Folic acid-CdTe quantum dot conjugates and their applications for cancer cell targeting
© Springer-Verlag 2010
Received: 25 April 2010
Accepted: 7 July 2010
Published: 31 July 2010
In this study, we report the preparation, luminescence, and targeting properties of folic acid-CdTe quantum dot conjugates. Water-soluble CdTe quantum dots were synthesized and conjugated with folic acid using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide-N-hydroxysuccinimide chemistry. The influence of folic acid on the luminescence properties of CdTe quantum dots was investigated, and no energy transfer between them was observed. To investigate the efficiency of folic acid-CdTe nanoconjugates for tumor targeting, pure CdTe quantum dots and folic acid-coated CdTe quantum dots were incubated with human nasopharyngeal epidermal carcinoma cell line with positive expressing folic acid receptors (KB cells) and lung cancer cells without expression of folic acid receptors (A549 cells). For the cancer cells with positive folate receptors (KB cells), the uptake for CdTe quantum dots is very low, but for folic acid-CdTe nanoconjugates, the uptake is very high. For the lung cancer cells without folate receptors (A549 cells), the uptake for folic acid-CdTe nanoconjugates is also very low. The results indicate that folic acid is an effective targeting molecule for tumor cells with overexpressed folate receptors.
Cancer is the second major cause of death in the USA, killing approximately half a million people in the USA alone every year (ACS 2009). Early detection and effective treatment provide the best hope for cancer patients. For example, early detection of cancer can avoid approximately 3–35% of cancer deaths (Colditz and Sellers 2006). Therefore, there is a crucial need to develop cancer-specific imaging probes for the early diagnosis of cancer. Cells are almost transparent to visible light thereby making their direct observation using a conventional microscope a challenge. Cells therefore need to be labeled with a fluorophore to enhance the contrast, thereby making imaging a simpler task. The major limitations in using fluorophores are the phenomena of photo-bleaching and blinking (Morgan et al. 2005), rendering them poor contrasting agents. To overcome these limitations, semiconductor nanocrystals, also known as quantum dots (QDs), have been extensively studied for the last decade. Some of the advantages of quantum dots over fluorescent probes are their efficient fluorescence, chemical stability, broad excitation bands, narrow emission bands, good water solubility, compatible surface chemistry, high photostability, and low photo-bleaching rate (Morgan et al. 2005; Chen 2008). Even though Cd2+ containing quantum dots have high cytotoxicity (Zhang et al. 2006) that limits their practical application, in cell imaging they have tremendous potential as sensitive, nano-scale probes for early detection of cancer (Morgan et al. 2005; Chen 2008; Chen et al. 2006a; Smith et al. 2006). The applications of quantum dots for cell imaging and animal model studies can not only obtain insightful information for cancer detection but also for drug delivery and targeting (Chen and Sun 2008). Tumor targeting may be accomplished by using a tumor-specific ligand, such as folic acid. Folates are low molecular weight pterin-based vitamins required by eukaryotic cells for one-carbon metabolism and de novo nucleotide synthesis. The folate receptor is a glycosylphosphatidylinositol-anchored, high-affinity membrane folate-binding protein that is overexpressed in a wide variety of human tumors, including more than 90% of ovarian carcinomas (Sudimack and Lee 2000; Wang and Low 1998). On the other hand, normal tissue distribution of the folate receptor is highly restricted, making it a useful marker for targeted drug delivery to tumors. This methodology is currently being used for the selective delivery of imaging and therapeutic agents to tumor tissues (Leamon and Low 2001). Folic acid, a high-affinity ligand for the folate receptor (Kd = ∼10–10 M), retains its receptor binding property when covalently derivatized by its gamma-carboxyl group. Studies have shown that folate conjugates are taken into receptor-bearing tumor cells via folate receptor-mediated endocytosis (Antony 1996). Folic acid is potentially superior to antibodies as a targeting ligand because of its small size; lack of immunogenicity; ready availability; and simple, well-defined conjugation chemistry (Wang and Low 1998).
The conjugation of folic acid to quantum dots or nanoparticles has been investigated and reported by several groups (Bharali et al. 2005; Yang et al. 2009; Hu et al. 2009; Manzoor et al. 2009). However, several questions regarding the application of quantum dot-folic acid conjugates for tumor targeting remain to be answered. For example, the mechanism responsible for quantum dot luminescence quenching is not yet clear. In addition, the targeting efficacy of folic acid-quantum dots to folate receptor positive (FR+) and folate-negative (FR−) tumors also needs to be identified. The present research is aimed to answer these questions.
2 Materials and methods
Cadmium perchlorate hydrate (Cd(ClO4)2.H2O), thioglycolic acid (TGA), aluminium telluride (Al2Te3), sulfuric acid (H2SO4), sodium hydroxide (NaOH), folic acid (FA), N-hydroxysuccinimide (NHS), 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC), Eagle's minimum essential medium (EMEM), F-12 K medium, fetal bovine serum (FBS; 10%), sterilized phosphate buffered saline (PBS), and Trypsin were purchased from Aldrich. Human nasopharyngeal epidermal carcinoma cell line - KB cells (ATCC number CCL-17) and human lung carcinoma cell line A549 cells (ATCC number CCL-185) were purchased from ATCC.
Thioglycolic acid-stabilized cadmium telluride quantum dots were synthesized by a method described in the literature (Gaponik et al. 2002; Joly et al. 2005; Liu et al. 2006). Briefly, 1.463 g (4.70 mmol) of Cd(ClO4)2.H2O was dissolved in 125 ml of deionized (DI) water, and 0.793 ml (11.4 mmol) of thioglyocolic acid was added to the above solution under stirring. The pH of the solution was adjusted to 11.5 by dropwise addition of 0.5 M NaOH. The solution was transferred to a three-necked flask and de-gassed by bubbling Ar gas for ∼10 min; 0.4 g of Al2Te3 was charged into a small three-necked flask. H2Te3 gas generated by the addition of 3 ml of 0.5 M H2SO4 to Al2Te3, was bubbled through the solution for ∼5 min. The solution turned orange in color due to the formation of the CdTe precursors. The CdTe precursor solution was refluxed at 100°C, under open-air conditions, with condenser attached to promote the growth of nanocrystals. Folic acid was conjugated to TGA-coated CdTe quantum dots using EDC-NHS chemistry (Hermanson 1996; Wang et al. 2002); 0.05 M EDC-NHS (EDC/NHS = 1:10) and 0.05 M QDs were mixed for 5 min. Then an equal molar ratio of 0.05 M folic acid was added to the above solution and stirred gently at room temperature over night. The unreacted chemicals (EDC, NHS, folic acid, and QDs) were removed by dialysis against pH adjusted (pH 11–12) de-ionized water for 1 day. The cut off molecular weight of the dialysis membrane was 12,000 Da.
The Fourier transform infrared (FTIR) spectra were measured on a Shimadzu RF-5301 attenuated total reflectance—FTIR spectrometer. The folic acid conjugation with QDs was monitored by a high performance liquid chromatography (HPLC) method performed on a Waters 600 Multisolvent Delivery System equipped with a Waters 2996 photodiode array detector and a Waters BioSuite 250 size-exclusion (SEC) column (300 × 7.5 mm, 13 μm). The optical absorption spectra were recorded on a Shimadzu 1501 ultraviolet–visible (UV–VIS) spectrophotometer. The luminescence emission spectra were measured using a Shimadzu RF-5301PC fluorescence spectrophotometer. Luminescence lifetimes were collected using the frequency-doubled output of a synchronously pumped picosecond (ps) dye laser operating at 610 nm. The doubled output was focused onto the samples and emission collected at right angle to the input. The emission was spectrally filtered, and the lifetime measured using time-correlated single photon counting. The instrument resolution was determined to be about 50 ps FWHM using a standard scattering material.
Cell culture and fluorescence imaging
Human nasopharyngeal epidermal carcinoma cell line—KB cells (ATCC number CCL-17) and human lung carcinoma cell line A549 cells (ATCC number CCL-185) were cultured in EMEM and F-12 K medium respectively, supplemented with 10% FBS at 37°C (5% CO2). The cellular uptake of the folic acid-conjugated quantum dots was studied as described by (Bharali et al. 2005). Briefly, glass cover slips were sterilized in ethanol for 1 h and air dried. The sterilized cover slips were placed in 6 well plates. Then, the KB and A549 cells were trypsinized and re-suspended in their corresponding growth media at a concentration of around 7.5 × 105 mL−1; 60 μl of the cell suspension was placed on the cover slip and 2 ml of corresponding complete medium was added. The 6 well plates were incubated at 37°C with 5% CO2 for 24 h. After 24 h of incubation, the media was removed and the cells were rinsed with PBS, and 2 ml of corresponding fresh media was added to the wells. Finally, 150 μl of quantum dots were added to one set of wells and 150-μl-conjugated quantum dots were added to the other sets of wells, and mixed properly. The plates were returned to the incubator. After 2 h, 4 h, and 8 h of incubation the plates were taken out of the incubator and rinsed several times with sterile PBS. The cover slips were carefully taken out of the wells, placed on glass slides, and covered with another sterile cover slip for fluorescence imaging observation which was conducted on a fluorescence imaging system with a Meiji Trinocular Fluorescent Microscope and a Pixera “Cooled” Digital Camera.
3 Results and discussion
Eluted by 0.1 M phosphate buffer containing 0.15 M NaCl (pH 7.4) at the flow rate of 1.0 mL/min, the retention time of folic acid on the SEC-HPLC column was 12.7 min with the peak UV absorbance at 281.6 nm. Interestingly, the QDs exhibited very weak UV signals without characteristic absorption peaks. A fluorescence detector would be more appropriate for the QD detection (Wan et al. 2004). As expected, the folate-conjugated QDs showed a UV peak at 10.4 min with a UV–VIS spectrum in the range of 210 to 400 nm similar to that of folic acid, indicating the successful formation of folate-QD nanoconjugates.
3.2 Luminescence properties
In Figs. 3 and 5 the CdTe emission shows a dual peak between 600 and 650 nm. In the QD-FA conjugates the stronger peak is near ∼640 nm while in CdTe QDs alone or mixed with FA the stronger peak is close to ∼610 nm. This is a very interesting phenomenon that might give some information about the luminescence quenching of the QDs after conjugation with folic acid. It has been reported that the emission from II-VI semiconductor quantum dots is actually a combination of surface defect and excitonic emission (Joly et al. 2005; Liu et al. 2006; Chung et al. 2006). Most likely, the long-wavelength side is mainly due to surface defects, and the short-wavelength side is mainly due to the excitons. That the QD-FA conjugates show a stronger peak close to ∼640 nm while CdTe alone or mixed with FA shows a stronger peak close to ∼610 nm implies that more surface defects are produced in the quantum dots during the conjugation. More surface defects quench the excitonic luminescence and enhance the luminescence long-wavelength component. This is in agreement with the conclusion from the lifetime measurements that the production of surface defects during conjugation with folic acid is a major cause of the luminescence quenching.
3.3 Tumor cell targeting observations
In summary, the luminescence and targeting properties of folic acid-CdTe quantum dot conjugates were studied and the interaction between folic acid and CdTe quantum dots was investigated. No energy transfer is observed from folic acid to CdTe quantum dots in CdTe-folic acid nanoconjugates and this is attributed to the fact that the luminescence lifetime of folic acid is much shorter than the lifetime of the CdTe quantum dots. The change of the pH values and possible damage of the quantum dot surface coating are likely the direct causes to the luminescence quenching of CdTe quantum dots after conjugation. The specific tumor targeting efficiency of the folic acid-CdTe nanoconjugates was evaluated by a comparative uptake study of the targeted and non-targeted QDs in KB cells (FR+) and A549 cells (FR−). The observations further demonstrate the promising potential of the folic acid-CdTe QDs for targeted tumor cell detetction.
Wei Chen would like to thank the support by the Startup funds from UTA, the NSF and DHS joint advanced research initiative program (2008-DN-077-ARI016-03), and the US Army Medical Research Acquisition Activity (USAMRAA) under Contract No.W81XWH-10-1-0234 and No. W81XWH-10-1-0279, and DOD HDTRA1-08-P-0034. Part of the research described was performed at the W.R. Wiley Environmental Molecular Sciences Laboratory, a national scientific user facility sponsored by the Department of Energy's Office of Biological and Environmental Research and located at the Pacific Northwest National Laboratory (PNNL). PNNL is operated by Battelle for the US Department of Energy under contract DE-AC06-76RLO1830.
- ACS (2009) Cancer Facts & Figures. American Cancer Society, Atlanta, GAGoogle Scholar
- Antony AC (1996) Folate receptors. Annu Rev Nutr 16:501–521View ArticleGoogle Scholar
- Bharali DJ, Lucey DW, Jayakumar H, Pudavar HE, Prasad PN (2005) Folate-receptor-mediated delivery of InP quantum dots for bioimaging using confocal and two-photon microscopy. J Am Chem Soc 127(32):11364–11371View ArticleGoogle Scholar
- Carbone L, Kudera S, Carlino E, Parak WJ, Giannini C, Cingolani R, Manna L (2006) Multiple wurtzite twinning in CdTe nanocrystals induced by methylphosphonic acid. J Am Chem Soc 128(3):748–755View ArticleGoogle Scholar
- Chen W (2008) Nanoparticle fluorescence based technology for biological applications. J Nanosci Nanotechnol 8(3):1019–1051View ArticleGoogle Scholar
- Chen W, Sun XK (2008) Luminescent nanoparticles for biological applications: imaging, therapy, and targeting strategies. In: Chen X (ed) Recent Advances of Bioconjugate Chemistry in Molecular Imaging Kerala. Research Signpost, IndiaGoogle Scholar
- Chen W, Grouquist D, Roark J (2002) Voltage tunable electroluminescence of CdTe nanoparticle light-emitting diodes. J Nanosci Nanotechnol 1(1):47–53View ArticleGoogle Scholar
- Chen Q, Ma Q, Wan Y, Su X, Lin Z, Jin Q (2005) Studies on fluorescence resonance energy transfer between dyes and water-soluble quantum dots. Luminescence 20(4–5):251–255View ArticleGoogle Scholar
- Chen W, Joly AG, Morgan NY (2006a) Optical Physics and Applications of Luminescent Nanoparticles. In: Balandin AA, Wang KL (eds) Handbook of Semiconductor Nanostructures and Devices, vol 2. American Scientific Publishers, Los Angeles, pp 295–334Google Scholar
- Chen W, Zhang J, Westcott SL, Joly AG, Malm JO, Bovin JO (2006b) The Origin of X-ray Luminescence from CdTe Nanoparticles in CdTe/BaFBr:Eu2+ Nanocomposite Phosphors. J Appl Phys 99(34302):34301–34305Google Scholar
- Chou PT, Chen CY, Cheng CT, Pu SC, Wu KC, Cheng YM, Lai CW, Chou YH, Chiu HT (2006) Spectroscopy and femtosecond dynamics of type-II CdTe/CdSe core-shell quantum dots. Chemphyschem 7(1):222–228View ArticleGoogle Scholar
- Chung I, Witkoskie JB, Cao J, Bawendi MG (2006) Description of the fluorescence intensity time trace of collections of CdSe nanocrystal quantum dots based on single quantum dot fluorescence blinking statistics. Phys Rev E Stat Nonlin Soft Matter Phys 73(1 Pt 1):011106View ArticleGoogle Scholar
- Clapp AR, Medintz IL, Mauro JM, Fisher BR, Bawendi MG, Mattoussi H (2004) Fluorescence resonance energy transfer between quantum dot donors and dye-labeled protein acceptors. J Am Chem Soc 126(1):301–310View ArticleGoogle Scholar
- Clapp AR, Medintz IL, Fisher BR, Anderson GP, Mattoussi H (2005) Can luminescent quantum dots be efficient energy acceptors with organic dye donors? J Am Chem Soc 127(4):1242–1250View ArticleGoogle Scholar
- Clegg RM (1996) Fluorescence resonance energy transfer. In: Wang X, Herman B (eds) Fluorescence imaging spectroscopy and microscopy. Wiley, New York, pp 179–252Google Scholar
- Colditz GA, Sellers TA (2006) E ET. Epidemiology—identifying the causes and preventability of cancer. Nat Rev Cancer 6:75View ArticleGoogle Scholar
- Gaponik N, Talapin DV, Rogach AL, Hoppe K, Shevchenko EV, Kornowski A, Eychmuller A, Weller H (2002) Thiol-capping of CdTe nanocrystals: an alternative to organometallic synthetic routes. J Phys Chem B 106(29):7177–7185View ArticleGoogle Scholar
- Hermanson GT (1996) Bioconjugate techniques. Academic, San DiegoGoogle Scholar
- Hu H, Xiong LQ, Zhou J, Li FY, Cao TY, Huang CH (2009) Multimodal-luminescence core-shell nanocomposites for targeted imaging of tumor cells. Chem Eur J 15:3577–3584View ArticleGoogle Scholar
- Joly AG, Chen W, McCready DE, Malm J-O, Bovin J-O (2005) Upconversion luminescence of CdTe nanoparticles. Phys Rev B 71(16):165304View ArticleGoogle Scholar
- Leamon CL, Low PS (2001) Folate-mediated targeting: from diagnostics to drug and gene delivery. Drug Discov Today 6:44View ArticleGoogle Scholar
- Lei Y, Tang H, Zhou C, Zhang T, Feng M, Zou B (2008) Incorporating fluorescent quantum dots in water soluble polymer. J Lumin 128(3):277–281View ArticleGoogle Scholar
- Liu Y, Chen W, Joly AG, Wang Y, Pope C, Zhang Y, Bovin JO, Sherwood P (2006) Comparison of water-soluble CdTe nanoparticles synthesized in air and in nitrogen. J Phys Chem B Condens Mater Surf Interfaces Biophys 110(34):16992–17000Google Scholar
- Manzoor K, Johny S, Thomas D, Setua S, Menon D, Nair S (2009) Bioconjugated luminescent quantum dots of doped ZnS: a cyto-friendly system for targeted cancer imaging. Nanotechnology 20(65102):65101–65113Google Scholar
- Morgan NY, English S, Chen WW, Chernomordik V, Russo A, Smith PD, Gandjbakhche A (2005) Real time in vivo non-invasive optical imaging using near-infrared fluorescent quantum dots. Acad Radiol 12:313–323View ArticleGoogle Scholar
- Smith AM, Dave S, Nie S, True L, Gao X (2006) Multicolor quantum dots for molecular diagnostics of cancer. Expert Rev Mol Diagn 6(2):231–244View ArticleGoogle Scholar
- Sudimack J, Lee RJ (2000) Targeted drug deliver via the folate receptor. Adv Drug Deliv Rev 41:147View ArticleGoogle Scholar
- Thomas AH, Lorente C, Capparelli AL, Pokhrel MR, Braun AM, Oliveros E (2002) Fluorescence of pterin, 6-formylpterin, 6-carboxypterin and folic acid in aqueous solution: pH effects. Photochem Photobiol Sci 1:421–426View ArticleGoogle Scholar
- Tyagi A, Penzkofer A (2010) Fluorescence spectroscopic behaviour of folic acid. Chem Phys 367:83–92View ArticleGoogle Scholar
- Wan Y, Wang LP, Lin BZ, Chen QD, Zhang H, Yang B, Su XG, Jin QH (2004) Studies on quantum dots synthesized in aqueos solution for biological labeling. Can J Anal Sci Spectros 49(2):1–6Google Scholar
- Wang S, Low PS (1998) Folate-mediated targeting of antineoplastic drugs, imaging agents, and nucleic acids to cancer cells. J Control Release 53:39View ArticleGoogle Scholar
- Wang SP, Mamedova N, Kotov NA, Chen W, Studer J (2002) Antigen/antibody immunocomplex from CdTe nanoparticle bioconjugates. Nano Lett 2(8):817–822View ArticleGoogle Scholar
- Yang C, Ding N, Xu Y, Qu X, Zhang J, Zhao C, Hong L, Lu Y, Xiang G (2009) Folate receptor-targeted quantum dot liposomes as fluorescence probes. J Drug Target 17(7):502–511View ArticleGoogle Scholar
- Zhang Y, Kohler N, Zhang M (2002) Surface modification of superparamagnetic magnetite nanoparticles and their intracellular uptake. Biomaterials 23:1553–1561View ArticleGoogle Scholar
- Zhang Y, Chen W, Zhang J, Pope JL, Chen G, Pope C (2006) In vitro and in vivo Toxicity of CdTe nanoparticles. J Nanosci Nanotechnol 7:497–503View ArticleGoogle Scholar