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Anticancer drug delivery systems based on noncovalent interactions between carbon nanotubes and lineardendritic copolymers
Mohsen Adeli,*a Farahman Hakimpoor,a Masoumeh Ashiri,a Roya Kabirib and Masoumeh Bavadia
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Received 29th December 2010, Accepted 8th February 2011 DOI: 10.1039/c0sm01550d Hybrid nanomaterial-based drug delivery systems (HNDDSs) consisting of carbon nanotubes and lineardendritic copolymers linked to anticancer drugs were synthesized and characterized. Polycitric acidpolyethylene glycolpolycitric acid (PCAPEGPCA) lineardendritic copolymers were used to solubilize and functionalize multi-walled carbon nanotubes (MWCNTs) by noncovalent interactions. There are two key features of HNDDSs: (a) functionalized MWCNTs as a biocompatible platform for the delivery of therapeutic agents and diagnostics, (b) PCAPEGPCA lineardendritic copolymers as water soluble, biocompatible and highly functional hybrid materials with a linear PEG block and two dendritic PCA blocks that improve the solubility and functionality of MWCNTs respectively. In this work, cisplatin (cis-diamminedichloroplatinum (CDDP)a platinum-based chemotherapy drug) was conjugated to the carboxyl functional groups of the dendritic blocks of PCAPEGPCA linear dendritic copolymers and then the prodrugs interacted with the MWCNTs noncovalently and HNDDSs were obtained. To prove the efcacy of the synthesized HNDDSs, they were subjected to endocytosis and released CDDP molecules inside murine colon adenocarcinoma tumor C26 cancer cells. Then it was proved that HNDDSs are able to kill cancer cells effectively. Release of the anticancer drug from HNDDSs was also investigated.

Introduction
Efforts to overcome the limitations in the progress of novel cancer therapies such as inefcient distribution, insolubility, the killing of healthy tissues, and the inability to cross cellular barriers that are often encountered in the administration step has led to an interest in HNDDSs. These systems present noteworthy opportunities to meet novel cancer therapy challenges and have effectively been used to deliver biologically active agents into living cells for biomedical applications.17 The use of HNDDSs for biomedical applications constitutes a new eld called nanomedicine which involves a multi-step process from the design, synthesis, in vitro experimentation and initial administration to cross the tissue endothelium barrier and introduction into the interstitial space of tissues, through the cell membrane into organelles of cells and even through the perinuclear membrane into the nucleus of cells. Among diverse classes of HNDDSs, those based on the carbon nanotubes have attracted particular attention as carriers for biologically relevant molecules due to their unique properties such as the ability to cross cell membranes and high surface area per unit weight for high drug loading.824 A varity of HNDDSs

a Department of Chemistry, Faculty of Science, Lorestan University, Khoramabad, Iran. E-mail: mohadeli@yahoo.com; Fax: (+98)6612202782; Tel: (+98)916-3603772 b NMR lab, Faculty of Chemistry, Tabriz University, Tehran, Iran

based on the carbon nanotubes have been recently prepared and tested in vitro and in vivo for delivery of drugs.2531 Poor water solubility and low functionality are two critical factors that inuence the performance of CNTs for biomedical applications. Therefore, in order to make HNDDSs based on CNTs these factors should be improved.3241 The solubility of CNTs can be developed by treatment with polymers either covalently or noncovalently. In the covalent method, polymers are grafted onto the surface of CNTs through chemical linkages. This method is very effective because the grafted polymer raises the solubility and functionality of the CNTs even with a low degree grafting, but a disadvantage of this method is the creation of defect sites on the side walls of the CNTs, where sp2-hybridized carbons are changed to sp3-hybridized ones, leading to a diminished conjugated p system and therefore the loss of useful optical and electronic properties.4250 The non-covalent method is based on the supramolecular interactions between CNTs and polymers and includes polymer wrapping or adsorption.51,52 In this method the structure of the CNTs does not change as much as in the covalent method, but its disadvantage is the low functionality of the nal product. Herein a new method for developing of both functionality and water solubility of the CNTs using lineardendritic copolymers without damaging their structure has been reported. Lineardendritic copolymers are hybrid materials consisting of linear and dendritic macromolecules.5358 Supramolecular interactions between a linear dendritic copolymer and a CNT should result in a hybrid
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material with improved solubility and functionality. PEG is a well-studied and useful polymer not only to synthesize variety of lineardendritic copolymers but to improve the processability, water solubility and long blood circulation of CNTs through supramolecular chemistry.5971 Hence supramolecular interactions between PEG, and dendritic, block(s) of lineardendritic copolymers and CNTs should lead to water soluble and highly functional hybrid materials. PCAPEGPCA lineardendritic copolymers are biocompatible, water soluble and highly functional ABA type lineardendritic copolymers that have been synthesized and characterized previously and their potential application as nanocarriers has been also investigated.7274 In this work, highly functional and water soluble hybrid materials consisting of PCAPEGPCA lineardendritic copolymers and MWCNTs were prepared and their potential application as anticancer drug delivery systems has been investigated.

Experimental section
Materials MWCNT were prepared by a chemical vapor deposition procedure in the presence of Co/Mo/MgO as catalyst at 900  C. They were puried and opened using sulfuric acid/nitric acid mixture according to reported methods in literature.4550 Cisplatin (cisdiamminedichloroplatinum (CDDP) (purity 99.9%) was purchased from Aldrich Chemical Co. Citric acid monohydrate (MW 210.14), polyethyleneglycol (MW 1000), AgNO3 and the other materials were purchased from Merck. The murine colon adenocarcinoma tumor C26 cancer cells were obtained from the National Cell Bank of Iran (NCBI) Pasteur Institute, Tehran, Iran. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) powder, Annexin-V FLUOS Staining Kit, was obtained from Sigma. RPMI 1640 modied medium, fetal bovine serum (FBS), and penicillin/streptomycin solution were obtained from Gibco Invitrogen (Carlsbad, California). Phosphoric acid used as the mobile phase in high-performance liquid chromatography (HPLC) was purchased from Merck. Deionized water was used in all experiments. Characterization H NMR spectra were recorded in water solution on a Bruker DRX 400 MHz apparatus with the solvent proton signal for reference. Infrared spectroscopy (IR) spectra were recorded using a Nicolet 320 FT-IR spectrophotometer. PCAPEGPCA linear dendritic copolymers were dissolved in tetrahydrofuran and then dropped on a tablet of KBr. Solvent was immediately evaporated and IR spectra was recorded. HNDDSs were prepared in water, then the solvent was evaporated and the obtained powder was mixed with anhydrous KBr to make tablet and record the IR spectra. Raman spectra were recorded by an Almega Thermo Nicolet Dispersive Raman Spectrometer. Photographs were prepared by a digital Kodak camera. Ultraviolet (UV) spectra were recorded on a Shimadzu (1650 PC). Thermogravimetric analysis (TGA) were carried out using a thermal analyzer (model: DSC 60, Shimadzu, Japan) under dynamic atmosphere of an inert gas (i.e. N2) at 30  C min1. The particle size and polydispersity were determined using Dynamic Light Scattering (DLS) zetasizer ZS, Malvern instruments.
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1

Solvent for DLS experiments was water and concentration of sample was 0.001 g mL1. Transmission electron microscopy (TEM) images were obtained using an LEO 912AB electron microscope with an accelerating voltage of 200 kV. Samples were dissolved in water and drop on holder. Then water was evaporated at room temperature and images were recorded. The morphology of nanomaterials was also investigated using a Philips XL30 scanning electron microscope (SEM) with 12 and 15 V accelerating voltages. The sample used for SEM observations were coated with a thin layer of gold. Surface imaging studies were performed using atomic force microscopy (AFM) to estimate surface morphology and particle size distribution. The samples were imaged on a glass holders with the aid of Dualscope/Rasterscope C26, DME, Denmark, using DS 95-50-E scanner with vertical z-axis resolution of 0.1 nm. A simple and reproducible reversed-phase high performance liquid chromatography (HPLC) with a Knauer liquid chromatograph (Smart line; Knauer, Berlin, Germany) equipped with an ultraviolet detector (Wellchrom, K-2600; Knauer) and a reverse-phase C18 column (Nucleosil H.P. 25 cm 0.46 cm internal diameter, pore size mm; Knauer) using isocratic elution with UV absorbance detection was developed and validated for determination of cisplatin loaded onto the nanomaterials. The mobile phase was 15 mM phosphoric acid solution and ow rate was 1.00 mL min1. The column efuent was detected at 210 nm. The retention time of free CDDP peak was appeared between 24 min and the run time was 15 min. Linear regression with an acceptable linear relationship between response (peak area) and concentration in the range of 1 to 64 mg mL1 was observed. The regression coefcient was 0.9999 and the linear regression equation was Y 34324X + 15334. Sample concentrations were calculated using the calibration curves. Preparation of MWCNT/PCAPEGPCA hybrid nanomaterials The PCAPEGPCA lineardendritic copolymers were prepared according to reported procedure in literature.56 In this work, a variety of lineardendritic copolymers were synthesized using different ratios of PEG to monohydrated citric acid (CA) (1 : 2, 1 : 5, 1 : 8, 1 : 10) and they were used for solublizing MWCNTs in aqueous solutions with different pHs (3, 5, 7, 9) adjusted using 0.001 M NaOH aqueous solution. PCAPEGPCA linear dendritic copolymers (0.01 g) were dissolved in 5 mL water and the resulted solution was stirred for 5 min at room temperature. Then pristine or opened MWCNTs (0.001 g) were added to this solution in a round bottom ask and mixture was stirred at room temperature for 5 min. The mixture was sonicated for 15 min and ltrated to remove impurities such as graphite and then it was kept at room temperature. Synthesize of CDDP prodrugs CDDP was conjugated to the PCAPEGPCA linear-dendritic copolymers according to the reported methods in the literature with a slight modication.75,76 CDDP (0.009 g, 0.03 mmol) was suspended in distilled water (10 mL) and silver nitrate (0.005 g, 0.03 mmol) ([AgNO3]/[CDDP] 1) was added to this suspension. The mixture was stirred in the dark at room temperature until
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AgCl appeared as a white precipitate. Afterwards the mixture was ltered through a 0.45 mm membrane to remove the AgCl precipitate and PCAPEGPCA (0.03 g) was added to this solution and the mixture was stirred at room temperature for 48 h. Finally solution was dialyzed in water for 12 h to obtain PCAPEGPCACDDP prodrugs. Preparation of MWCNT/PCAPEGPCACDDP The PCAPEGPCACDDP prodrug (0.0005 g) was dissolved in water (2 mL) in a round bottom ask and MWCNTs (0.002 g) were added to this solution. The mixture was stirred at room temperature for 5 min and then water was added to the resulting mixture to adjust the total volume to 5 mL. The mixture was sonicated for 15 min and the resulting suspension was kept in the dark at room temperature. Loading capacity The loading capacity of HNDDSs (to load CDDP molecules) was determined by high performance liquid chromatography (HPLC), the analysis was performed triplicate. A water solution of HNDDSs was prepared and ltered and it was added to an aqueous solution of CDDP. Then an aliquot of 20 mL of the mixture was analyzed by HPLC to detect the free (not loaded) CDDP concentration. The loading capacity of the polymer was calculated as below: LC (Ci Cf)/Ci 100 where: LC loading capacity, Ci the initial concentration of CDDP and Cf the nal concentration of CDDP. In vitro drug release The rate of release of CDDP molecules from MWCNT/PCA PEGPCACDDP was measured in HBS (pH 7.4) at 37  C by HPLC. The MWCNT/PCAPEGPCACDDP was dispersed in 9.5 mL HBS-buffered saline and decanted into tubes, then the tubes were placed in a CO2 incubator at 37  C. Sampling (250 mL each time) was performed at predetermined time intervals. Samples were injected directly to the HPLC system and analysed. Cell culture Murine colon adenocarcinoma tumor C26 cancer cells were cultivated in RPMI-1640 medium supplemented with 10% fetal bovine serum and 1% penicilinestreptomycin at 37  C in a humidied incubator with 5% CO2. The cells were maintained in an exponential growth phase by periodic subcultivation. Cytotoxicity assay In vitro cytotoxicity of the nanomaterials was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The cells (2500 cells/well) were seeded in 96-well plates. The nanomaterials were then added to the wells in triplicate and incubated for 72 h. After the incubation period, 20 mL of MTT dye (5 mg mL1 in PBS) was added to each well, and they were incubated in the dark at 37  C for 5 h. Then medium were removed and formazan crystals were dissolved in 200 mL
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dimethylsulfoxide (DMSO) and 20 mL of glycine buffer. Then the absorbance of each well was measured by an ELISA reader (Statfax2100 Awareness Technology, USA) at 570 nm. Cell viability was calculated using the following equation: Cell viability (%) (Ints/Intscontrol) 100 (1)

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where Ints is the colorimetric intensity of the cells incubated with the samples, and Intscontrol is the colorimetric intensity of the cells incubated with the medium only (positive control). It is notable that owing to the absorption of vitamins, amino acids and ions at the surfaces of nanoparticles, it is clear that the common in vitro examination method can yield erroneous cell viability values. Outlier detection All MTT experiments were performed in triplicate or more, with the results expressed as mean standard deviation; standard deviation values are indicated as error bars in the MTT results plots. The results were statistically processed for outlier detection using a T procedure using MINITAB software (Minitab Inc., State College, PA). One-way analysis of variance (ANOVA) with p < 0.05 was performed for each set of MTT assay test repeats. Outlier samples have then been excluded from the corresponding asset viability calculations. In this method, a T-ratio is calculated as follows X X T S

(2)

where X is the suspected outlier point (normally the smallest or  the largest value in a set of measurements), X is the sample mean, and S is the (estimated) standard deviation. If the calculated value of T is equal to or exceeds a critical value, the outlier point is removed with a signicance level of 0.05. In the latter case, assuming that the data were sampled from a normal distribution, there is at least a 95% chance that the suspected point is in fact far from other points.

Results and discussion

Two factors including poor water solubility and low functionality limit the biomedical applications of CNTs. Linear polymers are able to wrap around CNTs through noncovalent interactions to solubilize them in different solvents. Among the variety of linear polymers that are used to solublize the CNTs in aqueous solutions, PEG is the most promising and well studied polymer that develops both their water solubility and biocompatibility. However PEG cannot improve the functionality of CNTs as well as their solubility and biocompatibility. Dendrimers and dendritic polymers are a type of tree-like polymers with a high functionality that should develop the functionality of CNTs, but a challenge is their weak non-covalent interactions with CNTs. Lineardendritic copolymers such as PCAPEGPCA which are a hybrid of linear and dendritic polymers are the best candidates to develop the solubility and the functionality of CNTs by their linear and dendritic blocks respectively (Fig. 1). Fortunately interactions between the PCAPEGPCA copolymers, synthesized using different ratios of PEG/CA, and
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Fig. 2 Photograph of dispersed MWCNTs by PCAPEGPCA linear dendritic copolymer, synthesized using different PEG/CA ratios and in different pH. i) Fresh solutions; ii) solutions after four months; iii) solutions after 12 months. PEG/CA ratios a) 1/2; b) 1/5; c) 1/8; d) 1/10 and in 1) pH 3, 2) pH 5, 3) pH 7, 4) pH 9, 5) pH 11.

Fig. 1 Noncovalent interactions between lineardendritic copolymers and MWCNTs lead to hybrid nanomaterials.

carbon nanotubes led to the corresponding hybrid nanomaterials (MWCNT/PCAPEGPCA). They were completely soluble in water in different pHs and their aqueous solutions were stable over several months (Fig. 2). MWCNT/PCAPEGPCA hybrid nanomaterials in which PCAPEGPCA lineardendritic copolymers were synthesized using 1/8 and 1/10 PEG/CA ratios precipitated in highly acidic and basic solutions, assigned to the hydrolysis of esteric bonds of PCA blocks. This result conrms that in addition to PEG, the PCA blocks play a signicant role in the solubilization of MWCNTs in aqueous solutions. Herein, CDDP molecules were activated and then added to the aqueous solution of PCAPEGPCA lineardendritic copolymers and anticancer prodrugs were synthesized (Scheme 1). The synthesized prodrugs were then added to the carbon nanotubes to interact with them noncovalently (Fig. 1).
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Scheme 1 Coordination of CDDP molecules to the carboxyl functional groups of PCAPEGPCA lineardendritic copolymers.

Interactions between the MWCNTs and lineardendritic copolymers were investigated using IR, NMR and Raman spectroscopy. Two absorbance bands corresponded to the acidic and esteric carbonyl functional groups of PCA blocks of the lineardendritic copolymers are exhibited at 1700 and 1740 cm1 respectively (Fig. 3d). In the IR spectra of MWCNT/PCAPEG PCA hybrid nanomaterials both absorbance bands are shifted toward lower frequencies, proving that there are strong interactions between PCA blocks and MWCNTs. This result also shows that the strength of the C]O bonds is weakened. On the other hand the intensity of the absorbance band of the esteric carbonyl functional groups is decreased signicantly, due to the reduced polarity of these groups (Fig. 3e). Both observations prove that
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Fig. 4 1H NMR spectra of a) PCAPEGPCA,b) MWCNT/PCA PEGPCA and c) MWCNT/PCAPEGPCACDDP. Fig. 3 IR spectra of a) pristine MWCNT, b) opened MWCNT, c) PCA PEGPCA, d) MWCNT/PCAPEGPCA and e) MWCNT/PCAPEG PCACDDP.

there is electron transfer from the p system of the MWCNTs to the C]O bonds. The absorbance band of aliphatic CH bonds is shifted toward higher frequencies, conrming that the CH2O bonds of PEG are also interacting with the MWCNTs (Fig. 3c and 3d). The disappearance of the absorbance band of the acidic carbonyl functional groups shows that CDDP molecules are conjugated to the lineardendritic copolymers through coordination to their hydroxyl functional groups. The shifting of the absorbance bands of the esteric carbonyl functional groups toward lower frequencies can be assigned to their more effective interactions with the carbon nanotubes upon conjugation of the CDDP molecules to the hydroxyl functional groups of linear dendritic copolymers (Fig. 3e). Comparison of the 1H NMR spectra of PCAPEGPCA lineardendritic copolymers with the MWCNT/PCAPEGPCA hybrid nanomaterial shows that the peak surface area of theh PEG and PCA blocks does not change signicantly upon interaction with MWCNTs. Therefore it can be found that both PEG and PCA blocks are interacting equally with the MWCNTs (Fig. 4a and b). The peak surface area of the PCA blocks decreased much more than that of the PEG block upon coordination of the CDDP molecules to the carboxyl functional groups of the lineardendritic copolymers (Fig. 4c).
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This result proves that conjugation of the CDDP molecules to the lineardendritic copolymers increases interactions of the PCA blocks with the MWCNTs much more than that for PEG block which is in agreement with the results obtained from the IR spectra. Raman spectroscopy provides more interesting results concerning the interactions between the MWCNTs and the linear dendritic copolymers. Fig. 5 shows the Raman spectra of pristine MWCNTs, opened MWCNTs, MWCNT/PCAPEGPCA and MWCNT/PCAPEGPCACDDP. In the Raman spectrum of pristine MWCNTs, D- and G-bands are exhibited at 1351 and 1584 cm1 respectively, while the acid-treatment MWCNTs results in a substantial increase in the D mode and a decrease in the tangential G mode associated with sp2-hybridized carbons from the graphitic sidewalls. The latter is ascribed to the defects produced during the purication process. The Raman spectrum of the MWCNT/PCAPEGPCA hybrid nanomaterial does not show recognizable D- and G-bands, conrming that the noncovalent interactions between the MWCNT and lineardendritic copolymer are strong enough to change the structural pattern of the MWCNTs. D- and G-bands of MWCNTs appear after conjugation of the CDDP molecules to the functional groups of the lineardendritic copolymer a shift of the G-band toward higher frequencies is observed while the D-band is not shifted.
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Fig. 5 Raman spectra of a) pristine MWCNT, b) opened MWCNT, c) PCAPEGPCA, d) MWCNT/PCAPEGPCA and e) MWCNT/ PCAPEGPCACDDP.

This result conrms that defects created on the sidewalls of carbon nanotubes do not play an important role in the noncovalent interactions between the lineardendritic copolymers and the MWCNTs. On the other words interactions between carboxyl functional groups of the opened MWCNT and PCA blocks are not important as much as interactions, pp staking, between C]C bonds of MWCNT and C]O bonds of linear-dendritic copolymers. Based on previous studies,45,46 effective interactions between polymer and carbon nanotubes should lead to changes in their conformation. Therefore conformation and topology of MWCNT/PCA-PEG-PCA and MWCNT/PCA-PEG-PCACDDP hybrid nanomaterials was investigated in solution and solid state by DLS, TEM and AFM. DLS diagrams display unimodal size distributions with mean diameters of 237, 563 and 371 nm for PCAPEGPCA, MWCNT/PCAPEGPCA and MWCNT/PCAPEGPCA CDDP hybrid nanomaterials respectively. The large size of the PCAPEGPCA lineardendritic copolymers is ascribed to their self-assembly in aqueous solution.56
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While the length of carbon nanotubes is around several micrometres, the size (diameter) of the MWCNT/PCAPEG PCA hybrid nanomaterials measured by DLS is 563 nm (Fig. 6). Similar to linear polymers which are coiled in solution, the decreased diameter of the MWCNTs can be assigned to their looping due to the noncovalent interactions with the linear dendritic copolymers (Fig. 7). Conrming the results obtained by IR and NMR spectroscopy, the diameter of the MWCNT/PCA PEGPCA hybrid nanomaterials is even more decreased after the conjugation of CDDP molecules. Based on the DLS results, MWCNT/PCAPEGPCACDDP hybrid nanomaterials should have a very packed conformation or spherical structure in the solution state. AFM images show spherical topology with 200350 nm diameter for MWCNT/PCAPEGPCACDDP hybrid nanomaterials (Fig. 8a andc). Three phases relating to the three components of the hybrid nanomaterials can be clearly recognized in their phase contrast images (Fig. 8b and d). According to the SEM images, MWCNT/PCAPEGPCA CDDP hybrid nanomaterials have a spherical topology and are in a packed conformation. The average size of the spherical objects is around 200300 nm (Fig. 9a and b). Once again it can be deduced that noncovalent interactions between lineardendritic copolymers and MWCNTs are so strong that they change the conformation of the MWCNTs from extended to a closed or packed state. This result is highly signicant when carbon nanotubes are used for medical proposes. Because their size is decreased to 200300 nm which is desirable for an effective passive targeting. Fig. 10a shows a TEM image of a MWCNT which is coiled, due to the noncovalent interactions with the PCAPEGPCA lineardendritic copolymer. It can be seen that it is opening towards an extended conformation upon the impaction of the electron beam of the TEM. The uneven surface of the carbon nanotube is assigned to the association of the lineardendritic copolymers onto its surface (Fig. 10a). Based on our previous investigations the cavity of the polymer-functionalized carbon nanotubes are able to host nanoparticles of up to 15 nm in diameter, due to their high solubility and opened cavity.77 Since the size of an individual PCAPEGPCA lineardendritic copolymer is less than 10 nm,56,58 they can transfer conjugated CDDP molecules to the cavity of MWCNT/PCAPEGPCA hybrid nanomaterials (Fig. 10b). As is exhibited by the TEM images, the synthesized

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Fig. 6 DLS diagrams of a) PCAPEGPCA, b) MWCNT/PCAPEG PCA and c) MWCNT/PCAPEGPCACDDP.

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Fig. 7 Noncovalent interactions between lineardendritic copolymers and MWCNTs change their conformation from extended to closed in the solution state.

Fig. 10 TEM images of a) MWCNT/PCAPEGPCA and b) MWCNT/PCAPEGPCACDDP.

Fig. 8 AFM images of MWCNT/PCAPEGPCACDDP: i) topographic image, ii) and iii) phase-contrast images and iv) image prole of the marked particles in images i) and ii).

PCAPEGPCACDDP because of the coordination of the CDDP molecules to the carboxyl functional groups of the linear dendritic copolymers. If each CDDP molecule coordinates to two carboxyl functional groups, then the number of the conjugated CDDP molecules to each MWCNT/PCAPEGPCA unit can be estimated to be 6 (Fig. 11). There are two main weight-loss temperatures for the synthesized PCAPEGPCA lineardendritic copolymers, the rst at 130250  C is assigned to the decomposition of the PCA blocks and the second at 380410  C is related to the decomposition of the PEG block. The PCA and PEG contents for the PCAPEG PCA lineardendritic copolymer are 50% and 40% respectively. Diagram c in Fig. 12 shows that the TGA pattern for the PCAPEGPCA lineardendritic copolymer changes upon interaction with the carbon nanotubes, so that the weight-loss domains shift to higher temperatures. This is more sensible for a MWCNT/PCAPEGPCACDDP hybrid nanomaterial, proving that conjugation of CDDP molecules to the functional groups of PCAPEGPCA lineardendritic copolymers increase their interactions with MWCNTs (Fig. 12). In order to examine the potential application of hybrid nanomaterials in nanomedicine and to understand their limitations and capabilities as nano-excipients in biological systems short-term in vitro cytotoxicity tests were conducted on murine colon adenocarcinoma tumor C26 cancer cells. The time of incubation was 5 h.

Fig. 9 a) and b) SEM images of the MWCNT/PCAPEGPCACDDP hybrid nanomaterial.

hybrid nanomaterials transfer CDDP molecules not only by conjugation to the lineardendritic copolymers on their surface but also in their cavity. The zeta potential of the hybrid nanomaterials changes from 18.3 for MWCNT/PCAPEGPCA to 6.3 mV for MWCNT/
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Fig. 11 Zeta potential diagrams of a) PCAPEGPCA, b) MWCNT/ PCAPEGPCA and c) MWCNT/PCAPEGPCACDDP.

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Fig. 14 In vitro release of CDDP from MWCNT/PCAPEGPCA CDDP in HBS (pH 7.4) buffer at 37  C.

Fig. 12 TGA diagrams of a) opened MWCNT, b) PCAPEGPCA, c) MWCNT/PCAPEGPCA and d) MWCNT/PCAPEGPCACDDP.

A MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay showed not only any toxicity, up to 12.5 mg mL1, for the PCAPEGPCA lineardendritic copolymers but also an increase in the growth of incubated cells against untreated control cells (Fig. 13). Due to the citric acid backbone of linear dendritic copolymers, it can probably be used as the source of energy by the cells and therefore it leads to an increase in the growth and division of the cells. Hence any object associated with the PCAPEGPCA lineardendritic copolymers should insert into the cell metabolism and inuence the viability of the cells extensively. MWCNTs raise the rate of the transferring of the lineardendritic copolymers from the cell membrane leading to an increase in the cell growth (Fig. 13). Due to their complementary roles, the toxicity of the MWCNT/PCAPEGPCACDDP HNDDSs is even higher than that for the free drug. Because MWCNTs increase the rate of crossing of the cell membrane and PCAPEGPCA linear dendritic copolymer improve the water solubility of the MWCNTs and probably inserts them in the cell metabolism.

It should be always kept in mind that in the presence of an MTT assay that although equal concentrations of MWCNT/ PCAPEGPCACDDP HNDDSs and the free drug are compared, the loaded drug on HNDDSs is much lower than free drug concentration. Therefore considering the real concentration of the drug content in the drug delivery systems, their toxicity is much higher than that of the free drug. Fig. 14 shows the rate of the release of the CDDP molecules from the MWCNT/PCAPEGPCACDDP HNDDSs at 37  C and pH 7.4. The rate of the release is slow and the cumulative release percent after 168 h is around 40%. According to this result and MTT assay, HNDDSs transfer through the cell membrane rapidly but release the loaded drugs slowly. Hence these systems can be used for efcient cancer therapy with minimal side effects.

Conclusion
Noncovalent interactions between MWCNTs and PCAPEG PCA lineardendritic copolymers lead to polyfunctional hybrid nanomaterials. Both the linear and dendritic parts interact with the carbon nanotubes and their interaction is strong enough to change their conformation from extended to globular. Hybrid nanomaterials are able to transport small molecules such as anticancer drugs both by their functional groups and their cavity. MTT assays show high toxicity for HNDDSs due to the complementary roles of carbon nanotubes and lineardendritic copolymers.

Notes and references

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Fig. 13 Percentage survival of C26 cancer cells, assessed by the MTT assay, after exposure to free CDDP, opened MWCNT, PCAPEGPCA, MWCNT/PCAPEGPCA and MWCNT/PCAPEGPCACDDP at 12.5, 25 and 50 mg mL1 (n 3). P.C. is the positive control.

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