Many nanomaterials have been used as novel carriers for cancer therapy. Among them, carbon based-nanomaterials have been extensively used in biological applications. We reported platforms based on graphene oxide (GO), ordered mesoporous carbon (OMC) and carbon nanotubes (CNT) to conjugate with doxorubicin (DOX). The conjugation of DOX with carbon nanomaterial was investigated by UV-Vis spectroscopy, field emission scanning electron microscope (FE-SEM) and cyclic voltammetry (CV) methods. We showed that graphene oxide was a highly efficient matrix. Efficient loading of DOX, 89%, 78%, and 73.5% at pH 7.0 was seen onto GO, OMC and CNT, respectively. Upon pH 4. 0 after 15 h, 69%, 61% and 61% of DOX could be released from the DOX-GO, DOX-OMC and DOX-CNT, respectively, which illustrated the significant benefits of the developed approach for carbon nanomaterial applications. In addition, the study evaluated the cytotoxicity effect of DOX-GO, DOX-OMC and DOX-CNT on HT29 colon cancer cell lines. Cytotoxicity tests showed significantly higher toxicity of DOX/GO, DOX/OMC and DOX/CNT in comparison with GO, OMC and CNT against HT29 colon cancer cells with cell viability of 22%, 40% and 44% after 48 h for DOX-GO, DOX-OMC and DOX-CNT, respectively. Thus, developing nanohybrids based on carbon nanomaterial conjugated to DOX will remarkably enhance the anti-cancer activity.
Research Article
Conjugation of doxorubicin and carbon based-nanostructures for drug delivery against HT-29 colon cancer cells
https://doi.org/10.21203/rs.3.rs-2632275/v1
This work is licensed under a CC BY 4.0 License
Version 1
posted
You are reading this latest preprint version
Doxorubicin
Graphene oxide
Ordered mesoporous carbon
Carbon nanotubes
Colon cancer
One of the most critical categories in the discussion of drug delivery is the controlled release in the body, which in the traditional drug delivery system practically has no control over the time, location, and rate of drug release. In the field of cancer treatment, drug delivery systems play a critical role in increasing the effectiveness of treatment (Zhao et al. 2020; Amiri et al. 2021). With the rapid advances in nanotechnology, progress in nanomaterials engineering has created many hopes in the design and use of drug carriers, so these new drug carriers create much potential in improving drug packaging and targeting (Li et al. 2019; Rezaeifar et al. 2016). Nanomaterials, as one of the main structures in nanotechnology due to their small dimensions, unique physical and chemical properties, which have attracted the interest of many researchers in various fields, including the improvement of targeted drug delivery methods (Mitchell et al. 2021). In particular, hybrid nanocomposites have recently been investigated as promising systems for imaging and drug therapy applications (Patra et al. 2018). This aspect of nanomaterials is especially useful in cancer treatment, where the size of the drug delivery system is the key to targeting cancer cells by increasing permeability and inhibition (Manzano and Vallet-Regí 2020; Han et al. 2019). Nanoparticles must be biocompatible, biodegradable, non-inflammatory, and non-immunogenic. The purpose of designing drug delivery systems based on nanocomposites is to overcome the defects and disadvantages of conventional pharmaceutical formulations, increase the drug's effect by focusing on the target site, and reduce the amount of drug required (Begines et al. 2020; Zhang et al. 2013).
Carbon-based nanomaterials such as carbon nanotubes and graphene, due to their unique properties such as low toxicity, unique shape and geometry, high loading capacity, easy synthesis, and low cost, increased their use in the transfer of drugs, proteins, and genes (Debnath and Srivastava 2021; Maiti et al. 2019). The unique shape of carbon nanocarriers and the absence of these shapes in the morphology of the biological system of the human body is another advantage of using these nanocarriers in drug delivery (Sajjadi et al. 2021; Jampilek and Kralova 2021; Mohajeri et al. 2019; Mahajan et al. 2018). The main challenge of cancer treatment is separating cancerous cells from the body's normal cells (Chakraborty and Rahman 2012). Although chemotherapy plays a crucial role in cancer treatment, conventional chemotherapy has not been able to target only cancer cells and affects normal cells. However, using anti-cancer agents harms normal tissue (Lindley et al. 1999). Doxorubicin is a medicine from the class of anthracyclines. Anthracyclines (doxorubicin, daunorubicin, and idarubicin) are antibiotics with a wide range of clinical applications (Zhao et al. 2018). The use of Doxorubicin is limited for various reasons, including the drug's toxic effect, low stability in the blood circulation system, difficult penetration in the heterogeneous tissue of tumors, and the destruction of the drug by the decomposing enzymes in the body (Christidi and Brunham 2021). Despite the substantial and significant anti-cancer effects, due to the side effects of doxorubicin, including cardiomyopathy, and the occurrence of resistance to the drug, its use in treatment protocols is limited (Rawat et al. 2021). Drug release systems based on nanotechnology create a spectacular effect on cancer treatment. Advances in biomaterials and bio-engineering sciences are involved in the new approaches of nanoparticles, which may create a new window in improving cancer patients. Therefore, this study attempts to load the anti-cancer drug doxorubicin on carbon nanomaterials such as carbon nanotubes, ordered mesoporous carbon and graphene oxide by presenting a simple and cheap method. Also, the anti-cancer activity of these conjugates was investigated on HT-29 colon cancer cell lines.
Materials
Graphene oxide was purchased from Iranian Nano Materials Pioneers. Multi-walled carbon nanotubes (MWCNTs) were purchased from Shenzhen Nano-Technologies Port Co., Ltd. (China). Sulfuric acid (H2SO4), sodium hydroxide (NaOH), phosphoric acid (H3PO4), sodium dihydrogen phosphate (NaH2PO4), disodium hydrogen phosphate (Na2HPO4), sodium phosphate (Na3PO4), doxorubicin hydrochloride (C27H30ClNO11) and ordered mesoporous carbon (OMC) were purchased from Sigma-Aldrich and used as received.
Apparatus
Surface morphology and chemical composition of nanocomposite were examined by a scanning electron microscope of MIRATESCAN-XMU (Czech Republic) combined with EDS (energy-dispersive X-ray Spectroscopy) machine. UV-Vis spectra of samples were recorded by UV-Vis spectrophotometer (UV-1900, Shimadzu Co., Japan). Electrochemical measurements were performed with a potentiostat/galvanostat (Sama 500-c Electrochemical Analysis system, Sama, Iran). A conventional three-electrode configuration consisting of Ag|AgCl|KCl3M as the reference electrode, a platinum wire as auxiliary electrode and DOX-CNT, DOX-GO and DOX-OMC modified carbon paste electrode as working electrodes was employed.
Loading and on-demanded release experiments of DOX
1 mg of carbon nanostructures (GO, CNT and OMC) in 4 mL phosphate buffer solution (PBS, 0.1 M) with different pH was dispersed for 30 min at 25 0C. 1 mg of DOX was added to carbon nanostructures dispersed solution and continuously stirred for 3 h. The product was centrifuged with relative centrifugal force of 8000 g for 15 min. The collected pruduct washed with water and dried overnight at room temperature. The loaded percent of DOX on GO, CNT and OMC nanostructures was determined based on the standard curve of DOX (absorbance vs. concentration) at 488 nm with the concentration difference of DOX between the initial DOX solution (C0) and the supernatant solution (Cs) using the following equation:
Loading % = [(C0 –Cs)/C0]×100
The on-demand release was done by dialysis tubing. 1 mg of DOX-GO, DOX-CNT and DOX-OMC was dispersed in 4 mL of PBS (0.1 M), placed into dialysis tubing and dialyzed in 10 mL of PBS solution with pH 4.0 and 7.0. The release% of DOX was calculated with recording absorbance of PBS at 488.
Modification of carbon paste electrode with DOX-GO, DOX-CNT and DOX-OMC hybrids
The carbon paste electrode (CPE) was prepared based on previous reports (Zareyy et al. 2019; Bagheri Ladmakhi et al. 2020). The graphite powder plus paraffin hand-mixed until a uniformly wetted paste was obtained. Then the carbon paste was packed into a glass tube (with internal radius 3 mm). Electrical contact was made by a copper wire. The new surface of electrode was obtained by polishing it on a weighing paper. 1 mg of DOX-GO, DOX-CNT and DOX-OMC hybrids were added to 1 mL of water and sonicated for 30 min. Drop casting 5 µL of hybrids on CPE resulted in the DOX-GO/CPE, DOX-CNT/CPE and DOX-OMC/CPE used in this work.
Cell culture and treatment
The colon cell line (HT29) was cultured in RPMI 1640 medium containing 10% fetal serum, penicillin (100 µg/mL), streptomycin (100 µg/mL), and glutamine (0.002 M) for 72 h at room temperature. First, 3 mL of the culture medium containing 8000 cells were placed in plates containing wells and incubated in an atmosphere containing 5% CO2 for 24 h at 37°C. The DOX-GO, DOX-OMC and DOX-CNT suspensions were prepared and diluted with culture medium at different concentrations. Then 200 µL of DOX-GO, DOX-CNT and DOX-OMC were immediately added into the cells for 24 and 48 h. Cells not treated with DOX-GO, DOX-OMC, and DOX-CNT were used as controls.
Cytotoxicity assay
The toxicity of DOX-GO, DOX-CNT and DOX-OMC was evaluated on HT29 cells. The toxicity and viability of cells were measured by exposure to different times and concentrations of DOX-GO, DOX-CNT and DOX-OMC. After 24 and 48 h of exposure, the cells were washed, 200 µL of culture medium containing 0.5 mg of MTT was added to the well plates and incubated for 4 h at 37°C. The colored crystals of formazan were formed due to the metabolism of tetrazolium. The well plates were replaced with 150 µl of DMSO. After 30 min extraction at room temperature, the absorbance of the formazan solution is read at 570 nm. The light absorbance of each well plate is a criterion for survival of cells, recorded by an ELISA reader. Viability was calculated using the following equation:
%Viability = [(As – Ab) / (Ac-Ab)]×100 (2)
Where Ab, Ac and As are the absorbance of blank, control and sample solutions, respectively.
Characterization
The surface morphology of samples was evaluated by scanning electron microscope (SEM). Figure 1 shows FE-SEM images of GO, OMC, CNT, DOX-GO, DOX-OMC and DOX-CNT. As can be seen, GO has a wavy shape and wrinkled edge, while sphere shape particles were observed on the surface of the DOX-GO nanohybrid, indicating that GO sheets are decorated by DOX. In the case of OMC and CNT, the diameter of ordered mesoporous carbon and carbon nanotubes increased after loading with DOX, which confirms the decoration of OMC and CNT by DOX. It represents that -OH and -COOH groups in carbon nanostructures provide hydrogen bonds with -OH and -NH2 of DOX. In addition, there is π-π interaction between DOX and carbon nanostructure.
Figure 2 depicts cyclic voltammograms of free DOX, DOX-GO/CPE, DOX-OMC/CNT and DOX-OMC/CPE in the 0.1 M PBS (pH 7.0). DOX-GO/CPE and DOX-CNT/CPE in comparison with DOX-OMC/CPE, show well-defined redox noses (the anodic peak potential of around 400 mV and the cathodic peak potential of about 240 mV), related to the oxidation and reduction of DOX. Therefore, the presence of DOX on carbon nanomaterial is confirmed. The surface concentration of the electro-active DOX onto GO, OMC and CNT can be estimated using the equation (Hazhir et al. 2019):
Γ = Q/nFA (3)
Where Q is the charge consumed in coulombs, obtained from integrating the anodic (or cathodic) peak area in cyclic voltammogram under the background correction. The average value of (7.13 ± 0.57)×10− 11, (2.36 ± 0.93)×10− 11 and (6.97 ± 0.89)×10− 11 mol/cm2 was calculated for DOX-GO, DOX-OMC and DOX-CNT, respectively.
The confirmation of the formed DOX-GO, DOX-OMC and DOX-CNT nanohybrids was determined by UV-Vis spectroscopy in the range of 200–600 nm (Fig. 3a). DOX/GO nanohybrid shows distinct absorption peaks of DOX at around 240, 260, 300, and 500 nm, while the DOX absorption bands of DOX-OMC and DOX-CNT are weak and broad. Therefore, the UV-Vis spectrum confirms the formation of DOX-GO, DOX-OMC and DOX/CNT nanohybrids.
Loading and on-demanded release analysis
The loading% and release% of DOX is determined based on the standard curve of DOX by UV-Vis spectroscopy at 488 nm (Fig. 3b). The results demonstrated a plot (Fig. 3c) with a linear regression of A = 0.0285 + 0.027 [DOX] (R = 0.991). The loading% of DOX onto different interfaces (GO, OMC and CNT) was compared in 0.1 M PBS at different pH of PBS and shaking times. Figure 4a shows the pH effect of PBS at shaking time of 3 h on loading% of DOX on GO, OMC and CNT. As seen, the loading% on nanostructures increases from 5.0 to 7.0 and then a decrease is obtained at pH higher than 7.0. The pH dependent behavior may be due to the different degree of hydrogen-bonding interaction between DOX and carbon nanostructures under different pH conditions; whereas there is strongest hydrogen bonding interaction of carbon nanostructures with DOX in pH 7.0. Similar feature has been observed for the loading of anti-cancer drugs on carbon nanostructures reported in literatures (Hazhir et al. 2019; Samimi Tehrani et al. 2020). The effect of shacking time in range of 1–5 h on the loading% of DOX at pH 7.0 is shown in Fig. 4b. It can be seen, the surface of GO carrier is saturated at the shacking time of 3 h, while the surface of OMC and CNT carriers is saturated at 4 and 3 h, respectively. Moreover, the loading% onto the GO (89%) nanostructure in comparison with OMC (80%) and CNT (78%) nanocarriers was high in optimum condition due to stronger hydrogen bonds of GO (because of abundant functional groups) with DOX.
Figure 5 shows the release% of DOX from DOX-GO, DOX-OMC and DOX-CNT nanohybrids in 0.1 M PBS with pH 4.0 and 7.0 at different times. As shown, the release of DOX from carbon nanocarriers at pH 4.0 is more significant than at pH 7.0 because of the strong hydrogen bonding between DOX and carbon nanostructures at pH 7.0. Moreover, in acidic pH, DOX releases more than in a neutral environment because DOX is protonated in an acidic environment and its solubility increases in water. Moreover, the results showed that 50% of DOX was released from DOX-CNT after 5 h at pH 4.0, while 50% of DOX was released for DOX-GO and DOX-OMC after 10 h, indicating release rate of DOX from DOX-CNT is faster than DOX-GO and DOX-OMC. This was confirmed that there are strong bonds between of DOX and functional groups of GO and the release of DOX needs a long time. Also, it is suggested that DOX penetrates in porous structures of OMC and release% is low at low time in comparison with CNT and GO.
In vitro cytotoxicity evaluation
The cell toxicity of free DOX, DOX-GO, DOX-OMC and DOX-CNT with different concentrations on HT-29 colon cell lines in three time intervals of 24 and 48 h was evaluated by MTT assay to assess cell viability (Fig. 6). According to the results of MTT assay, we didn’t observe any significant toxicity for GO, OMC and CNT on HT-29 lines, while DOX-GO, DOX-OMC and DOX-CNT cause a significant decrease in the survival of the HT-29 cell lines depending on dosage and time. The results revealed a good cytocompatibility of GO, OMC and CNT which showed low apparent toxicity against HT-29 cells, even at higher concentrations. The treatment of free DOX showed most viability loss (22% and 13% for 24 h and 48 h to exposure, respectively). The higher toxicity of free DOX was caused by more rapid passive diffusion in vitro, while the DOX-GO, DOX-OMC and DOX-CNT nanohybrids internalized into cell mainly through less efficient endocytosis. Also, the results showed that the viability loss significantly increases within DOX-GO in comparison to DOX-CNT and DOX-OMC. The viability loss of DOX-GO is comparable to that reported by free DOX. It is suggested that DOX-GO in contact with the HT-29 cells has the high ability to penetrate and deliver DOX which can lead to cell death.
The half-maximal inhibitory concentration (IC50) of free DOX, DOX-GO, DOX-OMC and DOX-CNT after 24 and 48 h exposure against cell lines was illustrated in Table 1. For DOX-GO (because of rich functional groups on the surface) after 24 and 48 h exposure, the IC50 values were found to be lower than that observed for DOX-OMC and DOX-CNT. Result reveals that DOX and carbon nanostructures conjugate showed enhanced anti-cancer capability against HT-29 cell.
In the present work, we established DOX and carbon nanostructures (GO, OMC and CNT) conjugates and studied the anti-cancer activity of these conjugates on HT-29 colon cancer cell lines. The results showed that the GO, OMC and CNT nanostructures had low toxic effect on the cell lines. DOX-GO, DOX-OMC and DOX-CNT due to the DOX onto nanostructure lead to the significant viability loss and toxicity against HT-29 cell. The viability loss of DOX-GO is comparable to that reported by free DOX. The results suggest that the DOX-carbon nanostructures conjugates will pave the way for a new approach to prevent and treat cancer.
Acknowledgement
The authors are sincerely thankful for the research facilities provided by the Ayatollah Amoli Branch of the Islamic Azad University.
Funding
This research received no specific grant from any funding agency.
Ethics declarations
Competing interests
The Authors declare that they have no conflicts of interest.
Ethical approval
This research didn’t contain the experiments involved in animals or human participants, there is no need for ethical statement.
- Amiri M, Khazaeli P, Salehabadi A, Salavati-Niasari M (2021) Hydrogel beads-based nanocomposites in novel drug delivery platforms: Recent trends and developments. Adv Colloid Interf Sci 288:102316
- Bagheri Ladmakhi H, Chekin F, Fathi Sh, Raoof JB (2020) Electrochemical sensor based on magnetite graphene oxide/ordered mesoporous carbon hybrid to detection of allopurinol in clinical samples. Talanta 211:120759
- Begines B, Ortiz T, Pérez-Aranda M, Martínez G, Merinero M, Argüelles-Arias F, et al (2020) Polymeric nanoparticles for drug delivery: Recent developments and future prospects. Nanomater 10:1403
- Chakraborty S, Rahman T (2012) The difficulties in cancer treatment. Ecancermedicalscience 6:1
- Christidi E, Brunham LR (2021) Regulated cell death pathways in doxorubicin-induced cardiotoxicity. Cell Death Dis 12:1
- Debnath SK, Srivastava R (2021) Drug delivery with carbon-based nanomaterials as versatile nanocarriers: Progress and prospects. Front Nanotechnol 3:644564
- Han HJ, Ekweremadu C, Patel N (2019) Advanced drug delivery system with nanomaterials for personalised medicine to treat breast cancer. J Drug Deliv Sci Technol 52:1051
- Hazhir N, Chekin F, Raoof JB, Fathi Sh (2019) A porous reduced graphene oxide/chitosan-based nanocarrier as a delivery system of doxorubicin. RSC Adv 9:30729
- Jampilek J, Kralova K (2021) Advances in drug delivery nanosystems using graphene-based materials and carbon nanotubes. Mater 14:1059
- Li C, Wang J, Wang Y, Gao H, Wei G, Huang Y, et al (2019) Recent progress in drug delivery. Acta Pharma Sinica B 9:1145
- Lindley C, McCune JS, Thomason TE, Lauder D, Sauls A, Adkins S, et al (1999) Perception of chemotherapy side effects cancer versus noncancer patients. Cancer Pract 7:59
- Mahajan S, Patharkar A, Kuche K, Maheshwari R, Deb PK, Kalia K, et al (2018) Functionalized carbon nanotubes as emerging delivery system for the treatment of cancer. Int J Pharma 548:540
- Maiti D, Tong X, Mou X, Yang K (2019) Carbon-based nanomaterials for biomedical applications: a recent study. Front Pharmacol 9:1401
- Manzano M, Vallet-Regí M (2020) Mesoporous silica nanoparticles for drug delivery. Adv Funct Mater 30:1902634
- Mitchell MJ, Billingsley MM, Haley RM, Wechsler ME, Peppas NA, Langer R (2021) Engineering precision nanoparticles for drug delivery. Nat. Rev. Drug Discov 20:101
- Mohajeri M, Behnam B, Sahebkar A (2019) Biomedical applications of carbon nanomaterials: Drug and gene delivery potentials. J Cell Physiol 234:298
- Patra JK, Das G, Fraceto LF, Campos EVR, Rodriguez-Torres MDP, Acosta-Torres LS, et al (2018) Nano based drug delivery systems: recent developments and future prospects. J Nanobiotechnol 16:1
- Rawat PS, Jaiswal A, Khurana A, Bhatti JS, Navik U (2021) Doxorubicin-induced cardiotoxicity: An update on the molecular mechanism and novel therapeutic strategies for effective management. Biomed Pharma 139:111708
- Rezaeifar M, Mahmoudvand H, Amiri M (2016) Formulation and evaluation of diphenhydramine gel using different gelling agents. Der Pharma Chem 8:243
- Samimi Tehrani N, Masoumi M, Chekin F, Sharifzadeh Baei M (2020) Nitrogen doped porous reduced graphene oxide hybrid as a nanocarrier of imatinib anti-cancer drug. Russ J App Chem 93:1221
- Sajjadi M, Nasrollahzadeh M, Jaleh B, Soufi GJ, Iravani S (2021) Carbon-based nanomaterials for targeted cancer nanotherapy: Recent trends and future prospects. J Drug Target 29:716
- Zareyy B, Chekin F, Fathi Sh (2019) NiO/porous reduced graphene oxide as active hybrid electrocatalyst for oxygen evolution reaction. Russ J Electrochem 55:333
- Zhang G, Zeng X, Li P (2013) Nanomaterials in cancer-therapy drug delivery system, J Biomed Nanotechnol 9:741
- Zhao N, Woodle MC, Mixson AJ (2018) Advances in delivery systems for doxorubicin. J Nanomed & Nanotechnol 9:519
- Zhao Z, Ukidve A, Kim I, Mitragotri S (2020) Targeting strategies for tissue-specific drug delivery. Cell 181:151
No competing interests reported.