Gold nanoparticles decorated with monosaccharides and sulfated ligands as potential modulators of the lysosomal enzyme N-acetylgalactosamine-6-sulfatase (GALNS).

N-Acetylgalactosamine-6-sulfatase (GALNS) is an enzyme whose deficiency is related to the lysosomal storage disease Morquio A. For the development of effective therapeutic approaches against this disease, the design of suitable enzyme enhancers (i.e. pharmacological chaperones) is fundamental. The natural substrates of GALNS are the glycosaminoglycans keratan sulfate and chondroitin 6-sulfate, which mainly display repeating units of sulfated carbohydrates. With a biomimetic approach, gold nanoparticles (AuNPs) decorated with simple monosaccharides, sulfated ligands (homoligand AuNPs), or both monosaccharides and sulfated ligands (mixed-ligand AuNPs) were designed here as multivalent inhibitors of GALNS. Among the homoligand AuNPs, the most effective inhibitors of GALNS activity are the ß-D-galactoside-coated AuNPs. In the case of mixed-ligand AuNPs, ß-D-galactosides/sulfated ligands do not show better inhibition than the ß-D-galactoside-coated AuNPs. However, a synergistic effect is observed for α-D-mannosides in a mixed-ligand coating with sulfated ligands that reduced IC50 by one order of magnitude with respect to the homoligand α-D-mannoside-coated AuNPs. SAXS experiments corroborated the association of GALNS with ß-D-galactoside AuNPs. These AuNPs are able to restore the enzyme activity by almost 2-fold after thermal denaturation, indicating a potential chaperoning activity towards GALNS. This information could be exploited for future development of nanomedicines for Morquio A. The recent implications of GALNS in cancer and neuropathic pain make these kinds of multivalent bionanomaterials of great interest towards multiple therapies.

Novel Core-Shell Polyamine Phosphate Nanoparticles Self-Assembled from PEGylated Poly(allylamine hydrochloride) with Low Toxicity and Increased In Vivo Circulation Time.

An approach for reducing toxicity and enhancing therapeutic potential of supramolecular polyamine phosphate nanoparticles (PANs) through PEGylation of polyamines before their assembly into nanoparticles is presented here. It is shown that the number of polyethylene glycol (PEG) chains for polyamine largely influence physico-chemical properties of PANs and their biological endpoints. Poly(allylamine hydrochloride) (PAH) are functionalized through carbodiimide chemistry with three ratios of PEG molecules per PAH chain: 0.1, 1, and 10. PEGylated PAH is then assembled into PANs by exposing the polymer to phosphate buffer solution. PANs decrease size and surface charge with increasing PEG ratios as evidenced by dynamic light scattering and zeta potential measurements, with the ten PEG/PAH ratio PANs having practically zero charge. Small angle X-ray scattering (SAXS) proves that PEG chains form a shell around a polyamine core, which is responsible for the screening of positive charges. MTT experiments show that the screening of amine groups decreases nanoparticle toxicity, with the lowest toxicity for the 10 PEG/PAH ratio. Fluorescence correlation spectroscopy (FCS) proves less interaction with proteins for PEGylated PANs. Positron emission tomography (PET) imaging of 18 F labelled PANs shows longer circulation time in healthy mice for PEGylated PANs than non-PEGylated ones.

Hybrid Multivalent Jack Bean α-Mannosidase Inhibitors: The First Example of Gold Nanoparticles Decorated with Deoxynojirimycin Inhitopes.

Among carbohydrate-processing enzymes, Jack bean α-mannosidase (JBα-man) is the glycosidase with the best responsiveness to the multivalent presentation of iminosugar inhitopes. We report, in this work, the preparation of water dispersible gold nanoparticles simultaneously coated with the iminosugar deoxynojirimycin (DNJ) inhitope and simple monosaccharides (ß-d-gluco- or α-d-mannosides). The display of DNJ at the gold surface has been modulated (i) by using an amphiphilic linker longer than the aliphatic chain used for the monosaccharides and (ii) by presenting the inhitope, not only in monomeric form, but also in a trimeric fashion through combination of a dendron approach with glyconanotechnology. The latter strategy resulted in a strong enhancement of the inhibitory activity towards JBα-man, with a Ki in the nanomolar range (Ki = 84 nM), i.e., more than three orders of magnitude higher than the monovalent reference compound.

Therapeutic Efficacy of Novel Antimicrobial Peptide AA139-Nanomedicines in a Multidrug-Resistant Klebsiella pneumoniae Pneumonia-Septicemia Model in Rats.

Antimicrobial peptides (AMPs) have seen limited clinical use as antimicrobial agents, largely due to issues relating to toxicity, short biological half-life, and lack of efficacy against Gram-negative bacteria. However, the development of novel AMP-nanomedicines, i.e., AMPs entrapped in nanoparticles, has the potential to ameliorate these clinical problems. The authors investigated two novel nanomedicines based on AA139, an AMP currently in development for the treatment of multidrug-resistant Gram-negative infections. AA139 was entrapped in polymeric nanoparticles (PNPs) or lipid-core micelles (MCLs). The antimicrobial activity of AA139-PNP and AA139-MCL was determined in vitro The biodistribution and limiting doses of AA139-nanomedicines were determined in uninfected rats via endotracheal aerosolization. The early bacterial killing activity of the AA139-nanomedicines in infected lungs was assessed in a rat model of pneumonia-septicemia caused by extended-spectrum ß-lactamase-producing Klebsiella pneumoniae In this model, the therapeutic efficacy was determined by once-daily (q24h) administration over 10 days. Both AA139-nanomedicines showed equivalent in vitro antimicrobial activities (similar to free AA139). In uninfected rats, they exhibited longer residence times in the lungs than free AA139 (∼20% longer for AA139-PNP and ∼80% longer for AA139-MCL), as well as reduced toxicity, enabling a higher limiting dose. In rats with pneumonia-septicemia, both AA139-nanomedicines showed significantly improved therapeutic efficacy in terms of an extended rat survival time, although survival of all rats was not achieved. These results demonstrate potential advantages that can be achieved using AMP-nanomedicines. AA139-PNP and AA139-MCL may be promising novel therapeutic agents for the treatment of patients suffering from multidrug-resistant Gram-negative pneumonia-septicemia.

The B & B approach: Ball-milling conjugation of dextran with phenylboronic acid (PBA)-functionalized BODIPY.

Mechanochemistry is an emerging and reliable alternative to conventional solution (batch) synthesis of complex molecules under green and solvent-free conditions. In this regard, we report here on the conjugation of a dextran polysaccharide with a fluorescent probe, a phenylboronic acid (PBA)-functionalized boron dipyrromethene (BODIPY) applying the ball milling approach. The ball milling formation of boron esters between PBA BODIPY and dextran proved to be more efficient in terms of reaction time, amount of reactants, and labelling degree compared to the corresponding solution-based synthetic route. PBA-BODIPY dextran assembles into nanoparticles of around 200 nm by hydrophobic interactions. The resulting PBA-BODIPY dextran nanoparticles retain an apolar interior as proved by pyrene fluorescence, suitable for the encapsulation of hydrophobic drugs with high biocompatibility while remaining fluorescent.

Loading dendritic cells with gold nanoparticles (GNPs) bearing HIV-peptides and mannosides enhance HIV-specific T cell responses.

Gold nanoparticles (GNPs) decorated with glycans ameliorate dendritic cells (DC) uptake, antigen-presentation and T-cells cross-talk, which are important aspects in vaccine design. GNPs allow for high antigen loading, DC targeting, lack of toxicity and are straightforward prepared and easy to handle. The present study aimed to assess the capacity of DC to process and present HIV-1-peptides loaded onto GNPs bearing high-mannoside-type oligosaccharides (P1@HM) to autologous T-cells from HIV-1 patients. The results showed that P1@HM increased HIV-specific CD4+ and CD8+ T-cell proliferation and induced highly functional cytokine secretion compared with HIV-peptides alone. P1@HM elicits a highly efficient secretion of pro-TH1 cytokines and chemokines, a moderate production of pro-TH2 and significant higher secretion of pro-inflammatory cytokines such as TNF-α and IL-1ß. Thus, co-delivery of HIV-1 antigens and HM by GNPs is an excellent vaccine delivery system inducing HIV-specific cellular immune responses in HIV+ patients, being a promising approach to improve anti-HIV-1 vaccines.

Functional Single-Chain Polymer Nanoparticles: Targeting and Imaging Pancreatic Tumors in Vivo.

The development of tools for the early diagnosis of pancreatic adenocarcinoma is an urgent need in order to increase treatment success rate and reduce patient mortality. Here, we present a modular nanosystem platform integrating soft nanoparticles with a targeting peptide and an active imaging agent for diagnostics. Biocompatible single-chain polymer nanoparticles (SCPNs) based on poly(methacrylic acid) were prepared and functionalized with the somatostatin analogue PTR86 as the targeting moiety, since somatostatin receptors are overexpressed in pancreatic cancer. The gamma emitter 67Ga was incorporated by chelation and allowed in vivo investigation of the pharmacokinetic properties of the nanoparticles using single photon emission computerized tomography (SPECT). The resulting engineered nanosystem was tested in a xenograph mouse model of human pancreatic adenocarcinoma. Imaging results demonstrate that accumulation of targeted SCPNs in the tumor is higher than that observed for nontargeted nanoparticles due to improved retention in this tissue.

A quantitative study of the intracellular dynamics of fluorescently labelled glyco-gold nanoparticles via fluorescence correlation spectroscopy.

The dynamic behaviour of gold nanoparticles functionalised with glucose (Glc-Au NPs) has been studied here by means of fluorescence correlation spectroscopy (FCS). Meaningful data on the state of aggregation and dynamics of Glc-Au NPs fluorescently-labelled with HiLyte Fluor647 (Glc-Au-Hi NPs) in the intracellular environment were obtained. Moreover, the work presented here shows that FCS can be used to visualise the presence of single NPs or NP aggregates following uptake and to estimate, locally, NP concentrations within the cell. FCS measurements become possible after applying a "prebleaching" methodology, when the immobile NP fraction has been effectively removed and thus significant FCS data has been recorded. In this study, Glc-Au-Hi NPs have been incubated with HepG2 cells and their diffusion time in the intracellular environment has been measured and compared with their diffusion value in water and cell media.

A new ex vivo method to evaluate the performance of candidate MRI contrast agents: a proof-of-concept study.

BACKGROUND: Magnetic resonance imaging (MRI) plays an important role in tumor detection/diagnosis. The use of exogenous contrast agents (CAs) helps to improve the discrimination between lesion and neighbouring tissue, but most of the currently available CAs are non-specific. Assessing the performance of new, selective CAs requires exhaustive assays and large amounts of material. Accordingly, in a preliminary screening of new CAs, it is important to choose candidate compounds with good potential for in vivo efficiency. This screening method should reproduce as close as possible the in vivo environment. In this sense, a fast and reliable method to select the best candidate CAs for in vivo studies would minimize time and investment cost, and would benefit the development of better CAs. RESULTS: The post-mortem ex vivo relative contrast enhancement (RCE) was evaluated as a method to screen different types of CAs, including paramagnetic and superparamagnetic agents. In detail, sugar/gadolinium-loaded gold nanoparticles (Gd-GNPs) and iron nanoparticles (SPIONs) were tested. Our results indicate that the post-mortem ex vivo RCE of evaluated CAs, did not correlate well with their respective in vitro relaxivities. The results obtained with different Gd-GNPs suggest that the linker length of the sugar conjugate could modulate the interactions with cellular receptors and therefore the relaxivity value. A paramagnetic CA (GNP (E_2)), which performed best among a series of Gd-GNPs, was evaluated both ex vivo and in vivo. The ex vivo RCE was slightly worst than gadoterate meglumine (201.9 ± 9.3% versus 237 ± 14%, respectively), while the in vivo RCE, measured at the time-to-maximum enhancement for both compounds, pointed to GNP E_2 being a better CA in vivo than gadoterate meglumine. This is suggested to be related to the nanoparticule characteristics of the evaluated GNP. CONCLUSION: We have developed a simple, cost-effective relatively high-throughput method for selecting CAs for in vivo experiments. This method requires approximately 800 times less quantity of material than the amount used for in vivo administrations.

Glyconanoparticles as multifunctional and multimodal carbohydrate systems.

The quest for the construction of multivalent carbohydrate systems, with precise geometries that are highly efficient in interacting with carbohydrate binding proteins, has been a goal of synthetic chemists since the discovery of the multivalent nature of carbohydrate-mediated interactions. However, the control of the spatial and topological requirements for these systems is still a challenge. Glyconanoparticles (GNPs) are sugar-coated gold, iron oxide or semiconductor nanoparticles with defined thiol-ending glycosides that combine the multivalent presentation of carbohydrates (glycoclusters) with the special chemico-physical properties of the nano-sized metallic core. The possibility of attaching different types of carbohydrates and other molecules (such as luminescent probes, peptides, and magnetic chelates) onto the same gold nanoparticle in a controlled way (multifunctional GNPs), as well as modifying the core in order to obtain glyconanoparticles with magnetic or fluorescence properties (multimodal GNPs) makes this multivalent glyco-scaffold suitable for carrying out studies on carbohydrate-mediated interactions and applications in molecular imaging. In this review, we focus mainly on the rational design of glyconanoparticles as scaffolds for combining different ligands and survey the most recent examples of glyconanoparticles as both multivalent carbohydrate systems and probes for molecular imaging.

Multivalent glycoconjugates as anti-pathogenic agents.

Multivalency plays a major role in biological processes and particularly in the relationship between pathogenic microorganisms and their host that involves protein-glycan recognition. These interactions occur during the first steps of infection, for specific recognition between host and bacteria, but also at different stages of the immune response. The search for high-affinity ligands for studying such interactions involves the combination of carbohydrate head groups with different scaffolds and linkers generating multivalent glycocompounds with controlled spatial and topology parameters. By interfering with pathogen adhesion, such glycocompounds including glycopolymers, glycoclusters, glycodendrimers and glyconanoparticles have the potential to improve or replace antibiotic treatments that are now subverted by resistance. Multivalent glycoconjugates have also been used for stimulating the innate and adaptive immune systems, for example with carbohydrate-based vaccines. Bacteria present on their surfaces natural multivalent glycoconjugates such as lipopolysaccharides and S-layers that can also be exploited or targeted in anti-infectious strategies.

Glycosystems in nanotechnology: Gold glyconanoparticles as carrier for anti-HIV prodrugs.

The therapeutic approach for the treatment of HIV infection is based on the highly active antiretroviral therapy (HAART), a cocktail of antiretroviral drugs. Notwithstanding HAART has shown different drawbacks like toxic side effects and the emergence of viral multidrug resistance. Nanotechnology offers new tools to improve HIV drug treatment and prevention. In this scenario, gold nanoparticles are an interesting chemical tool to design and prepare smart and efficient drug-delivery systems. Here we describe the preparation and antiviral activity of carbohydrate-coated gold nanoparticles loaded with anti-HIV prodrug candidates. The nucleoside reverse transcriptase inhibitors abacavir and lamivudine have been converted to the corresponding thiol-ending ester derivatives and then conjugated to ~3 nm glucose-coated gold nanoparticles by means of "thiol-for-thiol" ligand place exchange reactions. The drugs-containing glyconanoparticles were characterized and the pH-mediated release of the drug from the nanoparticle has been determined. The antiviral activity was tested by evaluating the replication of NL4-3 HIV in TZM-bl infected cells. The proof-of-principle presented in this work aims to introduce gold glyconanoparticles as a new multifunctional drug-delivery system in the therapy against HIV.

Influence of ligand presentation density on the molecular recognition of mannose-functionalised glyconanoparticles by bacterial lectin BC2L-A.

Polyvalent carbohydrate-protein interactions play a key role in bio- and pathological processes, including cell-cell communication and pathogen invasion. In order to study, control and manipulate these interactions gold nanoparticles have been employed as a 3D scaffold, presenting carbohydrate ligands in a multivalent fashion for use as high affinity binding partners and a model system for oligosaccharide presentation at biomacromolecular surfaces. In this study, the binding of a series of mannose-functionalised gold nanoparticles to the dimeric BC2L-A lectin from Burkholderia cenocepacia has been evaluated. BC2L-A is known to exhibit a high specificity for (oligo)mannosides. Due to the unique structure and binding nature of this lectin, it provides a useful tool to study (oligo)saccharides presented on multivalent scaffolds. Surface plasmon resonance and isothermal titration calorimetric assays were used to investigate the effect of ligand presentation density towards binding to the bacterial lectin. We show how a combination of structural complementarities between ligand presentation and lectin architecture and statistical re-binding effects are important for increasing the avidity of multivalent ligands for recognition by their protein receptors; further demonstrating the application of glyconanotechnology towards fundamental glycobiology research as well as a potential towards biomedical diagnostics and therapeutic treatments.

Naturalized Dyes: A New Opportunity for the Wood Coloring.

Naturalized dyes (NDs) are innovative and eco-friendly synthetic compounds in which a chromophore is covalently linked to a natural sugar (e.g., lactose). The sugar moiety confers water-solubility and biocompatibility to the dye molecule as a whole. NDs have demonstrated potential application in dyeing textiles and leather. The purpose of this work was to demonstrate that selected NDs can be also applied to dye wood. To that aim, two NDs were tested to color beech and poplar wood. The NDs were applied as a simple aqueous solution or mixed with a waterborne, biogenic staining agent (commercially available Gemma U50). Moreover, the effect of the application of a biogenic waterborne top coat (commercially available Resina Plus U49) was also studied. Different methods were tested to investigate the potential application of these NDs to wood. The dyeing behavior was analyzed in terms of penetration into the substrate, covering capacity and color homogeneity through macro- and microscopic observations and colorimetric measurements. The color fastness to water washout and the color stability to light, in particular by exposing the wooden samples to artificial aging (UV radiations in a Solar Box), were also investigated. The NDs, when used as water solutions, were able to afford a homogeneous coating and a pleasant appearance on the wood surface, as well as a good color fastness to washout with water. Dissolving the dyes in the stain or applying the top coat generally resulted in even better color fastness to washout. However, all the application methods tested showed limited resistance to fading in the Solar Box, which therefore remains a drawback for this type of product.

Simple engineering of hybrid cellulose nanocrystal-gold nanoparticles results in a functional glyconanomaterial with biomolecular recognition properties.

Cellulose nanocrystal and gold nanoparticles are assembled, in a unique way, to yield a novel modular glyconanomaterial whose surface is then easily engineered with one or two different headgroups, by exploiting a robust click chemistry route. We demonstrate the potential of this approach by conjugating monosaccharide headgroups to the glyconanomaterial and show that the sugars retain their binding capability to C-type lectin receptors, as also directly visualized by cryo-TEM.

STD NMR study of the interactions between antibody 2G12 and synthetic oligomannosides that mimic selected branches of gp120 glycans.

The human immunodeficiency virus type-1 (HIV-1) is able to shield immunogenic peptide epitopes on its envelope spike (a trimer of two glycoproteins, gp120 and gp41) by presenting numerous host-derived N-linked glycans. Nevertheless, broadly neutralizing antibodies against gp120 and gp41 have been isolated from HIV-1-infected patients and provide protection against viral challenge in animal models. Among these, the monoclonal antibody 2G12 binds to clusters of high-mannose-type glycans that are present on the surface of gp120. These types of glycans have thus been envisaged as target structures for the development of synthetic agents capable of eliciting 2G12-like antibodies. High-resolution structural studies of 2G12 and chemically defined glycan-type ligands, including crystallographic data, have been performed to gain an insight into this interaction. Further studies are still required to design a carbohydrate-based vaccine for HIV. Our previous NMR studies highlighted different recognition modes of two branched synthetic oligosaccharides, a penta- and a heptamannoside, by 2G12 in solution. In order to clarify the underlying structural reasons for such different behaviors, we have herein "dissected" the branches into the linear tri- and tetra- oligomannosides by chemical synthesis and studied their interactions with 2G12 in solution by saturation transfer difference (STD) NMR spectroscopy. The results confirm the distinct preferences of 2G12 for the studied branches and afford explanations for the observed differences. This study provides important structural information for further ligand optimizations. Possible effects of structural modifications on the solvent-exposed end of the ligands are also discussed.

Multivalent gold glycoclusters: high affinity molecular recognition by bacterial lectin PA-IL.

Multivalent protein-carbohydrate interactions are involved in the initial stages of many fundamental biological and pathological processes through lectin-carbohydrate binding. The design of high affinity ligands is therefore necessary to study, inhibit and control the processes governed through carbohydrate recognition by their lectin receptors. Carbohydrate-functionalised gold nanoclusters (glyconanoparticles, GNPs) show promising potential as multivalent tools for studies in fundamental glycobiology research as well as biomedical applications. Here we present the synthesis and characterisation of galactose functionalised GNPs and their effectiveness as binding partners for PA-IL lectin from Pseudomonas aeruginosa. Interactions were evaluated by hemagglutination inhibition (HIA), surface plasmon resonance (SPR) and isothermal titration calorimetry (ITC) assays. Results show that the gold nanoparticle platform displays a significant cluster glycoside effect for presenting carbohydrate ligands with almost a 3000-fold increase in binding compared with a monovalent reference probe in free solution. The most effective GNP exhibited a dissociation constant (K(d)) of 50 nM per monosaccharide, the most effective ligand of PA-IL measured to date; another demonstration of the potential of glyco-nanotechnology towards multivalent tools and potent anti-adhesives for the prevention of pathogen invasion. The influence of ligand presentation density on their recognition by protein receptors is also demonstrated.

Dissecting the carbohydrate specificity of the anti-HIV-1 2G12 antibody by single-molecule force spectroscopy.

Broadly neutralizing anti-HIV-1 monoclonal antibody 2G12 exclusively targets a conserved cluster of high-mannose oligosaccharides present on outer viral envelope glycoprotein gp120. This characteristic makes the otherwise immunogenically "silent" glycan shield of gp120 a tempting target for drug and vaccine design. However, immune responses against carbohydrate-based mimics of gp120 have failed to provide immunization against HIV-1 infection, highlighting the need to understand the molecular events that determine immunogenicity better. In this work, the unbinding kinetics of the gp120-2G12 (k(0) = 0.002 ± 0.09 s(-1), x(++) = 1.5 ± 1.2 nm), Man(4)-2G12 (k(0) = 0.35 ± 0.32 s(-1), x(++) = 0.6 ± 0.2 nm), and Man(5)-2G12 interactions were measured by single-molecule force spectroscopy. To our knowledge, this is the first single-molecule study aimed at dissecting the carbohydrate-antibody recognition of the gp120-2G12 interaction. We were able to confirm crystallographic models that show both the binding of the linear Man(4) arm to 2G12 and also the multivalent gp120 glycan binding to 2G12. These results demonstrate that single-molecule force spectroscopy can be successfully used to dissect the molecular mechanisms underlying immunity.

Low-generation dendrimers with a calixarene core and based on a chiral C(2)-symmetric pyrrolidine as iminosugar mimics.

The preparation of low-generation dendrimers based on a simple calix[4]arene scaffold by insertion of the iminosugar-analogue C(2)-symmetric 3,4-dihydroxypyrrolidine is described. This methodology allows a rapid incorporation of a considerable number of iminosugar-like moieties in a reduced volume and in a well-defined geometry. The inclusion of alkali-metal ions (sodium and potassium) in the polar cavity defined by the acetamide moieties at the lower rim of the calixarene was demonstrated, which allows also the rigidification of the dendrimer structure and the iminosugar presentation in the clusters. The combination of the supramolecular properties of calixarenes with the advantage of a dendrimeric presentation of repetitive units opens up the possibility of generating well-defined multivalent and multifaceted systems with more complex and/or biologically relevant iminosugars.

Neutralization of ionic interactions by dextran-based single-chain nanoparticles improves tobramycin diffusion into a mature biofilm.

The extracellular matrix protects biofilm cells by reducing diffusion of antimicrobials. Tobramycin is an antibiotic used extensively to treat P. aeruginosa biofilms, but it is sequestered in the biofilm periphery by the extracellular negative charge matrix and loses its efficacy significantly. Dispersal of the biofilm extracellular matrix with enzymes such as DNase I is another promising therapy that enhances antibiotic diffusion into the biofilm. Here, we combine the charge neutralization of tobramycin provided by dextran-based single-chain polymer nanoparticles (SCPNs) together with DNase I to break the biofilm matrix. Our study demonstrates that the SCPNs improve the activity of tobramycin and DNase I by neutralizing the ionic interactions that keep this antibiotic in the biofilm periphery. Moreover, the detailed effects and interactions of nanoformulations with extracellular matrix components were revealed through time-lapse imaging of the P. aeruginosa biofilms by laser scanning confocal microscopy with specific labeling of the different biofilm components.