Intranasal Delivery of Anti-Apoptotic siRNA Complexed with Fas-Signaling Blocking Peptides Attenuates Cellular Apoptosis in Brain Ischemia.

Ischemic stroke-induced neuronal cell death leads to the permanent impairment of brain function. The Fas-mediating extrinsic apoptosis pathway and the cytochrome c-mediating intrinsic apoptosis pathway are two major molecular mechanisms contributing to neuronal injury in ischemic stroke. In this study, we employed a Fas-blocking peptide (FBP) coupled with a positively charged nona-arginine peptide (9R) to form a complex with negatively charged siRNA targeting Bax (FBP9R/siBax). This complex is specifically designed to deliver siRNA to Fas-expressing ischemic brain cells. This complex enables the targeted inhibition of Fas-mediating extrinsic apoptosis pathways and cytochrome c-mediating intrinsic apoptosis pathways. Specifically, the FBP targets the Fas/Fas ligand signaling, while siBax targets Bax involved in mitochondria disruption in the intrinsic pathway. The FBP9R carrier system enables the delivery of functional siRNA to hypoxic cells expressing the Fas receptor on their surface-a finding validated through qPCR and confocal microscopy analyses. Through intranasal (IN) administration of FBP9R/siCy5 to middle cerebral artery occlusion (MCAO) ischemic rat models, brain imaging revealed the complex specifically localized to the Fas-expressing infarcted region but did not localize in the non-infarcted region of the brain. A single IN administration of FBP9R/siBax demonstrated a significant reduction in neuronal cell death by effectively inhibiting Fas signaling and preventing the release of cytochrome c. The targeted delivery of FBP9R/siBax represents a promising alternative strategy for the treatment of brain ischemia.

Systemic Treatment with Fas-Blocking Peptide Attenuates Apoptosis in Brain Ischemia.

Apoptosis plays a crucial role in neuronal injury, with substantial evidence implicating Fas-mediated cell death as a key factor in ischemic strokes. To address this, inhibition of Fas-signaling has emerged as a promising strategy in preventing neuronal cell death and alleviating brain ischemia. However, the challenge of overcoming the blood-brain barrier (BBB) hampers the effective delivery of therapeutic drugs to the central nervous system (CNS). In this study, we employed a 30 amino acid-long leptin peptide to facilitate BBB penetration. By conjugating the leptin peptide with a Fas-blocking peptide (FBP) using polyethylene glycol (PEG), we achieved specific accumulation in the Fas-expressing infarction region of the brain following systemic administration. Notably, administration in leptin receptor-deficient db/db mice demonstrated that leptin facilitated the delivery of FBP peptide. We found that the systemic administration of leptin-PEG-FBP effectively inhibited Fas-mediated apoptosis in the ischemic region, resulting in a significant reduction of neuronal cell death, decreased infarct volumes, and accelerated recovery. Importantly, neither leptin nor PEG-FBP influenced apoptotic signaling in brain ischemia. Here, we demonstrate that the systemic delivery of leptin-PEG-FBP presents a promising and viable strategy for treating cerebral ischemic stroke. Our approach not only highlights the therapeutic potential but also emphasizes the importance of overcoming BBB challenges to advance treatments for neurological disorders.

Challenges and opportunities of CAR T-cell therapies for CLL.

Chimeric antigen receptor (CAR) T-cell therapies have transformed the treatment landscape of blood cancers. These engineered receptors which endow T cells with antibody-like target cell recognition combined with the typical T cell target cell lysis abilities. Introduced into the clinic in the 2010s, CAR T-cells have shown efficacy in chronic B lymphocytic leukemia (CLL), but a majority of patients do not achieve sustained remission. Here we discuss the current treatment landscape in CLL using small molecules and allogeneic stem cell transplantation, the niche CAR T-cells filled in this context, and what we have learned from biomarker and mechanistic studies. Several product parameters and improvements are introduced as examples of how the bedside-to-bench is translated into improved CAR T-cells for CLL. We hope to convey to our readers the crucial role translational medicine plays in transforming the treatment outcomes for patients with CLL and how this line of research is an essential component of modern medicine.

Spatial immunosampling of MRI-defined glioblastoma regions reveals immunologic fingerprint of non-contrast enhancing, infiltrative tumor margins.

Glioblastoma (GBM) treatment includes maximal safe resection of the core and MRI contrast-enhancing (CE) tumor. Complete resection of the infiltrative non-contrast-enhancing (NCE) tumor rim is rarely achieved. We established a safe, semi-automated workflow for spatially-registered sampling of MRI-defined GBM regions in 19 patients with downstream analysis and biobanking, enabling studies of NCE, wherefrom recurrence/progression typically occurs. Immunophenotyping revealed underrepresentation of myeloid cell subsets and CD8+ T cells in the NCE. While NCE T cells phenotypically and functionally resembled those in matching CE tumor, subsets of activated (CD69hi) effector memory CD8+ T cells were overrepresented. Contrarily, CD25hi Tregs and other subsets were underrepresented. Overall, our study demonstrated that MRI-guided, spatially-registered, intraoperative immunosampling is feasible as part of routine GBM surgery. Further elucidation of the shared and spatially distinct microenvironmental biology of GBM will enable development of therapeutic approaches targeting the NCE infiltrative tumor to decrease GBM recurrence.

Live imaging of SARS-CoV-2 infection in mice reveals that neutralizing antibodies require Fc function for optimal efficacy.

Neutralizing antibodies (NAbs) are effective in treating COVID-19, but the mechanism of immune protection is not fully understood. Here, we applied live bioluminescence imaging (BLI) to monitor the real-time effects of NAb treatment during prophylaxis and therapy of K18-hACE2 mice intranasally infected with SARS-CoV-2-nanoluciferase. Real-time imaging revealed that the virus spread sequentially from the nasal cavity to the lungs in mice and thereafter systemically to various organs including the brain, culminating in death. Highly potent NAbs from a COVID-19 convalescent subject prevented, and also effectively resolved, established infection when administered within three days. In addition to direct neutralization, depletion studies indicated that Fc effector interactions of NAbs with monocytes, neutrophils, and natural killer cells were required to effectively dampen inflammatory responses and limit immunopathology. Our study highlights that both Fab and Fc effector functions of NAbs are essential for optimal in vivo efficacy against SARS-CoV-2.

Live Imaging of SARS-CoV-2 Infection in Mice Reveals Neutralizing Antibodies Require Fc Function for Optimal Efficacy.

Neutralizing antibodies (NAbs) are effective in treating COVID-19 but the mechanism of immune protection is not fully understood. Here, we applied live bioluminescence imaging (BLI) to monitor the real-time effects of NAb treatment in prophylaxis and therapy of K18-hACE2 mice intranasally infected with SARS-CoV-2-nanoluciferase. We could visualize virus spread sequentially from the nasal cavity to the lungs and thereafter systemically to various organs including the brain, which culminated in death. Highly potent NAbs from a COVID-19 convalescent subject prevented, and also effectively resolved, established infection when administered within three days. In addition to direct Fab-mediated neutralization, Fc effector interactions of NAbs with monocytes, neutrophils and natural killer cells were required to effectively dampen inflammatory responses and limit immunopathology. Our study highlights that both Fab and Fc effector functions of NAbs are essential for optimal in vivo efficacy against SARS-CoV-2.

Targeted Delivery of Recombinant Heat Shock Protein 27 to Cardiomyocytes Promotes Recovery from Myocardial Infarction.

Ischemic heart disease, especially myocardial infarction (MI), is the leading cause of death worldwide. Apoptotic mechanisms are thought to play a significant role in cardiomyocyte death after MI. Increased production of heat shock proteins (Hsps) in cardiomyocytes is a normal response to promote tolerance and to reduce cell damage. Hsp27 is considered to be a therapeutic option for the treatment of ischemic heart disease due to its protective effects on hypoxia-induced apoptosis. Despite its antiapoptotic effects, the lack of strategies to deliver Hsp27 to the heart tissue in vivo limits its clinical applicability. In this study, we utilized an antibody against the angiotensin II type 1 (AT1) receptor, which is expressed immediately after ischemia/reperfusion in the heart of MI rats. To achieve cardiomyocyte-targeted Hsp27 delivery after ischemia/reperfusion, we employed the immunoglobulin-binding dimer ZZ, a modified domain of protein A, in conjunction with the AT1 receptor antibody. Using the AT1 receptor antibody, we achieved systemic delivery of ZZ-TAT-GFP fusion protein into the heart of MI rats. This approach enabled selective delivery of Hsp27 to cardiomyocytes, rescued cells from apoptosis, reduced the area of fibrosis, and improved cardiac function in the rat MI model, thus suggesting its applicability as a cardiomyocyte-targeted protein delivery system to inhibit apoptosis induced by ischemic injury.

Nose-to-Brain Delivery of Cancer-Targeting Paclitaxel-Loaded Nanoparticles Potentiates Antitumor Effects in Malignant Glioblastoma.

Glioblastoma multiforme (GBM) is an aggressive tumor with no curative treatment. The tumor recurrence after resection often requires chemotherapy or radiation to delay the infiltration of tumor remnants. Intracerebral chemotherapies are preferentially being used to prevent tumor regrowth, but treatments remain unsuccessful because of the poor drug distribution in the brain. In this study, we investigated the therapeutic efficacy of cancer-targeting arginyl-glycyl-aspartic tripeptide (RGD) conjugated paclitaxel (PTX)-loaded nanoparticles (NPs) against GBM by nose-to-brain delivery. Our results demonstrated that RGD-modified PTX-loaded NPs showed cancer-specific delivery and enhanced anticancer effects in vivo. The intranasal (IN) inoculation of RGD-PTX-loaded NPs effectively controls the tumor burden (75 ± 12% reduction) by inducing apoptosis and/or inhibiting cancer cell proliferation without affecting the G0 stage of normal brain cells. Our data provide therapeutic evidence supporting the use of intranasally delivered cancer-targeted PTX-loaded NPs for GBM therapy.

Intranasal delivery of cancer-targeting doxorubicin-loaded PLGA nanoparticles arrests glioblastoma growth.

Glioblastoma multiforme (GBM) is the most aggressive form of brain tumour and treatment is very challenging. Despite the recent advances in drug delivery systems, various approaches that allow sufficient deposition of anti-cancer drugs within the brain remain unsuccessful due to limited drug delivery throughout the brain. In this study, we utilised an intranasal (IN) approach to allow delivery of anti-cancer drug, encapsulated in PLGA nanoparticles (NPs). PLGA NPs were modified with the RGD ligand to enable Avß3 expressing tumour-specific delivery. IN delivery of RGD-conjugated-doxorubicin (DOX)-loaded-PLGA-nanoparticles (RGD-DOX-NP) showed cancer-specific delivery of NP and inhibition of brain tumour growth compared to the free-DOX or non-modified DOX-NP in the C6-implanted GBM model. Further, IN treatment with RGD-DOX-NP induces apoptosis in the tumour region without affecting normal brain cells. Our study provides therapeutic evidence to treat GBM using a non-invasive IN approach, which may further be translated to other brain-associated diseases.

A Positioning Device for the Placement of Mice During Intranasal siRNA Delivery to the Central Nervous System.

Intranasal (IN) drug delivery to the brain has emerged as a promising method to bypass the blood-brain barrier (BBB) for the delivery of drugs into the central nervous system (CNS). Recent studies demonstrate the use of a peptide, RVG9R, incorporating the minimal receptor-binding domain of the Rabies virus glycoprotein, in eliciting the delivery of siRNA into neurons in the brain. In this protocol, the peptide-siRNA formulation is delivered intranasally with a pipette in the dominant hand, while the anesthetized mouse is restrained by the scruff with the nondominant hand in a "head down-and-forward position" to avoid drainage into the lung and stomach upon inhalation. This precise gripping of mice can be learned but is not easy and requires practice and skill to result in effective CNS uptake. Furthermore, the process is long-drawn, requiring about 45 min for the administration of a total volume of ~20-30 µL of solution in 1-2 µL droplet volumes per inhalation, with 3-4 min rest periods between each inhalation. The objective of this study is to disclose a mouse positioning device that enables the appropriate placement of mice for efficient IN administration of the peptide-siRNA formulation. Multiple features are incorporated into the design of the device, such as four or eight positioning chairs with adjustable height and tilt to restrain anesthetized mice in the head down-and-forward position, enabling easy visualization of the mice's nares and a built-in heating pad to maintain the mice's body temperatures during the procedure. Importantly, the ability to treat four or eight mice simultaneously with RVG9R-siRNA complexes in this manner enables studies on a much quicker time scale, for the testing of an IN therapeutic siRNA approach. In conclusion, this device allows for appropriate and controlled mouse head positioning for IN application of RVG9R-siRNA and other therapeutic molecules, such as nanoparticles or antibodies, for CNS delivery.

Intranasal delivery of a Fas-blocking peptide attenuates Fas-mediated apoptosis in brain ischemia.

Ischemic stroke-induced neuronal cell death results in the permanent disabling of brain function. Apoptotic mechanisms are thought to play a prominent role in neuronal injury and ample evidence implicates Fas signaling in mediating cell death. In this study, we describe the neuroprotective effects of a Fas-blocking peptide (FBP) that by obstructing Fas signaling in cerebral ischemia inhibits apoptosis. Using an intranasal administration route in a rat model of focal cerebral ischemia, we demonstrate that nose-to-brain delivery of FBP after middle cerebral artery occlusion (MCAO) surgery results in the delivery and retention of FBP in Fas-expressing ischemic areas of the brain. A single intranasal administration of 2 mg/kg FBP resulted in significantly reduced neuronal cell death by inhibiting Fas-mediated apoptosis leading to decreased infarct volumes, reduced neurologic deficit scores and recovery from cerebral ischemia. Intranasally delivered FBP might be a promising strategy for the treatment of cerebral ischemic stroke.

Trileucine residues in a ligand-CPP-based siRNA delivery platform improve endosomal escape of siRNA.

siRNA entrapment within endosomes is a significant problem encountered with siRNA delivery platforms that co-opt receptor-mediated entry pathways. Attachment of a cell-penetrating peptide (CPP), such as nona-arginine (9R) to a cell receptor-binding ligand like the Rabies virus glycoprotein, RVG, allows effective siRNA delivery to the cytoplasm by non-endocytic pathways, but a significant amount of siRNA complexes also enters the cell by ligand-induced receptor endocytosis and remain localized in endosomes. Here, we report that the incorporation of trileucine (3 Leu) residues as an endo-osmolytic moiety in the peptide improves endosomal escape and intracellular delivery of siRNA. The trileucine motif did not affect early non-endosomal mechanism of cytoplasmic siRNA delivery but enhanced target gene silencing by >20% only beyond 24 h of transfection when siRNA delivery is mostly through the endocytic route and siRNA trapped in the endosomes at later stages were subject to release into cytoplasm. The mechanism may involve endosomal membrane disruption as trileucine residues lysed RBCs selectively under endosomal pH conditions. Interestingly <3 Leu or >3 Leu residues were not as effective, suggesting that 3 Leu residues are useful for enhancing cytoplasmic delivery of siRNA routed through endosomes.

Silencing CCR2 in Macrophages Alleviates Adipose Tissue Inflammation and the Associated Metabolic Syndrome in Dietary Obese Mice.

Adipose tissue macrophage (ATM)-mediated inflammation is a key feature contributing to the adverse metabolic outcomes of dietary obesity. Recruitment of macrophages to obese adipose tissues (AT) can occur through the engagement of CCR2, the receptor for MCP-1 (monocyte chemoattractant protein-1), which is expressed on peripheral monocytes/macrophages. Here, we show that i.p. administration of a rabies virus glycoprotein-derived acetylcholine receptor-binding peptide effectively delivers complexed siRNA into peritoneal macrophages and ATMs in a mouse model of high-fat diet-induced obesity. Treatment with siRNA against CCR2 inhibited macrophage infiltration and accumulation in AT and, therefore, proinflammatory cytokines produced by macrophages. Consequently, the treatment significantly improved glucose tolerance and insulin sensitivity profiles, and also alleviated the associated symptoms of hepatic steatosis and reduced hepatic triglyceride production. These results demonstrate that disruption of macrophage chemotaxis to the AT through cell-targeted gene knockdown strategies can provide a therapeutic intervention for obesity-related metabolic diseases. The study also highlights a siRNA delivery approach for targeting specific monocyte subsets that contribute to obesity-associated inflammation without affecting the function of other tissue-resident macrophages that are essential for host homeostasis and survival.

An anticoincidence-shielded gamma-ray spectrometer for analysis of low level environmental radionuclides.

We developed an ultralow-level background gamma-ray spectrometer, using active and passive shield devices at the same time. Cosmic-ray-induced background is suppressed by means of active shield devices consisting of plastic scintillating plates of 50mm thick and anti-coincidence electronic system. The observed background rate was 0.34 s(-1) (=0.12s(-1) per 100 cm(3) Ge volume) for energy regions between 50 and 3000 ke V. The detection efficiency curve for 10(3)ml Marinelli beaker samples is obtained over all the energy regions. The advantages of the method are demonstrated by measuring the activity of 137Cs in powdered milk sample prepared without taking any chemical procedure. The MDA for 137Cs is estimated to be (17+/-1.7)mBq at a confidence level of 95% and it is about a factor of 10 lower than the MDA obtained from the previous cryostat assembly with 10-cm thick lead shielding.