Latanoprost-loaded phytantriol cubosomes for the treatment of glaucoma.

Glaucoma is a degenerative optic neuropathy characterized by increased intraocular pressure that if untreated can result in blindness. Ophthalmological drug therapy is a challenge of great clinical importance due to the diversity of ocular biological barriers which commonly causes limited or no effectiveness for drugs delivered through the eye. In this work, we proposed the development of nanosized cubic liquid crystals (cubosomes) as a new drug carrier system for latanoprost, an anti-glaucoma drug. Latanoprost-loaded phytantriol cubosomes (CubLnp) were prepared using a top-down method. Latanoprost concentration in the formulations ranged from 0.00125% to 0.02% w/v. All cubosomes displayed an average size around 200 nm, a low polydispersity index of 0.1 and zeta potential values around -25 mV, with an encapsulation efficiency of about 90%. Structural studies revealed that cubosomes displayed a double-diamond surface, Pn3m cubic-phase structure, and was not affected by drug loading. Calorimetric studies revealed a fast and exothermic interaction between latanoprost and cubosomes. According to in vitro essays, latanoprost release from cubosomes was slow in time, evidencing a sustained release profile. Based on this behavior, the in vivo hypotensive intraocular effect was evaluated by means of the subconjunctival administration of CubLnp in normotensive rabbits. We obtained promising results in comparison with a marketed latanoprost formulation (0.005% w/v).

Preparation and Characterization of Stabilizer-Free Phytantriol-Based Water-in-Oil Internally Liquid Crystalline Emulsions.

Despite the widespread use of surfactants, there are known issues such as allergic reactions and formulation complications in their use as emulsion stabilizers. In this study, stabilizer-free water-in-oil (W/O) emulsions containing water, phytantriol, and almond oil were prepared by an ultra-turrax homogenizer, a standard laboratory equipment, and a high specialized high-shear device. Parameters such as mixing time, stirring rate, composition, order of addition of phases, and temperature were investigated to systematically optimize the preparation of the formulations through evaluating their accelerated physical stability by a centrifugal sedimentation technique. The liquid crystalline structure of the continuous phase was studied by small-angle X-ray scattering indicating a reverse hexagonal phase (H2). Microscopy images showed the emulsions prepared via high-shear method had smaller water droplets with more uniform shape and better dispersion as confirmed by Fourier-transform infrared-attenuated total reflection spectroscopy. Rheology studies showed a larger yield stress value for emulsions with higher content of phytantriol. Our results indicated that emulsions prepared by the high-shear device with higher amount of phytantriol were the most stable formulations. Applying the correct variables in the preparation of the stabilizer-free emulsions using ultra-turrax homogenizer, one could obtain similarly stable emulsions lacking the uniformity of the droplets.

Controlled coagulation and redispersion of thermoresponsive poly di(ethylene oxide) methyl ether methacrylate grafted cellulose nanocrystals.

HYPOTHESIS: Cellulose nanocrystals (CNCs) undergo precipitation in the presence of high concentrations of cationic surfactants in aqueous solutions. To avoid such behavior and/or to promote redispersion of CNC/surfactant mixtures, the CNC surface was grafted with poly di(ethylene oxide) methyl ether methacrylate, P(MEO2MA). EXPERIMENTS: CNC-g-P(MEO2MA) was characterized using the following techniques 13C solid-state nuclear magnetic resonance (13C SSNMR), Fourier-transform infrared spectroscopy - attenuated total reflection spectroscopy (FTIR-ATR) and thermal gravimetric analysis (TGA). Isothermal titration calorimetry (ITC), electrophoretic mobility, light scattering and high sensitivity differential scanning calorimetry (HSDSC) were used to study the interaction between CNC-g-P(MEO2MA) and ionic surfactants, dodecyltrimethylammonium bromide (C12TAB, cationic) and sodium dodecylsulfate (SDS, anionic) at temperatures below and above the LCST. FINDINGS: CNC-g-P(MEO2MA) underwent phase separation above its lower critical solution temperature (LCST ∼ 25 °C) and precipitated from solution as seen by HSDSC and transmittance experiments. When C12TAB was added to CNC-g-P(MEO2MA) it induced the precipitation that prevented the redispersion due to strong electrostatic interactions with the negative charges on the CNC surface. With increasing concentrations of SDS, the polymer phase transition temperature was increased, which can be used to redisperse the CNC complexes. By removing SDS from the mixture via dialysis, the CNC-g-P(MEO2MA) underwent subsequent phase transition.

Interactions and release of two palmitoyl peptides from phytantriol cubosomes.

Phytantriol cubosomes loaded with two palmitoyl peptides (Palpepcubes), namely GHKcube and GQPRcube, were prepared using an ultrasonication protocol. The Palpepcubes dimensions were characterized by dynamic light scattering (DLS) and cryo-transmission electron microscopy (cryo-TEM). Small-angle X-ray scattering (SAXS) analyses revealed that the bicontinuous cubic structure remained even at palmitoyl peptide contents as high as 5wt.%, with an increase in the cell parameter from approximately 6.5 to 7.2nm. Isothermal titration calorimetry (ITC) was used to elucidate the interactions between the blank cubosomes and the palmitoyl peptides, revealing an exothermic process of interaction. Moreover, the in vitro release of the palmitoyl peptides from the Palpepcubes was studied using a dialysis method coupled with liquid chromatography-mass spectrometry (LC/MS) technique, in which a sustained release of up to a few days was observed. Finally, the stability of the aqueous solutions of the palmitoyl peptides and the Palpepcubes kept at room temperature and at low temperature (4°C) was studied by LC/MS method, indicating that incorporation into cubosomes increases the peptide stability significantly.

Impact of preparation method and variables on the internal structure, morphology, and presence of liposomes in phytantriol-Pluronic(®) F127 cubosomes.

The formation of significant proportions of liposomes during the preparation of dispersed cubic phase particles presents a problem in trying to understanding cubosome behavior with a view to use in applications such as drug delivery. In this study, the variables impacting on liposome formation during cubosome production were interrogated. Bottom-up (BU) and top-down (TD) approaches were employed to prepare submicron sized liquid crystalline dispersions (cubosomes) of phytantriol in water with varying amounts of Pluronic(®)F127 (F127) as a stabilizer. In the BU approach, ethanol was used as a hydrotrope and was later removed using a rotary evaporator, whereas in the TD approach the bulk liquid gel was dispersed using ultrasonication. We aimed at finding the optimum ratio of phytantriol-to-F127 resulting in stable, liposome-free dispersions, whether this depends on the preparation method and the resulting morphology of the particles. The average particle size and zeta potential of the samples were measured using dynamic light scattering (DLS). Cryogenic transmission electron microscopy (Cryo-TEM) images showed a substantial number of liposomes in addition to cubosomes in the dispersion containing 4-1 (w/w) phytantriol-to-F127 prepared by the BU approach compared to very low liposome content with the TD approach. The effects of the amount of F127 in both approaches, amount of ethanol on the BU method and temperature on the TD method were investigated using small-angle X-ray scattering (SAXS). The cubosomes displayed cubic double-diamond (Pn3m) internal structure with a lattice parameter of approximately 6nm. In summary using the TD approach, with 4:1 phytantriol:F127 provided stable cubosome dispersion with minimal liposome co-existence.

Cellulose nanocrystal-poly(oligo(ethylene glycol) methacrylate) brushes with tunable LCSTs.

This paper reports on the synthesis of poly(oligoethylene glycol) methyl ether acrylate (POEGMA) grafted cellulose nanocrystals (CNCs) via surface initiated atom transfer radical polymerization (ATRP). An ATRP initiator (α-Bromoisobutyryl bromide) was covalently bonded to the surface of CNCs, followed by copolymerizing di(ethylene glycol) methyl ether methacrylate (MEO2MA) and oligoethylene glycol methyl ether methacrylate (OEGMA300) monomers from the surface using Cu(I)Br/2,2-dipyridal. Multiple POEGMA-g-CNC systems with varying MEO2MA/OEGMA300 content were synthesized, and they displayed a range of lower critical solution temperatures (LCSTs) in aqueous medium. µDSC endotherms and microstructural analysis indicated the collapse of POEGMA chains, followed by the aggregation of nanoparticles above their LCSTs. Cloud point measurements demonstrated a hysteresis in the heating and cooling of the POEGMA-g-CNC systems. It was found that the LCST of the nanoparticles could be tuned to between 23.8 to 63.8°C by adjusting the OEGMA300 content of the POEGMA brushes.

Synthesis of amine functionalized cellulose nanocrystals: optimization and characterization.

A simple protocol was used to prepare amine functionalized cellulose nanocrystals (CNC-NH2). In the first step, epichlorohydrin (EPH) was reacted with ammonium hydroxide to produce 2-hydroxy-3-chloro propylamine (HCPA). In the next step, HCPA was grafted to CNC using the etherification reaction in an organic solution media. Various reaction parameters, such as time, temperature, and reactant molar ratio were performed to determine the optimal reaction conditions. The final product (CNC-NH2(T)) was dialyzed for a week. Further purification via centrifugation yielded the sediment (CNC-NH2(P)) and supernatant (POLY-NH2). The presence of amine groups on the surface of modified CNC was confirmed by FTIR and the amine content was determined by potentiometric titration and elemental analysis. A high amine content of 2.2 and 0.6 mmol amine/g was achieved for CNC-NH2(T) and CNC-NH2(P), respectively. Zeta potential measurements confirmed the charge reversal of amine CNC from positive to negative when the pH was increased from 3 to 10. The flocculation of amine functionalized CNC due to its interactions with a negatively charged surfactant namely, sodium dodecyl sulfate (SDS) was investigated at pH 4. It showed promising results for applications, such as in flocculation of fine dispersions in water treatment. This simple and versatile synthetic method to produce high amine content CNC can be used for further conjugation as required for various applications.

Modified cellulose nanocrystal for vitamin C delivery.

Cellulose nanocrystal grafted with chitosan oligosaccharide (CNC-CSOS) was used to encapsulate vitamin C and prepare CNCS/VC complexes using tripolyphosphte via ionic complexation. The stability of vitamin C and the antioxidant activity of the CNCS/VC complexes were elucidated. The formation of the complex was confirmed using DSC and UV-vis spectrophotometry, and TEM was used to study the morphology of the complexes. The encapsulation efficiency of vitamin C at pH 3 and 5 was 71.6% ± 6.8 and 91.0 ± 1.0, respectively. Strong exothermic peaks observed in isothermal titration calorimetric (ITC) studies at pH 5 could be attributed to additional electrostatic interactions between CNC-CSOS and vitamin C at pH 5. The in vitro release of vitamin C from CNCS/VC complexes showed a sustained release of up to 20 days. The vitamin C released from CNCS/VC complex displayed higher stability compared with the control vitamin C solution, and this was also confirmed from the ITC thermograms. CNC-CSOS possessed a higher scavenging activity and faster antioxidant activity compared with its precursors, i.e., oxidized CNC and CSOS and their physical mixtures. Complexing vitamin C into CNC-CSOS particles yielded a dynamic antioxidant agent, where the vitamin C is released over time and displayed sustained antioxidant properties. Therefore, CNCS/VC can potentially be used in cosmeceutical applications as topical formulations.

Comparative release studies of two cationic model drugs from different cellulose nanocrystal derivatives.

Native cellulose nanocrystal (CNC), oxidized CNC (CNC-OX) and chitosan oligosaccharide grafted CNC (CNC-CSOS) were evaluated as potential drug delivery carriers for two model drug compounds, procaine hydrochloride (PrHy) and imipramine hydrochloride (IMI). The loading of PrHy and IMI was performed at pH 8 and 7, respectively. IMI displayed higher binding to CNC derivatives than PrHy. Drug selective membranes were prepared for each model drug and a drug selective electrode system was used to measure the drug concentration in the filtrate and release medium. Isothermal Titration Calorimetry (ITC) was used to elucidate the types of interactions between model drugs and CNC and its derivatives. The complexation between model drugs and CNC derivatives was confirmed by zeta potential and transmittance measurements. The binding and release of these drugs correlated with the nature and types of interactions that exist between the CNC and drug molecules.

Biodegradable and biocompatible polyampholyte microgels derived from chitosan, carboxymethyl cellulose and modified methyl cellulose.

Two biocompatible and biodegradable polyampholyte microgels, namely chitosan-carboxymethyl cellulose (CS-CMC) and chitosan-modified methyl cellulose (CS-ModMC) were synthesized by an inverse microemulsion technique. The CS-CMC microgel system was pH-responsive while the CS-ModMC system possessed both pH and thermo-responsive properties. For CS-CMC system, the number of -OCH2COOH and -NH2 groups was determined to be 1.5 and 1.1meq/g of microgel, respectively. In the pH range of 4-9, the zeta potential values varied from +10 to -40mV, while the hydrodynamic radius varied from 160nm in the swollen state (acidic and basic pH) to 110nm in the "collapse" state (neutral pH). Furthermore, TEM micrographs confirmed the swelling/deswelling behaviour of CS-CMC microgel particles at acidic, neutral and basic conditions. For CS-ModMC system, the number of -OCH2COOH and -NH2 groups was determined to be 0.8 and 0.6meq/g microgel, respectively. In the pH range of 4-9, the surface charge on the microgels varied from +25 to -60mV and the hydrodynamic radii were 190nm at low pH, 80nm at neutral pH, to 120nm at a high pH. In vitro drug release studies confirmed that CS-CMC microgels could encapsulate and release a model drug, thus they could potentially be used as biocompatible and biodegradable drug carriers.

Thiolated chitosan nanoparticles for enhancing oral absorption of docetaxel: preparation, in vitro and ex vivo evaluation.

The aim of this study was to prepare and evaluate mucoadhesive core-shell nanoparticles based on copolymerization of thiolated chitosan coated on poly methyl methacrylate cores as a carrier for oral delivery of docetaxel. Docetaxel-loaded nanoparticles with various concentrations were prepared via a radical emulsion polymerization method using cerium ammonium nitrate as an initiator. The physicochemical properties of the obtained nanoparticles were characterized by: dynamic light-scattering analysis for their mean size, size distribution, and zeta potential; scanning electron microscopy and transmission electron microscopy for surface morphology; and differential scanning calorimetry analysis for confirmation of molecular dispersity of docetaxel in the nanoparticles. Nanoparticles were spherical with mean diameter below 200 nm, polydispersity of below 0.15, and positive zeta potential values. The entrapment efficiency of the nanoparticles was approximately 90%. In vitro release studies showed a sustained release characteristic for 10 days after a burst release at the beginning. Ex vivo studies showed a significant increase in the transportation of docetaxel from intestinal membrane of rat when formulated as nanoparticles. Cellular uptake of nanoparticles was investigated using fluoresceinamine-loaded nanoparticles. Docetaxel nanoparticles showed a high cytotoxicity effect in the Caco-2 and MCF-7 cell lines after 72 hours. It can be concluded that by combining the advantages of both thiolated polymers and colloidal particles, these nanoparticles can be proposed as a drug carrier system for mucosal delivery of hydrophobic drugs.

Discriminated effects of thiolated chitosan-coated pMMA paclitaxel-loaded nanoparticles on different normal and cancer cell lines.

The aim of the present work was to prepare and characterize poly(methyl methacrylate) nanoparticles coated by chitosan-glutathione conjugate so as to encapsulate insoluble anticancer drugs. Nanoparticles were synthesized through radical polymerization of methyl methacrylate initiated by cerium (IV) ammonium nitrate. Paclitaxel (PTX), a model anticancer drug, was encapsulated in nanoparticles with a maximal encapsulation efficiency of 98.27%. These nanoparticles showed sustained in vitro release of the incorporated PTX (75% of the loaded dose was released in 10 days). All nanoparticles had positive charge and were spherical, with a size range of about 130-250 nm. The PTX-loaded nanoparticles showed cytotoxicity for NIH 3T3 and T47D breast carcinoma cells, along with no cytotoxicity for two colon cell lines (HT29, Caco2). FROM THE CLINICAL EDITOR: The aim of this work was to prepare and characterize poly(methyl methacrylate) nanoparticles coated by chitosan-glutathione conjugate in an effort to encapsulate Paclitaxel as a model of insoluble anticancer drugs. These nanoparticles showed sustained in vitro drug release.