Research Article, J Pharm Drug Deliv Res Vol: 12 Issue: 2
A Novel pH-sensitive Nanocomposite Based on Graphene Oxide for Improving Doxorubicin Release
Marziye Javaheri Kachousangi1*, Amir Shadboorestan4, Azam Shamsian1, Mohsen Amini3, Fatemeh Atyabi1 and Mohammad Hossein Ghahremani1,2
1Department of Pharmaceutical Nanotechnology, Tehran University of Medical Sciences, Tehran, Iran
2Department of Toxicology and Pharmacology, Tehran University of Medical Sciences, Tehran, Iran
3Department of Medicinal Chemistry, Tehran University of Medical Sciences, Tehran Iran
4Department of Toxicology, Tarbiat Modares University, Tehran, Iran
*Corresponding Author: Marziye Javaheri Kachousangi
Department of Pharmaceutical Nanotechnology, Tehran University of Medical Sciences, Tehran, Iran
Received date: 28 February, 2023, Manuscript No. JPDDR-22-70592;
Editor assigned date: 02 March, 2023, PreQC No. JPDDR-22-70592 (PQ);
Reviewed date: 16 March, 2023, QC No. JPDDR-22-70592;
Revised date: 23 March, 2023, Manuscript No. JPDDR-22-70592 (R);
Published date: 30 March, 2023, DOI: 10.4172/2325-9604.1000015
Citation: Kachousangi MJ, Shadboorestan A, Shamsian A, Amini M, Atyabi F, et al. (2023) A Novel pH-sensitive Nanocomposite Based on Graphene Oxide for Improving Doxorubicin Release. J Pharm Drug Deliv Res 12:2.
A novel nanocarrier for delivering chemotherapeutic drug, Doxorubicin (DOX), was fabricated using Graphene Oxide (GO) as a basic plane for conjugating and assembling other compounds. Previous studies immobilized DOX on the GO sheets with high efficiency however the release was very low and slow due to strong forces between DOX and GO. We attached DOX to GO-poly(ethyleneimine) 2KDa conjugate via a linker containing hydrazide bond. Hydrazide bond is sensitive to acidic pH and a significant increase in the efficiency of DOX release and cytotoxicity was observed in vitro. The MTT assay confirmed advantage of this nanocarrier in comparison to physical loading of DOX on GO sheets. Loading of DOX via hydrazide bond was as low as 4% versus near 75% physical loading of drug. While hydrazide bond containing nanocomposite was 4 to 6 fold more toxic than GO-DOX. The IC50 value for chemically bound DOX to hydrazide containing GO nano-composite was 9.5 µg/ml whereas the IC50 value for GODOX was 39 µg/ml after 72 h. As could be concluded from this study, nanocarriers based on hydrazide bond could be a good candidate for drug delivery.
Keywords: Functionalized graphene oxide, Polyethyleneimine, Doxorubicin, Hydrazide linker, pH-sensitive, Drug delivery
Breast cancer is among the most common cancers and the second leading cause of death in women . Treatment strategies are developed based on several factors such as type, stage and sensitivity of cancer to certain hormones as well as medical history of the patient.
Breast cancer is usually treated with surgery which may be followed by chemotherapy or radiotherapy or both [2,3]. The main groups of medications used for adjuvant breast cancer treatment after surgery are hormone blocking therapy for hormone receptor positive cancers, tumor targeted therapy which uses monoclonal antibodies like Trastuzumab, Bevacizumab, Lapatinib and chemotherapy with antineoplastic drugs to promote tumor cell destruction. Doxorubicin (DOX) is being used as desired chemotherapeutic drug in treatment of breast tumor cells. DOX interacts with DNA by intercalation and inhibition of topoisomerase II action and generation of free radicals.
They block DNA and RNA synthesis and cause strand scission. However, problems associated with DOX, as it is non-specifically distributed throughout the body and affects normal cells and organs.
Because of adverse side effects of chemotherapeutic drugs, scientists have tried to discover novel drug delivery systems for decades that reduce harmful effects and target tumors specifically. Nano-materials have found great applications in different fields as well as in medical diagnosis and treatment [4-10]. In drug delivery systems, different kinds of nanomaterials are being used including nanomicelles, liposomes carbon nanostructures, dendrimers and metallic nanoparticles [11-15]. In this regards, the nanocarriers can be used with passive targeting by enhanced permeation and retention effect (EPR) or they could be attached to convenient ligands or antibodies for active targeting [16,17].
Graphenic 2D nanostructures have been regarded and used since its discovery in 2004 in different fields of technology including nanomedicine [18-21]. However, because of high hydrophobic nature and high tendency for aggregation, graphene is not favorable for medical use and instead, oxidized graphene (graphene oxide) has been employed. Graphene Oxide (GO) has good water dispersion, high biocompatibility and the ability to form non-covalent and covalent bonds with different compounds simultaneously [22,23]. This makes GO a good choice for drug and gene delivery, imaging and sensing [24,25]. GO has both aromatic structures for π-π interaction with hydrophobic drugs or single strand genetic materials and oxygenated functional groups to form covalent bonds with different drugs or polymers [26-30].
Until now, DOX has been loaded on the GO planes through noncovalent interactions. This interactions include π-π interactions between aromatic rings of GO and DOX and hydrogen bonding. The tendency for adsorbing of DOX is high and GO is capable of loading DOX up to three times of its own weight. However, in spite of its high loading, release of DOX from GO planes is very low due to its strong interactions with GO surfaces.
Therefore, DOX was attached to GO surfaces through hydrazide linker which is sensitive to acidic environment of tumor. Although, the amount of hydrazide linked DOX was less than 5% versus about 75% through non-covalent interaction, IC50 decreased four times and this showed the preference of this method to non-covalent loading.
Materials and Methods
Doxorubicin hydrochloride was purchased from Exir Nano Sina, Iran. Maleimidopropionic acid hydrazide (MPH) was purchased from Apolloscientific, England. Industrial graphite (100-200 mesh) was purchased from an Iranian company. Sulfuric acid 98%, sodium nitrate, potassium permanganate, hydrochloric acid, sodium chloroacetate, N-Hydroxysuccinimide (NHS) and polyethyleneimine (PEI) 2 KDa were all from Merck, Germany. Pluronic F127 copolymer, N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC), 11-mercaptoundecanoic acid, dithiothreitol (DTT) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) were purchased from Sigma-Aldrich, Germany. Cell culture medium and reagents were all from Pan-Biotech (Germany).
Synthesis of graphene oxide
The lateral size of industrial graphite was between 100 to 200 micrometer and therefore, the size of graphite was reduced by dispersing 5 gram of it in 500 ml de-ionized water and probe sonicated for about 5 hours. Dispersion of graphite was dried in air and 4 g graphite was poured in three neck round bottom flask and 200 ml sulfuric acid 98% was added and stirred for 30 min. flask was replaced in ice bath and 2 g sodium nitrate was added slowly and flask was stirred for another 1 hour. After that, 16 g potassium permanganate was added slowly and the temperature was maintained at 4°C during this addition. The reaction was kept for 1 week. After this time, flask was put in an oil bath and temperature was set at 50°C and heated for 30 min and then, 100 ml deionized water was added and hydrogen peroxide was added drop wise until severe reactions with bubbles were stopped. The mixture was washed with 10% HCl solution and pH was raised to about 4 with repeated washing and centrifuge with deionized water. Then, the resulted graphite oxide was freeze dried and resulted powder was dispersed in deionized water and probesonicated until one layer graphene oxide was obtained .
Stabilization of graphene oxide layers
To stabilize the GO with pluronic F127, 100 mg graphene oxide was mixed in 100 ml water and sonicated until the layers were separated. Then, 1 mL pluronic F127 was added and sonicated and stirred overnight. Then, the mixture was centrifuged and washed 2 times with distilled water to remove excess F127 and lyophilized .
Synthesis of carboxylated nanographene oxide
To prepare carboxylated GO, 100 mg NGO was dispersed in 100 mL deionized water by bath sonication and then, 5 g NaOH was added and stirred for 2 h. Then, 5 g sodium chloroacetate was added and the mixture was sonicated for 2 hours followed by stirring for 24 h. The reaction mixture was centrifuged and washed with deionized water .
Synthesis of graphene oxide-polyethyleneimine conjugation
To synthesize the PEI-GO composite, 20 mg carboxylated GO stabilized by pluronic F127 was probe sonicated in 200 mL deionized water until a homogenized dispersion was obtained. Then 50 mg EDC was added and stirred for 2 h. Then, 50 mg NHS was added and incubated for 2 h. The reaction mixture was added slowly to 2 ml PEI 2 KDa (50 wt% in water) and stirred for up to 24 h. The resulted mixture was centrifuged and washed with deionized water and freeze dried .
Thiolation of GO-PEI conjugation
To add thiol bond to this nanocomposite, 40 mg 11- mercaptoundecanoic acid was dissolved in 30 mL methanol under argon. Then, 50 mg EDC was added and stirred for 2 h and 50 mg NHS was added and stirred for another 2 h. 75 mg GO-PEI nanocomposite was dispersed in methanol and sonicated and added to the reaction mixture and final reaction was stirred under argon for 12 h. Then, the mixture was centrifuged and washed twice with deionized water and freeze dried.
Cleavage of disulfide bonds
Thiol bonds are sensitive to oxidation and make disulfide bonds. To react with hydrazide bonds, free thiol groups are needed. To increase the output of reaction, obtained compound was reacted with Dithiothreitol (DTT). Obtained compound was sonicated and dispersed in methanol and 1 g NaOH was added. Then, 40 mg DTT was added under argon and stirred for 12 h. Then, reaction mixture was centrifuged and washed with deionized water and freeze dried.
Reaction of Maleimidopropionic acid Hydrazide (MPH) bond with thiolated PEI-GO conjugation
Last obtained compound was sonicated and dispersed in methanol and then, 13 mg MPH was added and stirred for 48 h under argon. Then centrifuged and washed with deionized water and freeze dried.
Reaction of doxorubicin with nanocarrier
To load the drug in resulted nanocarrier, 7 mg nanocarrier was probe sonicated and dispersed in PBS buffer with pH 8.5. Then, 28 mg DOX was added and stirred for 48 h. This reaction was done in the dark. Resulted mixture was centrifuged and washed with deionized water until the supernatant was colorless.
At first, 1 mL PBS with pH 5.5 and 7.5 was added to 1 mg nanocarrier loaded with drug in 2 microtubes and put in a shaker incubator at 37°C and centrifuged at determined time intervals of 0.5, 1, 2, 4, 8, 10, 24, 48 and 72 h then, supernatant was removed and replaced with new buffer. At the end, the absorptions of all samples were determined.
All steps of nanocarrier construction and loading of drug were characterized by FTIR spectra. FTIR spectra were obtained by using a Fourier Transform Infrared Spectroscopy (FT-IR) (Tensor 27, BRUKER). Thermogravimetric Analysis (TGA) was carried out on a Netzsch (TGA 209 F1) TG analyzer. TGA was used to characterize components of synthesized nanomaterials. X-ray Photoelectron Spectroscopy (XPS) was performed under applying the pulsed laser irradiation. The data were collected through a hemispherical analyzer supplied by an Al Kα x-ray source (hν=1486.6 eV) operating at a vacuum better than 10-7 Pa. For quantitative investigations, the XPS peaks were deconvoluted by using Gaussian components after a Shirley background subtraction. Elemental analysis and diagnosis the approximate amount of bounds were performed by XPS. Raman spectroscopy (Thermo Scientific Nicolet, USA) was used to analyze the quality of the produced GO. The morphology of GO was characterized by Field Emission Scanning Electron Microscopy (FESEM) (MIRA3TESCAN-XMU). Atomic Force Microscopy (AFM) images were acquired in a tapping mode by DME (DS 95) AFM scanner. AFM was used to detect the thickness and lateral size of constructed nanomaterials. The optical absorption was investigated by UV-visible absorption spectra (Lambda-35 UV-Vis spectrophotometer, Perkine-Elmer,USA). UV-Vis spectroscopy was used to monitor drug loading and release.
In vitro cytotoxicity assay
Typically, 104 MCF7 cells (National Cell Bank, Pasture Institute of Iran) were seeded in 96-well plates and cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin at 37°C under 5% CO2. After 24 h, the cells were incubated with 100 μL of different concentrations of different compounds in the culture medium. To prepare DOX samples, 10 mg DOX in 1 mL deionized water was used as the stock solution. Then, 100 μL of stock solution was taken and by adding 900 μL culture medium, 1 mg/mL stock solution of DOX was prepared and from this stock solution, 10 μM solution with the final volume of 100 μL was prepared and its concentration was halved in every step. For nanocarrier and nanocomposites with drug, 1 mg of each was dispersed in 1 ml deionized water and 100 μL was taken and the final concentration of 100 μg/mL was prepared and concentrations were halved in every step. After 24 h, 48 h and 72 h exposure, the cells were washed with 100 μL of PBS and incubated with 20 μL of 5 mg/mL MTT for 4 h at 37°C under 5% CO2. Finally, the insoluble purple formazan crystals produced by live cells were dissolved in 100 μL of Dimethyl Sulfoxide (DMSO). The plate was shaken until crystals were solved. Optical density of the produced stain was monitored at 570 nm, with 655 nm as a reference, using a microplate reader (Anthos 2020, UK). Cells without particle exposure were used as control.
Processing experimental data and plotting graphs were performed by GraphPad Prism software. The data were analyzed by one way analysis of variance (ANOVA). P-value<0.05 was considered significant.
Results and Discussion
Characterization of nanocarrier
Raman spectroscopy is a strong and non-invasive tool to recognize carbon materials. Peak at 1611 cm-1 (G-band) is related to E2 g mode and is attributed to the vibrations of carbon atoms in sp2 bonds. Peak at 1371 cm-1 (D-band) is related to A1 g mode and attributed to vibrations of carbon atoms with dangling bands at the edge of the sheet. G band is related to symmetric (graphitic) part and D band is because of distortions that were made in the graphitic planes. D/G ratio is 0.76 which confirms high oxidation of graphite. 2D peak at 2726 cm-1 is related to stacking of graphene oxide layers on each other (Figure 1a) .
Figure 1a: Raman spectra of GO.
The wide scan XPS spectrum of carboxylated GO (Figure 1b) shows 24% atomic ratio of oxygen at 539 ev and 76% atomic ratio of carbon at about 292 ev and this confirms highly oxidation of graphite. The core level high resolution C1s XPS of carboxylated GO is shown in Figure 1c. These spectra clearly suggest a considerable degree of oxidation and formation of GO. The deconvolution of C1s spectra shows four components that correspond to carbon atoms in different functional groups: The non-oxygenated C (284.93 eV) that covers the C=C bond of hybridized sp2 in the aromatic rings, the C in C-O bonds (285.79 eV) that involve the hydroxyl and epoxy groups, the C in C=O bonds (287.45 eV) that involve the carbonyl groups, and the C in OC= O (289.9) bonds that involve carboxyl groups . The wide scan XPS spectrum of PEI 2KDa (Figure 1d) shows 80.06% atomic ratio of carbon at 284 ev, 13.13% atomic ratio of oxygen at 531 ev and 6.81% atomic ratio of nitrogen at 399.47 ev. By comparison of this spectrum with the spectrum of carboxylated GO, it could be understood that conjugating PEI 2KDa to COOHGO added about 14% carbon and near 7% nitrogen to the nanocomposite and from this result, it could be concluded that about 20% PEI 2KDa was added to GO and since in the next steps hydrazide linker was added to amine groups and there are about 4.5% primary and secondary amines in 7% nitrogen of PEI 2KDa, approximate amount of DOX through hydrazide linker could be between 4-5%.
Figure 1b: Wide scan spectrums of carboxylated GO.
Figure 1c: Core level C1s XPS spectra of carboxylated GO.
Figure 1d: Wide scan spectrum of PEI 2KDa.
FESEM of GO is shown in Figure 2a-c. GO nanosheets are clearly obvious in these figures and it is completely recognizable that GO nano-sheets are composed of very few sheets. Smooth and distorted regions are observed which is attributed to aromatic and oxidized structures respectively and all of these reasons could confirm the formation of GO. Since GO nano-sheets were needed as a platform for conjugating other materials and cellular uptake, their lateral sizes should be decreased to few hundred nanometers and this was done by probe sonicators and SEM was shown in Figure 2d and as seen, was done successfully .
Figure 2: (a-c) FESEM images of graphene oxide, d) FESEM images of nanographene oxide.
Synthesis and chemical conjugation of all compounds including GO, carboxylated GO (COOH-GO), PEI-COOH GO, hydrazide conjugated to PEI-COOH-GO and COOH-GO-PEI-MPH-DOX were confirmed by FT-IR as shown in Figure 3a. strong and broad stretching band at 3200-3500 cm-1 due to O-H bond, strong C=O stretching peak at 1733 cm-1, C=C stretching peak at 1628 cm-1, stretching peak at 1400 cm-1 due to C-C bonds in aromatic rings, strong stretching peak at 1082 cm-1 due to C-O bond of ethers and C=C bending peak at 613 cm-1 in IR of GO confirmed the formation of GO. Strong stretching peak at 1741 cm-1 which appears in COOHGO spectrum, evidenced the covalent attachment of acetate to oxygenated groups and carboxyl groups were increased. Conjugation of PEI (2KDa) to GO was confirmed in COOH-GO-PEI spectrum through bending peak at 1632 cm-1 which shows up amide bond formation. In addition, asymmetric and symmetric C-H stretching bands of PEI at 2928 cm-1 and 2858 cm-1 confirmed the presence of PEI in GO nano-sheets. Attachment of MPH to PEI was confirmed by a stretching peak at 1384 cm-1 which is related to hydrazide bond and peak at 1033 cm-1 is related to ether bond of pluronic F127.
Conjugation of DOX with nanocarrier is confirmed by peaks at 1737 cm-1 and 1430 cm-1 which is related to C=N bond (conjugation of DOX with hydrazide bond).
Figure 3a: FT-IR spectra of GO, COOH-GO, COOH-GO-PEI, COOH-GO-PEI-MPH, COOH-GO-PEI-MPH-DOX.
TGA analysis was performed to investigate the degree of functionalization. TGA of graphite was shown in figure 6 and as expected from highly strong structure of graphite, until 900°C just 0.45% of its total mass was degraded and it is negligible. TGA of GO is shown in Figure 3b . TGA was done from 20°C to 900°C with the rate of 10°C per minute under nitrogen. First mass reduction was about 11.28% at 100Ì?C and it is as a result of evaporation of adsorbed water to graphene oxide. Next mass reduction was about 30% at 200°C and it is due to removing of CO, CO2 and H2O from graphene oxide. At this temperature, functional groups, which are more active, leave the structure and by increasing the temperature, this process continues until 900°C. The extent of mass reduction until 900°C was 48.65%. In Figure 3b, TGA of carboxylated GO was shown and since for carboxylation, acetate groups was added to hydroxyl and epoxy groups of the GO and did not change the structure of GO, it did not have obvious difference with the TGA of GO.
Figure 3b: TGA profile of graphite, GO, COOH-GO, COOH-GOPEI, PEI.
TGA of COOH-GO-PEI was shown in figure 3b. Degradation temperature for PEI 2KDa (50%wt of water) is from about 300°C until 400°C as shown in Figure 3b. With these evidences, conjugation of PEI to GO was confirmed and the extent of conjugation was about 25% wt of total mass by minimizing degradation of COOH-GO at that temperature range.
In Figure 3c, TGA of COOH-GO-PEI-MPH and COOH-GO-PEIMPH- DOX were shown which are very similar. Weight loss in two figures until 100°C relates to water evaporation, weight loss until 200°C shows loss of GO functional groups, losing weight until 300°C shows degradation of pluronic F127 and weight loss until 400°C is due to PEI degradation and other weight loss is due to gradual degradation of GO. In the TGA of COOH-GO-PEI-MPH-DOX, the extent of degradation is about 5% wt more than TGA of without DOX and this difference is related to hydrazide linked DOX.
Figure 3c: TGA profile of COOH-GO-PEI-MPH, COOH-GO-PEIMPH- DOX.
AFM of GO is shown in Figure 4A. Since GO is synthesized through Hummer method, the GO layers are not homogeneous and GO layers with different lateral sizes from below 100 nm until 2 μm could be seen but the monolayers of GO with thickness of 1 nm were detectable. According to AFM of COOHGO in Figure 4B, thickness of layers were from 1 nm until 4 nm and it shows stacking of some layers on each other but the lateral sizes of planes reduced to 300 nm and less due to probe sonication and carboxylation reaction. AFM image of COOH-GO-F127-PEI is shown in Figure 4C. The lateral size of nanocomposite is variable and it is until 300 nm but as a whole, the lateral sizes are more homogeneous and close to each other. Thickness of planes is from 3 nm until 15 nm. Due to addition of pluronic F127 and PEI (2KDa) on the two sides of the planes, the thickness of planes was increased and since the concentration of nanocomposite was high, the layers tend to aggregate on each other and thickness increased but it drastically decreased after reaction with DOX to 1.2 nm (Figure 4D) and this phenommenon is because of adding DOX on GO layers which is tightly bounded and decreased its thickness.
Figure 4: AFM images of (A) GO, (B) COOHGO, (C) COOHGOF127- PEI, (D) COOHGO-F127-PEI-MPH-DOX.
Loading and release of drug from nanocarrier
Doxorubicin (DOX) was used as a model chemotherapeutic drug. DOX is loaded on GO nanosheets through π-π stacking and hydrogen bonding with high drug loading capacity (between 200-300%) but drug release is very slow as a result of strong interactions between drug and GO. As a consequence, DOX was attached through hydrazide bond to PEI which hydrolyses in acidic pH of cancerous cells.
Entrapment efficiency and loading capacity of DOX in nanocarrier through non-covalent and covalent attachment are as follows:
Entrapment efficiency shows the percent of drug loading in nanocarrier: %EE=initial drug content free drug/initial drug content.
Loading capacity shows the drug content in nanocarrier: %LC=mass of loaded drug/mass of nanocarrier.
%Drug loading=mass of loaded drug/mass of nanocarrier+ mass of loaded drug
The extent of loaded drug was determined by UV-vis spectroscopy at 480 nm. Since the reaction between MPH and DOX is performed under basic condition, non-covalent immobilization of DOX on GO nano-sheets could not be avoided which this non-covalent loading is also done under similar situation .
The drug release profiles were evaluated on the basis of the results of drug absorption in the PBS buffer with pH 7.5 and pH 5.5 measured at specified time period with a UV-Vis spectroscopy. However, the difference between non-covalent and covalent bonding was not distinguished.
The content of release in pH 5.5 was two times more than release in pH 7.5 (P value<0.01) and from this, it was concluded that hydrazide bond is more unstable in acidic environment and a pH-sensitive nanocarrier was constructed (Figure 5) .
Figure 5: Release profiles of doxorubicin from COOH-GO-PEIMPH- DOX at pH 7.5 and pH 5.5. Error bars indicate mean ± standard error among the three independent experiments. P-value <0.01 indicates that there are significant differences between release of DOX from nanocarrier at pH 5.5 and 7.5.
From these results, it is clear that release of DOX from GO is very low and slow in spite of its high loading. Low release of DOX from GO has also been reported in other papers and in our research, attachment of DOX through pH-sensitive hydrazide linker is also added [41-42]. Release at pH 5.5 is twice as high as release at pH 7.5 and this result shows that this nanocarrier is pH sensitive.
The toxicity of compounds was tested on MCF7 cells (Figures 6-7).
In this case, the synthesized nanocarrier (COOH-GO-PEI-MPH), drug (DOX), conjugated nanocarrier with DOX (COOH-GO-PEI-MPHDOX) and non-covalently loaded nanocarrier with drug (GO-DOX) were included in the cytotoxicity studies. MTT of DOX was performed in the concentration of 0, 2, 4, 6, 8 and 10 μM in the three time intervals of 24 h, 48 h and 72 h (Figure 6). IC50 of DOX treatment after 24 h, 48 h and 72 h was 3.63 μM (1.97 μg/ml), 0.88 μM (0.48 μg/ml) and 0.69 μM (0.38 μg/ml), respectively.
Figure 6: Cytotoxicity of DOX in MCF7 cells. Cells were plated as indicated in methods and treated with different concentration of DOX. After 24, 48 and 72 hrs, MTT assay was performed and the results reported as Mean ± SD (n=4).
COOH-GO-PEI, COOH-GO-PEI-MPH-DOX and GO-DOX were prepared into different concentrations, 0.0, 1.5, 3.1, 6.25, 12.5, 25.0, 50.0 and 100 μg/mL and treated with MCF7 cell lines.
The nanocomposite of F127-COOHGO-PEI 2KDa showed toxicity equivalent to 35-40% at concentration of 10-20 μg/ml. Toxicity did not increase significantly with increasing concentration up to 100 μg/ml. Toxicity of nanocomposite was due to free amine groups of PEI 2KDa which decreased after conjugation.
As seen until now, GO could adsorb doxorubicin with high efficiency. It was shown that it could adsorb four fold of its weight. Its high tendency is due to aromatic rings of both and as a result π-π stacking. In addition, because of their O-H and COOH functional groups, they have hydrogen bonding. All of these reasons for binding make its release hard and as seen before, physical binding of DOX to GO is not satisfied. Therefore, we suggested chemical binding to increase its release and efficiency. IC50 of F127-COOHGOPEI2KDa- MPH-DOX was 72 μg/ml, 11 μg/ml and 9.5 μg/ml after 24 h, 48 h and 72 h. According to DL of 79% for F127-COOHGOPEI2KDa- MPH-DOX, amount of loaded drug in this nanocomposite was 59.882 μg/ml, 8.864 μg/ml and 7.505 μg/ml after 24 h, 48 h and 72 h at these IC50 doses. After matching with IC50 of DOX at that time intervals, the amount of released drug after 24 h, 48 h and 72 h was 3.3%, 5.4% and 5%. Release of DOX increased compared to experimental results.
IC50 of GO-DOX was 61 μg/ml, 59 μg/ml and 39 μg/ml. DL for GO-DOX was also 79%. According to DL of 79% for GO-DOX, amount of loaded drug in this nanocomposite was 46.61 μg/ml, 50.955 μg/ml and 30.02 μg/ml after 24 h, 48 h and 72 h at these IC50 doses.
After matching with IC50 of DOX at that time intervals, the amount of released drug after 24 h, 48 h and 72 h was 4.23%, 0.96% and 1.25%.
As could be seen from release data, released DOX from GO-DOX after 24 h was more than F127-COOHGO-PEI2KDa-MPH-DOX and therefore, IC50 for GO-DOX is lower while released DOX from F127-COOHGO-PEI2KDa-MPH-DOX was 4.44% and 3.75% higher than that of GO-DOX and XPS data confirmed these data which was shown earlier in XPS of GO-PEI 2KDa. It could be conclude from these results that hydrazide bond breakage had not been started after 24 h while by hydrazide bond breakage after 48 h and 72 h, the preference of hydrazide bond-containing nanocomposite to conventional nanocarrier was proved.
Nanocomposite with hydrazide bond is about 6 and 4 fold more toxic than physical GO-DOX whereas physical drug loading was 75% more than hydrazide covalent attachment (by 79% drug loading in fabricated nanocomposite, only about 4-5% was assigned to hydrazide bond and other was related to physical adsorption) (Figures 7a-c).
Considering the loading of DOX, the GO-PEI-MPH-DOX has shown comparable toxicity to soluble DOX (Figures 6 and 7), indicating the higher toxicity with low amount of DOX.
Figure 7: (A,B) The Cell viability evaluation of GO-PEI, GODOX and GO-PEI-MPH-DOX by MTT assays after (A) 24 h, (B) 48 h.
Figure 7c: 7C: 72 h. The cell viability was calculated as percent and presented as Mean ± SD (n=4). P value < 0.05 represents significant differences between treatments.
In comparison to previous papers, this nanocomposite has had higher cytotoxicity and this shows our success to design a system to improve DOX release [43-46].
The COOH-GO-PEI-MPH-DOX as a biocompatible drug delivery system was designed and synthesized. The comprehensive characterization techniques approve the synthesis and functionalization of GO nanosheets. In this research, physical binding of DOX to GO was compared to chemical binding via hydrazide bond.
Physical loading of DOX on GO nano-sheets is very high due to unique properties of GO. However, because of tight attachment of aromatic rings of DOX to GO planes, the release of DOX is not favored compared to its high. Therefore, hydrazide bond was used to enhance release and subsequently the cytotoxicity. The loading capacity in chemical binding was very lower than physical binding, however, the MTT assay showed higher cytotoxicity of GO-PEIMPH- DOX nanocomposite to GO-DOX. Release profiles at room temperature at different pH ranges didn’t show considerable differences, release was slow, low and pH dependent. They show more release in acidic environment. However, they showed better release in vitro. Loading of DOX via hydrazide bond was as low as 5% versus near 90% physical loading of drug. Instead, cytotoxicity via hydrazide bond was more than 4 to 6 fold compared to GO-DOX. This strongly shows primacy of hydrazide bond to physical attachment that has been studied in previous articles. Moreover, since hydrazide bond is hydrolyzed better in acidic environment, the release is better targeted to tumors. If we increase the density of hydrazide bond in GO sheets, loading of drug will be increased and final concentration of nanocomposite will be decreased. Sustained release nature of drug from GO in every cases of physical and chemical binding is favorable and attractive, since DOX is very toxic and can be easily cleared from body. The advantage of this drug delivery system is its high stability, protection of drug from degradation, prevention from distributing highly toxic drug in the body, delivering to the target sites and being sustained release to release drug over long time.
In regard to previous articles for using physical attachment of DOX to GO, hydrazide bond represents better candidate as it releases better in acidic environment and shows more toxicity despite about 85% lower loading. It could replace physical method that has been used in many articles. If the density of hydrazide bond in GO sheets is increased, DOX attachment will be increased and cytotoxicity will be more and it will be at lower concentration of nanocarrier.
• Graphene oxide (GO) was synthesized from industrial graphite.
• GO was carboxylated, formed amide bond with PEI (2KDa). Primary amine groups of PEI were thiolated and reacted with MPH.
• DOX was reacted with hydrazide bond of MPH.
• Every step was confirmed by suitable characterization technique.
• Drug loading and loading efficiency were calculated.
• Drug release was performed at room temperature at pH 5.5 and 7.5.
• MTT assay was performed to evaluate cytotoxicity for nanocarrier, drug, nanocarrier-MPH-drug and GO-DOX.
This research was made possible by grant No. 92-03-33-24378 from Tehran University of Medical Sciences. The Authors would also like to appreciate Sepideh Karoobi for her technical help.
Conflicts of Interest
There are no conflicts to declare.
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