Intracellular Delivery of Universal Proteins Using a Lysine Headgroup

Nov 13, 2013 - labeled bovine serum albumin, mouse anti-F actin antibody. [NH3], and ... delivered albumin and antibodies as confirmed by confocal las...
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Intracellular Delivery of Universal Proteins Using a Lysine Headgroup Containing Cationic Liposomes: Deciphering the Uptake Mechanism Satya Ranjan Sarker, Ryosuke Hokama, and Shinji Takeoka* Department of Life Science and Medical Bioscience, Graduate School of Advanced Science and Engineering, Waseda University (TWIns), Shinjuku-ku, Tokyo 162-8480, Japan S Supporting Information *

ABSTRACT: An amino acid-based cationic lipid having a TFA counterion (trifluoroacetic acid counterion) in the lysine headgroup was used to deliver functional proteins into human cervical cancer cells, HeLa, in the presence of serum. Proteins used in the study were fluorescein isothiocyanate (FITC) labeled bovine serum albumin, mouse anti-F actin antibody [NH3], and goat anti mouse IgG conjugated with FITC. The formation of liposome/protein complexes was confirmed using native polyacrylamide gel electrophoresis. Furthermore, the complexes were characterized in terms of their size and zeta potential at different pH values and found to be responsive to changes in pH. The highest delivery efficiency of the liposome/ albumin complexes was 99% at 37 °C. The liposomes effectively delivered albumin and antibodies as confirmed by confocal laser scanning microscopy (CLSM). Inhibition studies showed that the cellular uptake mechanism of the complexes was via caveolae-mediated endocytosis, and the proteins were subsequently released from either the early endosomes or the caveosomes as suggested by CLSM. Thus, lysine-based cationic liposomes can be a useful tool for intracellular protein delivery. KEYWORDS: amino acid-based cationic liposome, fluorescent labeled protein, antibody, intracellular protein delivery, endocytosis

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or Drosophila Antennapedia-derived penetrating peptide.9 However, there are many disadvantages of the current protein delivery vehicles such as low delivery efficiency, an inefficient release of the proteins from the endosomes to the cytosol,10−12 high cytotoxicity, and lack of a suitable vehicle that can deliver proteins with different size, charge and conformation.3 Therefore, there is a great demand for protein delivery carriers that can efficiently escort functional proteins into the cytosol or other subcellular organelles while retaining their biological activity. Few research groups have focused on the electrostatic complementarity between the positively charged cationic liposomes and the negatively charged proteins to make liposome/protein complexes that can deliver proteins intracellularly3,4 like the delivery of plasmid DNA and oligonucleotides. The preparation of liposome/protein complexes through electrostatic interaction is very simple, easy, and cost-effective. The complex formation between the proteins and the cationic liposomes is influenced by several factors including the surface

INTRODUCTION Intracellular delivery of functional proteins is an emerging topic in the field of biomedical research for the treatment of various disorders caused by dysfunctional proteins, vaccination, and the treatment of loss-of-function genetic diseases as well as certain cancers. The replacement of dysfunctional proteins is the safest and most direct approach for treating diseases because (i) no random or permanent genetic changes are required, (ii) the transient action of the therapeutic protein is highly specific, and (iii) there is less potential to interfere with normal biological processes leading to adverse effects.1,2 However, intracellular delivery of proteins is challenging because these molecules have many intrinsic limitations such as their large size, varying surface charges, and fragile tertiary structures,2 as well as impermeability to cell membranes due to electrostatic repulsions. Moreover, native proteins can undergo rapid degradation or inactivation in the presence of serum.1 To date, many protein delivery carriers have been reported such as cationic liposomes,3,4 polymeric nanoparticles,5 nanocapsules,6 and amphiphilic cationic peptides.7 The most common strategy for intracellular protein delivery is to fuse the desired protein to either protein transduction domains (PTDs),8 cell-penetrating peptides (CPPs) (e.g., HIV-1 transactivator of transcription (TAT) peptide), oligoarginines, © 2013 American Chemical Society

Received: Revised: Accepted: Published: 164

June 23, 2013 November 6, 2013 November 13, 2013 November 13, 2013 dx.doi.org/10.1021/mp400363z | Mol. Pharmaceutics 2014, 11, 164−174

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both FITC and BSA were determined using a UV−vis spectrophotometer (JASCO, Tokyo, Japan). First, a standard curve was prepared for BSA (excitation, 280 nm; emission, 335 nm) and FITC (excitation, 493 nm; emission, 535 nm) separately. Incorporation of FITC per BSA molecule was calculated using the F/M (molar ratio) and estimated to be 2. Preparation of the Liposome/Protein Complexes. To prepare the liposome/protein complexes, the dispersions of cationic liposomes (60−300 μg/mL) were mixed with the solution of albumin (15 μg/mL) and incubated at room temperature for 5 min (Supporting Information, Figure S1). The solutions were then diluted with an appropriate volume of Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum (FBS) to investigate the protein delivery efficiency as well as to decipher their cellular uptake mechanism. The complexes were also prepared from the cationic liposomes (30 μg/mL) and primary (2 μg/mL) as well as secondary (1 μg/mL) antibodies, respectively, through electrostatic interaction (Figure S2). Characterization of Proteins and the Liposome/ Protein Complexes at Different pH. All of the proteins (i.e., albumin, primary and secondary antibodies) used in the study were characterized in terms of their size and zeta potential at pH 7.4 (20 mM 4-(2-hydroxyethyl)piperazine-1ethanesulfonic acid (HEPES)). The liposome/protein complexes prepared in 20 mM HEPES buffer (pH 7.4) were diluted in the same buffer having different pH values such as 7.4 and 6.0. The liposome/albumin complexes were incubated for 30 min at room temperature and then characterized with regard to their size and zeta potential at different pH values using a Zetasizer (Nano-ZS, Malvem Instruments, Malvem, UK). Moreover, the size of the liposome/primary antibody and liposome/secondary complexes was also measured. The liposome/primary antibody and liposome/secondary antibody complexes were prepared at different ratios of lipid (30−300 μg/mL) to antibodies (10 μg/ mL) in 20 mM HEPES buffer (pH 7.4) and incubated at room temperature for 30 min before measuring their size using a dynamic light scattering spectrophotometer (Zetasizer NanoZS, Malvern Instruments, Malvern, UK). Confirmation of the Liposome/Protein Complexes by Native Polyacrylamide Gel Electrophoresis (Native PAGE). To confirm the liposome/protein complex formation, native PAGE was performed for the liposome/albumin and liposome/primary antibody complexes as representatives. Briefly, 12.7% acrylamide/bisacrylamide (0.75 M Tris-HCl, pH 8.8) and 3% acrylamide/bisacrylamide (0.25 M Tris-HCl, pH 6.8) gels were cast as stacking gel and separating gel, respectively. The solutions of albumin (1 μg) and primary antibody (1 μg) were mixed with the dispersions of 1 μg and 2 μg of lipid, respectively, followed by gentle mixing and incubation at room temperature for 5 min to allow the formation of the complexes. The complexes and 1 μg of each protein as positive control were loaded into the respective wells of the gel and run at 20 mA until the sample reached the separating gel. Finally, the gel was run at 30 mA for another 1 h and then stained with Coomassie Brilliant Blue overnight. The results were recorded and analyzed using a gel documentation unit. Measurement of Serum Stability and Protein Release Profile of the Liposome/Protein Complexes. To investigate the serum stability of the liposome/protein complexes in

charge, lipophilicity, hydrophobicity, as well as the size and conformation of the relevant protein.3,4 Therefore, many cationic lipids, that are successful in delivering plasmid DNA and oligonucleotides such as N-[1-(2,3-dioleoyloxy)propyl]N,N,N-trimethylammonium methyl sulfate (DOTAP), N-(2hydroxyethyl)-N,N-dimethyl-2,3-bis(tetradecyloxy)-1-propanaminium bromide (DMRIE), Trans-IT, FuGene 6, Transfast, LipofectAMINE, and Lipofectin, are inefficient to deliver proteins.3 In the previous study, we demonstrated that the lysine-based cationic aminolipid with a trifluoroacetic acid (TFA) counterion in the headgroup efficiently delivered plasmid DNA to a wide range of cell lines including HeLa, COS-7, PC-12, and SHSY-5Y cells along with mouse hippocampus primary cultured cells associated with low cytotoxicity in the presence of serum.13−15 This prompted the current study in which we explore whether the lysine-based cationic aminolipid with a TFA counterion in the headgroup can also deliver intact proteins into human cervical cancer cells, HeLa, in the presence of serum. Hence, we prepared cationic liposomes from only synthetic lipids without using any helper lipids such as dioleoylphosphatidylethanolamine (DOPE), cholesterol, and 1,2-dipalmitoyl-sn-glycero-3-phosphatidyl choline (DPPC). Fluorescent-labeled proteins such as FITC−BSA (∼67 kDa), mouse anti-F actin antibody [NH3], and goat anti mouse IgG conjugated with FITC were used as model proteins to make liposome/protein complexes prior to delivery. We have also used different drugs such as clathrin inhibitor chlorpromazine (CPZ), caveolin inhibitor nystatin (Nys), macropinocytosis inhibitor cytochalasin D (cyto D), and H+-ATPase inhibitor bafilomycin A1 that blocks acidification of lysosomes, to decipher the cellular uptake mechanism as well as the release profile of the liposome/protein complexes, respectively.

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MATERIALS AND METHODS Materials. The synthesis of 1,5-ditetradecyl-N-lysyl-N-tritylL-glutamate with a TFA counterion in the lysine headgroup has been described previously.14 FITC was purchased from Dojindo Laboratories (Kumamoto, Japan). BSA (∼66 kDa) was purchased from Sigma-Aldrich (St. Louis, MO). HeLa cells were purchased from American Type Culture Collection (Manassas, VA). Mouse anti-F actin primary antibody [NH3] (abbreviated as primary antibody) and goat antimouse IgG conjugated with FITC (abbreviated as secondary antibody) were purchased from Abcam (Cambridge, UK) and Molecular Probes (Invitrogen, Eugene, OR), respectively. Lysotracker red DND-99 and Lysotracker green DND-26 were purchased from Molecular Probes. Chlorpromazine, nystatin, cytochalasin D, and bafilomycin A1 were purchased from Sigma-Aldrich. Acrylamide/bis-acrylamide, ammonium persulfate (APS), and N,N,N′,N′-tetramethylethylenediamine (TEMED) were purchased from Bio-Rad Laboratories (Hercules, CA). Conjugation of FITC with Bovine Serum Albumin. First, 2 mg of bovine serum albumin (BSA) was dissolved in 1.0 mL of sodium carbonate solution (0.1 M, pH 9.0), and 1 mg/ mL fluorescein isothiocyanate (FITC) solution was prepared in dimethyl sulfoxide (DMSO) (0.12 mg of FITC in 120 μL of DMSO). The FITC solution was then slowly added to the BSA solution in a darkened laboratory upon gentle stirring and incubated at 4 °C overnight. The conjugated FITC−BSA (abbreviated as albumin) was separated from free FITC by gel filtration chromatography using a Sephadex G-25 column. After purification of conjugated FITC−BSA, the concentrations of 165

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The intracellular delivery of primary as well as secondary antibodies to HeLa cells using lysine-based cationic liposomes was also investigated by CLSM. Briefly, for live cell imaging, the solution of primary antibody (1 μg/mL) was mixed with the dispersion of cationic liposomes (30 μg/mL) and incubated for 5 min at room temperature to form the liposome/primary antibody complexes. The complexes (30:1 w/w) were then added to HeLa cells grown in a 35 mm glass bottom dish and incubated in an atmosphere of 5% CO2 at 37 °C. After 30 min incubation, old media was aspirated and liposome/secondary antibody complexes (30:1 w/w) were added to the cells and incubated for either a further 30 min or 24 h under the same conditions. The old media was then aspirated at specific time points and washed three times with prewarmed phosphate buffer saline containing heparan sulfate (20 U/mL). Finally, 1 mL of high glucose DMEM containing 10% FBS without phenol red was added to the cell culture dish and observed under CLSM. Moreover, a dispersion of the cationic liposome/ secondary antibody (30:1 w/w) complexes was also added to HeLa cells and incubated for 30 min before being observed under CLSM. The detailed protocols for actin filament staining using primary as well as secondary antibodies in fixed HeLa cells and other control experiments are given in the Supporting Information. Inhibition Study for the Cell Uptake Mechanism of the Liposome/Protein Complexes. Three endocytosis inhibitors, namely, chlorpromazine (clathrin inhibitor), nystatin (caveolin inhibitor), and cytochalasin D (actin inhibitor) were used at different concentrations to determine the cellular uptake mechanism of the liposome/protein complexes. HeLa cells were seeded into 6-well plates at a density of 1 × 105 cells/ well and incubated in an atmosphere of 5% CO2 at 37 °C overnight. Then the medium of each well of the cell culture dish was exchanged with different concentrations of inhibitors, such as chlorpromazine (5 and 10 μg/mL), cytochalasin D (5 and 10 μg/mL), and nystatin (12.5, 25, and 50 μg/mL) and incubated for 1 h under the same conditions. Liposome/ albumin complexes (200:15 w/w) were then added to each well of the 6-well plate and incubated under the same conditions for 2 h. After incubation, old media containing the complexes and inhibitors were aspirated and washed three times with prewarmed phosphate buffered saline containing the respective concentrations of the drugs and heparan sulfate (20 U/mL). The cells were then trypsinized and centrifuged at 1200 rpm for 3 min. The supernatant was aspirated, and the cell pellet was resuspended in 300 μL of PBS containing 1 wt % bovine serum albumin. Finally, the percentages of the fluorescent labeled cells were determined using a fluorescence activated cell sorter (BD FACS ARIA-II; BD Bioscience). The cellular uptake mechanism was also confirmed for the liposome/albumin complexes using CLSM. Briefly, 5 × 104 cells were seeded into a 35 mm glass bottom dish and incubated overnight. The old media was replaced with complete DMEM containing the respective inhibitors (chlorpromazine: 10 μg/mL; cytochalasin D: 10 μg/mL; and nystatin: 50 μg/mL) and incubated for 1 h. The liposome/ albumin (30:15 w/w) complexes were then added to the cells and incubated for another 1 h. Finally, the cells were observed under CLSM. Study of Protein Release from Endosomes. To study the release profile of proteins, HeLa cells (1 × 105 cells/well of a 6-well plate for FACS; 5 × 104 cells/35 mm glass bottom dish for CLSM) were incubated with a vacuolar type H+-ATPase

the presence of 10% FBS, the dispersions of cationic liposomes were mixed with the solution of FITC−BSA at different lipid/ protein ratios (60−300:15 w/w) and incubated at room temperature for 5 min. The solutions were then diluted with 2 mL of DMEM. Finally, 10% FBS was added to the complexes and incubated for 0, 0.5, 1, and 2 h at 37 °C, and the size of the complexes was measured using a dynamic light scattering spectrophotometer (Zetasizer Nano-ZS, Malvern Instruments, Malvern, UK). To determine the release profile of FITC−BSA from the liposome/FITC−BSA complexes in the presence of 10% FBS containing DMEM, the complexes were prepared and diluted with DMEM without phenol red, and the released amount of protein was measured prior to addition of FBS. Then 10% FBS was added to the complexes and incubated for 0.5, 1, and 2 h at 37 °C. The complexes were centrifuged at 45 000 rpm for 45 min. After centrifugation, supernatant was removed, and the liposome pellet was resuspended in DMEM without phenol red. Finally, the fluorescence intensity of the liposome pellet was measured using a spectrofluorometer (SHIMADZU, RF5300PC, Japan). Determination of the Protein Delivery Efficiency of Liposome/Albumin Complexes by Fluorescence Activated Cell Sorter (FACS). HeLa cells were utilized for evaluating the protein delivery efficiency of the liposome/ albumin complexes at 4 °C as well as 37 °C by a fluorescence activated cell sorter (FACS). The cells were seeded into a 6well plate at a density of 1 × 105 cells/well and incubated in an atmosphere of 5% CO2 at 37 °C overnight. The medium in each well of the cell culture dish was exchanged with the liposome/albumin complexes (60−300:15 w/w) in the presence of 10%, 20%, and 50% FBS and incubated for 2 h in an atmosphere of 5% CO2 at 37 °C. However, to evaluate the cell uptake efficiency at 4 °C, the complexes were prepared in high glucose complete DMEM and incubated at 4 °C for 2 h upon addition to the cells. In both cases, the old media was removed by aspiration, and the cells were then washed three times with prewarmed phosphate buffered saline containing heparan sulfate (20 U/mL) to remove any cell membrane bound liposome/protein complexes. The cells were then trypsinized with 500 μL of trypsin−ethylenediaminetetraacetic acid (EDTA) and centrifuged at 1200 rpm for 3 min. The supernatant was aspirated, and the cell pellet was resuspended in 300 μL of PBS containing 1 wt % bovine serum albumin. Finally, the percentages of the fluorescent labeled cells were determined using a fluorescence activated cell sorter (BD FACS ARIA-II; BD Bioscience, Oxford, UK). Intracellular Delivery of Proteins Confirmed by Confocal Laser Scanning Microscopy (CLSM). HeLa cells were seeded into a 35 mm glass bottom dish at a density of 5 × 104 cells and incubated in an atmosphere of 5% CO2 at 37 °C overnight. For live cell imaging, the medium of the cell culture dish was exchanged with the dispersion of liposome/albumin complexes (30:15 w/w) and incubated for 30 min, 2 h, and 24 h at 37 °C and only 30 min at 4 °C. The old media was then aspirated at specific time points and washed three times with prewarmed phosphate buffered saline containing heparan sulfate (20 U/mL). Finally, 1 mL of high glucose DMEM containing 10% FBS without phenol red was added to the cell culture dish and observed under confocal laser scanning microscopy (CLSM) (Fluoview, FV1000; Olympus Imaging America Inc., Center Valley, PA). 166

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inhibitor, bafilomycin A1 (50 nM and 100 nM), a known inhibitor for the acidification of lysosomes,16 for 1 h in an atmosphere of 5% CO2 and 37 °C. Then the liposome/albumin complexes (60−300:15 w/w for FACS; 30:15 w/w for CLSM) were added to the cells and incubated for 2 h and 30 min, respectively, for FACS analysis and CLSM, under the same conditions. Finally, the cells were prepared for the FACS analysis and CLSM observations as described previously.

Table 2. Size of the Liposome/Primary Antibody and Secondary Antibody Complexes concentration of lipid (μg/mL) 30 60 100 200 300

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RESULTS Characterization of Proteins and Liposome/FITC−BSA Complexes at Different pH Values. The zeta potentials of albumin, primary and secondary antibodies used in the study were −14 ± 3, −20 ± 2, and −11 ± 1 mV, respectively, at pH 7.4. Furthermore, the zeta potentials of the cationic liposomes were +60 and +65 mV at pH 7.4 and 6.0, respectively. The size of the cationic liposomes was 96 ± 40 and 85 ± 35 nm at pH 7.4 and 6.0, respectively. The liposomes were stable in terms of their structure and dispersibility at pH values between 6.0 and 7.4. The complex formation of the cationic liposomes with albumin was confirmed at pH 7.4 and 6.0 in 20 mM HEPES buffer. The size and zeta potential of the complexes were stable after 30 min incubation at room temperature. Table 1

concentration of lipid (μg/mL) 60 100 200 300

zeta potential (mV) 35 39 46 47

± ± ± ±

3 4 5 2

pH 6.0

size (nm) 883 792 579 357

± ± ± ±

502 401 345 347

zeta potential (mV) 51 49 50 48

± ± ± ±

2 5 3 2

size (nm) 672 349 150 119

± ± ± ±

472 421 263 197 126

± ± ± ± ±

77 88 19 10 5

liposome/secondary antibody (nm) 943 813 641 320 198

± ± ± ± ±

215 209 45 39 20

charged lysine-based cationic liposomes and the negatively charged proteins was confirmed by native polyacrylamide gel electrophoresis (native PAGE) (Figure S3). When the liposomes were complexed with the primary antibody at a weight ratio of 2:1, no band was evident (Figure S3; lane 1). By contrast, a band corresponding to the liposome/primary antibody complex (1:1 w/w) was visible (Figure S3; lane 2) that comigrated with the control primary antibody (Figure S3; lane 3). On the other hand, no band was detectable when the weight ratios of liposome/albumin complexes were 1:1 or 2:1 (Figure S3; lanes 4 and 5, respectively). However, there was a visible band for control albumin (Figure S3; lane 6). Presumably the interaction between the primary antibody and the liposomes is weak when compared to that of albumin. Consequently, the primary antibody requires a greater amount of lipid for the complex formation. These data are also consistent with a previous study that suggested the complex formation between the negatively charged proteins and the cationic liposomes is influenced by a number of factors in addition to the zeta potential, that is, lipophilicity, hydrophobicity, size, and the conformation of the protein of interest.3 Measurement of Size Stability and Protein Release Profile. The size of the liposome/FITC−BSA complexes was reduced after the addition of 10% FBS at all time points (0.5, 1, and 2 h) (Figure 1a). This could be due to the exchange of FITC−BSA with the serum proteins. The size of the complexes was also reduced with the increased concentration of lipid. The release of FITC−BSA from the liposome/protein complexes increased with the incubation period (0.5−2 h) at all lipid/FITC−BSA ratios (Figure 1b). Furthermore, as the amount of lipid increased, the rate of protein release from the liposome/FITC−BSA complexes also increased. Determination of the Protein Delivery Efficiency of Liposome/Albumin Complexes by FACS. A fluorescence activated cell sorter (FACS) was used to evaluate the protein delivery efficiency of the liposome/albumin complexes to HeLa cells in the presence of 10%, 20%, and 50% FBS. We analyzed the protein delivery efficiency at 4 °C as well as at 37 °C to investigate whether the cellular uptake process was temperature-dependent (Figure 2). The cell uptake efficiency reduced in the presence of 20% FBS when compared to that of the 10% FBS at all lipid/protein ratios (60−300:15 w/w). In the presence of both 10% and 20% FBS, the delivery efficiency increased with the increased concentration of lipid up to 200 μg/mL and thereafter got saturated. The highest percentage of the fluorescent labeled cells was 99% at 37 °C when the concentration of lipid and FBS was 200 μg/mL and 10%, respectively. Furthermore, the cell uptake efficiency reduced dramatically in the presence of 50% FBS at all lipid/FITC−BSA ratios. In the presence of 50% FBS, the cell uptake efficiency tended to decrease as the amount of lipid increased. However, almost none of the cells were fluorescent positive at 4 °C.

Table 1. Characterization of the Liposome/Protein Complexes at Different pH pH 7.4

liposome/primary antibody (nm)

414 253 96 73

summarizes the parameters of the complexes with different weight ratios of lipid to albumin (60−300:15 w/w). At pH 7.4, the zeta potential of the complexes increased from +35 to +47 mV with an increased amount of lipid when the complexes were incubated for 30 min at room temperature. However, the size of the complexes decreased from 883 to 357 nm with an increased amount of lipid. At pH 6.0, there was no significant change in the zeta potential of the complexes. However, the size of the complexes decreased sharply from 672 to 119 nm with an increased amount of lipid. When the liposomes were complexed with albumin at a 300:15 weight ratio, the negatively charged albumin was adsorbed on the positively charged surface of the liposomes thereby decreasing the zeta potential of the resulting complexes and maintaining their nonaggregated state. As the albumin ratio increased, the zeta potential of the complexes decreased. In this case, the surface-bound albumin acted as an aggregation point for the complexes. Table 2 summarizes the size of the liposome/primary antibody and liposome/secondary antibody complexes with different weight ratios of lipid to respective antibodies (30− 300:10 w/w). The size of both the liposome/primary antibody and liposome/secondary complexes decreased from 472 to 126 nm and 943 to 198 nm, respectively, with an increased amount of lipid. Confirmation of the Liposome/Protein Complexes by Native PAGE. The complex formation between the positively 167

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Figure 2. Intracellular protein (i.e., FITC−BSA) delivery efficiency of the lysine-based cationic liposomes in HeLa cells in the presence of 10%, 20%, and 50% FBS at 37 °C as analyzed by FACS. The cell uptake experiment was also performed at 4 °C. All of the experiments were performed three times on three different days. (NC: negative control; lipid: 60−300 μg; FITC−BSA: 15 μg). Negative control refers to only HeLa cells; there was no FITC−BSA, and liposome/protein complexes were used.

Figure 1. Determining the influence of serum proteins on the liposome/protein complexes. (a) The size of liposome/FITC−BSA complexes prepared at different lipid/protein ratios in the presence of 10% FBS and at different time points were measured. (b) FITC−BSA release profile from liposome/FITC−BSA complexes prepared at different lipid/protein ratios in the presence of 10% FBS and at different time points. Figure 3. Intracellular delivery of FITC−BSA (green) in HeLa cells using lysine-based cationic liposomes (in the presence of 10% FBS) as observed by CLSM at different time points. (a) After 30 min; (b) overlay of FITC−BSA and Lysotracker red DND-99. The white arrow indicates the colocalization FITC−BSA and Lysotracker red DND-99; (c) after 2 h; and (d) after 24 h of the addition of liposome/FITC− BSA complexes to HeLa cells. Scale bar: 10 μm.

Intracellular Delivery of Albumin by the Liposome/ Albumin Complexes. Confocal laser scanning microscopy (CLSM) was used to unravel the cellular uptake mechanism of the complexes and to detect the location of the fluorescent labeled proteins inside the cell. Albumin labeled with FITC was detected as green punctuates in the cytosol 30 min and 2 h after the delivery of the complexes to HeLa cells (Figure 3a and c). Furthermore, albumin was found to be colocalized with the Lysotracker red DND-99, which resulted in the yellow punctates in the cytosol (Figure 3b), indicating the entrapment of albumin in the endosomes. Interestingly, green punctates got diffused to the cytosolic space from endosomes after 24 h of the addition of complexes (Figure 3d). No intracellular delivery of albumin was detected when the respective complexes were incubated with the HeLa cells at 4 °C (Figure S4). Therefore, the liposome mediated cellular uptake is governed by an endocytosis pathway.

In Vitro Delivery of Antibodies by Liposome/Antibody Complexes. Liposome/antibody complexes delivered primary as well as secondary antibodies into live HeLa cells as shown by CLSM images (Figure 4). After 1 h of the delivery of primary antibody by liposome/primary antibody complexes, liposome/ secondary antibody complexes were also delivered to the same cells and incubated for another 1 h. Green punctates were then visible at the cell periphery as well as in the cytosol (Figure 4a). However, green punctates had spread evenly throughout the 168

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Figure 4. Delivery of mouse anti F-actin antibody [NH3] (primary antibody) and goat anti mouse IgG conjugated with FITC (secondary antibody) to HeLa cells at 37 °C using lysine-based cationic liposomes. (a) Primary antibody was complexed with cationic liposomes and delivered to live HeLa cells and incubated for 1 h. The cells were then treated with liposome/secondary antibody complexes and incubated for another 1 h. (b) HeLa cells were incubated for 24 h after the delivery of primary and secondary antibodies. (c) Secondary antibody was delivered using the cationic liposomes/secondary antibody complexes. (d) Primary antibody was delivered using the cationic liposomes/primary antibody complexes followed by fixation with 4% paraformaldehyde and stained with secondary antibody. (e) Conventional staining of the stress fibers of actin filaments using primary and secondary antibodies. (f) G-actin monomer was removed upon treatment with Triton-X 100 followed by fixation with 4% paraformaldehyde, and then conventional staining was performed. (g) The first primary antibody was delivered to live HeLa cells using cationic liposomes/primary antibody complexes and incubated for 1 h. The cells were then treated with Triton-X 100 to remove G-actin monomer and fixed using 4% paraformaldehyde. Finally, the cells were stained with the secondary antibody. Stress fibers of actin filaments are visible (indicated by arrow). Scale bar: 10 μm.

cytosolic space (Figure.4b), when the cells were incubated for 24 h. When only liposome/secondary antibody complexes were delivered into live HeLa cells, green punctates became visible after 2 h (Figure 4c). Furthermore, the liposome/primary antibody complexes were delivered to live HeLa cells. The cells were subsequently fixed with 4% paraformaldehyde and stained with secondary antibody, which resulted in the entire cellular region being stained green (Figure 4d). Therefore, we performed the conventional staining for the actin filaments of stress fibers. The results showed the entire cellular region as green due to the nonspecific binding of the primary antibody to

G-actin monomers (Figure 4e). To reduce the G-actin monomer concentration, HeLa cells were treated with TritonX 100 for 30 s before fixing with 4% paraformaldehyde, and conventional staining was performed. Using this protocol, actin filaments of stress fibers were successfully detected (Figure 4f). Moreover, liposome/primary antibody complexes were delivered into live HeLa cells and incubated for 1 h. The cells were then briefly treated with Triton-X 100 and stained with secondary antibody, which confirmed the presence of actin filaments of stress fibers (Figure 4g). 169

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Figure 5. (a) Cell uptake inhibition study for the liposome/FITC−BSA complexes by FACS. Three different drugs were under investigation, namely, clathrin inhibitor chlorpromazine (CPZ), macropinocytosis inhibitor cytochalasin D (Cyto D), and caveolin inhibitor nystatin (Nys) at different concentrations: CPZ (10 μg/mL); Cyto D (10 μg/mL); Nys (50 μg/mL). Concentration of lipid: 200 μg/mL. (b) Cell uptake inhibition study for the complexes (green) by CLSM. Concentration of lipid: 30 μg/mL; FITC−BSA: 15 μg/mL. Scale bar: 10 μm.

Inhibition Study for the Cellular Uptake Mechanism of the Liposome/Protein Complexes. To explore the endocytosis pathways involved in the cellular uptake of liposome/albumin complexes, HeLa cells were treated with different concentrations of the three different inhibitors, namely, chlorpromazine (clathrin-dependent endocytosis inhibitor), nystatin (caveolae-dependent endocytosis inhibitor), or cytochalasin D (macropinocytosis inhibitor). FACS data showed that more than 80% and 70% of cells were FITC positive in the presence of chlorpromazine and cytochalasin D, respectively. However, the cellular uptake efficiency was significantly inhibited (P < 0.015) in the presence of the caveolae inhibitor nystatin, with less than 30% of cells being FITC positive (Figure 5a). Data regarding dose dependence study of all three cell uptake inhibitors are described in the Supporting Information (Section S5). Furthermore, CLSM images detected FITC labeled albumin as green punctates within the cytosolic space of HeLa cells in the presence of both chlorpromazine and cytochalasin D (Figure 5b). No such punctuates were visible when the experiment was performed in the presence of nystatin. These results show that cellular uptake of the liposome/protein complexes took place via caveolae-mediated endocytosis. Study of Protein Release from Endosomes. To investigate whether the protein delivery mechanism involves the lysosomal pathway, cells were treated with bafilomycin A1 that inhibits lysosomal acidification. There was no inhibition of albumin release in the presence of 50 and 100 nM of bafilomycin A1 when the concentration of lipid was 60−300 μg/mL (Figure 6a). More specifically, more than 95% of the cells were fluorescent positive at 200 and 300 μg/mL of the

lipid. Furthermore, CLSM images confirmed the presence of albumin inside the cells (Figure 6b). Therefore, proteins from the liposome/protein complexes were released from either the early endosomes or the caveosomes and did not follow the lysosomal pathway. It is because bafilomycin A1 also inhibits acidification of endosomes and thereby prevents their maturation and fusion into the lysosomes.17 Once caveolaecoated vesicles are pinched off from the cell membrane, they can move to either early endosomes or caveosomes.18 Caveosomes are vesicles with neutral pH, and the mechanism for the release of the cargo molecules remains unknown.18

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DISCUSSION Cationic liposomes prepared from the cationic amino acidbased lipids have been reported to be highly efficient gene delivery vehicles for a variety of cell lines with low associated cytotoxicity.13,14,19,20 However, the intracellular delivery of proteins using amino acid-based cationic liposomes has not been reported to date. Unlike the formation of lipoplexes between the positively charged cationic liposomes and the negatively charged plasmid DNA, which rely only on the electrostatic interaction, the liposome/protein complexes are also stabilized by the van der Waals force and hydrophobic interaction.4 It is also known to be influenced by the net charge, lipophilicity, hydrophobicity, and conformation of the proteins of interest.3 For example, commercially available cationic lipid formulations, such as Bioporter, showed an extremely low efficiency of albumin delivery due to the presence of very few hydrophobic domains encrypted in its globular conformation.3 By contrast, our data showed that albumin made stable complexes with the lysine headgroup containing cationic 170

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Figure 6. (a) Endosomal release profile of FITC−BSA in the presence of vacuolar type H+-ATPase inhibitor bafilomycin A1, which inhibits acidification of lysosomes, in HeLa cells as analyzed by FACS. Lipid concentrations: 60−300 μg/mL; bafilomycin A1 concentrations: 50 nM (■ bar) and 100 nM (□ bar). (b) Endosomal release of FITC−BSA (green) as observed by CLSM. Concentration of lipid: 30 μg; FITC−BSA: 15 μg; bafilomycin A1 concentrations: (i) 50 nM and (ii) 100 nM. Scale bar: 10 μm.

potential of the complexes at pH 6.0 (Table 1), which corresponds to the conditions found in the early endosomes, is due to the presence of the TFA counterion in the lysine headgroup. The low pH brings about the release of negatively charged proteins from the complexes and results in the increased electrostatic repulsion among the cationic liposomes leading to the reduction of particle size within the endosomes. Furthermore, the cellular uptake efficiency of the liposome/ protein complexes should be influenced by their zeta potential and hydrodynamic size (Figure 2 and Table 1), because the positively charged complexes interact with the heparan sulfate proteoglycans of the negatively charged cell membrane through electrostatic interaction.21 Similarly, the size of liposome/ primary antibody and liposome/secondary antibody complexes was reduced with the increased amount of lipid (Table 2). This is because of the enhanced complexation of the antibodies with liposomes that results in the larger aggregates. Moreover, the particle size determines whether the complexes are taken up via clathrin-mediated endocytosis or caveolae-mediated endocytosis.22 We believe that the caveolae-mediated endocytosis is favorable for protein delivery because lysosomal degradation of the protein cargo is avoided. Instead, the cargo gets released from early endosomes or caveosomes in a process akin to

liposomes (Figure S3). Moreover, mouse anti F-actin primary antibody [NH3] also formed complexes with the cationic liposomes albeit at relatively high amounts of lipid by comparison to albumin. This arises because the net charge of the primary antibody (−20 mV) is more than that of FITC− BSA (−14 mV) (Table S1). Serum proteins also influence the size and stability of liposome/protein complexes (Figure 1). Serum proteins exchange with FITC−BSA of the liposome/ FITC−BSA complexes and are responsible for the smaller size of the complexes. The increased rate of protein release from the liposome/protein complexes over time could be due to the proteins adsorbed on the liposomal surface tending to release more easily when compared to the proteins present in aggregates of liposome/FITC−BSA complexes at low lipid/ protein ratios. On the other hand, preparation of the liposome/ protein complexes through electrostatic interaction is advantageous over the encapsulation process that is a very tedious and time-consuming process and has low encapsulation efficiency. By contrast, electrostatic interaction between the cationic liposomes and negatively charged proteins (Table S1) is very simple as well as highly efficient. The physicochemical properties of the liposomes are sensitive to changes in pH. For example, the elevated zeta 171

dx.doi.org/10.1021/mp400363z | Mol. Pharmaceutics 2014, 11, 164−174

Molecular Pharmaceutics

Article

Figure 7. Illustration of the cellular uptake mechanism of liposome/protein complexes. (1) Clathrin-mediated uptake; (2) caveolae-mediated uptake; (3) membrane fusion; and (4) macropinocytosis.

To confirm that proteins can be delivered to the cytosol without losing their biological activity, mouse anti F-actin antibody [NH3] as a primary antibody and goat anti mouse IgG conjugated with FITC as a secondary antibody were delivered to living cells to stain actin filaments of stress fibers. Delivery of these antibodies gave green punctates in the cytosol as well as in the perinuclear region after 2 h (Figure 4a), which spread uniformly upon incubation for 24 h (Figure 4b). However, no fibrous structure was confirmed due to the nonspecific binding of the primary antibody to G-actin. The primary antibody escaped successfully to the cytosol and bound not only to the F-actin filaments but also to the G-actin monomers. Pretreatment of the cells with Triton-X 100 to remove G-actin resulted in clear staining of the F-actin filaments (Figure 4f and g). These data suggested that both primary and secondary antibodies were successfully delivered to the cytosolic space without losing their biological activity. However, nonspecifically bound and unbound antibodies should be taken into consideration when performing immunological staining. Important cellular uptake pathways such as clathrin- and caveolae-dependent endocytosis are determined by the size of both the cargo molecules and the particles.26,27 For example, particles up to 200 nm are preferably and almost exclusively endocytosed via clathrin-coated pits and ultimately delivered to the lysosomes, while particles of 500 nm are taken up by the cells via caveolae and do not reach the lysosomal compartment.28 Complexes can also be taken inside the cells either via macropinocytosis or simple membrane fusion. Our data demonstrated that the size of the complexes was larger than

certain viruses (e.g., SV 40) and bacteria in eukaryotic cells.23−25 The cellular uptake efficiency of liposome/albumin complexes increases as the concentration of lipid increases in the presence of 10% and 20% FBS. The gradual enhancement in cellular uptake efficiency in the presence of 10% and 20% FBS as shown in Figure 2 can be explained in terms of the increased zeta potential and reduced particle size of the complexes. However, the cellular uptake efficiency reduced dramatically in the presence of 50% FBS because the presence of 50% FBS results in the exchange of FITC−BSA with serum proteins. The usual concentration of FBS for in vitro culturing of HeLa cells is 10%. We also hypothesize that 50% FBS influences the stability of liposome/FITC−BSA complexes, resulting in the reduced cell uptake efficiency. The cellular uptake efficiency at 4 °C was barely detectable, indicating the mechanism of uptake was endocytosis that requires energy. Albumin labeled with FITC was detected as green punctates in the cytosolic space, which is highly viscous and hydrophilic in nature, 30 min and 2 h after introducing the cells to the liposome/protein complexes (Figure 3a and c). The relatively slow release of free albumin into the cytosol is presumably due to the high molecular weight of the protein as well as the strong hydrophobic interaction between the cationic liposomes and albumin.4 Moreover, 24 h after the start of cellular uptake, the albumin had diffused uniformly to the cytosolic space (Figure 3d) without severe aggregation. These observations suggest that most of the albumin was molecularly released from either the endosomes or the caveosomes. 172

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Notes

500 nm at pH 7.4 and there was no influence on the cellular uptake efficiency in the presence of chlorpromazine and cytochalasin D. Moreover, the cellular uptake efficiency was significantly reduced (P < 0.015) in the presence of the caveolae inhibitor nystatin. Further evidence suggesting the complexes do not go through the lysosomal pathway came from our observation that the release of albumin is unaffected in the presence of bafilomycin A1. Rather, proteins were released from either the early endosomes or caveosomes without any denaturation. It is speculated that the presence of NH3+TFA− ionization states in the lysine headgroup of the cationic lipid makes it highly responsive to the early endosomal pH (i.e., pH 6.0). The complexes interact with the cell membrane via electrostatic interaction between the positively charged complexes and the negatively charged cell membrane. The complexes then get entry into the cell via caveolaemediated endocytosis (Figure 7). Once inside the cell, the endocytic caveolar carriers can fuse with either the early endosomes or the caveosomes. In the case of early endosomes, the fluorescent labeled proteins first get released from the internalized complexes inside the early endosomes. The liposomal membrane then fuses with the early endosomal membrane resulting in the release of fluorescent labeled proteins outside the endosomes. However, if the complexes go through caveosomes, which have a neutral pH, the mechanism of protein release is unknown.18 Taken together, our results suggest the cellular uptake mechanism for the lysine-based cationic liposome/protein complexes is caveolae-mediated endocytosis. The proteins are then released from either the early endosomes or the caveosomes.

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was partially supported by the GCOE “Practical chemical wisdom” and “High-Tech Research Centre” project for Waseda University: matching fund subsidy from MEXT, Japan.

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CONCLUSIONS Lysine-based cationic liposomes can be used for the intracellular delivery of negatively charged universal proteins through electrostatic complex formation. Proteins can also be delivered into living cells in the presence of serum with high efficiency. The cellular uptake of the complexes takes place through caveolae-dependent endocytosis, and the proteins are released from either the early endosomes or the caveosomes. Finally, amino acid-based cationic liposomes have great potential in protein therapy as delivery vehicles for the treatment of various protein deficiency or protein malfunction genetic disorders and cancers as well as to study the structure, function, and dynamics of specific intracellular proteins.

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ASSOCIATED CONTENT

* Supporting Information S

Diagrammatic presentation of the lysine-based cationic liposome/FITC-BSA complexes formation and the lysine-based cationic liposome/negatively charged antibody complexes formation, gel shift assay using native PAGE, delivery of FITC-BSA using lysine-based cationic liposomes/albumin complexes to HeLa cells at 4 °C, dose dependence study of different cell uptake inhibitors, and characterization of universal proteins in terms of their size and zeta potential. This material is available free of charge via the Internet at http://pubs.acs.org.

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