Evaluation of Novel B1R/B2R Agonists Containing TRIOZAN™ Nanoparticles for Targeted Brain Delivery of Antibodies in a Mouse Model of Alzheimer Disease.

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.

Facile design of autogenous stimuli-responsive chitosan/hyaluronic acid nanoparticles for efficient small molecules to protein delivery.

Easily assembled and biocompatible chitosan/hyaluronic acid nanoparticles with multiple stimuli-responsive ability are ideally suited for efficient delivery of therapeutic agents under specific endogenous triggers. We report a simple and versatile strategy to formulate oxidative stress and pH-responsive chitosan/hyaluronic acid nanocarriers with high encapsulation efficiencies of small drug molecules and nerve growth factor protein. This is achieved through invoking the dual role of a thioketal-based weak organic acid to disperse and functionalize low molecular weight chitosan in one-pot. Thioketal embedded chitosan/hyaluronic acid nanostructures respond to oxidative stress and show controlled release of quercetin, curcumin and NGF. Lowering the pH in the buffer solution led to higher quercetin release from NPs than at physiological pH, and mimicked the nanoparticle behavior in the environment of early to late endosomes. Curcumin and quercetin loaded NPs killed glioblastoma cells with high efficiency, and NGF-loaded nanoparticles retained biological activity of the protein and increased peripheral nerve outgrowth in explanted mouse dorsal root ganglia.

PEG-conjugated pyrrole-based polymers: one-pot multicomponent synthesis and self-assembly into soft nanoparticles for drug delivery.

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.

The susceptibility to chronic social defeat stress is related to low hippocampal extrasynaptic NMDA receptor function.

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.

Organotypic and primary neural cultures as models to assess effects of different gold nanostructures on glia and neurons.

Gold nanoparticles (AuNP) have unique physicochemical properties and have been used as delivery vehicles, contrast agents, and therapeutic compounds. Although the effects of AuNPs on peripheral tissues and immortalized cell lines have been extensively characterized, their effects on the central nervous system (CNS) are predominantly unknown. The main objective of the current study was to evaluate how AuNPs of varying sizes (1-100 nm), shapes (clusters, spheres, rods, flowers), and surfaces impact synaptic structures in the hippocampus, a brain structure often affected in neurodegeneration. Using a combination of organotypic hippocampal, as well as, primary neuronal, glial, and astrocytic cultures, we examined AuNPs impact on hippocampal dendritic spine density, internalization in various neural cells, and lysosomal status in astrocytes. Considering that neurons interact with astrocytes, and that lysosomes play a role in dendritic spine status, transcription factor TFEB and abundance of lysosomal marker, LAMP1 were evaluated. Both biomarkers were significantly increased in astrocytes exposed to AuNPs, suggesting that AuNPs not only enter lysosomes, but also increase lysosome biogenesis. Results from our studies show that AuNPs with poly(ethylene glycol) (AuNPs-PEG) or glutathione (AuNP-GSH) surfaces do not substantially decrease hippocampal dendritic spine density. Conversely, AuNPs coated with the detergent, CTAB, significantly decreased total spine density. Interestingly small gold nanoclusters (Au15(SG)13) with GSH reduced spine density, whereas larger gold nanoclusters (Au25(SG)18) with the same ligand did not. Thus, assessment of dendritic morphology, spine densities can reveal subtler changes of neural cells than cell death when exposed to nanoparticles, including AuNPs.

Unraveling Aqueous Self-Assembly of Telodendrimers to Shed Light on Their Efficacy in Drug Encapsulation.

Amphiphilic architectural polymers of tunable compositions self-assemble into soft nanoparticles of varied stability that is dependent on the number of poly(ethylene glycol) tails. Cryo-electron microscopy- and freeze-fracture-technique-based evaluations of their internal structure display morphologies unlike those of conventional block-copolymer-based micelles, with a uniform and homogeneous composition that strongly influences drug-specific encapsulation and release characteristics. The suberanilohydroxamic acid (SAHA) and Temozolomide drug combination (with or without telodendrimer loading) shows synergistic effects in glioblastoma, and curcumin-loaded DP3 telodendrimers reduce neurite loss in cisplatin-treated dorsal root ganglia explants.

Encapsulation and Delivery of Neutrophic Proteins and Hydrophobic Agents Using PMOXA-PDMS-PMOXA Triblock Polymersomes.

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.

Gold nanourchins and celastrol reorganize the nucleo- and cytoskeleton of glioblastoma cells.

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.

Dendritic Polyglycerol Sulfates in the Prevention of Synaptic Loss and Mechanism of Action on Glia.

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-ß (Aß42) 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 Aß42 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 Aß42, i.e., a direct interaction between dPGS and Aß42 species interfered with Aß 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.

Telodendrimers for Physical Encapsulation and Covalent Linking of Individual or Combined Therapeutics.

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.

Inhibition of carbonic anhydrase IX in glioblastoma multiforme.

Carbonic anhydrase IX (CAIX) is a transmembrane enzyme upregulated in several types of tumors including glioblastoma multiforme (GBM). GBM is among the most aggressive tumors among gliomas. Temozolomide (TMZ) therapy combined with surgical or radiation approaches is the standard treatment but not effective in long term. In this study we tested the treatment with acetazolamide (ATZ), an inhibitor of CAIX, alone or combined with TMZ. The experiments were performed in 2D and 3D cultures (spheroids) using glioblastoma U251N and human brain tumor stem cells (BTSCs). Several proteins implicated in tumor cell death were also investigated. The key results from these studies suggest the following: (1) Cell death of human glioblastoma spheroids and BTSC is significantly increased with combined treatment after 7 days, and (2) the effectiveness of ATZ is significantly enhanced against BTSC and U251N when incorporated into nano-carriers. Collectively, these results point toward the usefulness of nano-delivery of CAIX inhibitors and their combination with chemotherapeutics for glioblastoma treatment.

Asymmetric AB3 Miktoarm Star Polymers: Synthesis, Self-Assembly, and Study of Micelle Stability Using AF4 for Efficient Drug Delivery.

A simple and versatile methodology, which employs a combination of ring-opening polymerization and alkyne-azide click chemistry to synthesize amphiphilic AB3 miktoarm stars, is reported. Their aqueous self-assembly behavior was studied using dynamic light scattering, fluorescence, and asymmetrical flow field-flow fractionation (AF4). AB3 miktoarm stars form micelles which incorporate curcumin with high efficiency, and significantly reduce the viability of glioblastoma cells in spheroids. We demonstrate that AF4 is an effective technique to determine the size distribution of self-assembled structures exposed to a biological medium. The ease, with which asymmetric AB3 miktoarm polymers are constructed, provides a platform that can be widely employed to deliver a variety of lipophilic drugs.

Assessment of the developmental toxicity of nanoparticles in an ex vivo 3D model, the murine limb bud culture system.

The rapid growth of nanotechnological products for biomedical applications has exacerbated the need for suitable biological tests to evaluate the potential toxic effects of nanomaterials. The possible consequences of exposure during embryo and fetal development are of particular concern. The limb bud culture is an ex vivo 3D model in which growth, cell differentiation, and tissue organization occur and both molecular and functional endpoints can be quantitatively assessed. We employed this model to assess biochemical and morphological changes induced during organogenesis by two classes of nanostructured materials: quantum dot nanocrystals and organic polyglycerol sulfate dendrimers (dPGS). We show that quantum dots carrying mercaptopropionic acid (QD-MPA) on the surface, commonly used in biological studies, inhibit the development of limb buds from CD1 wildtype and Col2a1; Col10a1; Col1a1 triple transgenic fluorescent reporter mice, as revealed by changes in several morphological and biochemical markers. QD-MPA interfere with chondrogenesis and osteogenesis and disrupt the expression of COL10A1 and COL1A1, key markers of differentiation. In contrast, equivalent (3-100 nM) concentrations of dPGS do not adversely affect limb development. Neither QD-MPA nor dPGS-Cy5 alters the expression of several markers of cell proliferation or apoptosis. Collectively, these results suggest that murine limb buds in culture constitute a convenient, inexpensive and reliable developmental model for the assessment of the nanotoxicological effects of nanocrystals and polymers. In these 3D cultures, any effect that is observed can be directly ascribed to the nanostructures per se or a degradation component released from the complex nanostructure.

Quantum dot agglomerates in biological media and their characterization by asymmetrical flow field-flow fractionation.

The molecular composition of the biological environment of nanoparticles influences their physical properties and changes their pristine physicochemical identity. In order to understand, or predict, the interactions of cells with specific nanoparticles, it is critical to know their size, shape, and agglomeration state not only in their nascent state but also in biological media. Here, we use asymmetrical flow field-flow fractionation (AF4) with on-line multiangle light scattering (MALS), dynamic light scattering (DLS) and UV-Visible absorption detections to determine the relative concentration of isolated nanoparticles and agglomerates in the case of three types of semi-conductor quantum dots (QDs) dispersed in Dulbecco's Modified Eagle Media (DMEM) containing 10% of fetal bovine serum (DMEM-FBS). AF4 analysis also yielded the size and size distribution of the agglomerates as a function of the time of QDs incubation in DMEM-FBS. The preferred modes of internalization of the QDs are assessed for three cell-types, N9 microglia, human hepatocellular carcinoma cells (HepG2) and human embryonic kidney cells (Hek293), by confocal fluorescence imaging of live cells, quantitative determination of the intracellular QD concentration, and flow cytometry. There is an excellent correlation between the agglomeration status of the three types of QDs in DMEM-FBS determined by AF4 analysis and their preferred mode of uptake by the three cell lines, which suggests that AF4 yields an accurate description of the nanoparticles as they encounter cells and advocates its use as a means to characterize particles under evaluation.

Quantum dots induce heat shock-related cytotoxicity at intracellular environment.

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.

Caspase-1 activity in microglia stimulated by pro-inflammagen nanocrystals.

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-1ß 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.

Separation science: Principles and applications for the analysis of bionanoparticles by asymmetrical flow field-flow fractionation (AF4).

Field-flow fractionation is an analytical technique that allows the separation of particles over a size range, from a few nanometers to several microns in diameter. The separation takes place under mild conditions and is suited for the analysis of neutral or charged particles. A single measurement yields the size and concentration of each component of a mixture. However, developing a suitable fractionation method can be tedious and time-consuming. In this chapter, we present asymmetrical flow field-flow fractionation (AF4) conditions that have proven their reliability for the analysis of quantum dots and other nanoparticles in the 5-50 nm size range. Common pitfalls are emphasized together with strategies to overcome them.

Dual crosslinked hydrogel nanoparticles by nanogel bottom-up method for sustained-release delivery.

Polysaccharide-PEG hybrid nanogels (CHPOA-PEGSH) crosslinked by both covalent ester bonds and physical interactions were prepared by the reaction of a thiol-modified poly(ethylene glycol) (PEGSH) with acryloyl-modified cholesterol-bearing pullulan (CHPOA). Experimental parameters, including CHPOA concentration, the degree of acryloyl substitution of CHPOA, and the initial amounts of CHPOA and PEGSH, were modified in order to assess their effect on the size of the nanogels (50-150 nm) and on their degradation kinetics, monitored by dynamic light scattering (DLS) and asymmetrical flow field-flow fractionation (AF4) chromatography. Rhodamine-labeled nanogels were injected intravenously into mice and their concentration in blood was determined by a fluorescence assay as a function of post-injection time. The elimination half-life (t(1/2)) of CHPOA-PEGSH nanoparticles was about 15-fold longer (18 h) than that of CHP nanogels (1.2 h). The half-life enhancement of CHPOA-PEGSH was attributed to the presence of the crosslinker PEG chains, which prevent non-specific protein adsorption, and to the slow hydrolysis kinetics of the crosslinking esters in the biological milieu. The hybrid CHPOA-PEGSH nanogels are expected to be useful as injectable nanocarriers for drugs and proteins, in view of their low surface fouling and slow hydrolysis rate.

Short ligands affect modes of QD uptake and elimination in human cells.

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.

Lipopolysaccharide-QD micelles induce marked induction of TLR2 and lipid droplet accumulation in olfactory bulb microglia.

The intranasal entry of biological and artificial nanoparticles can induce inflammatory responses both locally and more widely in surrounding tissues. The aim of this study was to assess the microglia activation induced by nanoparticles with different surfaces in (i) a transgenic mouse (Toll-like receptor (TLR)-2-luciferase (Luc) reporter) which allowed the biophotonic imaging of microglial activation/innate immune response after intranasal delivery of nanoparticles and (ii) in microglial dispersed cells in vitro. Cadmium selenide nanoparticles (quantum dots, QD), surface-exchanged with lipopolysaccharide (LPS) to form micelles, were tested to assess microglia activation and lipid droplet formation in both model systems. In vivo imaging revealed a robust increase in the extent of microglial activation/TLR2 response, initially in the olfactory bulb, but also in other more caudal brain regions. The increased TLR2 expression was complemented with enhanced CD68 expression in activated microglia in the same regions. Intense in vitro microglial activation by LPS-QD micelles was accompanied by a significant enhancement of nitric oxide production and formation of large lipid droplets, suggesting the possibility of this organelle acting as an inflammatory biomarker in response to nanoparticles, and not simply as a storage site in fat tissues.