Ocular antibacterial chitosan-maleic acid hydrogels: In vitro and in vivo studies for a promising approach with enhanced mucoadhesion.

The aim was to design and evaluate a chitosan-based conjugate providing high mucoadhesiveness and antibacterial activity for ocular infections treatment. Chitosan was conjugated with maleic acid via amide bond formation and infrared spectroscopy. Furthermore, 2,4,6-Trinitrobenzene sulfonic acid (TNBS) allowed characterization and quantification of conjugated groups, respectively. Biocompatibility was tested via hemolysis assay and Hen's Egg-Chorioallantoic membrane test. Characterization of the pH and osmolarity of hydrogels was followed by mucoadhesion assessment utilizing rheology. In addition, antibacterial studies were carried out towards Escherichia coli by broth microdilution test and agar-disk diffusion assay. In vivo studies were carried out following the already established Draize test and determining pharmacokinetic profile of dexamethasone in aqueous humour. The conjugate exhibited a degree of modification of 50.05 % and no toxicity or irritability. Moreover, mucoadhesive properties were enhanced in 2.68-fold and 1.81-fold for elastic and viscous modulus, respectively. Furthermore, rheological synergism revealed the presence of a gel-like structure. Additionally, broth microdilution and agar disk diffusion studies exhibited enhancement in antibacterial activity. Finally, in vivo studies manifested that hydrogels were highly tolerated, evidencing promising characteristics of the developed conjugate. The conjugate presented promising antimicrobial, long lasting mucoadhesive features and highly improved pharmacokinetics, leading to a revolutionizing approach in the treatment of ocular bacterial infections.

Polymeric Nanoformulation of Zoledronic Acid Rescues Osteoblasts from the Harmful Effect of its Native Form: An In Vitro Investigation of Cytotoxic Potential on Osteoblasts and Osteosarcoma Cells.

Osteosarcoma (OS) is a malignant tumor, fatal for pediatric patients who do not respond to chemotherapy, alternative therapies and drugs can provide better outcomes. Zoledronic acid (Zol) belonging to the class of bisphosphonates (BPs) has a direct antitumor ability to prevent Ras GTPases modification and stimulate apoptosis. Despite advances in maintaining balance in skeletal events and direct anticancer properties, Zol causes cytotoxicity to normal healthy pre-osteoblast cells, hampering mineralization and differentiation. The study reports the preparation and evaluation of a nanoformulation that can diminish the existing drawbacks of native Zol. The cytotoxic effect is evaluated on bone cancer cells and healthy bone cells with three different cell lines namely, K7M2 (mouse OS cell line), SaOS2 (human OS cell line), and MC3T3E1 (healthy cell counterpart). It is observed that Zol nanoformulation is uptaken more (95%) in K7M2 whereas in MC3T3E1, the percent population internalizing nanoparticles (NPs) is 45%. Zol has a sustained release of 15% after 96 h from the NP which leads to a rescuing effect on the normal pre-osteoblast cells. In conclusion, it can be stated that Zol nanoformulation can be used as a good platform for a sustained release system with minimum side effects to normal bone cells.

Biopharmaceutical Assessment of Mesh Aerosolised Plasminogen, a Step towards ARDS Treatment.

Acute respiratory distress syndrome (ARDS) is a severe complication of lung injuries, commonly associated with bacterial, fungal and viral infections, including SARS-CoV-2 viral infections. ARDS is strongly correlated with patient mortality and its clinical management is very complex, with no effective treatment presently available. ARDS involves severe respiratory failure, fibrin deposition in both airways and lung parenchyma, with the development of an obstructing hyaline membrane drastically limiting gas exchange. Moreover, hypercoagulation is related to deep lung inflammation, and a pharmacological action toward both aspects is expected to be beneficial. Plasminogen (PLG) is a main component of the fibrinolytic system playing key roles in various inflammation regulatory processes. The inhalation of PLG has been proposed in the form of the off-label administration of an eyedrop solution, namely, a plasminogen-based orphan medicinal product (PLG-OMP), by means of jet nebulisation. Being a protein, PLG is susceptible to partial inactivation under jet nebulisation. The aim of the present work is to demonstrate the efficacy of the mesh nebulisation of PLG-OMP in an in vitro simulation of clinical off-label administration, considering both the enzymatic and immunomodulating activities of PLG. Biopharmaceutical aspects are also investigated to corroborate the feasibility of PLG-OMP administration by inhalation. The nebulisation of the solution was performed using an Aerogen® SoloTM vibrating-mesh nebuliser. Aerosolised PLG showed an optimal in vitro deposition profile, with 90% of the active ingredient impacting the lower portions of a glass impinger. The nebulised PLG remained in its monomeric form, with no alteration of glycoform composition and 94% of enzymatic activity maintenance. Activity loss was observed only when PLG-OMP nebulisation was performed under simulated clinical oxygen administration. In vitro investigations evidenced good penetration of aerosolised PLG through artificial airway mucus, as well as poor permeation across an Air-Liquid Interface model of pulmonary epithelium. The results suggest a good safety profile of inhalable PLG, excluding high systemic absorption but with good mucus diffusion. Most importantly, the aerosolised PLG was capable of reversing the effects of an LPS-activated macrophage RAW 264.7 cell line, demonstrating the immunomodulating activity of PLG in an already induced inflammatory state. All physical, biochemical and biopharmaceutical assessments of mesh aerosolised PLG-OMP provided evidence for its potential off-label administration as a treatment for ARDS patients.

Betaine- and L-Carnitine-Based Ionic Liquids as Solubilising and Stabilising Agents for the Formulation of Antimicrobial Eye Drops Containing Diacerein.

The therapeutic efficacy of topically administered drugs, however powerful, is largely affected by their bioavailability and, thus, ultimately, on their aqueous solubility and stability. The aim of this study was to evaluate the use of ionic liquids (ILs) as functional excipients to solubilise, stabilise, and prolong the ocular residence time of diacerein (DIA) in eye drop formulations. DIA is a poorly soluble and unstable anthraquinone prodrug, rapidly hydrolysed to rhein (Rhe), for the treatment of osteoarthritis. DIA has recently been evaluated as an antimicrobial agent for bacterial keratitis. Two ILs based on natural zwitterionic compounds were investigated: L-carnitine C6 alkyl ester bromide (Carn6), and betaine C6 alkyl ester bromide (Bet6). The stabilising, solubilising, and mucoadhesive properties of ILs were investigated, as well as their cytotoxicity to the murine fibroblast BALB/3T3 clone A31 cell line. Two IL-DIA-based eye drop formulations were prepared, and their efficacy against both Staphylococcus aureus and Pseudomonas aeruginosa was determined. Finally, the eye drops were administered in vivo on New Zealand albino rabbits, testing their tolerability as well as their elimination and degradation kinetics. Both Bet6 and Carn6 have good potential as functional excipients, showing solubilising, stabilising, mucoadhesive, and antimicrobial properties; their in vitro cytotoxicity and in vivo ocular tolerability pave the way for their future use in ophthalmic applications.

Bergamot Essential Oil: A Method for Introducing It in Solid Dosage Forms.

Bergamot essential oil (BEO) possess antimicrobial, antiproliferative, anti-inflammatory, analgesic, neuroprotective, and cardiovascular effects. However, it is rich in volatile compounds, e.g., limonene, that are susceptible to conversion and degradation reactions. The aim of this communication was to prepare a conjugate based on a quaternary ammonium chitosan derivative (QA-Ch) and methyl-ßCD (MCD), coded as BEO/QA-Ch-MCD, to encapsulate BEO in order to stabilize its volatile compounds, eliminate its unpleasant taste, and convert the oil in a solid dosage form. The obtained conjugate, BEO/QA-Ch-MCD, was highly soluble and had a percentage of extract association efficiency (AE %), in terms of polyphenols and limonene contents, of 22.0 ± 0.9 and 21.9 ± 1.2, respectively. Moreover, stability studies under UV stress in simulated gastric fluid showed that BEO/QA-Ch-MCD was more able to protect polyphenols and limonene from degradation compared to free BEO or BEO complexed with MCD (BEO/MCD). The complexation and subsequent lyophilization allowed the transformation of a liquid into a solid dosage form capable of eliminating the unpleasant taste of the orally administered oil and rendering the solid suitable to produce powders, granules, tablets, etc. These solid oral dosage forms, as they come into contact with physiological fluids, could generate nanosized agglomerates able to increase the stability of their active contents and, consequently, their bioavailability.

Jellyfish Polysaccharides for Wound Healing Applications.

Jellyfishes are considered a new potential resource in food, pharmaceutical and biomedical industries. In these latter cases, they are studied as source of active principles but are also exploited to produce marine collagen. In the present work, jellyfish skin polysaccharides (JSP) with glycosaminoglycan (GAG) features were extracted from Rhizostoma pulmo, a main blooming species of Mediterranean Sea, massively augmented by climate leaded "jellyfishication" of the sea. Two main fractions of R. pulmo JSP (RP-JSPs) were isolated and characterized, namely a neutral fraction (RP-JSP1) and a sulphate rich, negatively charged fraction (RP-JSP2). The two fractions have average molecular weights of 121 kDa and 590 kDa, respectively. Their sugar composition was evaluated through LC-MS analysis and the result confirmed the presence of typical GAG saccharides, such as glucose, galactose, glucosamine and galactosamine. Their use as promoters of wound healing was evaluated through in vitro scratch assay on murine fibroblast cell line (BALB/3T3 clone A31) and human keratinocytes (HaCaT). Both RP-JSPs demonstrated an effective confluency rate activity leading to 80% of scratch repair in two days, promoting both cell migration and proliferation. Additionally, RP-JSPs exerted a substantial protection from oxidative stress, resulting in improved viability of treated fibroblasts exposed to H2O2. The isolated GAG-like polysaccharides appear promising as functional component for biomedical skin treatments, as well as for future exploitation as pharmaceutical excipients.

Comparing Metabolomic and Essential Oil Fingerprints of Citrus australasica F. Muell (Finger Lime) Varieties and Their In Vitro Antioxidant Activity.

Comparative chemical analyses among peel and pulp essential oils (EOs) and methanolic extracts of four Citrus australasica varieties (Red, Collette, Pink Ice, and Yellow Sunshine), and the hybrid Faustrime, were performed using GC-MS and UHPLC-DAD-HR-Orbitrap/ESI-MS. Peel and pulp extracts were also analysed for their in vitro antioxidant activity on a Balb/3T3 clone A31 mouse embryo fibroblast cell line. The results of peel and pulp EOs were mainly characterised by monoterpenes and sesquiterpenes, respectively. All peels displayed a higher total phenol content (TPC) than pulps, and consequently a greater antioxidant activity. Collette peels and Pink Ice pulps showed the highest amount of identified flavonoids (e.g., luteolin, isosakuranetin, and poncirin derivatives). Collette and Red peels were rich in anthocyanins (delphinidin and petunidin glycosides), exhibiting the maximum protective activity against induced oxidative damage. In conclusion, finger lime fruits are good sources of health-promoting phytocomplexes, with the Red, Collette, and Pink Ice varieties being the most promising.

Thiolated 2-Methyl-ß-Cyclodextrin as a Mucoadhesive Excipient for Poorly Soluble Drugs: Synthesis and Characterization.

Thiolated cyclodextrins are structurally simple mucoadhesive macromolecules, which are able to host drugs and increase their apparent water solubility, as well as interact with the mucus layer prolonging drug residence time on the site of absorption. The aim of this study was to synthesize through green microwave-assisted process a freely soluble thiolated 2-methyl-ß-cyclodextrin (MßCD-SH). Its inclusion complex properties with dexamethasone (Dex), a poor water soluble drug, and mucoadhesive characteristics were also determined. The product was deeply characterized through NMR spectroscopy (2D COSY, 2D HSQC, 1D/2D TOCSY, and 1D ROESY), showing a thiolation degree of 67%, a selective thiolation on the C6 residues and a monomeric structure. The association constant of MßCD and MßCD-SH with Dex resulted in 2514.3 ± 32.3 M-1 and 2147.0 ± 69.3 M-1, respectively, indicating that both CDs were able to host the drug. Microrheological analysis of mucin in the presence of MBCD-SH showed an increase of complex viscosity, G' and G″, due to disulphide bond formation. The cytotoxicity screening on fibroblast BALB/3T3 clone A31 cells indicated an IC50 of 27.7 mg/mL and 30.0 mg/mL, for MßCD and MßCD-SH, respectively. Finally, MßCD-SH was able to self-assemble in water into nanometric structures, both in the presence and absence of the complexed drug.

Thiolated Hydroxypropyl-ß-cyclodextrin: A Potential Multifunctional Excipient for Ocular Drug Delivery.

The goal of this study was the design and evaluation of a thiolated cyclodextrin providing high drug solubilizing and mucoadhesive properties for ocular drug delivery. Hydroxypropyl-ß-cyclodextrin (HP-ß-CD) was thiolated via a microwave-assisted method, resulting in a degree of thiolation of 33%. Mucoadhesive properties of thiolated HP-ß-CD (HP-ß-CD-SH) were determined via rheological measurements and ex vivo studies on isolated porcine cornea. Due to thiolation of HP-ß-CD, a 2-fold increase of mucus viscosity and a 1.4-fold increase in residence time on isolated corneal tissue were achieved. After instillation, the mean precorneal residence time and AUC of dexamethasone (DMS) eye drops were 4-fold and 11.7-fold enhanced by HP-ß-CD-SH, respectively. Furthermore, in the presence of HP-ß-CD-SH, a constant high level of DMS in aqueous humour between 30 and 150 min after administration was observed. These results suggest that HP-ß-CD-SH is an excellent excipient for ocular formulations of poorly soluble drugs in order to prolong their ocular residence time and bioavailability.

Combination of Two Kinds of Medicated Microparticles Based on Hyaluronic Acid or Chitosan for a Wound Healing Spray Patch.

Olive leaves extract (OLE) has been extensively studied as antioxidant and antibiotic and these characteristics make it particularly interesting for use on wounds. For this reason, the aim of this study was to introduce OLE in microparticles (MP) of hyaluronic acid (MPHA-OLE) or chitosan (MPCs-OLE) to obtain a spray patch for the treatment of wounds in anatomical areas that are difficult to protect with traditional patches. The MP were characterized for particle size and ability to protect OLE from degradation, to absorb water from wound exudate, to control OLE release from MP. The MPHA and MPCs medicated or not and mixtures of the two types in different proportions were studied in vitro on fibroblasts by the scratch wound healing assay. The MP size was always less than 5 µm, and therefore, suitable for a spray patch. The MPCs-OLE could slow down the release of OLE therefore only about 60% of the polyphenols contained in it were released after 4 h. Both MPHA and MPCs could accelerate wound healing. A 50% MPHA-OLE-50% MPCs-OLE blend was the most suitable for accelerating wound healing. The MPHA-OLE-MPCs-OLE blends studied in this work were shown to have the characteristics suitable for a spray patch, thus giving a second life to the waste products of olive growers.

The Potential Role of Aerosolized Phosphodiesterase 3 Inhibitor Enoximone in the Management of Coronavirus Disease 2019 Hypoxemia: A Case Report.

Despite the various parenchymal presentation of coronavirus disease 2019 (COVID-19) pneumonia, the involvement of the vascular component, the reduction of perfusion in noninjured part of the lung and secondary right to left shunt play an important role in the genesis of the respiratory insufficiency. We present the case of a 72-year-old woman admitted to Livorno Hospital for severe respiratory insufficiency due to SARS-CoV-2 infection unresponsive to noninvasive in whom administration of nebulized phosphodiesterase 3 (PDE3) inhibitor enoximone was able to improve oxygenation avoiding tracheal intubation. Intravenous infusions of phosphodiesterase inhibitors are commonly used as pulmonary vasodilators in the management of pulmonary hypertension. This is the first case showing that inhaled route administration of PDE3 inhibitor enoximone could be important in the management of COVID-19 hypoxemia, to restore perfusion in noninjured part of the lung, improving oxygenation and avoiding risks of systemic infusion.

Correction: Piras, A.M., et al. Antibacterial, Antibiofilm, and Antiadhesive Properties of Different Quaternized Chitosan Derivatives. Int. J. Mol. Sci. 2019, 20, 6297.

The authors wish to make the following corrections to this paper [...].

Antivirulence Properties of a Low-Molecular-Weight Quaternized Chitosan Derivative against Pseudomonas aeruginosa.

The co-occurrence of increasing rates of resistance to current antibiotics and the paucity of novel antibiotics pose major challenges for the treatment of bacterial infections. In this scenario, treatments targeting bacterial virulence have gained considerable interest as they are expected to exert a weaker selection for resistance than conventional antibiotics. In a previous study, we demonstrated that a low-molecular-weight quaternized chitosan derivative, named QAL, displays antibiofilm activity against the major pathogen Pseudomonas aeruginosa at subinhibitory concentrations. The aim of this study was to investigate whether QAL was able to inhibit the production of relevant virulence factors of P. aeruginosa. When tested in vitro at subinhibiting concentrations (0.31-0.62 mg/mL), QAL markedly reduced the production of pyocyanin, pyoverdin, proteases, and LasA, as well as inhibited the swarming motility of three out of four P. aeruginosa strains tested. Furthermore, quantitative reverse transcription PCR (qRT-PCR) analyses demonstrated that expression of lasI and rhlI, two QS-related genes, was highly downregulated in a representative P. aeruginosa strain. Confocal scanning laser microscopy analysis suggested that FITC-labelled QAL accumulates intracellularly following incubation with P. aeruginosa. In contrast, the reduced production of virulence factors was not evidenced when QAL was used as the main polymeric component of polyelectrolyte-based nanoparticles. Additionally, combination of sub-MIC concentrations of QAL and tobramycin significantly reduced biofilm formation of P. aeruginosa, likely due to a synergistic activity towards planktonic bacteria. Overall, the results obtained demonstrated an antivirulence activity of QAL, possibly due to polymer intracellular localization and QS-inhibition, and its ability to inhibit P. aeruginosa growth synergizing with tobramycin.

Nanoparticles Based on Quaternary Ammonium Chitosan-methyl-ß-cyclodextrin Conjugate for the Neuropeptide Dalargin Delivery to the Central Nervous System: An In Vitro Study.

Peptide oral administration is a hard goal to reach, especially if the brain is the target site. The purpose of the present study was to set up a vehicle apt to promote oral absorption of the neuropeptide dalargin (DAL), allowing it to cross the intestinal mucosal barrier, resist enzymatic degradation, and transport drugs to the brain after crossing the blood-brain barrier. Therefore, a chitosan quaternary ammonium derivative was synthesized and conjugated with methyl-ß-cyclodextrin to prepare DAL-medicated nanoparticles (DAL-NP). DAL-NP particle size was 227.7 nm, zeta potential +8.60 mV, encapsulation efficiency 89%. DAL-NP protected DAL from degradation by chymotrypsin or pancreatin and tripled DAL degradation time compared to non-encapsulated DAL. Use of DAL-NP was safe for either Caco-2 or bEnd.3 cells, with the latter selected as a blood-brain barrier model. DAL-NP could also cross either the Caco-2 or bEnd.3 monolayer by the transepithelial route. The results suggest a potential DAL-NP ability to transport to the brain a DAL dose fraction administered orally, although in vivo experiments will be needed to confirm the present data obtained in vitro.

Repurposing of Plasminogen: An Orphan Medicinal Product Suitable for SARS-CoV-2 Inhalable Therapeutics.

The SARS-CoV-2 infection is associated with pulmonary coagulopathy, which determines the deposition of fibrin in the air spaces and lung parenchyma. The resulting lung lesions compromise patient pulmonary function and increase mortality, or end in permanent lung damage for those who have recovered from the COVID-19 disease. Therefore, local pulmonary fibrinolysis can be efficacious in degrading pre-existing fibrin clots and reducing the conversion of lung lesions into lasting scars. Plasminogen is considered a key player in fibrinolysis processes, and in view of a bench-to-bedside translation, we focused on the aerosolization of an orphan medicinal product (OMP) for ligneous conjunctivitis: human plasminogen (PLG-OMP) eye drops. As such, the sterile and preservative-free solution guarantees the pharmaceutical quality of GMP production and meets the Ph. Eur. requirements of liquid preparations for nebulization. PLG-OMP aerosolization was evaluated both from technological and stability viewpoints, after being submitted to either jet or ultrasonic nebulization. Jet nebulization resulted in a more efficient delivery of an aerosol suitable for pulmonary deposition. The biochemical investigation highlighted substantial protein integrity maintenance with the percentage of native plasminogen band > 90%, in accordance with the quality specifications of PLG-OMP. In a coherent way, the specific activity of plasminogen is maintained within the range 4.8-5.6 IU/mg (PLG-OMP pre-nebulization: 5.0 IU/mg). This is the first study that focuses on the technological and biochemical aspects of aerosolized plasminogen, which could affect both treatment efficacy and clinical dosage delivery. Increasing evidence for the need of local fibrinolytic therapy could merge with the availability of PLG-OMP as an easy handling solution, readily aerosolizable for a fast translation into an extended clinical efficacy assessment in COVID-19 patients.

Cell membrane coated nanocarriers - an efficient biomimetic platform for targeted therapy.

Targeted therapy approaches have become the core of modern translational science and as an intriguing field, it is the solution of the conventional drug delivery problems that were once unanswered. Traditional methods of delivering drugs and therapeutics faced issues of solubility, sustained release, not enough amount getting through the diseased site, for e.g a tumor. Various formulations of liposomes, polymers, dendrimers, etc have succeeded and made their way for clinical trials trying to enhance the pharmacokinetic and biodistribution of the drug. Many stealth coatings that include hydrophilic polymers (PEG, chitosan, polyacrylamides, etc) can act as a covering around the nanoparticle that can shield the surface from aggregation, opsonization and evade immune system, thus considered in Generally Recognized as Safe (GRAS) category. Several other polymers such as poly-2-oxazoline, polyethylene oxide, PEG-based surfactant (polysorbate-80), and zwitterionic phospholipids have also been tested for their antifouling properties. However, the polymer coating approach requires labor-intensive procedures and conjugation chemistries that often fail in mice model. Besides, due to immunogenicity and allergic reactions evoked by the PEG-coated nanoparticles, there was an urge to find biomimicking materials that can prove better as shielding agents which paved the way for cell membrane coated nanoparticles (CMCNPs) to come into the limelight. CMCNPs consist of a nanoparticle inner core covered by cell membrane that can be implicated in targeted drug delivery approaches, photothermal therapy, diagnosis or imaging making it a powerful theranostic tool. In this review, mode of preparation of CMCNPs, different sources of cell membranes (RBCs, WBCs, platelets, cancer cells, stem cells with some other unconventional sources) and nanoparticle cores that are employed have been thoroughly emphasized. In addition to this, advancements and limitations with respect to this newly emerging field have been focussed.

Quaternary Ammonium Chitosans: The Importance of the Positive Fixed Charge of the Drug Delivery Systems.

As a natural polysaccharide, chitosan has good biocompatibility, biodegradability and biosecurity. The hydroxyl and amino groups present in its structure make it an extremely versatile and chemically modifiable material. In recent years, various synthetic strategies have been used to modify chitosan, mainly to solve the problem of its insolubility in neutral physiological fluids. Thus, derivatives with negative or positive fixed charge were synthesized and used to prepare innovative drug delivery systems. Positively charged conjugates showed improved properties compared to unmodified chitosan. In this review the main quaternary ammonium derivatives of chitosan will be considered, their preparation and their applications will be described to evaluate the impact of the positive fixed charge on the improvement of the properties of the drug delivery systems based on these polymers. Furthermore, the performances of the proposed systems resulting from in vitro and ex vivo experiments will be taken into consideration, with particular attention to cytotoxicity of systems, and their ability to promote drug absorption.

2-Methyl-ß-cyclodextrin grafted ammonium chitosan: synergistic effects of cyclodextrin host and polymer backbone in the interaction with amphiphilic prednisolone phosphate salt as revealed by NMR spectroscopy.

Reduced molecular weight chitosan was quaternized with 2-chloro-N,N-diethylethylamine to obtain a water soluble derivative (N+-rCh). Methylated-ß-cyclodextrin (MCD), with 0.5 molar substitution, was covalently linked to N+-rCh through 1,6-hexamethylene diisocyanate spacer to give the derivatized ammonium chitosan N+-rCh-MCD. To shed light on the role of the cyclodextrin pendant in guiding binding interactions with amphiphilic active ingredients, corticosteroid prednisolone phosphate salt (PN) was considered. The deep inclusion of PN into cyclodextrin in PN/MCD model system was pointed out by analysis of 1H NMR complexation shifts, 1D ROESY spectra, and diffusion measurements (DOSY). By using proton selective relaxation rates measurements as investigation tool, the superior affinity of N+-rCh-MCD towards PN was demonstrated in comparison with parent ammonium chitosan N+-rCh.

Improvement of Peptide Affinity and Stability by Complexing to Cyclodextrin-Grafted Ammonium Chitosan.

Cyclodextrin-grafted polymers are attractive biomaterials that could bring together the host-guest complexing capability of pristine cyclodextrin and the pharmaceutical features of the polymeric backbone. The present paper is aimed at characterizing the potential application of ammonium-chitosan grafted with 2-methyl-ß-cyclodextrin (N+-rCh-MCD) as the functional macromolecular complexing agent for the oral administration of the neuropeptide dalargin (DAL). Specific NMR characterization procedures, along with UV and fluorescence techniques, as well as biological in vitro assessments have been performed. The results indicate that N+-rCh-MCD forms water-soluble complexes with DAL, with a prevalent involvement of Tyr or Phe over Leu and Ala residues. The association constant of DAL with the polymeric derivative is one order of magnitude higher than that with the pristine cyclodextrin (Ka: 2600 M-1 and 120 M-1, respectively). Additionally, N+-rCh-MCD shields DAL from enzymatic degradation in gastrointestinal in vitro models with a three-fold time delay, suggesting a future pharmaceutical exploitation of the polymeric derivative. Therefore, the greater affinity of N+-rCh-MCD for DAL and its protective effect against enzymatic hydrolysis can be attributed to the synergistic cooperation between cyclodextrin and the polymer, which is realized only when the former is covalently linked to the latter.

Antioxidant Effect of Cocoa By-Product and Cherry Polyphenol Extracts: A Comparative Study.

BACKGROUND: Recent studies have highlighted the importance of cherry and cocoa extracts consumption to protect cells from oxidative stress, paying particular attention to cocoa by-products. This study aims to investigate the protective effect of cocoa husk extract (CHE) and cherry extracts (CE) against ROS-induced oxidative stress in Human Umbilical Vein Endothelial Cells (HUVECs). METHODS: CE and CHE had antioxidant activity characterized by total polyphenols content (TPC). HUVECs were treated for 2 h and 24 h with increasing TPC concentrations of CE and CHE (5-10-25-50-100 µg Gallic Acid Equivalent (GAE)/mL) and then with H2O2 for 1 h. Cell viability and ROS production were evaluated. CE and CHE polyphenols permeability on excised rat intestine were also studied. RESULTS: CE and CHE showed a similar antioxidant activity (2.5 ± 0.01 mmol Fe2+/100 g FW (fresh weight) and 2.19 ± 0.09 mmol Fe2+/100 g FW, respectively, p > 0.05) whereas CHE had a higher TPC (7105.0 ± 96.9 mg GAE/100 g FW) than CE (402.5 ± 8.4 mg GAE/100 g), p < 0.05. The in vitro viability assay showed that both extracts were non-cytotoxic. CHE resulted in protection against ROS at lower concentrations than CE. CHE showed a 2-fold higher apparent permeability compared to CE. CONCLUSIONS: CHE represents a high-value antioxidant source, which is interesting for the food and pharmaceutical industries.