Hyaluronan-Metal Gold Nanoparticle Hybrids for Targeted Tumor Cell Therapy.

In this study, a novel multifunctional nanoplatform based on core-shell nanoparticles of spherical gold nanoparticles (AuNPs) capped with low and high molecular weight (200 and 700 kDa) hyaluronic acid (HA), was assembled via a green, one-pot redox synthesis method at room temperature. A multitechnique characterization approach by UV-visible spectroscopy, dynamic light scattering and atomic force microscopy pointed to the effective 'surface decoration' of the gold nanoparticles by HA, resulting in different grafting densities of the biopolymer chains at the surface of the metal nanoparticle, which in turn affected the physicochemical properties of the nanoparticles. Specifically, the spectral features of the gold plasmonic peak (and the related calculated optical size), the hydrodynamic diameter and the nanoparticle stability were found to depend on the molecular weight of the HA. The CD44-targeting capability of HA-functionalized gold nanoparticles was tested in terms of antibacterial activity and cytotoxicity. An enhanced inhibitory activity against both Gram-negative Escherichia coli and Gram-positive Staphylococcus aureus was found, with a HA molecular weight (MW)-dependent trend for the HA-capped AuNPs compared to the bare, glucose-capped AuNPs. Cell viability assays performed on two CD44-positive cell models, namely normal human umbilical vein endothelial (HUVEC) and prostate tumor (PC-3) cells, in comparison with neuroblastoma cells (SH-SY5Y), which do not express the CD44 receptor, demonstrated an increased cytotoxicity in neuroblastoma compared to prostate cancer cells upon the cellular treatments by HA-AuNP compared to the bare AuNP, but a receptor-dependent perturbation effect on cytoskeleton actin and lysosomal organelles, as detected by confocal microscopy. These results highlighted the promising potentialities of the HA-decorated gold nanoparticles for selective cytotoxicity in cancer therapy. Confocal microscopy imaging of the two human tumor cell models demonstrated a membrane-confined uptake of HA-capped AuNP in the cancer cells that express CD44 receptors and the different perturbation effects related to molecular weight of HA wrapping the metallic core of the plasmonic nanoparticles on cellular organelles and membrane mobility.

Collagenase-assisted wound bed preparation: An in vitro comparison between Vibrio alginolyticus and Clostridium histolyticum collagenases on substrate specificity.

Bacterial collagenase from the aerobic non-pathogenic Vibrio alginolyticus chemovar iophagus is an extracellular metalloproteinase. This collagenase preparation is obtained through a fermentation process and is purified chromatographically, resulting in a highly purified 82-kDa single-band protein that does not contain non-specific proteases or other microbial impurities. V. alginolyticus collagenase was added to a hyaluronan (HA)-based device to develop a novel debriding agent to improve the treatment of ulcers, necrotic burns, and decubitus in the initial phase of wound bed preparation. In this study, an in vitro biochemical characterisation of V. alginolyticus collagenase versus a commercial preparation from a Clostridium histolyticum strain on various dermal extracellular matrix (ECM) substrates was performed. V. alginolyticus collagenase demonstrated its ability to carry out the enzymatic cleavage of the substrate, allowing a selective removal of necrotic tissues while sparing healthy tissue, as reported in clinical studies and through routine clinical experience. in vitro tests under physiological conditions (pH, presence of Ca++, etc.) have demonstrated that V. alginolyticus collagenase exhibits very poor/limited non-specific proteolytic activity, whereas the collagenase preparation from C. histolyticum is highly active both on collagen and on non-collagenic substrates. This finding implies that while the V. alginolyticus enzyme is fully active on the collagen filaments that anchor the necrotic tissue to the wound bed, it does not degrade other minor, but structurally important, components of the dermal ECM. This feature could explain why collagenase preparation from V. alginolyticus has been reported to be much gentler on perilesional, healthy skin.

A new potential spreading factor: Streptomyces koganeiensis hyaluronidase. A comparative study with bovine testes hyaluronidase and recombinant human hyaluronidase of the HA degradation in ECM.

BACKGROUND: Recombinant human hyaluronidase has been used in the interstitial matrix to promote the dispersion of therapeutics. The production and isolation of an extracellular hyaluronidase from Streptomyces koganeiensis (rHyal_Sk) has recently been described. METHODS: The specificity of rHyal_Sk has been assessed against heparan sulfate, chondroitin sulfates and sulfated HAs. The oligomers generated by HA degradation have been investigated by MALDI-TOF MS analysis. rHyal_Sk has been compared with BTH and PH20 in vitro, against cross-linked HA (ACP) and HA-aggrecan complex, and in vivo, by means of a diffusion assay in nude mice. RESULTS: Depolymerization of HA by rHyal_Sk gave tetra-, hexa- and octasaccharides in high yields. The reaction mechanism and the high HA specificity were demonstrated. The in vivo diffusion assay, supported by the in vitro tests, evidenced an initially enhanced enzymatic activity of rHyal_Sk compared to BTH and PH20. CONCLUSIONS: rHyal_Sk, compared to BTH and PH20, showed higher substrate specificity and no inhibition from GAGs sulfate, together with a superior performance for HA depolymerization in ECM. As better predictive tests for the in vivo activity of hyaluronidase we developed two assays based on the degradation of ACP or of the HA-aggrecan complex. GENERAL SIGNIFICANCE: rHyal_Sk is a new potential spreading factor for intradermal drug administration. Hyaluronidases of distinct classes, that show equivalent activities in a common turbidimetric assay, could have different potencies and dose-efficacies in vivo which influences the therapeutic effect. The new proposed in vitro tests are designed to obtain a predictive characterization of the enzyme activity in vivo.

Copper(II) Complexes with Carnosine Conjugates of Hyaluronic Acids at Different Dipeptide Loading Percentages Behave as Multiple SOD Mimics and Stimulate Nrf2 Translocation and Antioxidant Response in In Vitro Inflammatory Model.

A series of copper(II) complexes with the formula [Cu2+Hy(x)Car%] varying the molecular weight (MW) of Hyaluronic acid (Hy, x = 200 or 700 kDa) conjugated with carnosine (Car) present at different loading were synthesized and characterized via different spectroscopic techniques. The metal complexes behaved as Cu, Zn-superoxide dismutase (SOD1) mimics and showed some of the most efficient reaction rate values produced using a synthetic and water-soluble copper(II)-based SOD mimic reported to date. The increase in the percentage of Car moieties parallels the enhancement of the I50 value determined via the indirect method of Fridovich. The presence of the non-functionalized Hy OH groups favors the scavenger activity of the copper(II) complexes with HyCar, recalling similar behavior previously found for the copper(II) complexes with Car conjugated using ß-cyclodextrin or trehalose. In keeping with the new abilities of SOD1 to activate protective agents against oxidative stress in rheumatoid arthritis and osteoarthritis diseases, Cu2+ interaction with HyCar promotes the nuclear translocation of erythroid 2-related factor that regulates the expressions of target genes, including Heme-Oxigenase-1, thus stimulating an antioxidant response in osteoblasts subjected to an inflammatory/oxidative insult.

Synergistic Effect of L-Carnosine and Hyaluronic Acid in Their Covalent Conjugates on the Antioxidant Abilities and the Mutual Defense against Enzymatic Degradation.

Hyaluronic acid (Hy) is a natural linear polymer that is widely distributed in different organisms, especially in the articular cartilage and the synovial fluid. During tissue injury due to oxidative stress, Hy plays an important protective role. All the beneficial properties of Hy make the polymer attractive for many biomedical uses; however, the low stability and short biological half-life limit Hy application. To overcome these problems, the addition of small antioxidant molecules to Hy solution has been employed to protect the molecular integrity of Hy or delay its degradation. Carnosine (ß-alanyl-L-histidine, Car) protects cells from the damage due to the reactive species derived from oxygen (ROS), nitrogen (RNS) or carbonyl groups (RCS). Car inhibits the degradation of hyaluronan induced by free radical processes in vitro but, like Hy, the potential protective action of Car is drastically hampered by the enzymatic hydrolysis in vivo. Recently, we conjugated Hy to Car and the derivatives (HyCar) showed protective effects in experimental models of osteoarthritis and rheumatoid arthritis in vivo. Here we report the antioxidant activity exerted by HyCar against ROS, RNS and RCS. Moreover, we tested if the covalent conjugation between Hy and Car inhibits the enzymatic hydrolysis of the polymer and the dipeptide backbone. We found that the antioxidant properties and the resistance to the enzymatic hydrolysis of Hy and Car are greatly improved by the conjugation.

Characterization of Bifidobacterium asteroides Isolates.

Bifidobacteria have long been recognized as bacteria with probiotic and therapeutic features. The aim of this work is to characterize the Bifidobacterium asteroides BA15 and BA17 strains, isolated from honeybee gut, to evaluate its safety for human use. An in-depth assessment was carried out on safety properties (antibiotic resistance profiling, ß-hemolytic, DNase and gelatinase activities and virulence factor presence) and other properties (antimicrobial activity, auto-aggregation, co-aggregation and hydrophobicity). Based on phenotypic and genotypic characterization, both strains satisfied all the safety requirements. More specifically, genome analysis showed the absence of genes encoding for glycopeptide (vanA, vanB, vanC-1, vanC-2, vanD, vanE, vanG), resistance to tetracycline (tetM, tetL and tetO) and virulence genes (asa1, gelE, cylA, esp, hyl).

The Role of Bifidobacteria in Predictive and Preventive Medicine: A Focus on Eczema and Hypercholesterolemia.

Bifidobacteria colonize the human gastrointestinal tract early on in life, their interaction with the host starting soon after birth. The health benefits are strain specific and could be due to the produced polysaccharides. The consumption of probiotics may prevent obesity, irritable bowel syndrome, eczema or atopic dermatitis, and asthma. Non-replicative strains of Bifidobacterium longum (NCC3001 and NCC2705) promote the differentiation of normal human epidermal keratinocytes (NHEKs), inducing a high expression of differentiation markers (keratin -KRT1-, and transglutaminase -TGM1-) and pro-regeneration markers (cathepsins), including ß-defensin-1, which plays an important role in modulating the cutaneous immune response. Strains belonging to the genera Bifidobacterium and Lactobacillus can increase tight-junction proteins in NHEKs and enhance barrier function. Bifidobacteria and lactobacilli may be used as prophylactic or therapeutic agents towards enteric pathogens, antibiotic-associated diarrhea, lactose intolerance, ulcerative colitis, irritable bowel syndrome, colorectal cancer, cholesterol reduction, and control of obesity and metabolic disorders. Bifidobacterium bifidum showed an in vitro capability of lowering cholesterol levels thanks to its absorption into the bacterial membrane. Several strains of the species Lactobacillus acidophilus, L. delbrueckii subsp. bulgaricus, L. casei, and L. gasseri led to a reduced amount of serum cholesterol due to their ability to assimilate cholesterol (in vitro). Lactococcus lactis KF147 and Lactobacillus plantarum Lp81 have also been shown to reduce cholesterol levels by 12%. Clarifying the specific health mechanisms of Bifidobacterium and Lactobacillus strains in preventing high-cost pathologies could be useful for delineating effective guidelines for the treatment of infants and adults.

Hyaluronan-carnosine conjugates inhibit Aß aggregation and toxicity.

Alzheimer's disease is the most common neurodegenerative disorder. Finding a pharmacological approach that cures and/or prevents the onset of this devastating disease represents an important challenge for researchers. According to the amyloid cascade hypothesis, increases in extracellular amyloid-ß (Aß) levels give rise to different aggregated species, such as protofibrils, fibrils and oligomers, with oligomers being the more toxic species for cells. Many efforts have recently been focused on multi-target ligands to address the multiple events that occur concurrently with toxic aggregation at the onset of the disease. Moreover, investigating the effect of endogenous compounds or a combination thereof is a promising approach to prevent the side effects of entirely synthetic drugs. In this work, we report the synthesis, structural characterization and Aß antiaggregant ability of new derivatives of hyaluronic acid (Hy, 200 and 700 kDa) functionalized with carnosine (Car), a multi-functional natural dipeptide. The bioactive substances (HyCar) inhibit the formation of amyloid-type aggregates of Aß42 more than the parent compounds; this effect is proportional to Car loading. Furthermore, the HyCar derivatives are able to dissolve the amyloid fibrils and to reduce Aß-induced toxicity in vitro. The enzymatic degradation of Aß is also affected by the interaction with HyCar.

Protective effect of a new hyaluronic acid -carnosine conjugate on the modulation of the inflammatory response in mice subjected to collagen-induced arthritis.

Several studies demonstrated the pharmacological actions of carnosine as well as hyaluronic acid (HA) during joint inflammation. In that regard, the aim of this study was to investigate the protective effect of a new HA -Carnosine conjugate (FidHycarn) on the modulation of the inflammatory response in mice subjected to collagen-induced arthritis (CIA). CIA was induced by two intradermal injections of 100 µl of an emulsion of collagen (CII) and complete Freund's adjuvant (CFA) at the base of the tail on day 0 and 21. At 35 day post CIA induction, the animals were sacrificed. CII injection caused erythema and edema in the hind paws, histological alterations with erosion of the joint cartilage as well as behavioral changes. Oral treatment with FidHycarn starting at the onset of arthritis (day 25) ameliorated the clinical signs, improved behavioral deficits as well as decreased histological and radiographic alterations. The degree of oxidative damage evaluated by inducible nitric oxide synthase (iNOS), nitrotyrosine, poly-ADP-ribose (PAR) expressions and malondialdehyde (MDA) levels, was also significantly reduced in Carnosine+HA association and FidHycarn treated mice. Moreover, the levels of proinflammatory cytokines and chemokines and cyclo-oxygenase COX-2 enzyme were also more significantly reduced by Carnosine+HA and FidHycarn compared to carnosine alone. However, interestingly, in some cases, the effects of FidHycarn were more important than Carnosine+HA association and not statistically different to methotrexate (MTX) used as positive control. Thus, the conjugation of Carnosine with HA (FidHycarn) could represent an interesting therapeutic strategy to combat arthritis disorders.

In Vivo Evaluation of a New Recombinant Hyaluronidase to Improve Gene Electro-Transfer Protocols for DNA-Based Drug Delivery against Cancer.

Cancer vaccines based on plasmid DNA represent a good therapeutic perspective, despite their low potency. Animal-derived hyaluronidases (Hyals) are employed in oncological clinical practice. Hyal has been also demonstrated to be a good enhancer of intramuscular Gene Electro-Transfer (GET) efficiency in anti-cancer preclinical protocols, with increased transfected cells and higher expression of the encoded genes. Nevertheless, the use of animal-derived Hyals results limited respect to their potentialities, since such preparations could be affected by low purity, variable potency and uncertain safety. To improve the delivery of intramuscular GET-based protocols in mouse, we investigated a new recombinant Hyal, the rHyal-sk, to assess in vivo safety and activity of this treatment at cellular and biochemical levels. We evaluated the cellular events and the inflammation chemical mediators involved at different time points after rHyal-sk administration plus GET. Our results demonstrated the in vivo safety and efficacy of rHyal-sk when injected once intramuscularly in association with GET, with no toxicity, good plasmid in-take ability, useful inflammatory response activation, and low immunogenicity. Following these findings, we would recommend the use of the new rHyal-sk for the delivery of DNA-based vaccines and immunotherapy, as well as into clinical practice, for tumor disease treatments.

Identification and characterization of a bacterial hyaluronidase and its production in recombinant form.

Hyaluronidases (Hyals) are broadly used in medical applications to facilitate the dispersion and/or absorption of fluids or medications. This study reports the isolation, cloning, and industrial-scale recombinant production, purification and full characterization, including X-ray structure determination at 1.45 Å, of an extracellular Hyal from the nonpathogenic bacterium Streptomyces koganeiensis. The recombinant S. koganeiensis Hyal (rHyal_Sk) has a novel bacterial catalytic domain with high enzymatic activity, compared with commercially available Hyals, and is more thermostable and presents higher proteolytic resistance, with activity over a broad pH range. Moreover, rHyal_Sk exhibits remarkable substrate specificity for hyaluronic acid (HA) and poses no risk of animal cross-infection.