Improved production of 76Br, 77Br and 80mBr via CoSe cyclotron targets and vertical dry distillation.

INTRODUCTION: The radioisotopes of bromine are uniquely suitable radiolabels for small molecule theranostic radiopharmaceuticals but are of limited availability due to production challenges. Significantly improved methods were developed for the production and radiochemical isolation of clinical quality 76Br, 77Br, and 80mBr. The radiochemical quality of the radiobromine produced using these methods was tested through the synthesis of a novel 77Br-labeled inhibitor of poly (ADP-ribose) polymerase-1 (PARP-1), a DNA damage response protein. METHODS: 76Br, 77Br, and 80mBr were produced in high radionuclidic purity via the proton irradiation of novel isotopically-enriched Co76Se, Co77Se, and Co80Se intermetallic targets, respectively. Radiobromine was isolated through thermal chromatographic distillation in a vertical furnace assembly. The 77Br-labeled PARP inhibitor was synthesized via copper-mediated aryl boronic ester radiobromination. RESULTS: Cyclotron production yields were 103 ±â€¯10 MBq∙µA-1∙h-1 for 76Br, 88 ±â€¯10 MBq∙µA-1∙h-1 for 80mBr at 16 MeV and 17 ±â€¯1 MBq∙µA-1∙h-1 for 77Br at 13 MeV. Radiobromide isolation yields were 76 ±â€¯11% in a small volume of aqueous solution. The synthesized 77Br-labeled PARP-1 inhibitor had a measured apparent molar activity up to 700 GBq/µmol at end of synthesis. CONCLUSIONS: A novel selenium alloy target enabled clinical-scale production of 76Br, 77Br, and 80mBr with high apparent molar activities, which was used to for the production of a new 77Br-labeled inhibitor of PARP-1. ADVANCES IN KNOWLEDGE: New methods for the cyclotron production and isolation of radiobromine improved the production capacity of 77Br by a factor of three and 76Br by a factor of six compared with previous methods. IMPLICATIONS FOR PATIENT CARE: Preclinical translational research of 77Br-based Auger electron radiotherapeutics, such as those targeting PARP-1, will require the production of GBq-scale 77Br, which necessitates next-generation, high-yielding, isotopically-enriched cyclotron targets, such as the novel intermetallic Co77Se.

PET Measures of D1, D2, and DAT Binding Are Associated With Heightened Tactile Responsivity in Rhesus Macaques: Implications for Sensory Processing Disorder.

Sensory processing disorder (SPD), a developmental regulatory condition characterized by marked under- or over-responsivity to non-noxious sensory stimulation, is a common but poorly understood disorder that can profoundly affect mood, cognition, social behavior and adaptive life skills. Little is known about the etiology and neural underpinnings. Clinical research indicates that children with SPD show greater prevalence of difficulties in complex cognitive behavior including working memory, behavioral flexibility, and regulation of sensory and affective functions, which are related to prefrontal cortex (PFC), striatal, and midbrain regions. Neuroimaging may provide insight into mechanisms underlying SPD, and animal experiments provide important evidence that is not available in human studies. Rhesus monkeys (N = 73) were followed over a 20-year period from birth into old age. We focused on a single sensory modality, the tactile system, measured at 5-7 years, because of its critical importance for nourishment, attachment, and social reward in development. Positron emission tomography imaging was conducted at ages 12-18 years to quantify the availability of the D1 and D2 subtypes of the DA receptor (D1R and D2R), and the DA transporter (DAT). Heightened tactile responsivity was related to (a) elevated D1R in PFC overall, including lateral, ventrolateral, medial, anterior cingulate (aCg), frontopolar, and orbitofrontal (OFC) subregions, as well as nucleus accumbens (Acb), (b) reduced D2R in aCg, OFC, and substantia nigra/ventral tegmental area, and (c) elevated DAT in putamen. These findings suggest a mechanism by which DA pathways may be altered in SPD. These pathways are associated with reward processing and pain regulation, providing top-down regulation of sensory and affective processes. The balance between top-down cognitive control in the PFC-Acb pathway and bottom-up motivational function of the VTA-Acb-PFC pathway is critical for successful adaptive function. An imbalance in these two systems might explain DA-related symptoms in children with SPD, including reduced top-down regulatory function and exaggerated responsivity to stimuli. These results provide more direct evidence that SPD may involve altered DA receptor and transporter function in PFC, striatal, and midbrain regions. More work is needed to extend these results to humans.

Radiochemical isolation method for the production of 52gMn from natCr for accelerator targets.

We report a novel, precipitation-based method for the isolation of Mn from Cr targets for cyclotron production of 52gMn. The separation produces no-carrier-added 52gMn with a decay corrected radiochemical yield of 85 ±â€¯3% and apparent molar activity for DOTA of 1.3 GBq/µmol. This method reduces stable metallic impurities in the purified 52gMn compared to previously reported chromatographic methods.

Simplified and automatable radiochemical separation strategy for the production of radiopharmaceutical quality 86Y using single column extraction chromatography.

We present a simplified, automatable single-column radiochemical separation method using the extraction chromatographic branched-DGA resin for the production of no-carrier-added 86Y with a radiochemical yield higher than 95%, an apparent molar activity of 1.4 ±â€¯0.4 Ci/µmol (DOTA) and 2.3 ±â€¯0.7 Ci/µmol (DTPA), and a run-to-run recycling efficiency of the isotopically-enriched target of 98 ±â€¯1%. These results enable the preparation of 86Y radiopharmaceuticals for 86Y/90Y-based cancer theranostic applications.

Evaluation of a chloride-based 89Zr isolation strategy using a tributyl phosphate (TBP)-functionalized extraction resin.

INTRODUCTION: The remarkable stability of the 89Zr-DOTA complex has been shown in recent literature. The formation of this complex appears to require 89Zr-chloride as the complexation precursor rather than the more conventional 89Zr-oxalate. In this work we present a method for the direct isolation of 89Zr-chloride from irradiated natY foils. METHODS: 89Zr, 88Zr, and 88Y were prepared by 16 MeV proton irradiation of natY foils and used for batch-extraction based equilibrium coefficient measurements for TBP and UTEVA resin. Radionuclidically pure 89Zr was prepared by 14 MeV proton-irradiation of natY foils. These foils were dissolved in concentrated HCl, trapped on columns of TBP or UTEVA resin, and 89Zr-chloride was eluted in <1 mL of 0.1 M HCl. For purposes of comparison, conventionally-isolated 89Zr-oxalate was converted to 89Zr-chloride by trapping, rinsing, and elution from a QMA cartridge into 1 M HCl. Trace metal analysis was performed on the resulting 89Zr products. RESULTS: Equilibrium coefficients for Y and Zr were similar between UTEVA and TBP resins across all HCl concentrations. Kd values of <10-1 mL/g were observed for Y across all HCl concentrations. Kd values of >103 mL/g were observed at HCl concentrations >9 M for Zr, falling to Kd values of <100 mL/g at low HCl concentrations. 89Zr-chloride was recovered from small columns of TBP in <1 mL of 0.1 M HCl with an overall recovery efficiency of 89 ±â€¯3% (n = 3). An average Y/Zr separation factor of 1.5 × 105 (n = 3) was obtained. Trace metal impurities, notably Fe, were higher in TBP-isolated 89Zr-chloride compared with 89Zr-chloride prepared using the conventional two-step procedure. CONCLUSION: TBP-functionalized resin appears promising for the direct isolation of 89Zr-chloride from irradiated natY targets. Excellent 89Zr recovery efficiencies were obtained, and chemical purity was sufficient for proof-of-concept chelation studies.

Characterization of the radiosynthesis and purification of [18F]THK-5351, a PET ligand for neurofibrillary tau.

This work characterizes the radiochemical synthesis, purification, and formulation of [18F]THK-5351, a tau PET radioligand, and develops an automated radiosynthesis routine (ELIXYS, Sofie Biosciences). Nucleophilic radiofluorination reaction was complete by 7min at 110°C with radiochemical yields proportional to precursor mass (0.1-0.5mg). Optimized HPLC purification produced radiotracer product with no chemical impurities observed on analytical HPLC in formulation. Automated radiosynthesis (ELIXYS), HPLC purification and formulation was completed in 86min producing formulated product suitable for human research use.

Preparation and in vivo characterization of 51MnCl2 as PET tracer of Ca2+ channel-mediated transport.

Manganese has long been employed as a T1-shortening agent in magnetic resonance imaging (MRI) applications, but these techniques are limited by the biotoxicity of bulk-manganese. Positron emission tomography (PET) offers superior contrast sensitivity compared with MRI, and recent preclinical PET studies employing 52gMn (t1/2: 5.6 d, ß+: 29%) show promise for a variety of applications including cell tracking, neural tract tracing, immunoPET, and functional ß-cell mass quantification. The half-life and confounding gamma emissions of 52gMn are prohibitive to clinical translation, but the short-lived 51Mn (t1/2: 46 min, ß+: 97%) represents a viable alternative. This work develops methods to produce 51Mn on low-energy medical cyclotrons, characterizes the in vivo behavior of 51MnCl2 in mice, and performs preliminary human dosimetry predictions. 51Mn was produced by proton irradiation of electrodeposited isotopically-enriched 54Fe targets. Radiochemically isolated 51MnCl2 was intravenously administered to ICR mice which were scanned by dynamic and static PET, followed by ex vivo gamma counting. Rapid blood clearance was observed with stable uptake in the pancreas, kidneys, liver, heart, and salivary gland. Dosimetry calculations predict that 370 MBq of 51Mn in an adult human male would yield an effective dose equivalent of approximately 13.5 mSv, roughly equivalent to a clinical [18F]-FDG procedure.

Radiomanganese PET Detects Changes in Functional ß-Cell Mass in Mouse Models of Diabetes.

The noninvasive measurement of functional ß-cell mass would be clinically valuable for monitoring the progression of type 1 and type 2 diabetes as well as the viability of transplanted insulin-producing cells. Although previous work using MRI has shown promise for functional ß-cell mass determination through voltage-dependent Ca2+ channel (VDCC)-mediated internalization of Mn2+, the clinical utility of this technique is limited by the cytotoxic levels of the Mn2+ contrast agent. Here, we show that positron emission tomography (PET) is advantageous for determining functional ß-cell mass using 52Mn2+ (t1/2: 5.6 days). We investigated the whole-body distribution of 52Mn2+ in healthy adult mice by dynamic and static PET imaging. Pancreatic VDCC uptake of 52Mn2+ was successfully manipulated pharmacologically in vitro and in vivo using glucose, nifedipine (VDCC blocker), the sulfonylureas tolbutamide and glibenclamide (KATP channel blockers), and diazoxide (KATP channel opener). In a mouse model of streptozotocin-induced type 1 diabetes, 52Mn2+ uptake in the pancreas was distinguished from healthy controls in parallel with classic histological quantification of ß-cell mass from pancreatic sections. 52Mn2+-PET also reported the expected increase in functional ß-cell mass in the ob/ob model of pretype 2 diabetes, a result corroborated by histological ß-cell mass measurements and live-cell imaging of ß-cell Ca2+ oscillations. These results indicate that 52Mn2+-PET is a sensitive new tool for the noninvasive assessment of functional ß-cell mass.

ImmunoPET Imaging of CTLA-4 Expression in Mouse Models of Non-small Cell Lung Cancer.

Cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) is expressed on the surface of activated T cells and some tumor cells, and is the target of the clinically approved monoclonal antibody ipilimumab. In this study, we investigate specific binding of radiolabeled ipilimumab to CTLA-4 expressed by human non-small cell lung cancer cells in vivo using positron emission tomography (PET). Ipilimumab was radiolabeled with 64Cu (t1/2 = 12.7 h) through the use of the chelator 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) to formulate 64Cu-DOTA-ipilimumab. CTLA-4 expression in three non-small cell lung cancer (NSCLC) cell lines (A549, H460, and H358) was verified and quantified by Western blot and enzyme-linked immunosorbent assays (ELISA). A receptor binding assay was utilized to monitor the binding and internalization of 64Cu-DOTA-ipilimumab in the NSCLC cell lines. Next, the biodistribution of 64Cu-DOTA-ipilimumab was mapped by longitudinal PET imaging up to 48 h after injection. Ex vivo biodistribution and histological studies were employed to verify PET results. By in vitro analysis, CTLA-4 was found to be expressed on all three NSCLC cell lines with A549 and H358 showing the highest and lowest level of expression, respectively. PET imaging and quantification verified these findings as the tracer accumulated highest in the A549 tumor model (9.80 ± 0.22%ID/g at 48 h after injection; n = 4), followed by H460 and H358 tumors with uptakes of 9.37 ± 0.26%ID/g and 7.43 ± 0.05%ID/g, respectively (n = 4). The specificity of the tracer was verified by injecting excess ipilimumab in A549 tumor-bearing mice, which decreased tracer uptake to 6.90 ± 0.51%ID/g at 48 after injection (n = 4). Ex vivo analysis following the last imaging session also corroborated these findings. 64Cu-DOTA-ipilimumab showed enhanced and persistent accumulation in CTLA-4-expressing tissues, which will enable researchers further insight into CTLA-4 targeted therapies in the future.

Radiolabeled pertuzumab for imaging of human epidermal growth factor receptor 2 expression in ovarian cancer.

PURPOSE: Human epidermal growth factor receptor 2 (HER2) is over-expressed in over 30% of ovarian cancer cases, playing an essential role in tumorigenesis and metastasis. Non-invasive imaging of HER2 is of great interest for physicians as a mean to better detect and monitor the progression of ovarian cancer. In this study, HER2 was assessed as a biomarker for ovarian cancer imaging using 64Cu-labeled pertuzumab for immunoPET imaging. METHODS: HER2 expression and binding were examined in three ovarian cancer cell lines (SKOV3, OVCAR3, Caov3) using in vitro techniques, including western blot and saturation binding assays. PET imaging and biodistribution studies in subcutaneous models of ovarian cancer were performed for non-invasive in vivo evaluation of HER2 expression. Additionally, orthotopic models were employed to further validate the imaging capability of 64Cu-NOTA-pertuzumab. RESULTS: HER2 expression was highest in SKOV3 cells, while OVCAR3 and Caov3 displayed lower HER2 expression. 64Cu-NOTA-pertuzumab showed high specificity for HER2 (Ka = 3.1 ± 0.6 nM) in SKOV3. In subcutaneous tumors, PET imaging revealed tumor uptake of 41.8 ± 3.8, 10.5 ± 3.9, and 12.1 ± 2.3%ID/g at 48 h post-injection for SKOV3, OVCAR3, and Caov3, respectively (n = 3). In orthotopic models, PET imaging with 64Cu-NOTA-pertuzumab allowed for rapid and clear delineation of both primary and small peritoneal metastases in HER2-expressing ovarian cancer. CONCLUSIONS: 64Cu-NOTA-pertuzumab is an effective PET tracer for the non-invasive imaging of HER2 expression in vivo, rendering it a potential tracer for treatment monitoring and improved patient stratification.

Surfactant-stripped naphthalocyanines for multimodal tumor theranostics with upconversion guidance cream.

Surfactant-stripped, nanoformulated naphthalocyanines (nanonaps) can be formed with Pluronic F127 and low temperature membrane processing, resulting in dispersed frozen micelles with extreme contrast in the near infrared region. Here, we demonstrate that nanonaps can be used for multifunctional cancer theranostics. This includes lymphatic mapping and whole tumor photoacoustic imaging following intradermal or intravenous injection in rodents. Without further modification, pre-formed nanonaps were used for positron emission tomography and passively accumulated in subcutaneous murine tumors. Because the nanonaps used absorb light beyond the visible range, a topical upconversion skin cream was developed for anti-tumor photothermal therapy with laser placement that can be guided by the naked eye.

ImmunoPET and Near-Infrared Fluorescence Imaging of Pancreatic Cancer with a Dual-Labeled Bispecific Antibody Fragment.

Dual-targeted imaging agents have shown improved targeting efficiencies in comparison to single-targeted entities. The purpose of this study was to quantitatively assess the tumor accumulation of a dual-labeled heterobifunctional imaging agent, targeting two overexpressed biomarkers in pancreatic cancer, using positron emission tomography (PET) and near-infrared fluorescence (NIRF) imaging modalities. A bispecific immunoconjugate (heterodimer) of CD105 and tissue factor (TF) Fab' antibody fragments was developed using click chemistry. The heterodimer was dual-labeled with a radionuclide (64Cu) and fluorescent dye. PET/NIRF imaging and biodistribution studies were performed in four-to-five week old nude athymic mice bearing BxPC-3 (CD105/TF+/+) or PANC-1 (CD105/TF-/-) tumor xenografts. A blocking study was conducted to investigate the specificity of the tracer. Ex vivo tissue staining was performed to compare TF/CD105 expression in tissues with PET tracer uptake to validate in vivo results. PET imaging of 64Cu-NOTA-heterodimer-ZW800 in BxPC-3 tumor xenografts revealed enhanced tumor uptake (21.0 ± 3.4%ID/g; n = 4) compared to the homodimer of TRC-105 (9.6 ± 2.0%ID/g; n = 4; p < 0.01) and ALT-836 (7.6 ± 3.7%ID/g; n = 4; p < 0.01) at 24 h postinjection. Blocking studies revealed that tracer uptake in BxPC-3 tumors could be decreased by 4-fold with TF blocking and 2-fold with CD105 blocking. In the negative model (PANC-1), heterodimer uptake was significantly lower than that found in the BxPC-3 model (3.5 ± 1.1%ID/g; n = 4; p < 0.01). The specificity was confirmed by the successful blocking of CD105 or TF, which demonstrated that the dual targeting with 64Cu-NOTA-heterodimer-ZW800 provided an improvement in overall tumor accumulation. Also, fluorescence imaging validated the PET imaging, allowing for clear delineation of the xenograft tumors. Dual-labeled heterodimeric imaging agents, like 64Cu-NOTA-heterodimer-ZW800, may increase the overall tumor accumulation in comparison to single-targeted homodimers, leading to improved imaging of cancer and other related diseases.

Chelator-Free Radiolabeling of Nanographene: Breaking the Stereotype of Chelation.

Macrocyclic chelators have been widely employed in the realm of nanoparticle-based positron emission tomography (PET) imaging, whereas its accuracy remains questionable. Here, we found that 64 Cu can be intrinsically labeled onto nanographene based on interactions between Cu and the π electrons of graphene without the need of chelator conjugation, providing a promising alternative radiolabeling approach that maintains the native in vivo pharmacokinetics of the nanoparticles. Due to abundant π bonds, reduced graphene oxide (RGO) exhibited significantly higher labeling efficiency in comparison with graphene oxide (GO) and exhibited excellent radiostability in vivo. More importantly, nonspecific attachment of 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA) on nanographene was observed, which revealed that chelator-mediated nanoparticle-based PET imaging has its inherent drawbacks and can possibly lead to erroneous imaging results in vivo.

Intrinsic and Stable Conjugation of Thiolated Mesoporous Silica Nanoparticles with Radioarsenic.

The development of new image-guided drug delivery tools to improve the therapeutic efficacy of chemotherapeutics remains an important goal in nanomedicine. Using labeling strategies that involve radioelements that have theranostic pairs of diagnostic positron-emitting isotopes and therapeutic electron-emitting isotopes has promise in achieving this goal and further enhancing drug performance through radiotherapeutic effects. The isotopes of radioarsenic offer such theranostic potential and would allow for the use of positron emission tomography (PET) for image-guided drug delivery studies of the arsenic-based chemotherapeutic arsenic trioxide (ATO). Thiolated mesoporous silica nanoparticles (MSN) are shown to effectively and stably bind cyclotron-produced radioarsenic. Labeling studies elucidate that this affinity is a result of specific binding between trivalent arsenic and nanoparticle thiol surface modification. Serial PET imaging of the in vivo murine biodistribution of radiolabeled silica nanoparticles shows very good stability toward dearsenylation that is directly proportional to silica porosity. Thiolated MSNs are found to have a macroscopic arsenic loading capacity of 20 mg of ATO per gram of MSN, sufficient for delivery of chemotherapeutic quantities of the drug. These results show the great potential of radioarsenic-labeled thiolated MSN for the preparation of theranostic radiopharmaceuticals and image-guided drug delivery of ATO-based chemotherapeutics.

Half-life of 51Mn.

The half-life of 51Mn was measured by serial gamma spectrometry of the 511-keV annihilation photon following decay by ß + emission. Data were collected every 100 seconds for 100,000-230,000 seconds within each measurement (n = 4). The 511-keV incidence rate was calculated from the 511-keV spectral peak area and count duration, corrected for detector dead time and radioactive decay. Least-squares regression analysis was used to determine the half-life of 51Mn while accounting for the presence of background contaminants, notably 55Co. The result was 45.59 ± 0.07 min, which is the highest precision measurement to date and disagrees with the current Nuclear Data Sheets value by over 6σ.

Spot-welding solid targets for high current cyclotron irradiation.

Zirconium-89 finds broad application for use in positron emission tomography. Its cyclotron production has been limited by the heat transfer from yttrium targets at high beam currents. A spot welding technique allows a three-fold increase in beam current, without affecting 89Zr quality. An yttrium foil, welded to a jet-cooled tantalum support base accommodates a 50µA proton beam degraded to 14MeV. The resulting activity yield of 48±4 MBq/(µA∙hr) now extends the outreach of 89Zr for a broader distribution.

Development of a novel linearly-filled Derenzo microPET phantom.

Positron emission tomography (PET) phantoms are used to calibrate PET scanners so that inter-scanner and inter-isotope comparison can be made between PET datasets. Hot rod style phantoms have a hole pattern, which is filled with a positron-emitting isotope and typically involves using two radioisotope reservoirs with the pattern created with channels in between. However, this configuration is difficult to fill and requires an excess of activity and volume. Here we present an alternative design, a phantom that is linearly filled-one channel at a time. The process of fabrication of prototypes of the design is described and PET images of the prototyped phantom are also shown for a variety of commonly used radioisotopes ((52)Mn, (64)Cu, (76)Br, (124)I). This design allows for a large reduction in isotope volume and required filling time making a quality assurance (QA) protocol safer, more efficient and less costly.

Uptake and retention of manganese contrast agents for PET and MRI in the rodent brain.

Manganese-enhanced magnetic resonance imaging (MRI) is an established neuroimaging method for signal enhancement, tract tracing, and functional studies in rodents. Along with the increasing availability of combined positron emission tomography (PET) and MRI scanners, the recent development of the positron-emitting isotope 52 Mn has prompted interest in the use of Mn2+ as a dual-modality contrast agent. In this work, we characterized and compared the uptake of systemically delivered Mn2+ and radioactive 52 Mn2+ in the rat brain for MRI and PET, respectively. Additionally, we examined the biodistribution of two formulations of 52 Mn2+ in the rat. In MRI, maximum uptake was observed one day following delivery of the highest MnCl2 dose tested (60 mg/kg), with some brain regions showing delayed maximum enhancement 2-4 days following delivery. In PET, we observed low brain uptake after systemic delivery, with a maximum of approximately 0.2% ID/g. We also studied the effect of final formulation vehicle (saline compared with MnCl2 ) on 52 Mn2+ organ biodistribution and brain uptake. We observed that the addition of bulk Mn2+ carrier to 52 Mn2+ in solution resulted in significantly reduced 52 Mn2+ uptake in the majority of organs, including the brain. These results lay the groundwork for further development of 52 Mn PET or dual Mn-enhanced PET-MR neuroimaging in rodents, and indicate several interesting potential applications of 52 Mn PET in other organs and systems. Copyright © 2016 John Wiley & Sons, Ltd.

Re-assessing the enhanced permeability and retention effect in peripheral arterial disease using radiolabeled long circulating nanoparticles.

As peripheral arterial disease (PAD) results in muscle ischemia and neovascularization, it has been claimed that nanoparticles can passively accumulate in ischemic tissues through the enhanced permeability and retention (EPR) effect. At this time, a quantitative evaluation of the passive targeting capabilities of nanoparticles has not been reported in PAD. Using a murine model of hindlimb ischemia, we quantitatively assessed the passive targeting capabilities of (64)Cu-labeled PEGylated reduced graphene oxide - iron oxide nanoparticles ((64)Cu-RGO-IONP-PEG) through the EPR effect using positron emission tomography (PET) imaging. Serial laser Doppler imaging was performed to monitor changes in blood perfusion upon surgical induction of ischemia. Nanoparticle accumulation was assessed at 3, 10, and 17 days post-surgery and found to be highest at 3 days post-surgery, with the ischemic hindlimb displaying an accumulation of 14.7 ± 0.5% injected dose per gram (%ID/g). Accumulation of (64)Cu-RGO-IONP-PEG was lowest at 17 days post-surgery, with the ischemic hindlimb displaying only 5.1 ± 0.5%ID/g. Furthermore, nanoparticle accumulation was confirmed by photoacoustic imaging (PA). The combination of PET and serial Doppler imaging showed that nanoparticle accumulation in the ischemic hindlimb negatively correlated with blood perfusion. Thus, we quantitatively confirmed that (64)Cu-RGO-IONP-PEG passively accumulated in ischemic tissue via the EPR effect, which is reduced as the perfusion normalizes. As (64)Cu-RGO-IONP-PEG displayed substantial accumulation in the ischemic tissue, this nanoparticle platform may function as a future theranostic agent, providing both imaging and therapeutic applications.

Accelerated Blood Clearance Phenomenon Reduces the Passive Targeting of PEGylated Nanoparticles in Peripheral Arterial Disease.

Peripheral arterial disease (PAD) is a leading global health concern. Due to limited imaging and therapeutic options, PAD and other ischemia-related diseases may benefit from the use of long circulating nanoparticles as imaging probes and/or drug delivery vehicles. Polyethylene glycol (PEG)-conjugated nanoparticles have shown shortened circulation half-lives in vivo when injected multiple times into a single subject. This phenomenon has become known as the accelerated blood clearance (ABC) effect. The phenomenon is of concern for clinical translation of nanomaterials as it limits the passive accumulation of nanoparticles in many diseases, yet it has not been evaluated using inorganic or organic-inorganic hybrid nanoparticles. Herein, we found that the ABC phenomenon was induced by reinjection of PEGylated long circulating organic-inorganic hybrid nanoparticles, which significantly reduced the passive targeting of (64)Cu-labeled PEGylated reduced graphene oxide-iron oxide nanoparticles ((64)Cu-RGO-IONP-PEG) in a murine model of PAD. Positron emission tomography (PET) imaging was performed at 3, 10, and 17 days postsurgical induction of hindlimb ischemia. At day 3 postsurgery, the nanoparticles displayed a long circulation half-life with enhanced accumulation in the ischemic hindlimb. At days 10 and 17 postsurgery, reinjected mice displayed a short circulation half-life and lower accumulation of the nanoparticles in the ischemic hindlimb, in comparison to the naïve group. Also, reinjected mice showed significantly higher liver uptake than the naïve group, indicating that the nanoparticles experienced higher sequestration by the liver in the reinjected group. Furthermore, photoacoustic (PA) imaging and Prussian blue staining confirmed the enhanced accumulation of the nanoparticles in the liver tissue of reinjected mice. These findings validate the ABC phenomenon using long circulating organic-inorganic hybrid nanoparticles upon multiple administrations to the same animal, which may provide valuable insight into the future clinical applications of nanoparticles for imaging and treatment of PAD.