Diego A. Rey

Single-Walled Carbon Nanotubes Deliver Peptide Antigen into Dendritic Cells and Enhance IgG Responses to Tumor-Associated Antigens

We studied the feasibility of using single-wall carbon nanotubes (SWNTs) as antigen carriers to improve immune responses to peptides that are weak immunogens, a characteristic typical of human tumor antigens. Binding and presentation of peptide antigens by the MHC molecules of antigen presenting cells (APCs) is essential to mounting an effective immune response. The Wilm’s tumor protein (WT1) is upregulated in many human leukemias and cancers and several vaccines directed at this protein are in human clinical trials. WT1 peptide 427 induces human CD4 T cell responses in the context of multiple human HLA-DR.B1 molecules, but the peptide has a poor binding affinity to BALB/c mouse MHC class II molecules. We used novel, spectrally quantifiable chemical approaches to covalently append large numbers of peptide ligands (0.4 mmol/g) onto solubilized SWNT scaffolds. Peptide-SWNT constructs were rapidly internalized into professional APCs (dendritic cells and macrophages) within minutes in vitro, in a dose dependent manner. Immunization of BALB/c mice with the SWNT–peptide constructs mixed with immunological adjuvant induced specific IgG responses against the peptide, while the peptide alone or peptide mixed with the adjuvant did not induce such a response. The conjugation of the peptide to SWNT did not enhance the peptide-specific CD4 T cell response in human and mouse cells, in vitro. The solubilized SWNTs alone were nontoxic in vitro, and we did not detect antibody responses to SWNT in vivo. These results demonstrated that SWNTs are able to serve as antigen carriers for delivery into APCs to induce humoral immune responses against weak tumor antigens.

Paradoxical glomerular filtration of carbon nanotubes

The molecular weight cutoff for glomerular filtration is thought to be 30–50 kDa. Here we report rapid and efficient filtration of molecules 10–20 times that mass and a model for the mechanism of this filtration. We conducted multimodal imaging studies in mice to investigate renal clearance of a single-walled carbon nanotube (SWCNT) construct covalently appended with ligands allowing simultaneous dynamic positron emission tomography, near-infrared fluorescence imaging, and microscopy. These SWCNTs have a length distribution ranging from 100 to 500 nm. The average length was determined to be 200–300 nm, which would yield a functionalized construct with a molecular weight of ∼350–500 kDa. The construct was rapidly (t1/2 ∼ 6 min) renally cleared intact by glomerular filtration, with partial tubular reabsorption and transient translocation into the proximal tubular cell nuclei. Directional absorption was confirmed in vitro using polarized renal cells. Active secretion via transporters was not involved. Mathematical modeling of the rotational diffusivity showed the tendency of flow to orient SWCNTs of this size to allow clearance via the glomerular pores. Surprisingly, these results raise questions about the rules for renal filtration, given that these large molecules (with aspect ratios ranging from 100:1 to 500:1) were cleared similarly to small molecules. SWCNTs and other novel nanomaterials are being actively investigated for potential biomedical applications, and these observations—that high aspect ratio as well as large molecular size have an impact on glomerular filtration—will allow the design of novel nanoscale-based therapeutics with unusual pharmacologic characteristics.

PET Imaging of Soluble Yttrium-86-Labeled Carbon Nanotubes in Mice

Background The potential medical applications of nanomaterials are shaping the landscape of the nanobiotechnology field and driving it forward. A key factor in determining the suitability of these nanomaterials must be how they interface with biological systems. Single walled carbon nanotubes (CNT) are being investigated as platforms for the delivery of biological, radiological, and chemical payloads to target tissues. CNT are mechanically robust graphene cylinders comprised of sp2-bonded carbon atoms and possessing highly regular structures with defined periodicity. CNT exhibit unique mechanochemical properties that can be exploited for the development of novel drug delivery platforms. In order to evaluate the potential usefulness of this CNT scaffold, we undertook an imaging study to determine the tissue biodistribution and pharmacokinetics of prototypical DOTA-functionalized CNT labeled with yttrium-86 and indium-111 (86Y-CNT and 111In-CNT, respectively) in a mouse model. Methodology and Principal Findings The 86Y-CNT construct was synthesized from amine-functionalized, water-soluble CNT by covalently attaching multiple copies of DOTA chelates and then radiolabeling with the positron-emitting metal-ion, yttrium-86. A gamma-emitting 111In-CNT construct was similarly prepared and purified. The constructs were characterized spectroscopically, microscopically, and chromatographically. The whole-body distribution and clearance of yttrium-86 was characterized at 3 and 24 hours post-injection using positron emission tomography (PET). The yttrium-86 cleared the blood within 3 hours and distributed predominantly to the kidneys, liver, spleen and bone. Although the activity that accumulated in the kidney cleared with time, the whole-body clearance was slow. Differential uptake in these target tissues was observed following intraveneous or intraperitoneal injection. Conclusions The whole-body PET images indicated that the major sites of accumulation of activity resulting from the administration of 86Y-CNT were the kidney, liver, spleen, and to a much less extent the bone. Blood clearance was rapid and could be beneficial in the use of short-lived radionuclides in diagnostic applications.

Gold hybrid nanoparticles for targeted phototherapy and cancer imaging

Gold and iron oxide hybrid nanoparticles (HNPs) synthesized by the thermal decomposition technique are bio-functionalized with a single chain antibody, scFv, that binds to the A33 antigen present on colorectal cancer cells. The HNP-scFv conjugates are stable in aqueous solution with a magnetization value of 44 emu g(-1) and exhibit strong optical absorbance at 800 nm. Here we test this material in targeting, imaging and selective thermal killing of colorectal cancer cells. Cellular uptake studies showed that A33-expressing cells take up the A33scFv-conjugated HNPs at a rate five times higher than cells that do not express the A33 antigen. Laser irradiation studies showed that approximately 53% of the A33-expressing cells exposed to targeted HNPs are killed after a six-minute laser treatment at 5.1 W cm(-2) using a 808 nm continuous wave laser diode while < 5% of A33-nonexpressing cells are killed. At a higher intensity, 31.5 W cm(-2), the thermal destruction increases to 99 and 40% for A33-expressing cells and A33 nonexpressing cells, respectively, after 6 min exposure. Flow cytometric analyses of the laser-irradiated A33 antigen-expressing cells show apoptosis-related cell death to be the primary mode of cell death at 5.1 W cm(-2), with increasing necrosis-related cell death at higher laser power. These results suggest that this new class of bio-conjugated hybrid nanoparticles can potentially serve as an effective antigen-targeted photothermal therapeutic agent for cancer treatment as well as a probe for magnetic resonance-based imaging.

Synthesis and Biodistribution of Oligonucleotide-Functionalized, Tumor-Targetable Carbon Nanotubes

Single-wall carbon nanotubes (SWNT) show promise as nanoscale vehicles for targeted therapies. We have functionalized SWNT using regioselective chemistries to confer capabilities of selective targeting using RGD ligands, radiotracing using radiometal chelates, and self-assembly using oligonucleotides. The constructs contained approximately 2-7 phosphorothioate oligonucleotide chains and 50-75 amines per 100 nm length of SWNT, based on a loading of 0.01-0.05 mmol/g and 0.3-0.6 mmol/g, respectively. Dynamic light scattering suggested the functionalized SWNT were well dispersed, without formation of large aggregates in physiologic solutions. The SWNT-oligonucleotide conjugate annealed with a complementary oligonucleotide sequence had a melting temperature of 54 degrees C. Biodistribution in mice was quantified using radiolabeled SWNT-oligonucleotide conjugates. Appended RGD ligands allowed for specific binding to tumor cells in a flow cytometric assay. The techniques employed should enable the synthesis of multifunctional SWNT capable of self-assembly in biological settings.

Combined strategy for the dispersion/dissolution of single walled carbon nanotubes and cellulose in water

Co-dispersion of native cellulose and single walled carbon nanotubes in water is demonstrated. The pH of the water should be between 6 and 10 for better dispersion. Raman spectra confirm debundling of nanotubes in water and FTIR spectra reveal that the co-solubility is likely caused through disruption of intramolecular hydrogen bonds in the cellulose by hydroxyl groups present on nanotubes surface and the creation of intermolecular hydrogen bonds between cellulose and carbon nanotubes. This is a very simple method for co-dispersion/dissolution of nanotubes and cellulose in water without covalent modifications and expands the repertoire of nanotube modification strategies that are amenable to biological applications.

Silica-deposited phospholipid nanotubules as a plausible drug targeting system

An aqueous dispersion of self-organized phospholipid tubules has been utilized as the template for silica-deposited nanotubules (∼0.5 μm thick and >10 μm long) by a sol–gel method. The formation of the hybrid tubules was mechanistically investigated by controlled sol–gel reaction. The incorporation of silica increases the mechanical and thermal stability of tubule geometry. After bioconjugating Ni2+-nitrilotriacetic acid (Ni-NTA) to the surface of chemically modified tubules containing primary amine groups, green fluorescent protein (GFP)-6 His and A33scFv-6 His were further bioconjugated in order to investigate a potential application of these hollow silica tubules as vehicle for targeted controlled release. The resulting tubules bound and internalized to SW1222 endothelial human colon carcinoma cells that express the A33 cell-surface glycoprotein more specifically than HT29 cells that do not express this antigen.

In vitro self-assembly of gold nanoparticle-coated poly(3-hydroxybutyrate) granules exhibiting plasmon-induced thermo-optical enhancements.

Polyhydroxyalkanoate (PHA) synthase attached to gold nanoparticles (AuNP) produce poly(3-hydroxybutyrate) (PHB) upon the addition of 3-hydroxybutyrate-CoA, and then coalesce to form micrometer-sized AuNP-coated PHB granules. These AuNP-coated PHB granules are potential theranostic agents that have enhanced imaging capabilities and are capable of heating upon near-infrared laser irradiation. The AuNP-coated PHB exhibited 11-fold enhancement in surface-enhanced Raman scattering over particles prior polymerization. Stained AuNP-coated PHB exhibited a 6-fold enhancement in fluorescence intensity as well as a 1.3-fold decrease in photobleaching rate compared to PHB granules alone. The granules were also shown to emit heat when illuminated at 808 nm with a 3.9-fold increase in heating rate compared to particles alone.