Development of finely tuned liposome nanoplatform for macrophage depletion.

BACKGROUND: Immunotherapy with clodronate-encapsulated liposomes, which induce macrophage depletion, has been studied extensively. However, previously reported liposomal formulation-based drugs (Clodrosome® and m-Clodrosome®) are limited by their inconsistent size and therapeutic efficacy. Thus, we aimed to achieve consistent therapeutic effects by effectively depleting macrophages with uniform-sized liposomes. RESULTS: We developed four types of click chemistry-based liposome nanoplatforms that were uniformly sized and encapsulated with clodronate, for effective macrophage depletion, followed by conjugation with Man-N3 and radiolabeling. Functionalization with Man-N3 improves the specific targeting of M2 macrophages, and radioisotope labeling enables in vivo imaging of the liposome nanoplatforms. The functionalized liposome nanoplatforms are stable under physiological conditions. The difference in the biodistribution of the four liposome nanoplatforms in vivo were recorded using positron emission tomography imaging. Among the four platforms, the clodronate-encapsulated mannosylated liposome effectively depleted M2 macrophages in the normal liver and tumor microenvironment ex vivo compared to that by Clodrosome® and m-Clodrosome®. CONCLUSION: The newly-developed liposome nanoplatform, with finely tuned size control, high in vivo stability, and excellent ex vivo M2 macrophage targeting and depletion effects, is a promising macrophage-depleting agent.

Circulation Time-Optimized Albumin Nanoplatform for Quantitative Visualization of Lung Metastasis via Targeting of Macrophages.

The development of molecular imaging probes to identify key cellular changes within lung metastases may lead to noninvasive detection of metastatic lesions in the lung. In this study, we constructed a macrophage-targeted clickable albumin nanoplatform (CAN) decorated with mannose as the targeting ligand using a click reaction to maintain the intrinsic properties of albumin in vivo. We also modified the number of mannose molecules on the CAN and found that mannosylated serum albumin (MSA) harboring six molecules of mannose displayed favorable pharmacokinetics that allowed high-contrast imaging of the lung, rendering it suitable for in vivo visualization of lung metastases. Due to the optimized control of functionalization and surface modification, MSA enhanced blood circulation time and active/passive targeting abilities and was specifically incorporated by mannose receptor (CD206)-expressing macrophages in the metastatic lung. Moreover, extensive in vivo imaging studies using single-photon emission computed tomography (SPECT)/CT and positron emission tomography (PET) revealed that blood circulation of time-optimized MSA can be used to discern metastatic lesions, with a strong correlation between its signal and metastatic burden in the lung.

Comparison Between Early and Late Retensioning of an Adjustable-Loop Cortical Suspension Device During Hamstring ACL Reconstruction.

BACKGROUND: Biomechanical studies have demonstrated significant loosening of the adjustable-loop device as compared with the fixed-loop device used in anterior cruciate ligament reconstruction. Retensioning of the adjustable loop has been recommended; however, the timing of the retensioning is unknown. HYPOTHESIS: Early (ER) and late retensioning (LR) will show similar gapping between the femoral tunnel and graft on follow-up magnetic resonance imaging (MRI) and similar clinical outcomes. STUDY DESIGN: Cohort study; Level of evidence, 3. METHODS: This study included 101 patients who underwent hamstring anterior cruciate ligament reconstruction using the adjustable-loop device for femoral fixation between June 2016 and January 2018. All patients a had follow-up MRI on postoperative day 1. Patients with revision surgery and those with reinjury after reconstruction were excluded. In the ER group, retensioning and knot tying of the initially tightened adjustable loop were performed after the flip of the button and before the graft was fixed at the tibia. In the LR group, retensioning and knot tying were performed after initial tightening of the adjustable loop and graft fixation at the tibial side. The tunnel-graft gap measured on multiplanar reformatted images of MRI scans was compared between the groups, as were clinical outcomes. RESULTS: The mean age of the patients at the time of surgery was 30.3 years (range, 14-61 years). ER and knot tying were performed in 56 patients and LR and knot tying in 45. Preoperative characteristics of the 2 groups showed no significant differences. The mean ± SD tunnel-graft gap was 1.5 ± 2.0 mm in the ER group and 5.4 ± 4.0 mm in the LR group (P < .001). There were no significant differences in clinical outcomes between the groups. CONCLUSION: ER and knot tying demonstrated less tunnel-graft gap than that of LR. However, there were no differences in clinical outcomes according to the timing of retensioning.

Performance Evaluation of SimPET-X, a PET Insert for Simultaneous Mouse Total-Body PET/MR Imaging.

PURPOSE: In this study, a small animal PET insert (SimPET-X, Brightonix Imaging Inc.) for simultaneous PET/MR imaging studies is presented. This insert covers an 11-cm-long axial field-of-view (FOV) and enables imaging of mouse total-bodies and rat heads. PROCEDURES: SimPET-X comprises 16 detector modules to yield a ring diameter of 63 mm and an axial FOV of 110 mm. The detector module supports four detector blocks, each comprising two 4 × 4 SiPM arrays coupled with a 20 × 9 array of LSO crystals (1.2 × 1.2 × 10 mm3). The physical characteristics of SimPET-X were measured in accordance with the NEMA NU4-2008 standard protocol. In addition, we assessed the compatibility of SimPET-X with a small animal-dedicated MRI (M7, Aspect Imaging) and conducted phantom and animal studies. RESULTS: The radial spatial resolutions at the center based on 3D OSEM without and with the warm background were 0.73 mm and 0.99 mm, respectively. The absolute peak sensitivity of the system was 10.44% with an energy window of 100-900 keV and 8.27% with an energy window of 250-750 keV. The peak NECR and scatter fraction for the mouse phantom were 348 kcps at 26.2 MBq and 22.1% with an energy window of 250-750 keV, respectively. The standard deviation of pixel value in the uniform region of an NEMA IQ phantom was 4.57%. The spillover ratios for air- and water-filled chambers were 9.0% and 11.0%, respectively. In the hot-rod phantom image reconstructed using 3D OSEM-PSF, all small rods were resolved owing to the high spatial resolution of the SimPET-X system. There was no notable interference between SimPET-X and M7 MRI. SimPET-X provided high-quality mouse images with superior spatial resolution, sensitivity, and counting rate performance. CONCLUSION: SimPET-X yielded a remarkably improved sensitivity and NECR compared with SimPETTM.

SimPET: a Preclinical PET Insert for Simultaneous PET/MR Imaging.

PURPOSE: SimPET/M7 system is a small-animal dedicated simultaneous positron emission tomography and magnetic resonance imaging (PET/MRI) scanner. The SimPET insert has been upgraded from its prototype with a focus on count rate performance and sensitivity. The M7 scanner is a 1-T permanent magnet-based compact MRI system without any cryogens. Here, we present performance evaluation results of SimPET along with the results of mutual interference evaluation and simultaneously acquired PET/MR imaging. PROCEDURES: Following NEMA NU 4-2008 standard, we evaluated the performance of the SimPET system. The M7 MRI compatibility of SimPET was also assessed by analyzing MRI images of a uniform phantom under different PET conditions and PET count rates with different MRI pulse sequences. Mouse imaging was performed including a whole-body 18F-NaF PET scan and a simultaneous PET/MRI scan with 64Cu-NOTA-ironoxide. RESULTS: The spatial resolution at center based on 3D OSEM without and with warm background was 0.7 mm and 1.45 mm, respectively. Peak sensitivity was 4.21 % (energy window = 250-750 keV). The peak noise equivalent count rate with the same energy window was 151 kcps at 38.4 MBq. The uniformity was 4.42 %, and the spillover ratios in water- and air-filled chambers were 14.6 % and 12.7 %, respectively. In the hot rod phantom image, 0.75-mm-diameter rods were distinguishable. There were no remarkable differences in the SNR and uniformity of MRI images and PET count rates with different PET conditions and MRI pulse sequences. In the whole-body 18F-NaF PET images, fine skeletal structures were well resolved. In the simultaneous PET/MRI study with 64Cu-NOTA-ironoxide, both PET and MRI signals changed before and after injection of the dual-modal imaging probe, which was evident with the exact spatiotemporal correlation. CONCLUSIONS: We demonstrated that the SimPET scanner has a high count rate performance and excellent spatial resolution. The combined SimPET/M7 enabled simultaneous PET/MR imaging studies with no remarkable mutual interference between the two imaging modalities.

Development of 99mTc-Labeled Human Serum Albumin with Prolonged Circulation by Chelate-then-Click Approach: A Potential Blood Pool Imaging Agent.

Technetium-99m-labeled human serum albumin (99mTc-HSA) has been utilized as a blood pool imaging agent in the clinic for several decades. However, 99mTc-HSA has a short circulation time, which is a critical shortcoming for a blood pool imaging agent. Herein, we developed a novel 99mTc-labeled HSA with a long circulation time using click chemistry and a chelator, 2,2'-dipicolylamine (DPA), (99mTc-DPA-HSA). Specifically, we examined the feasibility of copper-free strain-promoted alkyne-azide cycloaddition (SPAAC) for the incorporation of HSA to the [99mTc (CO)3(H2O)3]+ system by adopting a chelate-then-click approach. In this strategy, a potent chelate system, azide-functionalized DPA, was first complexed with [99mTc (CO)3(H2O)3]+, followed by the SPAAC click reaction with azadibenzocyclooctyne-functionalized HSA (ADIBO-HSA) under biocompatible conditions. Radiolabeling efficiency of azide-functionalized DPA (99mTc-DPA) was >98%. Click conjugation efficiency of 99mTc-DPA with ADIBO-HSA was between 76 and 99% depending on the number of ADIBO moieties attached to HSA. In whole-body in vivo single photon emission computed tomography images, the blood pool uptakes of 99mTc-DPA-HSA were significantly enhanced compared to those of 99mTc-HSA at 10 min, 2, and 6 h after the injection ( P < 0.001, 0.025, and 0.003, respectively). Furthermore, the blood activities of 99mTc-DPA-HSA were 8 times higher at 30 min and 10 times higher at 3 h after the injection compared to those of conventional 99mTc-HSA in ex vivo biodistribution experiment. The results exhibit the potential of 99mTc-DPA-HSA as a blood pool imaging agent and further illustrate the promise of the pre-labeling SPAAC approach for conjugation of heat-sensitive biological targeting vectors with [99mTc (CO)3(H2O)3]+.

Voxel-Based Dosimetry of Iron Oxide Nanoparticle-Conjugated 177Lu-Labeled Folic Acid Using SPECT/CT Imaging of Mice.

Several radiolabeled folic acid conjugates have been developed for targeted imaging and therapy. However, the therapeutic concept with radiolabeled folate conjugates has not yet been applied to clinical applications owing to the high renal absorbed dose. The effectiveness of targeted radionuclide therapy (TRT) depends primarily on the absorbed dose rate and on the total absorbed dose delivered to the tumor and to normal tissue. Owing to various limitations associated with organ level dosimetry, voxel-based dosimetry has become essential for the assessment of a more  accurate absorbed dose during TRT. In this study, we synthesized iron oxide nanoparticle (IONP)-conjugated radiolabeled folate (177Lu-IONP-Folate) and performed voxel-based dosimetry using SPECT/CT images of normal mice through direct Geant4 application for emission tomography (GATE) Monte Carlo (MC) simulation. We also prepared 177Lu-Folate and 177Lu-IONPs for the comparison of absorbed doses with that of 177Lu-IONP-Folate. In addition, we calculated the mean absorbed dose at the organ-level using the medical internal radiation dose (MIRD) schema. The radioactivities of all three radiotracers were mainly accumulated in the liver and kidneys immediately after injection. For the kidneys, the voxel-based absorbed doses obtained with 177Lu-IONP-Folate, 177Lu-Folate, and 177Lu-IONPs were 1.01 ± 0.17, 2.46 ± 0.50, and 0.52 ± 0.08 Gy/MBq, respectively. The renal absorbed dose decreased significantly (∼half) when 177Lu-IONP-Folate was used compared with when the 177Lu-Folate only was used. The mean absorbed dose values obtained at organ-level using the MIRD schema were comparable to voxel-based absorbed doses estimated with GATE MC. The voxel-based absorbed dose values obtained in this study of individualized activity show that the renal absorbed dose could be reduced to almost half with 177Lu-IONP-Folate. Therefore, 177Lu-IONP-Folate could be clinically applicable in the TRT of folate receptor-positive cancers in a personalized manner when using the voxel-based dosimetry method.

Analysis of ribonuclease activity in sub-nanoliter droplets by label-free fluorescence measurements.

We report the results of a label-free analysis of ribonuclease activity using droplet-based microfluidics. The ribonucleolytic activity of ribonucleases (RNases) plays a critical role in cellular functions such as development, survival, growth and differentiation. Altered ribonucleolytic activity and/or the expression level of the RNase A family are known to be associated with pancreatic, bladder, ovarian and thyroid cancers among others. For this reason, the RNase A family is a meaningful protein biomarker that can be used in the diagnosis of cancer and as a target for new drug screening. There are some successful traditional methods for analysing the RNase activity, such as radioactive label-based assay, methylene blue-based assay, gel zymography, as well as other more recently developed methods such as electrochemical assay and fluorescence resonance energy transfer (FRET). However, these methods require analytical samples with a volume ranging from microliters to milliliters, and are not suitable for high-throughput analysis. Therefore, we integrated ethidium bromide (EtBr), which intercalates the chemical itself to nucleic acid, to droplet-based microfluidics for a cost-effective, high-throughput analysis. Put simply, this method is dependent on the amount of intercalated EtBr molecules on RNA. Our assay also uses visible light that is harmless to humans, unlike previous methods that used harmful UV rays, to excite the EtBr molecules. Specifically, we monitored the ribonucleolytic activity of less than 10 nM RNase A in droplets of about 330 picoliters. Also, half the maximal inhibitory concentration (IC50) of the RNase inhibitor was successfully measured in the same volume of droplets at a frequency of 40 hertz.

In vivo bioluminescence imaging for leptomeningeal dissemination of medulloblastoma in mouse models.

BACKGROUND: The primary cause of treatment failure in medulloblastomas (MB) is the development of leptomeningeal dissemination (seeding). For translational research on MB seeding, one of the major challenges is the development of reliable experimental models that simulate the seeding and growth characteristics of MBs. To overcome this obstacle, we improved an experimental mouse model by intracisternal inoculation of human MB cells and monitoring with in vivo live images. METHODS: Human MB cells (UW426, D283 and MED8A) were transfected with a firefly luciferase gene and a Thy1.1 (CD90.1) marker linked with IRES under the control of the CMV promoter in a retroviral DNA backbone (effLuc). The MB-effLuc cells were injected into the cisterna magna using an intrathecal catheter, and bioluminescence images were captured. We performed histopathological analysis to confirm the extent of tumor seeding. RESULTS: The luciferase activity of MB-effLuc cells displayed a gradually increasing pattern, which correlated with a quantitative luminometric assay. Live imaging showed that the MB-effLuc cells were diffusely distributed in the cervical spinal cord and the lumbosacral area. All mice injected with UW426-effLuc, D283-effLuc and MED8A-effLuc died within 51 days. The median survival was 22, 41 and 12 days after injection of 1.2 × 10(6) UW426-effLuc, D283-effLuc and MED8A-effLuc cells, respectively. The histopathological studies revealed that the MB-effLuc cells spread extensively and diffusely along the leptomeninges of the brain and spinal cord, forming tumor cell-coated layers. The tumor cells in the subarachnoid space expressed a human nuclei marker and Ki-67. Compared with the intracerebellar injection method in which the subfrontal area and distal spinal cord were spared by tumor cell seeding in some mice, the intracisternal injection model more closely resembled the widespread leptomeningeal seeding observed in MB patients. CONCLUSION: The results and described method are valuable resources for further translational research to overcome MB seeding.

Angiogenin reduces immune inflammation via inhibition of TANK-binding kinase 1 expression in human corneal fibroblast cells.

Angiogenin (ANG) is reportedly multifunctional, with roles in angiogenesis and autoimmune diseases. This protein is involved in the innate immune system and has been implicated in several inflammatory diseases. Although ANG may be involved in the anti-inflammatory response, there is no evidence that it has direct anti-inflammatory effects. In this study we sought to determine whether ANG has an anti-inflammatory effect in human corneal fibroblasts (HCFs) exposed to media containing tumor necrosis factor-alpha (TNF-α). We found that ANG reduced the mRNA expression of interleukin-1 beta (IL-1ß), -6, -8 and TNF-α receptors (TNFR) 1 and 2. In contrast, ANG increased the mRNA expression of IL-4 and -10. Protein levels of TANK-binding kinase 1 (TBK1) were reduced by ANG in HCFs treated with TNF-α. Moreover, ANG diminished the expression of IL-6 and -8 and monocyte chemotactic protein- (MCP-) 1. The protein expression of nuclear factor-κB (NF-κB) was downregulated by ANG treatment. These findings suggest that ANG suppressed the TNF-α-induced inflammatory response in HCFs through inhibition of TBK1-mediated NF-κB nuclear translocation. These novel results are likely to play a significant role in the selection of immune-mediated inflammatory therapeutic targets and may shed light on the pathogenesis of immune-mediated inflammatory diseases.