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 inflammations, autoimmune diseases, and cancers. EVs are lipidic vesicles comprised of vesicles from endosomal origin called exosomes, microvesicles from membrane shedding, and apoptotic bodies from program 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, which can also be used as a biomarker 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 the EVs (herein called LEVs) derived from mouse breast cancer (4T1 tumors) and incorporated a near-infrared fluorescent dye to investigate their potential for cellular targeting and tumor delivery. Using the membrane extrusion, we have successfully hybridized both entities resulting in the formation of LEVs and characterized for their colloidal properties and stability over a period. While the EVs are broadly dispersed nano and micron-sized vesicles, LEVs were engineered as monodispersed with an average hydrodynamic size of 140±5. Using immuno-blot and ELISA, we monitored and quantified 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 found to be maximizing the LEVs uptake in tumors and organs like the liver, spleen, and lungs when compared to control PEGylated liposome 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.

Graphical Abstract

This investigation highlights the maximizing nanoparticle tumor delivery by hybridizing nanoparticles with tumor-derived-extracellular vesicles

INTRODUCTION

Extracellular vesicles (EVs) are protein-rich lipid bilayer-enclosed nanoparticles (NPs). They play a crucial role as intercellular communication mediators by transporting biological cargo such as proteins, lipids, and nucleic acids, between cells.^1–5 The body’s pathophysiological processes are modulated by interactions between EVs and recipient cells. Due to their potential intrinsic benefits, such as their suggested preferential accumulation to particular cells or organs and ability to cross biological barriers, research in EV-based drug delivery systems (DDS) has grown significantly in recent years and is well documented.^6–13 Despite their promising potential in the clinic, their clinical use and visualization as nanomedicine is limited due to the lack of standardization of EVs isolation, analysis, heterogeneity in size distribution, and colloidal stability over the ranges of ionic conditions.^14–17

EVs are classified according to their biogenesis, which also governs the properties of EVs. EVs released from endosomal origin are called exosomes which range from 30 to 200 nm in their hydrodynamic size. Similarly, other classes are microvesicles released from cell membranes and apoptotic bodies resulting from cells undergoing apoptosis.^5,13,18,19 Exosomes being the endosomal origin, are reached in endosome-associated proteins such as Rab GTPases, SNAREs, Annexins, flotillin, and tetraspanins (e.g. CD63, CD81, CD9).^3,20,21 Among these proteins, tetraspanins are abundant in exosomes and considered markers for exosomes. In drug delivery technology, exosomes are of great interest owing to their nano size and their resemblance with liposomal drug delivery systems and so are the other classes of EVs.

However, due to their low yield, it is difficult to isolate and store functional EVs in the numbers required for therapeutic delivery applications.^22–25 In order to separate EVs based on their size, density, and protein markers, several approaches have been investigated. These include ultracentrifugation, ultrafiltration, precipitation, immunoaffinity-based separation, and microfluidics-based procedures.^26–28 However, a significant problem is in the maintenance of the structural integrity and functional characteristics of EVs isolated using these procedures because naive EVs are not stable for prolonged periods due to precipitation and repeated freeze-thaw processes. Consequently, membrane fusion techniques have been reported in which EVs were hybridized with synthetic organic and inorganic nanoparticles to stabilize EVs’ biomarkers.²⁸ By using specific procedures, EVs and nanoparticles (such as polymers or liposomes conjugated with or loaded with biological agents) can be combined to create innovative drug delivery nanoplatforms or EV-based hybrid systems. The structural characteristics of EVs are analogous to liposomes, which renders EVs attractive for drug delivery. Since liposomes are phospholipid constructs, they are similar to the plasma membranes and have been widely used for efficient drug delivery. Therefore, producing EV-based hybrid systems represents a possible substitute for synthetic DDS.

Toward this endeavor, we have hybridized a synthetic liposome with the EVs derived from mouse breast cancer (4T1 cells) and incorporated a near-infrared (NIR) fluorescent dye to investigate its potential for cellular and in vivo tumor targeting. Resulting hybrid constructs herein named as LEVs. For the process, EVs were isolated from the cell supernatant and proceeded with a series of centrifugation to remove cell debris and large aggregates followed by filtration through a 200 nm filter to collect EVs of less than 200 nm. Considering the size classification recommended by the International Society for Extracellular Vesicles (ISEV) in Gothenburg, Sweden (April 2012),²⁹ these are exosomes, however, we cannot avoid the contamination from microvesicles and apoptotic bodies, therefore, we are comfortable naming them as EVs throughout this manuscript. Using the membrane extrusion, we have successfully hybridized both entities resulting in the formation of LEVs and characterized for their colloidal properties and stability over a period. We have monitored EVs-specific protein CD63, total protein concentration, and concentration of isolated EVs as handles to ensure its reproducibility throughout the experiments. For the first time, we have studied the drug delivery potentials of 4T1 LEVs in vitro and in vivo and are optimistic that this study can open future opportunities for drug delivery solutions that are superior in target recognition. Moreover, unlike most tumor models, 4T1 can spontaneously metastasize from the primary tumor in the mammary gland to multiple distant sites including lymph nodes, blood, liver, lung, brain, and bone, therefore, the present study further highlights the superiority of the hybrid construct in targeting both primary and metastatic sites.

MATERIAL AND METHODS

Materials

1,2-Dioctadecanoyl-sn-glycero-3-phospho-(1′-rac-glycerol) (sodium salt) (DSPG), (1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[succinyl(polyethyleneglycol)-2000] (ammonium salt)) DSPE-PEG (2000), Alexa- 488 and rhodamine-B-tagged phospholipids [Lα-phosphatidylethanolamine-N- (lissamine rhodamine B sulfonyl) (ammonium salt) (egg-transphosphatidylated, chicken)] were purchased from Avanti Polar Lipid Inc. (Alabaster, AL, USA). Mouse breast cancer cells (4T1) were purchased from ATCC (American Type Culture Collection, Manassas, VA, USA). Cholesterol was purchased from Fisher Scientific. The near IR fluorescent lipophilic DiR [(DiIC18(7) (1,1′-dioctadecyl-3,3,3′,3′-tetramethylindotricarbocyanine iodide)] was purchased from Invitrogen and used as received. Mouse monoclonal antibodies CD81 , CD63, and CD9 were purchased from Santa Cruz Biotechnology. Mouse CD63 ELISA Kit was purchased from abcam. Mouse IL-6 and TNF-α ELISA Kits were purchased from Invitrogen. All other reagents and chemicals were of analytical grade.

Cell lines and tumor model

Mouse breast cancer (4T1) and mouse macrophage (J774A.1) cell lines were obtained from ATCC. The J774A cell line was grown in Dulbecco’s Modified Eagle Medium (DMEM; Corning; Corning, NY, USA), and the 4T1 cell line in RPMI 1640 medium. Both media were supplemented with 10% heat-inactivated fetal bovine serum (FBS; Corning), and 1% penicillin-streptomycin solution (#SV30010; HyClone; Logan, UT, USA). The cells were kept at 37 °C in a humid environment with 5% CO₂. The cells employed throughout this research were between passages 5-8, and the cell culture media was changed every two to three days.

For tumor studies, six-week-old female immunocompetent C57BL6 mice were procured from the Jackson Laboratory (USA). All animal experiments and protocols were approved by the IACUC, University of Texas Health Sciences, Tyler, Texas. For tumor models, 1 × 10⁶ 4T1 cells in saline were implanted at the 5^th memory gland and the tumor growth was monitored periodically. Mice were fed with proper diet throughout the experiment.

Extracellular vesicle isolation

Extracellular vesicles (EVs) were harvested from 4T1 cells using centrifugation and filtration techniques.¹⁸ 4T1 cells were cultured in RPMI 1640 medium for 24 h at 37 C in a 5% CO₂ environment to harvest EVs. The cell supernatant media was collected and centrifuged at 1500 rpm for 15 min to remove cell debris. The supernatant was then collected and centrifuged again at 4000 rpm for 1 h to remove large aggregates. Finally, the supernatant was collected and filtered through 200 nm and was concentrated 10 times using a 10 kDa molecular weight cut-off (MWCO) Amicon filter (3500 rpm, 15 min). The concentrated EVs were stored at −80 ^°C for future use.

Synthesis of liposome

Liposome synthesis was carried out by thin film hydration technique followed by membrane extrusion as described by Pitchaimani et al.³⁰ Briefly, our test liposomes consist of DSPG and cholesterol dispersed in chloroform in a molar ratio of 70:30. The mixture was subjected to the rotatory evaporator and was recovered as a thin dried film (2.5 mg). The obtained thin film was hydrated by 1 mL DI water. It was then vortexed and sonicated (bath and probe) for proper mixing. The liposome solution was extruded using 400 and 200 nm polycarbonate membrane filters, respectively, to obtain nano-sized unilamellar liposomes. For the labeling, liposome and Alexa or DiR, (DSPE: Cholesterol: dye) were maintained at 65:30:5 molar ratio. For PEGylated liposomes, DSPG: DSPE-PEG: cholesterol: dye (55: 10: 30:5) molar composition was used. The use of PEG is essential in the control liposomes to enhance and prolong their colloidal stability to evaluate them in vitro and in vivo.

Engineered EVs, the LEVs preparation

A serial extrusion method was employed to fuse the EVs with Liposomes. In a typical LEVs engineering, a previously optimized dry film of lipid (DSPG and Cholesterol mixture) for a calculated number of liposomes was hydrated with known numbers of EVs at the ratio of 1:1. The numbers of particles were determined using Multiangle dynamic light scattering (MADLS). The volume of the mixture was adjusted to 1 ml with PBS. The mixture was then vortexed and sonicated using probe sonication (30% amplitude, 30 sec pulses on/off, for 2 min) under the ice for proper mixing. Thus, the formed multilamellar LEVs solution was extruded through 400 and 200 nm polycarbonate membrane filters, respectively, to get nano-sized unilamellar LEVs. The final LEVs were stored at 4^oC for future use. For Live animal imaging and biodistribution studies, as prepared DiR labeled LEVs (1mL) were concentrated using a 10 k molecular weight cut-off (MWCO) amicon filter (3000 rpm, 15 min) to 250 uL. The concentration of dye in LEVs and liposomes was maintained constant and was monitored spectrophotometrically and the droplets were imaged using an imaging system to ensure effective dye concentration for imaging.

Characteristic protein analysis

Total protein in EVs and LEVs was quantified using Bradford assay, SDS PAGE, and immune bolt experiments. For SDS PAGE, EVs and LEVs were mixed at a 1:1 ratio with Lameli buffer. All protein samples were denatured at 90 °C for 8 minutes, allowed to cool back down to room temperature to avoid melting the gel, and then loaded into a stain-free polyacrylamide gel for electrophoresis (4-20%) at a concentration of 20 μg total protein per lane. The SDS buffer was prepared by mixing 10x Tris/Glycine/SDS Buffer with D.I water at a ratio of 1:10. The gel was loaded into the electrophoresis chamber with the protein samples alongside a Protein standard. Electrophoresis was performed at 120 V for 60 minutes until the samples and the standard reached the end of the gel. The gel was imaged using a BioRad ChemiDoc Imaging System, after Coomassie blue staining.

Quantification of CD63 (characteristic exosome marker) and specific markers for 4T1 EVs such as CD81 and CD9 in isolated EVs from 4T1 cells was determined using respective ELISA kits according to the manufacturer’s instructions. The results obtained in our experiments were calculated by standard curve interpolation and expressed in pg/mL. CD63 concentration was maintained constant for all in vitro and in vivo experiments. For the analysis of other target proteins, immunoblots were used. Samples were drop-casted (4 μl droplet) in the activated membrane. After the droplets dried, blots were incubated with a blocking buffer for 30 minutes at room temperature. The antibody solutions of CD63 (exosome marker), CD81, and CD9 of final concentrations 200 μg/mL in blocking solution were prepared and replaced respectively. The antibody solution-treated membrane was incubated overnight at 4 ^°C under a rocking motion. After overnight incubation, the membrane was washed two times with wash buffer. Then, incubated with secondary antibody Horseradish peroxidase (hrp), 10 ul in 10 ml final volume (184 μg/mL) of blocking solution) for 1 hour at room temperature under rocking motion. The membrane was washed with buffer three times. The last wash was kept and removed before the addition of Cell signaling reagent (Reagent A and Reagent B) just before imaging. The membrane was kept in the reagent (10 ml) and gently shaken in a rocking motion for about 5 min in the dark and images of the membrane (x-ray imaging) were taken using Biorad chemiluminescence.

Colloidal characterization Studies

MADLS was used to assess the hydrodynamic diameter (Dh) and polydispersity index (PDI) of liposomes and LEVs using a Zetasizer Ultra Red (Malvern Panalytical Ltd., Malvern, UK). The MADLS at three different angles viz., 17°, 90°, and 173° angles was used to measure the concentration of NPs in a solution. The same device was used for electrophoretic light scattering (ELS) to study zeta potential. All colloidal properties were assessed at 1 mg/mL as calculated by lyophilization.

Monitoring hybridization process

The hybridization of liposomes and EVs producing LEVs was monitored by a fluorescence resonance energy transfer (FRET) study. FRET liposomes were synthesized as described in our publications.^11,18,31 FRET fluorophore lipids, NBD acting as an electron donor and Rh-B acting as an electron acceptor, were incorporated in a lipid mixture in the molar ratio of 1:7 resulting in the formation of FRET liposome. For fusion analysis, a ratio of 1:1 (EVs : FRET liposome) volume ratio was mixed, and bath sonicated for 5 min to initiate fusion. FRET liposomes, before and after fusion of EVs, were analyzed by fluorescence spectroscopy by exciting samples at 470 nm and measuring the emission spectra between 500 and 700 nm.

Cellular uptake study (Flow cytometry)

The cells were seeded at a density of 250,000 cells per well in a 12-well plate (in 2 mL of the medium) for the Alexa-labeled Liposomes and LEVs uptake study.³² The Liposomes and LEVs NPs were added at the required time points the following day. The cells were harvested by trypsin treatment and then resuspended in 1 mL of prewarmed BSA solution (1% in PBS, #P3688, Sigma-Aldrich). A 488 laser was used to activate the fluorescence in order to evaluate the samples using flow cytometry (CytoFLEX, Beckman Coulter, Brea, CA, USA). Using CytExpert Software (Beckman Coulter), data (50,000 total events per sample) were evaluated while cells were analyzed at a rate of about 200 events per second. The mean cell-associated fluorescence in the FITC channel was then calculated. Mean fluorescence intensity measurements were standardized to a control group that wasn’t given any treatment in order to depict the data. The internalization half-time values were then determined using the GraphPad Prism program (Dotmatics, Boston, MA, USA) based on the internalization time course by curve-fitting the data using the equation below:

$$F\left(t\right)=F_0+(F_{\text{plateau}}-F_0)(1-e^{-tk}),$$ (1)

where $F\left(t\right)$ is the cell fluorescence signal at time $t$, $F_0$ and $F_{\text{plateau}}$ are the initial fluorescence signal and the maximum signal, respectively, and $k$ is the internalization rate constant. The half-time of internalization was calculated as the ratio of ln 2 and $k$. ^33,34

Mouse breast cancer 4T1 cells were seeded at the bottom of 8-well glass-bottomed chambers (#C8-1.5H-N, Cellvis, Mountain View, CA, USA) at a density of 50,000 cells/well and left to adhere overnight. The medium was then changed to RPMI containing 100μL of 1mg/mL of both control Liposomes and LEVs that were Alexa 488-labeled at the desired time points. Following three PBS washing steps, the cells were fixed with prechilled 4% paraformaldehyde in PBS. A BioTek Lionheart FX Automated Microscope was used to examine the cellular uptake of both liposomes and LEVs by cancer cells in Alexa fields (visualized with FITC filter, excitation/emission wavelengths of 494/517 nm).

Biocompatibility

The biocompatible nature of the LEVs was investigated in 4T1 cells using the Cell Counting Kit-8 (CCK-8) cell viability assay. In brief, 4T1 cells were seeded in a 96-well plate at the density of 10,000 cells per well and incubated for 24 h. After confluence, cells were treated with different concentrations of LEVs (20–100 μg mL⁻¹) and incubated for 24 h. After incubation, the medium was removed and washed with 1X PBS, and 10 μL of CCK-8 reagent was added and incubated further for 4 h in the dark at 37 °C. The WST-8 (water-soluble tetrazolium salt) contained in the CCK-8 reagent can be reduced to a highly water-soluble yellow formazan by dehydrogenase in the mitochondria of the cell. The amount of formazan produced is proportional to the number of living cells. After 4 h incubation, the plates were analyzed for absorbance at 450 nm using a microplate reader.

In Vitro Cytokine Release Assay

The immunogenicity of the LEVs along with the control Liposomes were tested for its immunoregulatory potential using a standard cytokine release assay. In brief, 5 × 10⁵ J774A cells were seeded in a 12-well plate and treated with control Liposomes (100 μg mL⁻¹), and LEVs (100 μg mL⁻¹) at 37 °C for 24 h. After 24 h incubation, cell culture supernatants were collected and stored at −20 °C as small aliquots. For cytokine assay, samples were thawed and analyzed for proinflammatory cytokines, IL-6 and TNF-α, using Invitrogen IL-6 and TNF-α Mouse ELISA Kit in the 96-plate reader as per the manufacturer’s recommendations. For positive control, cells were dosed with 3 μg mL⁻¹ of lipopolysaccharide (LPS) for 24 h. The cytokines produced in each sample were calculated from standard curves using known concentrations of recombinant cytokines for each ELISA antibody kit.

Live animal imaging and biodistribution

All animal procedures were approved by IUCAC for the care and use of laboratory animals. 5−6-week-old female immunocompetent C57BL6 mice were purchased from Jackson Laboratory, USA. A tumor model was developed by injecting 1×10⁶ 4T1 cells at the 5^th memory gland of female C57BL6 mice. The length (L) and width (W) of each tumor were measured on two alternative days. When the tumor grew to ~5 mm³, animals were treated with DiR-labeled control liposomes and LEVs by tail vein injection at the protein dose of ~950pG of CD63 per mouse. 950pG of CD63 was calibrated from the EVs isolated from T-75 culture flask at ~85% confluency. DiR intensity in liposomes and LEVs were optimized spectrophotometrically to be equivalent. Whole-body images were acquired by the near-infra-red anima imaging system (Pearl Impulse Imaging System, LI-COR) under 2.0% isoflurane anesthesia at specified times (0 h-24 h) post-injection. After the final imaging session, 24 h post-injection, animals were euthanized following the IACUC protocol, and major organs were collected and imaged by the near-infra-red animal imaging system. The radiance (photon emission per unit area) of a region of interest (ROI) was acquired and used for NPs distribution analysis.

Live subject statement

Female immunocompetent C57BL6 mice (5–6 weeks old) were purchased from Jackson Laboratory, USA, each weighing about ~25 g, were housed in standard facilities, and were supplied with ad libitum access to food and water.

All animal experiments and protocols were performed in strict accordance with the NIH guidelines for the care and use of laboratory animals (National Institutes of Health (1985) Guide for the Care and Use of Laboratory Animals. NIH Publication Number 85-23, US Department of Health, Education and Welfare, Bethesda, MD). The animal protocols were approved by the Institutional Animal Care and Use Committee (IACUC) and Institutional Biosafety Committee (IBC), University of Texas at Tyler, Tyler Texas. The animals were treated according to the policies and guidelines of the University of Texas at Tyler Animal Care and Use Committee.

Statistical analysis

GraphPad Prism 7.0 software was used for statistical analysis among different groups using ANOVA method, followed by a student t-test. The results were considered statistically significant if the p-value was <0.05, with the significance levels denoted as **** for p < 0.0001, *** for p < 0.001, ** for p < 0.01 and * for p < 0.05.

RESULTS AND DISCUSSION

Synthesis and characterization of hybridized liposomes (LEVs)

Preparation of hybridized liposomes involves the isolation of extracellular vesicles (EVs) from mouse breast cancer (4T1) cells and surface infusion with synthetic liposomes as illustrated in Scheme 1. Isolated EVs were characterized using MADLS to capture size distribution heterogeneity at three different angles 17°, 90°, and 173°. MADLS laser striking from acute to obtuse angles can capture a large population of particles, therefore, distribution heterogeneity was maximally captured in this analysis, which shows EVs are polydisperse and size ranges from 10-150 nm with some micro aggregates (Figure 1A). Following the syringe extrusion, we have hybridized EVs with synthetic lipid constituents to form LEVs. These LEVs showed an average hydrodynamic diameter of 140 ± 5 nm with a polydispersity index (PDI) of 0.145± 0.02. From the symmetry in the distribution spectrum and narrow PDI, we can infer that LEVs are monodispersed in their hydrodynamic size distribution. Similarly, a control PEGylated liposome (without EVs) was also prepared and characterized as monodispersed with a hydrodynamic size of 120 ±6 nm in diameter. This difference in the size of PEGylated liposomes and LEVs can be attributed to the presence of proteins in EVs, LEVs are larger while using the same technique for their fabrication. The PEGylated liposome due to a negatively charged phospholipids and carboxylate group exhibited −31 ± 0.8 mV (Supplementary Figure S1). The zeta potential of the isolated EVs was found to be −15 ± 0.3 mV, however, when hybridized to form LEVs, the zeta potential significantly shifted to a negative value of −36 ± 0.6 mV, which can be attributed to the incorporation of negatively charged phospholipids (Supplementary Figure S1). This change in zeta potential with narrow ±0.6 mV is the indication of the formation of the hybrid system, which otherwise could have exhibited large ±SD. Next, we examined the colloidal stability of liposome formulations for up to 7 days at 4°C to further assess their stability. LEVs displayed superior stability than EVs in terms of PDI and size. In comparison to 1.0 to 0.39 (indication of polydisperse particles) for EVs, the PDI of LEVs changed during the course of 7 days from 0.14 to 0.16 (indication of monodisperse particles). In addition, LEVs showed less size variation than EVs. These characterization data demonstrate that the stability of EVs is improved when EVs are incorporated into the lipid bilayer skeleton as LEVs (Figure 1B).

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.

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.

While these colloidal properties suggested the formation of a hybrid construct, the LEVs, we further wanted to confirm this at a molecular level. Therefore, to understand the hybridization process, we employ a Fluorescence resonance energy transfer (FRET) technique. FRET study has been widely used to study membrane fusion.^35,36 For this study, a FRET liposome containing fluorescent donor l-a-Phosphatidylethanolamine-N-(4-nitro benzo-2-oxa-1,3-diazole) (Ammonium salt) (PE-NBD) (Fluorescent donor, λem = 525 nm) and l-a-Phosphatidylethanolamine-N- (lissamine rhodamine-B sulfonyl) (Ammonium salt) (PE-Rh-B) (Fluorescent acceptor, λem = 595 nm) in 1:7 M ratio was prepared in which DSPG and cholesterol are at a molar ratio of 70:30. This FRET liposome was hybridized with EVs at 1:1 number of particle and emission spectrum was recorded from 500 to 700 nm (Figure 2A). As shown in Figure 2A, after hybridization, the FRET effect was diminished and we observed independent emission of donor at 525 nm and acceptor at 595 nm, which can only happen when the distance between the FRET pair grows. This implies that EV contents were inserted into the liposome’s lipid bilayer, validating successful hybridization.

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.

SDS-page and immunoblot analysis were performed to confirm the presence of proteins after the hybridization process. SDS-PAGE analysis showed distinct protein bands in both EVs and LEVs as shown in Figure 2B. LEVs showed similar protein bands to that of EVs, signifying that the EVs’ protein content has been conserved through the hybridization process. The protein cargo of EVs is important for their unique characteristic. As shown in Figure 2C, dot-blot assay showed the presence of major EV marker proteins like transmembrane proteins (CD81, CD63, and CD9) in both EVs and LEVs which signifies the successful retention of major EV proteins through the hybridization process.

Cellular uptake and biocompatibility studies

Cellular internalization of LEVs was studied on mouse breast cancer (4T1), a parent cell from where the EVs are isolated, using fluorescence imaging and flow cytometry analysis. As shown in Figure 3, internalization of RhB labeled LEVs and bare liposome. We hypothesized that LEVs would target mother cancer cells and internalize more compared to control PEGylated liposomes. In these in vitro experiments, 4T1 cells were incubated with control PEGylated liposomes and LEVs for up to 4 h. Results showed minimal internalizations of control liposomes (Figure 3A) and maximum internalization into 4T1 cells (Figure 3A). Taken together, these microscopic results indicated the possibility that our newly designed LEVs have the potential to be used as a cancer-targeting system. Using ImageJ, the percent coverage of RhB staining was quantified to compare the differential uptake of LEVs and control liposomes (Figure 3B).

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.

To further confirm this finding with a more robust quantitative analysis, a flow cytometry assay was performed in 4T1 cells. Figure 4A shows the flow cytometry analysis on the 4T1 cell line showing the internalization of RhB-labeled liposome and LEVs, after 4 h of treatment. As expected, there was a maximum uptake of LEVs by 4T1 cells as compared to that of control liposomes. As can be seen in the fluorescence Vs count plot of 4T1 at 4 h, the fluorescence intensity of LEVs and liposome, the spectrum for LEVs shifted to the right compared to the liposome showing enhanced internalization (Supplementary Figure S3). With the confirmation of cellular internalization of both types of NP systems, we further analyzed their biocompatibility against 4T1 cells through cell viability assay (Figure 4B). Cell viability was determined after 24 h of NPs treatment. The observed results showed that even at higher lipid concentrations (200 μg/mL lipid concentration), NPs were not cytotoxic (Figure 4B) demonstrating their compatibility at a wide range of concentrations.

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.

Cytokine Expression

Cytokines signal through specific cell surface receptors to broadly regulate immune development, differentiation, proliferation, and survival, which can be pro-inflammatory or anti-inflammatory. These molecules are typically produced by various innate and adaptive immune cells and regulate immune responses in health and disease. Proinflammatory cytokines are primarily responsible for initiating an effective defense against exogenous materials, but overproduction of these mediators can be harmful and may ultimately lead to shock, multiple organ failure, and death. In contrast, anti-inflammatory cytokines are crucial for downregulating the exacerbated inflammatory process and maintaining homeostasis for the proper functioning of vital organs, but the excessive anti-inflammatory response may also result in the suppression of the body’s immune function. Therefore, our goal here is to understand the eliciting immune response using murine macrophage J774 cells by quantifying proinflammatory cytokine IL-6 and TNF-alpha to envision possibilities of macrophage activation due to LEVs. The rationale behind the use of macrophages is due to the fact that a tumor is made up of varieties of cells in which macrophage is one of the most abundant immune cells within tumors and performs a broad repertoire of functions via diverse phenotypes. These tumor-associated macrophages (TAMs) have profound effects on increases in angiogenesis, tumor invasion, and the depression of immunity, as a result, TAMs can be taken into consideration in tumor immunotherapy.^37–40 Aligned towards this direction, our experiments evident the elevation of Il-6 and TNF-alpha in the cell supernatant (Figure 5). IL-6 is responsible for promoting tumor angiogenesis, aggravating local inflammation, and helping stem cell self-renewal. TNF-alpha is produced by macrophages is in a large degree (Figure 5). It is responsible for many cellular functions such as apoptosis, angiogenesis, and immune cell recruitment and regulation including extracellular matrix remodeling. Production of these two cytokines was significantly higher in the LEVs treated cell as compared to that of Liposome and naïve 4T1-EVs (Figure 5). Results obtained further support the evidence of higher cellular uptake of LEVs (Figure 4) that elicit elevated cytokine secretion suggesting that these engineered LEVs could have great potential in tumor immune therapy. However, to pinpoint the underlying molecular mechanisms, a more detailed study using animal models is needed which is out of the scope of this pilot study.

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.

Live animal imaging and biodistribution

The tumor-targeting effects of LEVs and Liposomes in vivo were investigated via NIR fluorescence imaging using near infra-red live animal imaging system. Tumor-bearing mice were first established and were injected with DiR-labeled NPs (i.e., DiR-LEVs or DiR-Liposomes). Each mouse-bearing orthotopic breast tumor was injected with the same amount of DiR, which was optimized spectrophotometrically and confirmed by using the same live animal imaging system to ensure equivalent intensity for both groups. Figure 6 shows the live images of mice at different time points after the injection. All the images were obtained under the same imaging settings for better comparison. At 1 h after the injection, a considerable fluorescence signal was observed in the tumor region of the mice receiving DiR-LEVs (Figure 6A) and control liposomes (Figure 6B). Notably, after 3 and 6 h post-injection, we observed fluorescence diffusion throughout the body in the control liposome group (Figure 6B). Whereas a distinguishable fluorescence signal exists in the LEVs injected group (Figure 6A). After 24 h, we observed signal diffusion throughout the body in both groups with distinguishable tumor geometry in LEVs injected group. These results suggested a clear trend of EV’s protein influence in target recognition. These observations further give us a guideline that NPs systems are rapidly taken up by tumors and, therefore, are good candidates to devise as a targeted drug delivery system.

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.

Furthermore, for a more comprehensive investigation of the nanoparticles’ biodistribution, an ex vivo imaging of vital organ collects including tumors was carried out. After 24 h post-injection, animals were euthanized following an approved protocol, and the ex vivo imaging of respective vital organs was performed. Figure 7A and 7B shows the representative fluorescence images of tumors and organs/tissues from different groups. The LEV-treated group showed comparable tumor/organ fluorescence intensity notably stronger than that of the control liposome group, which was in accordance with the in vivo imaging results, mostly in reticuloendothelial system (RES) organs. Results indicated that both NPs could selectively accumulate in tumors and major RES organs like the lung, spleen, and liver. Further, intensity per gram tissue was plotted to map the NPs accumulation in the organ. Results in Figure 7C indicated that both types of NPs were found to be distributed in RES Organs. While going through each organ, we observed the retention of 4T1-LEV is higher in these major organs. Due to the small number of animals in this pilot study, it is difficult to access the significance level of NPs accumulation in each organ. Therefore, we run a student t-test to analyze differential tumor uptake. We found a significant difference in NPs tumor homing which could be due to the acquired properties from the parent cell. However, more studies are warranted to further analyze deep inside the response at a molecular level by blocking tumor surface receptors that are responsible for NPs capture. As in human breast cancer, 4T1 metastatic disease develops spontaneously from the primary tumor. Also, the progressive spread of 4T1 metastases to the draining lymph nodes and other organs including lymph nodes, blood, liver, lung, brain, and bone, is very similar to that of human mammary cancer. Among these, the lungs are highly targeted, which is also evident from ex-vivo imaging that we observed metastatic nodules (Figure 8). Considering the difficulties in delivering a drug to the lungs, we hope this hybrid system could be instrumental in maximizing drug delivery to the lungs.

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.

Conclusion

The goal of the presented hybrid system, liposome and EV hybridization, is to investigate the combined benefits when engineered as a drug delivery NP, which otherwise lacks such functional identities when used independently. Mouse 4T1-derived EVs were successfully hybridized with synthetic liposomes resulting in the formation of monodispersed LEVs with narrow PDI. These LEVs were also labeled with fluorescent dye for their tracking in cells and live animals without altering their colloidal properties. As observed from live animal imaging, LEVs showed an enhanced tumor-targeting effect as compared to that of control liposomes. In addition, tissue imaging further revealed tumor accumulation of LEVs to a higher extent than that of control PEGylate liposome. The increased in vivo tumor distribution of LEVs as compared with control liposomes could presumably be due to the functional properties of EVs that preferentially communicate with their parent cells. This communication was further evident from the tissue imaging where we observed metastatic nodules in the lungs as 4T1 tumors spontaneously metastasize to the lungs. Collectively, these results signaled the potential of LEVs in tumor targeting that encourages further investigation on the direction of the proposed hybrid system where synthetic NPs can be re-engineered with different EV types for respective target applications.