GQDs-MSNs nanocomposite nanoparticles for simultaneous intracellular drug delivery and fluorescent imaging
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Although number of stimuli-responsive drug delivery systems based on mesoporous silica nanoparticles (MSNs) have been developed, the simultaneous real-time monitoring of carrier in order to guarantee proper drug targeting still remains as a challenge. GQDs-MSNs nanocomposite nanoparticles composed of graphene quantum dots (GQDs) and MSNs are proposed as efficient doxorubicin delivery and fluorescent imaging agent, allowing to monitor intracellular localization of a carrier and drug diffusion route from the carrier.
Graphene quantum dots (average diameter 3.65 ± 0.81 nm) as a fluorescent agent were chemically immobilized onto mesoporous silica nanoparticles (average diameter 44.08 ± 7.18 nm) and loaded with doxorubicin. The structure, morphology, chemical composition, and optical properties as well as drug release behavior of doxorubicin (DOX)-loaded GQDs-MSNs were investigated. Then, the in vitro cytotoxicity, cellular uptake, and intracellular localization studies were carried out. Prepared GQDs-MSNs form stable suspensions exhibiting excitation-dependent photoluminescence (PL) behavior. These nanocomposite nanoparticles can be easily DOX-loaded and show pH- and temperature-dependent release behavior. Cytotoxicity studies proved that GQDs-MSNs nanocomposite nanoparticles are nontoxic; however, when loaded with drug, they enable the therapeutic activity of DOX via its active delivery and release. GQDs-MSNs owing to their fluorescent properties and efficient in vitro cellular internalization via caveolae/lipid raft-dependent endocytosis show a high potential for the optical imaging, including the simultaneous real-time optical tracking of the loaded drug during its delivery and release.
KeywordsGraphene quantum dots Mesoporous silica nanoparticles Nanocarriers Cellular uptake Drug delivery Real-time monitoring of drug release Bioimaging Nanomedicine
The development of theranostic nanoplatforms for simultaneous diagnosis and therapy, particularly in cancer treatment, is enabled by the combination of nanomaterials with different functionalities. Among a variety of multifunctional nanoplatforms, mesoporous silica-based nanostructures and nanocomposite materials has been recognized and widely studied as promising drug carriers owing to their mesoporous structure, their unique properties, such as large surface area and pore volume, high chemical stability, reactive surface, but also cell membrane-penetration ability and low cytotoxicity (Lee et al. 2011). Mesoporous silica nanoparticles (MSNs) have been employed so far as an efficient carrier of various therapeutics, but also imaging agents, including other functional nanostructured materials. It was proposed that MSNs-based drug delivery systems facilitate controlled delivery and release of anticancer drugs, thus enhancing their therapeutic efficiency along with diminishing their side effects in comparison to standard drug administration (Bharti et al. 2015). Up to date, many MSNs-based theranostic nanoplatforms for bioimaging, drug delivery, and therapy have been developed, taking the advantage of different nanoparticles as capping agents, such as, e.g., Fe3O4 (Lee et al. 2010), Au (Ma et al. 2012), CdS (Lai et al. 2003), or embedded into MSNs as a core, e.g., Fe3O4 (Yao et al. 2017a, b), but also paramagnetic ions (Gd3+, Mn2+) incorporation (Lin et al. 2004) or their chelates (Cao et al. 2015). Another approach also includes the advantage of different release stimuli, such as pH, redox potential, adenosine triphosphate gradient, enzymes, temperature, and multi-stimuli-responsive systems (Moreira et al. 2016).
Number of developed stimuli-responsive drug delivery systems based on MSNs have been developed so far; however, the simultaneous real-time monitoring of the drug carrier in order to guarantee proper drug targeting remains as a challenge. Despite the great potential of MSNs as an efficient drug carrier, these nanoparticles cannot itself emit a fluorescence signal allowing for their detection, however not only when the drug has been released, but also before the release, allowing to monitor intracellular localization of a carrier and diffusion route of the drug from the carrier. This issue has been addressed by the fluorescent labeling of MSNs by capping or encapsulation with fluorescence organic dyes, e.g., fluorescein isothiocyanite (Lu et al. 2009) up-conversion nanoparticles (Niu et al. 2014) and more recently with quantum dots (QDs) (Zhang et al. 2016; Yao et al. 2017a).
QDs are fluorescent nanocrystals, which are considered as an ideal fluorescent agent for bioimaging owing to their many superior properties compared to organic dyes (Resch-Genger et al. 2008). These properties are photostability, broad excitation wavelength, narrow emission, continuous and broad absorption spectra, and finally susceptibility to surface functionalization, including biomolecules. The most commonly used so far QDs contain heavy metals (e.g., CdHgTe, CdTeSe@CdZnS, CdSe@ZnS), which cause undesirable biological and environmental effects, and thus limit their use in biological applications. Graphene quantum dots (GQDs) have emerged as an alternative and a new class of QDs with fluorescent properties (Wen et al. 2015). GQDs are kind of zero-dimensional small graphene sheets fragments, in which the electronic transport is confined in three spatial dimensions. Due to the quantum confinement and edge effects, GQDs possess a non-zero band-gap, and therefore emit luminescence upon the excitation. GQDs are built-up from carbon, which is abundant in the biological systems; therefore, they are considered as a biocompatible nontoxic material. Moreover, GQDs show a molecule-like character and contain a number of carboxylic, epoxy and hydroxyl groups; therefore, they can be easily dissolved in water-based solvents and are easy for further functionalization. Finally, GQDs exhibit stable photoluminescence and superior resistance to photobleaching in comparison to traditional semiconductor QDs and organic dyes. Yao et al. (Yao et al. 2017a, b) reported on the GQDs as caps and local photothermal generators and magnetic mesoporous silica nanoparticles as drug carriers and magnetic thermoseeds, which exhibited strong synergetic effect, resulting in high efficiency to kill cancer cells. Later, Yao et al. (Yao et al. 2017b) reported on GQDs-capped MSNs with a potential for combined chemo- and photothermal cancer therapy, showing pH- and temperature-responsive doxorubicin release, and NIR-induced photothermal cytotoxicity. A similar system has been reported by Huang et al. (Huang et al. 2016), where GQDs-decorated MSNs have been prepared through the electrostatic interaction in contrast to previously mentioned studies, and have been successfully used for aspirin loading and its release. The imaging capacity of GQDs/MSNs system next to the drug delivery has been reported for the first time by Chen et at. (Chen et al. 2014), where GQDs were capped onto MSNs through an acid-cleavable acetal bond, and hence the acidic pH-triggered doxorubicin release from mesopores.
Summarizing, taking the advantage of both components properties, the combination of MSNs and GQDs may provide a new strategy for integrated nanocomposite system for an efficient optical bioimaging and drug delivery system, which is yet to be developed.
In order to address current challenges concerning the theranostic nanoplatform, such as increase of the efficient uptake and accumulation in cancer cells, effective drug delivery, and controlled release with simultaneous imaging and real-time monitoring capability, GQDs-MSNs nanocomposite nanoparticles were prepared by the immobilization of GQDs onto MSNs, which were then loaded with doxorubicin as a model anticancer drug. The release studies were performed in pH- and temperature-dependent manner. So far, most of the GQDs/MSNs-integrated nanoplatforms have been studied mainly for controlled drug delivery, and also for other therapeutic modalities, but their potential as bioimaging agent has been rather underestimated and dominated by therapeutic activities of the system. Therefore, further in this manuscript, the focus is on the detailed investigation on the cellular uptake and intracellular translocation of the GQDs-MSNs, which are the basis for the possibility of the simultaneous imaging and real-time monitoring not only of the drug but also the drug carrier, which has not been well presented until now. The efficiency of prepared GQDs-MSNs nanocomposite nanoparticles for the simultaneous drug delivery and release next to the bioimaging was evaluated using HeLa cancer cells as a model cellular system.
Materials and methods
Sodium citrate tribasic dehydrate (NaCitr, ≥ 99%), thiourea (TU, ≥ 99%), tetraethyl orthosilicate (TEOS, 98%), hexadecyltrimethylammonium bromide (CTAB, ≥ 99%), N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC), (3-aminopropyl) triethoxysilane (APTES, ≥ 98%), and phosphate-buffered saline (PBS) were purchased from Sigma-Aldrich. Ethanol (EtOH, 99.8%), methanol (MetOH, 99.8%), and hydrochloric acid (HCl, 35–38%) were purchased from POCH Basic. Dulbecco’s Modified Eagle’s medium (DMEM), Hanks Balanced Salt solution, fetal bovine serum (FBS), Trypsine-EDTA (0.25%), penicillin-streptomycin, glutaraldehyde solution, and agarose were purchased from Sigma-Aldrich and used as received. WST-1 Cell Proliferation Assay Kit was purchased from Clontech. LIVE/DEAD® Viability/Cytotoxicity Kit for mammalian cells was obtained from Thermo Fisher Scientific. Formaldehyde methanol free, osmium tetroxide solution, uranyl acetate, and Embed-It™ Low Viscosity Epoxy Kit were purchased from Polysciences.
Preparation of GQDs
GQDs were prepared via the hydrothermal method (Permatasari et al. 2016; Qu et al. 2014), with the minor modification, using NaCitr (instead of citric acid) as a carbon precursor and TU as a base. NaCitr and TU reagents were mixed in the molar ratio of 1:3 (1.3 g:1.15 g), respectively. The reaction was run in water-based (25 ml) solution of both reagents in Teflon-lined stainless autoclave at 180 °C for 8 h. The formation of GQDs was indicated by the color change of the reaction solution to the orange one. Obtained postreaction solution was then given to vacuum drying at 60 °C and pressure of 200 mbar in order to remove the excess water. The final product was precipitated by adding EtOH to the solution and collected by centrifugation (13.2 rpm, 15 min), and dried at 60 °C. The obtained solid material can be easily dispersed in water.
Preparation of MSNs
MSNs, as a carrier for GQDs, were synthesized using the previously reported template-directed sol-gel method, with the minor modifications (Nooney et al. 2002). In a typical used synthesis procedure 0.5 g CTAB, as the structure-directing agent, was dispersed in 240 ml of deionized water (H2Od) in a closed vessel under vigorous stirring at room temperature. After the solution became homogenous, its pH was adjusted to 11 with the 1 M (molar) NaOH solution and heated up to 80 °C. Subsequently, 2.5 ml of TEOS as the silicon source was dropwise added and the reaction solution was further stirred continuously for 3 h, until white precipitated was obtained. As-obtained product was collected by centrifugation (24,000 rpm, 15 min) and washing with MetOH and then dried overnight at 60 °C. In order to remove CTAB template, the final dried product was refluxed for 24 h in a mixture of 160 ml MetOH and 9 ml HCl (~37%). The obtained MSNs were centrifuged and washed with MetOH and H2Od, and finally dried at 60 °C.
Preparation GQDs-MSNs nanocomposite nanoparticles
Prior to the GQDs-MSNs nanocomposites preparation, as-prepared MSNs were firstly amino-functionalized. For this purpose, 0.04 g MSNs was dispersed in EtOH and then treated with 0.8 ml APTES under the stirring. After 24 h, the suspension was centrifuged (13.2 rpm, 15 min) and washed with EtOH repeatedly for three times, in order to remove unreacted APTES. The prepared GQDs were then covalently immobilized onto amino-functionalized MSNs (NH2-MSNs) with the use of carbodiimide crosslinker chemistry. The amino-functionalized MSNs were dispersed in PBS-based solution (pH = 5.8) of GQDs (1 mg/ml) and added with 0.2 ml of EDC linker solution (20 mg/ml). After 24 h, the unbound QDs were removed by successive centrifugation and washing with EtOH and H2Od.
DOX-loading and release studies
To investigate drug release behavior from prepared GQDs-MSNs nanocomposites, as well as MSNs, 0.5 mg DOX-loaded nanocarrier was dispersed in 2 ml of PBS with pH 4.5, 5.0, 6.5, and 7.4 and given to continuous shaking under dark conditions. The temperature of PBS solution was kept constant 37 or 50 °C. After predetermined time intervals, the samples were centrifuged, then supernatant samples were withdrawn and replaced with fresh PBS for continuous drug release. The amount of released DOX was determined spectrophotometrically at 485 nm.
Human cervical cancer cell line HeLa obtained from American Type Culture Collection (ATCC) and human fibroblast cell line MSU1.1 obtained from Prof. C. Kieda (CBM, CNRS, Orléans, France) were used for in vitro studies. Cells were cultured in a complete medium DMEM supplemented with 10% FBS, 100 units/ml penicillin, and 100 μg/ml streptomycin, and grown at 37 °C in humidified atmosphere containing 5% CO2.
In vitro cytotoxicity assays
In order to determinate the cytotoxicity of GQDs, MSNs, and GQDs-MSNs nanocomposite nanoparticles, as well as DOX-loaded nanocomposite nanoparticles, HeLa and MSU1.1 cells were treated with increasing concentration of nanoparticles (from 20 to 400 μg/ml) and incubated for 24 h at 37 °C under a 5% CO2 atmosphere. Cells without any treatment were used as a negative control. The effect of the GQDs on cell viability was determined by WST-1 assay according to manufacturer’s instructions. Briefly, 10 μl of WST-1 solution was added to each well of 96-well plate and further incubated. After 2 h, the absorbance was measured with a microplate reader (Anthos Zenyth 340rt) at 450 versus a 650 nm reference. The relative cell viability (%) was expressed as a percentage relative to the negative control. Data are reported as the average ± standard deviation (SD) of experiments performed in triplicate. The cytotoxicity analysis was also performed for chemical inhibitors of cellular uptake (see, Fig. S4 in ESM).
The effect of the nanoparticles on cell viability was also determined using Live/Dead assay kit and analyzed by InCell Analyzer apparatus (GE Healthcare). Briefly, cultured adherent cells in a 96-well plate, previously co-incubated for 24 h with nanoparticles solutions at increasing concentration mentioned before, were prepared. Cells non-treated and treated with 50 v/v% dimethyl sulfoxide were used as negative and positive control (DMSO), respectively. Cells were washed with PBS, then a Live/Dead Viability Kit (Life Technologies) composed of two fluorescent dyes, calcein-AM and ethidium homodimer (EthD-1), for the staining of live and dead cells were used. Thus, in live cells, green fluorescence derived from calcein was observed in cytoplasm, whereas EthD-1 enters dead cells and was observed as red fluorescence in nucleus. Three repetitions for each condition were carried out. The images were acquired from 20 fields from each well, and then statistically analyzed by InCell Developer Toolbox software. The total cell number was normalized to the non-treated control group. Data are expressed as the average ± standard deviation (SD) of three different experiments.
Cell uptake of GQDs- and GQDs-MSNs-imaging studies
To investigate the GQDs and GQDs-MSNs nanocomposites’ ability to cell penetration and their intracellular distribution, as well as to monitor DOX delivery and release, confocal microscopy was performed on cancer cell line (HeLa). Briefly, cells were plated on a chambered Lab-Tek dish (1 × 104 cells/well), grown overnight, and then were incubated at 37 °C with GQDs, GQDs-MSNs nanocomposites (20 μg/ml) for 3 or 24 h. The cells were then rinsed three times with PBS (pH 7.4). The distribution of GQDs, GQDs-MSNs nanocomposites and doxorubicin was analyzed using a confocal laser scanning microscope (CLSM, FV1000, Olympus). Colocalization of DOX with GDQ-MSN was analyzed via Pearson’s coefficient constant.
Tracking pathway of cellular uptake
In order to block energy-dependent mechanisms of GQDs-MSNs uptake, the grown HeLa cells were incubated at 4 °C for 1 h. Media was then replaced with cold serum-free DMEM containing 20 μg/ml of GQDs-MSNs and incubated for another 3 h at 4 °C. Afterward, cells were rinsed with PBS, maintained in phenol red-free medium, and imaged using a laser scanning confocal microscope (CLSM, Olympus FV1000).
The influence of different endocytic inhibitors on the cellular uptake of GQDs-MSNs was also assessed. Briefly, the seeded HeLa cells were incubated separately with (1) methyl-β-cyclodextrin (2,5 mg/ml), as an inhibitor of caveolae/lipid raft-dependent endocytosis; (2) chlorpromazine hydrochloride (5 μg/mL), as an inhibitor of clathrin-mediated endocytosis; and (3) wortmannin (150 ng/mL), as macropinocytosis inhibitor, for 1 h at 37 °C. Subsequently, cells were incubated with 20 μg/ml of GQDs-MSNs nanoparticles nanocomposite for 3 h and imaged as mentioned above. The concentration of different inhibitors were chosen based on performed earlier their cytotoxicity analysis (Fig. S2 in ESM).
The cellular uptake and distribution of GQDs-MSNs nanocomposites in cells was further analyzed by TEM (Jeol JEM-1400) according to Graham and Orenstein (Graham and Orenstein 2007) procedure with minor modification. Briefly, HeLa cells previously incubated with 100 μg/ml of GQDs or GQDs/MSNs nanocomposites for 4 h were fixed with 2.5% glutaraldehyde solution for 2 h and then postfixed in 1% osmium tetroxide for 1 h at room temperature. Then, samples were dehydrated through a graded series of ethanol concentrations (50, 70, 80, 90, 96, and 100%) and embedded in epoxy resin. Ultrathin sections were prepared using ultramicrotome (RMC PowerTome PT-XL), collected onto TEM grids, stained with 1% uranyl acetate, and imaged under Jeol JEM-1400 TEM.
Results and discussion
Preparation and characterization of GQDs and GQDs-MSNs
For the synthesis of GQDs, the bottom-up approach was chosen, as it is reported to yield better quality GQDs concerning their morphology, size distribution, and optical properties (Bacon et al. 2014). After the hydrothermal reaction, a bluish solution with blue emission under UV light was obtained. From the HRTEM images (Fig. S1a,b in ESM), a clear interplanar distance of about 0.35 nm can be found as marked particularly in the Fig. S1b, which corresponds to that of the (002) d-spacing of graphite (Qu et al. 2013) confirming that prepared GQDs have a graphite-like nature. As presented in the HRTEM images, the crystal structure of GQDs is strongly defected; hence, it is difficult to indicate accurate hexagonal ordering typical for graphene. Prepared GQDs are uniform in size (ranging from 1.5 to 7.6 nm), with an average particles diameter of 3.65 nm (Fig. S1c in ESM). AFM images (Fig. S1 e, f in ESM) confirm that uniform GQDs were prepared, as they show a topographic height of nanostructures up to 1.15 nm. Raman spectrum of prepared GQDs reveals prominent D, G, D’, and 2D bands at 1328.6 cm−1, 1575.8 cm−1, 1611.2 cm−1, and 2642.6 cm−1, respectively. G band is a result of in-plane vibrations of sp2 bonded carbon atoms, whereas D and D’ bands are due to the out of plane vibrations attributed to the presence of structural defects. Found ID/IG ratio is around 0.86, which indicates that sp2 bonds of the carbon are disrupted by the presence of oxygen-containing functional groups (originating from the precursor), and therefore prepared GQDs are considered as highly defected materials.
The XPS elemental analysis of prepared GQDs reveals the presence of carbon, oxygen, nitrogen, sulfur, and sodium. The survey and relevant core level C 1s, O 1 , N 1s spectra and S 2s, S 2p region spectra are given in Fig. S2 in ESM. C 1s core level spectrum is composed of three peaks at around 284.5 eV, 286.0 eV, and 288.2 eV that can be assigned to the sp2 C in graphene (C-C, C=C bonds), sp3 C in C-O, C-N bonds and O-C=O, respectively (Qu et al. 2013). The O 1s core level peaks at around 531.3 and 532.6 eV correspond to oxygen in states of C=O, C-O-C/C-OH, respectively (Qu et al. 2013). As shown, C=O-related oxygen states are dominating. The C=O groups are formed by the intramolecular dehydrogenation of carbon precursor, and the other C-O-C/C-OH are remaining surface and edge-related epoxy groups and carboxylic functional groups, resulting from the incomplete dehydrogenation and carbonization during the synthesis. Moreover, N 1s core level spanning from around 396.0 to 403.0 eV is clearly resolved, as well as S 2s and S 2p core levels at around 227.0 and 163.0 eV, respectively (Qu et al. 2013). Elemental XPS analysis reveals that prepared GQDs contain 72.34 at% of C, which is a high content in comparison to other results (Hao et al. 2015), 13.97 at% of O, 4.97 at% of N and 5.39 at% of S, hence confirming N and S-doping of GQDs.
These results confirm that obtained GQDs are rich in carbon, doped with N and S atoms, but still contain number of oxygen-containing functional groups and exhibit disordered and disrupted structure, what is likely for zero-dimensional (0D) nanostructures in comparison to two-dimensional (2-D) graphene nanosheets (Kozak et al. 2016).
Summary of the physicochemical properties: the average particle size (dTEM) and standard geometric deviation (σg), the surface area (SSA) and pore volume (V), zeta potential (ζ) results measured in water-based (H2Od) suspensions; DOX-loading and release efficiency (% DOXloaded, loading capacity in μg DOX/1 mg NPs, and release in %)
SSA (cm2/g)/V (cm3/g)
Zeta potential (mV)/std. dev.
442.33 / 1.06
−30.0 / 0.3
+34.1 / 0.7
−24.2 / 1.2
197.40 / 0.65
+31.1 / 0.8
−6.9 / 1.6
+30.8 / 1.8
Loading capacity (μg DOX/1 mg NPs)
Release at pH 5.0 after 48 h (%)
DOX-loading and release profile
To study the anticancer drug-loading and release efficiency, doxorubicin hydrochloride was non-covalently loaded onto prepared GQDs-MSNs nanocomposite nanoparticles, as well as onto un-modified MSNs as a control drug carrier. The advantage of the adsorption interaction due to the high surface area of mesoporous carrier, as well as electrostatic attraction was exploited for this purpose. The DOX-loading efficiency (% DOXloaded) was estimated spectrophotometrically to be 53.93 (which corresponds to the loading capacity of 269.7 μg/mg) and 78.88% (which corresponds to the loading capacity of 399.4 μg/mg) for GQDs-MSNs and MSNs, respectively. As expected, the drug-loading efficiency was higher in case of control un-modified MSNs, what can be assigned to their high surface area and pore volume, but also to the efficient electrostatic attraction between positively charged DOX molecules and negatively charged MSNs. When considering DOX-loading onto GQDs-MSNs, the drug-loading efficiency was reduced, firstly by their lower specific surface area and pore volume, which were blocked by the GQDs immobilized onto MSNs. Secondly, the drug-loading efficiency through the electrostatic attraction is unfavorable to take place owing to the positive charge of GQDs-MSNs. Results of the zeta potential measurements (see, Table 1) show that due to the DOX-loading the electrophoretic potential of DOX-GQD@MSN suspension does not change significantly and remain positive (+ 30.8 mV). Whereas for DOX-MSNs, the significant zeta potential decrease is observed; however, it remains negative value (− 6.9 mV). These results indicate that water-based suspensions of prepared DOX-GQDs-MSNs exhibit better stability than DOX-MSNs; hence, these nanocomposite nanoparticles can be considered as more preferred carrier for drug delivery application.
In vitro cytotoxicity
The loading of doxorubicin onto MSNs or GQDs-MSNs generally results in a decrease of cells viability in a concentration-dependent manner. In case of HeLa cells, the decrease after incubation with DOX-MSNs proceeds very rapidly from the 25 μg/ml nanoparticles concentration, and more than 80% of cells are indicated as death. Whereas, for DOX-GQDs-MSNs-treated cells a marked decrease in viability is observed at higher concentration of 250 μg/ml. This is in agreement with the presented above DOX-release profile, showing that the amount of released DOX from DOX-GQDs-MSNs is much lower than from DOX-MSNs, therefore justifying the need for higher nanocarrier concentration in this case, but not a higher dose of DOX itself.
In comparison, for human fibroblasts, the viability decrease is milder; however, for DOX-MSNs-treated cells, the 50% inhibition of the cell viability is observed already at the concentration of 10 μg/ml and then decline gradually. The cytotoxicity of DOX-GQDs-MSNs initially seems to be lower, but ultimately at higher concentrations (from 50 μg/ml) reaches higher value than for DOX-MSNs.
The WST-1 results on the cytotoxicity of prepared nanomaterials, reflecting the metabolic activity of cells, were then compared to plasma membrane integrity of cells, indicated by the intracellular esterase activity with the LIVE/DEAD assay (Fig. 5c, d). Here, two dyes were used, which quickly discriminates live from dead cells by simultaneous staining with green-fluorescent calcein-AM, indicating intracellular esterase activity and red-fluorescent ethidium homodimer-1, indicating a loss of plasma membrane integrity. In addition, the fluorescence images acquired with the InCell Apparatus (Fig. 5e) give an overview on the cell viability but also proliferation. In case of nanoparticles without doxorubicin, obtained results are comparable with those from WST-1 assay. The only difference is observed in case of fibroblasts, where at the highest concentration of MSNs and GQDs-MSNs the viability does not exceed 70%. Moreover, evaluation of DOX-MSNs and DOX-GQDs-MSNs cytotoxicity based on the cell membrane integrity analysis shows that these drug carriers are not as cells deleterious as it was presented above with the metabolic activity analysis. It is presented (particularly for fibroblasts), that even at the highest applied doses of 250 μg/ml, the viability was maintained at 80 and 60% for DOX-MSNs and DOX-GQDs-MSNs, respectively. However, as presented in Fig. 5e, the treatment with DOX-loaded nanoparticles has a strong effect on the cell proliferation, which is particularly the case of DOX-MSNs, in comparison to control non-treated cells. Therefore, found with these two assays differences in the cytotoxicity of prepared nanoparticles, arise from the fact that different aspects of cell functions were analyzed, cell viability and cell vitality, by live/dead assay and WST-1, respectively (Kwolek-Mirek and Zadrag-Tecza 2014). However, both are required for the estimation of the physiological state of a cell after exposure to various types of stressors, including nanoparticles. Whereas, the fluorescent staining distinguishes live from dead cells, the WST-1 assay gives the information about their ability to reproduction. Hence, the lower number of living cells upon DOX-MSNs and DOX-GQDs-MSNs indicates the action of successfully delivered and released doxorubicin. Further, both assays confirmed that conjugation of biocompatible GQDs with nontoxic MSNs, leads to the creation of low-cytotoxic GQDs-MSNs nanocomposite nanoparticles, making them particularly good candidates for drug-delivery system.
Intracellular localization and uptake mechanism of GQDs-MSNs
In order to evaluate the exact uptake mechanism of GQDs-MSNs, the inhibition of selected routes were performed. Results given in Fig. S5 (ESM) indicate that by lowering the temperature to 4 °C, the energy-dependent mechanisms is blocked. This is observed by lacking signal in cells, is comparison to control HeLa cells treated with 20 μg/ml GQDs-MSNs for 3 h at 37 °C, where the signal in cells is clear. Therefore, the endocytosis is considered as an exact uptake mechanism of studied nanoparticles. However, by using suitable inhibitors, different types of endocytosis were further investigated. Chlorpromazine is reported to specifically inhibit clathrin-dependent endocytosis (CDE), while methyl-β-cyclodextrin has been extensively used to inhibit clathrin-independent endocytosis (CIE), especially in caveolae/lipid raft-mediated endocytosis (Le et al. 2002; Parton and Richards 2003, dos Santos et al. 2011). Wortmannin, in turn, as covalent inhibitor of phosphoinositide 3-kinases (PI3Ks), plays a role in inhibition of micropinocytosis (Araki et al. 1996; Rupper et al. 2001). As can be seen in Fig. S5, chlorpromazine showed no inhibitory capacity in HeLa cells on studied nanoparticles uptake. Contrary to chlorpromazine, the MβCD inhibited the uptake of GQDs-MSNs into cells completely, suggesting the CIE as a main route of these nanoparticles internalization. Similar results were presented by Ekkapongpisit et al. (Ekkapongpisit et al. 2012), contrary to Hao et al. (Hao et al. 2012), who indicated that spherical mesoporous silica nanoparticles are internalized rather via the clathrin-mediated pathway. Interestingly, in our study, the wortmannin inhibits nanoparticles internalization partially, indicating that maybe larger nanoparticles or nanoparticles aggregates are internalized by micropinocytosis (Meng et al. 2011; Vollrath et al. 2013).
Intracellular distribution of doxorubicin
In order to investigate the colocalization of the GQDs-MSNs nanocomposite nanoparticles with DOX, the analysis was performed using the Coloc2 tool and the Pearson’s correlation coefficients were found. These coefficients are 0.77 and 0.44 for cells incubated with the 20 μg/ml of DOX-GQDs-MSNs for 3 and 24 h, respectively. In case of the cell exposure to 200 μg/ml of DOX-GQDs-MSNs for 3 and 24 h, found coefficients were 0.39 and 0.37, respectively. These results indicate that colocalization occurs and is more significant in case of shorter time of incubation, whereas longer time of incubation results in the decreased colocalization coefficient. This confirms the time-dependent release of DOX from the GQDs-MSNs nanocomposite nanoparticles.
Summarizing, DOX-GQDs-MSNs nanocomposite nanoparticles are found to be efficient in DOX delivery via their internalization in HeLa cancer cells but also allow for simultaneous real-time optical tracking of the DOX during its delivery and release.
In this study, we reported on the successful preparation of GQDs-MSNs nanocomposite nanoparticles as an efficient intracellular drug delivery system, but also simultaneously, as fluorescent agent for the optical imaging. Prepared GQDs-MSNs with an average particle size is below 50 nm and high-positive zeta potential form stable suspensions exhibiting excitation-dependent PL behavior. Moreover, they can be easily loaded with doxorubicin chosen as a model drug and show the pH- and temperature-dependent doxorubicin release behavior, due to weakening of the interaction between DOX molecules and GQDs-MSNs. The cytotoxicity assays confirmed that the conjugation of biocompatible GQDs with nontoxic MSNs, leads to the creation of GQDs-MSNs nanocomposite nanoparticles with negligible cytotoxicity, which may serve as a potential drug nanocarriers. Moreover, these assays also confirmed the therapeutic action of delivered and released doxorubicin. Further, the in vitro optical imaging efficacy of cells with GQDs-MSNs, resultant of their cellular internalization via caveloae/lipid raft-mediated endocytosis in HeLa cells, was proven with CLSM and confirmed with TEM imaging. The GQDs entrapment onto mesoporous silica nanoparticles structure resulted in the increasing fluorescent signal in comparison to bare GQDs, which could be related with the immobilization and aggregation effect, but also with their surface charge-related more effective intracellular penetration. Moreover, proposed GQDs-MSNs drug delivery systems enabled the simultaneous real-time optical tracking of the drug during its delivery and release, but also the monitoring of the penetration of the nanoparticles itself. These results indicate therefore that the GQDs-MSNs possess both strong enough fluorescence emission and internalization ability, what makes them a promising material for bioimaging and biolabeling.
Authors would like to thank Dr. Marcin Jarek and Dr. Mateusz Kempiński (Faculty of Physics, Adam Mickiewicz University in Poznań) for support during the XRD measurements and running XPS measurements, respectively.
This work was supported by the National Science Center, Poland (NCN) under research grant Miniatura 2017/01/X/ST5/00134. Partial support by the National Science Centre grant SONATA-BIS 6: 2016/22/E/ST3/00458 is also acknowledged. Teofil Jesionowski during the research was supported by the PUT research grant no. 03/32/DSPB/0806/2018.
Compliance with ethical standards
Conflict of interest
The authors declare that they have no conflict of interest.
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