Orthogonal proteogenomic analysis identifies the druggable PA2G4-MYC axis in 3q26 AML.

The overexpression of the ecotropic viral integration site-1 gene (EVI1/MECOM) marks the most lethal acute myeloid leukemia (AML) subgroup carrying chromosome 3q26 abnormalities. By taking advantage of the intersectionality of high-throughput cell-based and gene expression screens selective and pan-histone deacetylase inhibitors (HDACis) emerge as potent repressors of EVI1. To understand the mechanism driving on-target anti-leukemia activity of this compound class, here we dissect the expression dynamics of the bone marrow leukemia cells of patients treated with HDACi and reconstitute the EVI1 chromatin-associated co-transcriptional complex merging on the role of proliferation-associated 2G4 (PA2G4) protein. PA2G4 overexpression rescues AML cells from the inhibitory effects of HDACis, while genetic and small molecule inhibition of PA2G4 abrogates EVI1 in 3q26 AML cells, including in patient-derived leukemia xenografts. This study positions PA2G4 at the crosstalk of the EVI1 leukemogenic signal for developing new therapeutics and urges the use of HDACis-based combination therapies in patients with 3q26 AML.

The clonal hematopoiesis mutation Jak2V617F aggravates endothelial injury and thrombosis in arteries with erosion-like intimas.

BACKGROUND: Superficial plaque erosion causes many acute coronary syndromes. However, mechanisms of plaque erosion remain poorly understood, and we lack directed therapeutics for thrombotic complication. Human eroded plaques can harbor neutrophil extracellular traps (NETs) that propagate endothelial damage at experimental arterial lesions that recapitulate superficial erosion. Clonal Hematopoiesis of Indeterminate Potential (CHIP) denotes age-related clonal expansion of bone marrow-derived cells harboring somatic mutations in the absence of overt hematological disease. CHIP heightens the risk of cardiovascular disease, with the greatest increase seen in individuals with JAK2V617F. Neutrophils from mice and humans with JAK2V617F undergo NETosis more readily than Jak2WT (wild-type) cells. We hypothesized that JAK2V617F, by increasing propensity to NETosis, exacerbates aspects of superficial erosion. METHODS AND RESULTS: We generated Jak2V617F and Jak2WT mice with heterozygous Jak2V617F in myeloid cells. We induced areas of denuded endothelium that recapitulate features of superficial erosion and assessed endothelial integrity, cellular composition of the erosion, thrombosis rates, and response to ruxolitinib, a clinically available JAK1/2 inhibitor, in relation to genotype. Following experimental erosion, Jak2V617F mice have greater impairment of endothelial barrier function and increased rates of arterial thrombosis. Neointimas in Jak2V617F mice exhibit increased apoptosis, NETosis, and platelet recruitment. Jak2V617F mice treated with ruxolitinib show increased endothelial continuity and reduced apoptosis in the neointima comparable to levels in Jak2WT. CONCLUSIONS: These observations provide new mechanistic insight into the pathophysiology of superficial erosion, the heightened risk for myocardial infarction in JAK2V617F CHIP, and point the way to personalized therapeutics based on CHIP status.

Protein Corona Sensor Array Nanosystem for Detection of Coronary Artery Disease.

Coronary artery disease (CAD) is the most common type of heart disease and represents the leading cause of death in both men and women worldwide. Early detection of CAD is crucial for decreasing mortality, prolonging survival, and improving patient quality of life. Herein, a non-invasive is described, nanoparticle-based diagnostic technology which takes advantages of proteomic changes in the nano-bio interface for CAD detection. Nanoparticles (NPs) exposed to biological fluids adsorb on their surface a layer of proteins, the "protein corona" (PC). Pathological changes that alter the plasma proteome can directly result in changes in the PC. By forming disease-specific PCs on six NPs with varying physicochemical properties, a PC-based sensor array is developed for detection of CAD using specific PC pattern recognition. While the PC of a single NP may not provide the required specificity, it is reasoned that multivariate PCs across NPs with different surface chemistries, can provide the desirable information to selectively discriminate the condition under investigation. The results suggest that such an approach can detect CAD with an accuracy of 92.84%, a sensitivity of 87.5%, and a specificity of 82.5%. These new findings demonstrate the potential of PC-based sensor array detection systems for clinical use.

Unveiling the protein signature of the human osteosarcoma 3AB-OS cancer stem cell line.

In cancer research today, one of the major challenges is the eradication of cancer stem cells (CSCs) within the tumor mass. These cells play a crucial role in initiating, growing, and maintaining the tumor. Evidence has demonstrated the presence and significance of CSCs in the development and progression of osteosarcoma (OS). However, our understanding of the specific markers for OS stem cells remains limited. In this study, we aim to identify distinct biomarkers for this cell population by conducting a proteomic analysis comparing OS stem cells to their non-stem counterparts. Our investigation focuses on a particular cell line called 3AB-OS, which exhibits stem-like characteristics, and its differentiated parental cell line, MG63. Through this research, we discovered 63 proteins exclusively expressed in 3AB-OS cells. Applying an in silico bioinformatics approach, we determined that the majority of these proteins are associated with RNA metabolism. Additionally, we identified a potential correlation between the insulin-like growth factor-binding proteins (IGF2BPs) signaling pathway and the tumorigenic and stemness features observed in 3AB-OS cells.

Mice deficient for G-protein-coupled receptor 75 display altered presynaptic structural protein expression and disrupted fear conditioning recall.

There are a number of G-protein-coupled receptors (GPCRs) that are considered "orphan receptors" because the information on their known ligands is incomplete. Yet, these receptors are important targets to characterize, as the discovery of their ligands may lead to potential new therapies. GPR75 was recently deorphanized because at least two ligands appear to bind to it, the chemokine CCL5 and the eicosanoid 20-Hydroxyeicosatetraenoic acid. Recent reports suggest that GPR75 may play a role in regulating insulin secretion and obesity. However, little is known about the function of this receptor in the brain. To study the function of GPR75, we have generated a knockout (KO) mouse model of this receptor and we evaluated the role that this receptor plays in the adult hippocampus by an array of histological, proteomic, and behavioral endpoints. Using RNAscope® technology, we identified GPR75 puncta in several Rbfox3-/NeuN-positive cells in the hippocampus, suggesting that this receptor has a neuronal expression. Proteomic analysis of the hippocampus in 3-month-old GPR75 KO animals revealed that several markers of synapses, including synapsin I and II are downregulated compared with wild type (WT). To examine the functional consequence of this down-regulation, WT and GPR75 KO mice were tested on a hippocampal-dependent behavioral task. Both contextual memory and anxiety-like behaviors were significantly altered in GPR75 KO, suggesting that GPR75 plays a role in hippocampal activity.

Macrophage Cell Membrane-Cloaked Nanoplatforms for Biomedical Applications.

Biomimetic approaches utilize natural cell membrane-derived nanovesicles to camouflage nanoparticles to circumvent some limitations of nanoscale materials. This emergent cell membrane-coating technology is inspired by naturally occurring intercellular interactions, to efficiently guide nanostructures to the desired locations, thereby increasing both therapeutic efficacy and safety. In addition, the intrinsic biocompatibility of cell membranes allows the crossing of biological barriers and avoids elimination by the immune system. This results in enhanced blood circulation time and lower toxicity in vivo. Macrophages are the major phagocytic cells of the innate immune system. They are equipped with a complex repertoire of surface receptors, enabling them to respond to biological signals, and to exhibit a natural tropism to inflammatory sites and tumorous tissues. Macrophage cell membrane-functionalized nanosystems are designed to combine the advantages of both macrophages and nanomaterials, improving the ability of those nanosystems to reach target sites. Recent studies have demonstrated the potential of these biomimetic nanosystems for targeted delivery of drugs and imaging agents to tumors, inflammatory, and infected sites. The present review covers the preparation and biomedical applications of macrophage cell membrane-coated nanosystems. Challenges and future perspectives in the development of these membrane-coated nanosystems are addressed.

Nanoparticle protein corona evolution: from biological impact to biomarker discovery.

Nanoparticles exposed to biological fluids such as blood, quickly interact with their surrounding milieu resulting in a biological coating that results in large part as a function of the physicochemical properties of the nanomaterial. The large nanoparticle surface area-to-volume ratio further augments binding of biological molecules and the resulting biomolecular or protein corona, once thought of as problematic biofouling, is now viewed as a rich source of biological information that can guide the development of nanomedicines. This review gives an overview of the utility of the protein corona in proteomic profiling and discusses how a better understanding of nano-bio interactions can accelerate the clinical translation of nanomedicines and facilitate the identification of disease-specific biomarkers. With the FDA requirement of the protein corona analysis of nanoparticles in place, it is envisaged that analyzing the protein corona of nanoparticles on a case-by-case basis can provide highly valuable nano-bio interface information that can aid and improve their clinical translation.

Targeted delivery of protein arginine deiminase-4 inhibitors to limit arterial intimal NETosis and preserve endothelial integrity.

AIMS: Recent evidence suggests that 'vulnerable plaques', which have received intense attention as underlying mechanism of acute coronary syndromes over the decades, actually rarely rupture and cause clinical events. Superficial plaque erosion has emerged as a growing cause of residual thrombotic complications of atherosclerosis in an era of increased preventive measures including lipid lowering, antihypertensive therapy, and smoking cessation. The mechanisms of plaque erosion remain poorly understood, and we currently lack validated effective diagnostics or therapeutics for superficial erosion. Eroded plaques have a rich extracellular matrix, an intact fibrous cap, sparse lipid, and few mononuclear cells, but do harbour neutrophil extracellular traps (NETs). We recently reported that NETs amplify and propagate the endothelial damage at the site of arterial lesions that recapitulate superficial erosion in mice. We showed that genetic loss of protein arginine deiminase (PAD)-4 function inhibited NETosis and preserved endothelial integrity. The current study used systemic administration of targeted nanoparticles to deliver an agent that limits NETs formation to probe mechanisms of and demonstrate a novel therapeutic approach to plaque erosion that limits endothelial damage. METHODS AND RESULTS: We developed Collagen IV-targeted nanoparticles (Col IV NP) to deliver PAD4 inhibitors selectively to regions of endothelial cell sloughing and collagen IV-rich basement membrane exposure. We assessed the binding capability of the targeting ligand in vitro and evaluated Col IV NP targeting to areas of denuded endothelium in vivo in a mouse preparation that recapitulates features of superficial erosion. Delivery of the PAD4 inhibitor GSK484 reduced NET accumulation at sites of intimal injury and preserved endothelial continuity. CONCLUSIONS: NPs directed to Col IV show selective uptake and delivery of their payload to experimentally eroded regions, illustrating their translational potential. Our results further support the role of PAD4 and NETs in superficial erosion.

Analysis of the Human Plasma Proteome Using Multi-Nanoparticle Protein Corona for Detection of Alzheimer's Disease.

As the population affected by Alzheimer's disease (AD) grows, so does the need for a noninvasive and accurate diagnostic tool. Current research reveals that AD pathogenesis begins as early as decades before clinical symptoms. The unique properties of nanoparticles (NPs) may be exploited to develop noninvasive diagnostics for early detection of AD. After exposure of NPs to biological fluids, the NP surface is altered by an unbiased but selective and reproducible adsorption of biomolecules commonly referred to as the biomolecular corona or protein corona (PC). The discovery that the plasma proteome may be differentially altered during health and disease leads to the concept of disease-specific PCs. Herein, the disease-specific PCs formed around NPs in a multi-NPs platform are employed to successfully identify subtle changes in plasma protein patterns and detect AD (>92% specificity and ≈100% sensitivity). Similar discrimination power is achieved using banked plasma samples from a cohort of patients several years prior to their diagnosis with AD. With the nanoplatform's analytic ability to analyze pathological proteomic changes into a disease-specific identifier, this promising, noninvasive technology with implications for early detection and intervention could benefit not only patients with AD but other diseases as well.

Biomaterials and nanomedicine for bone regeneration: Progress and future prospects.

Bone defects pose a heavy burden on patients, orthopedic surgeons, and public health resources. Various pathological conditions cause bone defects including trauma, tumors, inflammation, osteoporosis, and so forth. Auto- and allograft transplantation have been developed as the most commonly used clinic treatment methods, among which autologous bone grafts are the golden standard. Yet the repair of bone defects, especially large-volume defects in the geriatric population or those complicated with systemic disease, is still a challenge for regenerative medicine from the clinical perspective. The fast development of biomaterials and nanomedicine favors the emergence and promotion of efficient bone regeneration therapies. In this review, we briefly summarize the progress of novel biomaterial and nanomedical approaches to bone regeneration and then discuss the current challenges that still hinder their clinical applications in treating bone defects.

Biomimetic Nanoparticles Potentiate the Anti-Inflammatory Properties of Dexamethasone and Reduce the Cytokine Storm Syndrome: An Additional Weapon against COVID-19?

Recent studies on coronavirus infectious disease 2019 (COVID-19) pathophysiology indicated the cytokine release syndrome induced by the virus as the main cause of mortality. Patients with severe COVID-19 infection present a systemic hyper inflammation that can lead to lung and multi-organ injuries. Among the most recent treatments, corticosteroids have been identified to be effective in mitigating these catastrophic effects. Our group has recently developed leukocyte-derived nanovesicles, termed leukosomes, able to target in vivo the inflamed vasculature associated with pathological conditions including cancer, cardiovascular diseases, and sepsis. Herein, to gain insights on the anti-inflammatory properties of leukosomes, we investigated their ability to reduce uncontrolled inflammation in a lethal model of lipopolysaccharide (LPS)-induced endotoxemia, recapitulating the cytokine storm syndrome observed in COVID-19 infection after encapsulating dexamethasone. Treated animals showed a significant survival advantage and an improved immune response resolution, as demonstrated by a cytokine array analysis of pro- and anti-inflammatory cytokines, chemokines, and other immune-relevant markers. Our results showed that leukosomes enhance the therapeutic activity of dexamethasone and better control the inflammatory response compared to the free drug. Such an approach could be useful for the development of personalized therapies in the treatment of hyperinflammation related to infectious diseases, including the ones caused by COVID-19.

Impact of plasma concentration of transferrin on targeting capacity of nanoparticles.

It is becoming increasingly accepted that various diseases have a capacity to alter the composition of plasma proteins. This alteration in protein composition may consequently change the targeting capacity of nanoparticles (NPs). In this study, the impact of a model targeting ligand's (i.e., Transferrin; Tf) concentration in human plasma on the targeting capacity of gold NPs (Au NPs), pre-conjugated with Tf, is investigated. Our findings demonstrate that the protein corona formation by both healthy and Tf depleted human plasma diminishes the targeting efficacy of Au NPs within human cancer cells despite a preservation of targeting ability by plasma with excess Tf (10-fold). Moreover, the plasma samples obtained from patients with various Tf levels (e.g., thalassemia major, sickle cell anemia, aplastic anemia, and iron deficiency anemia) have affected the accessibility of the targeting Tf in the corona layer and subsequently affected their targeting ability, which emphasizes the critical role of disease-specific protein corona on the efficacy of Au NPs. Ultimately, variations of protein concentration (e.g., due to disease occurrence and progress) in plasma affect its recruiting in corona formation, and in turn, affect the targeting and therapeutic efficacies of Au NPs.

Leukocyte-mimicking nanovesicles for effective doxorubicin delivery to treat breast cancer and melanoma.

In the last decades, several approaches were developed to design drug delivery systems to address the multiple biological barriers encountered after administration while safely delivering a payload. In this scenario, bio-inspired and bio-mimetic approaches have emerged as promising solutions to evade the mononuclear phagocytic system while simultaneously negotiating the sequential transport across the various biological barriers. Leukocytes freely circulate in the bloodstream and selectively target the inflamed vasculature in response to injury, infection, and cancer. Recently we have shown the use of biomimetic nanovesicles, called leukosomes, which combine both the physical and biological properties of liposomes and leukocytes, respectively, to selectively deliver drugs to the inflamed vasculature. Here we report the use of leukosomes to target and deliver doxorubicin, a model chemotherapeutic, to tumors in syngeneic murine models of breast cancer and melanoma. Exploiting the inflammatory pathway responsible for recruiting immune cells to the site of injury, leukosomes exhibited increased targeting of cancer vasculature and stroma. Furthermore, delivery of doxorubicin with leukosomes enabled significant tumor growth inhibition compared with free doxorubicin in both breast and melanoma tumors. This study demonstrates the promise of using biomimetic nanovesicles for effective cancer management in solid tumors.

Immunoengineering in glioblastoma imaging and therapy.

Patients diagnosed with glioblastoma have poor prognosis. Conventional treatment strategies such as surgery, chemotherapy, and radiation therapy demonstrated limited clinical success and have considerable side effects on healthy tissues. A central challenge in treating brain tumors is the poor permeability of the blood-brain barrier (BBB) to therapeutics. Recently, various methods based on immunotherapy and nanotechnology have demonstrated potential in addressing these obstacles by enabling precise targeting of brain tumors to minimize adverse effects, while increasing targeted drug delivery across the BBB. In addition to treating the tumors, these approaches may be used in conjunction with imaging modalities, such as magnetic resonance imaging and positron emission tomography to enhance the prognosis procedures. This review aims to provide mechanistic understanding of immune system regulation in the central nervous system and the benefits of nanoparticles in the prognosis of brain tumors. This article is characterized under: Diagnostic Tools > in vivo Nanodiagnostics and Imaging Nanotechnology Approaches to Biology > Cells at the Nanoscale Nanotechnology Approaches to Biology > Nanoscale Systems in Biology.

Macrophage-derived nanovesicles exert intrinsic anti-inflammatory properties and prolong survival in sepsis through a direct interaction with macrophages.

Despite numerous advances in medical treatment, sepsis remains one of the leading causes of death worldwide. Sepsis is characterized by the involvement of all organs and tissues as a consequence of blood poisoning, resulting in organ failure and eventually death. Effective treatment remains an unmet need and novel approaches are urgently needed. The growing evidence of clinical and biological heterogeneity of sepsis suggests precision medicine as a possible key for achieving therapeutic breakthroughs. In this scenario, biomimetic nanomedicine represents a promising avenue for the treatment of inflammatory diseases, including sepsis. We investigated the role of macrophage-derived biomimetic nanoparticles, namely leukosomes, in a lipopolysaccharide-induced murine model of sepsis. We observed that treatment with leukosomes was associated with significantly prolonged survival. In vitro studies elucidated the potential mechanism of action of these biomimetic vesicles. The direct treatment of endothelial cells (ECs) with leukosomes did not alter the gene expression profile of EC-associated cell adhesion molecules. In contrast, the interaction of leukosomes with macrophages induced a decrease of pro-inflammatory genes (IL-6, IL-1b, and TNF-α), an increase of anti-inflammatory ones (IL-10 and TGF-ß), and indirectly an anti-inflammatory response on ECs. Taken together, these results showed the ability of leukosomes to regulate the inflammatory response in target cells, acting as a bioactive nanotherapeutic.

Biohybrid Nanoparticles to Negotiate with Biological Barriers.

Incapability of effective cross-talk with biological environments has partly impaired the in vivo functionality of nanoparticles (NPs). Homing, biodistribution, and function of NPs could be engineered through regulating their interactions with in vivo niches. Inspired by communications in biological systems, endowing a "biological identity" to synthetic NPs is one approach to control their biodistribution, and immunonegotiation profiles. This synthetic-biological combination is referred to as biohybrid NPs, which comprise both i) engineerable, readily producible, and trackable synthetic NPs as well as ii) biological moieties with the capability to cross-talk with immunological barriers. Here, the latest understanding on the in vivo interactions of NPs, biological barriers they face, and emerging methods for quantitative measurements of NPs' biodistribution are reviewed. Some key biomolecules that have emerged as negotiators with the immune system in the context of cancer and autoimmunity, and their inspirations on biohybrid NPs are introduced. Critical design considerations for efficient cross-talk between NPs and innate and adaptive immunity followed by hybridization methods are also discussed. Finally, clinical translation challenges and future perspectives regarding biohybrid NPs are discussed.

Laser irradiation affects the biological identity and cellular uptake of plasmonic nanoparticles.

The biological identity of nanoparticles (NPs) is defined by a protein layer formed on their surface, called protein corona (PC), once they meet the biological milieu. Any change in the PC composition may influence the biological fate of NPs. The PC composition is strongly dependent on several parameters including the physicochemical properties of NPs, and biological and environmental factors. As one of the main features of plasmonic NPs is their capacity to induce local heating by laser irradiation, we hypothesized that laser irradiation may change the biological identity of NPs and therefore alter their biological fate. To test this hypothesis, here we investigated the effects of either simultaneous or sequential laser irradiation on the conformations of a few proteins selected from two main categories of plasma proteins (i.e. human serum albumin and human fibrinogen) on the surfaces of gold nanorods (AuNRs). The outcomes revealed a significant role of laser irradiation on conformational changes of fibrinogen compared to albumin. Moreover, the effects of plasmonic heating - at various times - on the achieved corona composition from interactions of AuNRs and human plasma with various concentrations were monitored. Consequently, the cellular uptake of the corona coated AuNRs was measured in two cell types: malignant (MCF-7) and normal (MCF-10A) breast cell lines. The results demonstrated a substantial reduction in the cellular uptake of AuNRs in response to an increase in the laser irradiation time, especially in MCF-10A. Our results may pave the way for a mechanistic understanding of the biological identity of plasmonic NPs which in turn can help their safe and efficient clinical translations.

Exosome-like Nanovectors for Drug Delivery in Cancer.

Cancer treatment still represents a formidable challenge, despite substantial advancements in available therapies being made over the past decade. One major issue is poor therapeutic efficacy due to lack of specificity and low bioavailability. The progress of nanotechnology and the development of a variety of nanoplatforms have had a significant impact in improving the therapeutic outcome of chemotherapeutics. Nanoparticles can overcome various biological barriers and localize at tumor site, while simultaneously protecting a therapeutic cargo and increasing its circulation time. Despite this, due to their synthetic origin, nanoparticles are often detected by the immune system and preferentially sequestered by filtering organs. Exosomes have recently been investigated as suitable substitutes for the shortcomings of nanoparticles due to their biological compatibility and particularly small size (i.e., 30-150 nm). In addition, exosomes have been found to play important roles in cell communication, acting as natural carriers of biological cargoes throughout the body. This review aims to highlight the use of exosomes as drug delivery vehicles for cancer and showcases the various attempts used to exploit exosomes with a focus on the delivery of chemotherapeutics and nucleic acids.

Design and Development of Biomimetic Nanovesicles Using a Microfluidic Approach.

The advancement of nanotechnology toward more sophisticated bioinspired approaches has highlighted the gap between the advantages of biomimetic and biohybrid platforms and the availability of manufacturing processes to scale up their production. Though the advantages of transferring biological features from cells to synthetic nanoparticles for drug delivery purposes have recently been reported, a standardizable, batch-to-batch consistent, scalable, and high-throughput assembly method is required to further develop these platforms. Microfluidics has offered a robust tool for the controlled synthesis of nanoparticles in a versatile and reproducible approach. In this study, the incorporation of membrane proteins within the bilayer of biomimetic nanovesicles (leukosomes) using a microfluidic-based platform is demonstrated. The physical, pharmaceutical, and biological properties of microfluidic-formulated leukosomes (called NA-Leuko) are characterized. NA-Leuko show extended shelf life and retention of the biological functions of donor cells (i.e., macrophage avoidance and targeting of inflamed vasculature). The NA approach represents a universal, versatile, robust, and scalable tool, which is extensively used for the assembly of lipid nanoparticles and adapted here for the manufacturing of biomimetic nanovesicles.