Photosensitization Priming of Tumor Microenvironments Improves Delivery of Nanotherapeutics via Neutrophil Infiltration

Remodeling of tumor microenvironments enables enhanced delivery of nanoparticles (NPs). This study shows that direct priming of a tumor tissue using photosensitization rapidly activates neutrophil infiltration that mediates delivery of nanotherapeutics into the tumor. A drug delivery platform is comprised of NPs coated with anti‐CD11b antibodies (Abs) that target activated neutrophils. Intravital microscopy demonstrates that the movement of anti‐CD11b Abs‐decorated NPs (NPs‐CD11b) into the tumor is mediated by neutrophil infiltration induced by photosensitization (PS) because the systemic depletion of neutrophils completely abolishes the nanoparticle tumor deposition. The neutrophil uptake of NPs does not alter neutrophil activation and transmigration. For cancer therapy in mice, tumor PS and photothermal therapy of anti‐CD11b Abs‐linked gold nanorods (GNRs‐CD11b) are combined to treat the carcinoma tumor. The result indicates that neutrophil tumor infiltration enhances nanoparticle cancer therapy. The findings reveal that promoting tumor infiltration of neutrophils by manipulating tumor microenvironments could be a novel strategy to actively deliver nanotherapeutics in cancer therapies.     

Adipose‐Derived Biogenic Nanoparticles for Suppression of Inflammation

Extracellular vesicles secreted from adipose‐derived mesenchymal stem cells (ADSCs) have therapeutic effects in inflammatory diseases. However, production of extracellular vesicles (EVs) from ADSCs is costly, inefficient, and time consuming. The anti‐inflammatory properties of adipose tissue‐derived EVs and other biogenic nanoparticles have not been explored. In this study, biogenic nanoparticles are obtained directly from lipoaspirate, an easily accessible and abundant source of biological material. Compared to ADSC‐EVs, lipoaspirate nanoparticles (Lipo‐NPs) take less time to process (hours compared to months) and cost less to produce (clinical‐grade cell culture facilities are not required). The physicochemical characteristics and anti‐inflammatory properties of Lipo‐NPs are evaluated and compared to those of patient‐matched ADSC‐EVs. Moreover, guanabenz loading in Lipo‐NPs is evaluated for enhanced anti‐inflammatory effects. Apolipoprotein E and glycerolipids are enriched in Lipo‐NPs compared to ADSC‐EVs. Additionally, the uptake of Lipo‐NPs in hepatocytes and macrophages is higher. Lipo‐NPs and ADSC‐EVs have comparable protective and anti‐inflammatory effects. Specifically, Lipo‐NPs reduce toll‐like receptor 4‐induced secretion of inflammatory cytokines in macrophages. Guanabenz‐loaded Lipo‐NPs further suppress inflammatory pathways, suggesting that this combination therapy can have promising applications for inflammatory diseases.     

Ceria Nanoparticles Meet Hepatic Ischemia‐Reperfusion Injury: The Perfect Imperfection

The mononuclear phagocyte system (MPS, e.g., liver, spleen) is often treated as a “blackbox” by nanoresearchers in translating nanomedicines. Often, most of the injected nanomaterials are sequestered by the MPS, preventing their delivery to the desired disease areas. Here, this imperfection is exploited by applying nano‐antioxidants with preferential liver uptake to directly prevent hepatic ischemia‐reperfusion injury (IRI), which is a reactive oxygen species (ROS)‐related disease. Ceria nanoparticles (NPs) are selected as a representative nano‐antioxidant and the detailed mechanism of preventing IRI is investigated. It is found that ceria NPs effectively alleviate the clinical symptoms of hepatic IRI by scavenging ROS, inhibiting activation of Kupffer cells and monocyte/macrophage cells. The released pro‐inflammatory cytokines are then significantly reduced and the recruitment and infiltration of neutrophils are minimized, which suppress subsequent inflammatory reaction involved in the liver. The protective effect of nano‐antioxidants against hepatic IRI in living animals and the revealed mechanism herein suggests their future use for the treatment of hepatic IRI in the clinic.     

Physicochemical and Biological Properties of Biomimetic Mineralo‐Protein Nanoparticles Formed Spontaneously in Biological Fluids

Recent studies indicate that mineral nanoparticles (NPs) form spontaneously in human body fluids. These biological NPs represent mineral precursors that are associated with ectopic calcifications seen in various human diseases. However, the parameters that control the formation of mineral NPs and their possible effects on human cells remain poorly understood. Here a nanomaterial approach to study the formation of biomimetic calcium phosphate NPs comparable to their physiological counterparts is described. Particle sizing using dynamic light scattering reveals that serum and ion concentrations within the physiological range yield NPs below 100 nm in diameter. While the particles are phagocytosed by macrophages in a size‐independent manner, only large particles or NP aggregates in the micrometer range induce cellular responses that include production of mitochondrial reactive oxygen species, caspase‐1 activation, and secretion of interleukin‐1β (IL‐1β). A comprehensive proteomic analysis reveals that the particle‐bound proteins are similar in terms of their identity and number, regardless of particle size, suggesting that protein adsorption is independent of particle size and curvature. In conclusion, the conditions underlying the formation of mineralo‐protein particles are similar to the ones that form in vivo. While mineral NPs do not activate immune cells, they may become pro‐inflammatory and contribute to pathological processes once they aggregate and form larger mineral particles.     

Glycocalyx‐Mimicking Nanoparticles for Stimulation and Polarization of Macrophages via Specific Interactions

Malignant tumors develop multiple mechanisms to impair and escape from antitumor immune responses, of which tumor‐associated macrophages that often show immunosuppressive phenotype (M2), play a critical role in tumor‐induced immunosuppression. Therefore, strategies that can reverse M2 phenotype and even enhance immune‐stimulation function of macrophage would benefit tumor immunotherapy. In this paper, self‐assembled glyco‐nanoparticles (glyco‐NPs), as artificial glycocalyx, have been found to be able to successfully induce the polarization of mouse primary peritoneal macrophages from M2 to inflammatory type (M1). The polarization change was evidenced by the decreased expression of cell surface signaling molecules CD206 and CD23, and the increased expression of CD86. Meanwhile, secretion of cytokines supported this polarization change as well. More importantly, this phenomenon is observed not only in vitro, but also in vivo. As far as we known, this is the first report about macrophage polarization being induced by synthetic nanomaterials. Moreover, preparation, characterization of these glyco‐NPs and their interaction with the macrophages are also demonstrated.     

Neutrophil‐Based Drug Delivery Systems

White blood cells (WBCs) are a major component of immunity in response to pathogen invasion. Neutrophils are the most abundant WBCs in humans, playing a central role in acute inflammation induced by pathogens. Adhesion to vasculature and tissue infiltration of neutrophils are key processes in acute inflammation. Many inflammatory/autoimmune disorders and cancer therapies have been found to be involved in activation and tissue infiltration of neutrophils. A promising strategy to develop novel targeted drug delivery systems is the targeting and exploitation of activated neutrophils. Herein, a new drug delivery platform based on neutrophils is reviewed. There are two types of drug delivery systems: neutrophils as carriers and neutrophil‐membrane‐derived nanovesicles. It is discussed how nanoparticles hijack neutrophils in vivo to deliver therapeutics across blood vessel barriers and how neutrophil‐membrane‐derived nanovesicles target inflamed vasculature. Finally, the potential applications of neutrophil‐based drug delivery systems in treating inflammation and cancers are presented.     

Comparative Evaluation of Intestinal Nitric Oxide in Embryonic Zebrafish Exposed to Metal Oxide Nanoparticles

Nanoparticle (NP) exposure may induce oxidative stress through generation of reactive oxygen and nitrogen species, which can lead to cellular and tissue damage. The digestive system is one of the initial organs affected by NP exposure. Here, it is demonstrated that exposure to metal oxide NPs induces differential changes in zebrafish intestinal NO concentrations. Intestinal NO concentrations are quantified electrochemically with a carbon fiber microelectrode inserted in the intestine of live embryos. Specificity of the electrochemical signals is demonstrated by NO‐specific pharmacological manipulations and the results are correlated with the 4,5‐diaminofluorescein‐diacetate (DAF‐FM‐DA). NPs are demonstrated to either induce or reduce physiological NO levels depending on their redox reactivity, type and dose. NO level is altered following exposure of zebrafish embryos to CuO and CeO₂ NPs at various stages and concentrations. CuO NPs increase NO concentration, suggesting an intestinal oxidative damage. In contrast, low CeO₂ NP concentration exposure significantly reduces NO levels, suggesting NO scavenging activity. However, high concentration exposure results in increased NO. Alterations in NO concentration suggest changes in intestinal physiology and oxidative stress, which will ultimately correspond to NPs toxicity. This work also demonstrates the use of electrochemistry to monitor in vivo changes of NO within zebrafish organs.     

A myeloperoxidase-responsive and biodegradable luminescent material for real-time imaging of inflammatory diseases

We report the synthesis and characterization of a myeloperoxidase (MPO)-responsive, biodegradable, and luminescent material derived from functionalized cyclodextrin. The nanoparticle based on this functional material shows notably high and sustainable luminescent signals upon triggering by inflammatory conditions with abnormally elevated reactive oxygen species and MPO. Activated neutrophils during inflammatory responses can be selectively imaged using this nanoprobe, with luminescent signals positively correlated to neutrophil counts. This nanoprobe enables in vivo precise quantification and tracking of the number and dynamics of neutrophils in both superficial and deep tissues in various mouse models of inflammatory diseases, including peritonitis, paw edema, colitis, and acute lung injury. In vitro and in vivo evaluations also demonstrate the safety and biocompatibility of this newly engineered material in its native and nanoparticle forms.     

Neutrophils Enable Local and Non-Invasive Liposome Delivery to Inflamed Skeletal Muscle and Ischemic Heart

Uncontrolled inflammation is a major pathological factor underlying a range of diseases including autoimmune conditions, cardiovascular disease, and cancer. Improving localized delivery of immunosuppressive drugs to inflamed tissue in a non-invasive manner offers significant promise to reduce severe side effects caused by systemic administration. Here, a neutrophil-mediated delivery system able to transport drug-loaded nanocarriers to inflamed tissue by exploiting the inherent ability of neutrophils to migrate to inflammatory tissue is reported. This hybrid system (neutrophils loaded with liposomes ex vivo) efficiently migrates in vitro following an inflammatory chemokine gradient. Furthermore, the triggered release of loaded liposomes and reuptake by target macrophages is studied. The migratory behavior of liposome-loaded neutrophils is confirmed in vivo by demonstrating the delivery of drug-loaded liposomes to an inflamed skeletal muscle in mice. A single low-dose injection of the hybrid system locally reduces inflammatory cytokine levels. Biodistribution of liposome-loaded neutrophils in a human-disease-relevant myocardial ischemia reperfusion injury mouse model after i.v. injection confirms the ability of injected neutrophils to carry loaded liposomes to inflammation sites. This strategy shows the potential of nanocarrier-loaded neutrophils as a universal platform to deliver anti-inflammatory drugs to promote tissue regeneration in inflammatory diseases.     

Tolerogenic Iron Oxide Nanoparticles in Type 1 Diabetes: Biodistribution and Pharmacokinetics Studies in Nonobese Diabetic Mice

Nanoparticle (NP) administration is among the most attractive approaches to exploit the synergy of different copackaged molecules for the same target. In this work, iron oxide NPs are surface‐engineered for the copackaging of the autoantigen proinsulin, a major target of adaptive immunity in type 1 diabetes (T1D), and 2‐(1′H‐indole‐3′‐carbonyl)‐thiazole‐4‐carboxylic acid methylester (ITE), a small drug conditioning a tolerogenic environment. Magnetic resonance imaging (MRI) combined with magnetic quantification are used to investigate NP biokinetics in nonobese diabetic (NOD) mice and control mice in different organs. Different NP biodistribution, with in particular enhanced kidney elimination and a stronger accumulation in the pancreas for prediabetic NOD mice, is observed. This is related to preferential NP accumulation in the pancreatic inflammatory zone and to enhancement of renal elimination by diabetic nephropathy. For both mouse strains, an MRI T2 contrast enhancement at 72 h in the liver, pancreas, and kidneys, and indicating recirculating NPs, is also found. This unexpected result is confirmed by magnetic quantification at different time points as well as by histological evaluation. Besides, such NPs are potential MRI contrast agents for early diagnosis of T1D.     

Increased oxidative stress in foam cells obtained from hemodialysis patients

Premature atherosclerosis represents the main cause of mortality among end‐stage renal disease patients (ESRD). Increased inflammation and oxidative stress are involved in initiation and progression of the atherosclerotic plaque. As foam cells are capable of producing significant amounts of inflammatory mediators and free radicals, we hypothesized that foam cells from uremic patients could produce more inflammation and oxidative stress than foam cells from normal people and be, somehow, involved in the accelerated atherosclerosis of uremia. To test this hypothesis, the levels of a few markers of inflammation and oxidative stress: Tumor necrosis factor‐α, inducible nitric oxide synthase, malondialdehyde, nitric oxide by‐products were measured in the supernatants of macrophage‐derived foam cells cultures from 18 hemodialysis patients and 18 apparently healthy individuals controls. Malondialdehyde levels in the supernatant of cell cultures (macrophages stimulated or not with native and oxidized lipoprotein) were significantly increased in uremic patients; no statistically significant difference was found between the supernatant concentrations of nitric oxide by‐products, inducible nitric oxide synthase activity, and tumor necrosis factor‐α between patients and controls. Our results, obtained with human macrophages and macrophage‐derived foam cells, are compatible with the theory that increased cellular oxidative stress and inflammatory activity in ESRD patients could accelerate the atherosclerotic process. The present culture protocol showed it is possible to use human mononuclear cells to evaluate the oxidative metabolism of foam cells, which are considered to be the initial step of atherosclerotic lesions.     

Anti‐inflammatory Properties of Cerium Oxide Nanoparticles

The valence and oxygen defect properties of cerium oxide nanoparticles (nanoceria) suggest that they may act as auto‐regenerative free radical scavengers. Overproduction of the free radical nitric oxide (NO) by the enzyme inducible nitric oxide synthase (iNOS) has been implicated as a critical mediator of inflammation. NO is correlated with disease activity and contributes to tissue destruction. The ability of nanoceria to scavenge free radicals, or reactive oxygen species (ROS), and inhibit inflammatory mediator production in J774A.1 murine macrophages is investigated. Cells internalize nanoceria, the treatment is nontoxic, and oxidative stress and pro‐inflammatory iNOS protein expression are abated with stimulation. In vivo studies show nanoceria deposition in mouse tissues with no pathogenicity. Taken together, it is suggested that cerium oxide nanoparticles are well tolerated in mice and are incorporated into cellular tissues. Furthermore, nanoceria may have the potential to reduce ROS production in states of inflammation and therefore serve as a novel therapy for chronic inflammation.     

Mammalian Cells Exhibit a Range of Sensitivities to Silver Nanoparticles that are Partially Explicable by Variations in Antioxidant Defense and Metallothionein Expression

While it is well known that there are interspecies differences in Ag sensitivity, differences in the cytotoxic responses of mammalian cells to silver nanoparticles (Ag NPs) are also observed. In order to explore these response outcomes, six cell lines, including epithelial cells (Caco‐2, NHBE, RLE‐6TN, and BEAS‐2B) and macrophages (RAW 264.7 and THP‐1) of human and rodent origin, are exposed to 20 nm citrate‐ and PVP‐coated Ag NPs with Au cores, as well as 20 nm citrate‐coated particles without cores. An MTS assay shows that while Caco‐2 and NHBE cells are resistant to particles over a 0.1–50 μg mL^?1 dose range, RAW 264.7, THP‐1, RLE‐6TN, and BEAS‐2B cells are more susceptible. While there are small differences in dissolution rates, there are no major differences in the cytotoxic potential of the different particles. However, differences in anti‐oxidant defense and metallothionein expression among different cell types are observed, which can partially explain differential Ag NP sensitivity. So, it is important to consider these differences in understanding the potential heterogeneous effects of nano Ag on mammalian biological systems.     

Disease‐Triggered Drug Release Effectively Prevents Acute Inflammatory Flare‐Ups,Achieving Reduced Dosing

For conditions with inflammatory flare‐ups, fast drug‐release from a depot is crucial to reduce cell infiltration and prevent long‐term tissue destruction. While this concept has been explored for chronic diseases, preventing acute inflammatory flares has not been explored. To address this issue, a preventative inflammation‐sensitive system is developed and applied to acute gout, a condition where millions of inflammatory cells are recruited rapidly, causing excruciating and debilitating pain. Rapid drug release is first demonstrated from a pH‐responsive acetalated dextran particle loaded with dexamethasone (AcDex‐DXM), reducing proinflammatory cytokines in vitro as efficiently as free drug. Then, using the air pouch model of gout, mice are pretreated 24 h before inducing inflammation. AcDex‐DXM reduces overall cell infiltration with decreased neutrophils, increases monocytes, and diminishes cytokines and chemokines. In a more extended prophylaxis model, murine joints are pretreated eight days before initiating inflammation. After quantifying cell infiltration, only AcDex‐DXM reduces the overall joint inflammation, where neither free drug nor a conventional drug‐depot achieves adequate anti‐inflammatory effects. Here, the superior efficacy of disease‐triggered drug‐delivery to prevent acute inflammation is demonstrated over free drug and slow‐release depots. This approach and results promise exciting treatment opportunities for multiple inflammatory conditions suffering from acute flares.     

CSF1R‐ and SHP2‐Inhibitor‐Loaded Nanoparticles Enhance Cytotoxic Activity and Phagocytosis in Tumor‐Associated Macrophages

Immune modulation of macrophages has emerged as an attractive approach for anti‐cancer therapy. However, there are two main challenges in successfully utilizing macrophages for immunotherapy. First, macrophage colony stimulating factor (MCSF) secreted by cancer cells binds to colony stimulating factor 1 receptor (CSF1‐R) on macrophages and in turn activates the downstream signaling pathway responsible for polarization of tumor‐associated macrophages (TAMs) to immunosuppressive M2 phenotype. Second, ligation of signal regulatory protein α (SIRPα) expressed on myeloid cells to CD47, a transmembrane protein overexpressed on cancer cells, activates the Src homology region 2 (SH2) domain ‐phosphatases SHP‐1 and SHP‐2 in macrophages. This results in activation of “eat‐me‐not” signaling pathway and inhibition of phagocytosis. Here, it is reported that self‐assembled dual‐inhibitor‐loaded nanoparticles (DNTs) target M2 macrophages and simultaneously inhibit CSF1R and SHP2 pathways. This results in efficient repolarization of M2 macrophages to an active M1 phenotype, and superior phagocytic capabilities as compared to individual drug treatments. Furthermore, suboptimal dose administration of DNTs in highly aggressive breast cancer and melanoma mouse models show enhanced anti‐tumor efficacy without any toxicity. These studies demonstrate that the concurrent inhibition of CSF1‐R and SHP2 signaling pathways for macrophage activation and phagocytosis enhancement could be an effective strategy for macrophage‐based immunotherapy.     

Functionalization of Carbon Nanoparticles Modulates Inflammatory Cell Recruitment and NLRP3 Inflammasome Activation

The inflammatory effects of carbon nanoparticles (NPs) are highly disputed. Here it is demonstrated that endotoxin‐free preparations of raw carbon nanotubes (CNTs) are very limited in their capacity to promote inflammatory responses in vitro, as well as in vivo. Upon purification and selective oxidation of raw CNTs, a higher dispersibility is achieved in physiological solutions, but this process also enhances their inflammatory activity. In synergy with toll‐like receptor (TLR) ligands, CNTs promote NLRP3 inflammasome activation and it is shown for the first time that this property extends to spherical carbon nano‐onions (CNOs) of 6 nm in size. In contrast, the benzoic acid functionalization of purified CNTs and CNOs leads to significantly attenuated inflammatory properties. This is evidenced by a reduced secretion of the inflammatory cytokine IL‐1β, and a pronounced decrease in the recruitment of neutrophils and monocytes following injection into mice. Collectively, these results reveal that the inflammatory properties of carbon NPs are highly dependent on their physicochemical characteristics and crucially, that chemical surface functionalization allows significant moderation of these properties.     

Targeting Neutrophils for Enhanced Cancer Theranostics

Improving tumor accumulation and delivery efficiency is an important goal of nanomedicine. Neutrophils play a vital role in both chemically mediating inflammatory response through myeloperoxidase (MPO) and biologically promoting metastasis during inflammation triggered by the primary tumor or environmental stimuli. Herein, a novel theranostic nanomedicine that targets both the chemical and biological functions of neutrophils in tumor is designed, facilitating the enhanced retention and sustained release of drug cargos for improved cancer theranostics. 5-hydroxytryptamine (5-HT) is equipped onto nanoparticles (NPs) loaded with photosensitizers and Zileuton (a leukotriene inhibitor) to obtain MPO and neutrophil targeting NPs, denoted as HZ-5 NPs. The MPO targeting property of 5-HT modified NPs is confirmed by noninvasive positron emission tomography imaging studies. Furthermore, photodynamic therapy is used to initiate the inflammatory response which further mediated the accumulation and retention of neutrophil targeting NPs in a breast cancer model. This design renders a greatly improved theranostic nanomedicine for efficient tumor suppression, and more importantly, inhibition of neutrophil-mediated lung metastasis via the sustained release of Zileuton. This work presents a novel strategy of targeting neutrophils for improved tumor theranostics, which may open up new avenues in designing nanomedicine through exploiting the tumor microenvironment.