- Open Access
Folic acid modified gelatine coated quantum dots as potential reagents for in vitro cancer diagnostics
© Gérard et al; licensee BioMed Central Ltd. 2011
Received: 9 August 2011
Accepted: 10 November 2011
Published: 10 November 2011
Gelatine coating was previously shown to effectively reduce the cytotoxicity of CdTe Quantum Dots (QDs) which was a first step towards utilising them for biomedical applications. To be useful they also need to be target-specific which can be achieved by conjugating them with Folic Acid (FA).
The modification of QDs with FA via an original "one-pot" synthetic route was proved successful by a range of characterisation techniques including UV-visible absorption spectroscopy, Photoluminescence (PL) emission spectroscopy, fluorescence life-time measurements, Transmission Electron Microscopy (TEM) and Dynamic Light Scattering (DLS). The resulting nanocomposites were tested in Caco-2 cell cultures which over-express FA receptors. The presence of FA on the surface of QDs significantly improved the uptake by targeted cells.
The modification with folic acid enabled to achieve a significant cellular uptake and cytotoxicity towards a selected cancer cell lines (Caco-2) of gelatine-coated TGA-CdTe quantum dots, which demonstrated good potential for in vitro cancer diagnostics.
Nanoparticles and especially quantum dots (QDs) have attracted much interest in recent years as potential diagnostics and drug delivery tools [1–3]. Thiol-stabilised CdTe semiconducting nanoparticles or quantum dots (QDs) present the particular advantage of being water-soluble and easy to functionalise [4, 5]. In addition it has been shown that protective coatings such as gelatine may provide substantial improvement of their luminescence efficiency and biocompatibility [6, 7]. They are therefore attractive for fluorescent bio-labelling, provided that they can be made specific to a target type of cell. In the present work, we have combined the improved biocompatibility provided by a gelatine coating with an increased uptake from cancerous cells over-expressing folic acid receptors. While the conjugation of folic acid (FA) to various nanoparticle types via a polymer spacer has been widely reported [8–13], here we describe a new, rapid, one-pot synthesis of folic acid-conjugated gelatine-coated TGA-capped CdTe QDs. The uptake of the resulting particles by cancer cells was assessed in Caco-2 cells which naturally over-express folate receptors (FR).
For clarity purposes, gelatine-coated TGA-capped CdTe will be referred to as QD(A), gelatine-coated TGA-capped CdTe QDs with incorporated FA as QD(B) and gelatine-coated TGA-capped CdTe to which FA was conjugated via 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide ( EDC) coupling as QD(C).
Results and Discussion
Synthesis and characterisation of folic acid-conjugated gelatine-coated CdTe QDs
Luminescence lifetime decay measurements.
Size of QDs as measured by TEM and DLS, and their zeta potential.
Hydrodynamic diameter (DLS by number) (nm)
Polydispersity index (PDI)
Zeta potential (mV)
Standard deviation of Zeta potential
4.2 (+/- 0.7)
4.8 (+/- 0.8)
4.9 (+/- 0.9)
QD (C) was prepared by treating QD (A) with EDC in order to covalently bound FA to gelatine. One side effect of the treatment is the cross-linking of gelatine through intra- and inter-molecular reactions of carboxylic groups with amino groups of the protein [15, 16]. This lead to reduced swellability of gelatine and hence a smaller hydrodynamic radius as confirmed by the present results, as well as to less carboxylic groups available on the surface. This explains why the surface charge was rather low despite the presence of FA molecules.
Biological testing of nanocomposites
Caco-2 cells were previously reported to not efficiently take up a variety of nanoparticles; however, since they are known to over-express folate receptors, the folic acid molecules present on the surface of particles were expected to significantly increase the uptake by these cells.
To confirm that the increased upatke was related to FA, a competition assay was performed with free FA. In the case of QD (C) internalisation was reduced by free FA to the same level as QD (A) alone. As expected the free FA molecules could block the cellular receptors and QD (C) could only be internalised by unspecific endocytosis. On the other hand, the uptake of QD (A) was raised by the presence of free FA almost to the level of QD (C) alone. In this case, free FA could bind to gelatine thus dragging the particles into the cells. The uptake of QD (B) was not significantly altered by free FA because the surface was probably already saturated in randomly orientated FA molecules. Overall it could be reasonably concluded that the increase in uptake was directly linked to the presence of FA on the surface of the particles. QD (B) also proved to be of very little interest for biological applications.
Cytotoxicity of FA modified QDs towards Caco-2 cells.
Thiol-stabilised aqueous CdTe QDs have been reported to be generally more toxic than ones produced through the organic route due to their lack of protective shell. Adding a layer of gelatine however was found to reduce their cytotoxicity  which is believed to arise mainly from the release of cadmium ions. Another critical aspect in QD toxicity is the size of the particles. In our study we used large, red-emitting QDs which have been reported to be less toxic than smaller ones, mostly because they are not able to penetrate as deep in the cell. The cytotoxicity of our QDs appeared to be related to their uptake rate to a certain extent. FA-modified QDs however tend to be more cytotoxic than bare gelatinated QDs, which may be explained by their blocking of the FA receptors thus depriving the cells from this essential nutrient. This make them potential candidates for targeted cancer therapy, but more in-depth biological studies would be required in order to guarantee good enough specificity.
In conclusion, all characterisation analyses that were carried out (UV-visible absorption spectroscopy, PL, DLS, zeta potential, fluorescence lifetime decay) pointed towards the effective modification of the gelatine-TGA CdTe QD surface with FA, using our approach. The most definite proof remains the competitive uptake of FA and QDs which demonstrated that variations were linked to the presence or absence of FA on the surface of particles. To some extent, the molecule can be incorporated to the gelatine shell; however the availability of FA for recognition was only obtained by covalent conjugation. We have thus developed a new potential assay for in vitro cancer diagnostic by identifying cells which highly express FR as it is the case for most carcinoma cells. This is also a proof of concept for a new facile, efficient, one-pot synthesis of functionalised QDs which could be used to create combined diagnostics and therapeutic tools.
Al2Te3 was purchased from Cerac Inc. All other chemicals for synthesis were purchased from Sigma-Aldrich. All synthetic procedures and sample preparation were performed using degassed Millipore water. Caco-2 cells were purchased from the European Cell Culture Collection (ECCC).
Synthesis of QD (A), (B) and (C)
QD (A), (B) and (C) were synthesised using a modification of the procedure previously reported by our group. Briefly, the gelatine coated QDs were prepared by passing H2Te gas through an aqueous basic solution containing Cd(ClO4)2, thioglycolic acid (TGA) stabilizer. The resultant mixture was heated under reflux for 2 hours. The solution was then cooled to 80°C and divided into three flasks, A, B and C. Folic acid (0.01 moles, 0.28 g) was added directly to Flask B and the solution was stirred for 15 min. EDC (0.1 g) and DMAP (0.1 g) were added to flask C and the solution was stirred for 15 mins to activate the QDs for conjugation. Folic acid (0.01 moles, 0.28 g) was then added, and the mixture was allowed to react for 15 min, while stirring. From each of the crude solutions A, B and C, different fractions were precipitated out using 2-isopropanol and centrifuging (3000 rpm, 10 mins). Unreacted materials were removed by purification on a Sephadex column.
Caco-2 cells were cultured in appropriate medium (500 mL Minimum Essential Medium (MEM) supplemented with 0.055 g of sodium pyruvate, 5 mL of a solution of penicillin (2 mM) and streptomycin (2 mM), 5 mL of 1 mM gentamicin and 100 mL of Fetal Bovine Serum (FBS)) at 37°C and in a 5% CO2 atmosphere. 80% confluent cell cultures were trypsinised and re-suspended in cell culture medium to a final concentration of 1.105 cells/mL and seeded on cover slips. After 24 h incubation allowing the cells to adhere to the substrate, half of the medium was removed from each dish and replaced by the same volume of serum-free medium. The cells were incubated for a further 4 h before the medium was aspirated out and replaced with 2 mL of QD suspension in Dubelcco's modified Phosphate Buffer Saline (DPBS) at a final concentration of 10-7 mol/L. After four more hours, the QD containing solution was aspirated out of the dishes and the cells were washed three times with PBS. They were then fixed with 70% ethanol and mounted on slides using Vectashield mounting media containing 4',6-diamidino-2-phenylindole (DAPI). For FA competition experiments, FA at a final concentration of 10-7 mol/L was added to the cell cultures along with QDs. Control cultures in DPBS without QDs, and with or without FA accordingly were also analysed.
Caco-2 cells were seeded as before and treated with QDs in the same conditions. After 4 h incubation, the QD containing solution was aspirated out of the dishes and the cells were washed three times with PBS. 50 μg of Calcein AM were dissolved in 50 μL of dimethyl sulfoxide (DMSO). The resulting 50 μL of solution were diluted in 10 mL of DPBS. 1 mL of dilute Calcein AM was added to each dish and incubated at room temperature for 30 min. The staining solution was aspirated out and the cell cultures were washed three times with PBS. Live cells, stained in green, were imaged using a confocal microscope, counted and compared to control cultures.
A Shimadzu UV-1601 UV-Visible Spectrophotometer was used to measure QD absorption spectra. Scans were carried out in the 300-700 nm range. A Varian - Cary Eclipse Fluorescence Spectrophotometer was used to determine the photoluminescence (PL) emission spectra of QDs. The excitation wavelength was 480 nm and the emission was detected in the range 490-700 nm. The Quantum Yields (QY) were calculated from the PL spectra using Rhodamine 6 G as a reference. Hydrodynamic radii and zeta potential of nanoparticles were measured on a Malvern Zetasizer Nano Series V5.10. Five measurements were usually taken for each sample, each made of 10 to 20 accumulations as optimised by the machine. Fluorescence lifetime decays were measured using time-correlated single photon counting (TCSPC) on a Flurolog 3 Horiba Jovin Yvon, with samples excited at 480 nm and decays measured to 10000 counts. Biexponential fitting was used to generate the decay curves. A Jeol 2100 Transmission Electron Microscope (TEM) was used to image the CdTe QDs. Sizes of the nanoparticles were measured using ImageJ software. An Olympus FV1000 Point-Scanning Confocal Microscope was used to examine the cells after staining with QDs and counter-staining with DAPI or Calcein AM. Sequential acquisition was used to acquire the two colour images which were overlaid and analysed using the Olympus Fluoview version 7B software.
The project was funded by Science Foundation Ireland and the Higher Education Authority. Cell lines were kindly provided by Dr Shona Harmon, School of Pharmacy and Pharmaceutical Science, Trinity College Dublin.
- Drbohlavova J, Adam V, Kizek R, Hubalek J: Quantum Dots - Characterization, Preparation and Usage in Biological Systems. International Journal of Molecular Sciences. 2009, 10: 656-673. 10.3390/ijms10020656.View ArticleGoogle Scholar
- Yong K-T, Roy I, Swihart MT, Prasad PN: Multifunctional nanoparticles as biocompatible targeted probes for human cancer diagnosis and therapy. Journal of Materials Chemistry. 2009, 19: 4655-4672. 10.1039/b817667c.View ArticleGoogle Scholar
- Choi HS, Liu W, Liu F, Nasr K, Misra P, Bawendi MG, Frangioni JV: Design considerations for tumour-targeted nanoparticles. Nat Nano. 2010, 5: 42-47. 10.1038/nnano.2009.314.View ArticleGoogle Scholar
- Byrne SJ, le Bon B, Corr SA, Stefanko M, O'Connor C, Gun'ko YK, Rakovich YP, Donegan JF, Williams Y, Volkov Y, Evans P: Synthesis, characterisation, and biological studies of CdTe quantum dot-naproxen conjugates. Chemmedchem. 2007, 2: 183-+. 10.1002/cmdc.200600232.View ArticleGoogle Scholar
- Gerhards C, Schulz-Drost C, Sgobba V, Guldi DM: Conjugating Luminescent CdTe Quantum Dots with Biomolecules. The Journal of Physical Chemistry B. 2008, 112: 14482-14491. 10.1021/jp8030094.View ArticleGoogle Scholar
- Prasad B, Nikolskaya N, Connolly D, Smith T, Byrne S, Gerard V, Gun'ko Y, Rochev Y: Long-term exposure of CdTe quantum dots on PC12 cellular activity and the determination of optimum non-toxic concentrations for biological use. Journal of Nanobiotechnology. 2010, 8: 7-10.1186/1477-3155-8-7.View ArticleGoogle Scholar
- Byrne SJ, Williams Y, Davies A, Corr SA, Rakovich A, Gun'ko YK, Rakovich YP, Donegan JF, Volkov Y: "Jelly Dots": Synthesis and Cytotoxicity Studies of CdTe Quantum Dot-Gelatin Nanocomposites. Small. 2007, 3: 1152-1156. 10.1002/smll.200700090.View ArticleGoogle Scholar
- Kim J-H, Jang HH, Ryou S-M, Kim S, Bae J, Lee K, Han MS: A functionalized gold nanoparticles-assisted universal carrier for antisense DNA. Chemical Communications. 2010, 46: 4151-4153. 10.1039/c0cc00103a.View ArticleGoogle Scholar
- Li KG, Chen JT, Bai SS, Wen X, Song SY, Yu Q, Li J, Wang YQ: Intracellular oxidative stress and cadmium ions release induce cytotoxicity of unmodified cadmium sulfide quantum dots. Toxicology in Vitro. 2009, 23: 1007-1013. 10.1016/j.tiv.2009.06.020.View ArticleGoogle Scholar
- Zeng RS, Zhang TT, Liu JC, Hu S, Wan Q, Liu XM, Peng ZW, Zou BS: Aqueous synthesis of type-II CdTe/CdSe core-shell quantum dots for fluorescent probe labeling tumor cells. Nanotechnology. 2009, 20: 8-Google Scholar
- Schroeder JE, Shweky I, Shmeeda H, Banin U, Gabizon A: Folate-mediated tumor cell uptake of quantum dots entrapped in lipid nanoparticles. Journal of Controlled Release. 2007, 124: 28-34. 10.1016/j.jconrel.2007.08.028.View ArticleGoogle Scholar
- Bharali DJ, Lucey DW, Jayakumar H, Pudavar HE, Prasad PN: Folate-receptor-mediated delivery of InP quantum dots for bioimaging using confocal and two-photon microscopy. Journal of the American Chemical Society. 2005, 127: 11364-11371. 10.1021/ja051455x.View ArticleGoogle Scholar
- Zhang Y, Huang N: Intracellular uptake of CdSe-ZnS/polystyrene nanobeads. Journal of Biomedical Materials Research Part B-Applied Biomaterials. 2006, 76B: 161-168. 10.1002/jbm.b.30347.View ArticleGoogle Scholar
- Doucette MM, Stevens VL: Folate Receptor Function Is Regulated in Response to Different Cellular Growth Rates in Cultured Mammalian Cells. J Nutr. 2001, 131: 2819-2825.Google Scholar
- Adhirajan N, Shanmugasundaram N, Babu M: Gelatin microspheres cross-linked with EDC as a drug delivery system for doxycyline: Development and characterization. Journal of Microencapsulation. 2007, 24: 659-671. 10.1080/02652040701500137.View ArticleGoogle Scholar
- Liang H-C, Chang W-H, Liang H-F, Lee M-H, Sung H-W: Crosslinking structures of gelatin hydrogels crosslinked with genipin or a water-soluble carbodiimide. Journal of Applied Polymer Science. 2004, 91: 4017-4026. 10.1002/app.13563.View ArticleGoogle Scholar
- Cartiera MS, Johnson KM, Rajendran V, Caplan MJ, Saltzman WM: The uptake and intracellular fate of PLGA nanoparticles in epithelial cells. Biomaterials. 2009, 30: 2790-2798. 10.1016/j.biomaterials.2009.01.057.View ArticleGoogle Scholar
- Neri S, Mariani E, Meneghetti A, Cattini L, Facchini A: Calcein-Acetyoxymethyl Cytotoxicity Assay: Standardization of a Method Allowing Additional Analyses on Recovered Effector Cells and Supernatants. Clin Diagn Lab Immunol. 2001, 8: 1131-1135.Google Scholar
- Bottrill M, Green M: Some aspects of quantum dot toxicity. Chemical Communications. 2011, 47: 7039-7050. 10.1039/c1cc10692a.View ArticleGoogle Scholar
- Lovrić J, Bazzi HS, Cuie Y, Fortin GRA, Winnik FM, Maysinger D: Differences in subcellular distribution and toxicity of green and red emitting CdTe quantum dots. Journal of Molecular Medicine. 2005, 83: 377-385. 10.1007/s00109-004-0629-x.View ArticleGoogle Scholar
- Kamen BA, Smith AK: A review of folate receptor alpha cycling and 5-methyltetrahydrofolate accumulation with an emphasis on cell models in vitro. Advanced Drug Delivery Reviews. 2004, 56: 1085-1097. 10.1016/j.addr.2004.01.002.View ArticleGoogle Scholar
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