Search

INTRODUCTION: Treatment options for cancer metastases, the primary cause of cancer mortality, are limited. The chemokine receptor CXCR4 is an attractive therapeutic target in cancer because it mediates metastasis by inducing cancer cell and macrophage migration. Here we engineered carrier-free CXCR4-targeting RNA-protein nanoplexes that not only inhibited cellular migration but also polarized macrophages to the M1 phenotype. MATERIALS AND METHODS: A CXCR4-targeting single-chain variable fragment (scFv) antibody was fused to a 3030 Da RNA-binding protamine peptide (RSQSRSRYYRQRQRSRRRRRRS). Self-assembling nanoplexes were formed by mixing the CXCR4-scFv-protamine fusion protein (CXCR4-scFv-RBM) with miR-127-5p, a miRNA shown to mediate M1 macrophage polarization. RNA-protein nanoplexes were characterized with regard to their physicochemical properties and therapeutic efficacy. RESULTS: CXCR4-targeting RNA-protein nanoplexes simultaneously acted as a targeting ligand, a macrophage polarizing drug, and a miRNA delivery vehicle. Our carrier-free, RNA-protein nanoplexes specifically bound to CXCR4-positive macrophages and breast cancer cells, showed high drug loading (~ 90% w/w), and are non-toxic. Further, these RNA-protein nanoplexes significantly inhibited cancer and immune cell migration (75 to 99%), robustly polarized macrophages to the tumor-suppressive M1 phenotype, and inhibited tumor growth in a mouse model of triple-negative breast cancer CONCLUSIONS: We engineered a novel class of non-toxic RNA-protein nanoplexes that modulate the tumor stroma. These nanoplexes are promising candidates for add-ons to clinically approved chemotherapeutics. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s12195-019-00589-w) contains supplementary material, which is available to authorized users.

Gap junctions are membrane channels found in all cells of the human body that are essential to cellular physiology. Gap junctions are formed from connexin proteins and are responsible for transfer of biologically active molecules, metabolites, and salts between neighboring cells or cells and their extracellular environment. Over the last few years, aberrant connexin 43 (Cx43) expression has been associated with cancer recurrence, metastatic spread, and poor survival. Here we provide an overview of the general structure and function of gap junctions and review their roles in different cancer types. We discuss new therapeutic approaches targeting Cx43 and potential new ways of exploiting gap junction transfer for drug delivery and anti-cancer treatment. The permeability of Cx43 channels to small molecules and macromolecules makes them highly attractive targets for delivering drugs directly into the cytoplasm. Cancer cells overexpressing Cx43 may be more permeable and sensitive to chemotherapeutics. Because Cx43 can either act as a tumor suppressor or oncogene, biomarker analysis and a better understanding of how Cx43 contextually mediates cancer phenotypes will be required to develop clinically viable Cx43-based therapies.

Extracellular vesicles are natural delivery systems widely implicated in cellular communication. However, to fully utilize these vehicles as nanocarriers, we must explore various methods to modify their applicability as drug delivery vehicles. In this review, we outline and discuss techniques to engineer extracellular vehicles for enhanced loading, targeting, circulation, and tracking. We highlight cutting-edge methods to amplify extracellular vesicle secretion and production and optimize storage conditions to improve their clinical suitability. Moreover, we focus on reverse engineering as an important step in controlling their biological function. By taking a reductionist approach to characterize and understand the individual components of these carriers, we can not only elucidate complex mechanisms of action but also advance the field through the creation of synthetic drug delivery vehicles. Finally, we propose current challenges and future directions of the field.

Background Titanium dioxide nanoparticles (TiO2 NPs) have a wide range of applications in several industrial and biomedical domains. Based on the evidence, the workers exposed to inhaled nanosized TiO2 powder are more susceptible to the risks of developing respiratory diseases. Accordingly, this issue has increasingly attracted the researchers’ interest in understanding the consequences of TiO2 NPs exposure. Regarding this, the present study was conducted to analyze the local effects of TiO2 NPs on allergic airway inflammation and their uptake in a mouse model of ovalbumin (OVA)-induced allergic airway inflammation. Methods For the purpose of the study, female BALB/c mice with or without asthma were intranasally administered with TiO2 NPs. The mice were subjected to histological assessment, lung function testing, scanning electron microscopy (SEM), inductively coupled plasma mass spectrometry (ICP-MS), and NP uptake measurement. In addition, T helper (Th) 1/Th2 cytokines were evaluated in the lung homogenate using the enzyme-linked immunosorbent assay. Results According to the results, the mice receiving OVA alone or OVA plus TiO2 NPs showed eosinophilic infiltrates and mucus overproduction in the lung tissues, compared to the controls. Furthermore, a significant elevation was observed in the circulating Th2 cytokines, including interleukin (IL)-4, IL-5, and IL-13 after NP exposure. The TiO2 NPs were taken up by alveolar macrophages at different time points. As the results of the SEM and ICP-MS indicated, TiO2 NPs were present in most of the organs in both asthmatic and non-asthmatic mice. Conclusion Based on the findings of the current study, intranasally or inhalation exposure to high-dose nanosized TiO2 particles appears to exacerbate the allergic airway inflammation and lead to systemic uptake in extrapulmonary organs. These results indicate the very important need to investigate the upper limit of intranasally or inhalation exposure to nanosized TiO2 particles in occupational and environmental health policy.

Myocardial infarction (MI) remains a major cause of mortality worldwide. Despite significant advances in MI treatment, many who survive the acute event are at high risk of chronic cardiac morbidity. Here we developed a cell-free therapeutic that capitalizes on the antifibrotic effects of micro(mi)RNA-101a and exploits the multi-faceted regenerative activity of mesenchymal stem cell (MSC) extracellular nanovesicles (eNVs). While the majority of MSC eNVs require local delivery via intramyocardial injection to exert therapeutic efficacy, we have developed MSC eNVs that can be administered in a minimally invasive manner, all while remaining therapeutically active. When loaded with miR-101a, MSC eNVs substantially decreased infarct size (12 ± 2.4% vs. 21.4 ± 5.7%) and increased ejection fraction (53.6 ± 7.6% vs. 40.3 ± 6.0%) and fractional shortening (23.6 ± 4.3% vs. 16.6 ± 3.0%) compared to control. These findings are significant as they represent an advance in the development of minimally invasive cardio-therapies.

Nanotechnology is showing promise in many medical applications such as drug delivery and hyperthermia. Nanoparticles administered to the respiratory tract cause local reactions and cross the blood-air barrier, thereby providing a means for easy systemic administration but also a potential source of toxicity. Little is known about how these effects are influenced by preexisting airway diseases such as asthma. Here, BALB/c mice are treated according to the ovalbumin (OVA) asthma protocol to promote allergic airway inflammation. Dispersions of polyethylene-glycol-coated (PEGylated) and citrate/tannic-acid-coated (citrated) 5 nm gold nanoparticles are applied intranasally to asthma and control groups, and (i) airway resistance and (ii) local tissue effects are measured as primary endpoints. Further, nanoparticle uptake into extrapulmonary organs is quantified by inductively coupled plasma mass spectrometry. The asthmatic precondition increases nanoparticle uptake. Moreover, systemic uptake is higher for PEGylated gold nanoparticles compared to citrated nanoparticles. Nanoparticles inhibit both inflammatory infiltrates and airway hyperreactivity, especially citrated gold nanoparticles. Although the antiinflammatory effects of gold nanoparticles might be of therapeutic benefit, systemic uptake and consequent adverse effects must be considered when designing and testing nanoparticle-based asthma therapies.

A limitation to using exosomes to their fullest potential is their limited secretion from cells, a major bottleneck to efficient exosome production and application. This is especially true for mesenchymal stem cell (MSCs), which can self-renew but have limited expansion capacity, undergoing senescence after only a few passages, with exosomes derived from senescent stem cells showing impaired regenerative capacity compared to young cells. Here we examined the effects of small molecule modulators capable of enhancing exosome secretion from MSCs. Treatment of MSCs with a combination of methyldopamine and norepinephrine robustly increased exosome production by three-fold without altering the ability of the MSC exosomes to induce angiogenesis, polarize macrophages to the anti-inflammatory phenotype, or inhibit fibrosis. These small molecule modulators provide a promising means to increase exosome production by MSCs.

Excessive or prolonged recruitment of inflammatory monocytes to damaged tissue can significantly worsen patient outcomes. Monocytes migrate to sites of tissue inflammation in response to high local concentrations of CCL2, a chemokine that binds to and signals through the CCR2 receptor. While the role of CCR2 in cellular migration is well studied, it is unclear how CCR2 inhibition affects macrophage polarization and if multivalency can increase downstream signaling effects. Using affinity selection with a phage library, we identified a novel scFv (58C) that binds specifically and with high affinity to the N-terminal domain of CCR2 (K(D) = 59.8 nM). The newly identified 58C-scFv bound to native CCR2 expressed on macrophages and MDA-MB-231 cells, inhibited migration, and induced a pro-inflammatory M1-phenotype in macrophages. The M1/M2 macrophage phenotype ratio for monomeric 58C-scFv was significantly increased over the negative control by 1.0×10(4)-fold (iNOS/Arg-1), 5.1×10(4)-fold (iNOS/Mgl2), 3.4×10(5)-fold (IL-6/Arg-1), and 1.7×10(6)-fold (IL-6/Mgl2). Multivalent display of 58C-scFv on liposomes further reduced migration of both cell types by 25 to 40% and enhanced M1 polarization by 200% over monomeric 58C-scFv. These studies demonstrate that CCR2 inhibition polarizes macrophages towards an inflammatory M1 phenotype and that multivalent engagement of CCR2 increases the effects of 58C-scFv on polarization and migration. These data provide important insights into the role of multivalency in modulating binding, downstream signaling, and cellular fate.

Mesenchymal stem cell (MSC)-derived exosomes mediate tissue regeneration in a variety of diseases including ischemic heart injury, liver fibrosis, and cerebrovascular disease. Despite an increasing number of studies reporting the therapeutic effects of MSC exosomes, the underlying molecular mechanisms and their miRNA complement are poorly characterized. Here we microRNA (miRNA)-profiled MSC exosomes and conducted a network analysis to identify the dominant biological processes and pathways modulated by exosomal miRNAs. At a system level, miRNA-targeted genes were enriched for (cardio)vascular and angiogenesis processes in line with observed cardiovascular regenerative effects. Targeted pathways were related to Wnt signaling, pro-fibrotic signaling via TGF-β and PDGF, proliferation, and apoptosis. When tested, MSC exosomes reduced collagen production by cardiac fibroblasts, protected cardiomyocytes from apoptosis, and increased angiogenesis in HUVECs. The intrinsic beneficial effects were further improved by virus-free enrichment of MSC exosomes with network-informed regenerative miRNAs capable of promoting angiogenesis and cardiomyocyte proliferation. The data presented here help define the miRNA landscape of MSC exosomes, establish their biological functions through network analyses at a system level, and provide a platform for modulating the overall phenotypic effects of exosomes.

The cell-penetrating trans-activator of transcription (TAT) is a cationic peptide derived from human immunodeficiency virus-1. It has been used to facilitate macromolecule delivery to various cell types. This cationic peptide is capable of crossing the blood-brain barrier and therefore might be useful for enhancing the delivery of drugs that target brain tumors. Here we test the efficiency with which relatively small (20 nm) micelles can be delivered by an intra-arterial route specifically to gliomas. Utilizing the well-established method of flow-arrest intra-arterial injection we compared the degree of brain tumor deposition of cationic TAT-decorated micelles versus neutral micelles. Our in vivo and post-mortem analyses confirm glioma-specific deposition of both TAT-decorated and neutral micelles. Increased tumor deposition conferred by the positive charge on the TAT-decorated micelles was modest. Computational modeling suggested a decreased relevance of particle charge at the small sizes tested but not for larger particles. We conclude that continued optimization of micelles may represent a viable strategy for targeting brain tumors after intra-arterial injection. Particle size and charge are important to consider during the directed development of nanoparticles for intra-arterial delivery to brain tumors.

TheE. colisingle stranded DNA binding protein (SSB) is essential to viability. It plays key roles in DNA metabolism where it binds to nascent single strands of DNA and to target proteins known as the SSB interactome. There are >2,000 tetramers of SSB per cell with perhaps 100-150 associated with genome at any one time, either at DNA replication forks or at sites of DNA repair. The remaining 1,900 tetramers could constantly diffuse throughout the cytosol or be associated with the inner membrane as observed for other DNA metabolic enzymes such as DnaA and RecA. To visualize SSB directly and to ascertain spatiotemporal changes in tetramer localization in response to DNA damage, SSB-GFP chimeras were visualized using a novel, super-resolution microscope optimized for visualization of prokaryotic cells. Results show that in the absence of DNA damage, SSB localizes to a small number of foci and the excess protein is observed associated with the inner membrane where it binds to the major phospholipids. Within five minutes following DNA damage, the vast majority of SSB disengages from the membrane and is found almost exclusively in the cell interior. Here, it is observed in a large number of foci, in discreet structures or, in diffuse form spread over the genome, thereby enabling repair events. In the process, it may also deliver interactome partners such as RecG or PriA to sites where their repair functions are required.

Intra-arterial (IA) drug delivery has been proposed for the treatment of a wide range of brain diseases, including malignant brain tumors. However, pharmacokinetic optimization for IA drug delivery to the brain remains a challenge. In this report, we apply and expand the well-established Dedrick model of IA drug delivery to the brain and test the effects of modifying drug and delivery parameters. These simulations show that altering the properties of candidate drugs and physiological variables can have profound effects on regional deposition after IA injections. We show that drug and physiological optimization aimed at rapid drug extraction and sustained retention is necessary to maximize regional deposition after of IA injections.

Harnessing exosomes as therapeutic drug delivery vehicles requires a better understanding of exosomal composition and their mode of action. A full appreciation of all the exosomal components (proteins, lipids, and RNA content) will be important for the design of effective exosome-based or exosome-mimicking drug carriers. In this review we describe the presence of rarely studied, non-coding RNAs that exist in high numbers in exosomes. We discuss the implications of the molecular composition and heterogeneity of exosomes on their biological and therapeutic effects. Finally, we highlight outstanding questions with regard to RNA loading into exosomes, analytical methods to sort exosomes and their sub-populations, and the effects of exosomal proteins and lipids on recipient cells. Investigations into these facets of exosome biology will further advance the field, could lead to the clinical translation of exosome-based therapeutics, and aid in the reverse-engineering of synthetic exosomes. Although synthetic exosomes are still an underexplored area, they could offer researchers a way to manufacture exosomes with highly defined structure, composition, and function.

A suitable aerosol droplet size and formulation output rate is essential for the therapy of lung diseases under application of nebulizers. The current study investigated the potential of amine-modified poly(vinyl alcohol)s as excipients for inhalation delivery. A change of conductivity (effective at0.1mg/ml) and viscosity (effective at0.1mg/ml) of samples that were supplemented with charge-modified polymers had a significant influence on the generated droplet size (shift from ~8 to ~4 μm) and formulation throughput rate (shift from ~0.2 to ~1.0 g/min), where polymers with a higher amine density (and molecular weight) showed an elevated activity. Biocompatibility assessment of polymers in A549 cells and an isolated lung model resulted in cell lysis and lung edema formation dependent on the type (degree of amine substitution) and dose of polymer applied. Suitable compositions and concentrations of amine-modified poly(vinyl alcohol)s were identified with respect to an optimized nebulizer performance and acceptable biocompatibility. Charge-modified polymers represent novel excipients with potential to improve inhalation therapy.

Myocardial infarction (MI) remains a major cause of mortality worldwide. Despite significant advances in MI treatment, many who survive the acute event are at high risk of chronic cardiac morbidity. Here we developed a cell-free therapeutic that capitalizes on the antifibrotic effects of micro(mi)RNA-101a and exploits the multi-faceted regenerative activity of mesenchymal stem cell (MSC) extracellular nanovesicles (eNVs). While the majority of MSC eNVs require local delivery via intramyocardial injection to exert therapeutic efficacy, we have developed MSC eNVs that can be administered in a minimally invasive manner, all while remaining therapeutically active. When loaded with miR-101a, MSC eNVs substantially decreased infarct size (9.2 ± 1.7% vs. 20.0 ± 6.5%) and increased ejection fraction (53.6 ± 7.6% vs. 40.3 ± 6.0%) and fractional shortening (23.6 ± 4.3% vs. 16.6 ± 3.0%) compared to control. These findings are significant as they represent an advance in the development of minimally invasive cardio-therapies.

Jinli Wang,1,2,* Min Jeong Seo,1,* Michael B Deci,1 Brian R Weil,3 John M Canty,3,4 Juliane Nguyen1,2 1Department of Pharmaceutical Sciences, School of Pharmacy, University at Buffalo, The State University of New York, Buffalo, NY, USA; 2Department of Biomedical Engineering, University at Buffalo, The State University of New York, Buffalo, NY, USA; 3Department of Medicine, Department of Physiology and Biophysics, Department of Biomedical Engineering, The Clinical and Translational Research Center, University at Buffalo, Buffalo, NY, USA; 4VA Western New York Healthcare System, Buffalo, NY, USA *These authors contributed equally tothis work Background: After myocardial infarction (MI), inflammatory cells infiltrate the infarcted heart in response to secreted stimuli. Monocytes are recruited to the infarct via CCR2 chemokine receptors along a CCL2 concentration gradient. While infiltration of injured tissue with monocytes is an important component of the reparatory response, excessive or prolonged inflammation can adversely affect left ventricular remodeling and worsen clinical outcomes.Materials and methods: Here, we developed poly(ethylene glycol) (PEG)-distearoylphosphatidylethanolamine (PEG-DSPE) micelles loaded with a small molecule CCR2 antagonist to inhibit monocyte recruitment to the infarcted myocardium. To specifically target CCR2-expressing cells, PEG-DSPE micelles were further surface decorated with an anti-CCR2 antibody.Results: Targeted PEG-DSPE micelles showed eight-fold greater binding to CCR2-expressing RAW 264.7 monocytes than plain, non-targeted PEG-DSPE micelles. In a mouse model of MI, CCR2-targeting PEG-DSPE micelles loaded with a CCR2 small molecule antagonist significantly decreased the number of Ly6Chigh inflammatory cells to 3% of total compared with PBS-treated controls. Furthermore, CCR2-targeting PEG-DSPE micelles significantly reduced the infarct size based on epicardial and endocardial infarct arc lengths.Conclusion: Both non-targeted and CCR2-targeting PEG-DSPE micelles showed a trend toward improving cardiac function. As such, PEG-DSPE micelles represent a promising cardiac therapeutic platform. Keywords: CCR2, inflammatory monocytes, micelles, myocardial infarction

Background: Advances in radiotherapy for thoracic cancers have resulted in improvement of survival. However, radiation exposure to the heart can induce cardiotoxicity. No therapy is currently available to inhibit these untoward effects. We examined whether a small tetrapeptide, N-acetyl-Ser-Asp-Lys-Pro (Ac-SDKP), can counteract radiation-induced cardiotoxicity by inhibiting macrophage-dependent inflammatory and fibrotic pathways. Methods and Results: After characterizing a rat model of cardiac irradiation with magnetic resonance imaging protocols, we examined the effects of Ac-SDKP in radiation-induced cardiomyopathy. We treated rats with Ac-SDKP for 18 weeks. We then compared myocardial contractile function and extracellular matrix by cardiac magnetic resonance imaging and the extent of inflammation, fibrosis, and Mac-2 (galectin-3) release by tissue analyses. Because Mac-2 is a crucial macrophage-derived mediator of fibrosis, we performed studies to determine Mac-2 synthesis by macrophages in response to radiation, and change in profibrotic responses by Mac-2 gene depleted cardiac fibroblasts after radiation. Cardiac irradiation diminished myocardial contractile velocities and enhanced extracellular matrix deposition. This was accompanied by macrophage infiltration, fibrosis, cardiomyocyte apoptosis, and cardiac Mac-2 expression. Ac-SDKP strongly inhibited these detrimental effects. Ac-SDKP migrated into the perinuclear cytoplasm of the macrophages and inhibited radiation-induced Mac-2 release. Cardiac fibroblasts lacking the Mac-2 gene showed reduced transforming growth factor β1, collagen I, and collagen III expression after radiation exposure. Conclusions: Our study identifies novel cardioprotective effects of Ac-SDKP in a model of cardiac irradiation. These protective effects are exerted by inhibiting inflammation, fibrosis, and reducing macrophage activation. This study shows a therapeutic potential of this endogenously released peptide to counteract radiation-induced cardiomyopathy.

Exosomes are small lipid vesicles secreted by a variety of cells. They were originally assumed to provide cells with the mean to dispose of unneeded proteins, nucleic acids, and lipids. However, exosomes are now known to play a role in intercellular communication. Cancer cells have been shown to secrete exosomes to not only modulate their immediate microenvironment, but also to exert effects in distant tissues and organs. To fully understand cancer exosomes and their function, it is important to elucidate their molecular composition. Here we provide an overview of exosome biogenesis, composition, and physicochemical properties. We also describe how their secretion from cancer cells can be affected by exposure to specific drugs.

Since their introduction in 1980, the number of advanced targeted nanocarrier systems has grown considerably. Nanocarriers capable of targeting single receptors, multiple receptors, or multiple epitopes have all been used to enhance delivery efficiency and selectivity. Despite tremendous progress, preclinical studies and clinically translatable nanotechnology remain disconnected. The disconnect in targeting efficacy may stem from poorly-understood factors such as receptor clustering, spatial control of targeting ligands, ligand mobility, and ligand architecture. Further, the relationship between receptor distribution and ligand architecture remains elusive. Traditionally, targeted nanocarriers were engineered assuming a "static" target. However, it is becoming increasingly clear that receptor expression patterns change in response to external stimuli and disease progression. Here, we discuss how cutting-edge technologies will enable a better characterization of the spatiotemporal distribution of membrane receptors and their clustering. We further describe how this will enable the design of new nanocarriers that selectively target the site of disease. Ultimately, we explore how the precision engineering of targeted nanocarriers that adapt to receptor dynamics will have the potential to drive nanotechnology to the forefront of therapy and make targeted nanomedicine a clinical reality. This article is categorized under: Therapeutic Approaches and Drug Discovery > Emerging Technologies Nanotechnology Approaches to Biology > Nanoscale Systems in Biology Biology-Inspired Nanomaterials > Lipid-Based Structures Biology-Inspired Nanomaterials > Protein and Virus-Based Structures.

The relative abundance of anionic lipids on the surface of endothelia and on glioma cells suggests a workable strategy for selective drug delivery by utilizing cationic nanoparticles. Furthermore, the extracellular pH of gliomas is relatively acidic suggesting that tumor selectivity could be further enhanced if nanoparticles can be designed to cationize in such an environment. With these motivating hypotheses the objective of this study was to determine whether nanoparticulate (20 nm) micelles could be designed to improve their deposition within gliomas in an animal model. To test this, we performed intra-arterial injection of micelles labeled with an optically quantifiable dye. We observed significantly greater deposition (end-tissue concentration) of cationizable micelles as compared to non-ionizable micelles in the ipsilateral hemisphere of normal brains. More importantly, we noted enhanced deposition of cationizable as compared to non-ionizable micelles in glioma tissue as judged by semiquantitative fluorescence analysis. Micelles were generally able to penetrate to the core of the gliomas tested. Thus we conclude that cationizable micelles may be constructed as vehicles for facilitating glioma-selective delivery of compounds after intraarterial injection.