Exploring near-infrared autofluorescence properties in parathyroid tissue: an analysis of fresh and paraffin-embedded thyroidectomy specimens.

Significance: Near-infrared autofluorescence (NIRAF) utilizes the natural autofluorescence of parathyroid glands (PGs) to improve their identification during thyroid surgeries, reducing the risk of inadvertent removal and subsequent complications such as hypoparathyroidism. This study evaluates NIRAF's effectiveness in real-world surgical settings, highlighting its potential to enhance surgical outcomes and patient safety. Aim: We evaluate the effectiveness of NIRAF in detecting PGs during thyroidectomy and central neck dissection and investigate autofluorescence characteristics in both fresh and paraffin-embedded tissues. Approach: We included 101 patients diagnosed with papillary thyroid cancer who underwent surgeries in 2022 and 2023. We assessed NIRAF's ability to locate PGs, confirmed via parathyroid hormone assays, and involved both junior and senior surgeons. We measured the accuracy, speed, and agreement levels of each method and analyzed autofluorescence persistence and variation over 10 years, alongside the expression of calcium-sensing receptor (CaSR) and vitamin D. Results: NIRAF demonstrated a sensitivity of 89.5% and a negative predictive value of 89.1%. However, its specificity and positive predictive value (PPV) were 61.2% and 62.3%, respectively, which are considered lower. The kappa statistic indicated moderate to substantial agreement (kappa = 0.478; P < 0.001 ). Senior surgeons achieved high specificity (86.2%) and PPV (85.3%), with substantial agreement (kappa = 0.847; P < 0.001 ). In contrast, junior surgeons displayed the lowest kappa statistic among the groups, indicating minimal agreement (kappa = 0.381; P < 0.001 ). Common errors in NIRAF included interference from brown fat and eschar. In addition, paraffin-embedded samples retained stable autofluorescence over 10 years, showing no significant correlation with CaSR and vitamin D levels. Conclusions: NIRAF is useful for PG identification in thyroid and neck surgeries, enhancing efficiency and reducing inadvertent PG removals. The stability of autofluorescence in paraffin samples suggests its long-term viability, with false positives providing insights for further improvements in NIRAF technology.

Leveraging thiol-functionalized biomucoadhesive hybrid nanoliposome for local therapy of ulcerative colitis.

Directly administering medication to inflamed intestinal sites for treating ulcerative colitis (UC), poses significant challenges like retention time, absorption variability, side effects, drug stability, and non-specific delivery. Recent advancements in therapy to treat colitis aim to improve local drug availability that is enema therapy at the site of inflammation, thereby reducing systemic adverse effects. Nevertheless, a key limitation lies in enemas' inability to sustain medication in the colon due to rapid peristaltic movement, diarrhea, and poor local adherence. Therefore, in this work, we have developed site-specific thiolated mucoadhesive anionic nanoliposomes to overcome the limitations of conventional enema therapy. The thiolated delivery system allows prolonged residence of the delivery system at the inflamed site in the colon, confirmed by the adhesion potential of thiolated nanoliposomes using in-vitro and in-vivo models. To further provide therapeutic efficacy thiolated nanoliposomes were loaded with gallic acid (GA), a natural compound known for its antibacterial, antioxidant, and potent anti-inflammatory properties. Consequently, Gallic Acid-loaded Thiolated 2,6 DALP DMPG (GATh@APDL) demonstrates the potential for targeted adhesion to the inflamed colon, facilitated by their small size 100 nm and anionic nature. Therapeutic studies indicate that this formulation offers protective effects by mitigating colonic inflammation, downregulating the expression of NF-κB, HIF-1α, and MMP-9, and demonstrating superior efficacy compared to the free GA enema. The encapsulated GA inhibits the NF-κB expression, leading to enhanced expression of MUC2 protein, thereby promoting mucosal healing in the colon. Furthermore, GATh@APDL effectively reduces neutrophil infiltration and regulates immune cell quantification in colonic lamina propria. Our findings suggest that GATh@APDL holds promise for alleviating UC and addressing the limitations of conventional enema therapy.

Patient-specific vascularized tumor model: Blocking monocyte recruitment with multispecific antibodies targeting CCR2 and CSF-1R.

Tumor-associated inflammation drives cancer progression and therapy resistance, often linked to the infiltration of monocyte-derived tumor-associated macrophages (TAMs), which are associated with poor prognosis in various cancers. To advance immunotherapies, testing on immunocompetent pre-clinical models of human tissue is crucial. We have developed an in vitro model of microvascular networks with tumor spheroids or patient tissues to assess monocyte trafficking into tumors and evaluate immunotherapies targeting the human tumor microenvironment. Our findings demonstrate that macrophages in vascularized breast and lung tumor models can enhance monocyte recruitment via CCL7 and CCL2, mediated by CSF-1R. Additionally, a multispecific antibody targeting CSF-1R, CCR2, and neutralizing TGF-ß (CSF1R/CCR2/TGF-ß Ab) repolarizes TAMs towards an anti-tumoral M1-like phenotype, reduces monocyte chemoattractant protein secretion, and blocks monocyte migration. This antibody also inhibits monocyte recruitment in patient-specific vascularized tumor models. In summary, this vascularized tumor model recapitulates the monocyte recruitment cascade, enabling functional testing of innovative therapeutic antibodies targeting TAMs in the tumor microenvironment.

Lens Regeneration: The Application of iSyTE and In Silico Approaches to Evaluate Gene Expression in Lens Organoids.

Several protocols have been established for the generation of lens organoids from embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs), and other cells with regenerative potential in humans or various animal models. It is important to examine how well the regenerated lens organoids reflect lens biology, in terms of its development, homeostasis, and aging. Toward this goal, the iSyTE database (integrated Systems Tool for Eye gene discovery; https://research.bioinformatics.udel.edu/iSyTE/ ), a bioinformatics resource tool that contains meta-analyzed gene expression data in wild-type lens across different embryonic, postnatal, and adult stages, can serve as a resource for comparative analysis. This article outlines the approaches toward effective use of iSyTE to gain insights into normal gene expression in the mouse lens, enriched expression in the lens, and differential gene expression in select mouse gene-perturbation cataract/lens defects models, which in turn can be used to evaluate expression of key lens-relevant genes in lens organoids by transcriptomics (e.g., RNA-sequencing (RNA-seq), microarrays, etc.) or other downstream methods (e.g., RT-qPCR, etc.).

Generation of Functional Lentoid Bodies from Human-Induced Pluripotent Stem Cells.

The pathological mechanisms of cataract remain largely unknown due to the lack of appropriate in vitro cellular models. We developed a stable in vitro system, namely, a "fried egg" differentiation method to generate functional lentoid bodies (LBs) from induced pluripotent stem cells (iPSCs). The iPSCs-derived LBs exhibited crystalline lens-like morphology and a transparent structure, and expressed lens-specific markers. TEM examination and optical analysis further demonstrated that it has the same cell arrangement structure and magnifying ability as lens.

Models of Photoreceptor Degeneration in Adult Zebrafish.

Zebrafish maintain a remarkable ability to regenerate their neural retina following rapid and extensive loss of retinal neurons. This is mediated by Müller glial cells (MG), which re-enter the cell cycle to produce amplifying progenitor cells that eventually differentiate into the lost retinal neurons. For example, exposing adult albino zebrafish to intense light destroys large numbers of rod and cone photoreceptors, which are then restored by MG-mediated regeneration. Here, we describe an updated method for performing these acute phototoxic lesions to adult zebrafish retinas. Next, we contrast this method to a chronic, low light lesion model that results in a more muted and sustained damage to photoreceptors and does not trigger a MG-mediated regeneration response. Thus, these two methods can be used to compare and contrast the genetic and morphological changes associated with acute and chronic methods of photoreceptor degeneration.

In Vivo Imaging of Rodent Retina in Retinal Disease.

High-quality imaging of the retina is crucial to the diagnosis and monitoring of disease, as well as for evaluating the success of therapeutics in human patients and in preclinical animal models. Here, we describe the basic principles and methods for in vivo retinal imaging in rodents, including fundus imaging, fluorescein angiography, optical coherence tomography, fundus autofluorescence, and infrared imaging. After providing a concise overview of each method and detailing the retinal diseases and conditions that can be visualized through them, we will proceed to discuss the advantages and disadvantages of each approach. These protocols will facilitate the acquisition of optimal images for subsequent quantification and analysis. Additionally, a brief explanation will be given regarding the potential results and the clinical significance of the detected abnormalities.

Bioactive nanocomposite hydrogel enhances postoperative immunotherapy and bone reconstruction for osteosarcoma treatment.

Osteosarcoma, a malignant bone tumor often characterized by high hedgehog signaling activity, residual tumor cells, and substantial bone defects, poses significant challenges to both treatment response and postsurgical recovery. Here, we developed a nanocomposite hydrogel for the sustained co-delivery of bioactive magnesium ions, anti-PD-L1 antibody (αPD-L1), and hedgehog pathway antagonist vismodegib, to eradicate residual tumor cells while promoting bone regeneration post-surgery. In a mouse model of tibia osteosarcoma, this hydrogel-mediated combination therapy led to remarkable tumor growth inhibition and hence increased animal survival by enhancing the activity of tumor-suppressed CD8+ T cells. Meanwhile, the implanted hydrogel improved the microenvironment of osteogenesis through long-term sustained release of Mg2+, facilitating bone defect repair by upregulating the expression of osteogenic genes. After 21 days, the expression levels of ALP, COL1, RUNX2, and BGLAP in the Vis-αPD-L1-Gel group were approximately 4.1, 5.1, 5.5, and 3.4 times higher than those of the control, respectively. We believe that this hydrogel-based combination therapy offers a potentially valuable strategy for treating osteosarcoma and addressing the tumor-related complex bone diseases.

Efficiently directing differentiation and homing of mesenchymal stem cells to boost cartilage repair in osteoarthritis via a nanoparticle and peptide dual-engineering strategy.

Mesenchymal stem cells (MSCs) are expected to be useful therapeutics in osteoarthritis (OA), the most common joint disorder characterized by cartilage degradation. However, evidence is limited with regard to cartilage repair in clinical trials because of the uncontrolled differentiation and weak cartilage-targeting ability of MSCs after injection. To overcome these drawbacks, here we synthesized CuO@MSN nanoparticles (NPs) to deliver Sox9 plasmid DNA (favoring chondrogenesis) and recombinant protein Bmp7 (inhibiting hypertrophy). After taking up CuO@MSN/Sox9/Bmp7 (CSB NPs), the expressions of chondrogenic markers were enhanced while hypertrophic markers were decreased in response to these CSB-engineered MSCs. Moreover, a cartilage-targeted peptide (designated as peptide W) was conjugated onto the surface of MSCs via a click chemistry reaction, thereby prolonging the residence time of MSCs in both the knee joint cavity of mice and human-derived cartilage. In a surgery-induced OA mouse model, the NP and peptide dual-modified W-CSB-MSCs showed an enhancing therapeutic effect on cartilage repair in knee joints compared with other engineered MSCs after intra-articular injection. Most importantly, W-CSB-MSCs accelerated cartilage regeneration in damaged cartilage explants derived from OA patients. Thus, this new peptide and NPs dual engineering strategy shows potential for clinical applications to boost cartilage repair in OA using MSC therapy.

Multilayered hydrogel scaffold construct with native tissue matched elastic modulus: A regenerative microenvironment for urethral scar-free healing.

The unsuitable deformation stimulus, harsh urine environment, and lack of a regenerative microenvironment (RME) prevent scaffold-based urethral repair and ultimately lead to irreversible urethral scarring. The researchers clarify the optimal elastic modulus of the urethral scaffolds for urethral repair and design a multilayered PVA hydrogel scaffold for urethral scar-free healing. The inner layer of the scaffold has self-healing properties, which ensures that the wound effectively resists harsh urine erosion, even when subjected to sutures. In addition, the scaffold's outer layer has an extracellular matrix-like structure that synergizes with adipose-derived stem cells to create a favorable RME. In vivo experiments confirm successful urethral scar-free healing using the PVA multilayered hydrogel scaffold. Further mechanistic study shows that the PVA multilayer hydrogel effectively resists the urine-induced inflammatory response and accelerates the transition of urethral wound healing to the proliferative phase by regulating macrophage polarization, thus providing favorable conditions for urethral scar-free healing. This study provides mechanical criteria for the fabrication of urethral tissue-engineered scaffolds, as well as important insights into their design.

Microenvironment matters: In vitro 3D bone marrow niches differentially modulate survival, phenotype and drug responses of acute myeloid leukemia (AML) cells.

Acute myeloid leukemia (AML) is a deadly form of leukemia with ineffective traditional treatment and frequent chemoresistance-associated relapse. Personalized drug screening holds promise in identifying optimal regimen, nevertheless, primary AML cells undergo spontaneous apoptosis during cultures, invalidating the drug screening results. Here, we reconstitute a 3D osteogenic niche (3DON) mimicking that in bone marrow to support primary AML cell survival and phenotype maintenance in cultures. Specifically, 3DON derived from osteogenically differentiated mesenchymal stem cells (MSC) from healthy and AML donors are co-cultured with primary AML cells. The AML cells under the AML_3DON niche showed enhanced viability, reduced apoptosis and maintained CD33+ CD34-phenotype, associating with elevated secretion of anti-apoptotic cytokines in the AML_3DON niche. Moreover, AML cells under the AML_3DON niche exhibited low sensitivity to two FDA-approved chemotherapeutic drugs, further suggesting the physiological resemblance of the AML_3DON niche. Most interestingly, AML cells co-cultured with the healthy_3DON niche are highly sensitive to the same sample drugs. This study demonstrates the differential responses of AML cells towards leukemic and healthy bone marrow niches, suggesting the impact of native cancer cell niche in drug screening, and the potential of re-engineering healthy bone marrow niche in AML patients as chemotherapeutic adjuvants overcoming chemoresistance, respectively.

Enhancing miR-19a/b induced cardiomyocyte proliferation in infarcted hearts by alleviating oxidant stress and controlling miR-19 release.

Fully restoring the lost population of cardiomyocytes and heart function remains the greatest challenge in cardiac repair post myocardial infarction. In this study, a pioneered highly ROS-eliminating hydrogel was designed to enhance miR-19a/b induced cardiomyocyte proliferation by lowering the oxidative stress and continuously releasing miR-19a/b in infarcted myocardium in situ. In vivo lineage tracing revealed that ∼20.47 % of adult cardiomyocytes at the injected sites underwent cell division in MI mice. In MI pig the infarcted size was significantly reduced from 40 % to 18 %, and thereby marked improvement of cardiac function and increased muscle mass. Most importantly, our treatment solved the challenge of animal death--all the treated pigs managed to live until their hearts were harvested at day 50. Therefore, our strategy provides clinical conversion advantages and safety for healing damaged hearts and restoring heart function post MI, which will be a powerful tool to battle cardiovascular diseases in patients.

An ultra-long-acting L-asparaginase synergizes with an immune checkpoint inhibitor in starvation-immunotherapy of metastatic solid tumors.

Metastasis stands as the primary contributor to mortality associated with tumors. Chemotherapy and immunotherapy are frequently utilized in the management of metastatic solid tumors. Nevertheless, these therapeutic modalities are linked to serious adverse effects and limited effectiveness in preventing metastasis. Here, we report a novel therapeutic strategy named starvation-immunotherapy, wherein an immune checkpoint inhibitor is combined with an ultra-long-acting L-asparaginase that is a fusion protein comprising L-asparaginase (ASNase) and an elastin-like polypeptide (ELP), termed ASNase-ELP. ASNase-ELP's thermosensitivity enables it to generate an in-situ depot following an intratumoral injection, yielding increased dose tolerance, improved pharmacokinetics, sustained release, optimized biodistribution, and augmented tumor retention compared to free ASNase. As a result, in murine models of oral cancer, melanoma, and cervical cancer, the antitumor efficacy of ASNase-ELP by selectively and sustainably depleting L-asparagine essential for tumor cell survival was substantially superior to that of ASNase or Cisplatin, a first-line anti-solid tumor medicine, without any observable adverse effects. Furthermore, the combination of ASNase-ELP and an immune checkpoint inhibitor was more effective than either therapy alone in impeding melanoma metastasis. Overall, the synergistic strategy of starvation-immunotherapy holds excellent promise in reshaping the therapeutic landscape of refractory metastatic tumors and offering a new alternative for next-generation oncology treatments.

Photoswitchable dynamics and RNAi synergist with tailored interface and controlled release reprogramming tumor immunosuppressive niche.

Immunosuppressive tumor microenvironment (ITM) severely limited the efficacy of immunotherapy against triple-negative breast cancer (TNBC). Herein, Apt-LPR, a light-activatable photodynamic therapy (PDT)/RNAi immune synergy-enhancer was constructed by co-loading miR-34a and photosensitizers in cationic liposomes (in phase III clinical trial). Interestingly, the introduction of tumor-specific aptamers creates a special "Liposome-Aptamer-Target" interface, where the aptamers are initially in a "lying down" state but transform to "standing up" after target binding. The interfacing mechanism was elaborately revealed by computational and practical experiments. This unique interface endowed Apt-LPR with neutralized surface potential of cationic liposomes to reduce non-specific cytotoxicity, enhanced DNase resistance to protect aptamers, and preserved target-binding ability for selective drug delivery. Upon near-infrared irradiation, the generated reactive oxygen species would oxidize unsaturated phospholipids to destabilize both liposomes and lysosomes, realizing stepwise lysosomal escape of miR-34a for tumor cell apoptosis and downregulation of PD-L1 to suppress immune escape. Together, tumor-associated antigens released from PDT-damaged mitochondria and endoplasmic reticulum could activate the suppressive immune cells to establish an "immune hot" milieu. The collaborative immune-enhancing strategy effectively aroused systemic antitumor immunity and inhibited primary and distal tumor progression as well as lung metastasis in 4T1 xenografted mouse models. The photo-controlled drug release and specific tumor-targeting capabilities of Apt-LPR were also visualized in MDA-MB-231 xenografted zebrafish models. Therefore, this photoswitchable PDT/RNAi immune stimulator offered a powerful approach to reprogramming ITM and reinforcing cancer immunotherapy efficacy.

Local delivery of carboplatin-loaded hydrogel and calcium carbonate enables two-stage drug release for limited-dose radiation to eliminate mouse malignant glioma.

Postoperative radiotherapy remains the gold standard for malignant glioma treatment. Clinical limitations, including tumor growth between surgery and radiotherapy and the emergence of radioresistance, reduce treatment effectiveness and result in local disease progression. This study aimed to develop a local drug delivery system to inhibit tumor growth before radiotherapy and enhance the subsequent anticancer effects of limited-dose radiotherapy. We developed a compound of carboplatin-loaded hydrogel (CPH) incorporated with carboplatin-loaded calcium carbonate (CPCC) to enable two-stage (peritumoral and intracellular) release of carboplatin to initially inhibit tumor growth and to synergize with limited-dose radiation (10 Gy in a single fraction) to eliminate malignant glioma (ALTS1C1 cells) in a C57BL/6 mouse subcutaneous tumor model. The doses of carboplatin in CPH and CPCC treatments were 150 µL (carboplatin concentration of 5 mg/mL) and 15 mg (carboplatin concentration of 4.1 µg/mg), respectively. Mice receiving the combination of CPH-CPCC treatment and limited-dose radiation exhibited significantly reduced tumor growth volume compared to those receiving double-dose radiation alone. Furthermore, combining CPH-CPCC treatment with limited-dose radiation resulted in significantly longer progression-free survival than combining CPH treatment with limited-dose radiation. Local CPH-CPCC delivery synergized effectively with limited-dose radiation to eliminate mouse glioma, offering a promising solution for overcoming clinical limitations.

Pulmonary delivery of silver nanoparticles prevents influenza infection by recruiting and activating lymphoid cells.

Silver nanoparticles (AgNPs) are a potential antiviral agent due to their ability to disrupt the viral particle or alter the virus metabolism inside the host cell. In vitro, AgNPs exhibit antiviral activity against the most common human respiratory viruses. However, their capacity to modulate immune responses during respiratory viral infections has yet to be explored. This study demonstrates that administering AgNPs directly into the lungs prior to infection can reduce viral loads and therefore virus-induced cytokines in mice infected with influenza virus or murine pneumonia virus. The prophylactic effect was diminished in mice with depleted lymphoid cells. We showed that AgNPs-treatment resulted in the recruitment and activation of lymphocytes in the lungs, particularly natural killer (NK) cells. Mechanistically, AgNPs enhanced the ability of alveolar macrophages to promote both NK cell migration and IFN-γ production. By contrast, following infection, in mice treated with AgNPs, NK cells exhibited decreased activation, indicating that these nanoparticles can regulate the potentially deleterious activation of these cells. Overall, the data suggest that AgNPs may possess prophylactic antiviral properties by recruiting and controlling the activation of lymphoid cells through interaction with alveolar macrophages.

Enhanced anticancer efficacy of TRAIL-conjugated and odanacatib-loaded PLGA nanoparticles in TRAIL resistant cancer.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) demonstrates unique characteristics in anticancer therapies as it selectively induces apoptosis in cancer cells. However, most cancer cells are TRAIL-resistant. Odanacatib (ODN), a cathepsin K inhibitor, is considered a novel sensitizer for cancer treatment. Combination therapy between TRAIL and sensitizers is considered a potent platform that improves TRAIL-based anticancer therapies beyond TRAIL monotherapy. Herein, we developed ODN loaded poly(lactic-co-glycolic) nanoparticles conjugated to GST-TRAIL (TRAIL-ODN-PLGA-NPs) to target and treat TRAIL-resistant cancer. TRAIL-ODN-PLGA-NPs demonstrated a significant increase in cellular uptake via death receptors (DR5 and DR4) on surface of cancer cells. TRAIL-ODN-PLGA-NPs exposure destroyed more TRAIL-resistant cells compared to a single treatment with free drugs. The released ODN decreased the Raptor protein, thereby increasing damage to mitochondria by elevating reactive oxygen species (ROS) generation. Additionally, Bim protein stabilization improved TRAIL-resistant cell sensitization to TRAIL-induced apoptosis. The in vivo biodistribution study revealed that TRAIL-ODN-PLGA-NPs demonstrated high location and retention in tumor sites via the intravenous route. Furthermore, TRAIL-ODN-PLGA-NPs significantly inhibited xenograft tumor models of TRAIL-resistant Caki-1 and TRAIL-sensitive MDA-MB-231 cells.The inhibition was associated with apoptosis activation, Raptor protein stabilizing Bim protein downregulation, Bax accumulation, and mitochondrial ROS generation elevation. Additionally, TRAIL-ODN-PLGA-NPs affected the tumor microenvironment by increasing tumor necrosis factor-α and reducing interleukin-6. In conclusion, we evealed that our formulation demonstrated synergistic effects against TRAIL compared with the combination of free drug in vitro and in vivo models. Therefore, TRAIL-ODN-PLGA-NPs may be a novel candidate for TRAIL-induced apoptosis in cancer treatment.

Single-dose of integrated bilayer microneedles for enhanced hypertrophic scar therapy with rapid anti-inflammatory and sustained inhibition of myofibroblasts.

Hypertrophic scar (HS) tends to raised above skin level with high inflammatory microenvironment and excessive proliferation of myofibroblasts. The HS therapy remains challenging due to dense scar tissue which makes it hard to penetrate, and the side effects resulting from intralesional corticosteroid injection which is the mainstay treatment in clinic. Herein, bilayer microneedle patches combined with dexamethasone and colchicine (DC-MNs) with differential dual-release pattern is designed. Two drugs loaded in commercially available materials HA and PLGA, respectively. Specifically, after administration, outer layer rapidly releases the anti-inflammatory drug dexamethasone, which inhibits macrophage polarization to pro-inflammatory phenotype in scar tissue. Subsequently, inner layer degrades sustainedly, releasing antimicrotubular agent colchicine, which suppresses the overproliferation of myofibroblasts with extremely narrow therapeutic window, and inhibits the overexpression of collagen, as well as promotes the regular arrangement of collagen. Only applied once, DC-MNs directly delivered drugs to the scar tissue. Compared to traditional treatment regimen, DC-MNs significantly suppressed HS at lower dosage and frequency by differential dual-release design. Therefore, this study put forward the idea of integrated DC-MNs accompany the development of HS, providing a non-invasive, self-applicable, more efficient and secure strategy for treatment of HS.

Highly BBB-permeable nanomedicine reverses neuroapoptosis and neuroinflammation to treat Alzheimer's disease.

The prevalence of Alzheimer's disease (AD) is increasing globally due to population aging. However, effective clinical treatment strategies for AD still remain elusive. The mechanisms underlying AD onset and the interplay between its pathological factors have so far been unclear. Evidence indicates that AD progression is ultimately driven by neuronal loss, which in turn is caused by neuroapoptosis and neuroinflammation. Therefore, the inhibition of neuroapoptosis and neuroinflammation could be a useful anti-AD strategy. Nonetheless, the delivery of active drug agents into the brain parenchyma is hindered by the blood-brain barrier (BBB). To address this challenge, we fabricated a black phosphorus nanosheet (BP)-based methylene blue (MB) delivery system (BP-MB) for AD therapy. After confirming the successful preparation of BP-MB, we proved that its BBB-crossing ability was enhanced under near-infrared light irradiation. In vitro pharmacodynamics analysis revealed that BP and MB could synergistically scavenge excessive reactive oxygen species (ROS) in okadaic acid (OA)-treated PC12 cells and lipopolysaccharide (LPS)-treated BV2 cells, thus efficiently reversing neuroapoptosis and neuroinflammation. To study in vivo pharmacodynamics, we established a mouse model of AD mice, and behavioral tests confirmed that BP-MB treatment could successfully improve cognitive function in these animals. Notably, the results of pathological evaluation were consistent with those of the in vitro assays. The findings demonstrated that BP-MB could scavenge excessive ROS and inhibit Tau hyperphosphorylation, thereby alleviating downstream neuroapoptosis and regulating the polarization of microglia from the pro-inflammatory M1 phenotype to the anti-inflammatory M2 phenotype. Overall, this study highlights the therapeutic potential of a smart nanomedicine with the capability of reversing neuroapoptosis and neuroinflammation for AD treatment.

Comparing spatial distributions of ALA-PpIX and indocyanine green in a whole pig brain glioma model using 3D fluorescence cryotomography.

Significance: ALA-PpIX and second-window indocyanine green (ICG) have been studied widely for guiding the resection of high-grade gliomas. These agents have different mechanisms of action and uptake characteristics, which can affect their performance as surgical guidance agents. Elucidating these differences in animal models that approach the size and anatomy of the human brain would help guide the use of these agents. Herein, we report on the use of a new pig glioma model and fluorescence cryotomography to evaluate the 3D distributions of both agents throughout the whole brain. Aim: We aim to assess and compare the 3D spatial distributions of ALA-PpIX and second-window ICG in a glioma-bearing pig brain using fluorescence cryotomography. Approach: A glioma was induced in the brain of a transgenic Oncopig via adeno-associated virus delivery of Cre-recombinase plasmids. After tumor induction, the pro-drug 5-ALA and ICG were administered to the animal 3 and 24 h prior to brain harvest, respectively. The harvested brain was imaged using fluorescence cryotomography. The fluorescence distributions of both agents were evaluated in 3D in the whole brain using various spatial distribution and contrast performance metrics. Results: Significant differences in the spatial distributions of both agents were observed. Indocyanine green accumulated within the tumor core, whereas ALA-PpIX appeared more toward the tumor periphery. Both ALA-PpIX and second-window ICG provided elevated tumor-to-background contrast (13 and 23, respectively). Conclusions: This study is the first to demonstrate the use of a new glioma model and large-specimen fluorescence cryotomography to evaluate and compare imaging agent distribution at high resolution in 3D.