Accepted Manuscript Hyaluronic acid-modified multiwalled carbon nanotubes for targeted delivery of doxorubicin into cancer cells Xueyan Cao, Lei Tao, Shihui Wen, Wenxiu Hou, Xiangyang Shi PII: DOI: Reference:
S0008-6215(14)00275-4 http://dx.doi.org/10.1016/j.carres.2014.06.030 CAR 6791
To appear in:
Carbohydrate Research
Received Date: Revised Date: Accepted Date:
25 April 2014 25 June 2014 27 June 2014
Please cite this article as: Cao, X., Tao, L., Wen, S., Hou, W., Shi, X., Hyaluronic acid-modified multiwalled carbon nanotubes for targeted delivery of doxorubicin into cancer cells, Carbohydrate Research (2014), doi: http:// dx.doi.org/10.1016/j.carres.2014.06.030
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Hyaluronic acid-modified multiwalled carbon nanotubes for targeted delivery of doxorubicin into cancer cells
Xueyan Cao, Lei Tao, Shihui Wen, Wenxiu Hou, Xiangyang Shi* College of Chemistry, Chemical Engineering and Biotechnology, Donghua University, Shanghai 201620, P. R. China
_________________________________________________ *To whom correspondence should be addressed. Phone: +86 21 67792656; Fax: +86 21 67792306-804. E-mail: [email protected]
1
Abstract Development of novel drug carriers for targeted cancer therapy with high efficiency and specificity is of paramount importance and has been one of the major topics in current nanomedicine. Here we report a general approach to using multifunctional multiwalled carbon nanotubes (MWCNTs) as a platform to encapsulate an anticancer drug doxorubicin (DOX) for targeted cancer therapy. In this approach, polyethyleneimine (PEI)-modified MWCNTs were covalently conjugated with fluorescein isothiocyanate (FI) and hyaluronic acid (HA). The formed MWCNT/PEI-FI-HA conjugates were characterized via different techniques and were used as a new carrier system to encapsulate the anticancer drug doxorubicin for targeted delivery to cancer cells overexpressing CD44 receptors. We show that the formed MWCNT/PEI-FI-HA/DOX complexes with a drug loading percentage of 72% are water soluble and stable. In vitro release studies show that the drug release rate under an acidic condition (pH 5.8, tumor cell microenvironment) is higher than that under physiological condition (pH 7.4). Cell viability assay demonstrates that the carrier material has good biocompatibility in the tested concentration range, and the MWCNT/PEI-FI-HA/DOX complexes can specifically target cancer cells overexpressing CD44 receptors and exert growth inhibition effect to the cancer cells. The developed HA-modified MWCNTs hold a great promise to be used as an efficient anticancer drug carrier for tumor-targeted chemotherapy.
Key words：Multiwalled carbon nanotubes; hyaluronic acid; doxorubicin; targeted cancer therapy
2
1. Introduction Although chemotherapy has been one of the most important methods to treat cancer, many anticancer drugs, like doxorubicin (DOX), not only kill the cancerous tissues, but also lead to adverse side-effect on normal tissues.1-2 In addition, many cancer drugs lack water solubility, significantly limiting their bioavailability. Therefore, it is essential to develop a drug delivery carrier with targeting specificity,3-5 good biocompatibility,6-8 sufficient stability,9-11 and long circulation time.12-13 Currently, a wide range of nanocarrier systems, including but not limited to liposomes,14 nanoparticles,15-16 nanogels,17 polymer micelles,18-19 and dendrimers20 have been developed to both improve the water solubility of the anticancer drug and achieve the specific targeting drug delivery. Carbon nanotubes (CNTs)21-23 have gained tremendous attention as a promising nanocarrier owing to their distinct characteristics, such as ultrahigh surface area, high drug-loading capability, effective transportation capability, and enhanced cellular uptake.24-26 For biomedical applications, the poor aqueous dispersibility and high aggregation tendency of pristine CNTs can be resolved by appropriate surface functionalization via either non-covalent or covalent modification strategies,27-28 rendering the CNTs water soluble and highly biocompatible.29-30 In our previous work, we have shown that polyethyleneimine (PEI) covalently linked onto the surface of acid-treated multiwalled carbon nanotubes (MWCNTs) can be further modified with different surface functional groups for biomedical applications.30-31 Our results reveal that the water-dispersibility and biocompatibility of PEI-modified MWCNTs can be significantly improved after further modification of the PEI amines with acetyl groups or polyethylene glycol moieties. For targeted drug delivery applications, the MWCNTs can be modified with amine-terminated poly(amidoamine) dendrimers pre-modified with fluorescein isothiocyanate (FI) and folic acid (FA).32-33 The developed multifunctional MWCNT-based delivery system can achieve a high drug 3
payload of anticancer drug doxorubicin (DOX) with a pH-responsive release property showing fast DOX release under acidic environment and slow release at a physiological pH condition. Importantly, the complexes were able to target cancer cells overexpressing high-affinity FA receptors and effectively inhibit the growth of the cancer cells. These prior successes lead us to hypothesize that the MWCNTs may be covalently modified with other targeting ligands for targeted cancer therapy applications. Hyaluronic acid (HA) is a linear polysaccharide consisting of repeating disaccharide units of D-glucuronic acid and N-acetyl-D-glucosamine.34-35 HA has unique and excellent physicochemical properties, such as biodegradability, biocompatibility, and nonimmunogenicity.36-37 Besides, it is also known that HA has a strong affinity to bind cell-specific surface markers such as cluster determinant 44 (CD44) receptors,38-39 which are overexpressed on the surface of several different cancer cells.39 Such interesting selectivity of HA to CD44 receptor-overexpressing cancer cells has shown a great application potential in cancer diagnosis and therapy.40-44 In this present study, we synthesized and characterized multifunctional MWCNTs modified with FI and HA (MWCNT/PEI-FI-HA) as a new anticancer drug carrier for target delivery of DOX to cancer cells overexpressing CD44 receptors. Acid-treated MWCNTs were first covalently modified with PEI, followed by sequential modification with FI and HA (Figure 1). The formed MWCNT/PEI-FI-HA conjugates were characterized using different methods. The loading of DOX onto the multifunctional MWCNTs and the release of DOX from the MWCNTs were monitored via UV-vis spectrometry. The targeted therapeutic efficacy of MWCNT/PEI-FI-HA/DOX complexes in vitro was investigated in detail.
2. Experimental 2.1. Materials 4
MWCNTs (diameter = 30-70 nm, length = 100 nm-2 µm) were synthesized and characterized in a previous report.45 HA (Mw = 31,200) was obtained from Zhenjiang Dong Yuan Biotechnology Corporation (Zhenjiang, China). Hyperbranced PEI (Mw≈25,000) and all other chemicals and solvents were purchased from Aldrich (St. Louis, Missouri) and used as received. HeLa cells (a human cervical carcinoma cell line) and L929 cells (a mouse fibroblast cell line) were from Institute of Biochemistry and Cell Biology, the Chinese Academy of Sciences (Shanghai, China). RPMI 1640 medium, DMEM medium, fetal bovine serum (FBS), penicillin, and streptomycin were purchased from Hangzhou Jinuo Biomedical Technology (Hangzhou, China). Regenerated cellulose dialysis membranes with molecular weight cut-off (MWCO) at 14,000 and 50,000 were acquired from Fisher (Pittsburgh, PA). Water used in all experiments was purified using a Milli-Q Plus 185 water purification system (Millipore, Bedford, MA) with resistivity higher than 18 M Ω • cm.
2.2. Synthesis of MWCNT/PEI-FI-HA conjugates The modification of MWCNTs with PEI to form MWCNT/PEI was adapted from our previous report.30 Briefly, acid-treated MWCNTs with carboxyl residues (120.0 mg) dispersed in DMSO (50 mL) were activated with EDC (120.0 mg) and NHS (62.0 mg) co-dissolved into DMSO (5 mL) under vigorous magnetic stirring. The reaction was continued for 3 h to activate the carboxyl residues of MWCNTs, followed by the addition of PEI solution (120 mg in 12 mL DMSO). The reaction mixture was sonicated for 2 d to obtain MWCNT/PEI conjugates. Finally, the DMSO and the excess of reactants and byproduct were removed from the reaction mixture by extensive dialysis against phosphate buffered saline (PBS) solution (3 times, 2 L) and water (5 times, 2 L) using a dialysis membrane with MWCO of 50, 000 for 3 d. The MWCNT/PEI conjugates were obtained following lyophilization. 5
Multifunctional MWCNTs (MWCNT/PEI-FI-HA) were synthesized by conjugating FI and HA onto the surface of MWCNT/PEI. Firstly, MWCNT/PEI (140.0 mg) was dispersed into DMSO (50 mL). Then, FI (4.85 mg) dissolved into DMSO (4 mL) was added into the DMSO solution of MWCNTs under vigorous magnetic stirring. The reaction was continued for 12 h to get the raw product of MWCNT/PEI-FI. Then, HA (120.0 mg) dissolved in DMSO (10 mL) was activated by EDC (120.0 mg in 5.0 mL DMSO) for 3 h and was added dropwise into the DMSO solution of MWCNT/PEI-FI under vigorous magnetic stirring. The reaction was continued for 48 h to obtain the raw product of MWCNT/PEI-FI-HA conjugates. Then, the MWCNT/PEI-FI-HA conjugates were purified and lyophilized according to the procedure used for preparation of MWCNT/PEI conjugates. 2.3. Characterization techniques 1
H NMR spectra of functionalized MWCNTs were recorded using a Bruker DRX 400 nuclear
magnetic resonance spectrometer. Samples were dispersed in D2O before measurements. UV-Vis spectra were collected on a PerkinElmer Lambda 25 UV-Vis spectrophotometer. Samples were dissolved in water before the experiments. Thermogravimetric analysis (TGA) was performed using a TG 209 F1 (NETZSCH Instruments Co., Ltd., Germany) thermogravimetric analyzer with a heating rate of 20 °C/min and a temperature range of 30–900 °C in air. Zeta potential measurements were carried out using a Zetasizer Nano ZS system (Malvern, UK) equipped with a standard 633 nm laser. The morphology of the MWCNTs was observed by transmission electron microscopy (TEM) using a JEOL 2010F analytical electron microscope (JEOL, Japan) operating at 200 kV. A 5 µL aqueous solution of samples (3 mg/mL) was dropped onto a carbon-coated copper grid and air dried before TEM analysis.
6
2.4. Loading of DOX onto MWCNT/PEI-FI-HA conjugates The loading of DOX onto the multifunctional MWCNTs was carried out under the optimal conditions as described in our previous work.33 Briefly, MWCNT/PEI-FI-HA dispersed in water (2 mg/mL, 5 mL) was mixed with an aqueous DOX solution (0.8 mg/mL, 5 mL) under magnetic stirring. The mixture was adjusted to have a pH value of 8.0. The mixture was stirred for 48 h at room temperature in dark. The formed MWCNT/PEI-FI-HA/DOX complexes were purified by repeated centrifugation (15000 rpm, 5 min) and redispersion in water having a pH value of 8.0 until the supernatant became colorless. The DOX payload was calculated according the method described in our previous work.33 The final purified MWCNT/DOX complexes were lyophilized and stored at -20 °C in dark before use.
2.5. Release of DOX from MWCNT/PEI-FI-HA/DOX complexes MWCNT/PEI-FI-HA/DOX complexes (0.88 mg) dispersed in PBS (2 mL, 40 mM, pH = 7.4) or acetate buffer (2 mL, 40 mM, pH = 5.8) buffer solution was placed in a dialysis bag with MWCO of 14,000 and dialyzed against the corresponding buffer medium (10 mL) at 37 oC. The experiment was done in triplicate. At the pre-designed time intervals (2, 4, 6, 8, 10, 24, 36, and 72 h, respectively), the outer phase buffer medium (1 mL) was taken out and the concentration of the released DOX was quantified by UV-vis spectroscopy as described in our previous work.33 The volume of the outer phase buffer medium was kept constant by replenishing the corresponding buffer solution (1 mL).
2.6. MTT cell viability measurements HeLa cells were continuously grown in DMEM cell culture medium supplemented with penicillin (100 units/mL), streptomycin (100 µ g/mL), and 10% heat-inactivated FBS. 7
MTT assay was used to assess the viability of the cells. Briefly, approximately 8×103 HeLa cells per well were seeded into a 96-well plate and cultured overnight to allow the cells to adhere onto the plate. Then, fresh FBS-free medium containing free DOX or MWCNT/PEI-FI-HA/DOX complexes with the same DOX concentration (0.5, 1, 2, and 4 µM, respectively) was added to each well. After 24 h incubation, MTT in PBS solution was added. The assays were carried out according to the manufacturer’s instructions. Mean and standard deviation for the triplicate wells were reported. For comparison, the multifunctional MWCNTs without DOX loading were also tested at equivalent MWCNT concentrations.
2.7.
HA-targeted
cellular
uptake
and
selective
antitumor
efficacy
of
MWCNT-PEI-FI-HA/DOX complexes To evaluate the HA-mediated targeted cellular uptake and selective antitumor efficacy of the MWCNT/PEI-FI-HA/DOX complexes, HeLa cells expressing high-level CD44 receptors42, 46 and L929 cells expressing low-level CD44 receptors42, 47 were used. L929 cells were regularly cultured in RPMI 1640 medium supplemented with penicillin (100 units/mL), streptomycin (100 µ g/mL), and 10% heat-inactivated FBS. Approximately 2 × 105 cells per well were seeded in 12-well plates the day before the experiments to allow the cells to adhere onto the plate. The cells were rinsed 3 times with PBS. Then free DOX, or MWCNT/PEI-FI-HA/DOX complexes with the same DOX concentration (1 µM) was separately added to either HeLa or L929 cells, and the cells were incubated for 2 h at 37 oC in the FBS-free medium. The cells were then washed with PBS for 3 times, treated by trypsin, and harvested by centrifugation at 1000 rpm for 5 min. Finally the harvested cells were resuspended in PBS and analyzed using a Becton Dickinson FACSCalibur flow cytometer equipped with an argon 8
laser (488 nm). The FL2-fluorescence of 1 × 10 4 cells was measured, and the mean fluorescence of the gated viable cells was quantified. To qualitatively confirm the specific cellular uptake of the MWCNT/PEI-FI-HA/DOX complexes, confocal microscopic imaging (Carl Zeiss LSM 700, Jena, Germany) was performed. Cover slips with a diameter of 14 mm were pretreated with 5% HCl, 30% HNO3, and 75% alcohol and then fixed in 12-well tissue culture plate. 4 × 10 4 HeLa or L929 cells were seeded into each well and cultured for about 24 h. Before imaging, the cells were treated with FBS-free medium containing MWCNT/PEI-FI-HA/DOX complexes for 2 h. Then the cells were rinsed with PBS for 3 times, fixed with glutaraldehyde (2.5%) for 15 min at 4 °C, and counterstained with Hoechst 33342 (1 µg/mL) for 15 min at 37 °C using a standard procedure. Finally, the cells were imaged using a 63 × oil-immersion objective lens. To evaluate the targeted cancer cell inhibition efficiency of the MWCNT/PEI-FI-HA/DOX complexes, approximately 8 × 103 cells per well were incubated in a 96-well plate overnight. Then, the FBS-free medium containing the MWCNT/PEI-FI-HA/DOX complexes (1 µM DOX) were added to HeLa or L929 cells. After 2 h incubation, the medium in wells containing the complexes was totally taken out and replenished with the same volume of FBS-containing fresh medium. The cells were then incubated for 24 h at 37 °C. After that, an MTT assay was used to quantify the viability of cells according to protocols described above.
2.8. Statistical analysis One way ANOVA statistical analysis was performed to evaluate the significance of the experimental data. 0.05 was selected as the significance level, and the data were indicated with (*) for p < 0.05, (**) for p < 0.01, and (***) for p < 0.001, respectively. 9
3. Results and discussion 3.1. Synthesis and characterization of MWCNT/PEI-FI-HA conjugates Similar to our previous work,30 water-soluble PEI-modified MWCNTs were first synthesized. The formed MWCNT/PEI was then sequentially modified with FI and HA via PEI-mediated covalent conjugation chemistry to form multifunctional MWCNT/PEI-FI-HA conjugates (Figure 1). The formation of MWCNT/PEI-FI-HA conjugates was first confirmed via
1
H NMR
spectroscopy (Figure 2). It is known that acid-treated MWCNTs do not display any salient features related to the proton signals,30 while after linked with PEI, the -CH2- proton signals at 1~3 ppm related to PEI are clearly observed in the spectrum of the MWCNT/PEI (Figure 2b).31 After modification of the PEI amines with FI (MWCNT/PEI-FI), the characteristic proton peaks at the region of 6.5~7.0 ppm related to FI can be seen (Figure 2c, enlarged spectrum can be seen in Figure S1, Support Information). Further modification of the amines of PEI with HA onto MWCNTs (MWCNT/PEI-FI-HA) led to the emergence of the characteristic proton peaks related to HA (Figures 2a and 2d). Due to the coating of HA onto the surface of the MWCNTs, the proton signals associated with FI can be hardly seen. UV-vis spectrometry was further used to characterize the MWCNT/PEI-FI-HA conjugates (Figure 3a). The apparent absorption peaks at 255 nm is associated with the typical absorption peak of MWCNTs, while the absorption peak at 500 nm is related to the typical FI absorption peak, suggesting the successful conjugation of FI moieties onto the MWCNTs. It should be noted that the MWCNT/PEI-FI-HA conjugates do not display the typical MWCNT-associated absorption feature at 255 nm, likely due to the slight alteration of the electronic structure of MWCNTs after modification of HA. However, the exact reason is still unclear now. 10
The surface modification of MWCNTs was further confirmed by zeta potential measurements (Table 1). The surface potential of the acid-treated MWNCTs (-46.2 ± 5.8 mV) became positive (40.9 ± 0.26 mV) after modification with PEI. Subsequent modification of the PEI amines with FI and HA moieties generated negatively charged multifunctional MWCNTs (-53.2 mV±3.26). The changes of zeta potential values reflected the successful surface modification of the MWCNTs. TGA was used to quantitatively characterize the conjugation of PEI and HA onto the surface of MWCNTs. Due to the fact that at the temperature of 421 °C, PEI has 100% weight loss (Figure S2, Support Information), while pristine MWCNTs have only 1% weight loss (Figure 4), we selected 421 °C as the inflection point to calculate the weight losses of MWCNTs after different modifications. It can be seen that at the inflection point (421 °C), MWCNT/PEI, MWCNT/PEI-FI and MWCNT/PEI-FI-HA have weight losses of 27.0%, 29.5% and 74.0%, respectively. Therefore, by subtracting the weight loss of unmodified MWCNTs, MWCNT/PEI, and MWCNT/PEI-FI, the grafting percentages of the PEI, FI, and HA were estimated to be 26.0%, 2.5%, and 44.5%, respectively. The morphology of the formed multifunctional MWCNTs was characterized by TEM (Figure 5). When compared with the pristine acid-treated MWCNTs,30 no appreciable changes or aggregation can be observed after the conjugation of PEI and FI-HA onto the surface of MWCNTs, suggesting that the conjugation was quite uniform. It should be noted that compared with the study related to dendrimer-modified MWCNTs used for DOX loading and delivery,33 the PEI modification strategy is also simple and flexible. Besides the fact that different functionalities (e.g., imaging agent FI and targeting moiety HA) are able to be covalently modified onto MWCNT/PEI, the cost of PEI is much lower than that of dendrimers. Therefore, the PEI modification chemistry is quite practical to generate multifunctional MWCNTs-based platform for drug delivery applications. 11
3.2. pH-dependent DOX loading and release As a model cancer drug, DOX is known to have pH-dependent hydrophilicity.48-49 At acidic pH conditions, DOX is protonated and quite water-soluble, which does not facilitate the hydrophobic π-π stacking interaction with the MWCNTs. In contrast, under basic condition, the deprotonated DOX is quite hydrophobic, which is beneficial for its effective π-π stacking interaction with the MWCNTs. Following the protocols described in our previous work,33 we were able to generate the MWCNT/PEI-FI-HA/DOX complexes under the optimal condition (except at pH 9.5), and the DOX loading percentage was calculated to be 72.0%. The lower DOX loading percentage of the MWCNT/PEI-FI-HA/DOX complexes than that of the MWCNT/dendrimer/DOX complexes may be due to the fact that the modifications of branched PEI and linear HA polymer are quite effective in occupying the surface of MWCNTs, thereby restricting the π-π stacking interaction between DOX and the MWCNTs. Likewise, the loading experiment was performed at pH 8.0, instead of 9.5, which may also contribute to the lower drug loading percentage. Note that at the pH 9.5, we were unable to get the stable MWCNT/PEI-FI-HA/DOX complexes. The formed MWCNT/PEI-FI-HA/DOX complexes were characterized with UV-Vis spectroscopy (Figure 3b). The apparent absorption peak at 481 nm (typical absorption peak of DOX) clearly indicates the successful payload of DOX onto the multifunctional MWCNTs. It should be noted that both MWCNT/PEI-FI-HA/DOX and MWCNT/PEI-FI-HA are quite colloidally stable in water, PBS, and cell culture medium and no precipitation occurs for at least 3 months. As expected, the release of DOX from MWCNT/PEI-FI-HA/DOX complexes should also be pH-dependent because of the pH-dependent π - π stacking interaction between DOX and MWCNTs. The release profile of DOX from the complexes was explored under two different pH conditions (pH 12
= 5.8 and 7.4), which represent the acidic tumor microenvironment and physiological environment, respectively (Figure 6). It can be seen that at the same time point, the DOX release rate is faster at pH 5.8 than at pH 7.4. After 48 h, 54.6% of DOX was released at the acidic pH (pH = 5.8), while only 40.8% of DOX was released at the physiological pH (pH = 7.4). The pH-responsive DOX release of the MWCNT/PEI-FI-HA/DOX complexes is beneficial for treating tumor site with slightly acidic pH microenvironment. The quite high DOX release at pH 7.4 is due to the partial protonation of DOX at this pH, which does not favor the π-π stacking interactions between DOX and MWCNTs. Furthermore, the relatively slow DOX release at pH 5.8 may be attributed to the fact that a small portion of DOX is covalently linked with the activated carboxyl groups of HA.
3.3. Therapeutic efficacy of MWCNT/PEI-FI-HA/DOX complexes The therapeutic efficacy of MWCNT/PEI-FI-HA/DOX complexes was tested using HeLa cells. After 24 h incubation of MWCNT/PEI-FI-HA/DOX complexes with cells, an MTT assay was performed to evaluate the viability of HeLa cells (Figure 7a). Similar to free DOX, MWCNT/PEI-FI-HA/DOX complexes were able to cause an obvious loss of cell viability when compared with the untreated control cells. This suggests that the encapsulation of DOX within the multifunctional MWCNTs does not compromise the therapeutic efficacy of the DOX drug. To exclude the possible toxicity of MWCNTs, the MWCNT/PEI-FI-HA without DOX loading was also tested (Figure 7b). It can be observed that at equivalent concentrations, MWCNT/PEI-FI-HA do not exhibit cytotoxicity to HeLa cells. Our data imply that the therapeutic efficacy of MWCNT/PEI-FI-HA/DOX complexes is solely related to the encapsulated DOX drug.
3.4. HA-targeted inhibition of cancer cells using MWCNT/PEI-FI-HA/DOX complexes 13
HA was selected as a targeting ligand to be conjugated onto the MWCNT/PEI surfaces for specific delivery of the DOX drug to cancer cells overexpressing CD44 receptors, such as ovarian, colon, stomach, and myelocytic blood cancer cells.31, 50 Considering the high cytotoxicity of MWCNT/PEI as
demonstrated
in
our
previous
work,30
we
tested
the
targeting
specificity
of
MWCNT/PEI-FI-HA/DOX complexes using HeLa cells with high-level CD44 receptor expression and the normal L929 cells with low-level CD44 receptor expression,42, 47 and the MWCNT/PEI-FI material without HA was not selected as control. Flow cytometry was used to quantify the cellular uptake of MWCNT/PEI-FI-HA/DOX complexes via the inherent red fluorescence signal of DOX after the cells were treated for 2 h (Figure 8). It can be seen that for HeLa cells, the treatment of the complexes results in a significant increase of fluorescence signal within the cells. However, for the normal L929 cells, the fluorescence signal is much lower under the same DOX concentrations. The cell population data further show that the percentage of HeLa cells having the uptake of the complexes is 7 times more than that of the L929
cells.
These
results
are
consistent
with
the
idea
that
HeLa
cells
uptake
MWCNT/PEI-FI-HA/DOX complexes presumably through the CD44-mediated endocytosis pathway. The cellular uptake of the MWCNT/PEI-FI-HA/DOX complexes was also confirmed via confocal microscopic imaging of the red fluorescence of DOX and the green fluorescence of the conjugated FI moiety. As shown in Figure 9, after 2 h incubation of the MWCNT/PEI-FI-HA/DOX complexes, only HeLa cells display significant red and green fluorescence signals, which is associated with the specific internalization of MWCNT/PEI-FI-HA/DOX complexes into the cytoplasm of the cells. In contrast, the L929 cells treated with the same complexes do not show appreciable fluorescence signals. Our results suggest that HA-modified MWCNT/PEI-FI-HA/DOX 14
complexes can afford targeted delivery of DOX into cancer cells overexpressing CD44 receptors. To prove the targeted therapeutic efficacy of the MWCNT/PEI-FI-HA/DOX complexes, after 2 h incubation of the complexes with cells, the medium was replaced with the same volume of DOX-free fresh medium, and then the cells were incubated for 24 h at 37 °C before MTT assay (Figure 10). It is clear that after treatment with the MWCNT/PEI-FI-HA/DOX complexes, the viability of HeLa cells were obviously decreased (about 50%), however, the L929 cells were almost all alive. This indicates that the HA-targeted MWCNT/DOX complexes enable targeted inhibition of the growth of cancer cells via receptor-mediated binding and intracellular uptake, in agreement with our previous results.8, 20, 33, 51-52
4. Conclusion In summary, we developed a general approach to forming HA-modified multifunctional MWCNTs for targeted delivery of DOX into cancer cells overexpressing CD44 receptors. Via the PEI-mediated conjugation chemistry, targeting ligand HA and imaging dye FI can be covalently modified onto the surface of MWCNTs with good water dispersibility. The formed MWCNT/PEI-FI-HA carrier is able to load DOX through π-π stacking interactions between DOX and MWCNTs, and display a pH-responsive DOX release behavior with a faster DOX release rate under an acidic pH condition. The formed MWCNT/PEI-FI-HA/DOX complexes have non-compromised therapeutic efficacy when compared to free DOX. Importantly, the multifunctional HA-targeted MWCNT/DOX complexes are able to specifically target HeLa cells overexpressing CD44 receptors, and exert specific inhibition effect to the cancer cells. The developed MWCNT/PEI-FI-HA nanocarrier may hold a great promise to be used for targeted therapy of different types of cancer cells.
15
Acknowledgements Xueyan Cao and Lei Tao equally contributed to this work. This research is financially supported by the Ph.D. Programs Foundation of Ministry of Education of China (20130075110004), the National Natural Science Foundation of China (21273032), the Program for Professor of Special Appointment (Eastern Scholar) at Shanghai Institutions of Higher Learning, and the Fundamental Research Funds for the Central Universities (for X. C. and X. S.).
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12. Nagahama, K.; Mori, Y.; Ohya, Y.; Ouchi, T. Biomacromolecules, 2007, 8, 2135-2141. 13. Yallapu, M. M.; Jaggi, M.; Chauhan, S. C. Drug Discov. Today, 2011, 16, 457-463. 14. Ogawara, K.-i.; Un, K.; Minato, K.; Tanaka, K.-i.; Higaki, K.; Kimura, T. Int. J. Pharm., 2008, 359, 234-240. 15. Podsiadlo, P.; Sinani, V. A.; Bahng, J. H.; Kam, N. W. S.; Lee, J.; Kotov, N. A. Langmuir, 2007, 24, 568-574. 16. Yu, M.; Jambhrunkar, S.; Thorn, P.; Chen, J.; Gu, W.; Yu, C. Nanoscale, 2013, 5, 178-183. 17. Gonçalves, M.; Maciel, D.; Capelo, D.; Xiao, S.; Sun, W.; Shi, X.; Rodrigues, J.; Tomás, H.; Li, Y. Biomacromolecules, 2014, 15, 492-499. 18. Cheng, J.; Teply, B. A.; Sherifi, I.; Sung, J.; Luther, G.; Gu, F. X.; Levy-Nissenbaum, E.; Radovic-Moreno, A. F.; Langer, R.; Farokhzad, O. C. Biomaterials, 2007, 28, 869-876. 19. Song, S.; Chen, F.; Qi, H.; Li, F.; Xin, T.; Xu, J.; Ye, T.; Sheng, N.; Yang, X.; Pan, W. Pharm Res, 2014, 31, 1032-1045. 20. Wang, Y.; Guo, R.; Cao, X.; Shen, M.; Shi, X. Biomaterials, 2011, 32, 3322-3329. 21. Liu, Z.; Tabakman, S.; Welsher, K.; Dai, H. Nano Res., 2009, 2, 85-120. 22. Prakash, S.; Malhotra, M.; Shao, W.; Tomaro-Duchesneau, C.; Abbasi, S. Adv. Drug Deliv. Rev., 2011, 63, 1340-1351. 23. Vashist, S. K.; Zheng, D.; Pastorin, G.; Al-Rubeaan, K.; Luong, J. H. T.; Sheu, F.-S. Carbon, 2011, 49, 4077-4097. 24. Bianco, A.; Kostarelos, K.; Prato, M. Curr. Opin. Chem. Biol., 2005, 9, 674-679. 25. Kam, N. W. S.; Jessop, T. C.; Wender, P. A.; Dai, H. J. Am. Chem. Soc., 2004, 126, 6850-6851. 26. Kam, N. W. S.; O'Connell, M.; Wisdom, J. A.; Dai, H. Proc. Natl. Acad. Sci. USA, 2005, 102, 11600-11605. 17
27. Adair, J. H.; Parette, M. P.; Altınoğlu, E. I.; Kester, M. ACS Nano, 2010, 4, 4967-4970. 28. Bhirde, A. A.; Patel, V.; Gavard, J.; Zhang, G.; Sousa, A. A.; Masedunskas, A.; Leapman, R. D.; Weigert, R.; Gutkind, J. S.; Rusling, J. F. ACS Nano, 2009, 3, 307-316. 29. Lacerda, L.; Bianco, A.; Prato, M.; Kostarelos, K. Adv. Drug Deliv. Rev., 2006, 58, 1460-1470. 30. Shen, M.; Wang, S. H.; Shi, X.; Chen, X.; Huang, Q.; Petersen, E. J.; Pinto, R. A.; Baker, J. R., Jr.;; Weber, W. J., Jr. J. Phys. Chem. C, 2009, 113, 3150-3156. 31. Wen, S.; Zhao, Q.; An, X.; Zhu, J.; Hou, W.; Li, K.; Huang, Y.; Shen, M.; Zhu, W.; Shi, X. Adv. Healthcare Mater., 2014, DOI: 10.1002/adhm.201300631. 32. Shi, X.; Wang, S. H.; Shen, M.; Antwerp, M. E.; Chen, X.; Li, C.; Petersen, E. J.; Huang, Q.; Weber, W. J., Jr.; Baker, J. R., Jr. Biomacromolecules, 2009, 10, 1744-1750. 33. Wen, S. H.; Liu, H.; Cai, H. D.; Shen, M. W.; Shi, X. Y. Adv. Healthcare Mater., 2013, 2, 1267-1276. 34. Bae, K. H.; Yoon, J. J.; Park, T. G. Biotechnol. Progr., 2006, 22, 297-302. 35. Jiang, G.; Park, K.; Kim, J.; Kim, K. S.; Oh, E. J.; Kang, H. G.; Han, S. E.; Oh, Y. K.; Park, T. G.; Hahn, S. K. Biopolymers, 2008, 89, 635-642. 36. Lapcik, L.; Lapcik, L.; De Smedt, S.; Demeester, J.; Chabrecek, P. Chem. Rev., 1998, 98, 2663-2684. 37. Necas, J.; Bartosikova, L.; Brauner, P.; Kolar, J. Vet. Med., 2008, 53, 397-411. 38. Aruffo, A.; Stamenkovic, I.; Melnick, M.; Underhill, C. B.; Seed, B. Cell, 1990, 61, 1303-1313. 39. Li, F.; Bae, B. C.; Na, K. Bioconjugate Chem., 2010, 21, 1312-1320. 40. Bhang, S. H.; Won, N.; Lee, T. J.; Jin, H.; Nam, J.; Park, J.; Chung, H.; Park, H. S.; Sung, Y. E.; Hahn, S. K.; Kim, B. S.; Kim, S. ACS Nano, 2009, 3, 1389-1398. 41. Li, J.; He, Y.; Sun, W.; Luo, Y.; Cai, H.; Pan, Y.; Shen, M.; Xia, J.; Shi, X. Biomaterials, 2014, 18
35, 3666-3677. 42. Ma, M.; Chen, H. R.; Chen, Y.; Zhang, K.; Wang, X.; Cui, X. Z.; Shi, J. L. J. Mater. Chem., 2012, 22, 5615-5621. 43. Datir, S. R.; Das, M.; Singh, R. P.; Jain, S. Bioconjug Chem, 2012, 23, 2201-2213. 44. Swierczewska, M.; Choi, K. Y.; Mertz, E. L.; Huang, X.; Zhang, F.; Zhu, L.; Yoon, H. Y.; Park, J. H.; Bhirde, A.; Lee, S.; Chen, X. Nano Lett, 2012, 12, 3613-3620. 45. Petersen, E. J.; Huang, Q.; Weber, W. J., Jr. Environ. Health Persp., 2008, 116, 496-500. 46. Li, F.; Park, S.-J.; Ling, D.; Park, W.; Han, J. Y.; Na, K.; Char, K. Journal of Materials Chemistry B, 2013, 1, 1678-1686. 47. Lim, E. K.; Kim, H. O.; Jang, E.; Park, J.; Lee, K.; Suh, J. S.; Huh, Y. M.; Haam, S. Biomaterials, 2011, 32, 7941-7950. 48. Ali-Boucetta, H.; Al-Jamal, K. T.; McCarthy, D.; Prato, M.; Bianco, A.; Kostarelos, K. Chem. Commun., 2008, 459-461. 49. Liu, Z.; Sun, X.; Nakayama-Ratchford, N.; Dai, H. ACS Nano, 2007, 1, 50-56. 50. Toole, B. P.; Wight, T. N.; Tammi, M. I. J. Biol. Chem., 2002, 277, 4593-4596. 51. Zhang, M.; Guo, R.; Wang, Y.; Cao, X.; Shen, M.; Shi, X. Int. J. Nanomed., 2011, 6, 2337-2349. 52. Zheng, L.; Zhu, J.; Shen, M.; Chen, X.; Baker, J. R., Jr.; Wang, S. H.; Zhang, G.; Shi, X. Med. Chem. Commun., 2013, 4, 1001-1005.
19
Table 1. Zeta potential values of multifunctional MWCNTs before and after surface modification. Materials
MWCNTs
MWCNT/PEI
Zeta potential (mV)
- 46.2 ± 5.8
+ 40.9 ± 0.26
20
MWCNT/PEI-FI-HA - 53.2 ± 3.26
Figure captions Figure 1. Schematic illustration of the synthesis of multifunctional MWCNTs. Figure 2.
1
H NMR spectra of HA (a), MWCNT/PEI (b), MWCNT/PEI-FI (c), and
MWCNT/PEI-FI-HA (d), respectively. Figure 3. UV-Vis spectra of functionalized MWCNTs (1 mg/mL) and MWCNTs/DOX complexes (1 mg/mL)
dispersed
in
water.
(a)
Pristine
acid-treated
MWCNTs,
MWCNT/PEI,
and
MWCNT/PEI-FI-HA, respectively. (b) Free DOX (0.005 mg/mL), MWCNT/PEI-FI-HA (0.3 mg/mL), and MWCNT/PEI-FI-HA/DOX (0.28 mg/mL, [DOX] = 0.052 mg/mL), respectively. Figure 4. TGA curves of the pristine acid-treated MWCNTs, MWCNT/PEI, MWCNT/PEI-FI, and MWCNT/PEI-FI-HA, respectively. Figure 5. TEM image of MWCNT/PEI (a) and MWCNT/PEI-FI-HA (b), respectively. Figure 6. Release profile of DOX from MWCNT/PEI-FI-HA/DOX complexes (1 mg/mL) under different pH conditions. Figure 7. MTT viability assay of HeLa cells treated with free DOX and MWCNT/PEI-FI-HA/DOX complexes at the DOX concentrations of 0-4 µM for 24 h (a), and DOX-free MWCNT/PEI-FI-HA at corresponding DOX concentrations of the complexes between 1.25-10 mg/L (b). Figure 8. Flow cytometry analysis of the uptake of MWCNT/PEI-FI-HA/DOX complexes at the DOX concentration of 2 µM and 4 µM by L929 (b, c) and HeLa (e, f) cells after treatment for 2 h. L929 and HeLa cells treated with PBS were used as controls (a, d). Mean fluorescence (g) and cellular population percentage (h) are also shown. Figure
9.
Confocal
microscopic
images
of
HeLa
and
L929
cells
treated
with
MWCNT/PEI-FI-HA/DOX complexes ([DOX] = 2 µM) for 2 h. HeLa and L929 cells treated with PBS were used as controls. The scale bar in each panel represents 20 µm. 21
Figure 10. MTT assay of the viability of HeLa and L929 cells after treatment with MWCNT/PEI-FI-HA complexes/DOX ([DOX] = 2 µM) for 2 h, followed by replacing the medium with DOX-free fresh medium and incubating the cells for additional 24 h.
22
Figure 1 Cao et al. 23
Figure 2 Cao et al. 24
Figure 3 Cao et al.
25
MWCNT MWCNT/PEI MWCNT/PEI-FI MWCNT/PEI-FI-HA
100
Weight (%)
80 60 40 20 0 200
400
600
800
o
Tempurature ( C)
Figure 4 Cao et al.
26
Figure 5 Cao et al.
27
100 pH = 7.4 pH = 5.8
DOX release (%)
80 60 40 20 0
0
10
20
30
40
50
60
70
Time (h)
Figure 6 Cao et al.
28
Figure 7 Cao et al.
29
Figure 8 Cao et al. 30
Figure 9 Cao et al.
31
120
HeLa L929
Cell viability (%)
100
***
80 60 40 20 0
2
0
DOX concentration (µM)
Figure 10 Cao et al.
32
33
Research highlights: Modification of PEI and FI-HA does not appreciably change the morphology of MWCNTs. MWCNT/PEI-FI-HA/DOX complexes display a pH-responsive drug release behavior. MWCNT/PEI-FI-HA carrier is cytocompatible in the given concentration range. MWCNT/PEI-FI-HA/DOX complexes can target cancer cells via receptor-mediated manner. MWCNT/PEI-FI-HA/DOX complexes display a specific therapeutic effect to cancer cells.
34
S0008-6215(14)00275-4 http://dx.doi.org/10.1016/j.carres.2014.06.030 CAR 6791
To appear in:
Carbohydrate Research
Received Date: Revised Date: Accepted Date:
25 April 2014 25 June 2014 27 June 2014
Please cite this article as: Cao, X., Tao, L., Wen, S., Hou, W., Shi, X., Hyaluronic acid-modified multiwalled carbon nanotubes for targeted delivery of doxorubicin into cancer cells, Carbohydrate Research (2014), doi: http:// dx.doi.org/10.1016/j.carres.2014.06.030
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Hyaluronic acid-modified multiwalled carbon nanotubes for targeted delivery of doxorubicin into cancer cells
Xueyan Cao, Lei Tao, Shihui Wen, Wenxiu Hou, Xiangyang Shi* College of Chemistry, Chemical Engineering and Biotechnology, Donghua University, Shanghai 201620, P. R. China
_________________________________________________ *To whom correspondence should be addressed. Phone: +86 21 67792656; Fax: +86 21 67792306-804. E-mail: [email protected]
1
Abstract Development of novel drug carriers for targeted cancer therapy with high efficiency and specificity is of paramount importance and has been one of the major topics in current nanomedicine. Here we report a general approach to using multifunctional multiwalled carbon nanotubes (MWCNTs) as a platform to encapsulate an anticancer drug doxorubicin (DOX) for targeted cancer therapy. In this approach, polyethyleneimine (PEI)-modified MWCNTs were covalently conjugated with fluorescein isothiocyanate (FI) and hyaluronic acid (HA). The formed MWCNT/PEI-FI-HA conjugates were characterized via different techniques and were used as a new carrier system to encapsulate the anticancer drug doxorubicin for targeted delivery to cancer cells overexpressing CD44 receptors. We show that the formed MWCNT/PEI-FI-HA/DOX complexes with a drug loading percentage of 72% are water soluble and stable. In vitro release studies show that the drug release rate under an acidic condition (pH 5.8, tumor cell microenvironment) is higher than that under physiological condition (pH 7.4). Cell viability assay demonstrates that the carrier material has good biocompatibility in the tested concentration range, and the MWCNT/PEI-FI-HA/DOX complexes can specifically target cancer cells overexpressing CD44 receptors and exert growth inhibition effect to the cancer cells. The developed HA-modified MWCNTs hold a great promise to be used as an efficient anticancer drug carrier for tumor-targeted chemotherapy.
Key words：Multiwalled carbon nanotubes; hyaluronic acid; doxorubicin; targeted cancer therapy
2
1. Introduction Although chemotherapy has been one of the most important methods to treat cancer, many anticancer drugs, like doxorubicin (DOX), not only kill the cancerous tissues, but also lead to adverse side-effect on normal tissues.1-2 In addition, many cancer drugs lack water solubility, significantly limiting their bioavailability. Therefore, it is essential to develop a drug delivery carrier with targeting specificity,3-5 good biocompatibility,6-8 sufficient stability,9-11 and long circulation time.12-13 Currently, a wide range of nanocarrier systems, including but not limited to liposomes,14 nanoparticles,15-16 nanogels,17 polymer micelles,18-19 and dendrimers20 have been developed to both improve the water solubility of the anticancer drug and achieve the specific targeting drug delivery. Carbon nanotubes (CNTs)21-23 have gained tremendous attention as a promising nanocarrier owing to their distinct characteristics, such as ultrahigh surface area, high drug-loading capability, effective transportation capability, and enhanced cellular uptake.24-26 For biomedical applications, the poor aqueous dispersibility and high aggregation tendency of pristine CNTs can be resolved by appropriate surface functionalization via either non-covalent or covalent modification strategies,27-28 rendering the CNTs water soluble and highly biocompatible.29-30 In our previous work, we have shown that polyethyleneimine (PEI) covalently linked onto the surface of acid-treated multiwalled carbon nanotubes (MWCNTs) can be further modified with different surface functional groups for biomedical applications.30-31 Our results reveal that the water-dispersibility and biocompatibility of PEI-modified MWCNTs can be significantly improved after further modification of the PEI amines with acetyl groups or polyethylene glycol moieties. For targeted drug delivery applications, the MWCNTs can be modified with amine-terminated poly(amidoamine) dendrimers pre-modified with fluorescein isothiocyanate (FI) and folic acid (FA).32-33 The developed multifunctional MWCNT-based delivery system can achieve a high drug 3
payload of anticancer drug doxorubicin (DOX) with a pH-responsive release property showing fast DOX release under acidic environment and slow release at a physiological pH condition. Importantly, the complexes were able to target cancer cells overexpressing high-affinity FA receptors and effectively inhibit the growth of the cancer cells. These prior successes lead us to hypothesize that the MWCNTs may be covalently modified with other targeting ligands for targeted cancer therapy applications. Hyaluronic acid (HA) is a linear polysaccharide consisting of repeating disaccharide units of D-glucuronic acid and N-acetyl-D-glucosamine.34-35 HA has unique and excellent physicochemical properties, such as biodegradability, biocompatibility, and nonimmunogenicity.36-37 Besides, it is also known that HA has a strong affinity to bind cell-specific surface markers such as cluster determinant 44 (CD44) receptors,38-39 which are overexpressed on the surface of several different cancer cells.39 Such interesting selectivity of HA to CD44 receptor-overexpressing cancer cells has shown a great application potential in cancer diagnosis and therapy.40-44 In this present study, we synthesized and characterized multifunctional MWCNTs modified with FI and HA (MWCNT/PEI-FI-HA) as a new anticancer drug carrier for target delivery of DOX to cancer cells overexpressing CD44 receptors. Acid-treated MWCNTs were first covalently modified with PEI, followed by sequential modification with FI and HA (Figure 1). The formed MWCNT/PEI-FI-HA conjugates were characterized using different methods. The loading of DOX onto the multifunctional MWCNTs and the release of DOX from the MWCNTs were monitored via UV-vis spectrometry. The targeted therapeutic efficacy of MWCNT/PEI-FI-HA/DOX complexes in vitro was investigated in detail.
2. Experimental 2.1. Materials 4
MWCNTs (diameter = 30-70 nm, length = 100 nm-2 µm) were synthesized and characterized in a previous report.45 HA (Mw = 31,200) was obtained from Zhenjiang Dong Yuan Biotechnology Corporation (Zhenjiang, China). Hyperbranced PEI (Mw≈25,000) and all other chemicals and solvents were purchased from Aldrich (St. Louis, Missouri) and used as received. HeLa cells (a human cervical carcinoma cell line) and L929 cells (a mouse fibroblast cell line) were from Institute of Biochemistry and Cell Biology, the Chinese Academy of Sciences (Shanghai, China). RPMI 1640 medium, DMEM medium, fetal bovine serum (FBS), penicillin, and streptomycin were purchased from Hangzhou Jinuo Biomedical Technology (Hangzhou, China). Regenerated cellulose dialysis membranes with molecular weight cut-off (MWCO) at 14,000 and 50,000 were acquired from Fisher (Pittsburgh, PA). Water used in all experiments was purified using a Milli-Q Plus 185 water purification system (Millipore, Bedford, MA) with resistivity higher than 18 M Ω • cm.
2.2. Synthesis of MWCNT/PEI-FI-HA conjugates The modification of MWCNTs with PEI to form MWCNT/PEI was adapted from our previous report.30 Briefly, acid-treated MWCNTs with carboxyl residues (120.0 mg) dispersed in DMSO (50 mL) were activated with EDC (120.0 mg) and NHS (62.0 mg) co-dissolved into DMSO (5 mL) under vigorous magnetic stirring. The reaction was continued for 3 h to activate the carboxyl residues of MWCNTs, followed by the addition of PEI solution (120 mg in 12 mL DMSO). The reaction mixture was sonicated for 2 d to obtain MWCNT/PEI conjugates. Finally, the DMSO and the excess of reactants and byproduct were removed from the reaction mixture by extensive dialysis against phosphate buffered saline (PBS) solution (3 times, 2 L) and water (5 times, 2 L) using a dialysis membrane with MWCO of 50, 000 for 3 d. The MWCNT/PEI conjugates were obtained following lyophilization. 5
Multifunctional MWCNTs (MWCNT/PEI-FI-HA) were synthesized by conjugating FI and HA onto the surface of MWCNT/PEI. Firstly, MWCNT/PEI (140.0 mg) was dispersed into DMSO (50 mL). Then, FI (4.85 mg) dissolved into DMSO (4 mL) was added into the DMSO solution of MWCNTs under vigorous magnetic stirring. The reaction was continued for 12 h to get the raw product of MWCNT/PEI-FI. Then, HA (120.0 mg) dissolved in DMSO (10 mL) was activated by EDC (120.0 mg in 5.0 mL DMSO) for 3 h and was added dropwise into the DMSO solution of MWCNT/PEI-FI under vigorous magnetic stirring. The reaction was continued for 48 h to obtain the raw product of MWCNT/PEI-FI-HA conjugates. Then, the MWCNT/PEI-FI-HA conjugates were purified and lyophilized according to the procedure used for preparation of MWCNT/PEI conjugates. 2.3. Characterization techniques 1
H NMR spectra of functionalized MWCNTs were recorded using a Bruker DRX 400 nuclear
magnetic resonance spectrometer. Samples were dispersed in D2O before measurements. UV-Vis spectra were collected on a PerkinElmer Lambda 25 UV-Vis spectrophotometer. Samples were dissolved in water before the experiments. Thermogravimetric analysis (TGA) was performed using a TG 209 F1 (NETZSCH Instruments Co., Ltd., Germany) thermogravimetric analyzer with a heating rate of 20 °C/min and a temperature range of 30–900 °C in air. Zeta potential measurements were carried out using a Zetasizer Nano ZS system (Malvern, UK) equipped with a standard 633 nm laser. The morphology of the MWCNTs was observed by transmission electron microscopy (TEM) using a JEOL 2010F analytical electron microscope (JEOL, Japan) operating at 200 kV. A 5 µL aqueous solution of samples (3 mg/mL) was dropped onto a carbon-coated copper grid and air dried before TEM analysis.
6
2.4. Loading of DOX onto MWCNT/PEI-FI-HA conjugates The loading of DOX onto the multifunctional MWCNTs was carried out under the optimal conditions as described in our previous work.33 Briefly, MWCNT/PEI-FI-HA dispersed in water (2 mg/mL, 5 mL) was mixed with an aqueous DOX solution (0.8 mg/mL, 5 mL) under magnetic stirring. The mixture was adjusted to have a pH value of 8.0. The mixture was stirred for 48 h at room temperature in dark. The formed MWCNT/PEI-FI-HA/DOX complexes were purified by repeated centrifugation (15000 rpm, 5 min) and redispersion in water having a pH value of 8.0 until the supernatant became colorless. The DOX payload was calculated according the method described in our previous work.33 The final purified MWCNT/DOX complexes were lyophilized and stored at -20 °C in dark before use.
2.5. Release of DOX from MWCNT/PEI-FI-HA/DOX complexes MWCNT/PEI-FI-HA/DOX complexes (0.88 mg) dispersed in PBS (2 mL, 40 mM, pH = 7.4) or acetate buffer (2 mL, 40 mM, pH = 5.8) buffer solution was placed in a dialysis bag with MWCO of 14,000 and dialyzed against the corresponding buffer medium (10 mL) at 37 oC. The experiment was done in triplicate. At the pre-designed time intervals (2, 4, 6, 8, 10, 24, 36, and 72 h, respectively), the outer phase buffer medium (1 mL) was taken out and the concentration of the released DOX was quantified by UV-vis spectroscopy as described in our previous work.33 The volume of the outer phase buffer medium was kept constant by replenishing the corresponding buffer solution (1 mL).
2.6. MTT cell viability measurements HeLa cells were continuously grown in DMEM cell culture medium supplemented with penicillin (100 units/mL), streptomycin (100 µ g/mL), and 10% heat-inactivated FBS. 7
MTT assay was used to assess the viability of the cells. Briefly, approximately 8×103 HeLa cells per well were seeded into a 96-well plate and cultured overnight to allow the cells to adhere onto the plate. Then, fresh FBS-free medium containing free DOX or MWCNT/PEI-FI-HA/DOX complexes with the same DOX concentration (0.5, 1, 2, and 4 µM, respectively) was added to each well. After 24 h incubation, MTT in PBS solution was added. The assays were carried out according to the manufacturer’s instructions. Mean and standard deviation for the triplicate wells were reported. For comparison, the multifunctional MWCNTs without DOX loading were also tested at equivalent MWCNT concentrations.
2.7.
HA-targeted
cellular
uptake
and
selective
antitumor
efficacy
of
MWCNT-PEI-FI-HA/DOX complexes To evaluate the HA-mediated targeted cellular uptake and selective antitumor efficacy of the MWCNT/PEI-FI-HA/DOX complexes, HeLa cells expressing high-level CD44 receptors42, 46 and L929 cells expressing low-level CD44 receptors42, 47 were used. L929 cells were regularly cultured in RPMI 1640 medium supplemented with penicillin (100 units/mL), streptomycin (100 µ g/mL), and 10% heat-inactivated FBS. Approximately 2 × 105 cells per well were seeded in 12-well plates the day before the experiments to allow the cells to adhere onto the plate. The cells were rinsed 3 times with PBS. Then free DOX, or MWCNT/PEI-FI-HA/DOX complexes with the same DOX concentration (1 µM) was separately added to either HeLa or L929 cells, and the cells were incubated for 2 h at 37 oC in the FBS-free medium. The cells were then washed with PBS for 3 times, treated by trypsin, and harvested by centrifugation at 1000 rpm for 5 min. Finally the harvested cells were resuspended in PBS and analyzed using a Becton Dickinson FACSCalibur flow cytometer equipped with an argon 8
laser (488 nm). The FL2-fluorescence of 1 × 10 4 cells was measured, and the mean fluorescence of the gated viable cells was quantified. To qualitatively confirm the specific cellular uptake of the MWCNT/PEI-FI-HA/DOX complexes, confocal microscopic imaging (Carl Zeiss LSM 700, Jena, Germany) was performed. Cover slips with a diameter of 14 mm were pretreated with 5% HCl, 30% HNO3, and 75% alcohol and then fixed in 12-well tissue culture plate. 4 × 10 4 HeLa or L929 cells were seeded into each well and cultured for about 24 h. Before imaging, the cells were treated with FBS-free medium containing MWCNT/PEI-FI-HA/DOX complexes for 2 h. Then the cells were rinsed with PBS for 3 times, fixed with glutaraldehyde (2.5%) for 15 min at 4 °C, and counterstained with Hoechst 33342 (1 µg/mL) for 15 min at 37 °C using a standard procedure. Finally, the cells were imaged using a 63 × oil-immersion objective lens. To evaluate the targeted cancer cell inhibition efficiency of the MWCNT/PEI-FI-HA/DOX complexes, approximately 8 × 103 cells per well were incubated in a 96-well plate overnight. Then, the FBS-free medium containing the MWCNT/PEI-FI-HA/DOX complexes (1 µM DOX) were added to HeLa or L929 cells. After 2 h incubation, the medium in wells containing the complexes was totally taken out and replenished with the same volume of FBS-containing fresh medium. The cells were then incubated for 24 h at 37 °C. After that, an MTT assay was used to quantify the viability of cells according to protocols described above.
2.8. Statistical analysis One way ANOVA statistical analysis was performed to evaluate the significance of the experimental data. 0.05 was selected as the significance level, and the data were indicated with (*) for p < 0.05, (**) for p < 0.01, and (***) for p < 0.001, respectively. 9
3. Results and discussion 3.1. Synthesis and characterization of MWCNT/PEI-FI-HA conjugates Similar to our previous work,30 water-soluble PEI-modified MWCNTs were first synthesized. The formed MWCNT/PEI was then sequentially modified with FI and HA via PEI-mediated covalent conjugation chemistry to form multifunctional MWCNT/PEI-FI-HA conjugates (Figure 1). The formation of MWCNT/PEI-FI-HA conjugates was first confirmed via
1
H NMR
spectroscopy (Figure 2). It is known that acid-treated MWCNTs do not display any salient features related to the proton signals,30 while after linked with PEI, the -CH2- proton signals at 1~3 ppm related to PEI are clearly observed in the spectrum of the MWCNT/PEI (Figure 2b).31 After modification of the PEI amines with FI (MWCNT/PEI-FI), the characteristic proton peaks at the region of 6.5~7.0 ppm related to FI can be seen (Figure 2c, enlarged spectrum can be seen in Figure S1, Support Information). Further modification of the amines of PEI with HA onto MWCNTs (MWCNT/PEI-FI-HA) led to the emergence of the characteristic proton peaks related to HA (Figures 2a and 2d). Due to the coating of HA onto the surface of the MWCNTs, the proton signals associated with FI can be hardly seen. UV-vis spectrometry was further used to characterize the MWCNT/PEI-FI-HA conjugates (Figure 3a). The apparent absorption peaks at 255 nm is associated with the typical absorption peak of MWCNTs, while the absorption peak at 500 nm is related to the typical FI absorption peak, suggesting the successful conjugation of FI moieties onto the MWCNTs. It should be noted that the MWCNT/PEI-FI-HA conjugates do not display the typical MWCNT-associated absorption feature at 255 nm, likely due to the slight alteration of the electronic structure of MWCNTs after modification of HA. However, the exact reason is still unclear now. 10
The surface modification of MWCNTs was further confirmed by zeta potential measurements (Table 1). The surface potential of the acid-treated MWNCTs (-46.2 ± 5.8 mV) became positive (40.9 ± 0.26 mV) after modification with PEI. Subsequent modification of the PEI amines with FI and HA moieties generated negatively charged multifunctional MWCNTs (-53.2 mV±3.26). The changes of zeta potential values reflected the successful surface modification of the MWCNTs. TGA was used to quantitatively characterize the conjugation of PEI and HA onto the surface of MWCNTs. Due to the fact that at the temperature of 421 °C, PEI has 100% weight loss (Figure S2, Support Information), while pristine MWCNTs have only 1% weight loss (Figure 4), we selected 421 °C as the inflection point to calculate the weight losses of MWCNTs after different modifications. It can be seen that at the inflection point (421 °C), MWCNT/PEI, MWCNT/PEI-FI and MWCNT/PEI-FI-HA have weight losses of 27.0%, 29.5% and 74.0%, respectively. Therefore, by subtracting the weight loss of unmodified MWCNTs, MWCNT/PEI, and MWCNT/PEI-FI, the grafting percentages of the PEI, FI, and HA were estimated to be 26.0%, 2.5%, and 44.5%, respectively. The morphology of the formed multifunctional MWCNTs was characterized by TEM (Figure 5). When compared with the pristine acid-treated MWCNTs,30 no appreciable changes or aggregation can be observed after the conjugation of PEI and FI-HA onto the surface of MWCNTs, suggesting that the conjugation was quite uniform. It should be noted that compared with the study related to dendrimer-modified MWCNTs used for DOX loading and delivery,33 the PEI modification strategy is also simple and flexible. Besides the fact that different functionalities (e.g., imaging agent FI and targeting moiety HA) are able to be covalently modified onto MWCNT/PEI, the cost of PEI is much lower than that of dendrimers. Therefore, the PEI modification chemistry is quite practical to generate multifunctional MWCNTs-based platform for drug delivery applications. 11
3.2. pH-dependent DOX loading and release As a model cancer drug, DOX is known to have pH-dependent hydrophilicity.48-49 At acidic pH conditions, DOX is protonated and quite water-soluble, which does not facilitate the hydrophobic π-π stacking interaction with the MWCNTs. In contrast, under basic condition, the deprotonated DOX is quite hydrophobic, which is beneficial for its effective π-π stacking interaction with the MWCNTs. Following the protocols described in our previous work,33 we were able to generate the MWCNT/PEI-FI-HA/DOX complexes under the optimal condition (except at pH 9.5), and the DOX loading percentage was calculated to be 72.0%. The lower DOX loading percentage of the MWCNT/PEI-FI-HA/DOX complexes than that of the MWCNT/dendrimer/DOX complexes may be due to the fact that the modifications of branched PEI and linear HA polymer are quite effective in occupying the surface of MWCNTs, thereby restricting the π-π stacking interaction between DOX and the MWCNTs. Likewise, the loading experiment was performed at pH 8.0, instead of 9.5, which may also contribute to the lower drug loading percentage. Note that at the pH 9.5, we were unable to get the stable MWCNT/PEI-FI-HA/DOX complexes. The formed MWCNT/PEI-FI-HA/DOX complexes were characterized with UV-Vis spectroscopy (Figure 3b). The apparent absorption peak at 481 nm (typical absorption peak of DOX) clearly indicates the successful payload of DOX onto the multifunctional MWCNTs. It should be noted that both MWCNT/PEI-FI-HA/DOX and MWCNT/PEI-FI-HA are quite colloidally stable in water, PBS, and cell culture medium and no precipitation occurs for at least 3 months. As expected, the release of DOX from MWCNT/PEI-FI-HA/DOX complexes should also be pH-dependent because of the pH-dependent π - π stacking interaction between DOX and MWCNTs. The release profile of DOX from the complexes was explored under two different pH conditions (pH 12
= 5.8 and 7.4), which represent the acidic tumor microenvironment and physiological environment, respectively (Figure 6). It can be seen that at the same time point, the DOX release rate is faster at pH 5.8 than at pH 7.4. After 48 h, 54.6% of DOX was released at the acidic pH (pH = 5.8), while only 40.8% of DOX was released at the physiological pH (pH = 7.4). The pH-responsive DOX release of the MWCNT/PEI-FI-HA/DOX complexes is beneficial for treating tumor site with slightly acidic pH microenvironment. The quite high DOX release at pH 7.4 is due to the partial protonation of DOX at this pH, which does not favor the π-π stacking interactions between DOX and MWCNTs. Furthermore, the relatively slow DOX release at pH 5.8 may be attributed to the fact that a small portion of DOX is covalently linked with the activated carboxyl groups of HA.
3.3. Therapeutic efficacy of MWCNT/PEI-FI-HA/DOX complexes The therapeutic efficacy of MWCNT/PEI-FI-HA/DOX complexes was tested using HeLa cells. After 24 h incubation of MWCNT/PEI-FI-HA/DOX complexes with cells, an MTT assay was performed to evaluate the viability of HeLa cells (Figure 7a). Similar to free DOX, MWCNT/PEI-FI-HA/DOX complexes were able to cause an obvious loss of cell viability when compared with the untreated control cells. This suggests that the encapsulation of DOX within the multifunctional MWCNTs does not compromise the therapeutic efficacy of the DOX drug. To exclude the possible toxicity of MWCNTs, the MWCNT/PEI-FI-HA without DOX loading was also tested (Figure 7b). It can be observed that at equivalent concentrations, MWCNT/PEI-FI-HA do not exhibit cytotoxicity to HeLa cells. Our data imply that the therapeutic efficacy of MWCNT/PEI-FI-HA/DOX complexes is solely related to the encapsulated DOX drug.
3.4. HA-targeted inhibition of cancer cells using MWCNT/PEI-FI-HA/DOX complexes 13
HA was selected as a targeting ligand to be conjugated onto the MWCNT/PEI surfaces for specific delivery of the DOX drug to cancer cells overexpressing CD44 receptors, such as ovarian, colon, stomach, and myelocytic blood cancer cells.31, 50 Considering the high cytotoxicity of MWCNT/PEI as
demonstrated
in
our
previous
work,30
we
tested
the
targeting
specificity
of
MWCNT/PEI-FI-HA/DOX complexes using HeLa cells with high-level CD44 receptor expression and the normal L929 cells with low-level CD44 receptor expression,42, 47 and the MWCNT/PEI-FI material without HA was not selected as control. Flow cytometry was used to quantify the cellular uptake of MWCNT/PEI-FI-HA/DOX complexes via the inherent red fluorescence signal of DOX after the cells were treated for 2 h (Figure 8). It can be seen that for HeLa cells, the treatment of the complexes results in a significant increase of fluorescence signal within the cells. However, for the normal L929 cells, the fluorescence signal is much lower under the same DOX concentrations. The cell population data further show that the percentage of HeLa cells having the uptake of the complexes is 7 times more than that of the L929
cells.
These
results
are
consistent
with
the
idea
that
HeLa
cells
uptake
MWCNT/PEI-FI-HA/DOX complexes presumably through the CD44-mediated endocytosis pathway. The cellular uptake of the MWCNT/PEI-FI-HA/DOX complexes was also confirmed via confocal microscopic imaging of the red fluorescence of DOX and the green fluorescence of the conjugated FI moiety. As shown in Figure 9, after 2 h incubation of the MWCNT/PEI-FI-HA/DOX complexes, only HeLa cells display significant red and green fluorescence signals, which is associated with the specific internalization of MWCNT/PEI-FI-HA/DOX complexes into the cytoplasm of the cells. In contrast, the L929 cells treated with the same complexes do not show appreciable fluorescence signals. Our results suggest that HA-modified MWCNT/PEI-FI-HA/DOX 14
complexes can afford targeted delivery of DOX into cancer cells overexpressing CD44 receptors. To prove the targeted therapeutic efficacy of the MWCNT/PEI-FI-HA/DOX complexes, after 2 h incubation of the complexes with cells, the medium was replaced with the same volume of DOX-free fresh medium, and then the cells were incubated for 24 h at 37 °C before MTT assay (Figure 10). It is clear that after treatment with the MWCNT/PEI-FI-HA/DOX complexes, the viability of HeLa cells were obviously decreased (about 50%), however, the L929 cells were almost all alive. This indicates that the HA-targeted MWCNT/DOX complexes enable targeted inhibition of the growth of cancer cells via receptor-mediated binding and intracellular uptake, in agreement with our previous results.8, 20, 33, 51-52
4. Conclusion In summary, we developed a general approach to forming HA-modified multifunctional MWCNTs for targeted delivery of DOX into cancer cells overexpressing CD44 receptors. Via the PEI-mediated conjugation chemistry, targeting ligand HA and imaging dye FI can be covalently modified onto the surface of MWCNTs with good water dispersibility. The formed MWCNT/PEI-FI-HA carrier is able to load DOX through π-π stacking interactions between DOX and MWCNTs, and display a pH-responsive DOX release behavior with a faster DOX release rate under an acidic pH condition. The formed MWCNT/PEI-FI-HA/DOX complexes have non-compromised therapeutic efficacy when compared to free DOX. Importantly, the multifunctional HA-targeted MWCNT/DOX complexes are able to specifically target HeLa cells overexpressing CD44 receptors, and exert specific inhibition effect to the cancer cells. The developed MWCNT/PEI-FI-HA nanocarrier may hold a great promise to be used for targeted therapy of different types of cancer cells.
15
Acknowledgements Xueyan Cao and Lei Tao equally contributed to this work. This research is financially supported by the Ph.D. Programs Foundation of Ministry of Education of China (20130075110004), the National Natural Science Foundation of China (21273032), the Program for Professor of Special Appointment (Eastern Scholar) at Shanghai Institutions of Higher Learning, and the Fundamental Research Funds for the Central Universities (for X. C. and X. S.).
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19
Table 1. Zeta potential values of multifunctional MWCNTs before and after surface modification. Materials
MWCNTs
MWCNT/PEI
Zeta potential (mV)
- 46.2 ± 5.8
+ 40.9 ± 0.26
20
MWCNT/PEI-FI-HA - 53.2 ± 3.26
Figure captions Figure 1. Schematic illustration of the synthesis of multifunctional MWCNTs. Figure 2.
1
H NMR spectra of HA (a), MWCNT/PEI (b), MWCNT/PEI-FI (c), and
MWCNT/PEI-FI-HA (d), respectively. Figure 3. UV-Vis spectra of functionalized MWCNTs (1 mg/mL) and MWCNTs/DOX complexes (1 mg/mL)
dispersed
in
water.
(a)
Pristine
acid-treated
MWCNTs,
MWCNT/PEI,
and
MWCNT/PEI-FI-HA, respectively. (b) Free DOX (0.005 mg/mL), MWCNT/PEI-FI-HA (0.3 mg/mL), and MWCNT/PEI-FI-HA/DOX (0.28 mg/mL, [DOX] = 0.052 mg/mL), respectively. Figure 4. TGA curves of the pristine acid-treated MWCNTs, MWCNT/PEI, MWCNT/PEI-FI, and MWCNT/PEI-FI-HA, respectively. Figure 5. TEM image of MWCNT/PEI (a) and MWCNT/PEI-FI-HA (b), respectively. Figure 6. Release profile of DOX from MWCNT/PEI-FI-HA/DOX complexes (1 mg/mL) under different pH conditions. Figure 7. MTT viability assay of HeLa cells treated with free DOX and MWCNT/PEI-FI-HA/DOX complexes at the DOX concentrations of 0-4 µM for 24 h (a), and DOX-free MWCNT/PEI-FI-HA at corresponding DOX concentrations of the complexes between 1.25-10 mg/L (b). Figure 8. Flow cytometry analysis of the uptake of MWCNT/PEI-FI-HA/DOX complexes at the DOX concentration of 2 µM and 4 µM by L929 (b, c) and HeLa (e, f) cells after treatment for 2 h. L929 and HeLa cells treated with PBS were used as controls (a, d). Mean fluorescence (g) and cellular population percentage (h) are also shown. Figure
9.
Confocal
microscopic
images
of
HeLa
and
L929
cells
treated
with
MWCNT/PEI-FI-HA/DOX complexes ([DOX] = 2 µM) for 2 h. HeLa and L929 cells treated with PBS were used as controls. The scale bar in each panel represents 20 µm. 21
Figure 10. MTT assay of the viability of HeLa and L929 cells after treatment with MWCNT/PEI-FI-HA complexes/DOX ([DOX] = 2 µM) for 2 h, followed by replacing the medium with DOX-free fresh medium and incubating the cells for additional 24 h.
22
Figure 1 Cao et al. 23
Figure 2 Cao et al. 24
Figure 3 Cao et al.
25
MWCNT MWCNT/PEI MWCNT/PEI-FI MWCNT/PEI-FI-HA
100
Weight (%)
80 60 40 20 0 200
400
600
800
o
Tempurature ( C)
Figure 4 Cao et al.
26
Figure 5 Cao et al.
27
100 pH = 7.4 pH = 5.8
DOX release (%)
80 60 40 20 0
0
10
20
30
40
50
60
70
Time (h)
Figure 6 Cao et al.
28
Figure 7 Cao et al.
29
Figure 8 Cao et al. 30
Figure 9 Cao et al.
31
120
HeLa L929
Cell viability (%)
100
***
80 60 40 20 0
2
0
DOX concentration (µM)
Figure 10 Cao et al.
32
33
Research highlights: Modification of PEI and FI-HA does not appreciably change the morphology of MWCNTs. MWCNT/PEI-FI-HA/DOX complexes display a pH-responsive drug release behavior. MWCNT/PEI-FI-HA carrier is cytocompatible in the given concentration range. MWCNT/PEI-FI-HA/DOX complexes can target cancer cells via receptor-mediated manner. MWCNT/PEI-FI-HA/DOX complexes display a specific therapeutic effect to cancer cells.
34
