Engineered targeting tLyp-1 exosomes as gene therapy vectors for efficient delivery of siRNA into lung cancer cells

Abstract

Natural exosomes can express specific proteins and carbohydrate molecules on the surface and hence have demonstrated the great potentials for gene therapy of cancer. However, the use of natural exosomes is restricted by their low transfection efficiency. Here, we report a novel targeting tLyp-1 exosome by gene recombinant engineering for delivery of siRNA to cancer and cancer stem cells. To reach such a purpose, the engineered tLyp-1-lamp2b plasmids were constructed and amplified in Escherichia coli. The tLyp-1-lamp2b plasmids were further used to transfect HEK293T tool cells and the targeting tLyp-1 exosomes were isolated from secretion of the transfected HEK293T cells. Afterwards, the artificially synthesized siRNA was encapsulated into targeting tLyp-1 exosomes by electroporation technology. Finally, the targeting siRNA tLyp-1 exosomes were used to transfect cancer or cancer stem cells. Results showed that the engineered targeting tLyp-1 exosomes had a nanosized structure (approximately 100 nm) and high transfection efficiency into lung cancer and cancer stem cells. The function verifications demonstrated that the targeting siRNA tLyp-1 exosomes were able to knock-down the target gene of cancer cells and to reduce the stemness of cancer stem cells. In conclusion, the targeting tLyp-1 exosomes are successfully engineered, and can be used for gene therapy with a high transfection efficiency. Therefore, the engineered targeting tLyp-1 exosomes offer a promising gene delivery platform for future cancer therapy.

Keywords: Engineering; Gene therapy; Lung cancer; Targeting tLyp-1exosomes; Transfection.

Graphical abstract

Fig. 1

Engineering of the targeting tLyp-1-lamp2b plasmid. Artificial tLyp-1 gene and lamp2b gene were synthesized by PCR using cDNA as templates. The resultant tLyp-1-lamp2b gene was recombined into pEGFP-C1 blank plasmid through digesting with Bgl II and BamH I enzymes, and then connected by T4 ligase to form the targeting tLyp-1-lamp2b plasmid.

Fig. 2

Identification of the targeting tLyp-1-lamp2b plasmid. (A) Colony PCR image by agarose gel electrophoresis for identification of targeting tLyp-1-lamp2b plasmids. M, 2000 bp ladder marker; 1–4, samples of tLyp-1-lamp2b plasmids. (B) PCR image by agarose gel electrophoresis for identification of the recombinant plasmid double digested with Bgl II and Sal I. M, 2000 bp ladder marker; 1–4, samples of tLyp-1-lamp2b plasmids. (C) Sequencing for tLyp-1 and part of lamp2b genes. The complete plasmid sequencing and comparison results are shown in supplementary.

Fig. 3

Transfection efficiency of the targeting tLyp-1-lamp2b plasmids in HEK293T cells. (A) Microscopy images of human embryonic kidney HEK293T cells. (a):image of non-transfected HEK293T cells under fluorescent microscope in bright field; (b): image of non-transfected HEK293T cells under fluorescent microscope in dark field; (c): image of transfected HEK293T cells under fluorescent microscope in bright field; (d): image of transfected HEK293T cells under fluorescent microscope in dark field. The results indicate that the green fluorescent protein is detected as a marker of the transfection, demonstrating a successful transfection in HEK293T cells using the targeting tLyp-1-lamp2b plasmids. (B) mRNA expression of lamp2b in HEK293T cells after transfection with targeting tLyp-1-lamp2b plasmids. Real-time qRT-PCR analysis was performed on HEK293T cells that were either non-transfected (control) or transfected (sample) with a plasmid encoding tLyp-1+Lamp2b. (C) Protein expression of lamp2b in HEK293T cells after transfection with targeting tLyp-1-lamp2b plasmids. Western blot analysis was performed on HEK293T cells that were either non-transfected (control) or transfected (sample) with a plasmid encoding tLyp-1+lamp2b. *P < 0.01 vs. control. Data are presented as mean ± SD (n = 3).

Fig. 4

Characterization of the engineered targeting tLyp-1 exosomes. (A) Size distribution and movement of targeting tLyp-1 exosomes. The targeting tLyp-1 exosomes were measured by nanoparticle tracking analysis (NTA) with NanoSight LM10-HSB instrument. (B) Image of Brownian motion of the tLyp-1 exosomes in PBS (pH7.4) solution. The targeting tLyp-1 exosomes were photographed by NTA system. (C) Representative TEM images of natural and engineered exosomes. (a): natural exosomes; (b): targeting tLyp-1 exosomes. (D) Uptake of the engineered targeting tLyp-1exosomes into A549 cells. Both natural exosomes and engineered exosomes were labeled with DiO dye (green fluorescence). (a): mean fluorescent intensity was measured by FACS. 1: natural exosomes; 2: targeting tLyp-1 exosomes. (b): the quantitative results of fluorescent intensity. Data are presented as mean ± SD (n = 3). *P < 0.05 vs natural exosomes.

Fig. 5

Functional verification of the targeting siRNA tLyp-1 exosomes in silencing SOX2 gene of human NSCLC cells. (A) Designed 3 kinds of sequences for siRNA according to human SOX2 siRNAs. (B) mRNA expression of SOX2 in A549 cells after treated with lipo3000 carrying siRNA, natural exosomes carrying siRNA or targeting tLyp-1 exosomes carrying siRNA. Real-time qRT-PCR analysis was performed on A549 cells that were treated with lipo3000, natural exosomes or targeting tLyp-1 exosomes which carried 3 kinds of siRNAs, respectively. The results demonstrate that siR2 has the highest silence efficiency. P < 0.05; (a): vs natural exosomes; (b): vs. lipo3000. Data are presented as mean ± SD (n = 3). (C) Images of human NSCLC A549 cells under fluorescence microscope. (a): image of transfected A549 cells with siR2 lipo3000 under fluorescent microscope in bright field; (b): image of transfected A549 cells with the targeting siR2 tLyp-1 exosomes under fluorescent microscope in bright field; (c): image of transfected A549 cells with siR2 lipo3000 under fluorescent microscope in dark field; d, image of transfected A549 cells with targeting tLyp-1 exosomes under fluorescent microscope in dark field. siRNA (siR2) was labeled with Cy3 and blue was set as an indicator of transfection efficiency by fluorescence microscopy. The results demonstrate a successful transfection in A549 cells using the targeting siR2tLyp-1 exosomes.

Fig. 6

Functional verification of the targeting siRNA tLyp-1 exosomes in silencing SOX2 gene of human NSCLC cells and in decreasing the stemness of NSCLC stem cells. (A) Microscopic image for the appearance of human non-small lung cell (NSCLC) A549 stem cell spheres. (a): NSCLC A549 cells; (b): NSCLC A549 stem cells. (B) Knocked-down SOX protein of NSCLC A549 stem cells by targeting siR2 tLyp-1 exosomes. The study was performed by Western blotting assay. The results demonstrate that the targeting siR2 tLyp-1 exosomes enables to silence SOX2 protein in A549 stem cells as compared to untreated A549 stem cells. (C) The decreased stemness of NSCLC A549 stem cells by targeting siR2 tLyp-1 exosomes. NSCLC A549 stem cells were treated by targeting siR2 tLyp-1 exosomes, stained with anti-CD44-FITC, anti-CD24-PE, and analyzed with FACScan flow cytometer. The identification of phenotypes demonstrate that the population of CD44+/CD24- cells are reduced by knocking-down SOX2 gene.

Fig. 7

Mechanism illustration of the engineered targeting exosomes for efficient delivery of siRNA into human cancer cells. The tLyp-1-lamp2b plasmid transfected HEK293T cells can secreted tumor targeting tLyp-1 exosomes. By electroporation technology, targeting tLyp-1 exosomes were loaded with siRNA. When targeting tLyp-1 exosome ruptured in cytoplasm, siRNA was loaded into the RNA-induced silencing complex (RISC). The sense (passenger) strand was degraded while the antisense (guide) strand directs RISC to mRNA that has a complementary sequence, thereby resulting in the silence of target gene. The targeting tLyp-1 exosomes demonstrate highly transfection efficiency in NSCLC cells and highly SOX2 gene silencing ability in NSCLC stem cells.