N-methyl-D-aspartate receptors (NMDARs) have been highly implicated in the pathogenesis and treatment of depression. While NMDARs can be found inside and outside glutamate synapses, it remains ...unclear if NMDARs at synaptic (sNMDAR) and extrasynaptic locations (exNMDAR) play different roles in the formation of depression-related behaviors. Using chronic social defeat stress (CSDS), an animal model for anxiety- and depression-related behaviors, we found that mice susceptible to CSDS exhibited low hippocampal exNMDAR function. Raising exNMDAR function by enhancing the release of glutamate from astrocytic cystine-glutamate antiporters or targeting extrasynaptic receptors with agonist-coated gold nanoparticles that cannot enter the synaptic cleft prevented social avoidance behavior in stressed mice. Interestingly, ketamine, which is a fast-acting antidepressant, exhibited stronger blockade to sNMDARs than to exNMDARs. These findings suggest that the susceptibility and resilience of mice toward CSDS is related to low and high exNMDAR function in the hippocampus, respectively. Enhancing exNMDAR function could be a novel treatment approach for mood and anxiety disorders.
The blood-brain barrier (BBB) is a major obstacle to the development of effective therapeutics for central nervous system (CNS) disorders, including Alzheimer's disease (AD). This has been ...particularly true in the case of monoclonal antibody (mAbs) therapeutic candidates, due to their large size. To tackle this issue, we developed new nanoformulations, comprising bio-based Triozan polymers along with kinin B1 and B2 receptor (B1R and B2R) peptide agonist analogues, as potent BBB-permeabilizers to enhance brain delivery of a new anti-C1q mAb for AD (ANX005). The prepared B1R/B2R-TRIOZAN™ nanoparticles (NPs) displayed aqueous solubility, B1R/B2R binding capacity and uniform sizes (~130-165 nm). The relative biodistribution profiles of the mAb loaded into these NPs versus the naked mAb were assessed in vivo through two routes of administrations (intravenous (IV), intranasal (IN)) in the Tg-SwDI mouse model of AD. At 24 h post-administration, brain levels of the encapsulated mAb were significantly increased (up to 12-fold (IV) and 5-fold (IN), respectively) compared with free mAb in AD brain affected regions, entorhinal cortex and hippocampus of aged mice. Liver uptakes remained relatively low with similar values for the nanoformulations and free mAb. Our findings demonstrate the potential of B1R/B2R-TRIOZAN™ NPs for the targeted delivery of new CNS drugs, which could maximize their therapeutic effectiveness.
In order to better understand nanoparticle uptake and elimination mechanisms, we designed a controlled set of small, highly fluorescent quantum dots (QDs) with nearly identical hydrodynamic size ...(8-10 nm) but with varied short ligand surface functionalization. The properties of functionalized QDs and their modes of uptake and elimination were investigated systematically by asymmetrical flow field-flow fractionation (AF4), confocal fluorescence microscopy, flow cytometry (FACS), and flame atomic absorption (FAA). Using specific inhibitors of cellular uptake and elimination machinery in human embryonic kidney cells (Hek 293) and human hepatocellular carcinoma cells (Hep G2), we showed that QDs of the same size but with different surface properties were predominantly taken up through lipid raft-mediated endocytosis, however, to significantly different extents. The latter observation infers the contribution of additional modes of QD internalization, which include X-AG cysteine transporter for cysteine-functionalized QDs (QD-CYS). We also investigated putative modes of QD elimination and established the contribution of P-glycoprotein (P-gp) transporter in QD efflux. Results from these studies show a strong dependence between the properties of QD-associated small ligands and modes of uptake/elimination in human cells.
Polymersomes are attractive nanocarriers for hydrophilic and lipophilic drugs; they are more stable than liposomes, tunable, and relatively easy to prepare. The copolymer composition and molar mass ...are critical features that determine the physicochemical properties of the polymersomes including the rate of drug release. We used the triblock-copolymer, poly(2-methyl-2-oxazoline)-block-poly-(dimethysiloxane)-block-poly(2-methyl-2-oxazoline) (PMOXA–PDMS–PMOXA), to form amphipathic polymersomes capable of loading proteins and small hydrophobic agents. The selected agents were unstable neurotrophins (nerve growth factor and brain-derived neurotrophic factor), a large protein CD109, and the fluorescent drug curcumin. We prepared, characterized, and tested polymersomes loaded with selected agents in 2D and 3D biological models. Curcumin-loaded and rhodamine-bound PMOXA–PDMS–PMOXA polymersomes were used to visualize them inside cells. N-Methyl-d-aspartate receptor (NMDAR) agonists and antagonists were also covalently attached to the surface of polymersomes for targeting neurons. Labeled and unlabeled polymersomes with or without loaded agents were characterized using dynamic light scattering (DLS), UV–vis fluorescence spectroscopy, and asymmetrical flow field-flow fractionation (AF4). Polymersomes were imaged and tested for biological activity in human and murine fibroblasts, murine macrophages, primary murine dorsal root ganglia, and murine hippocampal cultures. Polymersomes were rapidly internalized and there was a clear intracellular co-localization of the fluorescent drug (curcumin) with the fluorescent rhodamine-labeled polymersomes. Polymersomes containing CD109, a glycosylphosphatidylinositol-anchored protein, promoted cell migration in the model of wound healing. Nerve growth factor-loaded polymersomes effectively enhanced neurite outgrowth in dissociated and explanted dorsal root ganglia. Brain-derived neurotrophic factor increased dendritic spine density in serum-deprived hippocampal slice cultures. NMDAR agonist- and antagonist-functionalized polymersomes targeted selectively neurons over glial cells in mixed cultures. Collectively, the study reveals the successful incorporation into polymersomes of biologically active trophic factors and small hydrophilic agents that retain their biological activity in vitro, as demonstrated in selected central and peripheral tissue models.
Although caspase-1 is a key participant in inflammation, there is no sensitive assay to measure its enzymatic activity in real time in cells or animals. Here we describe a nanosensor for caspase-1 ...ratiometric measurements, consisting of a rhodamine-labeled, caspase-1 cleavable peptide linked to quantum dots (QDs). Microglia cells were stimulated by lipopolysaccharide (LPS) and by hybrid nanoparticles LPS-QDs. These stimuli activated caspase-1 in microglia monolayers and in the mouse brain, while a selected caspase inhibitor markedly reduced it. LPS-QDs entered into the lysosomal compartment and led to an enlargement of these cellular organelles in the exposed microglia. Both lysosomal swelling and mitochondrial impairment contributed to caspase-1 activation and to the consequent interleukin-1β release. The results from these studies highlight how the unique properties of QDs can be used to create versatile biotools in the study of inflammation in real time in vivo.
Dendritic polyglycerols (dPG), particularly dendritic polyglycerol sulfates (dPGS), have been intensively studied due to their intrinsic anti-inflammatory activity. As related to brain pathologies ...involving neuroinflammation, the current study examined if dPG and dPGS can (i) regulate neuroglial activation, and (ii) normalize the morphology and function of excitatory postsynaptic dendritic spines adversely affected by the neurotoxic 42 amino acid amyloid-β (Aβ
) peptide of Alzheimer disease (AD). The exact role of neuroglia, such as microglia and astrocytes, remains controversial especially their positive and negative impact on inflammatory processes in AD. To test dPGS effectiveness in AD models we used primary neuroglia and organotypic hippocampal slice cultures exposed to Aβ
peptide. Overall, our data indicate that dPGS is taken up by both microglia and astrocytes in a concentration- and time-dependent manner. The mechanism of action of dPGS involves binding to Aβ
, i.e., a direct interaction between dPGS and Aβ
species interfered with Aβ fibril formation and reduced the production of the neuroinflammagen lipocalin-2 (LCN2) mainly in astrocytes. Moreover, dPGS normalized the impairment of neuroglia and prevented the loss of dendritic spines at excitatory synapses in the hippocampus. In summary, dPGS has desirable therapeutic properties that may help reduce amyloid-induced neuroinflammation and neurotoxicity in AD.
Quantum dots (QDs) are semiconductor nanocrystals with unique optical properties. Different proteins or polymers are commonly bound to their surfaces to improve biocompatibility. However, such ...surface modifications may not provide sufficient protection from cytotoxicity due to photodegradation and oxidative degradation. In this study, the cytotoxic effects of QDs, CdTe, and CdSe/ZnS were investigated using cadmium-resistant cells. CdTe QDs significantly reduced cell viability, whereas, CdSe/ZnS treatment did not markedly decrease the cell number. CdTe QDs were cytotoxic in cadmium-resistant cells suggesting that internalized QDs degraded and cadmium ions contributed to the cytotoxic effects. CdTe QDs were consistently more cytotoxic than CdSe/ZnS QDs, but both QDs as well as cadmium ions activated heat shock protein 70B′ promoter. QDs themselves are likely to contribute to HSP70B′ promoter activation in cadmium-resistant cells, because CdSe/ZnS QDs do not release sufficient cadmium to activate this promoter.
The physicochemical properties and cytotoxicity of diverse gold nanoparticle (AuNP) morphologies with smooth surfaces have been examined extensively. Much less is known about AuNPs with irregular ...surfaces. This study focuses on the effects of gold nanourchins in glioblastoma cells. With limited success of monotherapies for glioblastoma, multimodal treatment has become the preferred regimen. One possible example for such future therapeutic applications is the combination of AuNPs with the natural cytotoxic agent celastrol. Here, we used complementary physical, chemical and biological methods to characterize AuNPs and investigate their impact on glioblastoma cells. Our results show that gold nanourchins altered glioblastoma cell morphology and reorganized the nucleo- and cytoskeleton. These changes were dependent on gold nanourchin surface modification. PEGylated nanourchins had no significant effect on glioblastoma cell morphology or viability, unless they were combined with celastrol. By contrast, CTAB-nanourchins adversely affected the nuclear lamina, microtubules and filamentous actin. These alterations correlated with significant glioblastoma cell death. We identified several mechanisms that contributed to the impact of AuNPs on the cytoskeleton and cell survival. Specifically, CTAB-nanourchins caused a significant increase in the abundance of Rock1. This protein kinase is a key regulator of the cytoskeleton. In addition, CTAB-nanourchins led to a marked decline in pro-survival signaling via the PI3 kinase-Akt pathway. Taken together, our study provides new insights into the molecular pathways and structural components altered by gold nanourchins and their implications for multimodal glioblastoma therapy.
Polyethylene glycol grafted pyrrole-based conjugated polymers are synthesized through a one-pot multicomponent methodology, the self-assemblies of which enable nanoparticle size-selective ...encapsulation of drug molecules and their sustained release. Efficient loading of curcumin through drug-nanoparticle core interactions is probed using FRET, and the inherently fluorescent nature of polypyrrole could be used to detect these nanocarriers intracellularly.
New therapeutics for glioblastoma multiforme and our ability to deliver them using efficient nanocarriers constitute topical areas of research. We report a comparative study of temozolomide and ...quercetin in the treatment of glioblastoma (GBM) in three-dimensions, and their incorporation into micelles obtained from synthetically articulated architectural copolymers, and a commercially available linear polymer poly(ethylene glycol)-poly(lactic-co-glycolic acid) (PEG-PLGA). A versatile synthetic methodology to telodendrimers, which can be easily adapted to the needs of other therapeutic interventions, is presented. These dendritic block copolymers self-assemble into micelles and offer a platform for single or combination drug therapy. Telodendrimer micelles loaded with quercetin did not exhibit superior cell killing effect over the free drug, but acetazolamide, an inhibitor carbonic anhydrase IX, significantly reduced GBM cell viability in 3D spheroids. Results from these studies show that high loading of drugs into telodendrimer micelles requires a physical fit between the biologically active agent and telodendrimer nanocarrier, and points toward new possibilities for incorporation of chemotherapeutic and other agents to enhance their effectiveness.
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