Membrane Fusion Mediated Intracellular Delivery of Lipid Bilayer Coated Mesoporous Silica Nanoparticles

Jian Yang, Jing Tu, Gerda E. M. Lamers, René C. L. Olsthoorn, and Alexander Kros*

DOI: 10.1002/adhm.201700759

mole cular weight biomole cules like pro- teins.

As a result, most studies show only low loading capacities and since the MSN pores are generally too small they offer only weak protection against pro- teases.

In addition, these inorganic nanoparticles are typically taken up into cells by endocytosis, which could be detri- mental to sensitive cargo.

Recently, we showed cytosolic delivery of low molecular weight dyes/drugs into cells via membrane fusion of liposomes with cell membranes induced by a com- plementary pair of coiled-coil (CC) lipo- peptides (denoted CP

K

and CP

E

, Scheme 1).

For this, these cholesterol–

poly(ethylene glycol)–peptide conjugates were inserted into the lipid bilayers (LBs) of both liposomes and live cells resulting in fusion between opposing membranes. Mechanistic studies revealed that coiled-coil formation between the complementary peptides E

/K

drives this process.

Furthermore, liposomes containing the anticancer drug doxorubicin could target and kill modified HeLa cells in a zebrafish xenograft model using the same approach.

In this contribution we studied whether coiled-coil mediated membrane fusion

could be used for the delivery of large inorganic nanoparticles like protein encap- sulated MSNs to cells (Figure 1a). Cytochrome-c (cytC) was chosen as a model protein as its cytosolic delivery activates the intrinsic apoptotic pathway. This allowed us to monitor the uptake of cytC loaded MSNs (MSNs/cytC) and the induction of apoptosis

as a measure of cytosolic activity of cytC.

2. Results and Discussions

2.1. cytC Encapsulation and Release Studies

For this study, a new type of MSNs featuring a cuboidal-like geometry (90 × 43 nm

) was synthesized (Figure 1b).

The nitrogen adsorption–desorption isotherms of MSNs exhib- ited the characteristic type IV isotherms

with a Brunauer–

Emmett–Teller (BET) surface area of 506 m

g

(Figure 1c).

The pore size distribution was found to be 10 ± 1 nm.

cytC (geometric size 2.6 × 3.2 × 3.3 nm

) is a small redox pro- tein that is present in the inner membrane of mitochondria and induces apoptosis when released into the cytosol.

The pore dimensions of MSNs are sufficiently large to accommodate cytC and the open disk-shaped mesostructure renders the encapsulation of cytC very efficient. Within less than 5 min more than 95% of cytochrome-c (cytC) was encapsulated into Protein delivery into the cytosol of cells is a challenging topic in the field of

nanomedicine, because cellular uptake and endosomal escape are typically inefficient, hampering clinical applications. In this contribution cuboidal mesoporous silica nanoparticles (MSNs) containing disk-shaped cavities with a large pore diameter (10 nm) are studied as a protein delivery vehicle using cytochrome-c (cytC) as a model membrane-impermeable protein. To ensure colloidal stability, the MSNs are coated with a fusogenic lipid bilayer (LB) and cellular uptake is induced by a complementary pair of coiled-coil (CC) lipo- peptides. Coiled-coil induced membrane fusion leads to the efficient cytosolic delivery of cytC and triggers apoptosis of cells. Delivery of these LB coated MSNs in the presence of various endocytosis inhibitors strongly suggests that membrane fusion is the dominant mechanism of cellular uptake. This method is potentially a universal way for the efficient delivery of any type of inorganic nanoparticle or protein into cells mediated by CC induced membrane fusion.

J. Yang, J. Tu, R. C. L. Olsthoorn, Prof. A. Kros

Department of Supramolecular & Biomaterials Chemistry Leiden Institute of Chemistry

Leiden University

Einsteinweg 55, Leiden 2300, RA, The Netherlands E-mail: a.kros@chem.leidenuniv.nl

G. E. M. Lamers Institute of Biology Leiden University

Sylviusweg 72, Leiden 2333, BE, The Netherlands

1. Introduction

Intracellular protein delivery holds promise for a range of biomedical applications,

such as cancer therapy,

vaccina- tion, and enzyme based therapeutics.

However, therapeutic proteins are susceptible to proteolysis and denaturation, limiting their efficacy in the body.

To solve the delivery problem, protein delivery systems based on for instance polymeric nanoparticles,

hydrogels,

or lipid-based nano- particles

have been developed. Also mesoporous silica nanoparticles (MSNs) have been studied as carriers for a variety of biomolecules including anticancer drugs, oligonucleo- tides, and proteins.

However, most MSNs used in drug delivery studies to date typically have pores with diameters up to 4 nm, thereby limiting their use as efficient carriers for high

© 2017 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA,

Weinheim. This is an open access article under the terms of the Creative

Commons Attribution-NonCommercial-NoDerivs License, which

permits use and distribution in any medium, provided the original work

is properly cited, the use is non-commercial and no modifications or

adaptations are made.

maximum cytC loading capacity of these cuboidal MSNs was determined to be 470 µg mg

MSNs (Figure 2a, black curve).

The zeta-potential of MSNs/cytC also changed by increasing the concentration of cytC (Figure 2a, red curve), reaching almost neutrality at saturating concentrations of cytC. At cytC:MSNs weight ratios of 1:8 or 1:4 the encapsulation efficiency (EE%) was quantitative, revealing the excellent protein encapsulation potential of these large-pore MSNs. Compared to native cytC, the encapsulated cytC in the MSNs revealed a slight broadening of the adsorption peak in the UV–Vis spectrum, but no blue shift was observed, suggesting that the interaction between the protein and pore surface did not change the structure of cytC (Figure S1a, Supporting Information).

Since cytC has a positive charge at pH 7.4 its adsorption is mainly driven by

groups on the surface of MSNs. Therefore, we studied the influence of ionic strength on the release profile of cytC. At high ionic strength (270 × 10

), 70.4% of cytC was released from MSNs/cytC which decreased to only 16.8% in low ionic strength (12 × 10

) buffer revealing the electrostatic nature of protein binding (Figure S1, Supporting Information). Thus, cytC can be loaded with high efficiency into MSNs at low ionic strength and subsequently released at conditions of high ionic strength (e.g., inside cells).

However, the decreased surface charge of MSNs upon cytC loading as evidenced by the near neutral zeta-potential induced aggregation of the nanoparticles. To increase their colloidal sta- bility MSNs/cytC were therefore decorated with a lipid bilayer.

After introduction of a fusogenic lipid bilayer composed of

Figure 1. Fusion between cells and lipid bilayer coated MSNs mediated by coiled-coil formation between CP

K

and CP

E

. a) CytC (orange) is encap-

sulated into MSNs (green) and coated with a lipid bilayer upon mixing with liposomes (light blue) which are decorated with lipopeptide CP

E

(red)

resulting in MSNs/cytC@CPE-LBs. Cells pretreated with CP

K

(dark blue) are mixed with MSNs/cytC@CPE-LBs resulting in membrane fusion and

concomitant delivery of cytC into the cytoplasm. b) TEM image of MSNs, scale bar = 200 nm. c) Nitrogen adsorption–desorption isotherms and pore

size distribution (inset).

1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dioleoyl- sn-glycero-3-phosphoethanolamine (DOPE), and cholesterol (2:1:1 molar ratio)

the observed hydrodynamic diameter by dynamic light scattering of the nanoparticles was ≈230 nm (polydispersity index = 0.251) (Figure 2b). After loading the MSNs with cytC, the zeta-potential shifted from −28.0 to

−11.6 mV. Application of the lipid bilayer onto the exterior sur- face of these particles resulted in a more negative zeta-potential (−20.5 mV) (Figure 2c). As a result the lipid coated nanoparti- cles were colloidal stable for at least one week. The presence of the lipid bilayer also reduced the premature release of cytC by a factor of ≈1.6 as the lipid bilayer acts as a physical bar- rier

thereby retaining the protein better within the MSNs (Figure 2d).

2.2. Intracellular Delivery of cytC

Previously, we reported that a pair of complementary lipo- peptides (i.e., CP

E

/CP

K

) designed to form coiled-coils was able to mediate the targeted fusion of membranes between liposomes and cells.

So far, this approach was used to deliver low molecular weight dyes and drugs into the cytosol and nucleus of live cells. To study the scope of this synthetic fusion system, we now were interested to study whether coiled-coil mediated fusion could be used to enhance the intracellular delivery of inorganic nanoparticles like MSNs. Typically, MSNs or lipid bilayer coated MSNs are

taken up by endocytosis,

which can be detrimental to the cargo.

By employing coiled-coil mediated delivery, the cellular uptake mechanism might be shifted from endocytosis to a direct cytosolic entry via membrane fusion. In order to enhance the intracellular delivery of MSNs/cytC, the nanoparticles were coated with a fusogenic lipid bilayer containing 1 mol%

CP

E

(MSNs/cytC@CPE-LBs). To induce nanoparticle uptake via membrane fusion, cells were pretreated with CP

K

and subsequently MSNs/cytC@CPE-LBs were added to the medium. Lipopeptides and lipid bilayer coated MSNs with or without CP

E

were well tolerated by HeLa cells as no signs of toxicity were observed (Figure S2, Supporting Information).

Confocal microscopy imaging revealed that the cytosol became fluorescent within 30 min, indicative of the efficient delivery of Atto488-labeled cytC inside the cytosol (Figure 3a). By contrast, when one or both of the lipopeptides were omitted, and thus coiled-coil mediated fusion cannot occur, only limited cellular uptake was observed (Figure S3, Supporting Information). The intracellular location of the MSNs upon CC mediated delivery was further investigated by transmission electron microscopy (TEM). It showed that a fraction of the MSNs was outside of the cell, some were entering into the cytoplasm while the majority already appeared inside the cytosol (Figure 3d,e).

Importantly, the MSNs appeared not to be localized in

endosomes or lysosomes, but were found in the cytosol. From

the TEM image it seemed that the MSNs were aggregated

(Figure 3e). A possible explanation might be that upon the

Figure 2. Characterization of MSNs/cytC. a) Loading capacity and zeta-potential of MSNs/cytC, with different initial cytC concentrations (0.5–4 mg mL

,

1 × 10

phosphate buffer (PB), pH 7.4). b) Dynamic light scattering (DLS) of MSNs and MSNs/cytC@CPE-LBs (1 × 10

PB, pH 7.4). c) Zeta-potential

of MSNs, MSNs/cytC, CPE-LBs, and MSNs/cytC@CPE-LBs (error bars represented zeta deviation, 1 × 10

PB, pH 7.4). d) In vitro release profiles of

MSNs and MSNs/cytC@CPE-LBs in phosphate buffered saline (PBS), pH 7.4. Error bars show the standard deviation of three independent experiments.

delivery into the cells, the MSNs lose their lipid bilayers and it is well-known that bare MSNs have a tendency to aggregate.

In comparison, the uptake efficiency of bare MSNs into cells is low (Figure S3c, Supporting Information). More importantly, in the control experiment bare MSNs were located in early endosomal compartments as evidenced by the presence of a membrane bilayer (Figure S4, Supporting Information). These results show that CC driven membrane fusion enhances the cellular uptake of these nanoparticles.

2.3. Endocytosis and Micropinocytosis Inhibitors Marginally Affect Delivery

To investigate the cellular uptake pathway, we repeated the lipid bilayer coated MSNs delivery in the presence of sev- eral well-known inhibitors using flow cytometry measure- ments (FACS) and confocal microscopy imaging.

In this study, wortmannin (Wor) was used as a micropinocy- tosis inhibitor as it blocks PI3-kinase,

while gen- istein (Gen) inhibits tyrosine-phosphorylation of Cav 1 and caveolin-dependent endocytosis.

Furthermore, chlor- promazine (Chl) was used as it blocks clathrin-dependent endocytosis,

and microtubule formation was inhibited by nocodazole (Noc). Uptake studies in the presence of these inhibitors give insight in the intracellular trafficking and internalization mechanisms involved in the uptake of the lipid bilayer coated MSNs.

Finally, as endocytosis of nanopar- ticles is an energy-dependent process, sodium azide (NaN

) was used to deplete the energy demands for endocytosis and restrict metabolic activity.

FACS analysis revealed that Gen, Wor, and Noc had no adverse effect on the delivery of fluorescently labeled cytC (Figure 4a), whereas in the presence of Chl and NaN

, uptake of nanoparticles containing cytC was slightly lowered to 90% as compared to the initial experiment in the absence of inhibitors.

To further study the role of CC formation on the mechanism of cellular uptake, we omitted the CP

K

pretreatment of HeLa cells in control experiments. Without inhibitors, the MSNs/

cytC uptake was less than 60% (Figure 4b, red column). In the presence of Chl or NaN

, the uptake of lipid bilayer coated MSNs was sharply reduced to 10% (Figure 4b, dark blue and blue columns). This clearly indicates that in this control experi- ment the nanoparticles are most likely taken up by a clathrin- dependent endocytosis pathway. Taken together these inhibi- tion studies strongly indicated that the dominant pathway for CC mediated MSN delivery is most likely via membrane fusion between the lipid bilayer coated MSNs and the cell membrane.

By contrast, when CC mediated delivery cannot occur due to the absence of the lipopeptides, the dominant and less efficient route of cellular uptake is via endocytosis.

2.4. Cell Apoptosis Induced by cytC after Delivery

It is well-known that an increase in cytC concentration in the

cytosol triggers caspase activation,

ultimately resulting in

apoptosis of the cell.

Therefore, the amount of apoptosis as

quantified by caspase activity can be used as a tool to confirm

the actual delivery of cytC in the cytosol of cells. 100% apoptosis

was induced by treatment with anti-Fas receptor antibody after

5 h. CC mediated delivery and bioactivity of cytC using lipid

Figure 3. Intracellular delivery of cytC by MSNs@CPE-LBs. Confocal images showing a) location of Atto488 labeled cytC, b) cell nuclei stained by

Hoechst, and e) overlay, scale bar = 25 µm. Intracellular uptake in the presence of inhibitors. c) CC mediated cellular uptake of fluorescently labeled

cytC in CP

K

pretreated HeLa cells. TEM images of d) delivered MSNs/cytC@CPE-LBs into CP

K

pretreated HeLa cells. e) MSNs/cytC.

bilayer-coated MSNs resulted in 55% of apoptosis after 48 h (Figure 5a, green column). By contrast, free cytC induced only 10% of apoptosis (Figure 5a, red column), without CC infor- mation cytC delivered by lipid bilayers induced around 20% of

apoptosis (Figure 5d, blue column). Optical microscopy imaging demonstrated the morphological changes of apoptotic HeLa cells versus healthy cells upon cytC delivery, supporting the caspase assay (Figure 5b,c). In summary, coiled-coil mediated membrane fusion enhanced the observed level of apoptosis, due to the increased uptake and release of cytC into the cytosol of cells. In control experiments where one or both of the lipopeptides were omitted only minimal levels of apoptosis (<10%) were observed, revealing that coiled-coil mediated delivery of MSNs@

LBs is more efficient when compared to delivery via endocytic pathways (Figure 5d–f).

3. Conclusions

Direct cytosolic delivery of cytC encapsu- lated MSNs decorated with a fusogenic lipid bilayer was achieved by targeted coiled-coil mediated membrane fusion with the cells while the uptake via endocytosis was mini- mized as shown by the inhibition cell uptake study. The uptake pathway and localization of MSNs/cytC in HeLa cells were confirmed by TEM and confocal imaging, and the release of functional cytC was demonstrated by its ability to trigger enhanced levels of apop- tosis. We believe that our coiled-coil-based membrane fusion system is suitable for the delivery of a wide range of proteins or high molecular weight compounds as long as these can be encapsulated in the pores of the MSNs. It is envisaged that this method is also suitable for the delivery of any (in)organic nanoparticles as long as it can be encapsu- lated in or decorated with a fusogenic lipid bilayer carrying the coiled coil peptide at its interface. Therefore, this method of delivery may have applications in the field of biomedi- cine and diagnostics.

Figure 4. Mechanistic cellular uptake study. Intracellular uptake of a lipid bilayer coated MSNs (MSNs@CPE-LBs) in the presence of endocytosis/

micropinocytosis inhibitors. a) Coiled-coil induced lipid bilayer coated MSNs delivery and b) control experiment in which CP

K

was omitted. Uptake of MSNs@CPE-LBs was studied in the absence (red bar) and presence of endocytosis inhibitors. Error bars show the standard deviation of three independent experiments.

Figure 5. Cytoplasmic cytC delivery induces apoptosis. a,d) Percentage of apoptotic cells as

measured by caspase activity, after 30 and 48 h. Image of HeLa cells b) treated with cytC and

c) MSNs@CPE-LBs where CC formation triggered apoptosis. e) CP

K

pretreated cells incu-

bated with MSNs/cytC@LBs. f) HeLa cells incubated with MSNs/cytC@CPE-LBs after 30 h

treatment. Apoptotic cells are rarely seen in (e) and (f). Caspase activity was determined after

30 and 48 h. Error bars are standard deviation of three independent experiments. Error bars

show the standard deviation of three independent experiments. Scale bar = 25 µm.

Conflict of Interest

The authors declare no conflict of interest.

Keywords

CC, intracellular delivery, lipid bilayers, membrane fusion, mesoporous silica nanoparticles

Received: June 19, 2017 Revised: August 10, 2017 Published online: September 25, 2017

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