The discovery of carbon nanotubes (CNTs) promised to revolutionize biomedical research by offeringunique performance attributes inherent in their unique mechanical, electrical, optical and magnetic properties [1-6]. CNTs are highly anisotropic cylindrical nanostructures with lengthsranging from several hundred nanometres to several micrometres, and diameters of 0.4–100 nm. CNTscan be classified as single-walled carbon nanotubes (SWNTs) or multi-walled carbon nanotubes (MWNTs), depending on the number of graphene layers which compose its structure.
CNTs are typically prepared using one of three methods: laser ablation , arc-discharge , and chemicalvapor deposition (CVD) . CVD, the most popular pathway of production, involves reacting a metal catalyst (such as nickel, cobalt with a hydrocarbon feedstock at high temperatures (>800º C) to produce CNT. Both SWNTs and MWNTs, are commercially available in varying degrees of purity. Pristine CNT (non-purified and/or non-functionalised) are completely insoluble in all solvents -- a property which has generated some health concerns in terms of toxicity . Therefore the functionalization of CNT is a key step towards novel biomedical applications. Advanced methods to chemical modification and functionalization have enabledsolvation and dispersalof CNTs in water [11-13]. Properly functionalized CNT have a high propensity to cross cell membranes. Various functionalized SWNTs have been shown to transport various bio-molecules across into living cells without cytotoxicity [14, 15]. Nanospearing or nanopenetration molecular delivery, which relies on the penetration of magnetic carbon nanotubes (mCNTs) into cells by magnetic field exposure, was also recently suggested by Cai et al . In this technique, a rotating magnetic field first guided CNTs to spear the cells. In a second step, a static field pulled CNT into the cells. The researchers have achieved unprecedented high molecular delivery efficiency into difficult-to-transfect cells such as primary mammalian neurons and B lymphocytes with high viability after transfection. In addition to providing a successful molecular delivery platform, CNTs have been also used for successful cellular labelling/tracking in recent years. CNTs coated with peptides have been reported to be successfully delivered and imaged in living human cervical cancer HeLa cells . More interestingly, CNT fluorescence was detected and imaged from living Drosophila larva by using near-infrared (NIR) with no adverse effects on the viability and growth of the larva . These recent findings prompted our group to investigate CNT-mediated labelling platforms for stem cell tracking.
One of potential applications of nanotechnologies in stem cell research is the noninvasive tracking of stem cells and progenitor cells transplanted
2.Materials and methods
2.1. Source and isolation of human CD34+HSPC
Peripheral blood leukapheresis product (LP) was obtained with the patients’ informed consent (in accordance with the institutional guidelines approved by the Human Research Ethic Board of the University of Alberta) before cryopreservation. CB was collected immediately after delivery in a sterilizedtube containing heparin (1000 IU/ml), and with the informed consent of the mother (in accordance with the institutional guidelines approved by the Health Research Ethics Board ofthe University of Alberta). Light density cells from CB and LP were obtained by Percoll density gradient centrifugation and enriched for CD34+cells by immunoaffinity selection with MACS paramagnetic beads (Miltenyi Biotec, Auburn, CA, USA), according to the manufacturer’s instructions . The purity of isolated CB and LP CD34+ cells were
2.2. Synthesis of FITC-mCNT
Single-walled mCNT containing Ni and Y at the tip, with an average diameter of 1.2–1.5 nm and a length of 2–5
Oxidation of the carbon nanotubes: The first oxidation step was carried out as described previously [3, 11, 22-24]. Briefly, the purchased carbon nanotubes (200 mg) were refluxed with 0.5 M HNO3 (100 ml) for 48 h to introduce carboxylic groups. After refluxing, the solution was diluted with deionized water, filtered over a 0.2 μm polycarbonate filter (Millipore) and washed several times with deionized water. The sample was collected and dried overnight in a vacuum oven at 80º C to obtain mCNT 2 (170 mg) (figure 1).
Reaction with thionyl chloride to give SWNT-COCl: A suspension of mCNT 2 (100 mg) in 20 ml of SOCl2, together with five drops of dimethylformamide (DMF), was stirred at 70º C for 24 h. The mixture was cooled and centrifuged at 2000 rpm for 30 min. The excess SOCl2 was decanted and the resulting black solid was washed with anhydrous THF (3 × 20 ml) and dried overnight in a vacuum oven at 80º C to give mCNT 3 (78 mg) (figure 1).
Coupling with 2-(2-(2-aminoethoxy)ethoxy)ethan amine: The mixture of mCNT 3 (50 mg) and anhydrous 2-(2-(2-aminoethoxy)ethoxy)ethanamine (120 ml) was heated at 100º C for 100 h. During this time, the liquid phase became dark. After cooling, the mixture was poured into methanol (100 ml) and centrifuged to give a black solid, which was washed several times with methanol. The resulting solid was dried overnight in a vacuum oven at 80º C to give mCNT 4 (42 mg) (figure 1).
Labeling with FITC: A suspension of the mCNT 4 (25 mg) and FITC (5 mg) in anhydrous DMF (10 ml) was stirred in the dark for 5 h. Then the reaction mixture was poured into anhydrous ethyl ether (40 ml) and centrifuged to give a black solid, which was washed with methanol until TLC (10% MeOH in dichloromethane) showed no free FITC left. The product was dried overnight in a vacuum oven at 80 ℃ to give mCNT 5 (23 mg) (figure 1).
2.3. Magnetic-field driven HSPC uptake of FITC-mCNT
CD34+cells with a density of 2
2.4. FACS analysis
The cells exposed to FITC-mCNT at different time pointsand concentrations were collected, extensively washed andthen fixed in 1% paraformaldehyde prior to FACS analysis(FACscan, Becton-Dickinson, San Jose, CA, USA) todetermine uptake efficiencies.
2.5. Confocal microscopy
CD34+cells were seeded at a density of 1*105cells/cm2 on cover slips previously coated with poly-L-lysine (10
2.6. Cell viability
Cell viability was measured by the trypan blue exclusion assay. CD34+cells werecollected and pelleted by centrifugation at 700
2.7. Colony-forming unit (CFU) assay
After exposure to the FITC-mCNT solution with a concentrationof 40
3. Results and discussion
In the present study, we examined the magnetic-field-driven uptake of FITC-mCNT into HSPC. FITC-mCNT with both fluorescent and magnetic properties were synthesized and freshly prepared for our uptake experiments as summarized in figure 1.In order to test ourfluorescent and magnetic FITC-mCNTs for labelling HSPC,CD34+cells obtained from LP were exposed to solutionsof different concentrations of FITC-mCNT or mCNT alone(10, 20 and 40
FITC reached 83%, 90% and100% in LP CD34+ cells at 6 h after uptake of FITC-mCNTwith 10, 20 and 40
To confirm that even less mature HSPC can efficiently uptake FITC-mCNT, we next studied the internalization of FITC-mCNT (40
Our rapid FITC-mCNT labelling of HSPC mightoffer a solution for the difficulty of tracking the movement of transplanted HSPC in patients.
To investigate the cytotoxicity of mCNT, we studied the HSPC viability after FITC-mCNT uptake by trypan blue exclusion assay. With the exposure of FITC-mCNT to various concentrations (10, 20 and 40
It has been reported that CNT has no adverse effect on macrophages. It was also not cytotoxic and has no significant effect on adipogenic, osteogenic or chondrogenic differentiation of hMSC . To investigate the long-term cytotoxicity effect of our magnetic-field-driven FITC-mCNT uptake and their impact on the differentiation of HSPC, we performed a colony unit assay (CFU) on FITC-mCNT-labelled HSPC 1, 3 and 6 h after uptake. After 14 days, colonies were identified and enumerated. No evidence was observed of cytotoxicity nor was the differentiation affected in FITC-mCNTlabelled HSPC because there was no difference in overall colony number or type (CFU-GM: colony-forming unit of granulocyte/macrophage; BFU-E: burst forming unit of erythrocyte; CFU-GEMM: colony-forming unit of granulocyte macrophage-erythroid megakaryocyte) between the FITC-mCNT labelled and the control HSPC (figure 7). These observations suggest that FITC-mCNT internalization is not only efficient and safe, but it also does not alter the HSPC’s properties, similar to the effect of COOH-functionalized single-walled CNT in human mesenchymal stem cells.
4. Conclusion and future outlook
In the current study, a highly efficient and safe mCNT-mediated labelling method for stem cells has been presented. For the efficacy of future stem cell-based therapies, it is crucial to image stem cells and their final location
The authors would like to acknowledge funding supports fromthe Canadian Institutes of Health Research (CIHR) and theIndustrial Research Assistance Program (IRAP) program of the National Research Council (NRC),Canada. The authors would also like to acknowledge thevaluable discussions with Dr Eric Swanson, the Director ofthe National Research Council (IRAP program), Edmonton, Canada.
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