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Nanoparticle-mediated gene therapy has found substantial clinical impact, primarily focused on lipid-based nanoparticles. In comparison with lipid nanoparticles, polymeric particles may have certain advantages such as increased biocompatibility and controlled release. Our prior studies have found that polymeric mesoscale nanoparticles exhibited specific targeting to the renal proximal tubules. Thus, in this study, we sought to identify formulation parameters that allow for development of polymeric mesoscale nanoparticles encapsulating functional mRNA for delivery into tubular epithelial cells.We evaluated particle uptake in vitro prior to exploring formulation parameters related to introduction of a primary mixture of polymer in acetonitrile and hydrophilic mRNA in water. Finally, we evaluated their functionality in a renal tubular epithelial cell line.We found that MNPs are endocytosed within 15 min and that the mesoscale nanoparticle formulation procedure was generally robust to introduction of a primary mixture and encapsulation of mRNA. These particles exhibited substantial uptake in renal cells in vitro and rapid ( 1 h) expression of a model mCherry fluorescent protein.We anticipate these findings having potential in the delivery of specific gene therapies for renal disorders and cancer.

Background: Chronic renal inflammation has been widely recognized as a major promoter of several forms of high blood pressure including salt-sensitive hypertension. In diabetes, IL (interleukin)-6 induces salt sensitivity through a dysregulation of the epithelial sodium channel. However, the origin of this inflammatory process and the molecular events that culminates with an abnormal regulation of epithelial sodium channel and salt sensitivity in diabetes are largely unknown. Methods: Both in vitro and in vivo approaches were used to investigate the molecular and cellular contributors to the renal inflammation associated with diabetic kidney disease and how these inflammatory components interact to develop salt sensitivity in db/db mice. Results: Thirty-four-week-old db/db mice display significantly higher levels of IL-1β in renal tubules compared with nondiabetic db/+ mice. Specific suppression of IL-1β in renal tubules prevented salt sensitivity in db/db mice. A primary culture of renal tubular epithelial cells from wild-type mice releases significant levels of IL-1β when exposed to a high glucose environment. Coculture of tubular epithelial cells and bone marrow-derived macrophages revealed that tubular epithelial cell-derived IL-1β promotes the polarization of macrophages towards a proinflammatory phenotype resulting in IL-6 secretion. To evaluate whether macrophages are the cellular target of IL-1β in vivo, diabetic db/db mice were transplanted with the bone marrow of IL-1R1 (IL-1 receptor type 1) knockout mice. db/db mice harboring an IL-1 receptor type 1 knockout bone marrow remained salt resistant, display lower renal inflammation and lower expression and activity of epithelial sodium channel compared with db/db transplanted with a wild-type bone marrow. Conclusions: Renal tubular epithelial cell-derived IL-1β polarizes renal macrophages towards a proinflammatory phenotype that promotes salt sensitivity through the accumulation of renal IL-6. When tubular IL-1β synthesis is suppressed or in db/db mice in which immune cells lack the IL-1R1, macrophage polarization is blunted resulting in no salt-sensitive hypertension.

Background: Renal T cells contribute importantly to hypertension, but the underlying mechanism is incompletely understood. We reported that CD8Ts directly stimulate distal convoluted tubule cells (DCTs) to increase NCC (sodium chloride co-transporter) expression and salt reabsorption. However, the mechanistic basis of this pathogenic pathway that promotes hypertension remains to be elucidated. Methods: We used mouse models of DOCA+salt (DOCA) treatment and adoptive transfer of CD8 + T cells (CD8T) from hypertensive animals to normotensive animals in in vivo studies. Co-culture of mouse DCTs and CD8Ts was used as in vitro model to test the effect of CD8T activation in promoting NCC-mediated sodium retention and to identify critical molecular players contributing to the CD8T-DCT interaction. Interferon (IFNγ)-KO mice and mice receiving renal tubule-specific knockdown of PDL1 were used to verify in vitro findings. Blood pressure was continuously monitored via radio-biotelemetry, and kidney samples were saved at experimental end points for analysis. Results: We identified critical molecular players and demonstrated their roles in augmenting the CD8T-DCT interaction leading to salt-sensitive hypertension. We found that activated CD8Ts exhibit enhanced interaction with DCTs via IFN-γ-induced upregulation of MHC-I and PDL1 in DCTs, thereby stimulating higher expression of NCC in DCTs to cause excessive salt retention and progressive elevation of blood pressure. Eliminating IFN-γ or renal tubule-specific knockdown of PDL1 prevented T cell homing into the kidney, thereby attenuating hypertension in 2 different mouse models. Conclusions: Our results identified the role of activated CD8Ts in contributing to increased sodium retention in DCTS through the IFNγ-PDL1 pathway. These findings provide a new mechanism for T cell involvement in the pathogenesis of hypertension and reveal novel therapeutic targets.

Repeated administration of cisplatin causes CKD. In previous studies, we reported that the kidney-secreted survival protein renalase (RNLS) and an agonist peptide protected mice from cisplatin-induced AKI.To investigate whether kidney-targeted delivery of RNLS might prevent cisplatin-induced CKD in a mouse model, we achieved specific delivery of a RNLS agonist peptide (RP81) to the renal proximal tubule by encapsulating the peptide in mesoscale nanoparticles (MNPs). We used genetic deletion of RNLS, single-cell RNA sequencing analysis, and Western blotting to determine efficacy and to explore underlying mechanisms. We also measured plasma RNLS in patients with advanced head and neck squamous cell carcinoma receiving their first dose of cisplatin chemotherapy.In mice with CKD induced by cisplatin, we observed an approximate 60% reduction of kidney RNLS; genetic deletion of RNLS was associated with significantly more severe cisplatin-induced CKD. In this severe model of cisplatin-induced CKD, systemic administration of MNP-encapsulated RP81 (RP81-MNP) significantly reduced CKD as assessed by plasma creatinine and histology. It also decreased inflammatory cytokines in plasma and inhibited regulated necrosis in kidney. Single-cell RNA sequencing analyses revealed that RP81-MNP preserved epithelial components of the nephron and the vasculature and suppressed inflammatory macrophages and myofibroblasts. In patients receiving their first dose of cisplatin chemotherapy, plasma RNLS levels trended lower at day 14 post-treatment.Kidney-targeted delivery of RNLS agonist RP81-MNP protects against cisplatin-induced CKD by decreasing cell death and improving the viability of the renal proximal tubule. These findings suggest that such an approach might mitigate the development of CKD in patients receiving cisplatin cancer chemotherapy.

Although nanotechnology often addresses biomedical needs, nanoscale tools can also facilitate broad biological discovery. Nanoscale delivery, imaging, biosensing, and bioreactor technologies may address unmet questions at the interface between chemistry and biology. Currently, many chemical biologists do not include nanomaterials in their toolbox, and few investigators develop nanomaterials in the context of chemical tools to answer biological questions. We reason that the two fields are ripe with opportunity for greater synergy. Nanotechnologies can expand the utility of chemical tools in the hands of chemical biologists, for example, through controlled delivery of reactive and/or toxic compounds or signal-binding events of small molecules in living systems. Conversely, chemical biologists can work with nanotechnologists to address challenging biological questions that are inaccessible to both communities. This Perspective aims to introduce the chemical biology community to nanotechnologies that may expand their methodologies while inspiring nanotechnologists to address questions relevant to chemical biology.

Fusion protein tags are widely used to capture and track proteins in research and industrial bioreactor processes. Quantifying fusion-tagged proteins normally requires several purification steps coupled with classical protein assays. Here, we developed a broadly applicable nanosensor platform that quantifies glutathione-S-transferase (GST) fusion proteins in real-time. We synthesized a glutathione-DNA-carbon nanotube system to investigate glutathione-GST interactions via semiconducting single-walled carbon nanotube (SWCNT) photoluminescence. We found that SWCNT fluorescence wavelength and intensity modulation occurred specifically in response to GST and GST-fusions. The sensor response was dependent on SWCNT structure, wherein mod(n - m, 3) = 1 nanotube wavelength and intensity responses correlated with nanotube diameter distinctly from mod(n - m, 3) = 2 SWCNT responses. We also found broad functionality of this sensor to diverse GST-tagged proteins. This work comprises the first label-free optical sensor for GST and has implications for the assessment of protein expression in situ, including in imaging and industrial bioreactor settings.

We developed an innovative therapy for ischemic acute kidney injury with discerning kidney-targeted delivery of a selective Toll-like receptor 9 (TLR9) antagonist in mice subjected to renal ischemia reperfusion injury. Our previous studies showed that mice deficient in renal proximal tubular TLR9 were protected against renal ischemia reperfusion injury demonstrating a critical role for renal proximal tubular TLR9 in generating ischemic acute kidney injury. Herein, we used 300–400 nm polymer-based mesoscale nanoparticles that localize to the renal tubules after intravenous injection. Mice were subjected to sham surgery or 30 minutes renal ischemia and reperfusion injury after receiving mesoscale nanoparticles encapsulated with a selective TLR9 antagonist (unmethylated CpG oligonucleotide ODN2088) or mesoscale nanoparticles encapsulating a negative control oligonucleotide. Mice treated with the encapsulated TLR9 antagonist either six hours before renal ischemia, at the time of reperfusion or 1.5 hours after reperfusion were protected against ischemic acute kidney injury. The ODN2088-encapsulated nanoparticles attenuated renal tubular necrosis, inflammation, decreased proinflammatory cytokine synthesis. neutrophil and macrophage infiltration and apoptosis, decreased DNA fragmentation and caspase 3/8 activation when compared to the negative control nanoparticle treated mice. Taken together, our studies further suggest that renal proximal tubular TLR9 activation exacerbates ischemic acute kidney injury by promoting renal tubular inflammation, apoptosis and necrosis after ischemia reperfusion. Thus, our studies suggest a potential promising therapy for ischemic acute kidney injury with selective kidney tubular targeting of TLR9 using mesoscale nanoparticle-based drug delivery.

Interpretation of a 3D seismic survey located on the western margin of the Northern North Sea Basin demonstrates how the propagation, overlap and linkage of two north–south-striking, en echelon normal fault segments exerted a powerful influence over prospective subtle stratigraphic traps. The relay ramp that formed between the segments appears to have focused sediment dispersal, controlled reservoir distribution and aided post-depositional petroleum migration. Integration of electrical well log data, root-mean-square (RMS)-amplitude analysis and biostratigraphy with seismic interpretation demonstrates that a series of elongate, linear, sand-prone (reservoir) channel complexes characterize the depositional slopes generated by fault growth. The combination of synsedimentary rotation of bedding due to fault propagation and associated footwall uplift led to erosion and truncation of a laterally extensive, older channelized system (Lower Sequence), the downdip parts of which extend beyond the relay ramp. Its subsequent drape by transgressive shales created the subtle stratigraphic trap that now hosts the Cladhan Field, with charge occurring because the sandstones belonging to the Lower Sequence extend as far as the active kitchen in the neighbouring (hanging-wall) depocentre situated downdip and to the east. In contrast, the exploration of a younger, Upper Sequence of sandstones has proven to be disappointing due to their more restricted distribution, lack of access to charge, and occurrence of faults that offset and breach the thin end of the stratigraphic wedge. The implication is that partially breached relay ramps not only provide a preferential site for synrift clastic reservoirs to develop but also form important migration pathways through which oil passed from a petroleum kitchen into a trap. Thematic collection: This article is part of the Under-explored plays and frontier basins of the UK continental shelf collection available at: http://www.lyellcollection.org/cc/under-explored-plays-and-frontier-basins-of-the-uk-continental-shelf

Nanomedicines, nanotechnologies applied to medicine, have been in clinical use for 25 years because of their potential to improve the therapeutic index of drugs. Nanotechnologies can also broaden the classes of therapies that can be administered to patients. They have been used in drug delivery strategies to improve drug bioavailability, solubility, stability, and tolerability, among other benefits. Nanoparticles can also enable the use of nucleic acid therapies and other macromolecular drugs by encapsulating normally delicate cargoes. While the majority of nanoparticles localize drugs predominantly to the liver or spleen, a bevy of recent progress has focused on targeting to the kidneys. This chapter focuses on the advances in drug delivery strategies for the treatment of renal diseases, including methods to improve drug specificity to the nephron. We highlight recent preclinical and clinical developments in nanomedicines and their potential for advancement in nephrology.

Photoluminescent single-walled carbon nanotubes (SWCNT) hold substantial potential for a variety of applications in biology and medicine. Improved preparation of such materials requires optimization of various parameters, including those pertaining to ultracentrifugation techniques for removing non-photoluminescent carbonaceous materials. In this work, we investigated single-stranded DNA (ssDNA)-SWCNT preparations, which are widely used and exhibit strong photoluminescence (PL). We found, however, that total PL is not well-described by SWCNT concentration, and that it is much more sufficiently described by a comparison of SWCNT E22 transition peaks with surrounding baseline absorbance from non-fluorescent carbonaceous material. We used this metric, defined as efficacy, in optimizing techniques for centrifugation and subsequent fractionation. We found that increased centrifugal forces removed substantial non-photoluminescent material, but also more SWCNT mass, yielding less-concentrated but more-pure fluorescent SWCNT samples. Thus, a tradeoff exists between decreased sensor material and increased sensor quality, one which might be considered for each novel SWCNT-based nanosensor construct. We anticipate these studies serving as a basis for improved applied nanosensor development.

Preclinical measurements of drug exposure to specific organs and tissues is normally performed by destructive methods. Tissue-specific measurements are important, especially for drugs with intractable dose-limiting toxicities, such as doxorubicin-mediated cardiotoxicity. We developed a method to rapidly quantify doxorubicin exposure to tissues within living organisms using an implantable optical nanosensor that can be interrogated noninvasively following surgical implantation. The near-infrared fluorescence of single-walled carbon nanotubes functionalized with DNA was found to respond to doxorubicin via a large and uniform red-shift. We found this to be common to DNA-intercalating agents, including anthracycline compounds such as doxorubicin. Doxorubicin was measured in buffer and serum, intracellularly, and from single nanotubes on a surface. Doxorubicin adsorption to the DNA-suspended nanotubes did not displace DNA but bound irreversibly. We incorporated the nanosensors into an implantable membrane which allowed cumulative detection of doxorubicin exposure in vivo. On implanting the devices into different compartments, such as subcutaneously and within the peritoneal cavity, we achieved real-time, minimally invasive detection of doxorubicin injected into the peritoneal cavity, as well as compartment-specific measurements. We measured doxorubicin translocation across the peritoneal membrane in vivo. Robust, minimally invasive pharmacokinetic measurements in vivo suggest the suitability of this technology for preclinical drug discovery applications.

The rapid, minimally-invasive detection of diverse analytes in vivo remains a goal for the diagnosis of cancer and related diseases. Toward this end, we are designing nanosensors based on the optical properties of single-walled carbon nanotubes (SWCNT) for use both in patients and at the bedside. SWCNT fluorescence is in the near-infrared window for biological imaging, is stable upon laser excitation, and responds rapidly to changes in its local environment. We are approaching the design of multiplexed implantable biomarker sensors through several routes, including developing novel bioconjugate chemistry approaches to antibodies and other molecular recognition elements to improve sensitivity of the sensors. We are also evaluating molecular tools to screen non-specific interactions within the local environment, improving the selectivity of these sensors. In parallel, we are using aqueous two-phase extraction (ATPE) methods to allow for separation of single-chirality SWCNT prior to bioconjugate chemistry approaches, allowing for specific detection of multiple analytes at once. Finally, we are exploring various hydrogel systems to allow for their immobilization upon injection into living systems and repeated monitoring of local biomarker concentrations. We expect these approaches to further bridge the translation of sensor devices toward clinical diagnostic tools. We hope to deploy SWCNT-based sensors to detect disease at early stages, evaluate disease response to treatment, and monitor disease progression. These aims will allow physicians to make rapid, informed decisions on patient prognosis and treatment.

We determined that renal proximal tubular (PT) NF-κB essential modulator (NEMO) plays a direct and critical role in ischemic acute kidney injury (AKI) using mice lacking renal PT NEMO and by targeted renal PT NEMO inhibition with mesoscale nanoparticle–encapsulated NEMO binding peptide (NBP MNP). We subjected renal PT NEMO–deficient mice, WT mice, and C57BL/6 mice to sham surgery or 30 minutes of renal ischemia and reperfusion (IR). C57BL/6 mice received NBP MNP or empty MNP before renal IR injury. Mice treated with NBP MNP and mice deficient in renal PT NEMO were protected against ischemic AKI, having decreased renal tubular necrosis, inflammation, and apoptosis compared with control MNP-treated or WT mice, respectively. Recombinant peptidylarginine deiminase type 4 (rPAD4) targeted kidney PT NEMO to exacerbate ischemic AKI in that exogenous rPAD4 exacerbated renal IR injury in WT mice but not in renal PT NEMO–deficient mice. Furthermore, rPAD4 upregulated proinflammatory cytokine mRNA and NF-κB activation in freshly isolated renal proximal tubules from WT mice but not from PT NEMO–deficient mice. Taken together, our studies suggest that renal PT NEMO plays a critical role in ischemic AKI by promoting renal tubular inflammation, apoptosis, and necrosis., Renal proximal tubular NFκB essential modulator (NEMO) regulates ischemic acute kidney injury by promoting renal tubular inflammation, apoptosis, and necrosis.

Carbon nanotube-based molecular probes, imaging agents, and biosensors in cells and in vivo continue to garner interest as investigational tools and clinical devices due to their unique photophysical properties. Surface chemistry modulation of nanotubes plays a critical role in determining stability and interaction with biological systems both in vitro and in vivo. Among the many parameters that influence the biological fate of nanomaterials, surface charge is particularly influential due to direct electrostatic interactions with components of the cell membrane as well as proteins in the serum, which coat the nanoparticle surface in a protein corona and alter nanoparticle–cell interactions. Here, we modulated functional moieties on a helical polycarbodiimide polymer backbone that non-covalently suspended the nanotubes in aqueous media. By derivatizing the polymer with either primary amine or carboxylic acid side chains, we obtained nanotube complexes that present net surface charges of opposite polarity at physiological pH. Using these materials, we found that the uptake of carbon nanotubes in these cells is highly dependent on charge, with cationic nanotubes efficiently internalized into cells compared to the anionic nanotubes. Furthermore, we found that serum proteins drastically influenced cell uptake of the anionic nanotubes, while the effect was not prominent for the cationic nanotubes. Our findings have implications for improved engineering of drug delivery devices, molecular probes, and biosensors.

Cisplatin-induced acute kidney injury (CI-AKI) is a significant co-morbidity of chemotherapeutic regimens. While this condition is associated with substantially lower survival and increased economic burden, there is no pharmacological agent to effectively treat CI-AKI. The disease is hallmarked by acute tubular necrosis of the proximal tubular epithelial cells primarily due to increased oxidative stress. In our prior work, we developed a highly-selective kidney-targeted mesoscale nanoparticle (MNP) that accumulates primarily in the renal proximal tubular epithelial cells while exhibiting no toxicity. Here, we found that MNPs exhibit renal-selective targeting in multiple mouse models of tumor growth with virtually no tumor accumulation. We then evaluated the therapeutic efficacy of MNPs loaded with the reactive oxygen species scavenger edaravone in a mouse model of CI-AKI. We found a marked and significant therapeutic effect with this approach as compared to free drug or empty control MNPs, including improved renal function, histology, and diminution of oxidative stress. These results indicated that renal-selective MNP edaravone delivery holds substantial potential in the treatment of acute kidney injury among patients undergoing cisplatin-based chemotherapy.

Upper Permian (Zechstein Supergroup) evaporites have a major control on structural styles and prospectivity in the UK Southern North Sea (SNS). They form the regional super-seal for the main Rotliegend Group (Leman Sandstone Formation) reservoir play fairway immediately beneath. The evaporites have highly variable thicknesses due to the syndepositional basin architecture, differential loading and post-depositional deformation through diapirism and salt withdrawal. The halokinetic activity leads to touchdown (welding) of the supra-salt section onto the sub-salt strata and the development of narrow (up to 15 km-wide) graben systems. The interpretation and depth conversion of well-calibrated, high-quality, 3D post-stack time-migrated (PSTM) seismic data along the southwestern margin of the basin show that a NW–SE-striking elongate extensional Dowsing Graben System transects the area. The graben is defined by a series of large, overlapping, en echelon listric growth faults, with oblique secondary planar faults, which sole-out on two main (deep and shallow) decollement levels in the Zechstein Supergroup and the Middle Triassic Rot Halite Member. Whilst its initial formation was related to Mesozoic extension, the graben system also displays a contractional overprint resulting from regional compression and structural inversion during the Cenozoic. Detailed mapping of the Zechstein Supergroup has revealed that the evolution of the extensional system was influenced by the ESE–WNW-striking anhydrite–carbonate Zechstein shelf-margin. The occurrence of variable-thickness, low-velocity sediments within the graben impacts seismic imaging and depth conversion, leading to prospective structures being overlooked; something that has implications for prospectivity in the SNS and other evaporite basins where similar graben occur.

Therapeutic outcomes in patients with prostate cancer are hindered by the inability to discern indolent versus aggressive disease. To address this problem, we developed a quantitative fluorescent nanosensor for the cancer biomarker urokinase plasminogen activator (uPA). We used the unique fluorescent characteristics of single-walled carbon nanotubes (SWCNT) to engineer an optical sensor that responds to uPA via optical bandgap modulation in complex protein environments. The sensing characteristics of this construct were modulated by passivation of the hydrophobic SWCNT surface with bovine serum albumin (BSA). The sensor enabled quantitative detection of known uPA concentrations in human blood products. These experiments potentiate future use of this technology as a rapid, point-of-care sensor for biomarker measurements in patient fluid samples. We expect that further work will develop a method to discern aggressive vs indolent prostate cancer and reduce overtreatment of this disease.

Direct targeting to the kidneys is a promising strategy to improve drug therapeutic index for the treatment of kidney diseases. We sought to investigate the renal selectivity and safety of kidney-targeted mesoscale nanoparticle technology. We found that direct intravenous administration of these particles resulted in 26-fold renal selectivity and localized negligibly in the liver or other organs. The nanoparticles targeted the renal proximal tubular epithelial cells, as evidenced by intravital microscopy and ex vivo imaging. Mice treated with the nanoparticles exhibited no negative systemic consequences, immune reaction, liver impairment, or renal impairment. The localization of material selectively to the renal tubules is uncommon, and this work portends the development of renal-targeted drugs for the treatment of kidney diseases.

Preclinical measurements of drug exposure to specific organs and tissues is normally performed by destructive methods. Tissue-specific measurements are important, especially for drugs with intractable dose-limiting toxicities, such as doxorubicin-mediated cardiotoxicity. We developed a method to rapidly quantify doxorubicin exposure to tissues within living organisms using an implantable optical nanosensor that can be interrogated noninvasively following surgical implantation. The near-infrared fluorescence of single-walled carbon nanotubes functionalized with DNA was found to respond to doxorubicin via a large and uniform red-shift. We found this to be common to DNA-intercalating agents, including anthracycline compounds such as doxorubicin. Doxorubicin was measured in buffer and serum, intracellularly, and from single nanotubes on a surface. Doxorubicin adsorption to the DNA-suspended nanotubes did not displace DNA but bound irreversibly. We incorporated the nanosensors into an implantable membrane which allowed cumulative detection of doxorubicin exposure

Optical nanosensors hold the promise of rapid, minimally-invasive longitudal monitoring of analytes within a patient or at the bedside. Such devices will allow for informed clinical intervention based on changes in disease state profiles. We are designing nanosensors based on the optical properties of single-walled carbon nanotubes (SWCNT) for use in both in vivo and ex vivo formats. SWCNT fluorescence is in the ‘tissue-transparent’ near-infrared spectrum, allowing for fluorescence measurements through biological tissue and fluids. Further, SWCNT fluorescence is photostable and responds rapidly, allowing for multiple measurements over time. We are engineering SWCNT-based sensors such that their fluorescence emission responds to multiple cancer biomarkers and inflammatory proteins. Various deployment formats include semipermeable implantable membranes, injectable hydrogel materials, and chip-based devices. Within each device, we are comparing sensor response with its physicochemical properties to optimize sensor functionality. Finally, we are deploying each sensor device in patient serum, other clinical tissues, and in live animal models of disease. For each, we are using custom-built near-infrared probe spectroscopy with rapid point-and-detect functionality. We expect these translational sensor devices to have potential as research tools or clinical diagnostics to detect disease at early stages, evaluate disease response to treatment, or monitor disease progression. These aims will allow physicians to make rapid, informed decisions on patient prognosis and treatment.

Implantable optical nanosensors hold the promise of rapid, minimally-invasive longitudal monitoring of analytes in a patient or animal. Such devices will allow for informed clinical intervention based on changes in drug, biomarker, or disease state profiles. We developed implantable nanosensors based on the optical properties of single-walled carbon nanotubes (SWCNT). SWCNT fluorescence is in the ‘tissue-transparent’ near-infrared spectrum, allowing for fluorescence measurements after implantation inside an animal. Further, SWCNT fluorescence is photostable, allowing for multiple measurements over time. We engineered separate SWCNT-based sensors such that their fluorescence emission responded to various analytes, including small molecule drugs as well as protein and nucleic acid biomarkers. Each sensor device was immobilized into a semi-permeable biocompatible membrane, allowing analyte penetration but retaining SWCNT inside. We implanted each sensor device into live mice, either in the peritoneal cavity or subcutaneously, and performed whole-mouse near-infrared fluorescence imaging to ensure the stability of the membrane and sensor brightness. We used a sensor implant specific for the chemotherapeutic doxorubicin to drug monitor transport within the animal. We used sensor implants specific for the ovarian cancer protein biomarker HE4 or for individual microRNA sequences to measure exogenous or cancer-generated quantities of each biomarker in mice. We expect these implantable sensors to have potential as research tools or clinical diagnostics to monitor pharmacokinetics or locally measure disease biomarkers in real-time.

Optical sensor technologies for vivo use often run into difficulties when attempting to relay quantitative information. Fluorescence transmission from within tissues may be variable depending on depth and tissue heterogeneity. Modulation of the fluorescence emission band position may mitigate some of these difficulties. We developed sensors that modulate the near-infrared emission of single-walled carbon nanotubes and organic color centers (carbon nanotubes with covalent sp3 defects). We developed methods to control solvatochromic behavior to improve sensor responses, and we developed tools to observe solvatochromism within live cells and live animals. We found that nanotube solvatochromism extends to variations of local electrostatic charge, induced by adsorption/desorption of polyelectrolytes on the nanotube surface, such as proteins and nucleic acids. We also found that dielectric and charge-mediated solvatochromism can be compounded to exacerbate the response. Upon triggered desorption of a polyelectrolyte, amphiphilic molecules can adsorb to the recently-uncovered nanotube surface. This work has enabled the detection of disease biomarkers, drugs, and metabolites, and nucleic acids, including virus RNA, facilitating improvements in disease detection, drug development, and biomedical research.

Acute kidney injury (AKI) develops in up to 30% of patients who receive cisplatin-based chemotherapy. In this setting, AKI can result in delays in completion of treatment and/or the need to switch to other potentially less effective therapeutic regimens. Despite its high incidence and associated increased morbidity and mortality, there is no effective pharmacologic intervention to treat AKI of any etiology, including AKI resulting from cisplatin or any other nephrotoxic treatments. In the case of cisplatin-induced AKI, it is also imperative that any pharmacologic intervention that could be effective for AKI will not interfere with the systemic chemotherapeutic effects of cisplatin. In prior studies (Nano Letters, 2015), we reported the development of mesoscale nanoparticles (MNPs), composed of poly(lactic-co-glycolic acid) and polyethylene glycol (PLGA-PEG) that localize to the kidneys with a high affinity, primarily to the proximal tubules (the site of injury in AKI), and persist at that location for up to a week. Herein, we encapsulated a small molecule reactive oxygen species scavenger, (edaravone) in the MNPs. Experiments were performed in male C57BL/6 mice with cisplatin-induced AKI (25 mg/kg IP). To assess therapeutic efficacy, tail vein injections of 50 mg/kg edaravone-containing MNPs, 50 mg/kg empty control MNPs, or 30 mg/kg free edaravone were performed 24 hours after cisplatin injection (n=6-8 per group). Mice were euthanized at 72 hours post-cisplatin administration, and kidney and serum were saved for analysis. In a separate group of mice bearing metastatic small cell lung cancer, fluorescent MNPs were injected to determine whether MNPs still localize to the kidneys rather than to the tumor as has been described for other types of nanoparticles. Mice receiving cisplatin had a ~5 fold increase in serum creatinine and severe acute tubular necrosis as compared to controls. In contrast to mice receiving cisplatin alone, mice receiving edaravone-containing MNPs along with cisplatin had normal creatinine and histology similar to controls. Neither treatment with free edaravone nor empty MNPs resulted in a significant improvement in renal function or severity of tubular necrosis in mice with cisplatin-induced AKI. In the presence of AKI, the distribution of MNPs was still highly selective to the renal proximal tubules. In mice with metastatic lung tumors, we determined that MNPs maintain their specific renal distribution and do not localize to tumors as has been described with other smaller nanoparticle systems. These studies demonstrate that edaravone-containing MNPs can be an effective treatment for cisplatin-induced AKI while avoiding the possible therapeutic abatement associated with tumor deposition of reactive oxygen species scavengers. We anticipate that these studies will constitute the basis for the development of novel strategies for the treatment and prevention of cisplatin-induced AKI in humans. Citation Format: Daniel A. Heller, Edgar Jaimes, Ryan Williams, Janki Shah. Renal tubule-targeted supportive care nanotherapy for cisplatin-induced acute kidney injury [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2032.

Implantable optical nanosensors hold the promise of rapid, minimally-invasive longitudal monitoring of analytes in a patient or animal. Such devices will allow for informed clinical intervention based on changes in drug, biomarker, or disease state profiles. We developed implantable nanosensors based on the optical properties of single-walled carbon nanotubes (SWCNT). SWCNT fluorescence is in the ‘tissue-transparent’ near-infrared spectrum, allowing for fluorescence measurements after implantation inside an animal. Further, SWCNT fluorescence is photostable, allowing for multiple measurements over time. We engineered separate SWCNT-based sensors such that their fluorescence emission responded to various analytes, including small molecule drugs as well as protein and nucleic acid biomarkers. Each sensor device was immobilized into a semi-permeable biocompatible membrane, allowing analyte penetration but retaining SWCNT inside. We implanted each sensor device into live mice, either in the peritoneal cavity or subcutaneously, and performed whole-mouse near-infrared fluorescence imaging to ensure the stability of the membrane and sensor brightness. We used a sensor implant specific for the chemotherapeutic doxorubicin to drug monitor transport within the animal. We used sensor implants specific for the ovarian cancer protein biomarker HE4 or for individual microRNA sequences to measure exogenous or cancer-generated quantities of each biomarker in mice. We expect these implantable sensors to have potential as research tools or clinical diagnostics to monitor pharmacokinetics or locally measure disease biomarkers in real-time.

Toxin B is one of the major virulence factors ofClostridium difficile, a bacterium that is responsible for a significant number of diarrhea cases in acute care settings. Due to the prevalence ofC. difficileinduced diarrhea, rapid and correct diagnosis is crucial in the disease management. In this study, we have employed a stringentin vitroselection method to identify single-stranded DNA molecular recognition elements (MRE) specific for toxin B. At the end of the 12-round selection, one MRE with high affinity(Kd=47.3 nM) for toxin B was identified. The selected MRE demonstrated low cross binding activities on negative targets: bovine serum albumin,Staphylococcus aureusalpha toxin,Pseudomonas aeruginosaexotoxin A, and cholera toxin ofVibrio cholera. A modified sandwich ELISA assay was developed utilizing the selected ssDNA MRE as the antigen capturing element and achieved a sensitive detection of 50 nM of toxin B in human fecal preparations.

Ovarian cancer biomarker detection using a novel nanosensor implant in live mice., Patients with high-grade serous ovarian carcinoma (HGSC) exhibit poor 5-year survival rates, which may be significantly improved by early-stage detection. The U.S. Food and Drug Administration–approved biomarkers for HGSC—CA-125 (cancer antigen 125) and HE4 (human epididymis protein 4)—do not generally appear at detectable levels in the serum until advanced stages of the disease. An implantable device placed proximal to disease sites, such as in or near the fallopian tube, ovary, uterine cavity, or peritoneal cavity, may constitute a feasible strategy to improve detection of HGSC. We engineered a prototype optical sensor composed of an antibody-functionalized carbon nanotube complex, which responds quantitatively to HE4 via modulation of the nanotube optical bandgap. The complexes measured HE4 with nanomolar sensitivity to differentiate disease from benign patient biofluids. The sensors were implanted into four models of ovarian cancer, within a semipermeable membrane, enabling the optical detection of HE4 within the live animals. We present the first in vivo optical nanosensor capable of noninvasive cancer biomarker detection in orthotopic models of disease.

Increasing use of single-walled carbon nanotubes (SWCNTs) will lead to their increased release into the environment. Previous work has shown negative effects of SWCNT on growth and survival of model organisms. The aim of the current study was to determine the effect of SWCNT well-dispersed by either DNA or sodium cholate (SC) on the unicellular green algae Chlamydomonas reinhardtii in stagnant water conditions. Growth measurements were taken up to ten days for algae treated with varied levels of DNA:SWCNT or SC:SWCNT or controls, and chlorophyll content after 10 days was determined. Results show no effect on either growth or chlorophyll content of algae at any concentration or duration. This is in contradiction to prior work showing toxicity of SWCNT to environmental model organisms.

Widespread use of the chlorotriazine herbicide, atrazine, has led to serious environmental and human health consequences. Current methods of detecting atrazine contamination are neither rapid nor cost-effective. In this work, atrazine-specific single-stranded DNA (ssDNA) molecular recognition elements (MRE) were isolated. We utilized a stringent Systematic Evolution of Ligands by Exponential Enrichment (SELEX) methodology that placed the greatest emphasis on what the MRE should not bind to. After twelve rounds of SELEX, an atrazine-specific MRE with high affinity was obtained. The equilibrium dissociation constant (Kd) of the ssDNA sequence is 0.62 ± 0.21 nM. It also has significant selectivity for atrazine over atrazine metabolites and other pesticides found in environmentally similar locations and concentrations. Furthermore, we have detected environmentally relevant atrazine concentrations in river water using this MRE. The strong affinity and selectivity of the selected atrazine-specific ssDNA validated the stringent SELEX methodology and identified a MRE that will be useful for rapid atrazine detection in environmental samples.

Bromacil is a widely used herbicide that is known to contaminate environmental systems. Due to the hazards it presents and inefficient detection methods, it is necessary to create a rapid and efficient sensing device. Towards this end, we have utilized a stringentin vitroselection method to identify single-stranded DNA molecular recognition elements (MRE) specific for bromacil. We have identified one MRE with high affinity (Kd=9.6 nM) and specificity for bromacil compared to negative targets of selection and other pesticides. The selected ssDNA MRE will be useful as the sensing element in a field-deployable bromacil detection device.

Patients with high-grade serous ovarian carcinoma (HGSC) exhibit poor 5-year survival rates, which may be significantly improved by early-stage detection. The US FDA-approved biomarkers for HGSC—CA-125 and HE4—do not generally appear at detectable levels in the serum until advanced stages of the disease. An implantable device placed proximal to disease sites, such as in or near the fallopian tube, ovary, uterine cavity, or peritoneal cavity, may constitute a feasible strategy to improve detection of HGSC. Furthermore, a sensor implant under the skin may allow real-time measurements after diagnosis, to monitor treatment for example, via interrogation using a wearable device. We engineered a prototype optical sensor composed of an antibody-functionalized carbon nanotube complex, which responds quantitatively to HE4 via modulation of the nanotube optical bandgap. The complexes measured HE4 with nanomolar sensitivity to differentiate disease from benign patient biofluids. The sensors were implanted into four models of ovarian cancer, within a semipermeable membrane, enabling the optical detection of HE4 within the live animals. We thus present the first in vivo optical nanosensor capable of noninvasive cancer biomarker detection in orthotopic models of disease. This technology may potentially be implanted locally in the uterine cavity, for point-of-care measurement, or under the skin, to then interface with a wearable device. Citation Format: Daniel A. Heller, Ryan M. Williams, Douglas A. Levine. Implantable nanosensor for non-invasive ovarian cancer biomarker detection with wearable interface [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1938.

Modulation of the carbon nanotube photoluminescence band position has implications for applications such as sensing and imaging. Carbon nanotube photoluminescence solvatochromism, the hypsochromic or bathochromic shifting of nanotube emission, can be induced via changes in the local dielectric environment. We developed methods to modulate nanotube solvatochromic behavior to improve sensor responses, and we developed tools to observe solvatochromism within live cells and, recently, live animals. We found that nanotube solvatochromism extends to variations of local electrostatic charge, induced by adsorption/desorption of polyelectrolytes on the nanotube surface, such as proteins and nucleic acids. We also found that dielectric and charge-mediated solvatochromism can be compounded to exacerbate the response. Upon triggered desorption of a polyelectrolyte, such as upon desorption of RNA upon hybridization, for instance, amphiphilic molecules can adsorb to the recently-uncovered nanotube surface. The adsorption displaces water to result in hypsochromic shifting, which can be additive, as polyelectrolyte desorption will also cause a hypsochromic shift. We found that the amphiphile may be a surfactant but, recently, we found that proteins can result in a similar response, facilitating the detection of viruses. We developed tools to image changes in nanotube emission in live cells (near-infrared hyperspectral microscopy) and, recently, live animals (preclinical, near-infrared hyperspectral imaging). Carbon nanotube solvatochromism-based sensors, in combination with spectral imaging tools, allow measurements of biomarkers, drugs, and metabolites in live cells and organisms, facilitating improvements in disease detection, drug development, and biomedical research.

Single-walled carbon nanotubes exhibit unique photophysical properties that enable the detection of biomarkers, metabolites, and drugs in living cells, tissues, and organisms. The intrinsic near-infrared photoluminescence of nanotubes exhibits unique photostability and sensitivity to the local environment to transduce molecular binding events via changes in the nanotube optical bandgap. We developed carbon nanotube-based optical sensors to detect metabolites and cancer biomarkers in vivo, in order to improve early cancer detection and accelerate drug discovery for cancer and allied diseases. We introduced carbon nanotubes into disease models via implantation within devices, as well as by intravenous injection. In all cases, we found that properly-functionalized single-walled carbon nanotubes exhibit no deleterious effects in live cells or in vivo at concentrations needed for measurement. Via injecting a single-chirality carbon nanotube species responsive to lipids, we found that lipid accumulation in liver Kupffer cell lysosomes tracks the progression of non-alcoholic fatty-liver disease (NAFLD) in a live murine model. Notably, we found that that lipid accumulation is persistent in the Western Diet model of NAFLD after resuming normal feeding, suggesting long-term effects of a poor diet. This finding demonstrates that single-walled carbon nanotubes enable the development of new tools to allow biologists to measure disease states in situ.

The intrinsic near-infrared photoluminescence (fluorescence) of single-walled carbon nanotubes exhibits unique photostability, narrow bandwidth, penetration through biological media, environmental sensitivity, and both chromatic variety and range. Biomedical applications exploiting this large family of fluorophores will require the spectral and spatial resolution of individual (n,m) nanotube species’ fluorescence and its modulation within live cells and tissues, which is not possible with current microscopy methods. We present a wide-field hyperspectral approach to spatially delineate and spectroscopically measure single nanotube fluorescence in living systems. This approach resolved up to 17 distinct (n,m) species (chiralities) with single nanotube spatial resolution in live mammalian cells, murine tissues ex vivo, and zebrafish endothelium in vivo. We anticipate that this approach will facilitate multiplexed nanotube imaging in biomedical applications while enabling deep-tissue optical penetration, exceptional photostability, and single-molecule resolution in vivo.

Nanomedicines have been the subject of great interest for the treatment, diagnosis, and research of disease; however, few specifically address kidney disorders. Nanotechnology can confer significant benefits to medicine, such as the targeted delivery of drugs to specific tissues. Nanomedicines in the clinic have increased drug solubility, reduced off-target side effects, and provided novel diagnostic tools. There is an increasing cohort of nanomaterials that may have implications for kidney disease. Here, we review nanomaterial properties that are potentially applicable to kidney research and therapy, and we highlight clinical areas of need that may benefit from kidney nanomedicines.

The early detection of cancer could lead to vastly improved patient outcomes. We aim to identify cancer biomarkers within the body at early disease stages, permitting detection before symptoms arise. We are developing implantable nanosensors, using the unique optical properties of carbon nanotubes, to facilitate non-invasive detection via optical detection through living tissues. The sensors could enable early detection of cancer in people at high risk for the disease, in successfully treated patients to monitor recurrence, or in patients who are undergoing treatment to inform clinical decisions.

The detection of biomarkers and drugs in live cells and animals would allow for more effective studies of the dynamics of cancer and treatment. Toward these ends, single-walled carbon nanotube photoluminescence is suitable for measurements in live cells and in vivo, and nanotube near-infrared emission exhibits sensitivity to the local environment via solvatochromic responses. We developed new optical instrumentation to image carbon nanotubes and to measure spectral changes of carbon nanotube photoluminescence in live cells and animals. Concomitantly, we developed new methods to exacerbate the nanotube emission response to produce large spectral shifts (> 14 nm) upon the binding of cancer biomarkers and chemotherapeutic drugs. The ability to improve measurements and control of carbon nanotube solvatochromism may improve sensitivity and selectivity of nanosensors and potentially expand the application of nanotube-based optical measurements to new classes of bioanalytes.

Prostate cancer is the most prevalent cancer in the Western male population and the second leading cause of cancer death in men, affecting over 10 million individuals. Present approaches to control the cancer mortality have focused on the detection of the cancer at early stages when it is still locally confined and may be curable. Identification of the prostate-specific antigen (PSA) has facilitated the early diagnosis of prostate cancer. However, PSA has limited specificity and sensitivity in appropriately detecting early stages of abnormal prostate growth. PSA levels fail to differentiate between indolent and aggressive cancers, do not correlate with tumor size, and cross-react with other serine proteases namely, glandular kallikreins 1 and 2. Besides cancer, its levels also increase in men with benign prostatic hyperplasia (BPH), prostatitis, and other non-malignancies. Additional prostate-specific genes and metabolites need to be identified to provide a better understanding of the molecular mechanisms of prostate physiology and pathophysiology. Novel markers for the diagnosis and development of new treatment modalities are urgently needed.

The intrinsic near-infrared photoluminescence (fluorescence) of single-walled carbon nanotubes exhibits unique photostability, narrow bandwidth, penetration through biological media, environmental sensitivity and both chromatic variety and range. Biomedical applications exploiting this large family of fluorophores will require the spectral and spatial resolution of individual (n,m) nanotube species’ fluorescence and its modulation within live cells and tissues, which is not possible with current microscopy methods. We present a wide-field hyperspectral approach to spatially delineate and spectroscopically measure single nanotube fluorescence in living systems. This approach resolved up to 17 distinct (n,m) species (chiralities) with single nanotube spatial resolution in live mammalian cells, murine tissues ex vivo and zebrafish endothelium in vivo. We anticipate that this approach will facilitate multiplexed nanotube imaging in biomedical applications while enabling deep-tissue optical penetration and single-molecule resolution in vivo.

We synthesized “mesoscale” nanoparticles, approximately 400 nm in diameter, which unexpectedly localized selectively in renal proximal tubules and up to 7 times more efficiently in the kidney than other organs. Although nanoparticles typically localize in the liver and spleen, modulating their size and opsonization potential allowed for stable targeting of the kidneys through a new proposed uptake mechanism. Applying this kidney targeting strategy, we anticipate use in the treatment of renal disease and the study of renal physiology.