EXOPLEXs: Chimeric Drug Delivery Platform from the Fusion of Cell

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EXOPLEXs: Chimeric Drug Delivery Platform from the fusion of Cell Derived Nanovesicles and Liposomes Wei Jiang Goh, Shui Zou, Choon Keong Lee, Yi-Hsuan Ou, Jiong-Wei Wang, Bertrand Czarny, and Giorgia Pastorin Biomacromolecules, Just Accepted Manuscript • Publication Date (Web): 27 Nov 2017 Downloaded from http://pubs.acs.org on November 27, 2017

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EXOPLEXs: Chimeric Drug Delivery Platform from the fusion of Cell Derived Nanovesicles and Liposomes Wei Jiang Goh

a, b

, Shui Zou b, Choon Keong Lee b, Yi-Hsuan Ou b, Jiong-Wei Wang

c ,d

,

Bertrand Czarny b, † and Giorgia Pastorin a, b, e *

a

NUS Graduate School for Integrative Sciences and Engineering, Centre for Life Sciences (CeLS), 28 Medical Drive, #05-01, Singapore 117456

b

Department of Pharmacy, National University of Singapore, Science Drive 2, S15#05, Singapore 117543

c

Department of Surgery, Yong Loo Lin School of Medicine, National University of Singapore, Singapore

d

Cardiovascular Research Institute (CVRI), National University Heart Centre Singapore (NUHCS) and National University Health System (NUHS)

e

NUSNNI-NanoCore, National University of Singapore, T-Lab Level 11, 5A Engineering Drive 1, Singapore 117580

KEYWORDS: Cell Derived Nanovesicles; Liposomes; EXOPLEXs; Hybrid Biosystems

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ABSTRACT Cell Derived Nanovesicles (CDNs) have been recently investigated as novel Drug Delivery Systems (DDS), due to the preservation of key features from the cell membrane of their precursor cells, which are responsible for an efficient cellular uptake by target cells. However, CDNs suffer from low drug loading efficiencies as well as challenges in functionalization compared to conventional DDS like liposomes. Here, we describe the first study proposing the fusion of CDNs with liposomes to form EXOPLEXs. We report the preservation of cell membranes from precursor cells similarly to CDNs, as well as high loading efficiencies of more than 65% with doxorubicin hydrochloride, a model chemotherapeutic drug. The doxorubicin loaded EXOPLEXs (DOX-EXO) also demonstrated a higher in vitro cell killing effect than liposomes, while EXOPLEXs alone did not show any remarkable cytotoxicity. Taken together, these results illustrate the potential of EXOPLEXs as a novel DDS for targeted delivery of chemotherapeutics.

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INTRODUCTION Drug delivery systems (DDS) aim to improve drug targeting to the desired site with minimum off-target effects1. Liposomes have been used as DDS to a large extent, being able to encapsulate several chemotherapeutics with better pharmacological profiles than the corresponding free drugs. A few of these liposomal formulations (e.g. DOXIL® , which consists of a long-circulating liposomal preparation containing doxorubicin and MYOCET®, its non-PEGylated form) have been approved by regulatory agencies and they are currently used clinically2. Liposomes have been studied extensively, and methods of characterization and drug loading have been well established3. Notably, high drug encapsulation efficiencies have been reported using liposomes4.

Despite the merits of liposomes as DDS, their use is not without limitations. The accumulation of liposomal formulation at the tumor site as a result of the Enhanced Permeability and Retention (EPR) effect is due to nanovesicles’ nano-metric dimensions and prolonged circulation in the bloodstream (in the case of “stealth” liposomes), allowing the liposomes to transverse the leaky vasculature over the time and deposit at the tumor site5. However, liposomes do not target the cancerous tissue per se, while the leakage of drug at the site can still lead to collateral damage of otherwise healthy tissue due to the lack of specificity. This, in turn, could be associated with limited cellular uptake inside cancer cells and concomitant side effects6. Efforts aimed at functionalizing liposomes’ surface with enzymes and/or proteins overexpressed in cancer to provide active targeting often incur complicated processing steps. Furthermore, lipid-based nanocarriers have been shown to suffer from increased reticuloendothelial system (RES) clearance due to the opsonization of complement proteins as a result of the hydrophilic surfaces

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of liposomes 7. In addition, the synthetic nature of liposomes may even result in the Complement Activation-Related Pseudo Allergies (CARPA) in certain patient populations8.

Cell Derived Nanovesicles (CDNs), on the other hand, are being increasingly investigated in a nascent field of drug delivery9-11. CDNs can be produced in a variety of methods, such as passage through microchannels10,

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or by a centrifugation-shearing approach, to produce

nano-sized vesicles from precursor cells in a cost-effective manner10. The key advantage of CDNs over liposomes is the preservation of the key components (e.g. proteins, lipids etc.) of the original parent cells, which are postulated to confer improved cellular uptake and reduced risk of immunogenicity in a similar fashion as endogenously produces exosomes12. In this study, CDNs were derived from monocytes, as they have shown to possess intrinsic targeting towards inflammation such as in tumors13.

Nonetheless, CDNs suffer from low drug loading efficiencies due to technical challenges in loading of vesicles of nano dimensions without substantially denaturing the proteins on their surface10. In addition, it is also challenging to functionalize the surface of CDNs in order to confer additional properties such as theranostic capabilities, additional targeting impetus or to further extend their in vivo circulation as compared to using functionalized lipids in the production of multi-functional liposomes14.

EXOPLEXs, the hybrid DDS from the fusion of CDNs and liposomes, are expected to overcome the limitations of both formulation systems. This proposed hybrid DDS is currently unprecedented in the literature. EXOPLEXs are postulated to possess key advantages including high drug loading, amendable for surface functionalization, capable of exploiting the EPR effect 4


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and also low immunogenicity. As a result of their high drug loading properties resulting from the liposomal component and the improved cellular uptake profile derived from the CDNs component, EXOPLEXs are anticipated to be administered in a clinical setting with a lower concentration of chemotherapeutic required in comparison to currently used liposomes (as they are more efficiently up taken by cells), with intrinsic targeting effects (as they preserve key surface markers from parent cells) and also with minimized risks of allergic reactions (as they derive from the patient’s own cells and thus are expected to be non-immunogenic). Once these results are confirmed, the still-enigmatic advantage of using nanomedicine over conventional treatments will become clear.

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EXPERIMENTAL METHODS Materials

U937 monocytes were a kind gift from Associate Professor Gigi Chiu, National University of Singapore (NUS). Spin columns were purchased from ThermoScientific, and were supplied with 10µm filters attached while hydrophilic 8 µm and 5 µm membrane filters were purchased from Merck Millipore and used as supplied. ThermoScientific microcentrifuge was used in the cell disruption process for the production of Cell Derived Nanovesicles (CDNs) from U937 monocytes.

Doxorubicin

hydrochloride

Dipalmitoylphosphatidylcholine

was

purchased

(DPPC),

from

Sigma

Aldrich.

1-oleoyl-2-[12-biotinyl

(aminododecanoyl)]-sn-glycero-3-phosphocholine (Biotinylated lipid) and cholesterol were purchased from Avanti lipids. Streptavidin-coated 96-well plates were purchased from ThermoScientific and used as supplied.

Cell Culture U937 cells were grown in RPMI 1640 culture medium supplemented with 10% FBS. Cells were grown to 80% confluence, before removing 2 x 107 cells, centrifuging and suspending in Phosphate Buffered Saline (PBS) twice. This cell density represents a standard batch of CDNs. These cells were subsequently used for CDNs production.

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Cell Derived Nanovesicles (CDNs) Production CDNs were produced as described in an earlier publication

10

. Briefly, a serial extrusion

combined with shear force method was adopted by fitting spin columns with 10 µm and 8 µm filter membranes and passing the cells through the membrane filters sequentially using a microcentrifuge. The cell suspension was centrifuged at 14,000G x 10 minutes in spin columns fitted with 10µm membrane filters, followed by 8µm membrane filters twice each. The subsequent dispersion was further purified using a Sephadex G50 size exclusion column equilibrated with 250 mM ammonium phosphate, and the CDNs fractions of sizes between 100 to 200 nm were collected. CDNs were quantified by protein concentration using a standard BCA protein assay kit and normalized to 500 µg/ml for subsequent use.

Liposomes production Liposomes were produced using the thin film hydration method 3. 12.5 mg of lipids, consisting of 95 mol% DPPC and 5 mol% Cholesterol, were dissolved in 1 ml of chloroform. The solvent was then evaporated to form a dry film. The lipid thin film was hydrated with 1 ml of 250 mM ammonium phosphate for 1 hour at 45°C. The lipid dispersion was sized down by passing it through an Avestin LiposoFast-BasicTM extruder fitted with two polycarbonate 100 nm membrane filters. A buffer exchange step was then performed by passing the liposome suspension through a G50 sephadex column previously equilibrated with PBS to obtain 2 ml of liposomes fraction, using PBS (pH 7.4) as the eluting buffer. 7



EXOPLEXs Production EXOPLEXs were produced by the fusion of CDNs and liposomes. A lipid thin film was produced as described for the production of liposomes. 1 ml of CDNs containing 500 µg/ml protein concentration was used to hydrate the lipid film. The CDNs encapsulated liposomes were fused by extruding through an Avestin LiposoFast-BasicTM extruder fitted with two polycarbonate 100 nm membrane filters. A buffer exchange step was then performed by passing the liposome suspension through a G50 sephadex column previously equilibrated with PBS to obtain 2 ml of EXOPLEXs fraction, using PBS (pH 7.4) as the eluting buffer. EXOPLEXs produced from 250 µg/ml, 500 µg/ml and 1,000 µg/ml protein concentration were labelled correspondingly as EXO 0.5, EXO 1 and EXO 2.

Characterization of CDNs, Liposomes and EXOPLEXs Hydrodynamic diameters and zeta potential of CDNs, liposomes and EXOPLEXs were assayed by Dynamic Light Scattering (DLS) using the Zetasizer Nano (Malvern Instruments). Protein concentration was determined using a standard protein assay kit (Pierce BCA and Pierce microBCA kit).

For cryo-TEM imaging, lacey Formvar/Carbon, 300 mesh Copper grids (TED PELLA INC., USA) were loaded with 30 µl of sample. After a blotting time of 2 seconds, TEM grids were subjected to plunge-freezing into liquid ethane using Gatan CP3 Cryo-plunger 3 system (Gatan, 8


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Pleasanton, CA). Cryo-grids were then transferred in liquid nitrogen and imaged with a Carl Zeiss TEM, LIBRA® 120 PLUS, provided with advanced Koehler illumination system and operated with accelerating voltage of 120KeV15. Images were recorded at a magnification between 80,000-10,000x.

Protein marker characterization of CDNs and EXOPLEXs CDNs and EXOPLEXs were assayed for characteristic protein markers that are commonly associated with U937 monocytes. We aimed to demonstrate that these key protein markers on U937 cells were preserved through the CDN production process, and further retained on the surface of EXOPLEXs. These include but are not limited to the Tetraspanins (CD9 & CD63) and multivesicular body protein markers (ALIX and TSG101). The protein markers were assayed using flow cytometry. Briefly, latex beads purchased from Sigma Aldrich (3.2 µm in size) were diluted in PBS. 100 µl of diluted latex beads were added to 200 µl of sample to be assayed. Samples were normalized in relation to protein concentration. The samples and beads were allowed to mix for 2 hours at room temperature, before adding 500 µl of 100 mM glycine. The beads were then centrifuged at 3,000G x 10 minutes and suspended in 1 ml of PBS twice to remove non-adsorbed samples. In addition, extensive washing cycles (3 times) by centrifugation and suspension in PBS mitigated the possibility of adsorption of liposomes onto CDNs that were adsorbed onto the beads in an event of a co-mixture. The primary antibody was added (1:500 dilution) and incubated on ice for 1 hour. The beads were again centrifuged at 3,000G for 10 9



minutes and suspended in PBS. Secondary antibodies conjugated with fluorescent probes were added (1:500) and incubated on ice for 1 hour. The beads were again centrifuged at 3,000G for 10 minutes and suspended in PBS. The beads were then analyzed via flow cytometry at the respective excitation and emission wavelengths (events capped at 50,000).

Proof -of-Fusion Assay EXOPLEXs and liposomes were biotinylated by addition of 0.6 mg of biotin to the lipid film during the respective production. The Proof-of-Fusion assay was adapted from the standard Enzyme Linked ImmunoSorbent Assay (ELISA) method. Briefly, 200 µl of 5% w/v skim milk in PBS was first added to each well in the streptavidin-coated 96 well plate as blocking agent. The plate was then put on a shaker for 1 hour at room temperature before washing with 300 µl of PBST (0.05% v/v PBS-Tween 20) thrice. 100 µl of the samples (Liposomes, extruded EXO 2, un-extruded EXO 2, and CDNs) were then added to each well and incubated on a shaker at room temperature for 2 hours before washing with PBST thrice. 100 µl of the primary antibody (Alix or CD63 in 0.5% w/v skim milk) was then added and placed on a shaker for other 2 hours before washing with PBST for three times. Finally, 100 µl of secondary antibody (Alexa Fluor 568 in 0.5% w/v skim milk) were added and placed on a shaker for 2 hours before washing with PBST thrice. The fluorescence readings were then obtained using a microplate plate reader at λex 578 nm and λem 603 nm, respectively.

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Doxorubicin loading of liposomes and EXOPLEXs Loading of doxorubicin into liposomes and EXOPLEXs was done using the remote loading method. Briefly, 1 ml of either liposomes or EXOPLEXs was added to an equal volume of 200 µg/ml doxorubicin and incubated at 37°C for 1 hour with constant stirring at 300 rpm. The unencapsulated doxorubicin was subsequently removed by passing the sample through a Sephadex G50 column equilibrated with PBS. Doxorubicin encapsulation efficiency was determined by measuring the absorbance at 480 nm and compared against a doxorubicin standard curve. Briefly, the samples were lyzed by addition of 1% Triton X (1% v/v in PBS) and centrifuged for 10 minutes at 15,000G. Absorbance of the supernatant was taken at 480 nm and compared against a doxorubicin concentration curve to determine its concentration.

Doxorubicin release studies Doxorubicin release from liposomes and EXOPLEXs was evaluated by measuring the drug release from a dialysis tubing (3500 MWCO Thermoscientific SnakeSkin™) at 37°C. All doxorubicin-loaded samples were first normalized to a doxorubicin concentration of 64 µg/ml using PBS. 1 ml of each formulation was dialyzed against 2 liters of PBS over 24 hours, with a change of dialysis buffer at 2 hours. At various time points (1 hour, 2 hours, 4 hours, 8 hours and 24 hours), 50 µl aliquots were withdrawn from the tube and quantified spectrophotometrically at λex 470 nm and λem 590 nm, respectively. Percentage of drug release at each time point was recorded and the results were plotted as percentage of drug released versus time. 11



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In Vitro Analysis of Cellular uptake 1 x 105 HeLa cells were added to a glass bottom petri dish and grown overnight at 37°C. 50 µl of free doxorubicin, doxorubicin loaded liposomes (Dox-Lip) and EXOPLEXs (Dox-Exo) were added to each dish and incubated for 1 and 6 hours, respectively. Samples were normalized to 64 µg/ml doxorubicin each. Hoechst 33342 was added as per manufacturer instructions for live cell visualization. HeLa cells with no treatment were used as control. Cells were subsequently washed twice with PBS, trypsinized and suspended in 500 µl of PBS, before analyzing using the BD LSR Fortessa Flow Cytometry Analyser. Excitation and emission wavelengths used for Hoechst 33342 and doxorubicin were 350 nm & 461 nm, and 470 nm & 590 nm, respectively.

MTT Cell Viability assay Cell viability of HeLa cells on exposure to free doxorubicin, doxorubicin loaded liposomes and EXOPLEXs

were

investigated

using

a

standard

3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay over 48 hours. Doxorubicin loaded samples were normalized to 64 µg/ml of doxorubicin. HeLa cells were seeded at a cell density of 1x104 cells/well and incubated overnight before addition of 25 µl of samples with 5 dilutions using PBS. After 48 hours incubation at 37°C, 20 µl of MTT reagent (2.5 mg/ml) was added to each well and incubated for 2 hours. The expanded medium was aspirated and washed thrice gently with PBS, before adding 100 µl of DMSO. Absorbance 12


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readings at 570 nm were recorded. HeLa cells with addition of 25 µl of sterile PBS were used as control. Triplicates were performed. Confocal Microscopy 1 x 105 HeLa cells were added to a glass bottom petri dish and grown overnight at 37°C. 50 µl of free doxorubicin, doxorubicin loaded liposomes and EXOPLEXs were added to each dish and incubated for 6 hours. Samples were normalized to 64 µg/ml doxorubicin each. Hoechst 33342 was added as per manufacturer’s instructions for live cell visualization. The cells were fixed by addition of 4% paraformaldehyde and incubating for 20 minutes at room temperature, before washing twice with sterile PBS. Imaging was performed on FluoView laser scanning confocal microscope. Excitation and emission wavelengths used for Hoechst 33342 and doxorubicin were 350 nm & 461 nm, and 470 nm & 590 nm, respectively. Images were processed by ImageJ software. Results are found in the supplementary section (Figure S1).

Statistical Analysis All statistical analyses were performed using IBM SPSS Version 21. Fluorescence intensities of fusion assays were compared using the one way ANOVA statistical test, with Bonferroni post hoc tests. P values less than 0.05 were considered to be significant.

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RESULTS Production of EXOPLEXs EXOPLEXs were prepared from the fusion of CDNs and liposomes. CDNs were prepared as described in an earlier publication10. A thin lipid film was produced, and an aqueous suspension of CDNs was used to hydrate the lipid thin film, thus encapsulating the CDNs within the so-formed giant liposomes (Figure 1).

Figure 1. EXOPLEXs are produced from the fusion of liposomes and cell-derived nanovesicles. Giant multi-lamellar liposomes were loaded with cell-derived nanovesicles, before being extruded through two 100 nm membranes. The reduction in size forces the mixing of membranes of the liposomes and nanovesicles, producing EXOPLEXs. EXOPLEXs retain the protein surface configuration of cell-derived nanovesicles, playing an important role in cellular uptake, while being amendable to changes in therapeutic cargo and further surface modifications from fusion with various types of lipids.

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Giant multi-lamellar liposomes, encapsulating CDNs, were then extruded through two 100 nm membrane filters, causing a physical size reduction and forcing the mixing of the lipid bilayer and the CDNs’ membranes. As a result, the lipid bilayer of the liposomes and the membrane layer of the CDNs are now fused. Drug loading could be achieved either by adding the drug together with the CDNs at the hydration step, or by remotely load it into the formed EXOPLEXs. The drug loaded EXOPLEXs could then be used for targeted drug delivery.

Characterization of EXOPLEXs

The hydrodynamic diameter, polydispersity index (PDI) and zeta potential of EXOPLEXs, as well as the precursor CDNs from U937 monocytes and control liposomes, were characterized as reported in Table 1. The size of EXOPLEXs was found to be similar to the precursor CDNs, suggesting that fusion between the CDNs encapsulated within the liposomes and liposomes themselves took place and also exhibited the same morphology (Figure S2). In addition, it was observed that the PDI for EXOPLEXs was lower than that of CDNs. A possible explanation is that the addition of lipids resulted in the dilution of protein corona at the surface of EXOPLEXs, resulting in less aggregation due to protein-protein interactions, and hence a lower PDI.

In order to demonstrate the extent of fusion, increasing concentrations of CDNs, produced from 1 x 107, 2 x 107 and 4x 107 cells/ml of U937 monocytes, were used to the same lipid concentration, corresponding to EXO 0.5, EXO 1 and EXO 2, respectively (i.e. half (EXO 0.5) and double (EXO 2) the concentration of a typical batch of 2 x 107 cells/ml, which is EXO 1).

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Lastly, both EXOPLEXs and CDNs were found to be neutral in charge, namely between -5 and +5 mV. Liposomes were used as a control.

Table 1. Physical characterization of CDNs and EXOPLEXs.

A

B

C

CDNs (Processed from 1x 107 cells/ml) CDNs (Processed from 2x 107 cells/ml) CDNs (Processed from 4x 107 cells/ml)

Z-average (nm)

PDI

Zeta potential (mV)

185 ± 5.6

0.64

-4.88 ± 1.60

157 ± 4.3

0.89

-3.15 ± 0.37

154 ± 5.5

0.80

-3.42 ± 0.36

D

EXOPLEXs (EXO 0.5) (Derived from A)

222 ± 8.2

0.36

+2.24 ± 0.19

E

EXOPLEXs (EXO 1) (Derived from B)

182 ± 6.7

0.40

+0.75 ± 0.12

F

EXOPLEXs (EXO 2) (Derived from C)

206 ± 6.3

0.27

+1.51 ± 0.06

G

Liposomes

185 ± 5.5

0.22

+0.43 ± 0.53

We were also interested at investigating if the protein markers on the CDNs’ surface were preserved in the production of EXOPLEXs. Characteristic protein markers of CDNs, such as the Tetraspanins (CD9 & CD63) and multivesicular body (MVB) (ALIX & TSG101) were assayed by adsorbing CDNs and EXOPLEXs onto latex beads and detecting via flow cytometry with 16


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addition of a secondary antibody conjugated with a fluorescence tag (Figure 2). Empty beads were used as control.

All CDNs and EXOPLEXs assayed with the selected protein markers exhibited a shift to the right, compared to the control (which consists of beads with either CDNs or EXOPLEXs absorbed onto the surface but without addition of the primary antibodies). This confirmed the prescence of these protein markers in our EXOPLEX samples. The detection of these protein markers on the surface of EXOPLEXs suggests that fusion indeed took place, otherwise the protein markers would have remained trapped within the liposomes and been undetected. Close similarities in the protein marker profiles of CDNs and EXOPLEXs were also observed, further proving that the protein markers on the CDNs are preserved in the production of EXOPLEXs.

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Figure 2. Flow cytometry analysis of protein markers on EXOPLEXs, as compared to their constituent CDNs. Red regions indicate beads only, and the green and blue lines indicate CDNs and EXOPLEXs, respectively.

EXOPLEXs’ Proof-of-Fusion While the reduction in size and prescence of protein markers characteristic of CDNs found on EXOPLEXs suggest that fusion has taken place, clear discrimination between liposomes/CDNs mixture and EXOPLEXs should be established. In a proof-of-fusion assay, we adapted the Enzyme Linked ImmunoSorbent Assay (ELISA) procedure to demostrate that fusion between CDNs and liposomes occurred as expected (Figure 3A). One of the advantages of EXOPLEXs is the ability to add or change the lipids used in their production. In this assay, we added biotinylated lipids, producing biotinylated EXOPLEXs. EXOPLEXs from 4 x 107 cells/ml of precursor U937 cells were used. Biotinylated liposomes 18


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were produced as a control. In order to elucidate that the fusion is a result of the extrusion process, unextruded EXOPLEXs were included as a negative control.

Figure 3. Proof-of-fusion assay between CDNs and liposomes to form EXOPLEXs. (A) Schematic of adapted ELISA process. (B) Theorectical results should the fusion take place. (C) Relative fluorescence readings demostrating that fusion took place. *** indicated p< 0.001.

The samples were added to a streptavidin-coated 96 well plate. Samples that attached to the plate as a result of the streptavidin-biotin interaction would include both biotinylated liposomes and EXOPLEXs. However, only EXOPLEXs that exhibited a successful fusion would show a fluorecence signal when probed for the protein markers Alix and CD63, respectively (Figure 3B). This hypothesis was corroborated by the actual fluorescence readings that showed a distinct increase in fluorescence for EXOPLEXs as compared to the other samples (Figure 3C). More importantly, unextruded EXOPLEXs did not show elevated fluorescence readings, further 19



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indicating that the fusion is a result of the extrusion process. A similar experiment, but using streptavidin coated magnetic beads instead of streptavidin coated 96 well plates, was performed and yielded the same observation. (Figure S3)

Doxorubicin loading and release Doxorubicin hydrochloride, a chemotherapeutic drug, was selected in this study as a prototype of small molecule drug, commonly used in the clinic for the treatment of solid tumours in the form of Doxil® or Myocet® 16. In addition, doxorubicin is fluorescent, and can be tracked and quantified easily.

Doxorubicin was loaded into the liposomes and EXOPLEXs via remote loading, according to prior methods established in the literature for liposomes

17

. Interestingly, similar loading

efficiencies, between 65% and 75%, were measured when doxorubicin was loaded into liposomes and EXOPLEXs produced from varying precursor cell densities (Figure 4A). This suggests that the fusion process does not negatively impact the loading potential of EXOPLEXs.

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Figure 4. (A) Doxorubicin hydrochloride loading efficiencies of various formulations. Dox-Lip: doxorubicin loaded liposomes; Dox-EXO 0.5: Doxorubicin loaded EXOPLEXs, derived from 0.5x standard batch of CDNs, Dox-EXO 1: Doxorubicin loaded EXOPLEXs, derived from 1x standard batch of CDNs & Dox-EXO 2. Doxorubicin loaded EXOPLEXs, derived from 2x standard batch of CDNs. 1x standard batch of CDNs were prepared from 2 x 107 U937 cells. (B) Doxorubicin release profiles at 37°C in PBS for 24 hours.

In a similar manner, the release profiles of doxorubicin from EXOPLEX formulations were comparable to those of liposomes (Figure 4B), also indicating that the inclusion of cell membranes from the CDNs component did not impact the drug release profile. More than 80% of the loaded drug was released over 24 hours in sink conditions at 37°C.

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In Vitro Studies

Figure 5. Flow cytometry results of cellular uptake of doxorubicin at 1 hour and 6 hours timepoints. Hoechst 33342 stain was added for live cell visualization.

The doxorubicin loaded liposomes and EXOPLEXs were then added to HeLa cells, in order to study the cellular uptake properties of EXOPLEXs vis-à-vis liposomes, and whether the inclusion of protein markers from the CDNs onto the EXOPLEXs contributed to improved cellular uptake, and therefore better cell killing effect when loaded with doxorubicin.

We added the doxorubicin loaded liposomes (DOX-Lip) and EXOPLEXs (DOX-EXO) to the HeLa cells, and checked for the percentage of cells that internalized the nanovesicles at 1 hour

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and 6 hours timepoints using flow cytometry (Figure 5). Hoechst 33342 stain was added for live cell visualization.

At the 1 hour timepoint, it was observed that doxorubicin had entered almost 99.5% of HeLa cells, but it was noticeably lower at about 20% in both DOX-Lip and DOX-EXO samples. This suggests that the doxorubicin had been encapsulated within the nanoparticles, because any free doxorubicin absorbed onto the EXOPLEXs’ or CDNs’ surface would quickly enter the cells, giving a similar profile as free doxorubicin. At this time point, the cellular uptake of both DOX-Lip and DOX-EXO were similar, at about 20%. Cell killing effect was also at its minimum.

Interestingly, at the 6 hour timepoint, the difference between DOX-Lip and DOX-EXO nanovesicles became more apparent. About 6.5% of HeLa cells showed they had internalized the DOX-Lip and were non-viable, whereas DOX-EXO nanoparticles showed an increasing percentage of HeLa cells that had taken up doxorubicin and were non-viable, with the highest being DOX-EXO 2 at 39.4%. This indicates that the inclusion of cell membrane component from U937 in the production of EXOPLEXs contributed to the improved cellular uptake and therefore better cell killing effect of cancerous HeLa cells. The prescence of doxorubicin inside the HeLa cells was further confirmed using fluorscence microscopy (Figure S1).

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To further investigate the effect of doxorubicin-mediated cytotoxicity over a longer period of time, a MTT assay was performed over 48 hours on HeLa cells, using free doxorubicin as the positive control (Figure 6). Empty EXOPLEXs were first tested on HeLa cells, and found to be non-cytotoxic to cells in the concentration range tested (Figure S4).

Figure 6. MTT cell viability assay on HeLa cells over 48 hours (* and ** indicates p

EXOPLEXs: Chimeric Drug Delivery Platform from the Fusion of Cell

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