	2.5	2.5	45.0
50.0	5.0	5.0	40.0
50.0	7.5	7.5	35.0
50.0	10.0	10.0	30.0
50.0	12.5	12.5	25.0
50.0	15.0	15.0	20.0

Fig.1 PAGE analysis on the formations of protamine-siRNA complexes Protamine-siRNA-1 complexes in (A) or protamine-siRNA-2 complexes in (B) were formed by protamine and siRNA by mass ratios of 15∶1, 20∶1, 25∶1, 30∶1, 35∶1, 40∶1, respectively. Lane 1: siRNA-1(A) or siRNA-2(B); lanes 2—7: protamine-siRNA complexes formed by mass ratios of protamine to siRNA-1(A) or siRNA-2(B) by 15∶1, 20∶1, 25∶ 1, 30∶ 1, 35∶ 1, 40∶ 1, respectively. Protamine-siRNA-1 complexes formed by a mass ratio of protamine to siRNA-1 by 20∶1(C). Lane 1: siRNA-1, lanes 2—7: protamine-siRNA-1 complexes formed by simultaneously increasing the protamine and siRNA-1 by 2.5, 5.0, 7.5, 10.0, 12.5, 15.0 μL, respectively.

Fig.2 PAGE analysis on the effects of different metal ions on the stabilities of protamine-siRNA complexes

Fig.3 TEM images of protamine-siRNA complexes formed by protamine and siRNA with the mass ratios of 20∶1(A) and 40∶1(B)

Fig.4 AFM visualizations of protamine-siRNA complexes with the mass ratios of protamine to siRNA 20∶1(A, toroid) and 40∶1(B, filamentous) and protamine proteins(white dots) in the filamentous protamine-siRNA complexes(C)

Scheme 1 Schematic illustrations for the interactions between protamine and siRNA in protamine-siRNA complexes of different morphologies

Fig.5 PAGE analysis on the formation of disulfide bonds in the protamine-siRNA complexes Lane 1: siRNA, lanes 2—7: protamine-siRNA complexes of filament morphologies treated using β-mercaptoethanol with a final concentration of 0, 5, 10, 15, 20 and 40 mmol/L, respectively.

Table 2 Particle sizes of the protamine-siRNA complexes

Mass ratio of protamine to siRNA	d(90)/nm	Mass ratio of protamine-siRNA	d(90)/nm
15∶1	217.5±6.7	30∶1	325.8±7.6
20∶1	220.0±7.2	35∶1	384.2±6.2
25∶1	275.5±5.0	40∶1	405.0±4.3

Fig.6 Phagocytosis of A375 melanoma cells on toroid(A) and fibrous(B) protamine-siRNA complexes under confocal fluorescence microscopy

Fig.7 Effects of protamine-siRNA complexes on the proliferation of A375 cells a. Control; b. lipoHigh; c. phospholipid vesicles; d. siRNA; e. spherical complexes; f. filamentous complexes.

References 27

[1]	Fire A., Xu S., Montgomery M. K., Kostas S. A., Drive S. E., Mello C. C., Nature,1998, 391(6669), 806-811
[2]	Pan X.F., Molecular Biology of Genetic Diseases, Chemical Industry Press, Beijing, 2014, 436-447
	(潘学峰. 基因疾病的分子生物学, 北京: 化学工业出版社, 2014, 436-447)
[3]	Peedicayil J., Indian J. Med. Res., 2006, 123(1), 17-24
[4]	Jiang N., Pan X. F., Biotechnology Bulletin,2015, 31(4), 105-119
	(姜楠,潘学峰. 生物技术通报, 2015, 31(4), 105-119)
[5]	Tatiparti K., Sau S., Kashaw S. K., Iyer A. K., Nanomaterials,2017, 7(4), 77-94
[6]	Rengaswamy V., Zimmer D., Süss R., Rössler J., J. Control Release,2016, 235, 319-327
[7]	Hao H. W., Zhen Y. S., Wang Z. C., Chen F. L., Xie X., Cell Biol. Int., 2013, 37, 860-864
[8]	Hutchison J. M., Rau D. C., DeRouchey J. E., Biophys. J., 2017, 113(9), 1925-1933
[9]	Warrant R. W., Kim S. H., Nature,1978, 271(5641), 130-135
[10]	Balhorn R., Genome. Biol., 2007, 8(9), 227-235
[11]	He H. N., Ye J. X., Liu E. G., Liang Q. L., Liu Q., Yang V. C., J. Control Release,2014, 193, 63-73
[12]	Rui P. M., Ostermeier G. C.,Krawetz S. A,.J. Biol. Chem., 2004, 279(50), 51862-51868
[13]	Cornetta K., Anderson W. F., J. Virol. Methods,1989, 23(2), 187-194
[14]	Pillaiyar T., Manickam M., Namasivayam V., J. Enzym. Inhib. Med. Ch., 2017, 32(1), 403-425
[15]	Hudjashov G., Villems R., Kivisild T., PloS One,2013, 8(9), e74307
[16]	Wang J. H., Pei Y. Y., Xu H. D., Li L. J., Wang Y. Q., Liu G. L., Qu Y., Zhang N., Biomed Rep., 2016, 5(1), 87-92
[17]	Elbashir S. M., Harborth J., Weber K., Tuschl T., Methods,2002, 26(2), 199-213
[18]	Minakshi P., Ranjan K., Kumar P., Prasad G., Adv. Anim. Vet. Sci., 2013, 1(4S), 20-23
[19]	Petrov A. S., Bowman J. C., Harvey S. C., Williams L. D., RNA,2011, 17(2), 291-298
[20]	Misra V. K., Draper D. E., P. Natl. Acad. Sci. USA,2001, 98(22), 12456-12461
[21]	Iii R. J. T., Draper D. E., Nucleic Acids Res., 2017, 45(8), 4733-4742
[22]	Koo H., Park I., Lee Y., Kim H. G., Jung J. H., Lee J. H., Kim Y., Kim J. H., Park J. W., J. Am. Chem. Soc., 2016, 138(36), 11664-11671
[23]	Schön P., Methods, 2016, 103, 25-33
[24]	Tusup M., Pascolo S., Methods in Molecular Biology,2017, 1499, 155-163
[25]	Rio D. C., Jr A. M., Hannon G. J., Nilsen T. W., Cold Spring Harbor Protocols,2010, 2010(6), pdb.prot5443
[26]	Gilmore J., Deguchi K., Takeyasu K., Microscopy and Imaging Science: Practical Approaches to Applied Research and Education, Formatex Research Center, Badajoz, 2017, 300-306
[27]	Bowman J. C., Lenz T. K., Hud N. V., Williams L. D., Curr. Opin. Struct.Biol., 2012, 22(3), 262-272