One of the important goals of cancer treatment in drug delivery systems (DSS) is to increase the concentration of the drug only around the tumor cells to prevent damage to normal cells. In this study, doxorubicin HCl (DOX) was imprinted by the cationic polymer, graphene quantum dots (GQD) Poly (methacrylic acid-co-diallyldimethylammonium chloride) (PMA-DDA-DOX). Chemical structure (PMA-DDA-DOX) was investigated and confirmed by using Fourier-transform infrared spectroscopy (FT-IR). Particle size and morphology of (PMA-DDA-DOX) was evaluated by Field Emission Scanning Electron Microscope (FE-SEM). Then the toxicity of (PMA-DDA-DOX) was investigated in vitro study. Also to evaluate the effectiveness of (PMA-DDA-DOX) in the living organism, Nanoparticles were injected into BALB/C mice containing 4T1 breast cancer cells. Then in vivo imaging was performed at the excitation wavelength of 400 nm and emission of 535 nm. The images showed that particles (PMA-DDA-DOX) had accumulated around the cancer cell.
Research Article
Graphene quantum dots coated cationic polymer for targeted drug delivery and imaging of breast cancer
https://doi.org/10.21203/rs.3.rs-1740614/v1
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Graphene quantum dots
Doxorubicin
Cationic polymer
in vivo imaging
Breast Cancer
Drug delivery
Breast cancer is one of the most common types of cancer is the leading cause of cancer death in women [1]. Most anticancer drugs are designed to kill cancer cells that divide rapidly, but these drugs can also Non-selective affect normal cells that have a natural growth [2]. In drug delivery systems (DDS), the drug accumulates around the target tissue to prevent damage to normal cells [3]. Cancer cells produce large amounts of lactate anions due to their cellular activity. This phenomenon causes negativity environment around tumor cells [4]. Using this biophysical phenomenon, cation carriers can accumulate around cancer cells without any molecular markers and release the drug to prevent damage to normal cells [5]. Cationic polymers are macromolecules that can have positive charges in the backbone or side chains. Most cationic polymers have one or more amine functional groups that can be protonated. These polymers are divided into three groups: Synthetic, semi-synthetic, and natural. Due to their physical and chemical properties and the possibility of modifying these properties, they have become a candidate for drug delivery [6]. Poly methacrylic acid-co-diallyldimethylammonium chloride is a cationic polymer with an amino group that can be protonated. Also, good water solubility and very low toxicity can be used as a safe drug carrier[7]. In recent years; various methods have been developed to improve DDS. However, these methods have drawbacks such as cell damage, toxicity, immunogenicity, inefficient drug delivery, limited use in the body, etc [8]. Imaging of cancerous tissues facilitates the diagnosis and handling of the disease, Semiconductor metallic quantum dots are used in many fields of medicine and bio-imaging like long-term and real-time imaging. However, the side effects of Semiconductor metallic quantum dots have forced researchers to find alternatives to these quantum dots [9]. Particles of graphene quantum dots with properties such as solubility in water, chemical and physical stability, particle size below 100 nm, high fluorescence properties, low toxicity, and excellent biocompatibility can be a suitable alternative to metallic quantum dots [10]. In this study, we synthesized a non-toxic nanocarrier that can deliver DOX to tumor cells and image them.
Chemicals, materials and media
Dimethylformamide (DMF), hydrochloric acid, Sodium hydroxide, silane a174, and Phosphate-buffered saline (PBS) were bought from Merck Company (Germany). Fetal Bovine Serum (FBS) and RPMI medium were purchased from Gibco Company (USA), MTT dye [3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide], dimethyl sulfoxide (DMSO), Graphene quantum dots, Azobisisobutyronitrile(AIBN), and Methacrylic acid 99% were procured from Sigma-Aldrich (USA). 4T1 a breast cancer cell line was prepared from the National Cell Bank of Iran Pasteur Institute (Iran). Diallyldimethylammonium Chloride (60% in Water) was purchased from Exir GmbH Company (Austria). Doxorubicin HCl was prepared from Actoverco Pharmaceutical Company (Iran). Tris buffer was prepared from AppliChem (Germany). Dialysis Tubing 28.6 mm inflated (Molecular Weight Cut-Off is 12000 to 14000 daltons) was obtained from scientific laboratories Company. All solutions were made with Deionized Water
Synthesis of reduced GQD (RGQD)
First, 100 mg of GQD was added to 5 ml of water and 2.5 ml of sodium hydroxide solution (0.1 M) and mixed at 300 rpm for 30 minutes, 20 mg of NABH4 (0.528mmol) was added, The pH was adjusted to 10 with a solution of hydrochloric acid or sodium hydroxide (0.1M) The above solution was mixed for 3 hours at 80 ° C under reflux conditions. The temperature of the solution was reduced to room temperature and the pH was adjusted to 7 with hydrochloric acid (0.1M) [11].
Synthesis of silane-RGQD
200 mg of silane a174 (0.805mmol) was added to the RGQD solution under nitrogen gas and mixed at room temperature at 300 rpm for 24 hours, then 50 ml of 96% ethanol was added to the solution. The mixture was centrifuged and the resulting precipitate was dried at room temperature[11].
Synthesis of PMA-DDA-DOX
Finally, silane-RGQD was added to 100 ml of DMF and sonicated for 15 minutes. Then 500 mg methacrylic acid (5.80 mmol), 70 mg DOX (0.120 mmol), 5.5 g diallyl dimethyl ammonium chloride (60% in water) (20.41 mmol anhydrous) and, 200 mg AIBN (1.21 mmol) was added to the above solution under nitrogen gas at the temperature of 70 to 75 Cº and reflux conditions. After 24 h, the temperature was reduced and the nitrogen gas was cut off, the resulting mixture was centrifuged and kept in 20 mL of 20 mmol TRIS buffer. Also, to evaluate the biological properties of PMA-DDA-DOX, a nanocomposite without DOX was once again synthesized, which was labeled PMA-DDA[7]. Preparation of PMA-DDA-DOX route was shown in figure 1.
Figure 1. Preparation of PMA-DDA-DOX.
Animals ethics statement
Mice with the following characteristics were prepared from Pasteur Institute (Iran). Female adult BALB /C to 8 weeks, Storage conditions: 12h light/12h dark and 24º C with enough water and food.
4T1 cancer cells
4T1 cancer cells were obtained from the tumor cell bank of Pasteur Institute (Iran) which were maintained according to supplier instructions. 4T1 cells were harvested at exponential growth phase from culture, rinsed with sterile PBS two times, counted, and resuspended in PBS before running subcutaneously on the female BALB/C mice’s right flank.
In vivo study
First, Mice were anesthetized with ketamine and xylazine (100 and 10 mg/kg/BW, respectively) by intraperitoneal injection. Then 200 μl of the viable cells with concentration (4×107/mL) were injected subcutaneously into BALB/C mice’s flank area. A 29 gauge syringe needle was used for injection
Nanocomposite characterization:
FT-IR spectroscopy
The structure of (PMA-DDA-DOX) was evaluated using FT-IR (FTIR; 6700 Thermo Nicolet) spectroscopy. After drying PMA-DDA-DOX at room temperature, PMA-DDA-DOX spectra were obtained using KBr pellets in the range of 400-4000 cm-1
FE-SEM analysis
The morphology and particle size of PMA-DDA-DOX were analyzed by FESEM (TESCAN Model: MIRA III, Czech Republic) with a field emission gun based on the standard protocol[12].
Evaluation of DOX release kinetics from PMA-DDA-DOX
To evaluate the release kinetics of DOX from PMA-DDA-DOX, a UV-Vis spectrometer (Varian-Cary100, Australia) was used. 50 mg DOX was exposed to 20 ml PMA-DDA-DOX for 5 hours, the solution was transferred to a dialysis tube, and every 4 hours; Adsorption of the solution around the dialysis tube was taken at a wavelength of 520 nm. (520nm was the λ maximum DOX determined by UV-Vis spectrometer) [13].
In vivo fluorescence imaging
A BALB / C mouse with a tumor size of 100 mm3 was randomly selected. After anesthesia, 200 μl of PMA-DDA-DOX was injected intraperitoneally. After two days, in vivo imaging (KODAK In-Vivo Imaging System FX Pro (Carestream Health Inc., New Haven, CT, USA)) was used to monitor the release of PMA-DDA-DOX. These images were taken at the excitation wavelength of 400 nm and the emission wavelength at 535 nm.
Cytotoxicity assay
To assess the toxicity of (PMA-DDA, PMA-DDA-DOX, and DOX), 4T1 cell line was cultured at 1 105 cell/well in 96-well plates for 24h under optimal conditions (37 , 5% CO2 in humidified incubator). In the next step, the growth media (10% FBS) was removed and the cells were washed two times with PBS. A new maintenance RPMI medium (10% FBS) containing 0.05, 0.5, 5, 50, and 500µg/mL was added to all samples, and the cells were incubated for 48h. Triple wells were analyzed for each concentration and column elution buffer was selected as the control. A 10μL solution of freshly prepared 5mg/mL MTT in PBS was added to each well and allowed to incubate for an additional 4 hours. The media was removed and DMSO was added at 100µL/well. Plates were shaken gently to facilitate formazan crystal solubilization. The absorbance was determined at 545 nm with a microplate reader (STAT FAX 2100, BioTek, Winooski, USA). The percentages of cell toxicity and half-maximal inhibitory concentration (IC50) were calculated [14]. The percentage of cell viability was calculated as follows:
Hemolysis assay
Fresh red blood cells (RBC) in a healthy person were used for this analysis. Then, 15% (vol/vol) suspension of RBCs was prepared and diluted 1: 20 in PBS. Next 100μL of a 2-fold serial dilution series of the agent (PMA-DDA) was added in triplicate to 100μL of RBCs suspension in a 96-well plate and the plate was incubated for 1 h at 37°C. After centrifugation at 3,000 rpm for 10 min, the percentage of hemolysis was determined by measuring the absorbance at the wavelength of 414 nm of the 150μL of the supernatant. Negative control and 100% hemolysis were determined in PBS buffer and the presence of 1% Triton X-100, respectively[15]. Finally, the percentage of hemolysis was calculated as follows:
Characterization of PMA-DDA-DOX
FTIR Spectrum
Five important peaks in the FTIR spectrum (Figure 2) were investigated, there is a broad peak of 3433.43 Corresponds to the O-H unsymmetrical stretch in GQD and poly-methacrylic acid-co-diallyldimethylammonium chloride, The peak of 2800 to 3000 is related to C-H unsymmetrical stretch of -CH3 and C-H symmetrical stretch of N-CH2 The 1700 absorption, located on the shoulder of the 1658.13 couriers belongs to the carbonyl group in GQD and poly-methacrylic acid-co-diallyldimethylammonium chloride. Another strong absorption in 1658 is related to O-H symmetrical deformation. The last strong absorption in 1474.42 was related to O-H unsymmetrical deformation [16, 17].
Figure 2. FTIR spectra of PMA-DDA-DOX
FE-SEM
FE-SEM analysis was performed to assess particle size and morphology. As shown in Figure 3, the particle shape was amorphous. Also, SEM images showed great variation in particle size. As shown in the figure, no aggregation occurred. The particle size distribution histogram was shown from the SEM images in Figure 3 despite the variety in particle size; most particle sizes are below 55 nm. Also, particles larger than 100 nm are artifacts that result from incomplete washing of the sample
Figure 3. FE-SEM image of PMA-DDA-DOX, A particle size distribution histogram determined from the SEM images
Evaluation of DOX release kinetics from PMA-DDA-DOX
The DOX release rate of PMA-DDA-DOX is shown in Figure 4, the rate of release was monitored for 72 hours. The release rate was faster in the first 24 hours. Then the release speed decreased. But overall, the release rate of DOX is slow and has a relatively gentle slope, which helps maintain the concentration of the drug around the cancer cells for a longer time.
Figure 4. DOX release profile from PMA-DDA-DOX at 520nm
In vivo fluorescence imaging
Tumor cells are located in the right flank of the mice, after 2 days of PMA-DDA-DOX injection In vivo fluorescence imaging was performed. Due to the fluorescence properties of PMA-DDA-DOX, these particles are identified in brighter regions in Figure 5.Most of these particles accumulate around tumor cells and accumulation around other tissues is very little
Figure 5. In vivo fluorescence images from two angles of BALB / C mouse containing 4T1 breast cancer cells taken two days after PMA-DDA-DOX injection.
Cytotoxicity assay and Hemolysis assay
In the cytotoxicity assay (Figure 6), three samples of PMA-DDA, PMA-DDA-DOX, and DOX were examined. PMA-DDA showed the lowest toxicity at a concentration of 500 μg / mL (The percentage of toxicity: 13.93%) and also the toxicity of PMA-DDA-DOX compared to DOX was lower. To investigate the potential lytic effects of PMA-DDA on Human erythrocytes, a hemolytic assay of erythrocytes was performed. Briefly, the destruction of red blood cells is called hemolysis. The percentage of degradation of Human erythrocytes at different concentrations of PMA-DDA was examined and shown in Figure 7, the hemolytic percentage at 500 μg / mL is only 9.5%, which is a small amount for a drug carrier.
Figure 6. The percentage of toxicity of DOX, PMA-DDA, and PMA-DDA-DOX in different concentrations (μg/mL). Error bars indicate standard deviation
Figure 7.The hemolytic percentage at various concentrations of synthesized PMA-DDA
First, the chemical structure of PMA-DDA-DOX was investigated by FTIR It turned out Poly methacrylic acid-co-diallyldimethylammonium chloride containing graphene quantum dots (PMA-DDA-DOX) were formed. In the next step, the size and morphology of PMA-DDA-DOX were investigated by FE-SEM analysis, Increasing particle size in drug nanocarriers may block blood vessels in contrast, decreasing particle size increases the efficiency of these particles[18]. Therefore, particle size and non-aggregation are very important the results of FE-SEM and histogram showed that most PMA-DDA-DOX particles are smaller than 55 nm which is a suitable size for a drug carrier. In Fig. 4, The slow-release gives time to PMA-DDA-DOX particles to reach tumor cells and release DOX at an almost uniform rate after reaching the tumor cells that the concentration of the drug is maintained around the target tissue for a longer time [19]. One of the most critical aspects of DDS is the delivery of the drug to the target tissue this factor is directly related to the treatment and reduction of drug side effects [20, 21]. DOX efficacy can be enhanced by selecting the appropriate drug carrier, such as PMA-DDA-DOX, Which significantly accumulates around tumor cells. PMA-DDA-DOX with stable release, increase the concentration of DOX around the tumor and kill cancer cells without damaging normal cells. In this way, the dose of the drug can be increased without increasing toxicity [22]. Due to the biological complexity of living organisms, the effects of drug release cannot be accurately evaluated in the laboratory, so Real-time monitoring of the in vivo drug release process has a vital significance. Fluorescence imaging has unique advantages for tracking the release of drugs in vivo[23] Cancer cells produce more lactate anion than normal cells this phenomenon causes the environment around the cancer cells to become negative. Positively charged nanoparticles can provide an electrostatic bond with these negative charges. As shown in Fig. 5, cationic polymers are accurate and efficient drug carriers. positive charged PMA-DDA-DOX particles have accumulated around the tumor plasma membrane[24]. PMA-DDA-DOX particles reach the tumor cells through the bloodstream; they bind electrostatically to the lactate anion around the cancer cells and then release DOX. This prevents the side effects of DOX and increases the concentration of the drug around the target tissue[25, 26]. These in vivo studies have shown that these particles have high efficiency and selectivity in the living organism. Also, these particles still maintain their electrostatic bond after two days. Also, these particles with their strong fluorescence give a clear picture of tumor expansion, which is shown in Fig. 5 (brighter parts) This property can be used in the early diagnosis and treatment of cancer[27]. PMA-DDA-DOX reaches tumor cells through the bloodstream so these particles mustn't be toxic. Cytotoxicity and hemolysis tests were performed to investigate this issue. The cytotoxicity test in Fig. 6, showed that PMA-DDA-DOX was less toxic than DOX on 4T1 cells. The hemolysis test in Fig. 7, showed that PMA-DDA less than 10% destroyed red blood cells at a concentration of 500 (µg / mL). The results of the cytotoxicity and hemolysis tests showed that PMA-DDA-DOX is a suitable choice for the drug delivery system. Also, GQD and poly-methacrylic acid-co-diallyl dimethyl ammonium chloride do not add any additional toxicity to DOX.
Drug delivery by using cationic polymers containing graphene quantum dots has great potential in the treatment and diagnosis of cancer. The PMA-DDA-DOX can carry a wide range of anticancer drugs and release the drug around tumor cells with minimal damage to normal cells. They are traceable in the body by their inherent fluorescence; this feature can be used to assess the extent of cancer. These particles with properties such as good solubility in water, low toxicity slow release of the drug, stable electrostatic connection with lactate anion surrounding tumor cells, portability and protection of drug compounds as well as fluorescence imaging of target tissue, can be good candidates for the drug delivery system. It can be expected that the development of drug delivery based on graphene quantum dot cationic polymer, can reduce the side effects of anticancer drugs and increase their effectiveness.
Availability of Data and Material
The data are available from the corresponding author upon sensible request.
Acknowledgments
We thank the staff of the Semnan University of Medical sciences, Drug Design and Bioinformatics Unit and Biotechnology Research Center from Pasteur Institute of Iran.
Funding
No funding was received for this study
Author information
Authors and Affiliations
Department of Chemistry, Science and Research Branch, Islamic Azad University, Tehran, Iran
Mahdi Mirzababaei & Kambiz Larijani & Zohreh Mirjafary
Departments of Medicinal Chemistry, School of Pharmacy, Damghan Branch, Islamic Azad University, Damghan, Iran
Hamid Hashemi-Moghaddam
Drug Design and Bioinformatics Unit, Department of Medical Biotechnology, Biotechnology Research Center, Pasteur Institute of
Iran, Tehran, Iran
Hamid Madanchi
Contributions
All authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by Mahdi Mirzababaei, Kambiz Larijani, Hamid Hashemi-Moghaddam, Zohreh Mirjafary and Hamid Madanchi. The first draft of the manuscript was written by Mahdi Mirzababaei. All authors read and approved the final manuscript.
Corresponding author
Correspondence to Hamid Hashemi-Moghaddam.
Ethics declarations
Ethics Approval and Consent to Participate
This study was conducted according to the guidelines of the declaration of Helsinki and approved by the ethics committee of Islamic Azad University- Science and Research Branch (protocol code IR.IAU.SRB.REC.1398.104)
Conflicts of Interests/Competing Interest
The authors declare that there is no conflict of interest.
Consent for publication
All the authors are consent to publishing the paper.
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