Liposomal Encapsulation of Citicoline for Ocular Drug Delivery.

Glaucoma represents a group of neurodegenerative diseases characterized by optic nerve damage and the slowly progressive death of retinal ganglion cells. Glaucoma is considered the second leading cause of irreversible blindness worldwide. Pharmaceutical treatment of glaucoma is critical because of the properties of the ocular barrier that limit the penetration of drugs, resulting in lower systemic bioavailability. This behavior causes the need of frequent drug administration, which leads to deposition of concentrated solutions on the eye, causing toxic effects and cellular damage to the eye. To overcome these drawbacks, novel drug-delivery systems, such as liposomes, can play an important role in improving the therapeutic efficacy of antiglaucomatous drugs. In this work, liposomes were synthesized to improve various aspects, such as ocular barrier penetration, bioavailability, sustained release of the drug, targeting of the tissue, and reduction in intraocular pressure. Citicoline (CDP-choline; cytidine 5'-diphosphocholine) is an important intermediate in the biosynthesis of cell membrane phospholipids, with neuroprotective and neuroenhancement properties, and it was used in the treatment on retinal function and neural conduction in the visual pathways of glaucoma patients. In this study, citicoline was loaded into the 1,2-dioleoyl-sn-glycerol-3-phosphocholine and cholesterol liposomal carrier to enhance its therapeutic effect. The citicoline encapsulation efficiency, drug release, and size analysis of the different liposome systems were investigated using dynamic light scattering, nuclear magnetic resonance, infrared spectroscopy, and ToF-SIMS experiments.

Determination of nano and microplastic particles in hypersaline lakes by multiple methods.

Microplastics and nanoplastics have a range of impacts on the aquatic environment and present major challenges to their mitigation and management. Their transport and fate depend on their composition, form, and the characteristics of the receiving environment. We explore the spatial and temporal dynamics of plastic particles in the world's second-largest hypersaline lake, combining information from microscopic, thermal gravimetric, and fractional methods. Studies on microplastic and nanoplastic pollution in these important environments are scarce, and there is limited understanding of their dynamics and fate. Our results for Urmia Lake (Iran) in 2016 and 2019 show a discrepancy in the composition and quantity of microplastics measured in river tributaries to the lake and the lake itself, suggesting an active microplastic sink. Potential sink mechanisms in hypersaline lakes are explored. The present study indicates that microplastics have different transport mechanisms and fate in these extreme environments, compared to lake and ocean environments.

Formulation of liposomes functionalized with Lotus lectin and effective in targeting highly proliferative cells.

BACKGROUND: Liposomes, used to improve the therapeutic index of new and established drugs, have advanced with the insertion of active targeting. The lectin from Lotus tetragonolobus (LTL), which binds glycans containing alpha-1,2-linked fucose, reveals surface regionalized glycoepitopes in highly proliferative cells not detectable in normally growing cells. In contrast, other lectins localize the corresponding glycoepitopes all over the cell surface. LTL also proved able to penetrate the cells by an unconventional uptake mechanism. METHODS: We used confocal laser microscopy to detect and localize LTL-positive glycoepitopes and lectin uptake in two cancer cell lines. We then constructed doxorubicin-loaded liposomes functionalized with LTL. Intracellular delivery of the drug was determined in vitro and in vivo by confocal and electron microscopy. RESULTS: We confirmed the specific localization of Lotus binding sites and the lectin uptake mechanism in the two cell lines and determined that LTL-functionalized liposomes loaded with doxorubicin greatly increased intracellular delivery of the drug, compared to unmodified doxorubicin-loaded liposomes. The LTL-Dox-L mechanism of entry and drug delivery was different to that of Dox-L and other liposomal preparations. LTL-Dox-L entered the cells one by one in tiny tubules that never fused with lysosomes. LTL-Dox-L injected in mice with melanoma specifically delivered loaded Dox to the cytoplasm of tumor cells. CONCLUSIONS: Liposome functionalization with LTL promises to broaden the therapeutic potential of liposomal doxorubicin treatment, decreasing non-specific toxicity. GENERAL SIGNIFICANCE: Doxorubicin-LTL functionalized liposomes promise to be useful in the development of new cancer chemotherapy protocols.

Phosphorylated xanthan gum-Ag(I) complex as antibacterial viscosity enhancer for eye drops formulation.

Topical instillation of eye drops represents the treatment of choice for many ocular diseases. Ophthalmic formulations must meet general requirements, i.e. pH, osmolality, transparency and viscosity to ensure adequate retention without inducing irritation and the development of eye infections. We developed a phosphorylated xanthan gum-Ag(I) complex (XGP-Ag) showing pH (pH = 7.1 ± 0.3) and osmolality values (311 ± 2 mOsm/kg) close to that of human tears (pH = 6.5-7.6 and 304 ± 23 mOsm/kg) thanks to the presence of phosphate moieties along the chain. The presence of phosphate groups covalently bound to the XG chains avoids their dispersion in fluid, thus reducing the risk of corneal calcification. 0.02% w/v XGP-Ag solution showed high transparency (higher than 95% along the entire visible range), adequate refractive index (1.334 ± 0.001) and viscosity in the range: γ 1 s-1-10,000 s- 1 (26.4 ± 0.8-2.1 ± 0.4 mPa·s). Its cytotoxicity and capability to hinder bacterial proliferation was also verified.

PVA/STMP based hydrogels as potential substitutes of human vitreous.

PVA based hydrogels were synthesised using, as crosslinking agent, trisodium trimetaphosphate (STMP) to obtain potential substitutes for the vitreous body of the eye. The hydrogels, obtained using different amounts of STMP, were characterised by Infrared Spectroscopy which confirmed the successful occurrence of crosslinking reaction. The mechanical spectra of the fully hydrated samples confirmed covalently crosslinked systems (i.e. G' > G''). The rheological analysis pointed out that only one of the hydrogels (PVA STMP 8:1) showed a behaviour similar to that of human vitreous. The hydrogel was also subjected to injection through a small needle, a procedure that is essential in the use of vitreous substitutes. Further analysis in terms of light transmittance, water content measurements, diffusion coefficient and cytotoxicity confirmed the applicability of such a hydrogel as vitreous substitute.

Covalent hyaluronic-based coating of magnetite nanoparticles: Preparation, physicochemical and biological characterization.

In this paper we report about the preparation, physicochemical and biological characterization of a magneto responsive nanostructured material based on magnetite nanoparticles (NP) coated with hyaluronic acid (HA). A synthetic approach, based on a Cu(I)-catalyzed azide-alkyne 1,3-dipolar cycloaddition "click" reaction between azido-functionalized magnetite NP and a derivative of hyaluronic acid bearing propargylated ferulic acid groups (HA-FA-Pg), was developed to link covalently the polymer layer to the magnetite NP. The functionalization steps of the magnetite NP and their coating with the HA-FA-Pg layer were monitored by Fourier Transform Infrared (FTIR) spectroscopy and Thermal Gravimetric Analysis (TGA) while Dynamic Light Scattering (DLS) and ζ-potential measurements were performed to characterize the aqueous dispersions of the HA-coated magnetite NP. Aggregation and sedimentation processes were investigated also by UV-visible spectroscopy and the dispersions of HA-coated magnetite NP were found significantly more stable than those of bare NP. Magnetization and zero field cooled/field cooled curves revealed that both bare and HA-coated magnetite NP are superparamagnetic at room temperature. Moreover, cytotoxicity studies showed that the coating with HA-FA-Pg significantly reduces the cytotoxicity of the magnetite NP providing the rational basis for the application of the HA-coated magnetite NP as healthcare material.

Enriched Gellan Gum hydrogel as visco-supplement.

Viscosupplementation, i.e. intra-articular injection of hyaluronic acid derivatives, is considered as the most effective treatment for patients with mild to moderate osteoarthritis. Even if hyaluronic acid is still considered as the gold standard, research is now focusing on the development of new products with enhanced injectability and yet reasonable viscoelastic behavior for OA treatment. A Gellan Gum (GG) hydrogel was synthesized and coated with crosslinked polyvinyl alcohol (PVA) to protect the polysaccharide from degradation during sterilization and improve its performance for the foreseen application. Thermal analyses indicated that mixed hydrogel showed a higher degree of structuring than the bare polysaccharide core without losing its swelling properties, thanks to the hydrophylicity of both coating and cross-linking agent. The PVA coating increased elastic and viscous moduli of the polysaccharide core conferring it a higher resistance to shear and compression and better thixotropic properties. Despite the double crosslinking, hydrogel was injectable. Cytocompatibility towards chondrocytes was verified.

Chemical characterization of liposomes containing nutraceutical compounds: Tyrosol, hydroxytyrosol and oleuropein.

Tyrosol, hydroxytyrosol and oleuropein are among the major phenolic compounds in fruits, leaves and oils from Olea europaea L. These natural antioxidants molecules revealed several beneficial effects on human health, but a low bioavailability and accessibility to targeted site. Liposomes are drug/nutraceutical delivery carriers, used for driving bioactive molecules to desired target tissues, decreasing potential side effects and protecting the encapsulated molecule from enzymatic metabolic processes. In this study, zwitterionic liposomes containing tyrosol, hydroxytyrosol and oleuropein were synthesized and characterized for their size and surface charge. Particular attention was devoted to the determination of encapsulation efficiency (EE%), quantifying the loaded Tyr, HTyr and Ole amount, by using three different techniques: direct UV spectrophotometry, High Performance Liquid Chromatography and Trolox Equivalent Antioxidant Capacity assay. The results revealed higher EE% for oleuropein. Cyto-toxicity and cyto-compatibility of liposomes were also tested on human chondrocyte cells.

Thixotropic PVA hydrogel enclosing a hydrophilic PVP core as nucleus pulposus substitute.

A thixotropic polyvinyl alcohol (PVA) hydrogel, containing a hydrophilic poly-vinyl pyrrolidone (PVP) core, was obtained in order to develop a preformed 3D network able to maintain injectability. PVA was mixed with PVP in two different molar ratios (1:1 and 1:3) and chemically cross-linked using trisodium trimetaphosphate (STMP), which is able to react only with PVA component. A combination of Time of Flight- Secondary Ion Mass Spectrometry (ToF-SIMS), elemental analysis and UV spectroscopy permitted to determine both the cross-linking arm length and the crosslinking degree. Hydrogels were characterized in terms of swelling pressurization, rheological and mechanical behaviour. In particular, the viscoelastic behaviour of the hydrogel was analysed in shear and compression stress under dynamic conditions and compared with the performance of healthy human nucleus pulposus. In conclusion, the study demonstrated that the scaffold obtained mixing PVA and PVP in a molar ratio 1:1 can be considered a promising material to be utilised in the replacement of nucleus pulposus.

Amidic alginate hydrogel for nucleus pulposus replacement.

Degeneration of intervertebral discs is the most common cause of back pain. The first phase of this degenerative process involves the nucleus pulposus (NP). A rapid recovery of this structure can prevent further degradation of the annulus fibrosus. A new amidic derivative of alginate (AAA) was developed to obtain a polysaccharide possessing some of the physical-chemical properties of Hyal (i.e. viscosity) without losing the rigidity of the native alginate structure. The modified polysaccharide was crosslinked using 1.3 diaminopropane as crosslinking agent. The hydrogel obtained was characterized in terms of water uptake and rheological behavior. In particular, the viscoelastic behavior of the hydrogel was determined in shear stress under dynamic conditions and compared with the behavior of nondegenerated human lumbar NP. We then assessed the effect of the AAA hydrogel on NHC (Normal Human Chondrocyte) cell viability and on the production of important extracellular matrix factors, such as glycosaminoglycans and Type II collagen. In conclusion, the results achieved in this study demonstrated that the amidic alginate-based scaffold is a promising material to be utilized in the replacement of NP.

The applicability of an amidated polysaccharide hydrogel as a cartilage substitute: structural and rheological characterization.

An amidic derivative of a carboxymethylcellulose-based hydrogel was obtained and characterized in terms of amidation degree. NMR studies and FT-IR imaging spectroscopy demonstrated that the reaction allowed a polymer to be obtained that was characterized by a regular distribution of amidic groups along the polysaccharide chains. Through this regularity, a homogenous three-dimensional scaffold was obtained, which maintained the thixotropic property of the linear polysaccharide.

Protective effect of quercetin and rutin encapsulated liposomes on induced oxidative stress.

Natural antioxidants show many pharmacological properties, but poor solubility and inability to cross cell membrane. Liposomes are biocompatible and phospholipid vesicles able to carry hydrophilic, hydrophobic, and amphiphilic molecules. This paper focus on the synthesis of anionic, cationic and zwitterionic liposomes, loaded with quercetin or rutin, and on the evaluation of their cytotoxicity and protective effects against oxidative stress. Chemical characterization was obtained by dynamic light scattering and z-potential experiments. In vitro cell behavior was evaluated by Neutral Red Uptake test. All liposomes, empty and loaded with antioxidants, are stable. The cytotoxicity of both quercetin and rutin encapsulated in zwitterionic and anionic liposomes is higher than that of their solutions. Quercetin and rutin loaded in cationic liposomes are able to inhibit the toxic effect of empty liposomes. The encapsulation of rutin at 5.0×10-5 and 5.0×10-4M, in zwitterionic and anionic liposomes, protects fibroblasts by H2O2 treatment, while the loading with quercetin does not have effect on improving cell viability. All data suggest that the tested liposomes are stable and able to include quercetin and rutin. The liposomes encapsulation of antioxidants makes easier their internalization by cells. Moreover, zwitterionic and anionic liposomes loaded with rutin protect cells by oxidative stress. Liposomes stability together with their good in vitro cytocompatibility, both empty and loaded with antioxidant molecules, makes these systems suitable candidates as drug delivery systems. Moreover, the encapsulation of rutin, is able to protect cells by oxidative stress.

Hyaluronic acid hydrogel added with ibuprofen-lysine for the local treatment of chondral lesions in the knee: in vitro and in vivo investigations.

The effect of hyaluronan-based hydrogel with a cross-linking degree of 50% (i.e., the amount of carboxylate groups involved in the cross-lining reaction) (Hyal 50%), loaded with an anti-inflammatory drug (ibuprofen-lysine) for local administration in the osteoarthritic knee was evaluated. The kinetic of drug release from Hyal 50% hydrogel was performed in vitro by a continuous flow system. The release was studied at three different rates (0.375, 0.073, and 0.005 mL/s), and for the slowest one a period of 8 days was necessary to receive the complete release of the drug. A chondral defect was caused in the right femoral medial condyle of 24 rabbits. Twelve animals were treated with Hyal 50%+ibuprofen-lysine and 12 with Hyal 50% by percutaneous injection. The treatments were repeated every 10 days, for three or five times. At 30 and 50 days, no significant differences were observed between the groups (mean score at 30 days: 4.4+/-0.6 in Hyal 50%-treated group and 5.1+/-0.7 in Hyal 50% +Ibuprofen-lysine-treated group; mean score at 50 days: 6.9+/-0.6 in Hyal 50%-treated group and 7.1+/-0.6 in Hyal 50%+ Ibuprofen-lysine-treated group). A significant difference was observed in bone mineral density of the total tibia of rabbits treated by Hyal 50% + Ibuprofen-lysine in comparison with those treated only by Hyal 50%. The positive effects of Hyal 50% on chondral lesions were maintained in the presence of ibuprofen-lysine with the adjunctive effect of an increased limb usage, most likely due to less pain and discomfort.

In vitro biocompatibility of new PVA-based hydrogels as vitreous body substitutes.

In order to synthesize injectable hydrogels suitable as vitreous body substitutes, a new method based on the use of trisodium trimetaphosphate (STMP) to cross-link PVA was recently proposed. Hydrogels with different molar ratios between STMP and PVA were realised. The aim of the present study was the evaluation of the biocompatibility of the different STMP/PVA hydrogels synthesised by analysing the effects of their in vitro interaction with cultures of mouse fibroblasts NIH3T3, primary human microvascular endothelial cells adult (HMVECad) and human lens cells. Cytotoxicity of hydrogels was first evaluated by analysing cell density and proliferation. Morphological and morphometric analysis of cell in contact with hydrogels was then performed using light microscopy and scanning electron microscopy, respectively. Moreover, cell adhesion and growth onto the hydrogels surface was evaluated and correlated to the amount of adsorbed proteins. At last, the biocompatibility of the sheared STMP/PVA 1:8 hydrogel was tested. The in vitro data of all the STMP/PVA hydrogels demonstrated their good biocompatibility, and indicated that the 1:8 sample was the most promising as vitreous body substitute.

A PVA/PVP hydrogel for human lens substitution: Synthesis, rheological characterization, and in vitro biocompatibility.

To overcome opacification and absence of accommodation of human lens substitutes a new poly(vinyl alcohol) (PVA)/poly(N-vinyl-2- pyrrolidinone) (PVP) based hydrogel (PPS31075) was realised. The Infrared Spectroscopy and the mechanical spectra confirmed the successful occurrence of crosslinking reaction. The rheological analysis pointed out a behavior comparable with that of young human lens in terms of complex shear modulus and accommodation capability. Further analysis in terms of optical properties, water content measurements, diffusion coefficient, cytotoxicity, and human capsular cell adhesion confirmed the applicability of such a hydrogel as potential human lens substitute.

Preparation and physico-chemical characterisation of microporous polysaccharidic hydrogels.

A new technique to obtain microporous hydrogels was realised. It permits us to obtain a microporous structure directly on the already cross-linked hydrogel. It consists in stratifying the already cross-linked hydrogel on to a filter with known porosity and forcing the CO2 bubbles, derived from the addition of HCl to a porogen salt (NaHCO3), to cross through the filter first and then the matrix. By changing the porosity of the filter, it was possible to modulate the porous morphology of the hydrogels. The polysaccharides selected were hyaluronane, alginate, and carboxymethylcellulose. The influence of the porous morphology on the physico-chemical properties of the gel has been evaluated by FT-IR, FRAP, calorimetric, water uptake, and rheological analysis.