Targeted Nanocarriers Co-Opting Pulmonary Intravascular Leukocytes for Drug Delivery to the Injured Brain.

Ex vivo-loaded white blood cells (WBC) can transfer cargo to pathological foci in the central nervous system (CNS). Here we tested affinity ligand driven in vivo loading of WBC in order to bypass the need for ex vivo WBC manipulation. We used a mouse model of acute brain inflammation caused by local injection of tumor necrosis factor alpha (TNF-α). We intravenously injected nanoparticles targeted to intercellular adhesion molecule 1 (anti-ICAM/NP). We found that (A) at 2 h, >20% of anti-ICAM/NP were localized to the lungs; (B) of the anti-ICAM/NP in the lungs >90% were associated with leukocytes; (C) at 6 and 22 h, anti-ICAM/NP pulmonary uptake decreased; (D) anti-ICAM/NP uptake in brain increased up to 5-fold in this time interval, concomitantly with migration of WBCs into the injured brain. Intravital microscopy confirmed transport of anti-ICAM/NP beyond the blood-brain barrier and flow cytometry demonstrated complete association of NP with WBC in the brain (98%). Dexamethasone-loaded anti-ICAM/liposomes abrogated brain edema in this model and promoted anti-inflammatory M2 polarization of macrophages in the brain. In vivo targeted loading of WBC in the intravascular pool may provide advantages of coopting WBC predisposed to natural rapid mobilization from the lungs to the brain, connected directly via conduit vessels.

Selective targeting of nanomedicine to inflamed cerebral vasculature to enhance the blood-brain barrier.

Drug targeting to inflammatory brain pathologies such as stroke and traumatic brain injury remains an elusive goal. Using a mouse model of acute brain inflammation induced by local tumor necrosis factor alpha (TNFα), we found that uptake of intravenously injected antibody to vascular cell adhesion molecule 1 (anti-VCAM) in the inflamed brain is >10-fold greater than antibodies to transferrin receptor-1 and intercellular adhesion molecule 1 (TfR-1 and ICAM-1). Furthermore, uptake of anti-VCAM/liposomes exceeded that of anti-TfR and anti-ICAM counterparts by ∼27- and ∼8-fold, respectively, achieving brain/blood ratio >300-fold higher than that of immunoglobulin G/liposomes. Single-photon emission computed tomography imaging affirmed specific anti-VCAM/liposome targeting to inflamed brain in mice. Intravital microscopy via cranial window and flow cytometry showed that in the inflamed brain anti-VCAM/liposomes bind to endothelium, not to leukocytes. Anti-VCAM/LNP selectively accumulated in the inflamed brain, providing de novo expression of proteins encoded by cargo messenger RNA (mRNA). Anti-VCAM/LNP-mRNA mediated expression of thrombomodulin (a natural endothelial inhibitor of thrombosis, inflammation, and vascular leakage) and alleviated TNFα-induced brain edema. Thus VCAM-directed nanocarriers provide a platform for cerebrovascular targeting to inflamed brain, with the goal of normalizing the integrity of the blood-brain barrier, thus benefiting numerous brain pathologies.

Combining vascular targeting and the local first pass provides 100-fold higher uptake of ICAM-1-targeted vs untargeted nanocarriers in the inflamed brain.

New advances in intra-arterial (IA) catheters offer clinically proven local interventions in the brain. Here we tested the effect of combining local IA delivery and vascular immunotargeting. Microinjection of tumor necrosis factor alpha (TNFα) in the brain parenchyma causes cerebral overexpression of Inter-Cellular Adhesion Molecule-1 (ICAM-1) in mice. Systemic intravenous injection of ICAM-1 antibody (anti-ICAM-1) and anti-ICAM-1/liposomes provided nearly an order of magnitude higher uptake in the inflamed vs normal brain (from ~0.1 to 0.8%ID/g for liposomes). Local injection of anti-ICAM-1 and anti-ICAM-1/liposomes via carotid artery catheter provided an additional respective 2-fold and 5-fold elevation of uptake in the inflamed brain vs levels attained by IV injection. The uptake in the inflamed brain of respective untargeted IgG counterparts was markedly lower (e.g., uptake of anti-ICAM-1/liposomes was 100-fold higher vs IgG/liposomes). These data affirm the specificity of the combined effect of the first pass and immunotargeting. Intravital real-time microscopy via cranial window revealed that anti-ICAM-1/liposomes, but not IgG/liposomes bind to the lumen of blood vessels in the inflamed brain within minutes after injection. This straightforward framework provides the basis for translational efforts towards local vascular drug targeting to the brain.

Biocompatible coupling of therapeutic fusion proteins to human erythrocytes.

Carriage of drugs by red blood cells (RBCs) modulates pharmacokinetics, pharmacodynamics, and immunogenicity. However, optimal targets for attaching therapeutics to human RBCs and adverse effects have not been studied. We engineered nonhuman-primate single-chain antibody fragments (scFvs) directed to human RBCs and fused scFvs with human thrombomodulin (hTM) as a representative biotherapeutic cargo (hTM-scFv). Binding fusions to RBCs on band 3/glycophorin A (GPA; Wright b [Wrb] epitope) and RhCE (Rh17/Hr0 epitope) similarly endowed RBCs with hTM activity, but differed in their effects on RBC physiology. scFv and hTM-scFv targeted to band 3/GPA increased membrane rigidity and sensitized RBCs to hemolysis induced by mechanical stress, while reducing sensitivity to hypo-osmotic hemolysis. Similar properties were seen for other ligands bound to GPA and band 3 on human and murine RBCs. In contrast, binding of scFv or hTM-scFv to RhCE did not alter deformability or sensitivity to mechanical and osmotic stress at similar copy numbers bound per RBCs. Contrasting responses were also seen for immunoglobulin G antibodies against band 3, GPA, and RhCE. RBC-bound hTM-scFv generated activated protein C (APC) in the presence of thrombin, but RhCE-targeted hTM-scFv demonstrated greater APC generation per bound copy. Both Wrb- and RhCE-targeted fusion proteins inhibited fibrin deposition induced by tumor necrosis factor-α in an endothelialized microfluidic model using human whole blood. RhCE-bound hTM-scFv more effectively reduced platelet and leukocyte adhesion, whereas anti-Wrb scFv appeared to promote platelet adhesion. These data provide a translational framework for the development of engineered affinity ligands to safely couple therapeutics to human RBCs.

Molecular engineering of antibodies for site-specific covalent conjugation using CRISPR/Cas9.

Site-specific modification of antibodies has become a critical aspect in the development of next-generation immunoconjugates meeting criteria of clinically acceptable homogeneity, reproducibility, efficacy, ease of manufacturability, and cost-effectiveness. Using CRISPR/Cas9 genomic editing, we developed a simple and novel approach to produce site-specifically modified antibodies. A sortase tag was genetically incorporated into the C-terminal end of the third immunoglobulin heavy chain constant region (CH3) within a hybridoma cell line to manufacture antibodies capable of site-specific conjugation. This enabled an effective enzymatic site-controlled conjugation of fluorescent and radioactive cargoes to a genetically tagged mAb without impairment of antigen binding activity. After injection in mice, these immunoconjugates showed almost doubled specific targeting in the lung vs. chemically conjugated maternal mAb, and concomitant reduction in uptake in the liver and spleen. The approach outlined in this work provides a facile method for the development of more homogeneous, reproducible, effective, and scalable antibody conjugates for use as therapeutic and diagnostic tools.

Site-Specific Modification of Single-Chain Antibody Fragments for Bioconjugation and Vascular Immunotargeting.

The conjugation of antibodies to drugs and drug carriers improves delivery to target tissues. Widespread implementation and effective translation of this pharmacologic strategy awaits the development of affinity ligands capable of a defined degree of modification and highly efficient bioconjugation without loss of affinity. To date, such ligands are lacking for the targeting of therapeutics to vascular endothelial cells. To enable site-specific, click-chemistry conjugation to therapeutic cargo, we used the bacterial transpeptidase, sortase A, to attach short azidolysine containing peptides to three endothelial-specific single chain antibody fragments (scFv). While direct fusion of a recognition motif (sortag) to the scFv C-terminus generally resulted in low levels of sortase-mediated modification, improved reaction efficiency was observed for one protein, in which two amino acids had been introduced during cloning. This prompted insertion of a short, semi-rigid linker between scFv and sortag. The linker significantly enhanced modification of all three proteins, to the extent that unmodified scFv could no longer be detected. As proof of principle, purified, azide-modified scFv was conjugated to the antioxidant enzyme, catalase, resulting in robust endothelial targeting of functional cargo in vitro and in vivo.

Rapid prediction of stem cell mobilization using volume and conductivity data from automated hematology analyzers.

BACKGROUND: Rapid analytics to predict circulating hematopoietic stem cells are valuable for optimal management of mobilization, particularly for the use of newer and costly mobilization agents such as plerixafor. STUDY DESIGN AND METHODS: We used stepwise, linear multiple regression modeling applied to cell population data collected by routine hematology analyzers (Beckman Coulter DxH 800) on patients undergoing autologous stem cell collection (n = 131). Beta coefficients were used to derive a formula for a stem cell index (SCI). We then tested the correlation of SCI with stem cell counts and performance of the SCI as a predictor of poor mobilization with external validation in a separate cohort (n = 183). RESULTS: The SCI correlated strongly with CD34 counts by flow cytometry (r = 0.8372 in the development cohort, r = 0.8332 in the external validation cohort) and compares favorably with other rapid stem cell enumerating technologies. In the external validation cohort, the SCI performed well as a predictor (receiver operating characteristic area under the curve, 0.9336) of poor mobilization (CD34 count < 10), with a sensitivity of 72% and a specificity of 93%. When prevalence of poor mobilization was 33%, this resulted in a positive predictive value of 83% and a negative predictive value of 87%. The SCI also showed promise in tracking responses to plerixafor administration. CONCLUSION: The findings demonstrate the utility of the cell population data collected by hematology analyzers to provide rapid data beyond standard complete blood counts, particularly for stem cell count prediction, requiring no additional reagents, specimen, or instrumentation.

Mechanism of Collaborative Enhancement of Binding of Paired Antibodies to Distinct Epitopes of Platelet Endothelial Cell Adhesion Molecule-1.

Monoclonal antibodies (mAbs) directed to extracellular epitopes of human and mouse Platelet Endothelial Cell Adhesion Molecule-1 (CD31 or PECAM-1) stimulate binding of other mAbs to distinct adjacent PECAM-1 epitopes. This effect, dubbed Collaborative Enhancement of Paired Affinity Ligands, or CEPAL, has been shown to enhance delivery of mAb-targeted drugs and nanoparticles to the vascular endothelium. Here we report new insights into the mechanism underlying this effect, which demonstrates equivalent amplitude in the following models: i) cells expressing a full length PECAM-1 and mutant form of PECAM-1 unable to form homodimers; ii) isolated fractions of cellular membranes; and, iii) immobilized recombinant PECAM-1. These results indicate that CEPAL is mediated not by interference in cellular functions or homophilic PECAM-1 interactions, but rather by conformational changes within the cell adhesion molecule induced by ligand binding. This mechanism, mediated by exposure of partially occult epitopes, is likely to occur in molecules other than PECAM-1 and may represent a generalizable phenomenon with valuable practical applications.

ICAM-1-targeted thrombomodulin mitigates tissue factor-driven inflammatory thrombosis in a human endothelialized microfluidic model.

Diverse human illnesses are characterized by loss or inactivation of endothelial thrombomodulin (TM), predisposing to microvascular inflammation, activation of coagulation, and tissue ischemia. Single-chain antibody fragment (scFv)/TM) fusion proteins, previously protective against end-organ injury in murine models of inflammation, are attractive candidates to treat inflammatory thrombosis. However, animal models have inherent differences in TM and coagulation biology, are limited in their ability to resolve and control endothelial biology, and do not allow in-depth testing of "humanized" scFv/TM fusion proteins, which are necessary for translation to the clinical domain. To address these challenges, we developed a human whole-blood, microfluidic model of inflammatory, tissue factor (TF)-driven coagulation that features a multichannel format for head-to-head comparison of therapeutic approaches. In this model, fibrin deposition, leukocyte adhesion, and platelet adhesion and aggregation showed a dose-dependent response to tumor necrosis factor-α activation and could be quantified via real-time microscopy. We used this model to compare hTM/R6.5, a humanized, intracellular adhesion molecule 1 (ICAM-1)-targeted scFv/TM biotherapeutic, to untargeted antithrombotic agents, including soluble human TM (shTM), anti-TF antibodies, and hirudin. The targeted hTM/R6.5 more effectively inhibited TF-driven coagulation in a protein C (PC)-dependent manner and demonstrated synergy with supplemental PC. These results support the translational prospects of ICAM-targeted scFv/TM and illustrate the utility of the microfluidic system as a platform to study humanized therapeutics at the interface of endothelium and whole blood under flow.

Drug delivery by erythrocytes: "Primum non nocere".

Red blood cells (RBCs) are naturally capable of transporting diverse cargoes throughout the circulatory system, both loaded to their surface or within their inner volume. Starting largely from the 1970s, diverse approaches for encapsulation into, and surface coupling onto, RBCs have been investigated as potential drug delivery systems. In the last decade, these efforts have yielded diverse strategies to load drugs and nanocarriers to RBCs, and to optimize their pharmacokinetics, distribution, and effects in the body. Several formulations of donor RBCs encapsulated with enzymes and drugs are currently undergoing clinical trials for treatment of oncologic and neurologic conditions. Newer approaches include design of drugs with an affinity to circulating RBCs, encapsulation into RBCs using membrane permeating compounds, and design of hybrid drug delivery systems combining synthetic components with fragments of RBC membranes. Notwithstanding the growing enthusiasm and optimism in RBC drug delivery, in this article we discuss potentially problematic issues of this biomedical concept, especially impairment of biocompatibility of the carrier RBCs, and other adverse and unintended effects. Rigorous and systematic analysis of the cautionary aspects described in this article should be further developed and extended in order to soberly gauge the risk/benefit balance of any given RBC-based drug delivery application. While there is little doubt that RBC drug delivery will ultimately flourish, focusing research efforts on approaches that are unlikely to cause adverse effects in patients will help to sooner bring this day.

Encapsulation of α-Particle-Emitting 225Ac3+ Ions Within Carbon Nanotubes.

UNLABELLED: (225)Ac(3+) is a generator of α-particle-emitting radionuclides with 4 net α-particle decays that can be used therapeutically. Targeting (225)Ac(3+) by use of ligands conjugated to traditional bifunctional chelates limits the amount of (225)Ac(3+) that can be delivered. Ultrashort, single-walled carbon nanotubes (US-tubes), previously demonstrated as sequestering agents of trivalent lanthanide ions and small molecules, also successfully incorporate (225)Ac(3+). METHODS: Aqueous loading of both (225)Ac(3+) ions and Gd(3+) ions via bath sonication was used to construct (225)Ac@gadonanotubes ((225)Ac@GNTs). The (225)Ac@GNTs were subsequently challenged with heat, time, and human serum. RESULTS: US-tubes internally loaded with both (225)Ac(3+) ions and Gd(3+) ions show 2 distinct populations of (225)Ac(3+) ions: one rapidly lost in human serum and one that remains bound to the US-tubes despite additional challenge with heat, time, and serum. The presence of the latter population depended on cosequestration of Gd(3+) and (225)Ac(3+) ions. CONCLUSION: US-tubes successfully sequester (225)Ac(3+) ions in the presence of Gd(3+) ions and retain them after a human serum challenge, rendering (225)Ac@GNTs candidates for radioimmunotherapy for delivery of (225)Ac(3+) ions at higher concentrations than is currently possible for traditional ligand carriers.

Nontranscriptional role of Hif-1α in activation of γ-secretase and notch signaling in breast cancer.

γ-Secretase is composed of four proteins that are obligatory for protease activity: presenilin, nicastrin, Aph1, and Pen-2. Despite the progress toward understanding the function of these individual subunits, there is no information available pertaining to the modulation of γ-secretase in response to environmental changes in cells. Here, we show that hypoxia upregulates γ-secretase activity through a direct interaction with Hif-1α, revealing an unconventional function for Hif-1α as an enzyme subunit, which is distinct from its canonical role as a transcription factor. Moreover, hypoxia-induced cell invasion and metastasis are alleviated by either γ-secretase inhibitors or a dominant-negative Notch coactivator, indicating that γ-secretase/Notch signaling plays an essential role in controlling these cellular processes. The present study reveals a mechanism in which γ-secretase can achieve temporal control through conditional interactions with regulatory proteins, such as Hif-1α, under select physiological and pathological conditions.

Self-assembly of carbon nanotubes and antibodies on tumours for targeted amplified delivery.

Single-walled carbon nanotubes (SWNTs) can deliver imaging agents or drugs to tumours and offer significant advantages over approaches based on antibodies or other nanomaterials. In particular, the nanotubes can carry a substantial amount of cargo (100 times more than a monoclonal antibody), but can still be rapidly eliminated from the circulation by renal filtration, like a small molecule, due to their high aspect ratio. Here we show that SWNTs can target tumours in a two-step approach in which nanotubes modified with morpholino oligonucleotide sequences bind to cancer cells that have been pretargeted with antibodies modified with oligonucleotide strands complementary to those on the nanotubes. The nanotubes can carry fluorophores or radioisotopes, and are shown to selectively bind to cancer cells in vitro and in tumour-bearing xenografted mice. The binding process is also found to lead to antigen capping and internalization of the antibody-nanotube complexes. The nanotube conjugates were labelled with both alpha-particle and gamma-ray emitting isotopes, at high specific activities. Conjugates labelled with alpha-particle-generating (225)Ac were found to clear rapidly, thus mitigating radioisotope toxicity, and were shown to be therapeutically effective in vivo.

Addition of plerixafor to mobilization regimens in autologous peripheral blood stem cell transplants does not affect the correlation of preharvest hematopoietic precursor cell enumeration with first-harvest CD34+ stem cell yield.

The CXCR4 antagonist plerixafor is increasingly used in the mobilization regimens for autologous peripheral blood stem cell (PBSC) transplantation. This agent may mobilize a different subset of the stem cell population than traditional regimens, such as growth factors (with and without chemotherapy). Thus, it is important to determine whether plerixafor has an effect on the utility of measurements used to predict the yield of CD34(+) cells, usually either preharvest peripheral blood CD34(+) enumeration by flow cytometry or hematopoietic precursor cell (HPC) enumeration by automated hematology analysis. Although HPC enumeration has a weaker correlation with first-harvest CD34(+) cell yield, this parameter still plays an important role in the timing of apheresis procedures for autologous PBSC transplantation because of its technical simplicity and low cost. In the present study, we retrospectively examined the correlation of HPC measurements with CD34(+) cell yields in patients with multiple myeloma and lymphoma undergoing autologous PBSC transplantation, and investigated how the mobilization regimen affected these results. We found that the correlation coefficients ranged from 0.5877 to 0.7668 and were not significantly impacted by differences in diagnosis or inclusion of plerixafor in the mobilization regimen. The predictive ability of HPC enumeration for various target yields was also examined, and receiver-operating characteristic curves were generated. An HPC cutoff of 20 should result in adequate initial CD34(+) cell yields (>2.5 × 10(6) cell/kg) in >80% of autologous donors with or without plerixafor. This study confirms the utility of HPC enumeration in prediction of adequate initial cell yields, and demonstrates that this utility is maintained regardless of whether or not plerixafor is included in the mobilization regimen.

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.

Imaging and treating tumor vasculature with targeted radiolabeled carbon nanotubes.

Single wall carbon nanotube (SWCNT) constructs were covalently appended with radiometal-ion chelates (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid [DOTA] or desferrioxamine B [DFO]) and the tumor neovascular-targeting antibody E4G10. The E4G10 antibody specifically targeted the monomeric vascular endothelial-cadherin (VE-cad) epitope expressed in the tumor angiogenic vessels. The construct specific activity and blood compartment clearance kinetics were significantly improved relative to corresponding antibodyalone constructs. We performed targeted radioimmunotherapy with a SWCNT-([(225)Ac]DOTA) (E4G10) construct directed at the tumor vasculature in a murine xenograft model of human colon adenocarcinoma (LS174T). The specific construct reduced tumor volume and improved median survival relative to controls. We also performed positron emission tomographic (PET) radioimmunoimaging of the tumor vessels with a SWCNT-([(89)Zr]DFO)(E4G10) construct in the same murine LS174T xenograft model and compared the results to appropriate controls. Dynamic and longitudinal PET imaging of LS174T tumor-bearing mice demonstrated rapid blood clearance (<1 hour) and specific tumor accumulation of the specific construct. Incorporation of the SWCNT scaffold into the construct design permitted us to amplify the specific activity to improve the signal-to-noise ratio without detrimentally impacting the immunoreactivity of the targeting antibody moiety. Furthermore, we were able to exploit the SWCNT pharmacokinetic (PK) profile to favorably alter the blood clearance and provide an advantage for rapid imaging. Near-infrared three-dimensional fluorescent-mediated tomography was used to image the LS174T tumor model, collect antibody-alone PK data, and calculate the number of copies of VE-cad epitope per cell. All of these studies were performed as a single administration of construct and were found to be safe and well tolerated by the murine model. These data have implications that support further imaging and radiotherapy studies using a SWCNT-based platform and focusing on the tumor vessels as the target.

Selective killing of tumor neovasculature paradoxically improves chemotherapy delivery to tumors.

Antiangiogenic therapies are frequently used with concomitantly administered cancer chemotherapy to improve outcomes, but the mechanism for the benefit of the combination is uncertain. We describe a mechanism by which a specific, cytotoxic antivascular agent causes vascular remodeling and improved chemotherapy results. By selectively killing tumor neovasculature using short-ranged α-particles targeted to vascular endothelial (VE)-cadherin on vascular endothelial cells (by use of 225Ac-labeled E4G10 antibody) we were able both to reduce tumor growth and to increase the efficacy of chemotherapy, an effect seen only when the chemotherapy was administered several days after the vascular targeting agent, but not if the order of administration was reversed. Immunohistochemical and immunofluorescence studies showed that the vasculature of 225Ac-E4G10-treated tumors was substantially depleted; the remaining vessels appeared more mature morphologically and displayed increased pericyte density and coverage. Tumor uptake and microdistribution studies with radioactive and fluorescent small molecule drugs showed better accumulation and more homogenous distribution of the drugs within 225Ac-E4G10-treated tumors. These results show that 225Ac-E4G10 treatment leads to ablation and improvement of the tumor vascular architecture, and also show that the resulting vascular remodeling can increase tumor delivery of small molecules, thus providing a process for the improved outcomes observed after combining antivascular therapy and chemotherapy. This study directly shows evidence for what has long been a speculated mechanism for antiangiogenic therapies. Moreover, targeting the vessel for killing provides an alternative mode of improving chemotherapy delivery and efficacy, potentially avoiding some of the drawbacks of targeting a highly redundant angiogenic pathway.

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 approximately 350-500 kDa. The construct was rapidly (t(1/2) approximately 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.

Conscripts of the infinite armada: systemic cancer therapy using nanomaterials.

The field of clinical nanomaterials is enlarging steadily, with more than a billion US dollars of funding allocated to research by US government agencies in the past decade. The first generation of anti-cancer agents using novel nanomaterials has successfully entered widespread use. Newer nanomaterials are garnering increasing interest as potential multifunctional therapeutic agents; these drugs are conferred novel properties, by virtue of their size and shape. The new features of these agents could potentially allow increased cancer selectivity, changes in pharmacokinetics, amplification of cytotoxic effects, and simultaneous imaging capabilities. After attachment to cancer target reactive-ligands, which interact with cell-surface antigens or receptors, these new constructs can deliver cytolytic and imaging payloads. The molecules also introduce new challenges for drug development. While nanoscale molecules are of a similar size to proteins, the paradigms for how cells, tissues and organs of the body react to the non-biological materials are not well understood, because most cellular and metabolic processes have evolved to deal with globular, enzyme degradable molecules. We discuss examples of different materials to illustrate interesting principles for development and future applications of these nanomaterial medicines with emphasis on the possible pharmacologic and safety hurdles for accomplishing therapeutic goals.

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.