Estimated Molecular Structure of a Carbon Nanotubec Molecular Heater Based on Binding Properties to a Target Protein

Abstract

Background

Carbon nanotubes exhibit strong absorbance in the near-infrared (NIR) region and are considered as potent candidates for hyperthermic therapy because they generate significant amounts of heat upon excitation with NIR light. We prepared a single-walled carbon nanotube (SWNT)/IgG complex to use as a “smart molecular heater” for hyperthermic therapy.

Purpose

The aim of the present study was to assess the binding efficiency of DNA-functionalized SWNT/IgG complexes to a target protein.

Methods

3 types of complexes with different lengths of spacer arm chain (13.5, 29, and 56 Å) linked to biotinylated IgG were prepared, and we evaluated the effect of the spacer arm length on the specificity, affinity, and capacity of binding to a target protein.

Results

Complexes with longer spacer lengths showed increased binding affinity to a target protein. This could be due to a reduction in steric hindrance by increasing the segmental flexibility of the spacer arm.

Conclusions

The results of this study suggested that DNA-functionalized SWNT/IgG complexes could act as a heating nano-device for hyperthermic cancer therapy, and the complexes can bind various types of tumor by modifiying the specific antibody.

Keywords

Carbon nanotubes DNA functionalization Complex Antibody Binding ability

Introduction

Carbon nanotubes have been widely studied as multifunctional nanomaterials due to their unique electronic, mechanical, and chemical properties (1-2-3-4-5). Furthermore, carbon nanotubes have received considerable interest from the biomedical field in areas such as drug and gene delivery, scaffolds for tissue engineering, biosensing, and diagnostics (6-7-8-9-10-11-12).

Hyperthermic therapy using carbon nanotubes has been suggested as an efficient strategy for cancer treatment (13-14-15-16-18). Carbon nanotubes exhibit strong absorbance in the near-infrared region (NIR) and are considered as potent candidates for hyperthermic therapy because they generate significant amounts of heat upon excitation with NIR light.

In our previous study (19), we prepared a model complex comprising a single-walled carbon nanotube (SWNT) and an IgG antibody to use the complex as a “smart molecular heater” for hyperthermic therapy in cancer treatment. When the complex of SWNT and IgG was employed as an exothermic nano-device for hyperthermic therapy, stable dispersibility and specific binding efficiency to the targeted protein in body fluid were essential factors. For the stable dispersibility of the complex in body fluid, we treated the complex with double-stranded DNA (20). This DNA-functionalized SWNT complex showed improved stability and solubility in culture medium. Furthermore, DNA functionalization did not cause changes in binding affinity between the immobilized antibody in the complex and the targeted protein (19).

To prepare the complex, biotinylated IgG was bound to streptavidin molecules immobilized on the SWNT via a streptavidin-biotin coupling reaction. From the kinetic analysis of the binding parameters, we considered that segmental mobility of biotinylated IgG could affect the binding efficiency (19). The SWNT/IgG complex prepared in a previous study had a spacer arm 13.5 Å in length between the biotinyl group and the IgG molecule. Although a longer spacer arm length could confer a more flexible nature to the biotinylated IgG, the effect of spacer arm length on the specific binding efficiency to the targeted protein was not clear.

The purpose of this study was to evaluate the effect of spacer arm length on the SWNT/IgG complex in terms of specific binding efficiency to the target protein. If the effect of the chemical structure of the spacer arm on the binding efficiency of the IgG is clarified, design of a suitable SWNT/IgG complex model with efficient specific binding ability and stable dispersibility in body fluids could be established. For this purpose, we designed 3 types of biotinylated IgG with different spacer arm lengths, and immobilized these IgG segments on the DNA-functionalized SWNT. Three different DNA-functionalized SWNT/IgG complexes were evaluated with regards to the effect of spacer arm length on the specificity, affinity, and capacity of binding to the targeted protein.

Materials and Methods

Preparation of DNA-functionalized SWNT/IgG (anti-human IgG antibody) complexes

DNA treatment of SWNT to achieve stable dispersibility and streptavidin binding to the DNA-functionalized SWNT was carried out in accordance with the method described previously. Briefly, SWNT (400 mg, CG100; South West Nanotechnology, Norman, OK, USA) was poured into a mixture of 300 mL of sulfonic acid and 100 mL of nitric acid, and the mixture was sonicated for 7 h. This acid-treated SWNT was collected by filtration (0.2 μm pore size polytetrafluoroethylene membrane). The DNA-functionalized SWNT was prepared by sonication of a mixture of acid-treated SWNT (10 mg) and 300 bp of double-stranded DNA (100 mg, derived from salmon testis; Maruha-Nichiro Corporation, Tokyo, Japan) in 10 mL of deionized water for 1 h. Streptavidin was attached covalently onto the DNA-functionalized SWNT via an N-hydroxysuccinimide and 1-ethyl-3(3-dimethylaminopropyl) carbodiimide hydrochloride coupling reaction to yield stable amide linkages. The DNA-functionalized SWNT/IgG complexes were prepared using a streptavidin-biotin reaction of the DNA-functionalized SWNT/streptavidin and biotinylated IgG.

Three different types of biotinylated IgG (anti-human IgG antibody, generated in donkey; Rockland Immunochemicals, Gilbertsville, PA, USA) were purchased from Medical & Biological Laboratories (Nagoya, Japan). These biotinylated IgGs had different spacer arm lengths between the IgG antibody molecule and the biotin site, corresponding to 13.5 Å, 29 Å, and 56 Å. Schematic chemical structures of these biotinylated IgG are shown in Figure 1.

Fig. 1

Chemical structure of biotinylated IgG segments with different lengths of spacer chain. The spacer chain lengths were 13.5 Å (A), 29 Å (B), and 56 Å (C). Each spacer arm reacted amino groups of anti-human IgG and formed amide bonds. Biotin group in the spacer arm can bind to avidin molecule bound to the SWNT.

The streptavidin-bound DNA-functionalized SWNT complex (3 mg) was dissolved in 3 mLl of distilled water, and 300 μg of the biotinylated IgG added. The mixture was gently stirred for 2 h at 5°C. The resulting solution was filtered using a polytetrafluoroethylene membrane (0.2 μm pore size) to collect the DNA-functionalized SWNT/IgG (anti-human IgG antibody) complex. The filtered complex was adjusted to a concentration of 500 μg/ml (based on amount of SWNT) in phosphate-buffered saline (containing 0.01 % n-dodecyl-b-D-maltoside [DDM]; Dojindo, Kumamoto, Japan) using ultraviolet absorbance. These DNA-functionalized SWNT/IgG complexes with spacer arms of 13.5 Å, 29 Å, and 56 Å in length were coded as DNA-functionalized SWNT/IgG complex (S), (M), and (L), respectively.

Measurement of binding ratio using a quartz crystal microbalance

The quartz crystal microbalance (QCM, Affinix C4; Initium, Chigasaki, Japan) contained a gold-coated quartz sensor with a fundamental frequency of 27 MHz. A frequency decrease of 1 Hz in the QCM system was calibrated as an increase in 0.03 ng (21, 22).

The QCM sensor was exposed to 100 μL of phosphate-buffered solution containing 1 μg of Protein G (recombinant; Thermo Scientific, Rockford, IL, USA) for 1 h at room temperature to reduce nonspecific binding of antibody molecules to the sensor surface. After rinsing with phosphate-buffered solution to remove unbound Protein G, the sensor was exposed to 500 μL of phosphate-buffered solution, and a human IgG solution (Chrom Pure, whole molecule; Jackson Immuno Research Laboratories, West Grove, PA, USA) was injected (final concentration 50 μg/ml). The amount of human IgG immobilized on the QCM sensor was determined from the frequency shift.

The human IgG-coated sensor was exposed to 500 μL of TE buffer containing 0.15 M NaCl and 0.01% DDM, and the DNA-functionalized SWNT/IgG complex (S) (final concentration 50 μg/ml) was injected. The amount of complex bound to the human IgG antibody was determined from the frequency shift. The amounts of DNA-functionalized SWNT/IgG complex (M) and (L) bound to the human IgG antibody were determined in the same manner as described above.

Detection of binding of the complexes to target protein

Ninety-six well polystyrene microplates (BD Biosciences, Franklin Lakes, NJ, USA) were incubated overnight at 4°C with 100 μL of 10 μg/ml human IgG (Chrom Pure, whole molecule; Jackson Immuno Research Laboratories). After washing with phosphate-buffered saline (PBS), the wells were blocked with 3% bovine serum albumin (BSA) dissolved in PBS containing 0.01% DDM for 30 min. Then, the solution was replaced with different concentrations of either biotinylated anti-human IgG antibodies (generated in donkey; Rockland Immunochemicals) or a complex of streptavidin-bound SWNT molecules and biotinylated IgG antibodies (SWNT/IgG complex), each of which was dissolved in PBS containing 1% BSA, 0.01% DDM, and DNA (1 mg/ml) (buffer 1). Three types of biotinylated IgG antibodies were used, which possessed spacers of different lengths between biotin and IgG. The SWNT-IgG complex was functionalized using DNA. After incubation at 37°C for 60 min to 90 min, the wells were washed 3 times with 0.05% Tween 30-containing PBS (buffer 2).

To detect the SWNT/IgG complex, the wells were treated for 1 h to 1.5 h each at room temperature with mouse monoclonal anti-streptavidin antibody (×1000, S3E11; Pierce Biotechnology, Rockford, IL, USA) and then goat monoclonal anti-mouse IgG antibody (light chain-specific, cross-reacted minimally with other species) conjugated with horseradish peroxidase (HRP) (×2000, Jackson Immuno Research Laboratories), both of which were dissolved in buffer 1. The wells were washed extensively with buffer 2 after each treatment. To detect the biotinylated antibodies without SWNT, streptavidin (1 μg/ml) was firstly bound to the biotin-side chain, and then incubated with the antibodies in the above order. Prior to the HRP assay, absorbance at 450 nm was measured as a blank (AbsBlank). Thereafter, the HRP assay was performed using 3, 3’, 5, 5’-tetramethylbenzidine (TMB) as a substrate and an excess amount of H₂O₂ (TMB substrate reagent set; BD Falcon). The reagents were incubated in the wells for 0 to 15 min, followed by stopping the reaction by adding 1 M phosphoric acid. The absorbance (AbsTMB) of the reaction mixture at 450 nm was measured, being dependent on the production of oxidized TMB. The difference value, AbsTMB - AbsBlank, was calculated to obtain the amount of oxidized TMB. The initial production rate of oxidized TMB was calculated on the basis of the Lambert-Beer law, and the molar extinction coefficient at 450 nm (e450) of oxidized TMB was reported as 5.9 × 10⁴ M⁻¹ cm⁻¹.

Statistics

All values are presented as means ± S.E.M (n, number of observations). Statistical analysis was performed using an unpaired t test. A P value less than .05 was considered to be statistically significant.

Results

Preparation of 3 types of DNA-functionalized SWNT/IgG complexes

The prepared DNA-functionalized SWNT/IgG complexes were easily dispersible in deionized water or PBS. The suspensions of these complexes were stable for at least 2 weeks at 4°C without aggregation or precipitation.

Measurement of binding ratios of DNA-treated SWNT/IgG complexes using QCM

The amount of immobilized human IgG antibody on the QCM sensor was 24.9 ± 1.24 ng (n = 7). Figure 2 plots the changes in frequency observed following immobilization of different types of complexes. Adsorption of DNA-functionalized SWNT/IgG complex (S) resulted in a decrease in frequency of 1016 Hz, whereas DNA-functionalized SWNT/IgG complex (M) and DNA-functionalized SWNT/IgG complex (L) showed larger decreases in frequency of 2800 Hz and 3577 Hz, respectively. DNA-functionalized SWNT/IgG complexes with a longer spacer lengths (107.3 ± 3.90 ng for DNA-functionalized SWNT/IgG complex (L)) showed greater amounts of human IgG antibody binding than those with a shorter spacer length (30.5 ± 3.70 ng for DNA-functionalized SWNT/IgG complex (S)).

Fig. 2

Typical frequency changes for DNA-functionalized SWNT/IgG complexes binding to immobilized human IgG to the quartz crystal microbalance sensor. (A) DNA-functionalized SWNT/IgG complex (S) with 13.5 Å of spacer arm length; (B) DNA-functionalized SWNT/IgG complex (M) with 29 Å of spacer arm length; and (C) DNA-functionalized SWNT/IgG complex (L) with 56 Å of spacer arm length.

Analysis of binding of the DNA-functionalized SWNT/IgG complex with different spacer arm lengths to a target molecule

Our previous binding analysis revealed that DNA functionalization to improve the dispersability of SWNT did not interfere with the binding affinity or capacity between the immobilized antibody and the targeted protein. Here, in the presence of DNA, we compared IgG binding for different spacer arm lengths to the target molecule.

Figure 3 shows typical data for the binding between 3 types of DNA-functionalized SWNT/IgG complexes and the target molecule. The upper panels indicate the concentration-dependent binding of the complex to its target. A striking difference in the saturation curve was seen with the smallest slope for DNA-functionalized SWNT/IgG (S) complex, which had the shortest spacer of 13.5 Å. From the Lineweaver-Burk plots of the data (Fig. 3B), the affinity constant (Km, SWNT) and maximum reaction (V_max, SWNT) were estimated (Tab. I). The estimated Hill coefficient (n_H) was close to unity, indicating that the reaction obeyed the Michaelis-Menten formula, and a reaction occurred between the DNA-functionalized SWNT/IgG complex and the target molecule (Tab. I). The Km value based on the concentration of the IgG (Km, IgG(SWNT)) for the DNA-functionalized SWNT/IgG with a spacer length of 13.5 Å was 7.7- and 11.7-fold higher than those for the 29.0 Å and 56 Å spacers, respectively (Tab. I). This suggested that a short spacer reduced the binding affinity.

Fig. 3

Binding of the DNA-functionalized SWNT/IgG complex to its specific target. (A) Typical saturation curve. (B) Lineweaver-Burk plots. The data derived from the saturation curve were fitted to the dashed line. The line represents 1/Vmax (obtained from ordinate intercept) and 1/Km (obtained from abscissa intercept). (a) DNA-functionalized SWNT/IgG complex (S) with 13.5 Å of spacer arm length; (b) DNA-functionalized SWNT/IgG complex (M) with 29 Å of spacer arm length; and (c) DNA-functionalized SWNT/IgG complex (L) with 56 Å of spacer arm length.

Table I

Binding affinity and capacity of swnt-bound, biotinylated IgG with different spacer lengths to a target molecule

Spacer length (Å)	Code	n _H	K_m,SWNT (mg/ml)	K_m,IgG(SWNT) (mol/l)	pK_m,IgG(SWNT)	V_{max, SWNT} (fmol/min)
13.5	SWNT/IgG complex (S)	0.992 ± 0.003	110.8 ± 8.9	(42.11 ± 3.40) × 10⁻⁸	6.38 ± 0.03	1325 ± 219
29.0	SWNT/IgG complex (M)	0.999 ± 0.001	14.6 ± 6.3	(5.54 ± 2.39) × 10⁻⁸	7.33 ± 0.17^*	1430 ± 120
59.0	SWNT/IgG complex (L)	0.993 ± 0.003	9.4 ± 4.7	(3.56 ± 1.78) × 10⁻⁸	7.55 ± 0.20^*	1248 ± 13

K_m values based on the concentration of IgG (K_m,IgG(SWNT)) were calculated according to Lineweaver-Burk plots and QCM data. The V_max for SWNT-bound IgG (V_{max, SWNT}) was normalized with the corresponding data for IgG itself (V_max,_IgG) to obtain the relative V_max value (V_{max, SWNT}/V_{max, IgG}). n_H = Hill coefficient; pK_m,IgG(SWNT) = -log K_m,IgG(SWNT).

P <.01

compared with the group with a spacer length of 13.5 Å (n = 3).

To elucidate whether a bulky structure of SWNT is prerequisite for the importance of the spacer length in determining the binding affinity, we measured the binding ability between IgGs themselves with different spacer lengths and a target molecule. The n_H value was close to unity. For the Km, the IgG value was the same among the 3 types of IgG molecules even if they possessed different spacer lengths (Tab. II), indicating the importance of distance from the bulky SWNT attached to IgG.

Table II

Binding affinity and capacity of biotinylated IgG with different spacer lengths to a target molecule

Spacer length (Å)	Code	n _H	K_m,SWNT (mg/ml)	K_m,IgG(SWNT) (mol/l)	p_{Km, IgG(SWNT)}	V_{max, SWNT} (fmol/min)
13.5	SWNT/IgG complex (S)	0.999 + 0.001	34.6 + 5.1	(2.30 + 0.34) × 10⁻¹⁰	9.65 + 0.06	1220 + 82
29.0	SWNT/IgG complex (M)	0.999 + 0.001	24.8 + 2.0	(1.66 + 0.14) × 10⁻¹⁰	9.78 + 0.04	1367 + 50
59.0	SWNT/IgG complex (L)	0.999 + 0.001	27.7 + 1.4	(1.85 + 0.09) × 10⁻¹⁰	9.73 + 0.02	1392 + 44

K_m values based on the concentration of the IgG (K_m,IgG) were calculated according to the Lineweaver-Burk plots and QCM data. n_H = Hill coefficient; pK_m,IgG = -log K_m,IgG. No significant difference compared with the group with a spacer length of 13.5 Å (n = 3).

Next, we compared the binding capacity of either the DNA-functionalized SWNT/IgG complex or IgG itself with the different spacer lengths (Tabs. I and II). The spacer length affected neither V_max nor the relative V_max (V_max, SWNT/V max, IgG) for the DNA-functionalized SWNT/IgG complex to that for IgG themselves.

Discussion

Our strategy centered on whether the SWNT/IgG complex could be used as a “smart molecular heater” nano-device for hyperthermic cancer therapy. The DNA-functionalized SWNT/IgG complex consists of three units, i.e., functionalized-SWNT, an antibody molecule, and a spacer arm chain between SWNT and the antibody molecule. Each unit contributed its own characteristics to the DNA-functionalized SWNT/IgG complex. In our previous studies, we prepared a model complex of SWNT and an IgG antibody, and we discussed the effect of each unit on dispersion stability in body fluid and selective binding to the targeted protein (19).

To achieve selective targeting to cells, we conjugated the DNA-functionalized SWNT with certain ligands that specifically recognize targeted proteins. This model complex system had an anti-human IgG antibody as a ligand and showed good specific binding ability for the targeted protein (human IgG). The antibody molecule covalently bound to SWNT via the streptavidin/biotin reaction. However, the QCM results of the previous study revealed that 1 biotinylated IgG molecule bound to 1 streptavidin molecule immobilized on SWNT. Although streptavidin has 4 sites for biotin recognition, 3 sites were reported not to contribute to further biotin recognition (23).

We believe that the segmental mobility of the biotinylated IgG molecule could affect the binding efficiency to streptavidin sites immobilized on SWNT (19). Because the biotinylated IgG used in the previous study had a spacer arm 13.5 Å in length, a longer spacer arm length could endow the biotinylated IgG with a more flexible nature. Therefore, we designed 3 types of biotinylated igG with different spacer arm lengths (13.5, 29 and 56 Å). As expected, DNA-functionalized SWNT/IgG complex (L) (with the longer spacer arm length) showed better specific binding ability for the targeted protein than DNA-functionalized SWNT/IgG complex (S) (with the shorter spacer arm length). The spacer arm chain consisted of polyethylene glycol, and each ethylene glycol unit would contribute to the segmental mobility of the spacer arm. Okahata et al (24) reported that the size of the targeted molecule and the spacer arm mobility reduced the steric hindrance of the DNA-hybridization reaction. These results indicated that the steric hindrance is relatively small when an antibody molecule is immobilized through a long spacer arm chain for the streptavidin-biotin interaction. From the results of binding affinity measurements, the Km value of DNA-functionalized SWNT/IgG complex (S) with a spacer arm length of 13.5 Å was 7.7- and 11.7-fold higher than those of DNA-functionalized SWNT/IgG complex (M) (29 Å) and DNA-functionalized SWNT/IgG complex (L) (56 Å), respectively (Tab. I). In contrast, the Km value of biotinylated IgGs for the targeted protein was the same among the 3 types of IgG molecules even if they possessed different spacer arm lengths (Tab. II). These results suggested that a longer spacer arm length increased the binding affinity to the targeted protein, and segmental mobility of the spacer arm length could be a key factor for good binding affinity of the complex.

Based on the results obtained from this study, we estimated the chemical structure model of the DNA-functionalized SWNT/IgG complex as illustrated in Figure 4. DNA functionalization leads to stable dispersibility of SWNT in body fluid (25, 26). By assuming that the carbon atoms of SWNT used in this study number 37,000, 1.73 molecules of IgG were immobilized on one DNA-treated SWNT/IgG complex (19). This complex had selective binding ability to the targeted tumor cell and would have exothermic properties triggered by NIR irradiation. NIR irradiation induces local heating of a tumor region and can trigger thermal cell death without damage to normal cells (27, 28). Furthermore, the temperature of the local heating could be regulated by tuning the NIR light intensity. The prepared DNA-functionalized SWNT/IgG complex could bind different targeted proteins by replacing a different type of biotinylated IgG attached to the SWNT. This finding suggested that if the DNA-functionalized SWNT/IgG complex can be modified with a specific antibody for other tumor cells, the complex could selectively bind to the corresponding tumor cells and act as a heating nano-device for hyperthermic cancer therapy.

Fig. 4

Estimated molecular structure of the DNA-functionalized SWNT/IgG complex based on the results of our works. The number of biotinylated IgG spacer arms was estimated from the results of our research. (A) SWNT (molecular heater platform); (B) double-strands DNA (stable solubilizer of SWNT); (C) Streptavidin (covalently bound to SWNT); (D) biotinylated terminal of the spacer arm (bound to Streptavidin); (E) specific IgG molecule; (F) biotinylated IgG spacer arm.

In summary, we prepared 3 types of DNA-functionalized SWNT/IgG complex with different spacer arm lengths between SWNT and the antibody molecule (13.5, 29, and 56 Å). The complex with a longer spacer length increased the binding affinity to a targeted protein, due to a reduction in steric hindrance of the spacer arm. The segmental mobility of the spacer arm could be a key factor for good binding affinity of the DNA-functionalized SWNT/IgG complex. These complexes have stable dispersibility and good selective binding ability to targeted proteins. The results of this study suggested that a DNA-functionalized SWNT/IgG complex with refined chemical structure can act as a “smart molecular heater” nano-device for hyperthermic cancer therapy.

Footnotes

Acknowledgement

The authors are grateful to Maruha-Nichiro Corporation, Ltd. for supplying salmon testes DNA. We would like to thank Dr. Tadao Fukushima (Center for Regenerative Medicine, Fukuoka Dental College), Dr. Jun Ohno (Pathology Section, Fukuoka Dental College), and Dr. Naotoshi Nakashima (Department of Applied Chemistry, Graduate School of Engineering, Kyushu University) for helpful discussions.

Financial support: This study was supported in part by CREST, JST (Japan Science and Technology Agency) and by a Grant-in-aid for strategic study base formation support business (S1001059) from the Japan Society for the Promotion of Science.

Conflict of interest: The authors report no conflicts of interest in this work.

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