pH-sensitive carbonate apatite as an intracellular protein transporter

Biomaterials 31 (2010) 1453–1459

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pH-sensitive carbonate apatite as an intracellular protein transporter Seiichi Tada a, Ezharul H. Chowdhury a, b, Chong-Su Cho c, Toshihiro Akaike a, * a

Department of Biomolecular Engineering, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, Yokohama 226-8501, Japan Faculty of Medicine and Health Science, International Medical University (IMU), No. 126, Jalan 19/155B, Bukit Jalil, 57000 Kuala Lumpur, Malaysia c Department of Agricultural Biotechnology, Seoul National University, Seoul 151-921, Republic of Korea b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 11 September 2009 Accepted 7 October 2009 Available online 24 October 2009

The transfer of specific proteins into living cells to enable the regulation of cell function or the tracking of the intracellular distribution of proteins is a desirable objective for offering a potential alternative to gene therapy. Here, protein/carbonate apatite complexes were successfully fabricated for intracellular delivery of functional proteins since the carbonate apatite being highly water solubility under an acidic condition could easily be dissolved in endosomes following endocytosis, thus releasing the electrostatically associated proteins in cytoplasm. In this study, we characterized protein/carbonate apatite complexes as an intracellular protein delivery system and we checked intracellular delivery of proteins by carbonate apatite nanoparticles in vitro. Fluorescently-labeled bovine serum albumin as a model protein was effectively delivered into nearly 100% of HeLa cells by the simple addition of protein/carbonate apatite complexes to the cells. Confocal microscopic imaging suggested the endosomal release of protein delivered with carbonate apatite. And intracellularly delivered ß-galactosidase did not lose its enzymatic activity. These results suggested that intracellular delivery system of protein using pH-sensitive carbonate apatite carrier with a very simple procedure will be a highly effective method to the biological and clinical researches. Ó 2009 Elsevier Ltd. All rights reserved.

Keywords: Carbonate apatite pH sensitive Protein delivery Biocompatibility Bioactivity Endosomal release

1. Introduction Because of poor penetration of general proteins into living cells, intracellular delivery of proteins is a challenging approach that can be applied both in basic research and in clinic. In molecular and cell biology, intracellular delivery of protein has mainly been conducted to regulate cellular functions and to track the intracellular distribution of desirable proteins [1–7] although DNA transfection is nowadays adapted for the same purpose. For tracking the intracellular behaviors of protein, expression of the protein fused to fluorescent protein like GFP has often been adopted although the GFP can interfere the function or intracellular localization of a target protein due to a relatively large molecular size of GFP (27 kDa) [8]. On the other hand, it is more effective to deliver protein labeled with low-molecular weight fluorescent dye into the cells for tracking intracellular behaviors of protein. Also, synthesis of artificial peptides or proteins containing unnatural amino acid was recently studied [9], although it is very hard to test the intracellular activity of these peptides or proteins made through a cell-free protein synthesis system [10]. Therefore,

* Corresponding author. Tel.: þ81 45 924 5790; fax: þ81 45 924 5815. E-mail address: [email protected] (T. Akaike). 0142-9612/$ – see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.biomaterials.2009.10.016

effective intracellular protein delivery method with the protein synthesized outside the cells should be required to overcome the above-mentioned limitations. Up to now, a large number of protein-based compounds have been used as therapeutic agents [11], although current therapeutic proteins are limited in extracellularly secreted protein such as insulin, which shows its effect mainly by binding to receptor proteins. If intracellular delivery method is adapted to drug formulation, not only secreted proteins but also proteins in cytoplasm can also apply to therapeutics [11]. Intracellular protein delivery will also be expected as the alternative to gene therapy [12,13]. Generally, there are three kinds of methods to deliver proteins into cells. Physical methods such as microinjection [1,3–5,14] and electroporation [15] have difficulty to be applied in vivo [16]. Another way is the use of carrier molecules such as cationic lipids and polymers that are able to enter cells through endocytosis via ionic interactions [17,18]. The protein loaded carriers are internalized into cells, escape the endo-lysosomes and finally enter the cytoplasm. However, very small amount of internalized protein loaded carriers escape into the cytosolic compartment and result in denaturation of proteins. Therefore, smart carriers should be used for the rapid escape of proteins from endo-lysosomes. The other is protein modification for membrane penetration such as protein

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Fig. 1. The amount of FITC-BSA adsorbed in CO3Ap particles. FITC-BSA/CO3Ap complex suspension was prepared by supersaturation of CaCl2 in bicarbonate-buffered DMEM at final concentration of 4.8 mM or 7.8 mM. FITC-BSA was added to DMEM at concentrations of 5, 10, 20, 30, 50, 100 mg/mL before complex formation. The amount of FITC-BSA adsorbed in 1 mL of CO3Ap suspension was measured by fluorescence measurement. Data shown are means S.D. (n ¼ 3)

cationization [19–21] and modification with cell penetrating peptide(CPP) [6–8,12,13,22–24]. Especially CPP-mediated protein internalization has attracted much interest for the last two decades. Proteins covalently or non-covalently associated with CPP become possible to enter cells although the mechanism of internalization by CPP is still controversial. Until now, there are many researches, which refer to the mechanism of CPP-mediated protein delivery, and some researches suggest that the mechanism may depend on cargo molecules and cell type [24]. In the majority of studies, CPP is covalently bound to N- or C-terminal of a protein by gene engineering technique, and therefore these types of methods are not suitable for commercially available proteins. In addition, CPP modification by recombinant gene engineering is neither applicable to some proteins whose functional sites locate in their N- or C-terminal. For such proteins, it is preferable to choose the intracellular delivery method without covalent modification. In our previous studies, efficient intracellular delivery of DNA as well as mRNA was obtained using pH-sensitive carbonate apatite (CO3Ap) as the carrier because the DNA and mRNA were adsorbed

Fig. 2. The particle size distribution of FITC-BSA/CO3Ap complexes. FITC-BSA/CO3Ap complexes were prepared from DMEM containing 50 mg/mL of FITC-BSA and 4.8 mM CaCl2, and measured by DLS spectrophotometer.

Fig. 3. The TEM image of FITC-BSA/CO3Ap complexes. FITC-BSA/CO3Ap complexes were prepared from DMEM containing 50 mg/mL of FITC-BSA and 7.8 mM CaCl2, and observed by transmission electron microscope.

to CO3Ap carriers and rapidly escaped from the endo-lysosomes due to the high water solubility of the carbonate apatite in acidic pH environment similar to endosomal pH [25–29]. In this study, we investigated intracellular delivery of proteins using pH-sensitive carbonate apatite. Many delivery systems using calcium phosphate particles have been reported mainly for DNA delivery, not for protein delivery [26,30–32]. Only a few have been reported on protein delivery system using hydroxyapatite particles [33–35]. However, their aim was not the intracellular delivery of proteins, but a controlled release of proteins in vivo. This study provides an efficient system for protein delivery into living cells with a very simple experimental procedure. Generally for intracellular delivery of protein, it is unavoidable to optimize the carriers according to various kinds of protein. We examined intracellular delivery of bovine serum albumin (BSA) as a model protein using pH-sensitive carbonate apatite for future functional protein delivery.

Fig. 4. Optical density of the suspension of CO3Ap particles and FITC-BSA/CO3Ap complexes against pH value.


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2. Materials and methods 2.1. Cell culture HeLa and NIH3T3 cell lines were cultured on tissue culture dishes in Dulbecco’s modified Eagle’s medium (Wako, Osaka, Japan) supplemented with 10% fetal bovine serum (FBS), 50 mg/mL penicillin and 50 mg/mL streptomycin at 37 C in a humidified 5% CO2-containing atmosphere. P19 cell lines were cultured on 0.1% (w/v) gelatincoated dishes in the same condition as other cell lines. 2.2. Fluorescent labeling of bovine serum albumin 500 mg of bovine serum albumin (Sigma, St. Louis, MO, USA) was added to 2 mL of 0.25 M bicarbonate buffer (pH 9.8). Nine mg of fluorescein isothiocyanate (Dojindo, Kumamoto, Japan) dissolved in 100 mL of DMSO was added to the protein solution and stirred slowly at 4 C overnight. Fluorescent-labeled BSA (FITC-BSA) was purified on a PD-10 column (GE Healthcare, Buckinghamshire, UK). Dye/protein ratio was calculated by measurement of fluorescence intensity (excitation, 485 nm; emission, 535 nm) with DTX 880 microplate reader (Beckman Coulter, Fullerton, CA, USA) and protein concentration with a Dc protein assay kit (Bio-Rad, Richmond, CA, USA), and determined to be 2.21. Prepared FITC-BSA was lyophilized and dissolved in PBS for experiments. 2.3. Preparation of CO3Ap particles and FITC-BSA/CO3Ap complexes We prepared two types of CO3Ap particles. One was obtained from DMEM containing 4.8 mM CaCl2 and the other was obtained from DMEM containing 7.8 mM CaCl2. At first, FITC-BSA (5–100 mg) was added to 1 mL of bicarbonate-buffered DMEM (Gibco, Invitrogen, Carlsbad, CA). Next, 3 or 6 mL of 1 M CaCl2 solution was added to the protein solution, resulting in 4.8 mM and 7.8 mM of the final concentration of CaCl2, respectively, and immediately mixed with vortex slowly. Mixed solution was incubated at 37 C for 30 min and FITC-BSA/CO3Ap complexes were obtained. For preparation of CO3Ap particles, bicarbonate-buffered DMEM prepared from powder was used just prior to experiments to avoid a change in carbonate concentration during storage. 2.4. Measurement of amount of protein loaded into CO3Ap particles One mL of FITC-BSA/CO3Ap complex suspensions obtained with low and high concentration of CaCl2 (4.8 mM and 7.8 mM, respectively) was centrifuged at 20,400 g for 15 min and the supernatant was removed. One mL of PBS was added to the particles and centrifuged again to remove free protein. Obtained particles were dissolved in 100 mL of 0.1 M O,O’-bis(2-aminoethyl)ethyleneglycol-N,N,N0 ,N’-tetraacetate (EGTA) solution. Additionally the solutions were diluted by adding 900 mL of PBS and fluorescence intensities (excitation, 485 nm; emission, 535 nm) using DTX 880 microplate reader (Beckman Coulter) was measured. The amounts of protein loaded into CO3Ap particles were calculated from the fluorescence intensity and a standard curve plotted as the concentration of FITC-BSA vs. the fluorescence intensity. 2.5. Particle size measurement with dynamic light scattering (DLS) photometer The distribution of particle size was measured using FPAR-1000 fiber-optics particle analyzer (Otsuka Electronics, Osaka, Japan) equipped with a 660 nm diode laser. The measurement was carried out by a scattering angle of 90 at 25 C. The size distribution was obtained by CONTIN method. 2.6. Observation of FITC-BSA/CO3Ap complexes with transmission electron microscope (TEM) FITC-BSA/CO3Ap complexes obtained with 50 mg/mL of FITC-BSA and high concentration of CaCl2 (7.8 mM) were observed with H-7500 transmission electron microscope (Hitachi, Tokyo, Japan) at an acceleration voltage of 80 kV. After loading of the particles on the copper grid with collodion membrane, particles were washed with 10 mM phosphate buffer (pH7.4) and dried in the air. 2.7. Estimation of the solubility of FITC-BSA/CO3Ap complexes in acidic solution

Fig. 5. Phase contrast and fluorescence microscopy images of the cells after delivery of FITC-BSA. HeLa, NIH3T3 and P19 cells were incubated with FITC-BSA/CO3Ap complexes prepared from DMEM containing 50 mg/mL of FITC-BSA and 7.8 mM CaCl2 for 4 h. After the incubation, the cells were washed with 5 mM EGTA solution to remove residual complexes and observed by fluorescence microscope. A. HeLa cells, B. NIH3T3 cells, and C. P19 cells. Bar indicates 50 mm.

At first, 200 mL of FITC-BSA/CO3Ap complex suspension was prepared from DMEM containing 50 mg/mL of FITC-BSA and 7.8 mM of CaCl2. And a pH of the suspension was decreased by adding 1 N HCl and 1 mL of the suspension at each pH was taken. Optical density at 320 nm of the collected samples was measured with SmartSpecÔ 3000 spectrophotometer (Bio-Rad). 2.8. Intracellular delivery of protein with CO3Ap nanoparticles Cells were seeded at 5.0 104 cells per well into 24 well plates on the day before intracellular delivery. One mL of FITC-BSA/CO3Ap complex suspension was prepared from DMEM containing 50 mg/mL of FITC-BSA. After the addition of fetal bovine

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Fig. 6. Vertical sectional views of HeLa cell after delivery of FITC-BSA. FITC-BSA was delivered into HeLa cells in the same way as the experiment of Fig. 5. After the delivery and removal of residual complexes, the cell membranes were stained with fluorescent dye DiI and observed by confocal laser scanning microscope. Yellow and pink lines indicate the position of vertical sectional views. Bar indicates 20 mm.

serum (FBS) to the suspension for a final concentration of 10% (v/v), the suspension was added to the rinsed cells and incubated at 37 C in a humidified 5% CO2containing atmosphere for 0.5–4 h. After incubation, the suspension was removed and the cells were washed with PBS three times. Additionally, the cells were washed with 5 mM of EGTA/PBS solution to remove the residual complexes completely and observed with fluorescent microscope. For confocal microscopy, HeLa cells were seeded at 1.5 104 cells per cm2 on 0.1% (w/v) collagen-coated glass coverslips (24 24 mm) on the day before intracellular delivery. FITC-BSA was added into HeLa cells in the same manner as mentioned above. After adding into the cells and removal of residual complexes, the cells were stained with VybrandtÒ DiI (Molecular Probes) according to the manufacturer’s protocol, fixed by treatment of 4% (v/v) formaldehyde at room temperature for 10 min and observed with FV300 confocal laser scanning microscope (Olympus, Tokyo, Japan). For the observation of endosomal escape of protein, FITC-BSA was added into HeLa cells on collagen-coated coverslips in the same manner as mentioned above. At 1 or 4 h after adding of the complexes into the cells, endosomes and lysosomes were stained with LysoTrackerÒ Red DND-99 (Molecular Probes) according to the manufacturer’s protocol and fixed with formaldehyde solution. Additionally, nuclei were stained with 40 ,6-diamino-2- -phenylindole (DAPI) and observed with A1 confocal laser scanning microscope (Nicon, Tokyo, Japan). 2.9. Flow cytometry analysis HeLa cells were seeded at 5.0 105 cells per 60 mm tissue culture dish on the day before intracellular delivery. Five mL of FITC-BSA/CO3Ap complex suspension prepared from DMEM containing 50 mg/mL of FITC-BSA was added into the cells. After the addition of FITC-BSA to the cells and removal of residual complexes, the cells were collected with 0.25% trypsin, 1.0 mM EDTA solution and washed with 5 mM EDTA solution and centrifugation. Additionally, the cells were washed again with PBS, centrifuged and fixed with 4% (v/v) formaldehyde solution. Before flow cytometry analysis, the cells were washed with PBS, centrifuged and then analysed with EPICS XL flow cytometer (Beckman Coulter). 2.10. Enzymatic activity staining for ß-galactosidase HeLa cells were seeded at 5.0 104 cells per well into 24 well plates on the day before intracellular delivery. One mL of ß-galactosidase/CO3Ap complex suspension was prepared from DMEM containing 20 mg/mL of Escherichia coli ß-galactosidase (Dojindo). After the addition of fetal bovine serum (FBS) to the suspension at a final concentration of 10% (v/v), the suspension was added to the rinsed cells and incubated for 4 h. After incubation, the residual complexes were removed with 5 mM EGTA solution. And then the cells were fixed with 1.25% (v/v) glutaraldehyde solution. After washing the cells, X-gal staining solution (50 mM Tris HCl solution at pH 7.5 containing 5 mM potassium ferrocyanate, 5 mM potassium ferricyanate, 15 mM sodium chrolide, 1 mM magnesium chrolide and 0.5 mg/mL of X-gal) was added to the cells and incubated at 37 C for 3 h. Then the cells were washed with PBS and observed with phase contrast microscope. 2.11. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay HeLa cells were seeded at 5.0 104 cells per well into 24 well plates on the day before intracellular delivery. FITC-BSA was added into HeLa cells in the same manner as mentioned above. After adding, one mL of fresh DMEM containing 0.15 mg/mL of MTT (Sigma) was added to the rinsed cells and the cells were incubated at 37 C for

3 h. Then medium was removed and 500 mL of DMSO was added to each well. After dissolving tetrazolium salt, the absorbance of the solutions at 570 nm with a reference wavelength of 630 nm were measured with a microplate reader. Cell viabilities were normalized to the absorbance of non-treated cells.

3. Results and discussion 3.1. Characterization of BSA/CO3Ap complexes For efficient intracellular delivery, CO3Ap particles must form stable complexes with protein, have small size enough to be endocytosed by cells and should release protein specifically from the endo-lysosomes into the cytosolic compartment. At first, loading of BSA into CO3Ap particles against concentration of BSA was performed (Fig. 1). The results indicated that loading content of BSA into CO3Ap particles significantly increased with an increase of BSA concentration due to ionic interaction between BSA and the CO3Ap particles obtained by simple mixing of BSA-containing DMEM with CaCl2 solution. And more amount of BSA was loaded to the CO3Ap particles obtained with high concentration of CaCl2 (7.8 mM) than the CO3Ap particles obtained with low concentration of CaCl2 (4.8 mM). DLS measurement showed that average particle sizes of BSA/CO3Ap complexes (CaCl2: 4.8 mM) as shown in Fig. 2 were 369.1 66.0 nm, although BSA/ CO3Ap complexes obtained with high concentration of CaCl2 (7.8 mM) had bigger particle sizes of 593.0 93.4 nm with a little aggregates. This result suggests that higher Ca concentration led to enlargement of particle sizes and/or more amount of particles due to higher supersaturation and resulted in higher BSA loading. The morphological observation of BSA/CO3Ap complexes by TEM imaging revealed that the particles are compact and spherical shapes with particle sizes about 500 nm as shown in Fig. 3. Recent advances in pH-sensitive degradable carrier development have introduced new dimensions for intracellular delivery of therapeutics such as genes and proteins [36,37] because these carriers easily degrade in the pH condition of the endo-lysosomes and the therapeutics rapidly escape into the cytosol without denaturation of the therapeutics. Fig. 4 shows the optical density of BSA/CO3Ap complexes and CO3Ap particles themselves as an indicator of particle existence. The results indicated that the optical density of BSA/CO3Ap complexes and CO3Ap themselves rapidly decreased with a decrease of pH, suggesting that the BSA/CO3Ap complexes were completely dissolved in the condition below pH 7.0 and the BSA can be rapidly escaped from the endo-lysosomes into the cytosol.


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3.2. Cellular uptake of BSA/CO3Ap complexes

positive

negative

count

A

96

0 100

80

104

positive

negative

:0h :0.5h :1h :2h :4h

count

B

101 102 103 fluorescence intensity

0 103 101 102 fluorescence intensity

C

120

Delivered cells (%)

100

100

104

To investigate the fate of BSA/CO3Ap complexes in vitro by standard characterization method, fluorescently-labeled BSA was used. Fig. 5A shows phase contrast and fluorescent microscopy images of HeLa cells after incubation of FITC-BSA/CO3Ap complexes and FITC-BSA at 37 C for 4 h. As shown in Fig. 5A, the cells incubated with FITC-BSA/CO3Ap complexes showed much fluorescence intensities whereas the cells incubated with only FITC-BSA showed less fluorescence ones. Similar results were obtained in other cell lines, NIH3T3 (mouse fibroblast) and P19 (mouse embryonic carcinoma) as shown in Fig. 5B and C. These results suggest that FITC-BSA was successfully delivered into the cells due to the complex formation between CO3Ap with FITC-BSA. On the other hand, only with fluorescent microscopy, it is difficult to confirm whether FITC-BSA is delivered inside the cell membrane or outside, just on the membrane surface. In order to confirm the distribution of intracellularly delivered FITC-BSA by CO3Ap, confocal microscopy was conducted. Fig. 6 shows confocal microscope images of the HeLa cells after incubation of FITC-BSA/ CO3Ap complexes at 37 C for 4 h. The cell membrane was stained with fluorescent dye, DiI. The results revealed that fluorescence was mainly originated within the cell interior, but not on the cell membrane surface, suggesting that almost all BSA was internalized in the cells. The fluorescence intensity of the cells was measured for the efficiency of FITC-BSA delivery into the cells with flow cytometry. As shown in Fig. 7A, the cells incubated with complexes showed strong fluorescence with 36.8 and 96.8% of the delivery efficiency for 4.8 and 7.8 mM CaCl2, respectively, whereas FITC-BSA was not able to internalize in the cells without CO3Ap. Interestingly, the complexes obtained with high concentration of CaCl2 provided higher efficiency of protein delivery into the cells compared with complexes obtained with low concentration of CaCl2 probably due to the more amount of complex formation in the condition of more CaCl2. The percentages of protein delivered cells by FITC-BSA/CO3Ap complexes were plotted against incubation time in Fig. 7B and C. The results indicated that the percentages of delivered cells were 57.3, 84.3, 96.2 and 99.0% for 0.5, 1, 2, 4 h incubation, respectively, suggesting that BSA was rapidly internalized with time, especially within 1 h after addition of complexes and BSA was internalized into almost all cells within 4 h. Therefore, it is suggested that CO3Ap particles successfully and efficiently delivered FITC-BSA into cells.

80 3.3. Endosomal release of FITC-BSA from FITC-BSA/CO3Ap complexes into cytoplasm

60 40 20 0

0h

0.5 h

1h

2h

4h

Fig. 7. Flow cytometry analysis of intracellular delivery of FITC-BSA. A. HeLa cells were incubated with FITC-BSA/CO3Ap complexes prepared from DMEM containing 50 mg/mL of FITC-BSA and CaCl2 (4.8 mM or 7.8 mM) for 4 h. After the incubation and removal of residual complexes, the cells were collected, fixed with formaldehyde and analyzed by flow cytometry. Black: non-treaed, blue: 50 mg/mL FITC-BSA, green: FITC-BSA/CO3Ap (50 mg/mL FITC-BSA, 4.8 mM CaCl2), red: FITC-BSA/CO3Ap (50 mg/mL FITC-BSA, 7.8 mM CaCl2). B. HeLa cells were incubated with FITC-BSA/CO3Ap complexes prepared from DMEM containing 50 mg/mL of FITC-BSA and 7.8 mM CaCl2 for each incubation time, 0, 0.5, 1, 2, 4 h. After the incubation and removal of residual complexes, the cells were collected, fixed with formaldehyde and analyzed by flow cytometry. C. The delivery efficiency of FITC-BSA with CO3Ap particles analyzed by flow cytometry in Fig. 7B. Data shown are means S.D. (n ¼ 3)

Once FITC-BSA/CO3Ap complexes enter the cells by endocytosis, the endosomes fuse with the cell lysosomes which may lead to degradation of the internalized BSA in the lysosomes [37]. To avoid the fate of lysosome degradation, it is very important to guide endosomal release of the internalized BSA into the cytoplasm. Fig. 8 shows confocal microscopy of the HeLa cells after incubation of FITC-BSA/CO3Ap complexes at 37 C for 4 h. Endosomes/lysosomes were stained with LysoTrackerÒ Red. As shown in the figure, after 1 h incubation with FITC-BSA/CO3Ap complexes, most of FITC-BSA fluorescence was co-localized in stained endosomes and/or lysosomes although after 4 h, only a few dots of FITC-BSA fluorescence was co-localized in stained endo-lysosomes. Moreover, some cells showed homogeneous fluorescence of FITC-BSA diffused into the cytosol, suggesting that many of the intracellularly delivered FITCBSA escaped from endo-lysosomes.

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Fig. 8. Endosomal release of FITC-BSA delivered into HeLa cells with CO3Ap particles. HeLa cells were incubated with FITC-BSA/CO3Ap complexes prepared from DMEM containing 50 mg/mL of FITC-BSA and 7.8 mM CaCl2 for 1 and 4 h. After the delivery and removal of residual complexes, the cells were fixed with formaldehyde. Endosomes and lysosomes were stained with LysotrackerÒ Red and nuclei were stained with DAPI. The cells were observed by confocal laser scanning microscope. Blue: DAPI, green: FITC-BSA, and Red: LysoTrackerÒ Red. Bar indicates 10 mm.

3.4. Enzymatic activity of intracellularly delivered protein During intracellular delivery and release of proteins to cytoplasm, there might be the possibility that delivered protein could be degraded or denatured, and thus its activity was lost. In order to confirm whether the delivered protein keeps its activity or not,

ß-galactosidase was used to facilitate the detection of its biological activity following delivery into HeLa cells because its activity can easily be detected with colorimetric substrate X-gal. As shown in Fig. 9, positive staining as the indication of ß-galactosidase activity in the HeLa cells was observed after 4 h at 37 C for the ß-galactosidase-loaded CO3Ap nanoparticles whereas ß-galactosidase

Fig. 9. Confirmation of enzymatic activity of ß-galactosidase intracellularly delivered with CO3Ap particles. HeLa cells were incubated with ß-galactosidase/CO3Ap complexes prepared from DMEM containing 20 mg/mL of ß-galactosidase and 7.8 mM CaCl2 for 4 h. After the delivery and removal of residual complexes, the cells were stained with X-gal. Bar indicates 50 mm.


activity was not found for only the ß-galactosidase itself, indicating that the delivered protein is sufficiently intact to retain its enzymatic activity. 3.5. Cytotoxicity of BSA/CO3Ap complexes In order to assess the cytotoxicity of FITC-BSA/CO3Ap complexes, MTT assay was conducted on HeLa cells after 4 h incubation with FITC-BSA/CO3Ap complexes. The complexes showed no significant cytotoxicity to HeLa cells (data not shown). 4. Conclusion We established an intracellular delivery system of protein using pH-sensitive carbonate apatite particles. Complexes of carbonate apatite with fluorescently-labeled BSA were dissolved in acidic solution and resulted in endosomal escape of proteins after uptake of the complexes into HeLa cells. Delivered proteins into HeLa cells by carbonate apatite particles did not show loss of their enzymatic activity. FITC-BSA/carbonate apatite complexes also showed no significant cytotoxicity in HeLa cells. Thus, carbonate apatite particle-mediated intracellular delivery system of protein is suggested as a highly effective method with a very simple experimental protocol. This intracellular protein delivery system using pH-sensitive carbonate apatite carrier will become a promising method in various fields of biology including molecular biology, cellular biology and tissue engineering. Acknowledgement This research is supported by grants from the Ministry of Education, Sports, Science and Technology of Japan. S. Tada is also supported by the Grant-in-Aid for Global COE program. Appendix Figures with essential color discrimination. Figs. 6–9 in this article are difficult to interpret in black and white. The full color images can be found in the on-line version, at doi:10.1016/j. biomaterials.2009.10.016. References [1] Tjian R, Fey G, Graessmann A. Biological activity of purified simian virus 40 T antigen proteins. Proc Natl Acad Sci U S A 1978;75(3):1279–83. [2] Richardson WD. Introducing proteins into cultured animal cells. J Cell Sci 1988;91(3):319–22. [3] Wang YL. Exchange of actin subunits at the leading edge of living fibroblasts: possible role of treadmilling. J Cell Biol 1985;101(2):597–602. [4] Yu CL, Tsai MH, Stacey DW. Cellular ras activity and phospholipid metabolism. Cell 1988;52(1):63–71. [5] McClung JK, Kletzien RF. Analysis of BHK cell growth kinetics after microinjection of catalytic subunit of cyclic AMP-dependent protein kinase. Mol Cell Biol 1984;4(6):1079–85. [6] Lissy NA, Davis PK, Irwin M, Kaelin WG, Dowdy SF. A common E2F-1 and p73 pathway mediates cell death induced by TCR activation. Nature 2000;407:642–5. [7] Jo D, Liu D, Yao S, Collins RD, Hawiger J. Intracellular protein therapy with SOCS3 inhibits inflammation and apoptosis. Nat Med 2005;11:892–8. [8] Bastiaens PIH, Pepperkok R. Observing proteins in their natural habitat: the living cell. Trends Biochem Sci 2000;25:631–7. [9] Xie J, Schultz PG. A chemical toolkit for proteins–an expanded genetic code. Nat Rev Mol Cell Biol 2006;7:775–82. [10] Spirin AS. High-throughput cell-free systems for synthesis of functionally active proteins. Trends Biotechnol 2004 Oct;22:538–45.

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[11] Stolnik S, Shakesheff K. Formulations for delivery of therapeutic proteins. Biotechnol Lett 2009;31:1–11. [12] Schwarze SR, Ho A, Vocero-Akbani A, Dowdy SF. In vivo protein transduction: delivery of a biologically active protein into the mouse. Science 1999;285:1569–72. [13] Takenobu T, Tomizawa K, Matsushita M, Li ST, Moriwaki A, Lu YF, et al. Development of p53 protein transduction therapy using membrane-permeable peptides and the application to oral cancer cells. Mol Cancer Ther 2002;1:1043–9. [14] Brustugum OT, Fladmark KE, Døskeland SO, Orrenius S, Zhivotovsky B. Apotosis induced by microinjection of cytochrome c is caspase-dependent and inhibited by Bcl-s. Cell Death Differ 1998;5:660–8. [15] Fenton M, Bone N, Sinclair AJ. The efficient and rapid import of a peptide into primary B and T lymphocytes and a lymphoblastoid cell line. J Immunol Methods 1998;212:41–8. [16] Luo D, Saltzman WM. Synthetic DNA delivery systems. Nat Biotechnol 2000;18:33–7. [17] Lee ALZ, Wang Y, Ye W, Yoon HS, Chan SY, Yang Y. Efficient intracellular delivery of functional proteins using cationic polymer core/shell nanoparticles. Biomaterials 2008;29:1224–34. [18] Lee Y, Ishii T, Cabral H, Kim HJ, Seo JH, Nishiyama N, et al. Charge-conversional polyionic complex micelles-efficient nanocarriers for protein delivery into cytoplasm. Angew Chem Int Ed Engl 2009;48:5309–12. [19] Ryser HJP, Hancock R. Histones and basic polyamino acids stimulate the uptake of albumin by tumor cells in culture. Science 1965;150:501–3. [20] Futami J, Maeda T, Kitazoe M, Nukui E, Tada H, Seno M, et al. Preparation of potent cytotoxic ribonucleases by cationization: enhanced cellular uptake and decreased interaction with ribonuclease inhibitor by chemical modification of carboxyl groups. Biochemistry 2001;40:7518–24. [21] McNaughton BR, Cronican JJ, Thompson DB, Liu DR. Mammalian cell penetration, siRNA transfection, and DNA transfection by supercharged proteins. Proc Natl Acad Sci U S A 2009;106:6111–6. [22] Fawell S, Seery J, Daikh Y, Moore C, Chen LL, Pepinsky B, et al. Tat-mediated delivery of heterologous proteins into cells. Proc Natl Acad Sci U S A 1994;91:664–8. [23] Futaki S, Suzuki T, Ohashi W, Yagami T, Tanaka S, Ueda K, et al. Arginine-rich peptides. An abundant source of membrane-permeable peptides having potential as carriers for intracellular protein delivery. J Biol Chem 2001;276:5836–40. [24] Stewart KM, Horton KL, Kelley SO. Cell-penetrating peptides as delivery vehicles for biology and medicine. Org Biomol Chem 2008;6:2242–55. [25] Chowdhury EH, Akaike T. A bio-recognition device developed onto nanocrystals of carbonate apatite for cell-targeted gene delivery. Biotechnol Bioeng 2005;90:414–21. [26] Chowdhury EH, Maruyama A, Kano A, Nagaoka M, Kotaka M, Hirose S, et al. pH-sensing nano-crystals of carbonate apatite: effects on intracellular delivery and release of DNA for efficient expression into mammalian cells. Gene 2006;376:87–94. [27] Zohra FT, Chowdhury EH, Akaike T. High performance mRNA transfection through carbonate apatite-cationic liposome conjugates. Biomaterials 2009;30:4006–13. [28] Chowdhury EH. pH-sensitive nano-crystals of carbonate apatite for smart and cell-specific transgene delivery. Expert Opin Drug Deliv 2007;4:193–6. [29] Chowdhury EH. Self-assembly of DNA and cell-adhesive proteins onto pH-sensitive inorganic crystals for precise and efficient transgene delivery. Curr Pharm Des 2008;14:2212–28. [30] Graham FL, van der Eb AJ. Transformation of rat cells by DNA of human adenovirus 5. Virology 1973;54(2):536–9. [31] Jordan M, Schallhorn A, Wurm FM. Transfecting mammalian cells: optimization of critical parameters affecting calcium-phosphate precipitate formation. Nucleic Acids Res 1996;24:596–601. [32] Bisht S, Bhakta G, Mitra S, Maitra A. pDNA loaded calcium phosphate nanoparticles: highly efficient non-viral vector for gene delivery. Int J Pharm 2005;288:157–68. [33] Paul W, Sharma CP. Development of porous spherical hydroxyapatite granules: application towards protein delivery. J Mater Sci Mater Med 1999;10:383–8. [34] Midy V, Hollande E, Rey C, Dard M, Ploue¨t J. Adsorption of vascular endothelial growth factor to two different apatitic materials and its release. J Mater Sci Mater Med 2001;12:293–8. [35] Matsumoto T, Okazaki M, Inoue M, Yamaguchi S, Kusunose T, Toyonaga T, et al. Hydroxyapatite particles as a controlled release carrier of protein. Biomaterials 2004;25:3807–12. [36] Panyam J, Zhow WZ, Prabra S, Sahoo SK, Labhasewar V. Rapid endo-lysosomal escape of poly(DL-lactide-co-glycolic) nanoparticles; implications for drug and gene delivery. FASEB J 2002;16:1217–26. [37] Kim NWS, Dai H. Carbon nanotubes as intracellular protein transporters: generality and biological functionality. J Am Chem Soc 2005;127:6021–6.