Cellular Engineering with Membrane Fusogenic Liposomes to

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Cellular Engineering with Membrane Fusogenic Liposomes to Produce Functionalized Extracellular Vesicles Junsung Lee, Hyoungjin Lee, Unbyeol Goh, Jiyoung Kim, Moonkyoung Jeong, Jean Lee, and Ji-Ho Park ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b01315 • Publication Date (Web): 08 Mar 2016 Downloaded from http://pubs.acs.org on March 12, 2016

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ACS Applied Materials & Interfaces

Cellular Engineering with Membrane Fusogenic Liposomes to Produce Functionalized Extracellular Vesicles Junsung Lee,a,b,c,1 Hyoungjin Lee,a,c,1 Unbyeol Goh,a,c,1 Jiyoung Kim,a,c Moonkyoung Jeong,a,c Jean Lee,a,c and Ji-Ho Parka,b,c,d,*

a

Department of Bio and Brain Engineering, bGraduate School of Medical Science and

Engineering, cInstitute for Health Science and Technology, and dInstitute for the Nanocentury, Korea Advanced Institute of Science and Technology (KAIST), Daejeon, 34141, Republic of Korea.

1

These authors contributed equally to this work.

*Address correspondence to [email protected]

ACS Paragon Plus Environment

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ABSTRACT

Engineering of extracellular vesicles (EVs) without affecting biological functions remains a challenge, limiting the broad applications of EVs in biomedicine. Here, we report a method to equip EVs with various functional agents, including fluorophores, drugs, lipids, and bioorthogonal chemicals, in an efficient and controlled manner by engineering parental cells with membrane fusogenic liposomes, while keeping the EVs intact. As a demonstration of how this method can be applied, we prepared EVs containing azide-lipids, and conjugated them with targeting peptides using copper-free click chemistry to enhance targeting efficacy to cancer cells. We believe that this liposome-based cellular engineering method will find utility in studying the biological roles of EVs and delivering therapeutic agents through their innate pathway. .

KEYWORDS: cellular engineering, click chemistry, drug delivery, extracellular vesicle, liposome


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Extracellular vesicles (EVs), including exosomes and microvesicles, are secreted from most cell types and transferred to neighboring or distant cells.1,2 Many studies have reported that such EVmediated intercellular communications play a critical role in various pathophysiological processes, including those involved with the immune system and cancer.3-6 Recently, their endogenous origin and ability to deliver biological cargo to target cells have prompted efforts to engineer them for therapeutic applications.7-9 Using EVs for drug delivery presents some benefits in that they can recognize specific organs when derived from certain tumor cells,10-12 and can be immunologically inert when derived from immature dendritic cells.7 EVs also have the innate ability to package RNA species, which expands the spectrum of therapeutic agents to nucleic acid-based drugs such as mRNA and miRNA.4,10 Furthermore, there have been attempts to arm EVs with therapeutic agents and along with targeting ligands to enhance their delivery to target diseases.7,13 Likewise, development of a method to engineer EVs is in demand, and doing so without altering their endogenous properties is vital for their biomedical applications. Current methods for engineering EVs are divided into largely two approaches, loading exogenous agents directly to isolated EVs by co-incubation14,15 or electroporation7,13, and genetic engineering of parental cells to produce modified EVs.7,13,16 However, direct loading of isolated EVs could cause membrane damage, aggregation and low yield of EVs.9,17,18 In addition, this technique is restricted to certain types of cargo depending on which procedure is used, which limits membrane permeable or embeddable compounds to co-incubation, and hydrophilic molecules to electroporation.14,15 In the case of genetic engineering of parental cells, it requires modification of the EV-specific membrane proteins by fusion of functional proteins or peptides, which is likely to compromise the original physiological functions of membrane proteins in the EVs.7,13,19,20 Additionally, neither of these previous methods allows simultaneous loading of


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multiple compounds with disparate characteristics. In order to address these limitations, we here report a liposome-based cellular engineering method that enables EVs to be functionalized in an efficient and controlled manner by engineering parental cells via membrane fusogenic liposomes (Figure 1a). We demonstrate that EVs can be equipped with various functional agents, including fluorophores, drugs, lipids, and bio-orthogonal chemicals using this method without modification of the native proteins and lipids. Furthermore, we show that clickable EVs can be prepared by incorporating azide-lipid conjugates through liposome-based cellular engineering, and be utilized to provide specific targeting capability to cancer cells by conjugation with targeting ligands using copper-free click chemistry. EVs are known to be formed by the encapsulation of the cell’s cytosolic contents with its membrane. To leverage this mechanism of EV biogenesis for efficient loading of functional agents, we used membrane fusogenic liposomes (MFLs) to deliver lipophilic and hydrophilic agents into the cellular membrane and cytosol, respectively, by fusion of the liposomal and plasma membranes (Figure 1a). Based on previous reports,21 various liposomal formulations were tested for membrane fusogenicity and cargo loading efficiency into EVs. Among them, the L2 formulation showed the most efficient packaging of lipophilic cargo in EVs (Figures S1 and S2), and was thus selected as the MFL to be used for all subsequent experiments. Highly cationic liposomes (referred to here as non-fusogenic liposomes, NFLs) that enter cells via endocytosis, the conventional pathway of nanoparticle uptake, were prepared alongside MFLs for comparison (Table 1 and Figure S3).


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First, we evaluated the subcellular distribution of lipophilic and hydrophilic cargoes concurrently delivered via synthetic liposomes and their subsequent encapsulation into EVs. Cancer cells were treated with liposomes co-loaded with lipophilic dye, DiD and hydrophilic dye, carboxyfluorescein (CF) for 30 min. Confocal microscopy revealed that MFLs efficiently delivered DiD to the plasma membrane and CF into the cytosol, while NFLs transferred both cargoes into subcellular compartments, presumably early endosomes (Figure 1b). Two days after liposome treatment, EVs secreted from the cells were purified from the culture supernatant using an established ultracentrifugation protocol for EV isolation. MFL treatment to the parental cells allowed more efficient packaging of both DiD and CF into the secreted EVs, compared to treatments of NFL and their free form (Figure 1c). These results suggest that lipophilic and hydrophilic compounds can be co-packaged efficiently into the secreted EVs when delivered to parental cells via MFLs to avoid endosomal entrapment and lysosomal degradation. Hypothesizing that the phospholipids could also be delivered to the plasma membrane via MFLs and subsequently loaded into the membrane of EVs, we selected five fluorescent phospholipids of various lengths and saturations of hydrocarbon chains,22 each containing a primary amine group for further conjugation with functional cargo (Figure S4), and tested to observe which one showed the most efficient translocation into the EVs after delivering to parental cells via MFLs. Cancer cells were treated with liposomes containing fluorescent lipids for 30 min. Two days after liposome treatment, EVs secreted from the cells were isolated and their fluorescence was measured. Among the five lipids, we found a significantly marked enrichment of diphytanoylphosphatidylethanolamine (DPhPE) in the EVs compared to other lipids (Figure 1d), likely due to its high chemical stability in the cellular membrane.23 The DPhPE lipid was thus used for all subsequent experiments to incorporate functional lipids into


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the EVs. Similarly to the lipophilic agents, MFLs delivered fluorescent lipids selectively to plasma membranes (Figure 1e). In addition, MFL treatment exhibited more efficient incorporation of the lipids into the secreted EVs, compared to treatments of NFL and the lipids themselves (Figure 1f). The amount of lipids incorporated in the membrane of EVs was not proportional to that of intracellular lipids delivered by MFLs (Figure S5), implying that cells control the dose of intracellular packaging of cargo in the EVs. Cell engineering with synthetic liposomes to produce functionalized EVs did not influence the production, physicochemical and biological properties of EVs, or viability of parental cells (Figures S6-8). Next, to expand the scope of agents that can be attached to EVs, we produced versatile EVs that can be customized with any desired functional agent without modifying the membrane proteins by using copper-free click chemistry (Figure 2a). This bioorthogonal chemistry is based on the rapid and selective reaction of dibenzocyclooctyne (DBCO) groups with azides in physiological temperature and pH ranges.24 Confocal microscopy revealed that cells treated with MFLs containing azide-lipids (azide-MFLs) followed by treatment of DBCO-carboxyrhodamine (DBCO-CR) showed CR fluorescence predominantly on the plasma membrane, indicating efficient incorporation of azide-lipids into the plasma membrane (Figure 2b). Furthermore, the EVs produced from the cells treated with azide-MFLs showed significantly marked fluorescence when reacted with DBCO-CR directly (Figure 2c). These results illustrate that the EVs containing azide-lipids prepared through liposome-based cellular engineering could be decorated easily with various functional moieties by using copper-free click chemistry. We next tested whether EVs can be packaged efficiently with chemotherapeutics through liposome-based cellular engineering and further delivered to target cells according to their innate transport mechanism. Paclitaxel (PTX), a hydrophobic anti-cancer drug, and tirapazamine (TPZ),


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a hydrophilic agent that becomes cytotoxic selectively in hypoxic conditions, were chosen as therapeutic materials. Transwell experiments were performed to evaluate therapeutic effects of drug-loaded EVs (Figure 3a). Cancer cells were plated on the transwell filter and treated with drug-loaded liposomes for 30 min. The treated cells on the transwell filter were then coincubated with the lower chamber onto which fresh cancer cells were plated, and incubated for 48 h to allow production of drug-loaded EVs and their subsequent transfer to the cells in the lower chamber. The cell viability in both the upper filter and lower chamber was then evaluated using MTT assay. In both the TPZ and PTX treatments, the cells in the upper filter exhibited similar levels of viability regardless of liposome and cell type, whereas the cells in the lower chamber displayed significantly lower viability when co-incubated with the MFL-treated cells in the upper filter (Figures 3b and 3c). The B16F10 cells in the lower chamber exhibited higher viability than the MDA-MB-231 cells after MFL treatment regardless of the type of therapeutic agent. TPZ treatment in normoxic conditions did not induce any significant cytotoxicity (Figure S9). In addition, the EV-depleted supernatant did not induce any significant cytotoxicity toward the cells in the lower chamber (Figure S10), indicating that the EVs carry the therapeutic cargo to the cells. Collectively, these results suggest that selective delivery of therapeutic cargoes to each subcellular compartment (TPZ to cytosol and PTX to plasma membrane) via MFLs can lead to their efficient incorporation into the secreted EVs and the resulting EVs can be utilized for EV-mediated drug delivery. Delivery efficiencies of the cargo via EVs can vary depending on the type of cells that produce and receive the EVs. Thus, surface functionalization of EVs with targeting ligands can often help their selective delivery to target cells. In order to facilitate EV-mediated delivery particularly to cancer cells, regardless of the type of parental cells, we utilized the azide-EVs


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produced from the cells treated with azide-MFLs for bio-orthogonal reaction of the tumortargeting peptide conjugate, DBCO-CGKRK,25 onto them. We tested this targeted delivery of EVs with B16F10 cells in which relatively less efficient intercellular delivery of EVs was observed (Figures 3b and 3c). In a transwell system, B16F10-derived EVs were functionalized simultaneously with DiD and azide-lipids by engineering parental cells in the upper filter with MFLs co-loaded with the same materials (Figure 4a). These EVs produced from the cells in the upper filter were then conjugated in situ with DBCO-CGKRK pre-incubated in the lower chamber, and subsequently introduced to fresh B16F10 cells in the lower chamber. As expected, a higher quantity of the EVs conjugated with CGKRK through click chemistry was found in the lower chamber cells compared to other EVs (Figures 4b and S11). Likewise, therapeutic efficacy of B16F10-derived EVs loaded with PTX was also significantly enhanced after bioorthogonal conjugation of targeting ligands to the EV surface (Figure 4c). Taken together, these experiments demonstrated that our method produces versatile EVs that can simultaneously be loaded with various therapeutic cargoes in their interior and modified with numerous functional moieties on their exterior. Surface modification of EVs with exogenously incorporated lipids does not require alteration of the membrane proteins that primarily determine their biological functions, which sets this method apart from the previous approaches in which the targeting peptides are fused or conjugated to the exosomal membrane proteins.7,13 In this work, we reported a liposome-based cellular engineering method to equip EVs with an arsenal of functional agents, which was inspired by the process of the biogenesis of EVs. This method allowed us to engineer EVs with single or multiple functional agents in an efficient and controlled manner without compromising their native transmembrane proteins and lipids. We particularly prepared clickable EVs using this method and decorated them with targeting


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moieties through bio-orthogonal chemistry, in order to allow their specific delivery to cancer cells. This method has advantages over loading exogenous agents directly to isolated EVs by coincubation or electroporation, and genetic engineering of parental cells to produce modified EVs; it minimizes the aggregation and damage to EVs that might occur during the previously mentioned methods for EV engineering. The perspective of this method would be that it allows the potential use of self-originated EVs in situ by transforming the EVs within the body by delivering functional agents to the parental cells via synthetic liposomes. Therefore, it would eliminate the need for isolation of EVs from specific types of cells. We believe that this liposome-based cellular engineering method has great potential to help utilize EVs as selforiginated drug carriers for targeted therapy.

ASSOCIATED CONTENT Supporting Information. This material is available free of charge via the Internet at http://pubs.acs.org. Figure S1-S11; Membrane fusogenicity of liposome formulations composed with DMPC, PEGPE and DOTAP and their incorporation efficiency of cargo in extracellular vesicles (EVs); Membrane fusogenicity of liposome formulations composed with different base lipids and their incorporation efficiency of cargo in EVs; Membrane fusogenicity of MFLs and NFLs on cancer cells; Types and chemical structures of fluorescent phospholipids used in this study to engineer EVs; Fluorescence quantification of NBD-DPhPE lipids delivered to the cells and subsequently packaged into the EVs after MFL treatments at various concentrations; Cell viability of MDAMB-231 and B16F10 after incubation with synthetic liposomes for 30 min at various lipid


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concentrations; Amount of functional EVs produced from the cells treated with synthetic liposomes loaded with various functional agents; Physicochemical and biological properties of functional EVs produced by engineering parental cells with synthetic liposomes loaded with functional agents; Therapeutic effects of tirapazamine(TPZ)-loaded EVs in the normoxic condition that are produced from the upper filter cells treated with TPZ-loaded liposomes; EVmediated delivery of drugs; Fluorescent microscopic images of lower chamber cells treated with EVs that were produced from the upper filter cells treated with various DiD-loaded MFLs followed by in situ click conjugation with targeting peptide DBCO-CGKRK (PDF)

AUTHOR INFORMATION Corresponding Author *E-mail: [email protected] Notes The authors declare no competing financial interest.

ACKNOWLEDGMENTS This work was supported by the Basic Science Research Program (Grant No. NRF2015R1A1A1A05001420 and NRF-2015R1A2A2A04005760), and the Global Frontier Project (Grant No. NRF-2015M3A6A4045544) through the National Research Foundation funded by the Ministry of Science, ICT & Future Planning, Republic of Korea.


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Figure 1 Liposome-based cellular engineering for efficient intracellular packaging of cargo in extracellular vesicles. (a) Schematic showing a liposome-based cellular engineering method to produce functional extracellular vesicles (EVs) by engineering parental cells with membrane fusogenic liposomes loaded with functional hydrophilic or/and lipophilic agents. (b) Confocal microscopic images of cancer cells treated with synthetic liposomes co-loaded with hydrophilic cargo carboxyfluorescein (CF, green) and lipophilic cargo DiD (red). (c) Incorporation efficiency of cargo in EVs produced from the cells treated with cargo-loaded liposomes or free cargo. (d) Incorporation efficiency of lipids in EVs produced from the cells treated with MFLs containing various fluorescent lipids. (e) Confocal microscopic images of cells treated with synthetic liposomes containing fluorescent NBD-DPhPE lipids (green). (f) Incorporation efficiency of NBD-DPhPE lipids in the EVs produced from the cells treated with lipid-loaded liposomes or free lipids. MFL and NFL denote membrane fusogenic liposomes and non-fusogenic liposomes, respectively. Nuclei were stained with Hoechst (blue). Scale bar indicates 5 µm. Data represent averages ± S.D. [n = 4; NS, not significant, *P < 0.05, ** P < 0.01, and ***P < 0.001 by twoway ANOVA followed by Bonferroni post test.].


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Figure 2 Liposome-based cellular engineering to produce versatile EVs for customized surface functionalization. (a) Schematic showing a liposome-based cellular engineering method to produce versatile EVs that can be decorated with various functional agents (X) using copper-free click chemistry by engineering parental cells with MFLs containing azide-lipids. (b) Confocal microscopic images of cells treated with MFLs containing azide-lipids (azide-MFLs), followed by treatment with DBCO-carboxyrhodamine 110 (DBCO-CR, green). (c) Fluorescence quantification of CR-EVs in equal quantities of EVs treated with DBCO-CR after collecting EVs secreted from the cells with MFLs containing azide-lipids. Nuclei were stained with Hoechst (blue). Scale bar indicates 5 µm. Data represent averages ± S.D. [n = 4; ***P < 0.001 by unpaired t-test].


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Figure 3 Therapeutic effects of EVs produced by liposome-based engineering of parental cells. (a) Schematic showing a transwell system to directly treat fresh cells in the lower chamber (L) with EVs produced from cells engineered with cargo-loaded MFLs in the upper filter (U). (b) Therapeutic effects of TPZ-loaded EVs produced from the upper filter cells treated with TPZloaded liposomes. (c) Therapeutic effects of PTX-loaded EVs in the hypoxic condition produced from the upper filter cells with PTX-loaded liposomes. TPZ and PTX denote tirapazamine and paclitaxel, respectively. Data represent averages ± S.D. [n = 5-6; NS, not significant, *P < 0.05, **P < 0.01, and ***P < 0.001 by two-way ANOVA].


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Figure 4 Targeted therapeutic effects of EVs functionalized by liposome-based engineering of parental cells and subsequent click conjugation with targeting peptides. (a) Schematic showing a transwell system to treat fresh cells in the lower chamber (L) with EVs produced from cells engineered with azide-lipid-incorporated MFLs in the upper filter (U) followed by in situ conjugated with targeting peptide DBCO-CGKRK (D-CGKRK) using copper-free click chemistry. (b) Fluorescent quantification of lower chamber cells treated with EVs that were produced from the upper filter cells treated with various DiD-loaded MFLs followed by in situ click conjugation with D-CGKRK. (c) Therapeutic effects of PTX-loaded EVs that were produced from the upper filter cells treated with various PTX-loaded MFLs followed by conjugation with the D-CGKRK. Data represent averages ± S.D. [n = 5-6; **P < 0.01 by oneway ANOVA].


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Table 1 Lipid compositions and physical properties of synthetic liposomes used in this study. Lipid composition (molar ratio) Nanoparticlea

Hydrodynamic sizeb (nm)

Surface chargec (mV)

DMPC

PEG-PE

DOTAP

MFL

76.15

3.85

20

115.7

17.5

NFL

80

0

20

120.4

44.8

a

MFL and NFL denote membrane fusogenic liposomes and non-fusogenic liposomes, respectively.

b

Mean hydrodynamic sizes of the liposomes based on dynamic light scattering measurements (n = 3).

c

Mean surface charges of the liposomes based on zeta-potential measurements (n = 3).


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Table of Contents Graphic


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Cellular Engineering with Membrane Fusogenic Liposomes to

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