Surface Modification and Characterization of Cellulose Nanocrystal for Biomedical Applications

There is an ever-increasing desire to develop novel materials that could control the release of active compounds and increase their stability. Replacing petroleum-based synthetic polymers with sustainable materials has many advantages, such as reducing the dependence on fossil fuels, and diminishing environmental pollution. Recently, cellulose nanocrystal (CNC) obtained by acid hydrolysis of cellulose fibres has gained a lot of interest. The high mechanical strength, large and negatively charged surface area, and the presence of several hydroxyl groups that allow for modification with different functionalities make CNC an excellent candidate for various applications in the biomedical field. This thesis explores (i) the surface modification and characterization of modified CNC and (ii) the biomedical applications of these novel sustainable nanomaterials. In the first part, amine functionalized CNC was prepared. Ammonium hydroxide was reacted with epichlorohydrin (EPH) to produce 2-hydroxy-3-chloro propylamine (HCPA), which was then grafted to CNC using an etherification reaction. A series of reactions were carried out to determine the optimal conditions. The final product (CNC-NH2(T)) was dialyzed for one week. Further purification via centrifugation yielded the sediment (CNC-NH2(P)) and supernatant (POLY-NH2). The presence of amine groups was confirmed by FT-IR 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 negative to positive when the pH was decreased from 10 to 3. TEM images showed similar structural properties of the nanocrystals along with some minor aggregation. This simple, yet effective synthesis method can be used for further conjugation as required for various biomedical applications. Moreover, the surface of CNC was modified with chitosan oligosaccharide (CSos). First, the primary alcohol groups of CNC were selectively oxidized to carboxyl groups using the catalyst, 2,2,6,6- tetramethylpiperidine-1-oxyl radical (TEMPO), and were then reacted with the amino groups of CSos via the carbodiimide reaction using N-hydroxysuccinimide (NHS) and 1-Ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC). The appearance of C=O peak in FT-IR spectrum of oxidized CNC (CNC-OX), accompanied by calculations based on potentiometric titration revealed that CNC was successfully oxidized with a degree of oxidation of 0.28. The grafting of CSos on oxidized CNC was confirmed by the following observations: (i) the reduction of the C=O peak in FT-IR of CNC-CSos and the appearance of new amide peaks; (ii) the significant reduction of the carbonyl peak at 175 ppm in the 13C NMR spectrum for CNC-CSos; (iii) a higher decomposition temperature in TGA of CNC-CSos; (iv) a positive zeta potential of CNC-CSos at acidic pH; and (v) a degree of substitution of 0.26, which is close to the DO (0.28), indicating that 90% of COOH groups on CNC-OX were involved in the formation of amide bonds with CSos. TEM and AFM studies also revealed a completely diff erent morphology for CNC-CSos. In the second part, the potential of exploiting CNCs as delivery carriers for two cationic model drugs, procaine hydrochloride (PrHy) and imipramine hydrochloride (IMI), were investigated. IMI displayed a higher binding to CNC derivatives compared to PrHy. Isothermal titration calorimetry (ITC), transmittance and zeta potential measurements were used to elucidate the complexation between model drugs and CNC samples. It was observed that the more dominant exothermic peak observed in the ITC isotherms leading to the formation of larger particle-drug complexes could explain the increased binding of IMI to CNC samples. Drug selective membranes were prepared for each model drug that displayed adequate stability and rapid responses. Different in vitro release profiles at varying pH conditions were observed due to the pH responsive properties of the systems. Both drugs were released rapidly from CNC samples due to the ion-exchange e ffect, and CNC-CSos displayed a more sustained release profile. Furthermore, the antioxidant properties of CNC samples and the potential of CNC-CSos as a carrier for the delivery of vitamin C was investigated. CNC-CSos/vitamin C complexes (CNCS/VC) were formed between CNC-CSos and vitamin C via ionic complexation using sodium tripolyphosphate (TPP). The complexation was confirmed via DSC and UV-Vis absorbance measurements. TEM images showed complexes with a size of approximately 1 micron. The encapsulation efficiency of vitamin C was higher (91%) at pH 5 compared to pH 3 (72%). The in vitro release of vitamin C from CNCS/VC complexes exhibited a sustained release of up to 3 weeks, with the released vitamin C displaying higher stability compared to a control vitamin C solution. Antioxidant activity and kinetics of various CNC samples were studied using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay. CNC-CSos possessed a higher scavenging activity and faster antioxidant activity compared to its precursors, CNC-OX and CSos, and their physical mixture. Therefore, by loading vitamin C into CNC-CSos particles, a dynamic antioxidant system was produced. Vitamin C can be released over a prolonged time period displaying enhanced and sustained antioxidant properties since the carrier CNC-CSos also possesses antioxidant properties. As a result of this doctoral study, knowledge on the surface modification of CNC with amine groups and CSos have been advanced. The in vitro drug release and antioxidant studies suggest that systems comprising of CNC could be further explored as potential carriers in biomedical applications.4 month

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

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 5 wt.%, with an increase in the cell parameter from approximately 6.5 to 7.2 nm. 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 degrees C) was studied by LC/MS method, indicating that incorporation into cubosomes increases the peptide stability significantly1176067FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULO - FAPESP2014/24305-8; 2015/25406-

Impact Of Preparation Method And Variables On The Internal Structure, Morphology, And Presence Of Liposomes In Phytantriol-pluronic (r) F127 Cubosomes

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)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 (R) 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 6 nm. In summary using the TD approach, with 4:1 phytantriol:F127 provided stable cubosome dispersion with minimal liposome co-existence. (C) 2016 Elsevier B.V. All rights reserved.145845853Sao Paulo Research Foundation (FAPESP) [2014/24305-8]Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP

Synthesis Of Amine Functionalized Cellulose Nanocrystals: Optimization And Characterization

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)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. (C) 2015 Elsevier Ltd. All rights reserved.4094855CelluForceAboraNanoCFINSERCCoordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)CAPES [PVE 128/2013

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).Fil: Bessone, Carolina del Valle. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Unidad de Investigación y Desarrollo en Tecnología Farmacéutica. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Unidad de Investigación y Desarrollo en Tecnología Farmacéutica; ArgentinaFil: Akhlaghi, Seyedeh Parinaz. Universidade Estadual de Campinas; BrasilFil: Tartara, Luis Ignacio. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Unidad de Investigación y Desarrollo en Tecnología Farmacéutica. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Unidad de Investigación y Desarrollo en Tecnología Farmacéutica; ArgentinaFil: Quinteros, Daniela Alejandra. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Unidad de Investigación y Desarrollo en Tecnología Farmacéutica. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Unidad de Investigación y Desarrollo en Tecnología Farmacéutica; ArgentinaFil: Loh, Watson. Universidade Estadual de Campinas; BrasilFil: Allemandi, Daniel Alberto. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Unidad de Investigación y Desarrollo en Tecnología Farmacéutica. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Unidad de Investigación y Desarrollo en Tecnología Farmacéutica; Argentin