A pan-cancer agent for screening, resection and wound monitoring via NIR and SWIR imaging.

Fluorescence guided surgery (FGS) facilitates real time tumor delineation and is being rapidly established clinically. FGS efficacy is tied to the utilized dye and provided tumor contrast over healthy tissue. Apoptosis, a cancer hallmark, is a desirable target for tumor delineation. Here, we preclinically in vitro and in vivo, validate an apoptosis sensitive commercial carbocyanine dye (CJ215), with absorption and emission spectra suitable for near infrared (NIR, 650-900nm) and shortwave infrared (SWIR, 900-1700nm) fluorescence imaging (NIRFI, SWIRFI). High contrast SWIRFI for solid tumor delineation is demonstrated in multiple murine and human models including breast, prostate, colon, fibrosarcoma and intraperitoneal colorectal metastasis. Organ necropsy and imaging highlighted renal clearance of CJ215. SWIRFI and CJ215 delineated all tumors under ambient lighting with a tumor-to-muscle ratio up to 100 and tumor-to-liver ratio up to 18, from 24 to 168 h post intravenous injection with minimal uptake in healthy organs. Additionally, SWIRFI and CJ215 achieved non-contact quantifiable wound monitoring through commercial bandages. CJ215 provides tumor screening, guided resection, and wound healing assessment compatible with existing and emerging clinical solutions.

Surgical radioguidance with beta-emitting radionuclides; challenges and possibilities: A position paper by the EANM.

Radioguidance that makes use of ß-emitting radionuclides is gaining in popularity and could have potential to strengthen the range of existing radioguidance techniques. While there is a strong tendency to develop new PET radiotracers, due to favorable imaging characteristics and the success of theranostics research, there are practical challenges that need to be overcome when considering use of ß-emitters for surgical radioguidance. In this position paper, the EANM identifies the possibilities and challenges that relate to the successful implementation of ß-emitters in surgical guidance, covering aspects related to instrumentation, radiation protection, and modes of implementation.

Current clinical applications of Cerenkov luminescence for intraoperative molecular imaging.

BACKGROUND: Cerenkov luminescence imaging (CLI) is a new emerging technology that can be used for optical imaging of approved radiotracers, both in a preclinical, and even more recently, in a clinical context with rapid imaging times, low costs, and detection in real-time (Grootendorst et al. Clin Transl Imaging 4(5):353-66, 2016); Wang et al. Photonics 9(6):390, 2022). This brief review provides an overview of clinical applications of CLI with a focus on intraoperative margin assessment (IMA) to address shortcomings and provide insight for future work in this application. METHODS: A literature review was performed using PubMed using the search words Cerenkov luminescence imaging (CLI), intraoperative margin assessment (IMA), and image-guided surgery. Articles were selected based on title, abstract, content, and application. RESULTS: Original research was summarized to examine advantages and limitations of CLI compared to other modalities for IMA. The characteristics of Cerenkov luminescence (CL) are defined, and results from relevant clinical trials are discussed. Prospects of ongoing clinical trials are reviewed, along with technological advancements related to CLI. CONCLUSION: CLI is a proven method for molecular imaging and shows feasibility for determining intraoperative margins if future work involves establishing quantitative approaches for attenuation and scattering, depth analysis, and radiation safety for CLI at a larger scale.

Membrane-derived particles shed by PSMA-positive cells function as pro-angiogenic stimuli in tumors.

Cell membrane-derived particles (Mp) are rounded membrane-enclosed particles that are shed from tumor cells. Mp are formed from tumor membranes and are capable of tumor targeting and immunotherapeutic agents because they share membrane homology with parental cells; thus, they are under consideration as a drug delivery vehicle. Prostate-specific membrane antigen (PSMA), a transmembrane glycoprotein with enzymatic functionality, is highly expressed in Mp and extracellular vesicles (EV) from prostate cancer (PCa) with poor clinical prognosis. Although PSMA expression was previously shown in EV and Mp isolated from cell lines and from the blood of patients with high-grade PCa, no pathophysiological effects have been linked to PCa-derived Mp. Here, we compared Mp from PSMA-expressing (PSMA-Mp) and PSMA-non-expressing (WT-Mp) cells side by side in vitro and in vivo. PSMA-Mp can transfer PSMA and new phenotypic characteristics to the tumor microenvironment. The consequence of PSMA transfer to cells and increased secretion of vascular endothelial growth factor-A (VEGF-A), pro-angiogenic and pro-lymphangiogenic mediators, with increased 4E binding protein 1 (4EBP-1) phosphorylation.

A human antibody against pathologic IAPP aggregates protects beta cells in type 2 diabetes models.

In patients with type 2 diabetes, pancreatic beta cells progressively degenerate and gradually lose their ability to produce insulin and regulate blood glucose. Beta cell dysfunction and loss is associated with an accumulation of aggregated forms of islet amyloid polypeptide (IAPP) consisting of soluble prefibrillar IAPP oligomers as well as insoluble IAPP fibrils in pancreatic islets. Here, we describe a human monoclonal antibody selectively targeting IAPP oligomers and neutralizing IAPP aggregate toxicity by preventing membrane disruption and apoptosis in vitro. Antibody treatment in male rats and mice transgenic for human IAPP, and human islet-engrafted mouse models of type 2 diabetes triggers clearance of IAPP oligomers resulting in beta cell protection and improved glucose control. These results provide new evidence for the pathological role of IAPP oligomers and suggest that antibody-mediated removal of IAPP oligomers could be a pharmaceutical strategy to support beta cell function in type 2 diabetes.

Vascularized tumor on a microfluidic chip to study mechanisms promoting tumor neovascularization and vascular targeted therapies.

The cascade of events leading to tumor formation includes induction of a tumor supporting neovasculature as a primary hallmark of cancer. Developing vasculature is difficult to evaluate in vivo but can be captured using microfluidic chip technology and patient derived cells. Herein, we established an on chip approach to investigate the mechanisms promoting tumor vascularization and vascular targeted therapies via co-culture of metastatic renal cell carcinoma spheroids and endothelial cells in a 3D environment. Our model permitted real-time, high-resolution observation and assessment of tumor-induced angiogenesis, where endothelial cells sprout towards the tumor and mimic a vascular network. Bevacizumab, an angiogenic inhibitor, disrupted interactions between vessels and tumors, destroying the vascular network. The on chip approach enabled assessment of endothelial cell biology, vessel's functionality, drug delivery, and molecular expression of PSMA. Finally, observations in the vascularized tumor on chip permitted direct and conclusive quantification of this therapy in weeks as opposed to months in a comparable animal model. Teaser: Vascularized tumor on microfluidic chip provides opportunity to study targeted therapies and improves preclinical drug discovery.

Ambient Light Resistant Shortwave Infrared Fluorescence Imaging for Preclinical Tumor Delineation via the pH Low-Insertion Peptide Conjugated to Indocyanine Green.

Shortwave infrared (900-1,700 nm) fluorescence imaging (SWIRFI) has shown significant advantages over visible (400-650 nm) and near-infrared (700-900 nm) fluorescence imaging (reduced autofluorescence, improved contrast, tissue resolution, and depth sensitivity). However, there is a major lag in the clinical translation of preclinical SWIRFI systems and targeted SWIRFI probes. Methods: We preclinically show that the pH low-insertion peptide conjugated to indocyanine green (pHLIP ICG), currently in clinical trials, is an excellent candidate for cancer-targeted SWIRFI. Results: pHLIP ICG SWIRFI achieved picomolar sensitivity (0.4 nM) with binary and unambiguous tumor screening and resection up to 96 h after injection in an orthotopic breast cancer mouse model. SWIRFI tumor screening and resection had ambient light resistance (possible without gating or filtering) with outstanding signal-to-noise ratio (SNR) and contrast-to-noise ratio (CNR) values at exposures from 10 to 0.1 ms. These SNR and CNR values were also found for the extended emission of pHLIP ICG in vivo (>1,100 nm, 300 ms). Conclusion: SWIRFI sensitivity and ambient light resistance enabled continued tracer clearance tracking with unparalleled SNR and CNR values at video rates for tumor delineation (achieving a tumor-to-muscle ratio above 20). In total, we provide a direct precedent for the democratic translation of an ambient light resistant SWIRFI and pHLIP ICG ecosystem, which can instantly improve tumor resection.

Simultaneous quantitative imaging of two PET radiotracers via the detection of positron-electron annihilation and prompt gamma emissions.

In conventional positron emission tomography (PET), only one radiotracer can be imaged at a time, because all PET isotopes produce the same two 511 keV annihilation photons. Here we describe an image reconstruction method for the simultaneous in vivo imaging of two PET tracers and thereby the independent quantification of two molecular signals. This method of multiplexed PET imaging leverages the 350-700 keV range to maximize the capture of 511 keV annihilation photons and prompt γ-ray emission in the same energy window, hence eliminating the need for energy discrimination during reconstruction or for signal separation beforehand. We used multiplexed PET to track, in mice with subcutaneous tumours, the biodistributions of intravenously injected [124I]I-trametinib and 2-deoxy-2-[18F]fluoro-D-glucose, [124I]I-trametinib and its nanoparticle carrier [89Zr]Zr-ferumoxytol, and the prostate-specific membrane antigen (PSMA) and infused PSMA-targeted chimaeric antigen receptor T cells after the systemic administration of [68Ga]Ga-PSMA-11 and [124I]I. Multiplexed PET provides more information depth, gives new uses to prompt γ-ray-emitting isotopes, reduces radiation burden by omitting the need for an additional computed-tomography scan and can be implemented on preclinical and clinical systems without any modifications in hardware or image acquisition software.

Organ-on-chip systems as a model for nanomedicine.

Nanomedicine is giving rise to increasing numbers of successful drugs, including cancer treatments, molecular imaging agents, and novel vaccine formulations. However, traditionally available model systems offer limited clinical translation and, compared to the number of preclinical studies, the approval rate of nanoparticles (NPs) for clinical use remains disappointingly low. A new paradigm of modeling biological systems on microfluidic chips has emerged in the last decade and is being gradually adopted by the nanomedicine community. These systems mimic tissues, organs, and diseases like cancer, on devices with small physical footprints and complex geometries. In this review, we report studies that used organ-on-chip approaches to study the interactions of NPs with biological systems. We present examples of NP toxicity studies, studies using biological NPs such as viruses, as well as modeling biological barriers and cancer on chip. Organ-on-chip systems present an exciting opportunity and can provide a renewed direction for the nanomedicine community.

Phase 1 Trial of Antibody NI006 for Depletion of Cardiac Transthyretin Amyloid.

BACKGROUND: Transthyretin amyloid (ATTR) cardiomyopathy is a progressive and fatal disease caused by misfolded transthyretin. Despite advances in slowing disease progression, there is no available treatment that depletes ATTR from the heart for the amelioration of cardiac dysfunction. NI006 is a recombinant human anti-ATTR antibody that was developed for the removal of ATTR by phagocytic immune cells. METHODS: In this phase 1, double-blind trial, we randomly assigned (in a 2:1 ratio) 40 patients with wild-type or variant ATTR cardiomyopathy and chronic heart failure to receive intravenous infusions of either NI006 or placebo every 4 weeks for 4 months. Patients were sequentially enrolled in six cohorts that received ascending doses (ranging from 0.3 to 60 mg per kilogram of body weight). After four infusions, patients were enrolled in an open-label extension phase in which they received eight infusions of NI006 with stepwise increases in the dose. The safety and pharmacokinetic profiles of NI006 were assessed, and cardiac imaging studies were performed. RESULTS: The use of NI006 was associated with no apparent drug-related serious adverse events. The pharmacokinetic profile of NI006 was consistent with that of an IgG antibody, and no antidrug antibodies were detected. At doses of at least 10 mg per kilogram, cardiac tracer uptake on scintigraphy and extracellular volume on cardiac magnetic resonance imaging, both of which are imaging-based surrogate markers of cardiac amyloid load, appeared to be reduced over a period of 12 months. The median N-terminal pro-B-type natriuretic peptide and troponin T levels also seemed to be reduced. CONCLUSIONS: In this phase 1 trial of the recombinant human antibody NI006 for the treatment of patients with ATTR cardiomyopathy and heart failure, the use of NI006 was associated with no apparent drug-related serious adverse events. (Funded by Neurimmune; NI006-101 ClinicalTrials.gov number, NCT04360434.).

Translatable Drug-Loaded Iron Oxide Nanophore Sensitizes Murine Melanoma Tumors to Monoclonal Antibody Immunotherapy.

Macrophages comprise a significant portion of the immune cell compartment within tumors and are known contributors to tumor pathology; however, cancer immunotherapies targeting these cells are not clinically available. The iron oxide nanoparticle, ferumoxytol (FH), may be utilized as a nanophore for drug delivery to tumor-associated macrophages. We have demonstrated that a vaccine adjuvant, monophosphoryl lipid A (MPLA), can be stably captured within the carbohydrate shell of ferumoxytol without chemical modification of either the drug or the nanophore. This drug-nanoparticle combination (FH-MPLA) activated macrophages to an antitumorigenic phenotype at clinically relevant concentrations. In the immunotherapy-resistant B16-F10 model of murine melanoma, FH-MPLA treatment induced tumor necrosis and regression in combination with agonistic α-CD40 monoclonal antibody therapy. FH-MPLA, composed of clinically approved nanoparticle and drug payload, represents a potential cancer immunotherapy with translational relevance. FH-MPLA may be useful as an adjunctive therapy to existing antibody-based cancer immunotherapies which target only lymphocytic cells, reshaping the tumor immune environment.

Radiances of Cerenkov-Emitting Isotopes on the IVIS.

Cerenkov (or Cherenkov) luminescence occurs when charged particles exceed the phase velocity of a given medium. Cerenkov has gained interest in preclinical space as well as in clinical trials for optical visualization of numerous radionuclides. However, Cerenkov intensity has to be inferred from alternative databases with energy emission spectra, or theoretical fluence estimates. Here we present the largest experimental dataset of Cerenkov emitting isotopes recorded using the IVIS optical imaging system. We report Cerenkov measurements spanning orders of magnitude normalized to the activity concentration for 21 Cerenkov emitting isotopes, covering electron, alpha, beta minus, and positron emissions. Isotopes measured include Carbon-11, Fluorine-18, Phosphorous-32, Scandium-47, Copper-64, Copper-67, Gallium-68, Arsenic-72, Bromine-76, Yttrium-86, Zirconium-89, Yttrium-90, Iodine-124, Iodine-131, Cerium-134, Lutetium-177, Lead-203, Lead-212, Radium-223, Actinium-225, and Thorium-227. We hope this updating resource will serve as a rank ordering for comparing isotopes for Cerenkov luminescence in the visible window and serve as a rule of thumb for comparing Cerenkov intensities in vitro and in vivo. Methods: All Cerenkov emitting radionuclides were either produced at Memorial Sloan Kettering Cancer Center (Carbon-11, 11 C; Fluorine-18, 18 F; Iodine-124, 124 I), from commercial sources such as Perkin Elmer (Phosphorous-32, 32 P; Yttrium-90, 90 Y), Bayer (Radium-223, 223 Ra, Xofigo), 3D-Imaging (Zirconium-89, 89 Zr), Nuclear Diagnostic Products (Iodine-131, 131 I), or from academic collaborators at Washington University at St. Louis (Copper-64, 64 Cu), University of Wisconsin (Bromine-76, 76 Br), MD Anderson Cancer Center (Yttrium-86, 86 Y), Brookhaven National Laboratory (Arsenic-72, 72 As; Thorium-227, 227 Th), or Oak Ridge National Laboratory (Cerium-134, 134 Ce, Actinium-225, 225 Ac), and Viewpoint Molecular Targeting (Lead-203, 203 Pb; Lead 212, 212 Pb). All isotopes were diluted in triplicate on a black bottomed corning 96 well plate to several activity concentrations ranging from 0.1-250 µCi in 100-200 µL of Phosphate Buffered Saline. Cerenkov imaging was acquired on a single Perkin-Elmer Spectrum In-Vivo Imaging System (IVIS) at field of view c with exposures ranging up to 15 minutes or lower provided no part of the image intensity was saturated, or that the activity significantly changed during the exposure. Experimental radiances on the IVIS were calculated from regions of interest drown over each 96 well, and then normalized for the activity present in the well, and the volume the isotope was diluted into.

Detection of Shortwave-Infrared Cerenkov Luminescence from Medical Isotopes.

Medical radioisotopes produce Cerenkov luminescence (CL) from charged subatomic particles (ß+/-) traveling faster than light in dielectric media (e.g., tissue). CL is a blue-weighted and continuous emission, decreasing proportionally to increasing wavelength. CL imaging (CLI) provides an economic PET alternative with the advantage of also being able to image ß- and α emitters. Like any optical modality, CLI is limited by the optical properties of tissue (scattering, absorption, and ambient photon removal). Shortwave-infrared (SWIR, 900-1700 nm) CL has been detected from MeV linear accelerators but not yet from keV medical radioisotopes. Methods: Indium-gallium-arsenide sensors and SWIR lenses were mounted onto an ambient light-excluding preclinical enclosure. An exposure and processing pipeline was developed for SWIR CLI and then performed across 6 radioisotopes at in vitro and in vivo conditions. Results: SWIR CL was detected from the clinical radioisotopes 90Y, 68Ga, 18F, 89Zr, 131I, and 32P (biomedical research). SWIR CLI's advantage over visible-wavelength (VIS) CLI (400-900 nm) was shown via increased light penetration and decreased scattering at depth. The SWIR CLI radioisotope sensitivity limit (8.51 kBq/µL for 68Ga), emission spectrum, and ex vivo and in vivo examples are reported. Conclusion: This work shows that radioisotope SWIR CLI can be performed with unmodified commercially available components. SWIR CLI has significant advantages over VIS CLI, with preserved VIS CLI features such as radioisotope radiance levels and dose response linearity. Further improvements in SWIR optics and technology are required to enable widespread adoption.

Comprehensive evaluation of human-derived anti-poly-GA antibodies in cellular and animal models of C9orf72 disease.

Hexanucleotide G4C2 repeat expansions in the C9orf72 gene are the most common genetic cause of amyotrophic lateral sclerosis and frontotemporal dementia. Dipeptide repeat proteins (DPRs) generated by translation of repeat-containing RNAs show toxic effects in vivo as well as in vitro and are key targets for therapeutic intervention. We generated human antibodies that bind DPRs with high affinity and specificity. Anti-GA antibodies engaged extra- and intra-cellular poly-GA and reduced aggregate formation in a poly-GA overexpressing human cell line. However, antibody treatment in human neuronal cultures synthesizing exogenous poly-GA resulted in the formation of large extracellular immune complexes and did not affect accumulation of intracellular poly-GA aggregates. Treatment with antibodies was also shown to directly alter the morphological and biochemical properties of poly-GA and to shift poly-GA/antibody complexes to more rapidly sedimenting ones. These alterations were not observed with poly-GP and have important implications for accurate measurement of poly-GA levels including the need to evaluate all centrifugation fractions and disrupt the interaction between treatment antibodies and poly-GA by denaturation. Targeting poly-GA and poly-GP in two mouse models expressing G4C2 repeats by systemic antibody delivery for up to 16 mo was well-tolerated and led to measurable brain penetration of antibodies. Long-term treatment with anti-GA antibodies produced improvement in an open-field movement test in aged C9orf72450 mice. However, chronic administration of anti-GA antibodies in AAV-(G4C2)149 mice was associated with increased levels of poly-GA detected by immunoassay and did not significantly reduce poly-GA aggregates or alleviate disease progression in this model.

High-throughput screening of caterpillars as a platform to study host-microbe interactions and enteric immunity.

Mammalian models of human disease are expensive and subject to ethical restrictions. Here, we present an independent platform for high-throughput screening, using larvae of the tobacco hornworm Manduca sexta, combining diagnostic imaging modalities for a comprehensive characterization of aberrant phenotypes. For validation, we use bacterial/chemical-induced gut inflammation to generate a colitis-like phenotype and identify significant alterations in morphology, tissue properties, and intermediary metabolism, which aggravate with disease progression and can be rescued by antimicrobial treatment. In independent experiments, activation of the highly conserved NADPH oxidase DUOX, a key mediator of gut inflammation, leads to similar, dose-dependent alterations, which can be attenuated by pharmacological interventions. Furthermore, the developed platform could differentiate pathogens from mutualistic gastrointestinal bacteria broadening the scope of applications also to microbiomics and host-pathogen interactions. Overall, larvae-based screening can complement mammals in preclinical studies to explore innate immunity and host-pathogen interactions, thus representing a substantial contribution to improve mammalian welfare.

Prospective testing of clinical Cerenkov luminescence imaging against standard-of-care nuclear imaging for tumour location.

In oncology, the feasibility of Cerenkov luminescence imaging (CLI) has been assessed by imaging superficial lymph nodes in a few patients undergoing diagnostic 18F-fluoro-2-deoxyglucose (18F-FDG) positron emission tomography/computed tomography (PET/CT). However, the weak luminescence signal requires the removal of ambient light. Here we report the development of a clinical CLI fiberscope with a lightproof enclosure, and the clinical testing of the setup using five different radiotracers. In an observational prospective trial (ClinicalTrials.gov identifier NCT03484884 ) involving 96 patients with existing or suspected tumours, scheduled for routine clinical FDG PET or 131I therapy, the level of agreement of CLI with standard-of-care imaging (PET or planar single-photon emission CT) for tumour location was 'acceptable' or higher (≥3 in the 1-5 Likert scale) for 90% of the patients. CLI correlated with the concentration of radioactive activity, and captured therapeutically relevant information from patients undergoing targeted radiotherapy or receiving the alpha emitter 223Ra, which cannot be feasibly imaged clinically. CLI could supplement radiological scans, especially when scanner capacity is limited.

Identification of alternative protein targets of glutamate-ureido-lysine associated with PSMA tracer uptake in prostate cancer cells.

Prostate-specific membrane antigen (PSMA) is highly overexpressed in most prostate cancers and is clinically visualized using PSMA-specific probes incorporating glutamate-ureido-lysine (GUL). PSMA is effectively absent from certain high-mortality, treatment-resistant subsets of prostate cancers, such as neuroendocrine prostate cancer (NEPC); however, GUL-based PSMA tracers are still reported to have the potential to identify NEPC metastatic tumors. These probes may bind unknown proteins associated with PSMA-suppressed cancers. We have identified the up-regulation of PSMA-like aminopeptidase NAALADaseL and the metabotropic glutamate receptors (mGluRs) in PSMA-suppressed prostate cancers and find that their expression levels inversely correlate with PSMA expression and are associated with GUL-based radiotracer uptake. Furthermore, we identify that NAALADaseL and mGluR expression correlates with a unique cell cycle signature. This provides an opportunity for the future study of the biology of NEPC and potential therapeutic directions. Computationally predicting that GUL-based probes bind well to these targets, we designed and synthesized a fluorescent PSMA tracer to investigate these proteins in vitro, where it shows excellent affinity for PSMA, NAALADaseL, and specific mGluRs associated with poor prognosis.

Biomedical Applications of Lanthanide Nanomaterials, for Imaging, Sensing and Therapy.

The application of nanomaterials made of rare earth elements within biomedical sciences continues to make significant progress. The rare earth elements, also called the lanthanides, play an essential role in modern life through materials and electronics. As we learn more about their utility, function, and underlying physics, we can contemplate extending their applications to biomedicine. This particularly applies to diagnosis and radiation therapy due to their relatively unique features, such as an ultra-wide Stokes shift in the luminescence, variable magnetism and potentially tunable properties, due to the library of lanthanides available and their multivalent oxidation state chemistry. The ability to prepare nanomaterials of relatively smaller sizes has increased the likelihood of use in vivo. In this review, we summarize the different emerging applications of nanoparticles with rare earth elements as the host or doped elements for biomedical applications in the past three to four years, especially in the area of imaging and disease diagnosis. Researchers have made progress in utilizing surfactants and polymers to modify the surface of lanthanide nanoparticles to enhance biocompatibility. At the same time, specific antibodies and proteins can also be conjugated to these nanoparticles to increase targeting efficiency for specific tumor models. Finally, in the near-infrared II imaging window, lanthanide nanoparticles have been shown to exhibit extraordinary bright emission, which is an exciting development for image-guided surgery.

The ancillary effects of nanoparticles and their implications for nanomedicine.

Nanoparticles are often engineered as a scaffolding system to combine targeting, imaging and/or therapeutic moieties into a unitary agent. However, mostly overlooked, the nanomaterial itself interacts with biological systems exclusive of application-specific particle functionalization. This nanoparticle biointerface has been found to elicit specific biological effects, which we term 'ancillary effects'. In this Review, we describe the current state of knowledge of nanobiology gleaned from existing studies of ancillary effects with the objectives to describe the potential of nanoparticles to modulate biological effects independently of any engineered function; evaluate how these effects might be relevant for nanomedicine design and functional considerations, particularly how they might be useful to inform clinical decision-making; identify potential clinical harm that arises from adverse nanoparticle interactions with biology; and, finally, highlight the current lack of knowledge in this area as both a barrier and an incentive to the further development of nanomedicine.

Small, Seeding-Competent Huntingtin Fibrils Are Prominent Aggregate Species in Brains of zQ175 Huntington's Disease Knock-in Mice.

The deposition of mutant huntingtin (mHTT) protein aggregates in neurons of patients is a pathological hallmark of Huntington's disease (HD). Previous investigations in cell-free and cell-based disease models showed mHTT exon-1 (mHTTex1) fragments with pathogenic polyglutamine (polyQ) tracts (>40 glutamines) to self-assemble into highly stable, ß-sheet-rich protein aggregates with a fibrillar morphology. HD knock-in mouse models have not been extensively studied with regard to mHTT aggregation. They endogenously produce full-length mHTT with a pathogenic polyQ tract as well as mHTTex1 fragments. Here, we demonstrate that seeding-competent, fibrillar mHTT aggregates can be readily detected in brains of zQ175 knock-in HD mice. To do this, we applied a highly sensitive FRET-based protein amplification assay that is capable of detecting seeding-competent mHTT aggregate species down to the femtomolar range. Furthermore, we show that fibrillar structures with an average length of ∼200 nm can be enriched with aggregate-specific mouse and human antibodies from zQ175 mouse brain extracts through immunoprecipitations, confirming that such structures are formed in vivo. Together these studies indicate that small, fibrillar, seeding-competent mHTT structures are prominent aggregate species in brains of zQ175 mice.