Target Delivery and Cell Imaging Using Hyaluronic Acid

Article pubs.acs.org/molecularpharmaceutics

Target Delivery and Cell Imaging Using Hyaluronic AcidFunctionalized Graphene Quantum Dots Abdullah-Al-Nahain,† Jung-Eun Lee,‡ Insik In,§ Haeshin Lee,∥ Kang Dae Lee,⊥ Ji Hoon Jeong,*,‡ and Sung Young Park*,@ †

Department of Green Bio Engineering, Korea National University of Transportation, Chungju 380-702, Republic of Korea School of Pharmacy, Sungkyunkwan University, 300 Cheoncheon-dong, Jangan-gu, Suwon, Gyeonggi-do 440-746, Republic of Korea § Department of Polymer Science and Engineering, Korea National University of Transportation, Chungju 380-702, Republic of Korea ∥ Department of Chemistry, KAIST, Daejeon 305-701, Republic of Korea ⊥ Department of Otolaryngology-Head and Neck Surgery, College of Medicine, Kosin University, Busan, Republic of Korea @ Department of Chemical and Biological Engineering, Korea National University of Transportation, Chungju 380-702, Republic of Korea ‡

S Supporting Information *

ABSTRACT: This work demonstrates the way to achieve efficient and target specific delivery of a graphene quantum dot (GQD) using hyaluronic acid (HA) (GQD-HA) as a targeting agent. HA has been anchored to a GQD that accepts the fascinating adhesive properties of the catechol moiety, dopamine hydrochloride, conjugated to HA, which was confirmed by X-ray photoelectron spectroscopy. Transmission electron microscopy revealed a particle size of ∼20 nm, and the fluorescence spectra revealed significant fluorescence intensity even after the anchoring of HA. The prepared GQD-HA was applied to CD44 receptor overexpressed tumor-bearing balb/c female mice, and the in vivo biodistribution investigation demonstrated more bright fluorescence from the tumor tissue. In vitro cellular imaging, via a confocal laser scanning microscope, exhibited strong fluorescence from CD44 overexpressed A549 cells. Both in vivo and in vitro results showed the effectiveness of using HA as targeting molecule. The loading and release kinetics of the hydrophobic drug doxorubicin from a GQD under mildly acidic conditions showed that a GQD can be considered as a novel drug carrier, while the nontoxic behavior from the MTT assay strongly supports the identification of GQD-HA as a biocompatible material. KEYWORDS: graphene quantum dots, hyaluronic acid, cell imaging, target delivery, in vivo

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Compared to the reported semiconductors, nanosized fluorescent particles made from carbon are highly beneficial because of biocompatible behavior under physiological conditions, which is greatly desirable for biological relevance.17−20 As a corollary, carbon dots, carbon nanocrystals, etc., represent excellent examples. The graphene quantum dot (GQD) reported by Juan et al. is an excellent one that belongs to QDs prepared from carbon as the source.21 Recent studies report that the GQD is less toxic and exhibits stable strong fluorescence along with electrical and thermal conductivity.17,19 To be a potential fluorescent probe for diagnosis, it is necessary for the nanoparticles to reach the final destination. The presence of different receptors on the membrane of cancer cells has offered an opportunity to search for the potential route

INTRODUCTION At present, a massive amount of research has been devoted to the development of various fluorescent materials to study the monitoring of living cells in biology and medical diagnostics.1As imaging agents in biological fields, fluorescent dyes have been widely reported in cases where traditional organic dyes played a vital role a few years ago, but the photoinstability, low quantum yield, and high cost of these dyes create opportunities for finding better materials to replace them.2,3 Interestingly, semiconductor “quantum dots” (QDs) made from nanoscale aggregates of elements such as cadmium and selenium are widely reported as excellent cell structure illuminators because of their bright and stable fluorescence.4−6 In most cases, to achieve stable and better molecular imaging, a coating of QDs is applied by using various ligands or organic compounds,7,8 but interaction between the coating agent and QDs may lead to photoinstability.9,10 On the other hand, their inherent toxicity because of the release of cadmium, even at lower concentrations, restricts many possible biological applications.11−16 © XXXX American Chemical Society

Received: April 15, 2013 Revised: August 21, 2013 Accepted: September 5, 2013

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dx.doi.org/10.1021/mp400219u | Mol. Pharmaceutics XXXX, XXX, XXX−XXX

Molecular Pharmaceutics

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for the delivery of imaging agents.22 Installation of a receptor binding molecule on the GQD thus can enhance the efficiency of the cells. A large number of receptor binding molecules are widely reported, such as antibodies, antibody fragments, peptides, and DNA−RNA aptamers, which have been investigated well. Among them, hyaluronic acid (HA) is extensively used as a major ligand because of its biocompatible, biodegradable, and nontoxic behavior, and this can easily bind to the CD44 (hyaluronan) receptor with high affinity.23−26After binding to the target site, the target molecule must be stable enough until it exhibits its desired objective. Various strategies have been followed to apply HA as target molecules, but in many cases, the procedures are complex and time-consuming. To overcome these limitations, the adhesive behavior of catechol can be a novel way to introduce target molecules onto different materials.27−29 Catechol, the side chain of an unusual amino acid of 3,4-dihydroxy-L-phenylalanine (L-DOPA or DN), which is found at high levels in adhesion materials of marine mussels, is unique as an adhesive for surface modification and biomedical devices. Under alkaline conditions, the quinone form of catechol is formed through self-polymerization with the accompanied oxidation of catechol groups and able to show adherent properties on a wide range of substrates.30,31 In this work, we anchored HA to a GQD as target molecule to deliver them efficiently. To attach HA, DN was conjugated to the HA backbone because of its adhesive nature. GQD-HA was characterized by dynamic light scattering (DLS), fluorescence, and X-ray photoelectron spectroscopy (XPS) investigation. The successful delivery of GQD-HA was evaluated using balb/c female mice as the in vivo model and A549 cells in vitro. To determine if the GQD is an imaging agent, we also examined it as an anticancer drug carrier using doxorubicin (DOX) as a model drug. An in vitro cytotoxicity assay was conducted to evaluate the toxicity of GQD-HA.

synthesized by following a previous report with a slight modification.32 Degrees of substitution (DS) were determined using 1H nuclear magnetic resonance spectroscopy (Bruker AVANCE 400). The degree of dopamine substitution for HADN was 1.65%.32 Preparation of GQD-HA. To prepare GQD-HA, 100 mg of GQD was dispersed in 10 mL of TBS in a glass vial. In another vial, 100 mg of HA-DN was dissolved in 10 mL of TBS. The HA-DN solution was then mixed with the GQD solution and allowed to stir for 24 h in the dark at 60 °C. Finally, the solution was purified following dialysis (molecular weight cutoff of 3500) and freeze-dried to yield the GQD-HA powder. Characterization. The prepared samples were characterized using dynamic light scattering (Zetasizer Nano, Malvern), UV−visible spectroscopy (Optizen 2120 UV, Mecasys Co. Ltd.), transmission electron microscopy, XPS using Omicrometer ESCALAB (Omicrometer, Taunusstein, Germany), and fluorescence spectroscopy (FluoroMate, Scinco Co. Ltd.). The fluorescence investigation of GQD and GQD-HA was conducted via excitation at 318 nm, where the concentration of GQD and GQD-HA was 0.5 mg/mL in phosphate-buffered saline (PBS) at pH 7.4 (scan speed of 600 nm/min, integration time of 100 ms, response time of 8.0 s, and voltage of 800 V). Besides this, the absorbance via the UV−vis spectrophotometer was also collected using 0.5 mg/mL GQD and GQD-HA in PBS at pH 7.4. The quantum yield (QY) of GQD was measured in reference to anthracene (QY of 27% with excitation at 340 nm).33,34 Different concentrations of anthracene in ethanol were prepared. The absorbance of anthracene was determined at the excitation wavelength. Following a similar procedure, the GQD and GQD-HA solutions were prepared in water and the absorbance was measured at 280 nm, yielding an absorbance between 0.01 and 0.1 nm. Fluorescence spectra of all solutions were recorded with a Scinco fluorometer at an excitation of 318 nm within the emission range from 350 to 650 nm. The integrated fluorescence intensity is the area under the fluorescence curve. The quantum yield was calculated following the established equation.35,36 Loading of DOX onto GQD-HA. Doxorubicin loading was conducted using a different volume of DOX (1 mg/mL dissolved in DMSO) at a fixed concentration of GQD-HA in PBS at pH 7.4 while the mixture was vigorously stirred for 24 h in the dark. Unbound DOX and DMSO were removed by dialysis for 12 h against PBS, during which the external medium was renewed two times during dialysis. The DOX loading efficiency (DLE) was determined by UV absorbance. The absorbance at 480 nm was detected with a UV spectrophotometer (Optizen 2120UV, Mecasys Co. Ltd.). Various concentrations of DOX were prepared, and the absorbance at 480 nm was measured to obtain the calibration curve to calculate the DLE. DOX Release Profile. The release behavior of DOX from GQD-HA was evaluated at pH 7.4 in PBS (10 mmol) and pH 5.0 in acetate-buffered saline (10 mmol) in dialysis chambers. The chambers were immersed in 30 mL of release medium at pH 7.4 and 5.0, respectively. Finally, the samples were placed in a shake incubator at 37 °C for the whole experimental period. To determine the amount of DOX released, a 3 mL solution from the outside released medium was taken, and via measurement of the absorbance using UV spectroscopy, the amount of drug released was calculated.

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EXPERIMENTAL SECTION Materials. Hyaluronic acid (230 kDa) was purchased from LifeCore. Dopamine hydrochloride (DN), 1-ethyl-3-[3(dimethylamino)propyl]carbodiimide hydrochloride (EDC), doxorubicin hydrochloride, and 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide (MTT) were purchased from Sigma-Aldrich. N-Hydroxysuccinimide (NHS) was purchased from Merck. Concentrated H2SO4, nitric acid, and sodium bicarbonate were purchased from Sigma Aldrich Korea. Pitchbased carbon fiber was purchased from Cytech. Penicillinstreptomycin, fetal bovine serum (FBS), a 0.25% (w/v) trypsin/0.03% (w/v) EDTA solution, and RPMI-1640 medium were purchased from Gibco BRL (Carlsbad, CA). Preparation of Graphene Quantum Dots (GQDs). Graphene quantum dots were prepared following the same procedure reported by Juan et al.21 Briefly, 300 mg of carbon fiber was treated in 60 mL of concentrated H2SO4 and 20 mL of concentrated HNO3. The mixture was sonicated for 2 h, and then the reaction was allowed to proceed for 24 h while the mixture was being vigorously stirred at 120 °C. After the reaction had been completed, the mixture was poured into 800 mL of water and the pH of the solution was adjusted to 8.0 via addition of sodium bicarbonate. Dialysis was performed for 3 days using the dialysis membrane (molecular weight cutoff of 3500), and after the sample had been freeze-dried, the dried powder form of GQDs was obtained. Synthesis of Dopamine-Conjugated Hyaluronic acid (HA-catechol). Dopamine-conjugated HA (HA-DN) was B



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In Vitro Cytotoxicity. The in vitro cytotoxicity investigation was conducted following our previous report.37,38 MDCK as CD44 negative and A549 as CD44 overexpressed cell lines were chosen. Both cell lines were cultured in RPMI supplemented with 10% fetal bovine serum (FBS), 100 units/ L penicillin, and 100 μg/mL streptomycin. The cells were then incubated for 2 days in a humidified 5% CO2-containing balanced air incubator at 37 °C. The medium was changed three times during the incubation period. The cytotoxicity of these cells was measured using the 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide (MTT) assay method; 200 μL of cells, at a density of 2 × 104 cells/well, were placed in each well of a 96-well plate. Then the cells were incubated for 24 h at 37 °C in a humidified 5% CO2-containing atmosphere. After that, the medium was removed and the cells were incubated with different concentrations of GQD and GQD-HA. To evaluate the cell killing efficiency of the loaded DOX, cells were also treated with different concentrations of DOX and DOX-loaded GQD-HA at which an equivalent concentration of DOX was present. As a blank, there were only the cells. The cells were then incubated like before for an additional 24 h. Then the medium containing the drug was removed, and a total of 20 μL of a stock solution containing 15 mg of MTT in 3 mL of PBS was added and incubated for an additional 4 h. Finally, 180 μL of MTT solubilizing agents was added to the cell, which was then properly shaken for 15 min. The absorbance was measured at a wavelength of 570 nm. The relative cell viability was measured by comparing the control well containing only the cell. In Vitro Imaging and Evaluation of Cellular Uptake. By using the same type of cells, an in vitro imaging investigation was conducted; 2 × 105 cells/well, for each cell type, were placed in an eight-well plate. All cells were treated with same concentration (0.1 mg/mL) of GQD and GQD-HA. After the cells had been treated with the sample, the cells were incubated for 4 h at 37 °C in a humidified 5% CO2-containing atmosphere. Finally, the cells were washed several times with PBS, and fresh culture medium was added. The cells were imaged by using an LSM510 confocal laser scanning microscope (Carl Zeiss) with an argon blue laser light at 488 nm with a 505 nm emission filter at a magnification of 20×. Evaluation of cellular uptake was performed following a similar method reported by Chunsoo et al.39 To quantify the cellular uptake of GQD and GQD-HA, both MDCK and A549 cells (2 × 105 cells/well) were selected and seeded in a 12-well plate. After incubation for 24 h, the medium was removed and cells were treated with 0.1 mg/mL GQD and GQD-HA. Cells were then allowed to incubate for 4 h. After that, the samplecontaining medium was removed and cells were washed several times with PBS. Using 1% Triton X-100 (Sigma-Aldrich), cells were lysed. The relative amounts of accumulated GQD and GQD-HA within the cell interior were determined by measuring the fluorescence intensity at an excitation wavelength of 318 nm using a microplate reader fluorescence spectrophotometer (Varioskan Flash, Thermo). In Vivo Bioimaging. Balb/c mice were properly raised and tended, and when they reached 6 weeks of age, they were considered for experimental investigation. To evaluate the targeted delivery of GQD-HA, tumors were grown on the back of the mice using A549 cells. After 10 days, when the tumor volume reached ∼100 mm3, both samples were intravenously injected. To investigate the efficient delivery of GQD to tumor tissue and its biodistribution, GQD and GQD-HA were

injected intravenously at concentrations of 5 and 10 mg/kg of body weight, respectively, through the tail vein (by considering the same intensity, two different doses have been chosen). After 2 h, the mice were sacrificed and their organs dissected. As a control, only PBS was injected. Fluorescence images were captured using an OPTIX MX3 imaging system (ART Inc., Montreal, QC). Statistical Analysis. Statistical analysis of the accumulation of GQD and GQD-HA was conducted with a paired t test with a two-tailed distribution, and differences were considered statistically significant at p < 0.05. The data are expressed as means ± the standard deviation (n = 3).

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RESULTS AND DISCUSSION GQD has attracted greater interest in the biomedical field because of to its bioimaging behavior. Though there are so many routes to synthesize GQD easily, it is difficult to achieve better efficacy when it is applied in biological fields, especially in cellular imaging because of the possibility of it being taken up by normal cells along with cancer cells. To demonstrate the efficient delivery of GQD following receptor-mediated endocytosis to tumor tissue, HA has been anchored by accepting the adhesive properties of catechol under mildly alkali conditions.40 Installation of HA can aid in increasing the bright fluorescence from in vivo and in vitro investigations because HA will act as a targeting molecule to allow more accumulation of GQD in the cancer cells. The preparation of GQD-HA and the mechanism of entrance into the cancer cell accompanied by the DOX release behavior have been illustrated in Scheme 1. Fluorescence spectra showed that the prepared GQD shows its highest fluorescence within the visible region, with a maximum at 440 nm.21 After reduction of GQD by HA-DN, fluorescence was also observed without a change in the peak position. The quantum yield of GQD was 2.86% considering anthracene as a standard, whereas it was 1.32% for GQDHA.35,36 It is seen that after reduction with HA-DN, the fluorescence intensity was reduced at the same concentration. This is due to the presence of fewer GQDs in GQD-HA at the same concentration. The size of the as-prepared GQD was 5− 12 nm, as determined by DLS (Figure S1 of the Supporting Information). After the sample had been anchored with HADN, the size was within the range of 35−55 nm (Figure 1b). As a nanoparticle intended to be a biological fluorescent probe and drug carrier, the increase in particle size is strong enough to use. The TEM image shows the size of GQD-HA around 20 nm (Figure 1c). Figure 1d shows a digital photograph of the prepared GQD and GQD-HA under UV excitation at 365 nm, where strong blue luminescence is observed in both cases. UV− vis spectroscopy shows the absorbance peak at 280 nm for GQD (Figure S2 of the Supporting Information).21 As we have introduced HA as a targeting agent onto GQD, an XPS investigation was conducted to ensure the anchoring of HA-DN on GQD. The narrow-scan XPS C1s core-level spectrum of GQD can be curve-fit into three peaks components with binding energy at 284.6, 286.5, and 288.2 eV that can be attributed to C−C, C−O, and CO species, respectively (Figure 2a). For GQD-HA, the increase in intensity at 286.5 eV has been marked; this peak is assigned to C−O indicating the anchoring of HA-DN to GQD.32,41−43 Furthermore, a highresolution XPS investigation also confirmed the conjugation of dopamine to HA by showing the N1S peak at ∼399.8 eV C



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Scheme 1. Target Delivery of GQDs Using HA and Subsequent Release of the Drug from the Surfaces of GQD in a Tumor Cell Environment

originating from the amide bond and that at 402.0 eV for the -NH2 group of HA.44,45 Before examining the in vivo targeting capability of GQD and the effectiveness of using HA as a target molecule to deliver GQD, we studied the selective uptake of GQD-HA following the CLSM investigation. All investigated cells were incubated with both GQD and GQD-HA for 4 h. Figure 3 shows the fluorescence behavior of GQD and GQD-HA from MDCK and A549 cells. Inadequate fluorescence intensity has been observed from MDCK cells incubated with GQD and GQD-HA (Figure 3a). Figure 3b provides the evidence of bright fluorescence from A549 cells for GQD-HA compared to GQD. Accumulation of GQD and GQD-HA was further evaluated in terms of fluorescence intensity.39 Figure 3c also shows a negligible amount of fluorescence intensity for GQD and GQD-HA from MDCK cells (0.3 ± 1.41% and 1.52 ± 5.14%, respectively) where considerable fluorescence (11.05 ± 7.54%) was assigned to A549 cells when cells were incubated with GQD-HA (Figure 3d). Using findings from the confocal and cellular uptake investigation, it is clearly indicated that a larger amount of GQD-HA was taken up by A549 cancer cells. For only GQD was there no targeting agent; small amounts of GQD were allowed to enter into both types of cells, wherein due to the presence of HA more GQD-HA entered the cell cytoplasm via receptor-mediated endocytosis-based target delivery.46,47 The enhanced permeability and retention effect (EPR) might also play a vital role in the accumulation of more GQD-HA in cancer cells.48,49

Figure 1. (a) Fluorescence intensity of GQD and GQD-HA with excitation at 318 nm. (b) Particle size of GQD-HA determined by DLS. (c) TEM image of GQD-HA. (d) Digital photographs of GQD and GQD-HA under a UV lamp (excitation at 365 nm).

The results of in vitro imaging encouraged us to verify the in vivo biodistribution and tumor specific delivery of GQD-HA. GQD (5 mg/kg of body weight) and GQD-HA (10 mg/kg of D



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Figure 2. C1s XPS spectra of (a) GQD and (b) GQD-HA. (c) Narrow scan N1s spectra of GQD-HA.

Figure 3. (a and b) CLSM images of GQD and GQD-HA in (a) MDCK and (b) A549 cells after incubation for 4 h. The left column shows the bright field images, the middle column the gray images, and the right column the merged images. The scale bar is 50 μm. (c and d) Quantitative analysis of cellular accumulation of (c) GQD and (d) GQD-HA in MDCK and A549 cells after incubation for 4 h.

HA compared to GQD in Figure 4a. The strong fluorescence from the tumor indicates the targeted accumulation of GQDHA to the tumor site. Panels b and c of Figure 4 provide the qualitative and quantitative biodistribution profiles of GQD and GQD-HA, respectively, from the dissected organs (liver,

body weight) were administered following tail vein injection of A549 cell tumor-bearing balb/c female mice. Considering the similar fluorescence intensity, we have used different concentrations of GQD and GQD-HA. After 2 h, considerable fluorescence signals were detected from the tumor for GQDE



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molecules onto the GQD-HA surface. A fluorescence spectroscopy investigation was further conducted to determine the fluorescence behavior of DOX. Figure S3b of the Supporting Information shows the strong quenching behavior of DOX, confirming the successful loading of doxorubicin onto the surfaces of GQD. Loading of DOX was quantitatively assayed by UV spectroscopy by measuring the absorbance at 480 nm. The result indicated ∼75% DOX was loaded onto the surfaces of GQD. Figure 5 shows the release of DOX from the surfaces of GQD-HA at different pH values. From Figure 5, one can clearly

Figure 5. Release of DOX from DOX-loaded GQD-HA.

see that a burst release of 42% was found within 6 h, which was increased to almost 60% within 12 h under mildly acidic accommodation (pH 5.0).53 It is also found that almost all drugs were released within 48 h from the GQD-HA matrix. This release behavior of DOX is advantageous with regard to the tumor as the cancerous tissues have a slightly acidic environment. When the DOX-loaded GQD-HA is internalized into the cancer cell, the acidic environment of the cancer cell induces DOX molecules to be released from the surface of the carrier. This is due to the increased hydrophilicity of doxorubicin at lower pH values via protonation of the NH2 group of DOX, and this subsequently weakens the hydrophobic interaction between the GQD surface and DOX.54,55 Next, an in vitro toxicity experiment was conducted to analyze the toxic behavior of GQD-HA. Concentrationdependent toxicity of GQD against all investigated cells was found. At lower concentrations, GQD shows a considerable amount of cell viability against both types of MDCK and A549 cells, but at higher concentrations, a significant toxicity was traced (Figure 6a,b). Interestingly, the toxicity was reduced when HA-DN was anchored onto the surface of GQD. This is due to the biocompatible behavior of HA.23−26 Finally, we examined the cytotoxic behavior of DOX released from GQDHA, where an equal amount of DOX was applied for the sake of comparison. A significant amount of cytotoxicity was found depending on the DOX concentration (Figure 6c). It is found that free DOX shows more toxicity than loaded DOX. The reason may be that free DOX directly entered the cell following diffusion by spending a short period of time while DOX-loaded GQD-HA followed endocytosis, and to release from the surface of GQD-HA, a comparatively long time was taken.

Figure 4. (a) In vivo fluorescence images of GQD-HA in mice after tail vein injection. (b) Ex vivo images of liver, kidney, spleen, heart, and tumor after dissection. (c) Normalized intensity from dissected organs.

kidney, spleen, and lung) and tumor tissue. The fluorescence behavior of the observed organs indicates that GQD-HA significantly accumulated in the tumor tissue and also in liver and kidney. It is reported that there are a large number of leaky blood vessels surrounding the tumor tissue.47 As a result, GQDHA particles preferentially accumulated in the tumor tissue where the CD44 receptors attracted the HA of GQD to bind more frequently. It is seen that GQD has accumulated in liver and kidney to a greater degree than GQD-HA. Accumulation of GQD in the highly perfused liver may be the reason for the combined effect of circulating blood passing through liver and uptake of nanosize GQD by the reticuloendothelial system.49,50 Besides this, the fluorescence intensity from kidneys indicates rapid excretion of GQD over GQD-HA. Though GQD can be applied as an imaging agent, we anticipated that the surface of prepared GQD (like that of graphene oxide) can be an excellent raised area for gathering hydrophobic drug molecules onto it via π−π interaction.51,52 Figure S3a of the Supporting Information shows the absorbance peak at 480 nm indicating the loading of DOX F



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delivery systems, while the nontoxic properties of GQD-HA assure the biocompatible behavior. All the admirable findings confirm the accomplishment of delivering GQD as an in vivo fluorescent probe and in vitro drug transporter.

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ASSOCIATED CONTENT

S Supporting Information *

UV−vis and fluorescence data. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. Telephone: +82-31-290-7783. Fax: +82-31-292-8800. *E-mail: [email protected]. Telephone: +82-(0)43-841-5225. Fax: +82-(0)43-841-5220. Author Contributions

A.A.-N. and J.-E.L. contributed equally to this work. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by Grant N00140008 from the industrial infrastructure program for fundamental technologies funded by the Ministry of Trade, Industry & Energy, Grant R0001435 from the MOTIE, and the Fusion Research R&D Program of the Korea Research Council for Industrial Science & Technology (2012, 2013).

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REFERENCES

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Figure 6. In vitro cytotoxicity of GQD and GQD-HA following the MTT assay: (a) MDCK cells, (b) A549 cells, and (c) DOX-loaded GQD-HA in A549 cells.

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CONCLUSION In conclusion, we have successfully linked the target molecule to GQD using a convenient method picking up the advantages of the adhesive manner of DOPA. The bright fluorescence from the tumor-bearing investigated mice argues that GQD was advent to the final destination, while the strong fluorescence from in vitro imaging of CD44-overexpressed A549 cells powerfully supports the success of the in vivo investigation. The release of hydrophobic DOX from GQD surfaces acknowledges that GQD can be applied as a strong drug transporter in drug G



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Target Delivery and Cell Imaging Using Hyaluronic Acid

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