Investigation of Bundled and Debundled Functionalized Single Walled Carbon Nanotubes (SWNT) as Vaccine Adjuvants

Brandon Keeler*, Tarek Fadel**, Tarek Fahmy**

*

Yale School of Undergraduate Studies, **Department of Biomedical Engineering, Yale University, P.O. Box 208284, New Haven, Connecticut 06520
Abstract:

Functionalized single walled carbon nanotubes (SWNT) are an emerging nanoparticle that has potential for being a biomaterial for immunotherapeutic applications. To understand these particles and study their effectiveness as a vaccine adjuvant, we first characterized both bundled and debundled SWNT observing size, morphology, protein adsorption, and cytotoxicity. Bundled SWNT are approximately 500 nm in diameter and we could debundle these SWNT to as small as 20 nm, making them ideal for internalization of macrophages and dendritic cells. We showed that b SWNT and d SWNT can both adsorb high concentrations of protein antigen, due to their high surface area. These nanoparticles were also shown to be nontoxic at lower concentrations, making them a practical biomaterial to be used in the body. We demonstrated that the high concentration of an antigen adsorbed onto the surface of these SWNT elicits an enhanced inflammatory response after presenting them to bone marrow dendritic cells. Lastly, data illustrated enhanced T cell stimulation. SWNT show a lot of promise as an aid to make better, more effectives vaccines. Introduction: Single walled carbon nanotubes (SWNT) are revolutionary nanoparticles that have been used in recent studies in the field of immunology. These nanotubes are composed of single graphite sheets that are wrapped into cylindrical tubes, which are known to have very high tensile strength.[1] Because of their morphology, nanotubes also have a considerably higher surface area than other nanoparticles (up to 1190 m2/g).[2] SWNTs extremely high tensile strength and surface

area make them perfect for biological applications: for example, vaccine delivery systems[3], adoptive cell transfer therapy[4], bioimaging[5], and biosensing[6]. SWNT tend to bundle due to Van der Waal forces between the carbon atoms. Using ultrasonication, which applies high power sound energy to agitate particles, we can debundle these SWNT into smaller particles. Making these nanoparticles even smaller, increases their ability to be internalized by immune system cells, such as dendritic cells. We can use the diversity in sizes of SWNT to find an optimal vaccine adjuvant. What we are most interested in is using SWNT in vaccine development to elicit an enhanced immune response than traditional vaccines. In traditional vaccines, antigens that are characteristic of a certain disease are injected into the patient and ultimately interact with the immune system cells causing an immune response. There are many specific mechanisms that occur during this immune response. One specific mechanism involves the dendritic cell (DC). These phagocytic cells scan their surroundings by taking up extracellular fluid. Once they have detected an antigen, a release of tumor necrosis factor alpha (TNF-) causes the DCs to travel to the nearest lymph node, causing an inflammatory response. Once in the lymph nodes, stored major histocompatibility complexes (MHC) from the DCs can expose the antigens to surrounding T cells. The T cells are then stimulated, activated and release cytokines, which go on to further help the immune system destroy these antigen. Once these immune system cells have fought off the antigen, some of the cells differentiate into memory cells that remember this specific antigen. These memory cells are more numerous and easier to activate than nave cells and remain in the body for long periods of time to protect the patient against the return of that antigen in the form of the actual disease.

Due to SWNTs high surface area, high concentrations of bioactive peptides, drugs, and protein antigens can be adsorbed onto its surface[1]. By functionalizing the SWNT with HNO 3[7], not only does it increase the solubility of the SWNT, making them more biocompatible[1], but even higher concentrations of these molecules can be adsorbed onto their surface[2]. We can take advantage of this by adsorbing antigens onto the surface of SWNT and present them to dendritic cells to be endocytosed. Then we can present the dendritic cells to T cells, mimicking the mechanism previously explained. We want to see if the high local concentration of antigens on the SWNT would inhibit a greater immune response than simply antigens in solution. In this paper we will first characterize both the bundled and debundled SWNT by studying their size, morphology, ability to adsorb protein and cytotoxicity. Then we will use a model of an immune response to look at SWNTs effectiveness in enhancing the immunogenicity of an antigen to be used for a vaccine.
Figure 1

Transmission electron microscopy (TEM) images of (left) b SWNT (middle) d SWNT with OVA (right) b SWNT starting to debundle. SWNT were negatively stained with uric acid.

Debundling of Single Walled Carbon Nanotubes (SWNT): We initially debundled the functionalized carbon nanotube bundles to observe and compare bundled SWNT (b SWNT) and debundled SWNT (d SWNT) and establish a protocol for debundling our particles. All nanotubes were previously functionalized with 3M HNO3. Three 5 ug/ml SWNT solutions were prepared using a stock solution of nanotubes in 15 mL phosphate buffer solution (PBS) in test tubes. Surfactant Tween 20 was added to prevent SWNTs from reaggregated into bundles and protein Ovalbumin (OVA) was added in order to view protein adsorption on nanotubes under TEM. Solutions were separated into 6 groups by amount of surfactant dispersant, and presence of protein. Test tubes were ultrasonicated for 2 minutes at 150 watts. Following ultrasonication, 150 l of tween 20 was added to the first test tube, 1.5 ml was added to the second, and no Tween 20 was added to the third; giving concentrations of 1%, 10%, and 0% Tween 20 respectively. Then each test tube was ultrasonicated for an additional 4 minutes in an ice bath to prevent overheating. Then 5 ml from each test tube was transferred to 3 corresponding test tubes, in which 2.5 mg of ovalbumin was added to each; giving us a 500ug/ml solution of OVA. Bundled SWNT (b SWNT) and debundled SWNT (d SWNT) with OVA were imaged using transmission electron microscopy (TEM). [Figure 1] SWNT bundles were as large as 500nm in diameter. A single nanotube (or nanoneedle) is about 1-5 nm in diameter. Though we could not debundle the SWNT completely, we were able to get the particle size down to as low as 20 nm in diameter. SWNT were successfully debundled as illustrated in the figure.

Figure 2

Adsorption isotherm of Ovalbumin on dSWNT and bSWNT

Determining SWNT ability to adsorb protein antigen: Next, we were interested in measuring SWNTs ability to adsorb protein on their surface. Ovalbumin was used because it is known to stimulate an allergic reaction and inhibit an immune response. 400l of 500g/ml solution of Ovalbumin was serially diluted at 200l in PBS solution in 8 eppendorf (ED) tubes. 200l of 100g/ml SWNT were added to each eppendorf tube. ED tubes were tumbled for 1 hour to ensure coating of protein on SWNT. Then SWNT + protein solutions were centrifuged for 20-25 minutes at 13,200 rpm. Supernatant from each ED tube was extracted and concentration of protein was measured using BCA and Micro BCA protein assays(reading absorbance at 562 nm). Then mass balance was used to calculate concentration of protein adsorbed onto the SWNT. The same procedure was followed for d SWNT of equal concentration. D SWNT were prepared by ultrasonicating SWNT for 6 minutes at 150 watts. Both bundled and debundled SWNT share similar protein adsorption capacities. B SWNTs slightly higher curve could be due to the structure of the bundle themselves, trapping protein like a net or

small discrepancies in concentration from the extraction of supernatant process. Moles of protein added versus moles of protein adsorbed were graphed and fitted using a one phase exponential association. [Figure 2] This shows that debundling these particles wont have a large affect on their ability to hold high concentrations of proteins on its surface. Knowing this, we can use a range of particles sizes without compromising protein concentration.
Table 1

Illustrating number of viable cells at different concentrations of SWNT

Cytotoxicity of SWNT on murine RAW (macrophages) cells: Cytotoxicity of SWNT was tested to see how toxic these particles were to cells and to determine an appropriate concentration of SWNT to use for experiments involving cells. 50g/ml solutions of b SWNT, d SWNT and d SWNT with OVA were prepared in sterile PBS. To avoid contamination of cells, SWNT were exposed to UV for 20-25 minutes, then resuspended in RPMI cell culture media(with 10% FBS and 2% penstrep). Splenocytes were extracted from wild type

mice and RAW cells were isolated using negative selection. Cells were counted and diluted to 2x105cells/ml in 20 ml. 100l of cells were added to two 96 well plates. SWNT were serially diluted at 100 l in two separate 96 well plates and then transferred to each well containing RAW cells. 5% sodium azide, which is known to kill cells, was used as a negative control and cells alone were used as the positive control. Cells were incubated for overnight at 37C and 5% CO2. 20 l CellTiter-Blue was added to each well. Florescence was measured using a spectrophotometer to measure viable cells. At the concentrations used (up to 50g/ml), SWNT did not show high levels of toxicity.[Table 1] Number of viable cells were fairly close to positive control. Due to SWNTs adsorption capacity, higher local concentrations of antigens can be presented to the immune system using relatively low concentrations of SWNT, making a safe and effective vaccine.
Figure 3

Graph of cytokine release with various SWNT treatment: TNF- (top left), IL-12 (top right), IL-2 (bottom left), INF-(bottom right)

Vaccine Experiment

Next, we looked at SWNT effectiveness in eliciting an immune response. A vaccine experiment was implemented to emulate how the immune system reacts to foreign antigens. Bone marrow dendritic cells (BMDC) were extracted from wild type mouse femurs. Next, splenocytes were extracted from OT-1 and OT-2 type mice spleens. OT-1 and OT-2 mice are genetically modified to have ovalbumin specific CD8+ cells or cytotoxic lymphocytes and CD4+ cells or helper T cells, respectively. Negative selection was used to isolate T cells from splenocytes and T cells were added to BMDCs. Equal concentrations of the following conditions were added to BMDCs and T cells in two 96 well plates: OVA alone, bSWNT, d SWNT, d SWNT without OVA, and serum free d SWNT. After 4 hours plate 1 was centrifuged for 5 minutes at 1000 rpm and supernatant was extracted. Using a sandwich enzyme-linked immunosorbent assay (ELISA), concentrations of TNF- and interleukin 12 (IL-12) were measured.[Figure 3] Absorbance was read at 450nm. After 48 hours, IL-2 and interferon gamma (INF) cytokines were measured in the same manner. Based on TNF- release, we can see that both debundled and bundled SWNT caused a greater inflammatory response than OVA alone. This also points to a heightened upregulation of MHC class 1 and 2 complexes. Using dSWNT alone as a negative control, we know that the nanoparticles themselves dont cause an inflammatory reaction[8], but the way in which the antigen is presented to the cells is completely responsible for this heightened response. No release of IL-12 was detected. A longer waiting period time might be required before a release of IL -12 occurs. IL-2 and IFN- are cytokines released by CD8+ and CD4+ cells upon stimulation and activation. IL-2 was shown to be present in equally high concentrations for all treatments except SWNT alone, showing high CD4+ stimulation. This could possibly be due to saturation of OVA and represent maximum IL-2 release. As for CD8+ cells, higher levels of IL-2 was associated with

the presence of both d SWNT and b SWNT than with OVA alone and no IL-2 for d SWNT alone. This again demonstrates higher stimulation of CD8+ cells with the aid of SWNT. However, the release of IL-2 is much lower with CD8+ cells than with CD4+ cells. This is because endocytosed antigens are normally displayed on MHC class 2 complexes, which interact solely with CD4+ cells. However, sometimes a process called cross-presentation occurs, in which he endocytosed antigen is displayed on a MHC class 1 complex and is presented to CD8+. This process of crosspresentation does not occur as frequently. Looking at IFN-, we see the same general trend in cytokine release. The discrepancy between release in CD4+ and CD8+ cells is due to the fact that CD8+ cells represent a greater cellular source of IFN-[9]. It is not certain why d SWNT in OT-1 group showed less cytokine release than OVA alone. The vaccine experiment was very useful in proving that SWNT do indeed augment the immunogenicity of an antigen. However, this experiment has room for optimization. These two cells play a critical role in the immune system. The release of these and many other cytokines are responsible for the proliferation of many immune system cells and activation of a multiple of mechanisms that help our bodies fight off harmful diseases. Conclusion Previous papers have tried different methods to use SWNT in vaccine delivery, using cell epitopes[8] and stimulatory antibodies[2], but we took a different approach than most of the available literature. Also, these papers deal with SWNT bundles only and do not investigate the use of debundled SWNT in their studies. Through our experiment we have successfully proved that SWNT can improve vaccines efficiency. This higher stimulation and proliferation of T cells ultimately leads to a greater immune

response within the patient. The most important aspect of this immune response is the establishment of memory cells to build immunity and protect a patient for long periods of time from many harmful diseases. Acknowledgements This work was supported by the Howard Hughes Medical Institute (HHMI), by National Human Genome Research Institute (NHGRI), by Yale College, and most importantly the STARS program for all of their teachings, support, and assistance. We would like to acknowledge Erin Steenblock for advice; Aimon Iftikhar for her help in obtaining BCA data; Dr. Christoph Rahner for use of TEM; Lindsay Soh, Betsy A. Smith, and Dr. K. P. for helpful comments on manuscript. References 1. Alberto Bianco: Applications of carbon nanotubes in drug delivery. Chem Bio 2005, 9:674679
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