Engineered Cell-Derived Vesicles Displaying Targeting Peptide and Functionalized with Nanocarriers for Therapeutic microRNA Delivery to Triple-Negative Breast Cancer in Mice

Abstract

Polymeric nanocarriers (PNCs) can be used to deliver therapeutic microRNAs (miRNAs) to solid cancers. However, the ability of these nanocarriers to specifically target tumors remains a challenge. Alternatively, extracellular vesicles (EVs) derived from tumor cells show homotypic affinity to parent cells, but loading sufficient amounts of miRNAs into EVs is difficult. Here, it is investigated whether uPAR-targeted delivery of nanococktails containing PNCs loaded with therapeutic antimiRNAs, and coated with uPA engineered extracellular vesicles (uPA-eEVs) can elicit synergistic antitumor responses. The uPA-eEVs coating on PNCs increases natural tumor targeting affinities, thereby enhancing the antitumor activity of antimiRNA nanococktails. The systemic administration of uPA-eEV-PNCs nanococktail shows a robust tumor tropism, which significantly enhances the combinational antitumor effects of antimiRNA-21 and antimiRNA-10b, and leads to significant tumor regression and extension of progression free survival for syngeneic 4T1 tumor-bearing mice. In addition, the uPA-eEV-PNCs-antimiRNAs nanococktail plus low dose doxorubicin results in a synergistic antitumor effect as evidenced by inhibition of tumor growth, reduction of lung metastases, and extension of survival of 4T1 tumor-bearing mice. The targeted combinational nanococktail strategy could be readily translated to the clinical setting by using autologous cancer cells that have flexibility for ex vivo expansion and genetic engineering.

Keywords: antisense miRNA; cell free cancer therapeutics; engineered nanococktails; targeted combinational cancer therapy; triple-negative breast cancer.

Figure 1.

Schematic illustration strategy of 4T1-engineered extracellular vesicles (eEVs) that display uPAR targeting uPA or scrambled uPA (Sc-uPA) peptide-functionalized PLGA-nanocarriers separately loaded with antimiRNA-21 or antimiRNA-10b.

Figure 2.

(a) Schematic illustration shows the pcPUR-uPA vector map with PDGFR transmembrane domain fused uPA or negative control scrambled-uPA (Sc-uPA) insert sequence used to engineer the 4T1 cancer cells to display uPAR targeting uPA or Sc-uPA peptide as a C-terminal fusion protein with PDGFR. (b) DLS revealing comparative size distribution of 4T1-eEVs isolated from cells engineered to display uPA and Sc-uPA (n=3). (c) TEM image of eEVs isolated from 4T1 cancer cells engineered to express uPA peptide (n=3). Insert figure within image ‘c’ shows the spherical shaped proteo-lipid vesicles of eEVs isolated from 4T1 cancer cells engineered to express uPA peptide at higher magnification.

Figure 3.

Proteomic analysis of 4T1-eEVs. (a) Venn diagram shows the comparison of protein identifications (4T1 and SKBR3) found in our study and reported by Vesiclepedia and ExoCarta. (b) Total protein signal from 41T-eEVs and SKBR3-eEVs by category. (c-d) Abundant cancer cell adhesion proteins identified in 4T1-eEVs. Z-score iBAQ quantification values are shown by color. Gene ontology (GO) analysis of identified proteins from 4T1-eEVs sample. Gene enrichment was performed by GO cellular component (e), GO biological process (f), molecular function (g), and reactome pathway analysis (h). Protein enrichment is represented as −log10 of p-value after Bonferroni correction.

Figure 4.

(a) Schematic diagram shows the uPA and Sc-uPA peptide displaying 4T1-eEVs functionalized PLGA-PEG nanocarriers loaded with antimiRNA-21 and antimiRNA-10b. (b) Characterization of 4T1-eEV functionalized PLGA-PEG nanocarriers by DLS analysis shows the comparative size distribution of 4T1-eEVs and eEVs functionalized nanoformulations (n=3). (c) Zeta sizer-based surface charge analysis shows the comparative charge analysis between 4T1-eEVs and eEVs functionalized nanoformulations (n=3). (d) Transmission electron micrograph shows the PLGA nanocarrier and 4T1-eEVs coated PLGA-PEG NPs. Negative-stain electron microscopy was performed to visualize the eEVs on the PLGA-PEG NCs. Scale bars, 100 nm. Insert figures within this panel showing the higher magnification images of hybrid polymeric nanocore and the proteo-lipid shell of eEV-PLGA. (e) Confocal laser scanning microscopy images showing the hybrid nano polymeric core (DiD) and shell (NBD-PC-green) (Scale bar, 1 μm). Investigation on the uPAR targeted uptake of uPA peptide displaying 4T1-eEV functionalized PLGA nanocarriers. Insert figures within this panel showing the higher magnification image of proteo-lipid shell of eEVs labelled with NBD-PC, polymeric nanocore labeled with DiD, and the merged image of core-shell hybrid (n=3). (f) Fluorescence microscopy images show the uPAR targeting capability of uPA peptide displaying 4T1-eEV functionalized PLGA-PEG nanocarriers loaded with antimiRNA-21-Cy5, (g) quantitative data shows the uPAR targeting efficiency of uPA peptide displaying 4T1-eEVs functionalized PLGA-PEG nanocarriers. The fluorescence images were acquired with an excitation at 650 nm and emission at 670 nm using a Celigo Imaging Cytometer (Nexcelom Bioscience, LLC, MA) (n=3). Incubation of the eEVs-uPA-PLGA-PEG-antimiRNA-21-Cy5 with scrambled siRNA treated 4T1 cells showed significantly higher uptake when compared to Sc-uPA-PLGA-PEG-antimiRNA-21-Cy5 NPs, and this uptake effect was abrogated in the uPAR siRNA treated 4T1 cells. In the siRNA-treated group, incubation of eEVs with uPA and Sc-uPA-PLGA-PEG-antimiRNA-21-Cy5 NPs showed relatively equal uptake (not significant), and showed comparatively higher uptake than uncoated PLGA-PEG-antimiRNA-21-Cy5 NPs. In vitro cytotoxicity assay. (h) CCK8 assay showing that the targeted combinational therapy, namely cocktail mixture of 4T1-eEV-uPA-PLGA-PEG-antimiRNA-21 and 4T1-eEV-uPA-PLGA-PEG-antimiRNA-10b with a combination of low dose DOX resulting in a significant antiproliferative effect compared to cells treated with free DOX (2.8-fold) and control cells. The uPAR targeted combinational nanococktail mixtures (uPA-4T1-eEVs-PNCs-AntimiRs) with low dose DOX caused significantly higher cytotoxicity (2.2-fold, p< 0.01) in 4T1 cells, when compared to non-uPAR targeted Sc-uPA-nanococktail formulations with low dose DOX. Similarly, treatment with uPAR targeted individual nanoformulations eEV-uPA-PLGA-PEG-antimiRNA-21 or eEVs-uPA-PLGA-PEG-antimiRNA-10b nanoformulations alone in combination with low dose DOX produced considerable cytotoxicity in 4T1 cells, compared to untreated control 4T1 cells and low dose DOX alone treatments. (t-test, *p<0.01, **p<0.001, and ***p<0.0001).

Figure 5.

In vivo biodistribution and uPAR targeted accumulation of eEVs functionalized PLGA-PEG-antimiRNA nanococktail in syngeneic 4T1 subcutaneous tumors in nude mice (nu/nu). (a) In vivo fluorescence imaging showed the whole-body biodistribution and 4T1-tumor specific accumulation of ICG labeled Sc-uPA and uPA nanococktail formulations administered via tail vein injection on Days 0, 6 and 12, and imaged on Days 2, 7 and 15 using a Lago (Spectral Imaging system) in vivo imaging system. (b) Photoacoustic imaging of 4T1 tumors for the accumulation of ICG labeled eEV-uPA-PLGA-PEG-antimiRNAs on Day 16. (c) Ex vivo fluorescence imaging showed the uPAR mediated 4T1 tumor-specific accumulation of eEV-uPA-PLGA-PEG-antimiRNA-21 nanococktail formulations on Day 17. The uPA and Sc-uPA nanococktail formulations injected nude mice were sacrificed on Day 17, and the organs (liver, spleen, kidneys, heart, lungs, and brain) were collected for ex vivo imaging. (d) The biodistribution and tumor-specific accumulation of ICG, antimiRNA-21, and antimiRNA-10b in nude mice injected with uPA and Sc-uPA functionalized eEV-PLGA-PEG-antimiRNA-21 nanococktail formulations. Lago fluorescence imaging and TaqMan real-time qRT-PCR were used for organ-specific biodistribution based on the quantification of ICG and antimiRNAs. The endogenous expression of sno234 was used as an internal control to normalize the qRT-PCR results. We used 5 animals (n = 5) in each group while repeating the experiment twice in the study (n = 10).

Figure 6.

In vivo biodistribution and uPAR targeted accumulation of eEVs functionalized PLGA-PEG-antimiRNA nanococktail in syngeneic BALB/cJ mice bearing 4T1 subcutaneous tumors. (a) In vivo fluorescence imaging shows the whole-body biodistribution and 4T1-tumor specific accumulation of ICG labeled uPA and Sc-uPA nanococktail formulations administered via tail vein on Days 0, 6 and 12, and imaged on Days 2, 7 and 15 using a Lago (Spectral Imaging system) imaging system for ICG fluorescence. (b) Ex vivo fluorescence imaging shows the uPAR mediated 4T1 tumor specific accumulation of uPA nanococktail formulations in BALB/cJ mice bearing syngeneic 4T1 subcutaneous tumors. The uPA and Sc-uPA nanococktail formulations injected nude mice were sacrificed on Day 17, and the organs (liver, spleen, kidneys, heart, lungs, and brain) were collected for ex vivo imaging analysis. (c) The biodistribution and tumor specific accumulation of ICG, antimiRNA-21, and antimiRNA-10b delivered using uPA and Sc-uPA nanococktail formulations injected in BALB/cJ mice bearing 4T1 subcutaneous tumors. Lago fluorescence imaging and Taqman real-time qRT-PCR were used for the organ-specific biodistribution based on the quantification of ICG and antimiRNAs. The endogenous expression of sno234 was used as an internal control to normalize qRT-PCR results. We used 5 animals (n = 5) in each group while repeating the experiment twice in the study (n = 10).

Figure 7.

(a) Survival curves of mice bearing 4T1 tumors given different treatments (Saline; Low dose DOX alone; Sc-uPA nanococktail; and uPA nanococktail). We used 10 animals in each group for assessing the survival rate (n=10). (b) 4T1 tumor growth kinetics after different treatments. (c) Body weight of mice receiving different treatments. (d) Ex vivo images of tumors excised one month after treatments. (e) Excised tumor weights were measured a month after treatments. (f) The number of metastatic nodules in the lungs after different treatments. (g) Histologic assessments of lungs and tumors using H&E staining in mice. Top panel: representative images of lung tissues in various treatment groups showed the metastatic nodules at the end of the experiment. Figure inserts within this panel showing the respective low magnification images of lung metastatic foci of 4T1 tumor under different treatment conditions. Bottom panels: representative H&E staining of tumor sections in various treatment groups. Scale bars, 250 μm. Data are means ± SD. Statistical significance was calculated by t-test (* p< 0.05, ** p< 0.01, *** p< 0.001).