In vivo targeting of intratumor regulatory T cells using PEG-modified single-walled carbon nanotubes.

Recent evidence regarding the role of regulatory T cells (Treg) in tumor development has suggested that the manipulation of Treg function selectively in the tumor microenvironment would be a desirable immunotherapy approach. Targeting intratumor immune populations would reduce side effects on peripheral healthy cells and increase antitumor efficacy of immunotherapies. However, no current approaches are available which enable selective in vivo targeting of intratumor Treg or other immune cell subpopulations. Herein, we investigated the ability of ligands against Treg-specific receptors to drive selective internalization of PEG-modified single-walled carbon nanotubes (PEG-SWCNTs) into Treg residing in the tumor microenvironment. We focused our attention on the glucocorticoid-induced TNFR-related receptor (GITR), as it showed higher overexpression on intratumor vs peripheral (i.e., splenic) Treg compared to other reported Treg-specific markers (folate receptor 4, CD103, and CD39). Ex vivo investigations showed that the Treg targeting efficiency and selectivity of PEG-SWCNTs depended on incubation time, dose, number of ligands per nanotube, and targeted surface marker. In vivo investigations showed that PEG-SWCNTs armed with GITR ligands targeted Treg residing in a B16 melanoma more efficiently then intratumor non-Treg or splenic Treg. The latter result was achieved by exploiting a combination of passive tumor targeting due to enhanced tumor vascular permeability, naturally increased intratumor Treg vs effector T cell (Teff) ratio, and active targeting of markers that are enriched in intratumor vs splenic Treg. We also found that PEG-SWCNTs loaded with GITR ligands were internalized by Treg through receptor-mediated endocytosis and transported into the cytoplasm and nucleus ex vivo and in vivo. This is the first example of intratumor immune cell targeting and we hope it will pave the way to innovative immunotherapies against cancer.

[1]  Andrew Tsourkas,et al.  Effect of ligand density, receptor density, and nanoparticle size on cell targeting. , 2013, Nanomedicine : nanotechnology, biology, and medicine.

[2]  Stefano Bellucci,et al.  Nanoparticles and Nanodevices in Biological Applications , 2009 .

[3]  V. Kuchroo,et al.  Adenosine generation catalyzed by CD39 and CD73 expressed on regulatory T cells mediates immune suppression , 2007, The Journal of experimental medicine.

[4]  N. Bottini,et al.  Cell-type specific and cytoplasmic targeting of PEGylated carbon nanotube-based nanoassemblies. , 2008, Journal of nanoscience and nanotechnology.

[5]  V. C. Moore,et al.  Band Gap Fluorescence from Individual Single-Walled Carbon Nanotubes , 2002, Science.

[6]  A. Rudensky,et al.  Regulatory T cell lineage specification by the forkhead transcription factor foxp3. , 2005, Immunity.

[7]  D. Scheinberg,et al.  Synthesis and biodistribution of oligonucleotide-functionalized, tumor-targetable carbon nanotubes. , 2008, Nano letters.

[8]  Hongjie Dai,et al.  Multiplexed multicolor Raman imaging of live cells with isotopically modified single walled carbon nanotubes. , 2008, Journal of the American Chemical Society.

[9]  T. Whiteside.  What are regulatory T cells (Treg) regulating in cancer and why? , 2012, Seminars in cancer biology.

[10]  Warren C W Chan,et al.  Nanoparticle-mediated cellular response is size-dependent. , 2008, Nature nanotechnology.

[11]  H. Dai,et al.  PEG branched polymer for functionalization of nanomaterials with ultralong blood circulation. , 2009, Journal of the American Chemical Society.

[12]  M. Colombo,et al.  Tumor-induced expansion of regulatory T cells by conversion of CD4+CD25- lymphocytes is thymus and proliferation independent. , 2006, Cancer research.

[13]  D. Mougiakakos,et al.  Regulatory T cells in cancer. , 2010, Advances in cancer research.

[14]  N. Zeps,et al.  Tumor-infiltrating FOXP3+ T regulatory cells show strong prognostic significance in colorectal cancer. , 2009, Journal of clinical oncology : official journal of the American Society of Clinical Oncology.

[15]  B. Chauffert,et al.  Tumor cells convert immature myeloid dendritic cells into TGF-β–secreting cells inducing CD4+CD25+ regulatory T cell proliferation , 2005, The Journal of experimental medicine.

[16]  Yasuhiko Tabata,et al.  Blood clearance and biodistribution of polymer brush-afforded silica particles prepared by surface-initiated living radical polymerization. , 2012, Biomacromolecules.

[17]  N. Bottini,et al.  Carbon nanotube-based nanocarriers: the importance of keeping it clean. , 2010, Journal of nanoscience and nanotechnology.

[18]  L. Terracciano,et al.  High frequency of tumor‐infiltrating FOXP3+ regulatory T cells predicts improved survival in mismatch repair‐proficient colorectal cancer patients , 2010, International journal of cancer.

[19]  M. Prato,et al.  Targeting carbon nanotubes against cancer. , 2012, Chemical communications.

[20]  Weibo Cai,et al.  Circulation and long-term fate of functionalized, biocompatible single-walled carbon nanotubes in mice probed by Raman spectroscopy , 2008, Proceedings of the National Academy of Sciences.

[21]  J. Shimizu,et al.  Stimulation of CD25+CD4+ regulatory T cells through GITR breaks immunological self-tolerance , 2002, Nature Immunology.

[22]  Bing Yan,et al.  Endosomal leakage and nuclear translocation of multiwalled carbon nanotubes: developing a model for cell uptake. , 2009, Nano letters.

[23]  J. Karp,et al.  Nanocarriers as an Emerging Platform for Cancer Therapy , 2022 .

[24]  Stanislaus S. Wong,et al.  Functionalized single-walled carbon nanotubes as rationally designed vehicles for tumor-targeted drug delivery. , 2008, Journal of the American Chemical Society.

[25]  E. Jaffee,et al.  Recruitment of latent pools of high-avidity CD8+ T cells to the antitumor immune response , 2005, The Journal of experimental medicine.

[26]  H. Levitsky,et al.  Natural Regulatory T Cells and De Novo-Induced Regulatory T Cells Contribute Independently to Tumor-Specific Tolerance1 , 2007, The Journal of Immunology.

[27]  S. Sakaguchi,et al.  Control of immune responses by antigen-specific regulatory T cells expressing the folate receptor. , 2007, Immunity.

[28]  D. Scheinberg,et al.  Tumor Targeting with Antibody-Functionalized, Radiolabeled Carbon Nanotubes , 2007, Journal of Nuclear Medicine.

[29]  Omar K. Yaghi,et al.  In vivo fluorescence imaging in the second near-infrared window with long circulating carbon nanotubes capable of ultrahigh tumor uptake. , 2012, Journal of the American Chemical Society.

[30]  Zhuang Liu,et al.  Drug delivery with carbon nanotubes for in vivo cancer treatment. , 2008, Cancer research.

[31]  Nunzio Bottini,et al.  Surface polyethylene glycol conformation influences the protein corona of polyethylene glycol-modified single-walled carbon nanotubes: potential implications on biological performance. , 2013, ACS nano.

[32]  Nicholas Melosh,et al.  Shape matters: intravital microscopy reveals surprising geometrical dependence for nanoparticles in tumor models of extravasation. , 2012, Nano letters.

[33]  R K Jain,et al.  Physiologically based pharmacokinetic modeling: principles and applications. , 1983, Journal of pharmaceutical sciences.

[34]  Sanjiv S Gambhir,et al.  A pilot toxicology study of single-walled carbon nanotubes in a small sample of mice. , 2008, Nature nanotechnology.

[35]  G. Dranoff,et al.  Combining immunotherapy and targeted therapies in cancer treatment , 2012, Nature Reviews Cancer.

[36]  Hari Singh Nalwa,et al.  Medical applications of nanoparticles in biological imaging, cell labeling, antimicrobial agents, and anticancer nanodrugs. , 2011, Journal of biomedical nanotechnology.

[37]  F. Toma,et al.  Degree of chemical functionalization of carbon nanotubes determines tissue distribution and excretion profile. , 2012, Angewandte Chemie.

[38]  M. Colombo,et al.  Regulatory T-cell inhibition versus depletion: the right choice in cancer immunotherapy , 2007, Nature Reviews Cancer.

[39]  J. Ellwart,et al.  CD103 is a hallmark of tumor‐infiltrating regulatory T cells , 2011, International journal of cancer.

[40]  Nunzio Bottini,et al.  PEG-modified carbon nanotubes in biomedicine: current status and challenges ahead. , 2011, Biomacromolecules.

[41]  Zhuang Liu,et al.  A route to brightly fluorescent carbon nanotubes for near-infrared imaging in mice. , 2009, Nature nanotechnology.

[42]  V. Muzykantov,et al.  Multifunctional Nanoparticles: Cost Versus Benefit of Adding Targeting and Imaging Capabilities , 2012, Science.