Microbubble-encapsulated Cobalt Nitrato Complexes for Ultrasound-triggerable Nitric Oxide Delivery.

Cobalt complexes exhibit versatile reactivity with nitric oxide (NO), enabling their utilization in applications ranging from homogeneous catalysis to NO-based modulation of biological processes. However, the coordination geometry around the cobalt center is complex, the therapeutic window of NO is narrow, and controlled NO delivery is difficult. To better understand the complexation of cobalt with NO, we prepared four cobalt nitrato complexes and present a structure-property relationship for ultrasound-triggerable NO release. We hypothesized that modulation of the coordination geometry by ligand-modification would improve responsiveness to mechanical stimuli, like ultrasound. To enable eventual therapeutic testing, we here first demonstrate the in vitro tolerability of [Co(ethylenediamine)2(NO)(NO3)](NO3) in A431 epidermoid carcinoma cells and J774A.1 murine macrophages, and we subsequently show successful encapsulation of the complex in poly(butyl cyanoacrylate) microbubbles. These hybrid Co-NO-containing microbubbles may in the future aid in ultrasound imaging-guided treatment of NO-responsive vascular pathologies.

Mechanisms and Barriers in Nanomedicine: Progress in the Field and Future Directions.

In recent years, steady progress has been made in synthesizing and characterizing engineered nanoparticles, resulting in several approved drugs and multiple promising candidates in clinical trials. Regulatory agencies such as the Food and Drug Administration and the European Medicines Agency released important guidance documents facilitating nanoparticle-based drug product development, particularly in the context of liposomes and lipid-based carriers. Even with the progress achieved, it is clear that many barriers must still be overcome to accelerate translation into the clinic. At the recent conference workshop "Mechanisms and Barriers in Nanomedicine" in May 2023 in Colorado, U.S.A., leading experts discussed the formulation, physiological, immunological, regulatory, clinical, and educational barriers. This position paper invites open, unrestricted, nonproprietary discussion among senior faculty, young investigators, and students to trigger ideas and concepts to move the field forward.

Histopathological biomarkers for predicting the tumour accumulation of nanomedicines.

The clinical prospects of cancer nanomedicines depend on effective patient stratification. Here we report the identification of predictive biomarkers of the accumulation of nanomedicines in tumour tissue. By using supervised machine learning on data of the accumulation of nanomedicines in tumour models in mice, we identified the densities of blood vessels and of tumour-associated macrophages as key predictive features. On the basis of these two features, we derived a biomarker score correlating with the concentration of liposomal doxorubicin in tumours and validated it in three syngeneic tumour models in immunocompetent mice and in four cell-line-derived and six patient-derived tumour xenografts in mice. The score effectively discriminated tumours according to the accumulation of nanomedicines (high versus low), with an area under the receiver operating characteristic curve of 0.91. Histopathological assessment of 30 tumour specimens from patients and of 28 corresponding primary tumour biopsies confirmed the score's effectiveness in predicting the tumour accumulation of liposomal doxorubicin. Biomarkers of the tumour accumulation of nanomedicines may aid the stratification of patients in clinical trials of cancer nanomedicines.

Mass Spectrometry Reveals Molecular Effects of Citrulline Supplementation during Bone Fracture Healing in a Rat Model.

Bone fracture healing is a complex process in which specific molecular knowledge is still lacking. The citrulline-arginine-nitric oxide metabolism is one of the involved pathways, and its enrichment via citrulline supplementation can enhance fracture healing. This study investigated the molecular effects of citrulline supplementation during the different fracture healing phases in a rat model. Microcomputed tomography (µCT) was applied for the analysis of the fracture callus formation. Matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) and liquid-chromatography tandem mass spectrometry (LC-MS/MS) were used for lipid and protein analyses, respectively. µCT analysis showed no significant differences in the fracture callus volume and volume fraction between the citrulline supplementation and control group. The observed lipid profiles for the citrulline supplementation and control group were distinct for the different fracture healing stages. The main contributing lipid classes were phosphatidylcholines (PCs) and lysophosphatidylcholines (LPCs). The changing effect of citrulline supplementation throughout fracture healing was indicated by changes in the differentially expressed proteins between the groups. Pathway analysis showed an enhancement of fracture healing in the citrulline supplementation group in comparison to the control group via improved angiogenesis and earlier formation of the soft and hard callus. This study showed the molecular effects on lipids, proteins, and pathways associated with citrulline supplementation during bone fracture healing, even though no effect was visible with µCT.

Nanoscale engineering of gold nanostars for enhanced photoacoustic imaging.

Photoacoustic (PA) imaging is a diagnostic modality that combines the high contrast resolution of optical imaging with the high tissue penetration of ultrasound. While certain endogenous chromophores can be visualized via PA imaging, many diagnostic assessments require the administration of external probes. Anisotropic gold nanoparticles are particularly valued as contrast agents, since they produce strong PA signals and do not photobleach. However, the synthesis of anisotropic nanoparticles typically requires cytotoxic reagents, which can hinder their biological application. In this work, we developed new PA probes based on nanostar cores and polymeric shells. These AuNS were obtained through one-pot synthesis with biocompatible Good's buffers, and were subsequently functionalized with polyethylene glycol, chitosan or melanin, three coatings widely used in (pre)clinical research. Notably, the structural features of the nanostar cores strongly affected the PA signal. For instance, despite displaying similar sizes (i.e. 45 nm), AuNS obtained with MOPS buffer generated between 2 and 3-fold greater signal intensities in the region between 700 and 800 nm than nanostars obtained with HEPES and EPPS buffers, and up to 25-fold stronger signals than spherical gold nanoparticles. A point source analytical model demonstrated that AuNS synthesized with MOPS displayed greater absorption coefficients than the other particles, corroborating the stronger PA responses. Furthermore, the AuNS shell not only improved the biocompatibility of the nanoconstructs but also affected their performance, with melanin coating enhancing the signal more than 4-fold, due to its own PA capacity, as demonstrated by both in vitro and ex vivo imaging. Taken together, these results highlight the strengths of gold nanoconstructs as PA probes and offer insights into the design rules for the nanoengineering of new nanodiagnostic agents.

RGD-coated polymeric microbubbles promote ultrasound-mediated drug delivery in an inflamed endothelium-pericyte co-culture model of the blood-brain barrier.

Drug delivery to central nervous pathologies is compromised by the blood-brain barrier (BBB). A clinically explored strategy to promote drug delivery across the BBB is sonopermeation, which relies on the combined use of ultrasound (US) and microbubbles (MB) to induce temporally and spatially controlled opening of the BBB. We developed an advanced in vitro BBB model to study the impact of sonopermeation on the delivery of the prototypic polymeric drug carrier pHPMA as a larger molecule and the small molecule antiviral drug ribavirin. This was done under standard and under inflammatory conditions, employing both untargeted and RGD peptide-coated MB. The BBB model is based on human cerebral capillary endothelial cells and human placental pericytes, which are co-cultivated in transwell inserts and which present with proper transendothelial electrical resistance (TEER). Sonopermeation induced a significant decrease in TEER values and facilitated the trans-BBB delivery of fluorescently labeled pHPMA (Atto488-pHPMA). To study drug delivery under inflamed endothelial conditions, which are typical for e.g. tumors, neurodegenerative diseases and CNS infections, tumor necrosis factor (TNF) was employed to induce inflammation in the BBB model. RGD-coated MB bound to and permeabilized the inflamed endothelium-pericyte co-culture model, and potently improved Atto488-pHPMA and ribavirin delivery. Taken together, our work combines in vitro BBB bioengineering with MB-mediated drug delivery enhancement, thereby providing a framework for future studies on optimization of US-mediated drug delivery to the brain.

mRNA Sonotransfection of Tumors with Polymeric Microbubbles: Co-Formulation versus Co-Administration.

Despite its high potential, non-viral gene therapy of cancer remains challenging due to inefficient nucleic acid delivery. Ultrasound (US) with microbubbles (MB) can open biological barriers and thus improve DNA and mRNA passage. Polymeric MB are an interesting alternative to clinically used lipid-coated MB because of their high stability, narrow size distribution, and easy functionalization. However, besides choosing the ideal MB, it remains unclear whether nanocarrier-encapsulated mRNA should be administered separately (co-administration) or conjugated to MB (co-formulation). Therefore, the impact of poly(n-butyl cyanoacrylate) MB co-administration with mRNA-DOTAP/DOPE lipoplexes or their co-formulation on the transfection of cancer cells in vitro and in vivo is analyzed. Sonotransfection improved mRNA delivery into 4T1 breast cancer cells in vitro with co-administration being more efficient than co-formulation. In vivo, the co-administration sonotransfection approach also resulted in higher transfection efficiency and reached deeper into the tumor tissue. On the contrary, co-formulation mainly promoted transfection of endothelial and perivascular cells. Furthermore, the co-formulation approach is much more dependent on the US trigger, resulting in significantly lower off-site transfection. Thus, the findings indicate that the choice of co-administration or co-formulation in sonotransfection should depend on the targeted cell population, tolerable off-site transfection, and the therapeutic purpose.

Nanomedicine Tumor Targeting.

Nanomedicines are extensively explored for cancer therapy. By delivering drug molecules more efficiently to pathological sites and by attenuating their accumulation in healthy organs and tissues, nanomedicine formulations aim to improve the balance between drug efficacy and toxicity. More than 20 cancer nanomedicines are approved for clinical use, and hundreds of formulations are in (pre)clinical development. Over the years, several key pitfalls have been identified as bottlenecks in nanomedicine tumor targeting and translation. These go beyond materials- and production-related issues, and particularly also encompass biological barriers and pathophysiological heterogeneity. In this manuscript, the author describes the most important principles, progress, and products in nanomedicine tumor targeting, delineates key current problems and challenges, and discuss the most promising future prospects to create clinical impact.

Hybrid ultrasound and photoacoustic contrast agent designs combining metal phthalocyanines and PBCA microbubbles.

Photoacoustic (PA) imaging is an emerging diagnostic technology that combines the penetration depth of ultrasound (US) imaging and the contrast resolution of optical imaging. Although PA imaging can visualize several endogenous chromophores to obtain clinically-relevant information, multiple applications require the administration of external contrast agents. Metal phthalocyanines have strong PA properties and chemical stability, but their extreme hydrophobicity requires their encapsulation in delivery systems for biomedical applications. Hence, we developed hybrid US/PA contrast agents by encapsulating metal phthalocyanines in poly(butyl cyanoacrylate) microbubbles (PBCA MB), which display acoustic response and ability to efficiently load hydrophobic drugs. Six different metal chromophores were loaded in PBCA MB, showing greater encapsulation efficiency with higher chromophore hydrophobicity. Notably, while the US response of the MB was unaffected by the loading of the chromophores, the PA characteristics varied greatly. Among the different formulations, MB loaded with zinc and cobalt naphthalocyanines showed the strongest PA contrast, as a result of high encapsulation efficiencies and tunable optical properties. The strong US and PA contrast signals of the formulations were preserved in biological environment, as demonstrated by in vitro imaging in serum and whole blood, and ex vivo imaging in deceased mice. Taken together, these findings highlight the advantages of combining highly hydrophobic PA contrast agents and polymeric MB for the development of contrast agents for hybrid US/PA imaging, where different types of information (structural, functional, or potentially molecular) can be acquired by combining both imaging modalities.

Microbiota modulation by dietary oat beta-glucan prevents steatotic liver disease progression.

Background & Aims: Changes in gut microbiota in metabolic dysfunction-associated steatotic liver disease (MASLD) are important drivers of disease progression towards fibrosis. Therefore, reversing microbial alterations could ameliorate MASLD progression. Oat beta-glucan, a non-digestible polysaccharide, has shown promising therapeutic effects on hyperlipidemia associated with MASLD, but its impact on gut microbiota and most importantly MASLD-related fibrosis remains unknown. Methods: We performed detailed metabolic phenotyping, including assessments of body composition, glucose tolerance, and lipid metabolism, as well as comprehensive characterization of the gut-liver axis in a western-style diet (WSD)-induced model of MASLD and assessed the effect of a beta-glucan intervention on early and advanced liver disease. Gut microbiota were modulated using broad-spectrum antibiotic treatment. Results: Oat beta-glucan supplementation did not affect WSD-induced body weight gain or glucose intolerance and the metabolic phenotype remained largely unaffected. Interestingly, oat beta-glucan dampened MASLD-related inflammation, which was associated with significantly reduced monocyte-derived macrophage infiltration and fibroinflammatory gene expression, as well as strongly reduced fibrosis development. Mechanistically, this protective effect was not mediated by changes in bile acid composition or signaling, but was dependent on gut microbiota and was lost upon broad-spectrum antibiotic treatment. Specifically, oat beta-glucan partially reversed unfavorable changes in gut microbiota, resulting in an expansion of protective taxa, including Ruminococcus, and Lactobacillus followed by reduced translocation of Toll-like receptor ligands. Conclusions: Our findings identify oat beta-glucan as a highly efficacious food supplement that dampens inflammation and fibrosis development in diet-induced MASLD. These results, along with its favorable dietary profile, suggest that it may be a cost-effective and well-tolerated approach to preventing MASLD progression and should be assessed in clinical studies. Impact and Implications: Herein, we investigated the effect of oat beta-glucan on the gut-liver axis and fibrosis development in a mouse model of metabolic dysfunction-associated steatotic liver disease (MASLD). Beta-glucan significantly reduced inflammation and fibrosis in the liver, which was associated with favorable shifts in gut microbiota that protected against bacterial translocation and activation of fibroinflammatory pathways. Together, oat beta-glucan may be a cost-effective and well-tolerated approach to prevent MASLD progression and should be assessed in clinical studies.

Perilipin 5 deletion protects against nonalcoholic fatty liver disease and hepatocellular carcinoma by modulating lipid metabolism and inflammatory responses.

The molecular mechanisms underlying the transition from nonalcoholic fatty liver disease (NAFLD) to hepatocellular carcinoma (HCC) are incompletely understood. During the development of NAFLD, Perilipin 5 (PLIN5) can regulate lipid metabolism by suppressing lipolysis and preventing lipotoxicity. Other reports suggest that the lack of PLIN5 decreases hepatic injury, indicating a protective role in NAFLD pathology. To better understand the role of PLIN5 in liver disease, we established mouse models of NAFLD and NAFLD-induced HCC, in which wild-type and Plin5 null mice were exposed to a single dose of acetone or 7,12-dimethylbenz[a]anthracene (DMBA) in acetone, followed by a 30-week high-fat diet supplemented with glucose/fructose. In the NAFLD model, RNA-seq revealed significant changes in genes related to lipid metabolism and immune response. At the intermediate level, pathways such as AMP-activated protein kinase (AMPK), signal transducer and activator of transcription 3 (STAT3), c-Jun N-terminal kinase (JNK), and protein kinase B (AKT) were blunted in Plin5-deficient mice (Plin5-/-) compared to wild-type mice (WT). In the NAFLD-HCC model, only WT mice developed liver tumors, while Plin5-/- mice were resistant to tumorigenesis. Furthermore, only 32 differentially expressed genes associated with NALFD progession were identified in Plin5 null mice. The markers of mitochondrial function and immune response, such as the peroxisome proliferator-activated receptor-γ, coactivator 1-α (PGC-1α) and phosphorylated STAT3, were decreased. Lipidomic analysis revealed differential levels of some sphingomyelins between WT and Plin5-/- mice. Interestingly, these changes were not detected in the HCC model, indicating a possible shift in the metabolism of sphingomelins during carcinogenesis.

C-C chemokine receptor type 7 (CCR7) regulates hepatic CD8 + T cell homeostasis and response to acute liver injury.

BACKGROUND AND AIMS: Acute liver failure (ALF) is a rare but life-threatening condition, and DILI, particularly acetaminophen toxicity, is the leading cause of ALF. Innate immune mechanisms further perpetuate liver injury, while the role of the adaptive immune system in DILI-related ALF is unclear. APPROACH AND RESULTS: We analyzed liver tissue from 2 independent patient cohorts with ALF and identified hepatic T cell infiltration as a prominent feature in human ALF. CD8 + T cells were characterized by zonation toward necrotic regions and an activated gene expression signature. In murine acetaminophen-induced liver injury, intravital microscopy revealed zonation of CD8 + but not CD4 + T cells at necrotic areas. Gene expression analysis exposed upregulated C-C chemokine receptor 7 (CCR7) and its ligand CCL21 in the liver as well as a broadly activated phenotype of hepatic CD8 + T cells. In 2 mouse models of ALF, Ccr7-/- mice had significantly aggravated early-phase liver damage. Functionally, CCR7 was not involved in the recruitment of CD8 + T cells, but regulated their activation profile potentially through egress to lymphatics. Ccr7-/- CD8 + T cells were characterized by elevated expression of activation, effector, and exhaustion profiles. Adoptive transfer revealed preferential homing of CCR7-deficient CD8 + T cells to the liver, and depletion of CD8 + T cells attenuated liver damage in mice. CONCLUSIONS: Our study demonstrates the involvement of the adaptive immune system in ALF in humans and mice. We identify the CCR7-CCL21 axis as an important regulatory pathway, providing downstream protection against T cell-mediated liver injury.

Sonogenetics for Monitoring and Modulating Biomolecular Function by Ultrasound.

Ultrasound technology, synergistically harnessed with genetic engineering and chemistry concepts, has started to open the gateway to the remarkable realm of sonogenetics-a pioneering paradigm for remotely orchestrating cellular functions at the molecular level. This fusion not only enables precisely targeted imaging and therapeutic interventions, but also advances our comprehension of mechanobiology to unparalleled depths. Sonogenetic tools harness mechanical force within small tissue volumes while preserving the integrity of the surrounding physiological environment, reaching depths of up to tens of centimeters with high spatiotemporal precision. These capabilities circumvent the inherent physical limitations of alternative in vivo control methods such as optogenetics and magnetogenetics. In this review, we first discuss mechanosensitive ion channels, the most commonly utilized sonogenetic mediators, in both mammalian and non-mammalian systems. Subsequently, we provide a comprehensive overview of state-of-the-art sonogenetic approaches that leverage thermal or mechanical features of ultrasonic waves. Additionally, we explore strategies centered around the design of mechanochemically reactive macromolecular systems. Furthermore, we delve into the realm of ultrasound imaging of biomolecular function, encompassing the utilization of gas vesicles and acoustic reporter genes. Finally, we shed light on limitations and challenges of sonogenetics and present a perspective on the future of this promising technology.

Role of Surface Curvature in Gold Nanostar Properties and Applications.

Gold nanostars (AuNSs) are nanoparticles with intricate three-dimensional structures and shape-dependent optoelectronic properties. For example, AuNSs uniquely display three distinct surface curvatures, i.e. neutral, positive, and negative, which provide different environments to adsorbed ligands. Hence, these curvatures are used to introduce different surface chemistries in nanoparticles. This review summarizes and discusses the role of surface curvature in AuNS properties and its impact on biomedical and chemical applications, including surface-enhanced Raman spectroscopy, contrast agent performance, and catalysis. We examine the main synthetic approaches to generate AuNSs, control their morphology, and discuss their benefits and drawbacks. We also describe the optical characteristics of AuNSs and discuss how these depend on nanoparticle morphology. Finally, we analyze how AuNS surface curvature endows them with properties distinctly different from those of other nanoparticles, such as strong electromagnetic fields at the tips and increased hydrophilic environments at the indentations, together making AuNSs uniquely useful for biosensing, imaging, and local chemical manipulation.

Nanomedicine - Immune System Interactions: Limitations and Opportunities for the Treatment of Cancer.

Nanoparticles interact with immune cells in many different ways. These interactions are crucially important for determining nanoparticles' ability to be used for cancer therapy. Traditionally, strategies such as PEGylation have been employed to reduce (the kinetics of) nanoparticle uptake by immune cells, to endow them with long circulation properties, and to enable them to exploit the Enhanced Permeability and Retention (EPR) effect to accumulate in tumors. More recently, with immunotherapy becoming an increasingly important cornerstone in the clinical management of cancer, ever more research efforts in academia and industry are focusing on specifically targeting immune cells with nanoparticles. In this chapter, we describe the barriers and opportunities of immune cell targeting with nanoparticles, and we discuss how nanoparticle-based drug delivery to specific immune cell populations in tumors as well as in secondary myeloid and lymphoid organs (such as bone marrow, lymph nodes, and spleen) can be leveraged to boost the efficacy of cancer immunotherapy.

CCL2 chemokine inhibition primes the tumor vasculature for improved nanomedicine delivery and efficacy.

Blood vessel functionality is crucial for efficient tumor-targeted drug delivery. Heterogeneous distribution and perfusion of angiogenic blood vessels contribute to suboptimal accumulation of (nano-) therapeutics in tumors and metastases. To attenuate pathological angiogenesis, an L-RNA aptamer inhibiting the CC motif chemokine ligand 2 (CCL2) was administered to mice bearing orthotopic 4T1 triple-negative breast cancer tumors. The effect of CCL2 inhibition on tumor blood vessel functionality and tumor-targeted drug delivery was evaluated via multimodal and multiscale optical imaging, employing fluorophore-labeled polymeric (10 nm) and liposomal (100 nm) nanocarriers. Anti-CCL2 treatment induced a dose-dependent anti-angiogenic effect, reflected by a decreased relative blood volume, increased blood vessel maturity and functionality, and reduced macrophage infiltration, accompanied by a shift in the polarization of tumor-associated macrophages (TAM) towards a less M2-like and more M1-like phenotype. In line with this, CCL2 inhibitor treatment improved the delivery of polymers and liposomes to tumors, and enhanced the antitumor efficacy of free and liposomal doxorubicin. Together, these findings demonstrate that blocking the CCL2-CCR2 axis modulates TAM infiltration and polarization, resulting in vascular normalization and improved tumor-targeted drug delivery.

Engineering Mesoscopic 3D Tumor Models with a Self-Organizing Vascularized Matrix.

Advanced in vitro systems such as multicellular spheroids and lab-on-a-chip devices have been developed, but often fall short in reproducing the tissue scale and self-organization of human diseases. A bioprinted artificial tumor model is introduced with endothelial and stromal cells self-organizing into perfusable and functional vascular structures. This model uses 3D hydrogel matrices to embed multicellular tumor spheroids, allowing them to grow to mesoscopic scales and to interact with endothelial cells. It is shown that angiogenic multicellular tumor spheroids promote the growth of a vascular network, which in turn further enhances the growth of cocultivated tumor spheroids. The self-developed vascular structure infiltrates the tumor spheroids, forms functional connections with the bioprinted endothelium, and can be perfused by erythrocytes and polystyrene microspheres. Moreover, cancer cells migrate spontaneously from the tumor spheroid through the self-assembled vascular network into the fluid flow. Additionally, tumor type specific characteristics of desmoplasia, angiogenesis, and metastatic propensity are preserved between patient-derived samples and tumors derived from this same material growing in the bioreactors. Overall, this modular approach opens up new avenues for studying tumor pathophysiology and cellular interactions in vitro, providing a platform for advanced drug testing while reducing the need for in vivo experimentation.

Nanomaterial-based contrast agents.

Medical imaging, which empowers the detection of physiological and pathological processes within living subjects, has a vital role in both preclinical and clinical diagnostics. Contrast agents are often needed to accompany anatomical data with functional information or to provide phenotyping of the disease in question. Many newly emerging contrast agents are based on nanomaterials as their high payloads, unique physicochemical properties, improved sensitivity and multimodality capacity are highly desired for many advanced forms of bioimaging techniques and applications. Here, we review the developments in the field of nanomaterial-based contrast agents. We outline important nanomaterial design considerations and discuss the effect on their physicochemical attributes, contrast properties and biological behaviour. We also describe commonly used approaches for formulating, functionalizing and characterizing these nanomaterials. Key applications are highlighted by categorizing nanomaterials on the basis of their X-ray, magnetic, nuclear, optical and/or photoacoustic contrast properties. Finally, we offer our perspectives on current challenges and emerging research topics as well as expectations for future advancements in the field.