|Home | About | Journals | Submit | Contact Us | Français|
Biomedical applications of carbon nanotubes have attracted much attention in recent years. Here, we summarize our previously developed protocols for functionalization and bioconjugation of single-walled carbon nanotubes (SWNTs) for various biomedical applications including biological imaging; using nanotubes as Raman, photoluminescence and photoacoustic labels; sensing using nanotubes as Raman tags and drug delivery. Sonication of SWNTs in solutions of phospholipid-polyethylene glycol (PL-PEG) is our most commonly used protocol of SWNT functionalization. Compared with other frequently used covalent strategies, our non-covalent functionalization protocol largely retains the intrinsic optical properties of SWNTs, which are useful in various biological imaging and sensing applications. Functionalized SWNTs are conjugated with targeting ligands, including peptides and antibodies for specific cell labeling in vitro or tumor targeting in vivo. Radio labels are introduced for tracking and imaging of SWNTs in real time in vivo. Moreover, SWNTs can be conjugated with small interfering RNA (siRNA) or loaded with chemotherapy drugs for drug delivery. These procedures take various times ranging from 1 to 5 d.
Carbon nanotubes with various unique physical and chemical properties have shown interesting applications in many fields, including biomedicine1,2. Functionalized carbon nanotubes with water solubility and biocompatibility are able to cross cell membranes, shuttling a wide range of biologically active molecules including drugs, proteins, DNA and RNA into cells3–7. The cytotoxicity of carbon nanotubes is largely dependent on their surface functionalization, with minimal toxic effects for well-functionalized, serum-stable nanotubes1,8. We have shown that after intravenous injection into mice, well-functionalized single-walled carbon nanotubes (SWNTs) accumulated in reticuloendothelial systems (RES) are slowly excreted, mainly through the biliary pathway, without exhibiting obvious side effects to the treated mice9,10. Recently, in vivo cancer treatment in an animal model has been realized by carbon nanotube-based drug delivery11.
Carbon nanotubes are classified as single-walled carbon nanotubes (SWNTs) and multi-walled carbon nanotubes (MWNTs), depending on the number of graphene layers from which a nanotube is composed. SWNTs are quasi-one-dimensional quantum wires with sharp densities of electronic states (electronic DOS) at the van Hove singularities and generally have more attractive unique intrinsic optical properties than MWNTS. SWNTs can be used as optical tags for biomedical detection and imaging12–18. Ultra-sensitive ex vivo protein sensing with a detection limit as low as 1 fM using SWNT Raman tags has been achieved using the resonance Raman scattering property of SWNTs and surface-enhanced Raman scattering (SERS)14. The Raman scattering, near-infrared (NIR) photoluminescence and high optical absorbance of SWNTs have all been used for biomedical molecular imaging in vitro and in vivo15–18. Thus, carbon nanotube bioconjugates are promising nanomaterials for biomedical applications.
SWNTs may have various potential advantages over other nanomaterials in different areas of nanobiotechnology. As an optical tag in biological imaging, SWNTs can be used in Raman, NIR fluorescence and photoacoustic imaging of cells and animals12,13,15–18. Multimodality optical imaging could thus be achieved using SWNTs as the contrast agent. Quantum dots or surface-enhanced Raman scattering gold/silver nanoparticles normally only have a single imaging functionality19,20. In contrast to widely used fluorescent quantum dots, carbon nanotubes contain no heavy metals and thus have a safer chemical composition. In the area of drug delivery, SWNT-based siRNA delivery works for a large range of cells, including notoriously ‘hard-to-transfect’ human T cells4, which are inert to conventional liposomal transfection agents. SWNTs can be efficiently loaded with aromatic chemotherapy drugs such as doxorubicin (DOX) through supramolecular π–π stacking21, obtaining an ultra-high loading capacity superior to other drug carriers including liposomes and micelles. Moreover, the high optical absorbance of SWNTs can also be used in photothermal therapy22,23, which may potentially be combined with chemotherapy11,21 and gene therapy4,24, both delivered by SWNTs to treat cancer in a more efficient manner.
For biomedical applications, raw hydrophobic SWNTs must be functionalized to afford water solubility and biocompatibility. Our previous studies have uncovered that the behaviors of SWNTs in biological systems in vitro (such as cellular uptake) and in vivo (such as blood circulation time and biodistribution) are highly dependent on their surface chemistry4,9,25. Developing proper surface functionalization on SWNTs is thus the most critical step to produce nanotube bioconjugates for a desired application. There are two major types of functionalization protocols for SWNTs: covalent reactions or non-covalent coating by amphiphilic molecules on nanotubes. Various covalent functionalization reactions, such as oxidation26,27 of nanotubes and 1,3-dipolar cycloaddition28 on the nanotube sidewalls, have been developed to produce water-soluble nanotubes useful in certain biomedical applications such as drug delivery2. Although covalent chemical reactions often allow stable functionalization on carbon nanotubes, the properties of SWNTs are degraded when the nanotube sidewall is damaged, dramatically decreasing the Raman scattering and NIR fluorescence signals of SWNTs1. Therefore, covalently functionalized carbon nanotubes have been widely used in drug and gene delivery2,29, but are usually not ideal for sensing and imaging applications1. In contrast, the structure and optical properties of SWNTs are largely maintained when non-covalent functionalization is used. However, the stability and biocompatibility of many non-covalently functionalized SWNTs are not satisfactory. For example, SWNTs solubilized in small-molecule surfactants (e.g., sodium dodecyl sulfate, SDS) will aggregate and precipitate if excess coating molecules are removed. An ideal functionalization should impart SWNTs with high water solubility, biocompatibility, minimal damage of nanotube structure and functional groups available for further bioconjugation.
Our group has developed systematic protocols for SWNT functionalization and bioconjugation in the past few years. Raw SWNTs are non-covalently functionalized by amphiphilic polymers, such as phospholipid-poly(ethylene glycol) (PL-PEG)6,22. Functionalized SWNTs have excellent stability in the aqueous phase and are highly biocompatible. Targeting ligands including antibodies and peptides can be conjugated to SWNTs to recognize specific cell receptors, yielding targeted SWNT bioconjugates useful for biological sensing14 and imaging15–18. We have also developed a protocol to label SWNTs with radioactive isotopes to track and image nanotubes in vivo by positron emission tomography (PET). In addition, SWNT-based siRNA transfection can be achieved by conjugating siRNA to SWNTs through a cleavable disulfide bond4,6. Furthermore, aromatic drug molecules can be non-covalently loaded onto SWNTs by simple mixing for drug delivery21.
Here, we systematically summarize the nanotube functionalization and bioconjugation protocols developed and used in our previous studies. Although our bioconjugation strategies apply for a wide range of biomolecules, only a few model systems are chosen to illustrate those protocols. Arg–Gly–Asp (RGD) peptide and Herceptin anti-Her2 antibody are used as targeting ligands. 64Cu is reported as an example of radiolabeling SWNTs. Anti-CXCR4 siRNA is chosen for siRNA conjugation and delivery. Finally, DOX is shown as an aromatic drug, loaded onto SWNTs for drug delivery. These detailed protocols should be beneficial to scientists interested in further developing biological applications of novel nanomaterials.
SWNTs are non-covalently functionalized by sonication of raw, hydrophobic nanotubes in water solutions of amphiphilic polymers (e.g., PL-PEG)6,22. The hydrophobic lipid chains of PL-PEG are strongly anchored onto the nanotube surface, whereas the hydrophilic PEG chain affords SWNT water solubility and biocompatibility. After removal of excess PL-PEG molecules, functionalized SWNTs show excellent stability in various aqueous phases including water, physiological buffers (e.g., phosphate buffered saline, PBS), cell medium and whole serum. The concentration of a SWNT solution can be determined by its optical density at 808 nm measured by a UV–VIS–NIR spectrometer with a weight extinction coefficient of 0.0465 mg l–1 cm–1 (dividing the optical density at 808 nm by the extinction coefficient gives the concentration)22. The length distribution of functionalized SWNTs can be determined by an atomic force microscope (AFM). Those non-covalently functionalized SWNTs retain their Raman and NIR fluorescence properties and are useful in biological detection and imaging applications. The functional group (e.g., amine) on the PEG terminal is available for further bioconjugation (Fig. 1).
Targeting ligands including antibodies and peptides can be conjugated to SWNTs to recognize specific cell receptors (Fig. 2). Herceptin is a monoclonal antibody that binds specifically to the Her2/neu receptor over-expressed on a wide range of human breast cancer cells30. The RGD peptide targets integrin αvβ3 receptors that are upregulated on fast-growing tumor vasculature cells and many types of cancer cells31. The antibody Herceptin is first thiolated by Traut's reagent following standard protocols15,16 and used immediately after purification. The Traut's reagent reacts with amino groups on the antibody and produces active thiol groups useful for bioconjugation. Thiolated RGD peptide synthesized following a previously published protocol32 is used directly. The thiolated antibody or peptide should be protected from oxidation by adding EDTA to prevent heavy metal-catalyzed oxidization, or Tris-(2-carboxyethyl)phosphine hydrochloride (TCEP) as a reducing agent, during the conjugation with nanotubes. Maleimide groups are introduced onto SWNTs by reacting PL-PEG-amine-functionalized SWNTs with a sulfosuccinimidyl 4-N-maleimidomethyl cyclohexane-1-carboxylate (Sulfo-SMCC) bifunctional linker. The activated SWNTs are then reacted with thiolated antibodies or peptides, obtaining targeted SWNT bioconjugates, which can be used in multiplexed Raman spectroscopic imaging15,18, ultra-sensitive Raman detection of proteins14, NIR fluorescence imaging16, photoacoustic imaging17 and targeted photothermal therapy22,23. The targeting ability of SWNT bioconjugates (e.g., SWNT-RGD) can be characterized by in vitro Raman spectroscopic imaging experiments, in order to examine the staining of integrin αvβ3-positive U87MG cells and αvβ3-negative MCF-7 cells incubated with the SWNT-RGD conjugate.
To image and track SWNTs in vivo by PET, SWNTs are labeled with a radioactive isotope (Fig. 3)25. PET imaging provides three-dimensional distribution information of radiolabeled nanotubes in live mice in real time. To obtain RGD-conjugated radiolabeled SWNTs, SWNTs are first reacted with a mixture of sulfo-SMCC and N-hydroxysuccinimide (NHS) activated 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) and then conjugated to RGD-SH. After removal of excess reagents, 64Cu radioactive isotope can then be complexed to the DOTA rings on the SWNTs to achieve radiolabeling. The radiolabeled, targeted SWNT bioconjugate can then be used for in vivo PET imaging of mice bearing integrin αvβ3-positive, e.g., U87MG human glioblastomas tumors25. A total of 5–10 million of U87MG cells should be injected subcutaneously on the shoulder of a nude mouse. The mice can be used 2–3 weeks after tumor inoculation. PET imaging should be carried out at 0.5, 2, 4, 6 and 24 h post injection (p.i.). Mice may be killed at 24 h p.i. when the blood circulation of nanotubes has ended.
The intracellular molecular delivery ability of SWNTs can be used for siRNA transfection4,6. In this example we chose to use CXCR4, a chemokine receptor that has an important role in the entry of HIV virus into human T cells33. SWNTs are first reacted with a bifunctional linker, Sulfosuccinimidyl 6-(3’-[2-pyridyldithio]-propionamido)hexanoate (Sulfo-LC-SPDP) and then conjugated with thiolated siRNA through a cleavable disulfide bond (Fig. 4). Two CXCR4 siRNAs with different sequences and a control luciferase siRNA are used. Dithiothreitol (DTT) is used to cleave any disulfide bond formed during storage of thiolated siRNA and removed by a NAP-5 column before the conjugation of siRNA with SWNTs. The SWNT–siRNA conjugates should be sterilized by centrifugation before cell incubation. Once transported into cells through endocytosis, siRNA is released from SWNTs by sulfide cleavage and then binds to CXCR4 mRNA to induce gene silencing. The CXCR4 receptor expression on CEM.NKR cells, a human T-cell line inert to commercial cationic liposome transfection agents34, is knocked down after cells are incubated with the SWNT–siRNAanti-CXCR4 conjugate for 3 d. The CXCR4 expression levels of cells can be determined by labeling cells with Phycoerythrin (PE)-anti-CXCR4 antibody and flow cytometry (FACS) measurement with dead cells excluded by propidium iodide (PI) staining. Control experiments using commercial cationic liposome-based transfection agents to transfect CEM.NKR with CXCR4 siRNA showed no obvious gene silencing effect. The mis-matched siRNA sequence (luciferase siRNA) also did not affect the CXCR4 expression.
SWNTs, with all atoms exposed on their surface, have an ultra-high surface area available for binding of aromatic molecules through supramolecular π–π stacking21. Functionalized SWNTs with or without targeting ligands can be loaded with DOX, an aromatic chemotherapy drug used for various types of cancers, by simply mixing the two solutions at a slightly basic pH. Excess unloaded DOX can be removed by filtration. The optical absorption of SWNT–DOX at 490 nm after subtraction of the SWNT absorption (at the same nanotube concentration) can be used to calculate the DOX concentration and loading in the SWNT–DOX complex21. On the basis of the UV–VIS–NIR absorption spectra, up to 4 g of DOX can be loaded on 1 g of SWNTs. The toxicity of SWNT–DOX is lower than that of free DOX but can be enhanced when conjugated with a targeting ligand such as RGD peptide for targeted drug delivery. Toxicity assays can be carried out by testing cell viabilities after incubating cells with free DOX, SWNT–DOX and SWNT–RGD–DOX at series of DOX concentrations using a CellTiter 96 kit (Promega). Samples should be measured in triplicate and the cell viability should be determined compared with that of the untreated control, defined as 100% viable.
Thiolated RGD (RGD-SH) peptide is prepared following a previously reported protocol32.
Dissolve 1.2 mg of RGD-SH in 200 μl of water (~10 mM). Store the RGD-SH solution at –20 °C in small aliquots to avoid too many freeze–thaw cycles. The solution can be stable for up to 6 months if used and stored properly.
Dissolve siRNA purchased from Dharmacon in the desired amount of RNase-free water to reach a siRNA concentration of 100 μM. Store siRNA solution at 20 °C in small aliquots to avoid too many freeze–thaw cycles. The solution can be stable for up to 6 months if used and stored properly.
Dissolve 8.4 g of sodium bicarbonate in 200 ml water. The solution can be stored at room temperature (~22 °C) in a plastic bottle and stable for 6 months.
Dissolve 1.0 g of sodium hydroxide in 250 ml water. The solution can be stored at room temperature in a plastic bottle and stable for 6 months. ! CAUTION Sodium hydroxide is a corrosive strong base. Wear goggles, lab coat and face mask during experiments.
Dissolve 1.64 g of sodium acetate in 200 ml of water. Add 22 μl of acetic acid. Add 1 g of Chelex 100 beads into the buffer to avoid heavy metal ion contamination. Sodium acetate buffer can be stored for 6 months at room temperature. ! CAUTION Acetic acid is evaporative and corrosive. Wear goggles, lab coat and face mask during experiments. Handle acetic acid inside a hood.
Culture U87MG cells in DMEM (low glucose) supplemented with 10% (vol/vol) FBS and 1% (vol/vol) penicillin–streptomycin at 37 °C. Culture MCF-7 cells in DMEM (high glucose) supplemented with 10% (vol/vol) FBS and 1% (vol/vol) penicillin–streptomycin at 37 °C. Culture BT474 cells in DMEM (high glucose) supplemented with 5 g l–1 of glucose, 10% (vol/vol) FBS and 1% (vol/vol) penicillin–streptomycin at 37 °C. Culture CEM.NKR cells in RPMI-1640 supplemented with 10% (vol/vol) FBS at 37 °C. All cells are cultured in 5% CO2 atmosphere.
Inject 5 × 106 U87MG cells subcutaneously into athymic nude mice (on their shoulders). Wait for 3–4 weeks before imaging, until tumor sizes reach 300–500 mm3. ! CAUTION Please obtain appropriate training regarding animal handling and have animal protocols in place before performing animal studies.
Confocal Raman spectroscopic imaging is carried out using a Horiba Jobin Yvon Raman confocal microscope with a 785-nm laser (80 mW) as the excitation light source. A × 50 objective was used for imaging with ~1 μm laser spot size. A 1-mm pin pole was applied to restrict the spatial resolution in z-axis to ~1 μm. Each Raman spectroscopic map contains at least 100 × 100 spectra with a 0.5-s integration time for each spectrum.
FACS measurement is carried out using a Becton Dickinson FACScan. A 488-nm laser is used as the excitation light source. Channels 1, 2 and 3 collect green, yellow and red fluorescence, respectively. A flow rate of 200–400 cells per s is used in the measurement.
Troubleshooting advice can be found in Table 1.
After Step 5, PL-PEG-functionalized SWNTs should have excellent water solubility and are stable in various biological solutions without any visible aggregation after a long period of incubation time (weeks) (Fig. 5). Atomic force microscope (AFM) images of SWNTs show that the lengths of SWNTs range from 50 to 300 nm, with an average of ~100 nm.
In 6A(vii), confocal Raman spectroscopic images of cells stained by SWNT–RGD should exhibit strong SWNT Raman signals on integrin αvβ3-positive U87MG cells but not on αvβ3-negative MCF-7 cells (Fig. 6a). Nonspecific binding of SWNTs to MCF-7 cells should be minimal. The ratio of Raman signals on positive versus negative cells is >40 (Fig. 6b). Raman imaging of BT474 (Her2-positive) and MCF-7 (Her2-negative) cells after staining with SWNT-Herceptin (Step 6B) should give very similar results (data not shown). Those targeted SWNT bioconjugates may also be used in ultra-sensitive protein microarray detection14.
In Step 6C(ix), PET images (Fig. 7) at 6 h p.i. should show high tumor uptake (10–15%ID/g) in U87MG tumor bearing mice injected with SWNT–RGD (functionalized by PL-PEG5000)25. Radiolabeled SWNTs without RGD conjugation should show a reduced uptake in the tumor (3–5%ID/g) in comparison with RGD-conjugated SWNTs. Tumor uptake will be significantly reduced if mice are pre-injected with a high dose of free RGD peptide before injection of SWNT–RGD (4–6%ID/g). Control integrin αvβ3-negative HT29 tumors should have a lower uptake of SWNT–RGD (3–5%ID/g) (Fig. 7d).
In Step 6D(vii), as shown in Figure 8, SWNT only and SWNT–siRNAluc mis-matched control-treated CEM.NKR cells show normal CXCR4 expressions4. The CXCR4 expression should be significantly reduced after treatment with SWNT–siRNACXCR4 (two sequences) compared with untreated cells. The RNAi effect should range from 70 to 90%. Other types of commercial cationic liposome-based siRNA transfection agents do not show significant siRNA transfection effects to CEM.NKR cells because human T cells are well known to be difficult to transfect.
In Step 6E, the SWNT–DOX solution is reddish in color because of the UV–VIS absorption of DOX stacked onto SWNTs (Fig. 9a). The DOX absorption peak at 490 nm after subtraction of SWNT absorption at this wavelength is used to determine DOX concentration (Fig. 9b). In Step 6E(iv), SWNT–DOX has a lower toxicity than free DOX. Conjugation of RGD enhances the toxicity of DOX-loaded SWNTs to integrin αvβ3-positive U87MG cells but not to αvβ3-negative MCF-7 cells (Fig. 9c,d)21.
The multiple projects involved here were supported by a Stanford Graduate Fellowship, a Stanford Bio-X grant, CCNE-TR at Stanford University, NIH-NCI R01 CA135109-02 and Ensysce Biosciences Inc. Drs Nadine Wong Shi Kam, Sarunya Bangsaruntip, Xiaowu Tang, Xiaoming Sun, Xiaoyuan Chen, Weibo Cai and Ms Nozomi Nakayama have also contributed in the development of this protocol.