α-Acylamino-ß-lactone N-Acylethanolamine-hydrolyzing Acid Amidase Inhibitors Encapsulated in PLGA Nanoparticles: Improvement of the Physical Stability and Protection of Human Cells from Hydrogen Peroxide-Induced Oxidative Stress.

N-Acylethanolamine acid amidase (NAAA) is an N-terminal cysteine hydrolase that preferentially catalyzes the hydrolysis of endogenous lipid mediators such as palmitoylethanolamide, which has been shown to exhibit neuroprotective and antinociceptive properties by engaging peroxisome proliferator-activated receptor-α. A few potent NAAA inhibitors have been developed, including α-acylamino-ß-lactone derivatives, which are very strong and effective, but they have limited chemical and plasmatic stability, compromising their use as systemic agents. In the present study, as an example of a molecule belonging to the chemical class of N-(2-oxo-3-oxetanyl)amide NAAA inhibitors, URB866 was entrapped in poly(lactic-co-glycolic acid) nanoparticles in order to increase its physical stability. The data show a monomodal pattern and a significant time- and temperature-dependent stability of the molecule-loaded nanoparticles, which also demonstrated a greater ability to effectively retain the compound. The nanoparticles improved the photostability of URB866 with respect to that of the free molecule and displayed a better antioxidant profile on various cell lines at the molecule concentration of 25 µM. Overall, these results prove that the use of polymeric nanoparticles could be a useful strategy for overcoming the instability of α-acylamino-ß-lactone NAAA inhibitors, allowing the maintenance of their characteristics and activity for a longer time.

Recent Advances of Taxol-Loaded Biocompatible Nanocarriers Embedded in Natural Polymer-Based Hydrogels.

The discovery of paclitaxel (PTX) has been a milestone in anti-cancer therapy and has promoted the development and marketing of various formulations that have revolutionized the therapeutic approach towards several malignancies. Despite its peculiar anti-cancer activity, the physico-chemical properties of PTX compromise the administration of the compound in polar media. Because of this, since the development of the first Food and Drug Administration (FDA)-approved formulation (Taxol®), consistent efforts have been made to obtain suitable delivery systems able to preserve/increase PTX efficacy and to overcome the side effects correlated to the presence of some excipients. The exploitation of natural polymers as potential materials for drug delivery purposes has favored the modulation of the bioavailability and the pharmacokinetic profiles of the drug, and in this regard, several formulations have been developed that allow the controlled release of the active compound. In this mini-review, the recent advances concerning the design and applications of natural polymer-based hydrogels containing PTX-loaded biocompatible nanocarriers are discussed. The technological features of these formulations as well as the therapeutic outcome achieved following their administration will be described, demonstrating their potential role as innovative systems to be used in anti-tumor therapy.

Lysyl oxidase engineered lipid nanovesicles for the treatment of triple negative breast cancer.

In the field of oncology research, a deeper understanding of tumor biology has shed light on the role of environmental conditions surrounding cancer cells. In this regard, targeting the tumor microenvironment has recently emerged as a new way to access this disease. In this work, a novel extracellular matrix (ECM)-targeting nanotherapeutic was engineered using a lipid-based nanoparticle chemically linked to an inhibitor of the ECM-related enzyme, lysyl oxidase 1 (LOX), that inhibits the crosslinking of elastin and collagen fibers. We demonstrated that, when the conjugated vesicles were loaded with the chemotherapeutic epirubicin, superior inhibition of triple negative breast cancer (TNBC) cell growth was observed both in vitro and in vivo. Moreover, in vivo results displayed prolonged survival, minimal cytotoxicity, and enhanced biocompatibility compared to free epirubicin and epirubicin-loaded nanoparticles. This all-in-one nano-based ECM-targeting chemotherapeutic may provide a key-enabling technology for the treatment of TNBC.

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.

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.

Phosphoprotein-based biomarkers as predictors for cancer therapy.

Disparities in cancer patient responses have prompted widespread searches to identify differences in sensitive vs. nonsensitive populations and form the basis of personalized medicine. This customized approach is dependent upon the development of pathway-specific therapeutics in conjunction with biomarkers that predict patient responses. Here, we show that Cdk5 drives growth in subgroups of patients with multiple types of neuroendocrine neoplasms. Phosphoproteomics and high throughput screening identified phosphorylation sites downstream of Cdk5. These phosphorylation events serve as biomarkers and effectively pinpoint Cdk5-driven tumors. Toward achieving targeted therapy, we demonstrate that mouse models of neuroendocrine cancer are responsive to selective Cdk5 inhibitors and biomimetic nanoparticles are effective vehicles for enhanced tumor targeting and reduction of drug toxicity. Finally, we show that biomarkers of Cdk5-dependent tumors effectively predict response to anti-Cdk5 therapy in patient-derived xenografts. Thus, a phosphoprotein-based diagnostic assay combined with Cdk5-targeted therapy is a rational treatment approach for neuroendocrine malignancies.

Long noncoding RNA SNHG12 integrates a DNA-PK-mediated DNA damage response and vascular senescence.

Long noncoding RNAs (lncRNAs) are emerging regulators of biological processes in the vessel wall; however, their role in atherosclerosis remains poorly defined. We used RNA sequencing to profile lncRNAs derived specifically from the aortic intima of Ldlr -/- mice on a high-cholesterol diet during lesion progression and regression phases. We found that the evolutionarily conserved lncRNA small nucleolar host gene-12 (SNHG12) is highly expressed in the vascular endothelium and decreases during lesion progression. SNHG12 knockdown accelerated atherosclerotic lesion formation by 2.4-fold in Ldlr -/- mice by increased DNA damage and senescence in the vascular endothelium, independent of effects on lipid profile or vessel wall inflammation. Conversely, intravenous delivery of SNHG12 protected the tunica intima from DNA damage and atherosclerosis. LncRNA pulldown in combination with liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis showed that SNHG12 interacted with DNA-dependent protein kinase (DNA-PK), an important regulator of the DNA damage response. The absence of SNHG12 reduced the DNA-PK interaction with its binding partners Ku70 and Ku80, abrogating DNA damage repair. Moreover, the anti-DNA damage agent nicotinamide riboside (NR), a clinical-grade small-molecule activator of NAD+, fully rescued the increases in lesional DNA damage, senescence, and atherosclerosis mediated by SNHG12 knockdown. SNHG12 expression was also reduced in pig and human atherosclerotic specimens and correlated inversely with DNA damage and senescent markers. These findings reveal a role for this lncRNA in regulating DNA damage repair in the vessel wall and may have implications for chronic vascular disease states and aging.

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.

Rapamycin-Loaded Biomimetic Nanoparticles Reverse Vascular Inflammation.

RATIONALE: Through localized delivery of rapamycin via a biomimetic drug delivery system, it is possible to reduce vascular inflammation and thus the progression of vascular disease. OBJECTIVE: Use biomimetic nanoparticles to deliver rapamycin to the vessel wall to reduce inflammation in an in vivo model of atherosclerosis after a short dosing schedule. METHODS AND RESULTS: Biomimetic nanoparticles (leukosomes) were synthesized using membrane proteins purified from activated J774 macrophages. Rapamycin-loaded nanoparticles were characterized using dynamic light scattering and were found to have a diameter of 108±2.3 nm, a surface charge of -15.4±14.4 mV, and a polydispersity index of 0.11 +/ 0.2. For in vivo studies, ApoE-/- mice were fed a high-fat diet for 12 weeks. Mice were injected with either PBS, free rapamycin (5 mg/kg), or rapamycin-loaded leukosomes (Leuko-Rapa; 5 mg/kg) once daily for 7 days. In mice treated with Leuko-Rapa, flow cytometry of disaggregated aortic tissue revealed fewer proliferating macrophages in the aorta (15.6±9.79 %) compared with untreated mice (30.2±13.34 %) and rapamycin alone (26.8±9.87 %). Decreased macrophage proliferation correlated with decreased levels of MCP (monocyte chemoattractant protein)-1 and IL (interleukin)-b1 in mice treated with Leuko-Rapa. Furthermore, Leuko-Rapa-treated mice also displayed significantly decreased MMP (matrix metalloproteinases) activity in the aorta (mean difference 2554±363.9, P=9.95122×10-6). No significant changes in metabolic or inflammation markers observed in liver metabolic assays. Histological analysis showed improvements in lung morphology, with no alterations in heart, spleen, lung, or liver in Leuko-Rapa-treated mice. CONCLUSIONS: We showed that our biomimetic nanoparticles showed a decrease in proliferating macrophage population that was accompanied by the reduction of key proinflammatory cytokines and changes in plaque morphology. This proof-of-concept showed that our platform was capable of suppressing macrophage proliferation within the aorta after a short dosing schedule (7 days) and with a favorable toxicity profile. This treatment could be a promising intervention for the acute stabilization of late-stage plaques.

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.

Preclinical three-dimensional colorectal cancer model: The next generation of in vitro drug efficacy evaluation.

Colorectal cancer (CRC), the third most common cancer diagnosed in both men and women in the United States, shows a highly ineffective therapeutic management. In these years neither substantial improvements nor new therapeutic approaches have been provided to patients. Performing the early lead discovery phases of new cancer drugs in cellular models, resembling as far as possible the real in vivo tumor environment, may be more effective in predicting their future success in the later clinical phases. In this review, we critically describe the most representative bioengineered models for anticancer drug screening in CRC from the conventional two-dimensional models to the new-generation three-dimensional scaffold-based ones. The scaffold aims to replace the extracellular matrix, thus influencing the biomechanical, biochemical, and biological properties of cells and tissues. In this scenario, we believe that reconstitution of tumor condition is mandatory for an alternative in vitro methods to study cancer development and therapeutic strategies.

Microfluidic Assembly of Liposomes with Tunable Size and Coloading Capabilities.

Liposomes used for the delivery of pharmaceuticals have difficulties scaling up and reaching clinical translation as they suffer from batch-to-batch variability. Here, we describe a microfluidic approach for creating reproducible, homogenous nanoparticles with tunable characteristics. These nanoparticles of sizes ranging from 30 to 500 nm are rapidly self-assembled by controlling the flow rates of ethanol and aqueous streams. This method of microfluidic assembly allows for the efficient encapsulation of both hydrophobic and hydrophilic drugs in the lipid bilayer and particle core, respectively, either separately or in combination.

Roles of PAD4 and NETosis in Experimental Atherosclerosis and Arterial Injury: Implications for Superficial Erosion.

RATIONALE: Neutrophils likely contribute to the thrombotic complications of human atheromata. In particular, neutrophil extracellular traps (NETs) could exacerbate local inflammation and amplify and propagate arterial intimal injury and thrombosis. PAD4 (peptidyl arginine deiminase 4) participates in NET formation, but an understanding of this enzyme's role in atherothrombosis remains scant. OBJECTIVE: This study tested the hypothesis that PAD4 and NETs influence experimental atherogenesis and in processes implicated in superficial erosion, a form of plaque complication we previously associated with NETs. METHODS AND RESULTS: Bone marrow chimeric Ldlr deficient mice reconstituted with either wild-type or PAD4-deficient cells underwent studies that assessed atheroma formation or procedures designed to probe mechanisms related to superficial erosion. PAD4 deficiency neither retarded fatty streak formation nor reduced plaque size or inflammation in bone marrow chimeric mice that consumed an atherogenic diet. In contrast, either a PAD4 deficiency in bone marrow-derived cells or administration of DNaseI to disrupt NETs decreased the extent of arterial intimal injury in mice with arterial lesions tailored to recapitulate characteristics of human atheroma complicated by erosion. CONCLUSIONS: These results indicate that PAD4 from bone marrow-derived cells and NETs do not influence chronic experimental atherogenesis, but participate causally in acute thrombotic complications of intimal lesions that recapitulate features of superficial erosion.

Biomimetic nanoparticles with enhanced affinity towards activated endothelium as versatile tools for theranostic drug delivery.

Activation of the vascular endothelium is characterized by increased expression of vascular adhesion molecules and chemokines. This activation occurs early in the progression of several diseases and triggers the recruitment of leukocytes. Inspired by the tropism of leukocytes, we investigated leukocyte-based biomimetic nanoparticles (i.e., leukosomes) as a novel theranostic platform for inflammatory diseases. Methods: Leukosomes were assembled by combining phospholipids and membrane proteins from leukocytes. For imaging applications, phospholipids modified with rhodamine and gadolinium were used. Leukosomes incubated with antibodies blocking lymphocyte function-associated antigen 1 (LFA-1) and CD45 were administered to explore their roles in targeting inflammation. In addition, relaxometric assessment of NPs was evaluated. Results: Liposomes and leukosomes were both spherical in shape with sizes ranging from 140-170 nm. Both NPs successfully integrated 8 and 13 µg of rhodamine and gadolinium, respectively, and demonstrated less than 4% variation in physicochemical features. Leukosomes demonstrated a 16-fold increase in breast tumor accumulation relative to liposomes. Furthermore, quantification of leukosomes in tumor vessels demonstrated a 4.5-fold increase in vessel lumens and a 14-fold increase in vessel walls. Investigating the targeting mechanism of action revealed that blockage of LFA-1 on leukosomes resulted in a 95% decrease in tumor accumulation. Whereas blockage of CD45 yielded a 60% decrease in targeting and significant increases in liver and spleen accumulation. In addition, when administered in mice with atherosclerotic plaques, leukosomes exhibited a 4-fold increase in the targeting of inflammatory vascular lesions. Lastly, relaxometric assessment of NPs demonstrated that the incorporation of membrane proteins into leukosomes did not impact the r1 and r2 relaxivities of the NPs, demonstrating 6 and 30 mM-1s-1, respectively. Conclusion: Our study demonstrates the ability of leukosomes to target activated vasculature and exhibit superior accumulation in tumors and vascular lesions. The versatility of the phospholipid backbone within leukosomes permits the incorporation of various contrast agents. Furthermore, leukosomes can potentially be loaded with therapeutics possessing diverse physical properties and thus warrant further investigation toward the development of powerful theranostic agents.

Inflammation and Cancer: In Medio Stat Nano.

Cancer treatment still remains a challenge due to the several limitations of currently used chemotherapeutics, such as their poor pharmacokinetics, unfavorable chemical properties, as well as inability to discriminate between healthy and diseased tissue. Nanotechnology offered potent tools to overcome these limitations. Drug encapsulation within a delivery system permitted i) to protect the payload from enzymatic degradation/ inactivation in the blood stream, ii) to improve the physicochemical properties of poorly water-soluble drugs, like paclitaxel, and iii) to selectively deliver chemotherapeutics to the cancer lesions, thus reducing the off-target toxicity, and promoting the intracellular internalization. To accomplish this purpose, several strategies have been developed, based on biological and physical changes happening locally and systemically as a consequence of tumorigenesis. Here, we will discuss the role of inflammation in the different steps of tumor development and the strategies based on the use of nanoparticles that exploit the inflammatory pathways in order to selectively target the tumor-associated microenvironment for therapeutic and diagnostic purposes.

Vascular Inflammation: A Novel Access Route for Nanomedicine.

Despite an improved understanding of its pathophysiology and a wide range of new treatments, cardiovascular disease (CVD) remains a serious public health issue and the number one cause of mortality in the United States. Conditions that promote chronic systemic inflammation, such as obesity, cancer, and autoimmune and infectious diseases, are now known to play an important role in promoting CVD by inducing the expression of endothelial adhesion molecules and chemokines; these in turn promote leukocyte adherence and infiltration, which initiates and spurs the progression of CVD. In response to this new understanding, researchers are evaluating the potential cardiovascular benefits of new-generation therapies based on endogenous molecules with anti-inflammatory properties. Similarly, targeted approaches that leverage the phenotypic differences between non-inflamed and inflamed endothelia have the potential to selectively deliver therapeutics and decrease the morbidity and mortality of CVD patients. In this review, we discuss the role of inflammation in CVD and explore the therapeutic potential of targeting inflamed vasculature through conventional and biomimetic approaches.

Effects of the protein corona on liposome-liposome and liposome-cell interactions.

A thorough understanding of interactions occurring at the interface between nanocarriers and biological systems is crucial to predict and interpret their biodistribution, targeting, and efficacy, and thus design more effective drug delivery systems. Upon intravenous injection, nanoparticles are coated by a protein corona (PC). This confers a new biological identity on the particles that largely determines their biological fate. Liposomes have great pharmaceutical versatility, so, as proof of concept, their PC has recently been implicated in the mechanism and efficiency of their internalization into the cell. In an attempt to better understand the interactions between nanocarriers and biological systems, we analyzed the plasma proteins adsorbed on the surface of multicomponent liposomes. Specifically, we analyzed the physical properties and ultrastructure of liposome/PC complexes and the aggregation process that occurs when liposomes are dispersed in plasma. The results of combined confocal microscopy and flow cytometry experiments demonstrated that the PC favors liposome internalization by both macrophages and tumor cells. This work provides insights into the effects of the PC on liposomes' physical properties and, consequently, liposome-liposome and liposome-cell interactions.