Inorganic Chemistry Communications 159 (2024) 111814
Available online 29 November 2023
1387-7003/© 2023 Elsevier B.V. All rights reserved.
Short communication
Novel carbon quantum dots incorporated polyacrylic acid/polyethylene glycol pH-sensitive nanoplatform for drug delivery
Mehrab Pourmadadi
a, Alireza Tajiki
b, Majid Abdouss
b,*, Alireza Beig Mohammadi
c, Zelal Kharaba
d,e, Abbas Rahdar
f,*, Ana M. Díez-Pascual
g,*aDepartment of Chemical Engineering, Faculty of Engineering, University of Tehran, Tehran 1417935840, Iran
bDepartment of Chemistry, Amirkabir University of Technology, Tehran, Iran
cDepartment of Physics, Humboldt-Universit¨at zu Berlin, Newtonstr. 15, 12489 Berlin, Germany
dDepartment of Clinical Pharmacy, College of Pharmacy, Al Ain University, Abu Dhabi, United Arab Emirates
eFaculty of Medical Sciences, Newcastle University, Newcastle Upon Tyne, UK
fDepartment of Physics, Faculty of Sciences, University of Zabol, Zabol 538-98615, Iran
gUniversidad de Alcal´a, Facultad de Ciencias, Departamento de Química Analítica, Química Física e Ingeniería Química, Ctra. Madrid-Barcelona, Km. 33.6, 28805, Alcal´a de Henares, Madrid, Spain
A R T I C L E I N F O Keywords:
Targeted drug delivery Breast cancer
Double emulsion synthesis Carbon quantum dots Hydrogel
A B S T R A C T
Quercetin (Qc) is a safe plant metabolite that plays an effective role as a chemical inhibitor against tumor cells and in preventing the progression of many cancers. In this research, carbon quantum dots (CQDs) dispersed in a pH-sensitive hydrogel comprising polyacrylic acid (PAA) and polyethylene glycol (PEG) were prepared with the aim to enhance the effectiveness and bioavailability of Qc drug. The PAA-PEG-CQDs-Qc nanoplatform was synthesized using the double emulsion method in order to control its size and shape. Numerous tests have been carried out to characterize the novel nanocarrier including FTIR, XRD, FE-SEM, DLS, and zeta potential, which corroborated its successful synthesis as well as its stability in a physiological environment. Very high drug loading and encapsulation efficiencies (47% and 89%, respectively) were attained, ascribed to hydrogen bonding of Qc with PAA and PEG as well as π-π interactions with the CQDs. Further, a sustained and selective drug release in acidic conditions similar to the tumor site was found. MTT and flow cytometry were used to assess the cytotoxicity of PAA, PAA-PEG, PAA-PEG-CQDs and PAA-PEG-CQD-Qc on the MCF-7 cancer cell line. Experi- mental results revealed that the drug-loaded nanocarrier had considerably higher cytotoxicity than the free drug, as well as increased apoptotic cell death, due to a synergistic effect of all the nanocomposite components in preventing the survival of cancer cells.
1. Introduction
The increase in the side effects of the use of chemical drugs and the long-standing position of medicinal plants in the health of societies has caused a global focus on the identification of medicinal plants and the investigation of their active compounds with the aim of formulating and producing effective and low-risk drugs [1,2]. Quercetin (Qc) can be mentioned among the effective plant substances, which is found as a flavonoid compound in most fruits, vegetables, leaves and seeds [3]. It is advised as an antioxidant for cancer prevention because of its pro- apoptotic and anti-cancer properties, which also boost the anti-tumor effects of chemotherapy drugs and synergistically increase the toxicity of medications against cancer cells [4]. Several studies have shown that
Qc inhibits breast, colon, prostate, ovarian, and lung cancer cells [5–8].
In addition, it has excellent antiproliferative action against most ma- lignancies by sensitizing cancer cells to chemotherapeutic treatments [9]. Besides to its anticancer role, this drug has medicinal advantages and prospective involvement in antibacterial, antiviral, antidiabetic, and anti-inflammatory activity, as well as a favorable function for the heart and blood vessels [10].
The common methods of drug administration have disadvantages such as weak bioavailability, low in-vivo stability, poor solubility, non- specific function, and in order to improve the performance, the use of nanocarriers in order to incorporate medicinal compounds and deliver them to the target tissues is of interest to researchers [11,12]. Mean- while, encapsulation method traps the drug in the carriers, which
* Corresponding authors.
E-mail addresses: [email protected] (M. Abdouss), [email protected] (A. Rahdar), [email protected] (A.M. Díez-Pascual).
Contents lists available at ScienceDirect
Inorganic Chemistry Communications
journal homepage: www.elsevier.com/locate/inoche
https://doi.org/10.1016/j.inoche.2023.111814
Received 9 August 2023; Received in revised form 17 November 2023; Accepted 25 November 2023
protects them from oxidation, isomerization and decomposition, and also increases the half-life of the drugs in a period of time and causes the controlled and continuous delivery of them to the target tissues [13–15].
Tumor vessels are extremely permeable and leaky due to an irregular basement membrane and a low number of endothelial progenitor cells.
The gap size between the endothelial cells in tumors varies from 100 to 780 nm, depending on its nature, which differs from the gap between normal cells, which is between 5 and 10 nm [16]. Polyethylene glycol (PEG) is a polymer frequently utilized to coat nanoparticles for size tuning. Also, this polymer specifically delays the function of the retic- uloendothelial system, has low toxicity, and is excreted intact from the kidneys or feces due to its low immunogenicity [17]. For example, Liu et al. fabricated PEG functionalized MoS2 quantum dots for pH responsive doxorubicin delivery to malignant glioblastoma tumor cell lines (U251). The PEGylated MoS2 quantum dots showed increased biological stability and reduced cytotoxicity toward normal cells. In other words, by coating MoS2 quantum dots, which are inherently toxic, with PEG, its excellent fluorescent and traceable properties have been used in the drug delivery system [18].
Given the capacity of carboxyl groups to form strong hydrogen bonds with mucosaccharide chains, polyacrylic acids (PAA) have excellent mucoadhesive characteristics. Acrylic acid derivatives are easily swollen by water absorption and in addition to this hydrophilic nature, their crosslinking structure makes them a good candidate for controlled drug delivery systems [19]. The 3D structure of this polymer has made it biologically neutral, a structure that is less common in other linear polymers. Hydrogels obtained from PAA and its copolymers are stimulus-responsive because they are very sensitive to environmental conditions [20]. These pH sensitive polymers, are very suitable for drug release in the acidic conditions of tumors and cause the specific function of the drug carrier. Amini-Fazl et al. introduced a magnetic hydrogel nanocarrier containing chitosan, PAA and Fe3O4 for 5-Fluorouracil administration in the colon and rectal. They demonstrated that their biocompatible platform released the drug in a regulated manner and can enhance the drug dosage at the specific sites by applying an external magnetic field [21].
Carbon quantum dots (CQDs) are a group of carbon nanomaterials that have very good fluorescence properties. Metal quantum dots cannot be used in biological systems due to their high toxicity, but CQDs are chemically and optically stable, highly biocompatible, easy to prepare, and have low toxicity, which are used as biological labels and cell im- aging agents [22]. Further, they can be used for photothermal-enhanced programmed tumor therapy [23]. Depending on the precursor materials used in the synthesis of CQDs, different functional groups such as carboxyl, amine and thiol are created on their surfaces and subsequently it becomes possible to further functionalize and covalently connect them to biological and chemical substances for various biomedical applica- tions [24]. Samimi et al. employed nitrogen doped carbon quantum dots for both therapeutic and diagnosis purposes of cancer. The NCQDs resulted from hydrothermal method, were conjugated with quinic acid as a targeting agent in order to specific delivery of gemcitabine to breast tumor cells [25].
In this research, a biocompatible hydrogel nanocarrier based on PAA, PEG and CQDs was designed and loaded with quercetin drug using the water-in-oil emulsification method. CQDs were chosen with the aim to increase drug absorption and loading of the nanocomposite. The resulting PAA-PEG-CQDs-Qc platform was characterized via FE-SEM imaging, DLS and Zeta potential, XRD and FT-IR analyses. The loading capacities and release profiles were evaluated in physiological and tumor conditions, in-vitro release and subsequently, drug release data from the platform were fitted to different kinetics models. The MTT assay was performed in order to assess the behavior of the developed platform towards cancer cells. Furthermore, flow cytometry test were carried out to show the cell death induction mechanism of the platform towards MCF-7, a type of human breast cancer cells. The results demonstrated the effective release of quercetin under pH stimulation
from the designed nanoplatform and its specific targeting at the tumor site.
2. Materials and methods 2.1. Materials
Diammonium citrate 98 %, Poly(acrylic acid) sodium salt (Mw ~ 5,100), glacial acetic acid ≥99 %, Poly(ethylene glycol) average Mw ~ 8,000, SPAN 80, dimethyl sulfoxide (DMSO), phosphate-buffered saline (PBS) tablets, and dialysis membrane with 12 Kda cut-off, all were prepared from Merck company (Germany). Quercetin ≥ 99 % was sourced from Sigma Aldrich Company (USA). Food grade hazelnut oil was purchased from a local store. Thermo Fisher Scientific Inc. (USA) supplied the 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium bromide (MTT). The American Type Culture Collection Company (USA) supplied the MCF-7 cell line and L929 cell lines.
2.2. Physicochemical characterization of the nanoparticles
A Zetasizer Nano-ZS device (UK) operating at a wavelength of 633 nm was used to measure the particle size distribution, zeta potential, and polydispersity index (PDI) of the synthesized nanoparticles. The morphology of freeze-dried nanoparticles were determined using a Tescan MIRA3 (Czech Republic) field emission scanning electron mi- croscope (FE-SEM). A drop of the lyophilized nanoparticles was placed on the holder and subsequently coated with gold, and the surface characteristics and shape of the nanoparticles were examined. Trans- mission electron microscopy (TEM) images were acquired using a JEOL model JEM 2010 microscope at an acceleration voltage of 200 kV.
Specimens were prepared by drop-coating the sample dispersion onto an amorphous carbon-coated 300-mesh copper grid. FTIR spectra were recorded in the wavenumber range of 400–4000 cm−1 using Bruker Alpha device (USA). X-ray diffraction (XRD) experiments were per- formed with a Phillips Xpert device (Netherlands), and in all experi- ments, a CuKα monochromatic beam with a wavelength of 1.542 A… ˜ was used.
2.3. Fabrication of PAA-PEG-CQDs nanocarrier and drug loading CQDs were prepared based on the method described earlier [26]. For this purpose, 1 g of diammonium citrate was magnetically dissolved in 40 ml deionized water at room temperature. The resulting solution poured into a steel hydrothermal reactor containing an inner chamber made of Teflon and subsequently placed in an electric oven for 16 h at 160 ◦C. Finally, the resulting mixture was freeze-dried in order to obtain CQDs in powder form.
The absorption and fluorescent properties of the CQDs were char- acterized (Fig. S1) via UV–Vis and fluorescence measurements. In the UV–Vis spectrum, two absorption peaks appear at 275 and 412 nm. The first can be attributed to the carbonic core center while the second is ascribed to surface or molecule center [26]. Under 420 nm excitation, the CQDs led to green emission at 540 nm. The excitation spectrum obtained by monitoring the emission at 540 nm is in agreement with the results found in the absorption spectrum. Further, the QCDs showed excitation-dependent emission. When the excitation wavelength was increased from 360 to 500 nm, the emission peak shifted from 530 to 570 nm. This behavior could be ascribed to the non-uniform particle size, as confirmed by the TEM images, which ranges between 2 and 8 nm (Fig. S2), and shows an average value of 4.5 nm. The CQDs display a quasi-spherical shape, and are crystalline in nature, with a lattice parameter of 0.22 nm, which agrees with (100) lattice fringes of graphite.
For the preparation of the nanocomposites, 0.4 g of PAA was added to 40 ml of 2 % (v/v) acetic acid at room temperature, and it was completely dissolved on a magnetic stirrer, yielding a homogenous
solution containing 1 % (w/v) PAA. The solution was then treated with 0.2 g of PEG powder until it was fully dissolved and then placed in an ultrasonic bath for 10 min. Then 40 mg of the CQDs were added to the solution and thoroughly homogenized on the stirrer. Subsequently, Qc was loaded, and the concentration of the drug in the final hydrogel was 5 μg/ml. Finally, a PAA-PEG CQDs hydrogel containing Qc was ach- ieved. Then, 0.2 % (v/v) of SPAN 80 surfactant was added to the hydrogel, and 10 ml of the PAA-PEG-CQD-Qc hydrogel covered with SPAN 80 surfactant was added drop by drop to 30 ml of the hydrophobic hazelnut oil phase under vigorous magnetic stirring to form a spherical nanocarrier with the drug in the hydrophobic phase. After 10 min, the same volume of hydrophobic phase, namely 30 ml of distilled water, was added dropwise to the emulsion and stirred for another 15 min; then it was removed from the stirrer to enable the layers separation. The drug nanocarrier was extracted from the aqueous phase by separating the oil phase using a sampler, and then the mixture was centrifuged at 4500 rpm for 10 min. A freeze dryer was used to pulverize each sample after the different stages.
2.4. Cell viability assessment
In order to investigate the effects of the developed nanocomposites as well as their individual components and drugs on human breast cancer cells (MCF-7), and normal fibroblast cell lines (L929), 105 cells were placed on the plates and cultured for 24 h. Then, the cells were treated with the same concentration of various substances at a time interval of 72 h. After finishing the treatment, the suspended cells were settled via centrifugation at a speed of 2,000 rpm. Subsequently, they were rinsed with phosphate-buffered saline (PBS), and placed in an incubator at 37 ◦C with a culture medium containing 3-(4,5-Dimethylthiazol-2-yl)- 2,5-diphenyltetrazolium bromide (MTT) salt and 5 % CO2 for 3 h.
During this process, due to the mitochondrial activity of living cells, MTT salt is reduced to blue formazan crystals that have different ab- sorption wavelengths [27]. Finally, the culture media were replaced by 200 µl dimethyl sulfoxide (DMSO) to dissolve the formazan crystals and subsequently all samples were analyzed using an ELISA reader device (Stat Fax 2100 Microplate Reader, USA).
2.5. Cell apoptosis analysis
Flow cytometry was performed using a flow cytometer device (BD Biosciences, USA), and an annexin V kit to differentiate between apoptosis and necrosis of the MCF-7 cells after treatment with the developed nanocomposites. Breast cancer cells were initially incubated in a 6-well plate at a density of 5 ×103 cells per vessel, and allowed to multiply for 24 h. Next, the cells were treated with the free drug, PAA, PAA-PEG, PAA-PEG-CQD, and PAA-PEG-CQD-Qc in PBS medium (pH = 7.4). Following centrifugation, the floating sections were removed, and the leftovers were resuspended in 100 μl of annexin V-FITC in PBS.
Moreover, propidium iodide (PI) as a fluorescent intercalating agent was added to the Annexin V FITC to label the cells. Finally, the samples were all incubated in the dark for 15 min at 37 ◦C [28].
2.6. Loading of quercetin drug
Firstly, 40 ml of ethanol were mixed with 10 mg of QC, and the liquid mixture was homogenized for 10 min in an ultrasonic bath. Then, 10 mg of each nanocarrier was added to the resulting solution followed by storage under dark conditions at room temperature for 24 h. Subse- quently, it was centrifuged for 10 min at a speed of 6,000 rpm to settle the carrier along with the absorbed drug. Finally, the supernatant so- lution was separated and analyzed by UV–Vis spectrophotometry at a wavelength of 380 nm. The loading efficiency (LE) and encapsulation efficiency (EE) were calculated using Equations (1) and (2), respectively.
LE%=TotalQC− FreeQc
TotalNanocarrier ×100 (1)
EE%=TotalQC− FreeQc
TotalQc ×100 (2)
2.7. Drug release study
Drug release experiments for PAA-PEG-CQDs-Qc nanocomposite were performed in two saline phosphate buffer media containing 50 % w/w ethanol (pH =5.4, similar to tumor environment and pH =7.4, similar to physiological environment). A certain amount of each nano- carrier was placed in a dialysis bag (Mw cutoff of 12,000 g/mol), which was then submerged in 30 ml of phosphate buffer containing 20 % ethanol v/v and stored at 37 ◦C in a shaker incubator for 96 h. At certain periods of time (0.5, 1, 2, 3, 6, 12, 24, 48, 72 and 96 h), the dialysis bag surrounding each solution was sampled, and replaced with the same amount of fresh buffer. The percentage of absorption of each sample was determined using a UV–Vis spectrophotometer, under the same condi- tions mentioned in the drug loading section.
3. Results and discussion 3.1. FT-IR experiments
In order to investigate the functional groups of the developed nanocomposites and the individual components, FT-IR spectra were recorded, and they are compared in Fig. 1a. In the spectrum of neat PAA, a broad band appeared at 3000–3730 cm−1 ascribed to the O–H stretching vibrations of the carboxylic acid groups. The bands at 1125 and 1641 cm−1 are also related to C-O and C =O stretching vibrations of carboxylic acids, respectively [19]. In the spectrum of PEG, the peak at 2888 cm−1 is attributed to the stretching of the aliphatic C–H bonds of polymer. Other characteristic peaks at 844, 960, 1153, 1280, 1346, and 1469 cm−1 were carefully assigned in former studies [29]. The spectrum of CQDs shows different peaks that correspond to functional groups located on their surface. The broad peak centered at 3450 cm−1 corre- sponds to the overlapping of N–H and O–H stretching vibrations, and that 2927 cm−1 to C–H stretching vibrations. Also the bands at 1640, 1380 and 1265 cm−1 can be related to amide C =O, amide III and C-O-C stretching, in agreement with results reported earlier [26]. The use of the ammonium salt of citric acid as the synthesis source of the CQDs has led to the formation of nitrogen containing functional groups [30]. In the spectra of PAA-PEG and PAA-PEG-CQDs nanocomposites, the char- acteristic peaks of each component can be observed albeit with reduced intensity and slightly shifted to lower wavenumber, indicative of the strong interaction between the nanocomposite components via hydrogen bonding. After loading the drug, additional small shifts are detected, ascribed to the interaction between the OH groups of Qc and the OH, COOH and CO-NH groups of PEG, PAA and the CQDs, respec- tively. Further, new peaks are found in the spectrum of PAA-PEG-CQD- Qc at 1354 and 1407 cm−1 which are associated with C-OH de- formations and C = C stretching vibrations of the aromatic rings in flavonoids, which corroborates the successful preparation of the final drug delivery system [31].
3.2. XRD experiments
The crystalline structure of the synthesized nanocomposites was examined using X-ray diffraction (Fig. 1b). In fact, the XRD patterns are unique for each material, and can prove their presence in a nano- composite. In the diffractogram of CQDs, a broad and weak peak appears centered at 2θ =25◦, which corresponds to the reflection of the (002) plane of the aromatic rings, similar to that found in neat graphite; this further corroborates the successful synthesis of amorphous carbon
quantum dots [32]. In the pattern of PEG, the sharp peaks at 2θ =19.2 and 23.4◦correspond to the (120) and (032) crystal planes of the high crystalline polymer [33]. Upon addition of PAA and CQDs, which have amorphous character, the crystallinity of PEG is reduced. The sharp characteristic peaks of PEG are shifted to higher angles, which can be ascribed to interstitial placement of the above components between PEG crystal planes, thus indicative of the successful formation of the nano- composites. Also, the intense peaks that are typical of PEG prevent the appearance of broad and weak peaks of the other amorphous materials in the diffractogram of the nanocomposites. An additional shift is observed upon addition of Qc, which further confirms the successful drug loading.
3.3. FE-SEM images
FE-SEM analysis was utilized to examine the surface morphology, size and aggregation state of the developed nanocomposites. Fig. 2 (a and b) shows images at different magnifications. Aggregates of spherical nanocarriers with a narrow size distribution are detected. Their size is in the range of 180–300 nm, with an average value of 230 nm. This dem- onstrates the effective preparation of the PAA-PEG-CQDs-Qc nano- carriers using the W-O-W technique with particular shape and size induction. The morphology of PAA-PEG-CQDs-Qc nanocarriers was also examined via TEM analysis (Fig. S2). The images corroborate the spherical shape of the nanocarriers, which showed darker areas likely corresponding to the CQDs.
3.4. Zeta potential and DLS results
The magnitude of the zeta potential indicates the degree of electro- static repulsion adjacent particles. When the zeta potential decreases, the attraction overcomes the repulsion and the particles are coagulated, while particles with high zeta potential are electrically stable. According to Fig. 2c, the zeta potential for the PAA-PEG-CQDs-Qc drug delivery system is − 45 mV, which indicates the high stability of this platform in fluids similar to the body, without aggregation, which is an essential feature. Also, the presence of oxygenated functional groups in the structure of the polymers, especially PAA in its sodium salt form, causes the surface charge of the prepared nanocomposite to be negative.
DLS analysis is a technique based on laser light interaction with particles. The scattering and fluctuations in the light intensity due to the Brownian motion of the nanoparticles in solution are analyzed, and provide information about the particle size distribution. As can be observed in Fig. 2d, the particle size of PAA-PEG-CQDs-Qc nano- composite is in the range of 200 to 330 nm, with an average diameter of 247 nm, in good agreement with the observations from SEM and TEM analyses. According to references, nanoparticles with a size of less than 100 nm cannot selectively affect cancer cells due to their ability to pass through the walls of healthy cells [34]. On the other hand, the optimal size for enhanced permeability and retention (EPR) effect has been re- ported to be 100–200 nm. However, the developed platforms do not necessarily need to enter the cancer tissues and, at the same time, they are small enough to be able to deliver the drug around the cancer cells.
In fact, previously many groups have found that larger sized nano- particles are better for tumor targeting [35]. Thus, a trade-off arises Fig. 1. FT-IR spectra (a) and XRD patterns (b) of the nanocomposites and the different components.
between “size increase/higher tumor targeting” versus “size decrease/
lower tumor targeting”. The increase in the nanoparticle size prolongs systemic blood circulation and thus EPR-based targeting. Therefore, the prepared nanocarrier has favorable conditions with regard to particle size. Also, the polydispersity index (PDI) for this nanocarrier has been found to be 0.2, which corroborates its narrow size distribution [19].
3.5. Drug loading evaluations
The LE% and EE% of Qc from PAA-PEG-CQDs platform were calcu- lated as described in section 2.6 and are collected in Table 1. The results demonstrated that the developed platform improved the drug absorp- tion significantly compared to previous studies. For example, Sharma et al. designed modified PEG-Poly(lactide) nanoparticles for the delivery of Qc drug to breast cancer cells, and attained an EE% of 62.8 % [36].
Also, Bashir and co-workers fabricated a Qc delivery platform based on karaya gum natural polysaccharide which polymerized in the presence of acrylic acid to form a hydrogel. The resulting co-polymer of PAA, showed an EE% lower than 88 % under different conditions [37]. The increased drug EE% for PAA-PEG-CQDs platform is due to the hydrogen bonding of Qc with the hydrogel containing PAA and PEG. In addition, the π-π stacking interactions between the aromatic rings of quercetin and the CQDs play a key role in enhancing both the LE% and EE% in the Fig. 2.(a, b) FE-SEM images at different scales, (c) zeta potential and (d) DLS results for PAA-PEG-CQDs-Qc nanocomposite.
Table 1
LE% and EE% of quercetin for different platforms.
Platform LE% EE%
PAA-PEG/Qc 36 78
PAA-PEG-CQDs/Qc 47 89
PAA-PEG-CQDs platform.
3.6. In-vitro studies on the release and its kinetics
Drug release tests for PAA-PEG-CQDs-Qc nanocomposite were per- formed at 37 ◦C and in 50 % phosphate buffer/ethanol solution at different pH values. Fig. 3 shows the cumulative release percentage over time. Clearly, the drug is released faster within the first 12 h, which is likely due to the adsorption of more Qc on the surface of the nanocarrier by weak van der Waals forces. Moreover, the concentration gradient enhances the release rate at the beginning of the discharge due to the absorption of large quantities of Qc on the nanocarrier. The burst release found within the initial hours after the drug carrier is delivered to the intended site may be beneficial in the treatment process [38]. The highest drug release under physiological conditions was 80 % in 96 h whereas it was 96.5 % for the same period in an acidic environment similar to cancer cells, demonstrating that the drug lasts longer under neutral conditions. This behavior also causes minimum damage to normal cells and subsequently selective action of the platform against cancer cells. Given that the slope of the curve approaches zero, the drug released at pH 7.4 after 96 h may be ignored since the unreleased drug is linked to the carrier via strong interactions and after this period, it is released only by changing the ionic strength of the solution. Since Qc drug is also connected to the carrier via hydrogen bonds, when the pH of the environment is decreased, the hydronium ion competes with the functional groups on the surface of the carrier to form a hydrogen bond with the drug, and consequently the released amount increases. Overall, under in-vivo conditions, the designed platform showed higher release in an acidic environment compared to the physiological pH of the body.
Considering the acidity of the environment of cancer cells, it is expected that the PAA-PEG-CQDs system behaves similarly in-vivo and at the cellular level, which provides the possibility of specific delivery of Qc to cancer cells.
In vitro release patterns were analyzed using several kinetic models, including zero-order, first-order, Higuchi, and Korsmeyer-Peppas models, with the goal of gaining insight about the mechanism of drug release from the nanocomposites. Fig. 4 displays the fitting results of the release data with the above mentioned kinetic models and their corre- lation coefficients and related equations are indicated next to each curve. According to R2 values, the release of quercetin from the PAA- PEG-CQDs nanocomposite followed the Korsmeyer-Peppas model. In this model, matching 60 % of the initial data of the drug release profile is enough to draw the curve [39]. The Korsmeyer-Peppas equation is as
follows (equation (3):
logMt
M∞
=logK+nlogt (3)
where Mt/M∞ is the accumulated Qc released, K is the rate constant and n is the release exponent. The n values were calculated to be 0.69 and 0.59 respectively for physiological and tumor environment conditions, respectively, which imply that the Qc release from PAA-PEG-CQDs platform follows a non-Fickian diffusion process.
3.7. Cell viability investigations
In order to investigate the viability of MCF-7 cancer cells and normal L929 cells upon treatment with the designed drug delivery system, as well as the possible cytotoxicity of the platforms against normal and cancer cells, MTT tests were performed. The analysis of the cytotoxicity results for the different platforms (Fig. 5) compared to the control sample with different p-values demonstrates a considerable reduction in cancer cell viability upon addition of different components. PAA induced minimal inhibition against MCF-7 cells, indicating its role as a biocompatible carrier [40]. With the incorporation of other compo- nents, the cell viability decreased, by about 20 and 30 % in the PAA- PEG, and PAA-PEG-CQDs platforms, respectively, and finally reached the minimum value (about 42 % reduction) in the system loaded with the drug (PAA-PEG-CQD-Qc). In fact, the simultaneous presence of components in the final platform causes a synergistic effect in prevent- ing the survival of cancer cells, and this effect is maximized with the incorporation of the drug. In addition, among drug-free platforms, PAA- PEG-CQDs has the lowest survival rate of cancer cells, because of the presence of the CQDs and their ability to pass through the wall of cancer cells due to their small size and induction of cytotoxicity. Also, the decrease in cell viability in the vicinity of PAA-PEG-CQDs-Qc platform in contrast to the free Qc shows the positive effects of the prolonged release of drug in the designed system compared to the direct exposure of the drug to cancer cells. Fig. 5 also shows the viability of the L929 cells in the presence of the different platforms. The percentage of cell viability for PAA, PAA-PEG and PAA-PEG-CQDs, and PAA-PEG-CQDs-Qc is very similar to that of the control sample, which indicates the biocompati- bility of the materials used in the drug delivery system and their non- toxicity to healthy cells. However, when quercetin is used individu- ally, it exhibits considerable toxicity in contrast to its application within a drug delivery system. Notably, PAA-PEG-CQDs platform, which fa- cilitates a gradual and sustained drug release, effectively limits the excessive buildup of the drug’s concentration near cells, thereby diminishing its harmful effects.
3.8. Flow cytometry experiments
Fig. 6 depicts the histograms related to the control group (absence of drug delivery platforms) and in the presence of nanocomposites and their individual components to investigate the death of MCF-7 cells. The four regions in each histogram represent necrotic cells (Q1), late apoptosis (Q2), live cells (Q3), and early apoptosis (Q4), respectively, which can provide valuable information about the mechanism of cell death induction by the various platforms. In the process of apoptosis or programmed cell death, dangerous cells are eliminated without damaging the cells or surrounding tissues. In fact, one of the most effective ways to eliminate cancer cells without side effects is by trig- gering the process of apoptosis [41]. In agreement with the results ob- tained from the MTT test, the percentage of live cells compared to the control sample, PAA had the least change and in other platforms it had a downward trend and finally reached the minimum value in contact with PAA-PEG-CQDs-Qc. Also, the total percentage of cell death from apoptosis mechanism in the final platform compared to other cases reached a maximum which proves the improvement of drug Fig. 3.Release curves of Qc from PAA-PEG-CQDs nanocarrier (n = 3) at
different pH values. Inset: magnified zone of the release curves for the first 6 h.
performance by using PAA-PEG-CQDs nanocarrier. Quercetin and similar drugs, when used alone, promote cell death through necrosis for various reasons such as low bioavailability, low solubility, and so forth.
However, when incorporated into a drug delivery system, mainly the mechanism of their function changes towards programmed cell death and apoptosis. This issue has also been observed in the flow cytometry studies performed on the PAA-PEG-CQDs platform.
4. Conclusion
In this research, a hydrogel nanocomposite based on PAA, PEG and CQDs was designed as a pH-sensitive nanocarrier to stabilize, increase solubility, bioavailability and improve the efficacy of Qc anticancer drug. The results show that the PAA-PEG-CQDs nanocarrier has signif- icantly increased LE and EE compared to PAA-PEG platform, ascribed to π-π interactions between CQDs and Qc. Drug release data indicated that in environments with a pH close to cancer cells, PAA-PEG-CQDs nano- carrier is able to release more drug compared to physiological pH and has a selective function with less side effects. Also, the results of cell toxicity studies showed that Qc encapsulated in PAA-PEG-CQDs has a higher cytotoxicity than the free drug against breast cancer cells.
Consequently, this study shows that the PAA-PEG-CQDs nanocarrier has the potential to be used as a pH-sensitive and biocompatible drug de- livery system for the targeted delivery of Qc to cancer cells.
Funding
Financial support from the Community of Madrid within the framework of the Multiyear Agreement with the University of Alcal´a in the line of action “Stimulus to Excellence for Permanent University Professors”, Ref. EPU-INV/2020/012, is gratefully acknowledged.
CRediT authorship contribution statement
Mehrab Pourmadadi: Data curation, Formal analysis. Alireza Tajiki: Data curation, Writing – original draft. Majid Abdouss:
Fig. 4. Fitting plots of drug release data with the indicated kinetic models. The plots include the fitting equations and the correlation coefficient values.
Fig. 5.Viability assessments of MCF-7 and L929 cells after exposure to different platforms using MTT. *, **,*** and ## signs represent p-value <
0.001, p-value <0.01, p-value <0.05, and p-value <0.001, respectively.
Supervision, Writing – review & editing. Alireza Beig Mohammadi:
Data curation, Writing – original draft. Zelal Kharaba: Investigation, Methodology, Resources. Abbas Rahdar: Writing – review & editing.
Ana M. Díez-Pascual: Writing – review & editing, Funding acquisition.
Declaration of competing interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Data availability
Data will be made available on request.
Acknowledgment
Abbas Rahdar thanks from University of Zabol for funding (UOZ-GR- 8906).
Appendix A. Supplementary material
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.inoche.2023.111814.
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