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. 2022 Feb 19;12(4):696.
doi: 10.3390/nano12040696.

Affibody Modified G-quadruplex DNA Micelles Incorporating Polymeric 5-Fluorodeoxyuridine for Targeted Delivery of Curcumin to Enhance Synergetic Therapy of HER2 Positive Gastric Cancer

Chao Zhang  1   2 , Shuangqing Fu  1 , Fanghua Zhang  1 , Mengnan Han  1 , Xuming Wang  1 , Jie Du  1 , Honglei Zhang  1 , Wei Li  1
Affiliations

Affibody Modified G-quadruplex DNA Micelles Incorporating Polymeric 5-Fluorodeoxyuridine for Targeted Delivery of Curcumin to Enhance Synergetic Therapy of HER2 Positive Gastric Cancer

Chao Zhang et al. Nanomaterials (Basel). .

Abstract

Combination chemotherapy is emerging as an important strategy for cancer treatment with decreased side effects. However, chemotherapeutic drugs with different solubility are not easy to realize co-delivery in traditional nanocarriers. Herein, an affibody modified G-quadruplex DNA micellar prodrug (affi-F/GQs) of hydrophilic 5-fluorodeoxyuridine (FUdR) by integrating polymeric FUdRs into DNA strands is developed for the first time. To achieve synergistic efficacy with hydrophobic drugs, curcumin (Cur) is co-loaded into affi-F/GQs micelles to prepare the dual drug-loaded DNA micelles (Cur@affi-F/GQs), in which affibody is employed as a targeting moiety to facilitate HER2 receptor-mediated uptake. Cur@affi-F/GQs have a small size of approximately 130 nm and exhibit excellent stability. The system co-delivers FUdR and Cur in a ratiometric manner, and the drug loading rates are 21.1% and 5.6%, respectively. Compared with the physical combination of FUdR and Cur, Cur@affi-F/GQs show higher cytotoxicity and greater synergistic effect on HER2 positive gastric cancer N87 cells. Surprisingly, Cur@affi-F/GQs significantly enhance the expression and activity of apoptosis-associated proteins in Bcl-2/Bax-caspase 8, 9-caspase 3 apoptotic pathway, which is the main factor in the death of tumor cells induced by FUdR. Overall, this nanoencapsulation is a promising candidate for the targeted co-delivery of drugs with significant differences in solubility.

Keywords: 5-fluorodeoxyuridine; DNA micelles; G-quadruplex; affibody; curcumin; synergetic therapy.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Scheme A1
Scheme A1
Synthesis route of 5-fluorodeoxyuridine phosphoramidite.
Scheme A2
Scheme A2
Strategy for preparing the cF13-affibody.
Figure A1
Figure A1
1H NMR spectrum of 5-fluorodeoxyuridine phosphoramidite.
Figure A2
Figure A2
13C NMR spectrum of 5-fluorodeoxyuridine phosphoramidite.
Figure A3
Figure A3
Characterization of purified affibody. The crude lysate of E. coli expression and purified affibody were loaded onto a 12% SDS-PAGE gel with protein weight standards. Lane 1, crude lysate of E. coli expression; Lane 2, purified affibody.
Figure A4
Figure A4
GPC traces and molar mass distribution characteristics of affibody and cF13-affibody.
Figure A5
Figure A5
TEM images of affi-F/GQs and Cur@affi-F/GQs.
Figure A6
Figure A6
Fluorescence spectra of pyrene in different concentrations of affi-F/GQs solutions.
Scheme 1
Scheme 1
(A) Design of FUdR-containing DNA conjugates for construction of affi-F/GQs. (B) Illustration of affi-F/GQs formed by Hoogsteen hydrogen bond, π-π stacking and hydrophobic force. (C) Preparation of Cur@affi-F/GQs co-loaded with FUdR and Cur through physical encapsulation. (D) Illustration of Hoogsteen hydrogen bond.
Figure 1
Figure 1
(A) Mass spectrometry of cF13-NH2, cF10G6-Chl. (B) cF13-NH2 and cF10G6-Chl were assayed by 10% denaturing polyacrylamide gel. (C) UV-vis analysis of affibody, cF13-NH2 and cF13-affibody. (D) cF13-NH2 and cF13-affibody were assayed by 2% agarose gel.
Figure 2
Figure 2
(A) Self-assembly of G-quadruplex DNA micelles were assayed by 2% agarose gel. (Lane from left to right: cF10G6, cF10G6-Chl, cF10T6-Chl and affi-F/GQs). (B) CD spectra of cF10G6-Chl, cF10T6-Chl and affi-F/GQs. (C) Size distribution of affi-F/GQs. (D) AFM image of affi-F/GQs.
Figure 3
Figure 3
(A) CMC determined by pyrene fluorescence probe assay. (B) Stability study of affi-F/GQs in different pH, DNase I treatment time and BSA concentrations.
Figure 4
Figure 4
(A) UV-vis spectra of Cur@affi-F/GQs, Cur and affi-F/GQs. (B) Standard curve of Cur and determination of Cur content in Cur@affi-F/GQs. (C) Size distribution of Cur@affi-F/GQs. (D) AFM image of Cur@affi-F/GQs.
Figure 5
Figure 5
(A) Cur release profile of Cur@affi-F/GQs in PBS (pH 7.4) and acetate buffer (pH 4.5). (B) FUdR release behavior in DNase II solution. (C) Cur release profile in DNase II solution.
Figure 6
Figure 6
(A) Fluorescent microscopic photographs of N87 and MGC 803 cells treated with FAM-labelled F/GQs and affi-F/GQs, respectively, for 1 h (relative FAM = 5 µM). (B) Colocalization studies of affi-F/GQs in N87 cells after incubation with lysosome tracker for 0.5 h. (C) Cur release from Cur@affi-F/GQs in N87 cells at predetermined times. Scale bar: 20 µm.
Figure 7
Figure 7
(A) In vitro cytotoxicity of free FUdR, F/GQs and affi-F/GQs after 48 h of co-incubation in N87 and MGC 803 cells, respectively. (B) Cytotoxicity test of N87 cells treated with FUdR and Cur alone, FUdR/Cur (6:1) (a physical mixture of FUdR and Cur at a molar ratio of 6:1) and Cur@affi-F/GQs (the same ratio of FUdR/Cur) for 48 h, respectively. (C) IC50 values of FUdR and Cur in gastric cancer N87 and MGC 803 cells. (D) Synergetic effect analysis of FUdR/Cur (6:1) and Cur@affi-F/GQs in N87 cells.
Figure 8
Figure 8
(A) Western blot of Bcl 2 and Bax in gastric cancer cells treated with free FUdR, F/GQs and affi-F/GQs, respectively. (B) Western blot of Bcl 2 and Bax in N87 cells treated with FUdR and Cur alone, FUdR/Cur (6:1) and Cur@affi-F/GQs, respectively. (C) Ratio of Bax/Bcl-2 in gastric cancer cells. (D) The activity of caspase 3, 8 and 9 in N87 cells treated with free FUdR, F/GQs and affi-F/GQs, respectively. (E) The activity of caspase 3, 8 and 9 in MGC 803 cells treated with free FUdR, F/GQs and affi-F/GQs, respectively. (F) The activity of caspase 3, 8 and 9 in N87 cells treated with FUdR and Cur alone, FUdR/Cur (6:1) and Cur@affi-F/GQs, respectively. (No significant difference (NS), ** p < 0.01 and *** p < 0.001).
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