Improved delivery of miR-1296 loaded cationic nanoliposomes for effective suppression of triple negative breast cancer

Abstract

Nowadays, microRNA is considered an attractive strategy for the effective treatment of cancer. A significant delivery of microRNA for cancer therapy remains a significant obstacle to target cancer cells. The restoring microRNA-1296 (miR-1296) has immense therapeutic efficacy in triple-negative breast cancer (TNBC). TNBC is an aggressive subtype of breast tumors with the progression of malignant transformation. This study aimed to develop a cationic nanoliposome that can serve as a miR-1296 carrier and studied its efficiency in TNBC. The efficacy of miR-1296 liposomes was evaluated on its apoptotic effect, cellular uptake, and potential chemotherapy sensitization in the TNBC cell line (MDA-MB-231). For in vitro viability study, the apoptotic effect was performed to validate protein expression using Alamar blue kit and western blot. The transfection of miR-1296 into TNBC cells was also investigated using cisplatin as a TNBC resistance drug. The fluorescent miR-1296-cy3 liposome was used for cellular uptake study. The miR-liposome was successfully prepared with a particle size of 123.6 ± 1.3 nm and encapsulation efficiency of 94.33%. A dose of 0.5 uM has significantly reduced the viability of MDA-MB-231 to be 33.45%±5.29 (P < 0.001). This result was validated by down-expression of CCND1, and PARP1, the miR-1296 receptor, and apoptosis marker. The image of the miR-1296-cy3 liposome showed cytoplasmic intracellular localization. It was found high sensitization of TNBC cell line for miR-1296 liposome compared to cisplatin (P < 0.001). Future in vivo research may answer questions concerning safety and stability. This study demonstrates that miR-191 liposomes may have promising clinical applications for TNBC therapy.

Keywords: Cellular uptake; Liposomes; Triple-negative breast cancer; miR-1296.

Fig. 1

Characterization of NL-miR-1296. (A) Particle size, PDI, zeta potential and EE% characterization of NL-miR-1296. (B) Transmission electron microscopy of plain NL, and NL-miR-1296 (C) TEM images. (D) present scanning electron microscope overview image of prepared NL-miR-1296; (E) 1% agarose gel used to test the complexation of miR-1296 with NL at different n/p ratios. Different n/p ratios used 0.5–10, where 0 represent free miR-1296 only. All wells contain the same amount of miR-1296 (0.65 μg) but various NL part. The same n/p ratios were used but with NL were permeabilized with 0.5% triton. NL-miR-1296 were allowed 30 min to complex before running. Run was for 40 min at 80 V using TBE buffer. Ethidium bromide (0.5 μg/mL) was used to stain the gel; (F) Characterization of fresh NL-miR-1296 with different n/p ratios. Data represent mean ± SEM; (G) In vitro release of miR-1296 from NL-miR-1296. The release was evaluated in pH 7.4 using TE buffer, at 37 °C. Free miR-1296 was tested for integrity purposes. Points represent averages ± SEM.

Fig. 2

Intracellular uptake of NL-miR-1296-cy3 in MDA-MB-231. About 100 thousand cells were seeded in coverslips in a 6-well cell culture plate. Next day, cells were treated with NL-miR-1296-cy3 in serum-free media (green). After 2,5,7, and 26 h, coverslips were fixed using 3.7% formaldehyde. Nuclei stained with DAPI (blue). Scale bars = 10 µm.

Fig. 3

. Viability, chemotherapy-sensitization, and stability analysis of NL-miR-1296. (A) MDA-MB-231 viability after treatment with different concentrations (0.12–3 μM) of NL-miR-1296, or NL-miR-NC (B) Cells transfected using Lipofectamine. (C) Safety of nanoliposomes was analyzed using different n/p ratios (D) NL-miR-1296 sensitizes MDA-MB-231 to cisplatin treatment at a dose of as low as 1 μM. (E) A dose–response curve of cisplatin treatment in MDA-MB-231 cells (D) Down-expression of CCND1, and PARP1 in NL-miR-1296 treated MDA-MB-231 cells.

Fig. 4

Effect of serum, temperature and time on NL-miR-231 stability. Serum stability was tested using 1% agarose gel. (A) simply samples of NL-miR-1296 (n/p ratio of 3) or free miR-1296 was incubated in 1:1 v/v of FBS (final concentration 50%) at 37 °C for 1, 3, and 24 h. (B) samples of the same NL-miR-1296 incubated in FBS for different periods were treated with 0.5%TritonX-100 just before the run and presented next to free miR incubated at the same time and temperature without FBS (denoted by §).

Fig. 5

Effect of storage time and temperature on physical and biological activity of NL-miR-1296. (A) Interplay between stability and biological activity of NL-miR-1296 on TNBC at different time and storage temperatures. MDA-MB-231 cells were seeded in 24-well plate for 24 h. After 40 or 80 days, cells were transfected with NL-miR-1296 stored at different temperatures. Media was changed after 5 h, and viability was tested using Alamar Blue after 48 h and compared to untreated cells. Freshly prepared NL-miR-1296 was used transfect the cells as described above to represent 0-time point. Physical characterization of NL-miR-1296 stored in different temperatures at different time points was presented as (B) particle size, (C) PDI, and (D) zeta potential. Data represent the mean ± SEM of two independent studies (n = 6). (Multiple student t-test).