Maximizing liposome tumor delivery by hybridizing with tumor-derived extracellular vesicles

Abstract

Extracellular vesicles (EVs) have gained widespread interest due to their potential in the diagnosis and treatment of inflammation, autoimmune diseases, and cancers. EVs are lipidic vesicles comprising vesicles of endosomal origin called exosomes, microvesicles from membrane shedding, and apoptotic bodies from programmed cell death membrane blebbing that carry complex sets of cargo from their cells of origin, including proteins, lipids, mRNA, and DNA. EVs are rich in integrin proteins that facilitate intrinsic cellular communication to deliver their cargo contents and can also be used as biomarkers to study respective cellular conditions. Within this background, we hypothesized that when these EVs are hybridized with synthetic liposomes, it would help navigate the hybrid construct in the complex biological environment to find its target. Toward this endeavor, we have hybridized a synthetic liposome with EVs (herein called LEVs) derived from mouse breast cancer (4T1 tumors) cells and incorporated a rhodamine-B/near-infrared fluorescent dye to investigate their potential for cellular targeting and tumor delivery. Using membrane extrusion, we have successfully hybridized both entities resulting in the formation of LEVs and characterized their colloidal properties and stability over a period. While EVs are broadly dispersed nano- and micron-sized vesicles, LEVs are engineered as monodispersed with an average hydrodynamic size of 140 ± 5. Using immunoblotting and ELISA, we monitored and quantified the EV-specific protein CD63 and other characteristic proteins such as CD9 and CD81, which were taken as a handle to ensure the reproducibility of EVs and thus LEVs. These LEVs were further challenged with mice bearing orthotopic 4T1 breast tumors and the LEV uptake was found to be maximum in tumors and organs like the liver, spleen, and lungs when compared to control PEGylated liposomes in live animal imaging. Likewise, the constructs were capable of finding lung metastasis as observed in ex vivo imaging. We anticipate that this study can open avenues for drug delivery solutions that are superior in target recognition.

Figure 1.

Physiochemical characterization of extracellular vesicles (EVs) derived from mouse breast cancer 4T1 cell using multi-angle dynamic light scattering at three different angles. (A) Size distribution of Engineered EVs with synthetic liposomes (LEVs), Liposomes, and EVs (B) Stability of Liposomes and LEVs over the period in terms of PDI and size, respectively.

Figure 2.

(A) Fluorescence Resonance Energy Transfer (FRET) study showing successful hybridization of EVs and liposomes. FRET study was conducted using fluorescent donor NBD (kem = 525 nm) and fluorescent acceptor RhB (λem = 595 nm) at an excitation wavelength of 470 nm. Fusion of two vesicles results in the increase in the distance between FRET pairs resulting the fluorescent donor’s (NBD’s) characteristic peak at 525 nm (blue curve). (B) SDS PAGE analysis of bulk protein in EVs and LEVs. Both samples were concentrated to get distinct protein bands (C) A qualitative Immunoblot (Dot blot) showing characteristic CD63, CD9, and CD81 in EVs and LEVS.

Figure 3.

Cellular internalization studies. Liposomes and LEVs labeled with Rh-B were incubated with 4T1 cells. (A) Fluorescence image showing cellular internalization of RhB labeled liposomes and LEVs after 1.5 h, 3 h, and 4 h incubation. Results showed minimal internalizations of control liposomes and maximum internalization of LEVs into 4T1 cells. (B) Fluorescence quantification of micrographs using ImageJ analysis in different periods (1.5 h, 3 h, and 4 h). DAPI (blue) was used to visualize the cell nuclei. Cells were fixed and examined using Fluorescence microscopy. Scale bar 100 μm. Data represent mean ± SD; n = 3; ** = p<0.01 and * = p<0.05. MFI = Mean Fluorescence Intensity.

Figure 4.

Cellular uptake studies using flow cytometry. The 4T1 cells were incubated with liposomes and LEVs labeled Rh-B and time courses of NP uptake were assessed. (A) The cell-associated fluorescence signals at various periods of incubation. (B) Biocompatibility study of liposomes and LEVs (20–200 μg/mL) against 4T1 cells after 24 h incubation. Data represent mean ± SD; n = 3; **** = p<0.0005.

Figure 5.

In vitro immunogenicity of liposomes and LEVs in J774.A cell was assessed by evaluating the proinflammatory cytokines (IL-6 and TNF-Alpha) released after 24 h incubation. LPS (lipopolysaccharide) was used as the standard for comparison. Data represent mean ± SD; n = 3; **** = p<0.0005.

Figure 6.

Live animal imaging: In vivo NIR fluorescence imaging of 4T1 tumor-bearing mice injected with DiR-loaded LEVs (A) and Liposomes (B). Each mouse was administered intravenously the same concentration of DIR that was optimized by respective fluorescence intensities. The fluorescence images were obtained at different time points after the injection under the same imaging settings. The tumor region of each mouse was indicated by an arrow mark.

Figure 7.

Each group with three animals was injected with respective NPs labeled with DIR dye. (A) Ex-vivo imaging of tumor tissues and organs (including brain, spleen, kidney, heart, lung, tumor, liver) 24 h after injection with control liposomes-DIR. (B) Ex-vivo imaging of tumor tissues and organs (including brain, spleen, kidney, heart, lung, tumor, liver) 24 h after injection with LEVs-DIR. (C) Intensity per gram tissue was plotted to map the NPs accumulation in tumor tissue and organs. Data represent mean ± SD; n = 3; * = p<0.05.

Figure 8.

Ex vivo imaging of lung tissue after 24 hr post-injection capturing nanoparticles distribution in lungs (n=3). Enlarged lung images showing metastatic consequences of 4T1 tumors.

Scheme 1.

Schematic representation of the fabrication of hybrid exosome. (A) Isolation of extracellular vesicles (EVs) from 4T1cells (B) Hybridization of cancer cell derived extracellular vesicles (EVs) with synthetic liposome using membrane extrusion.