Preparation and Characterisation of Highly Loaded Fluorescent Chitosan Nanoparticles

Chitosan (CS) nanoparticles have been developed as a versatile drug delivery system to transport drugs, genes, proteins, and peptides into target sites. Demands on fluorescent nanoparticles have increased recently due to various applications in medical and stem-cell-based researches. In this study, fluorescent CS nanoparticles were prepared by a mild method, namely, complex coacervation. Entrapment efficiency of sulforhodamine (SR101) loaded into CS nanoparticles was investigated to evaluate their capacity in incorporating fluorescent molecule. Particle size of produced fluorescent nanoparticles was in the range of 600–700 nm, and their particle size was highly dependent on the CS molecular weight as well as concentration. A high entrapment efficiency of SR101 into CS nanoparticles could also be obtained when it was dissolved in methanol. In conclusion, highly loaded fluorescent CS nanoparticles could be easily prepared using complex coacervation method and therefore can be applied in various medical researches

Development of Chitosan Nanoparticles as a Stable Drug Delivery System for Protein/siRNA

Chitosan nanoparticles (CS NPs) exhibit good physicochemical properties as drug delivery systems. The aim of this study is to determine the modulation of preparative parameters on the physical characteristics and colloidal stability of CS NPs. CS NPs were fabricated by ionic interaction with dextran sulphate (DS) prior to determination of their storage stability. The smallest CS NPs of 353 ± 23 nm with a surface charge of +56.2 ± 1.5 mV were produced when CS and DS were mixed at pH 4 and with a DS : CS mass ratio of 0.5 : 1. An entrapment efficiency of 98% was achieved when BSA/siRNA was loaded into the nanoparticles. The results also showed that particle size and surface charge of CS NPs were slightly changed up to 2 weeks when stored at 4 ∘ C. Greater particle size and surface charge were obtained with increasing the concentration of DS. In conclusion, NPs were sufficiently stable when kept at 4 ∘ C and able to carry and protect protein

In Vitro

Mixed micelles of Pluronic F127 and D-α-tocopheryl polyethylene glycol 1000 succinate (TPGS) in different molar ratios (10 : 0, 7 : 3, 5 : 5, and 3 : 7) were prepared to characterize this system as nanocarriers for targeted delivery of chemotherapeutic agents. Their size, zeta potential, critical micelle concentration, drug loading content, entrapment efficiency, drug release, cytotoxicity, and stability in serum were evaluated in vitro by using doxorubicin as the model anticancer drug. The micellar sizes ranged from 25 to 35 nm. The 7 : 3 and 5 : 5 micellar combinations had lower critical micelle concentrations ( M) than the 10 : 0 combination ( M). The entrapment efficiencies of the 7 : 3, 5 : 5, and 3 : 7 micellar combinations were 72%, 88%, and 69%, respectively. Doxorubicin release was greater at acidic tumour pH than at normal physiological pH. The doxorubicin-loaded mixed micelles showed greater percent inhibition and apoptosis activity in human breast adenocarcinoma (MCF-7) and acute monocytic leukaemia (THP-1) cell lines than free doxorubicin did. The mixed micelles were also stable against aggregation and precipitation in serum. These findings suggest that Pluronic F127-TPGS mixed micelles could be used as nanocarriers for targeted anticancer-drug delivery

Preparation, characterization and in vitro release study of BSA-loaded double-walled glucose-poly(lactideco- glycolide) microspheres

The aim of this study was to prepare a model protein, bovine serum albumin (BSA) loaded double-walled microspheres using a fast degrading glucose core, hydroxyl-terminated poly(lactide-co-glycolide) (Glu- PLGA) and a moderate-degrading carboxyl-terminated PLGA polymers to reduce the initial burst release and to eliminate the lag phase from the release profile of PLGA microspheres. The double-walled microspheres were prepared using a modified water-in-oil-in-oil-in-water (w/o/o/w) method and singlepolymer microspheres were prepared using a conventional water-in-oil-in-water (w/o/w) emulsion solvent evaporation method. The particle size, morphology, encapsulation efficiency, thermal properties, in vitro drug release and structural integrity of BSA were evaluated in this study. Double-walled microspheres prepared with Glu-PLGA and PLGA polymers with a mass ratio of 1:1 were non-porous, smooth-surfaced, and spherical in shape. A significant reduction of initial burst release was achieved for the double-walled microspheres compared to single-polymer microspheres. In addition, microspheres prepared using Glu- PLGA and PLGA polymers in a mass ratio of 1:1 exhibited continuous BSA release after the small initial burst without any lag phase. It can be concluded that the double-walled microspheres made of Glu-PLGA and PLGA polymers in a mass ratio of 1:1 can be a potential delivery system for pharmaceutical proteins

Controlled release of lysozyme from double-walled poly(Lactide-Co-Glycolide) (PLGA) microspheres

Double-walled microspheres based on poly(lactide-co-glycolide) (PLGA) are potential delivery systems for reducing a very high initial burst release of encapsulated protein and peptide drugs. In this study, double-walled microspheres made of glucose core, hydroxyl-terminated poly(lactide-co-glycolide) (Glu-PLGA), and carboxyl-terminated PLGA were fabricated using a modified water-in-oil-in-oil-in-water (w1/o/o/w2) emulsion solvent evaporation technique for the controlled release of a model protein, lysozyme. Microspheres size, morphology, encapsulation efficiency, lysozyme in vitro release profiles, bioactivity, and structural integrity, were evaluated. Scanning electron microscopy (SEM) images revealed that double-walled microspheres comprising of Glu-PLGA and PLGA with a mass ratio of 1:1 have a spherical shape and smooth surfaces. A statistically significant increase in the encapsulation efficiency (82.52 ± 3.28%) was achieved when 1% (w/v) polyvinyl alcohol (PVA) and 2.5% (w/v) trehalose were incorporated in the internal and external aqueous phase, respectively, during emulsification. Double-walled microspheres prepared together with excipients (PVA and trehalose) showed a better control release of lysozyme. The released lysozyme was fully bioactive, and its structural integrity was slightly affected during microspheres fabrication and in vitro release studies. Therefore, double-walled microspheres made of Glu-PLGA and PLGA together with excipients (PVA and trehalose) provide a controlled and sustained release for lysozyme

Development of nanocarriers for siRNA based on cationic polymers

A diverse range of viral and non-viral strategies has been developed more than a decade for a gene delivery such as plasmid DNA (pDNA) and oligodeoxynucleotides (ODN). Recently, the development has been extended to a newly discovered class of molecule, small interfering RNA (siRNA). The use of cationic and biodegradable polymeric particles has been widely utilised for delivery of these moieties. This study therefore, aimed to develop and investigate cationic polymers mainly chitosan and polyethylenimine (PEI) as well as their derivative particles; PLGA-PEI and -chitosan nanoparticles as siRNA carriers. Additionally, TAT-peptide has also been investigated as one of the delivery systems for siRNA. Certain process and formulation parameters have been extensively studied with regards of physical and biological properties of the above systems to obtain an optimal delivery system. Physical properties particularly particle size, surface charge as well as particle morphology have been found to be influenced by certain parameters such as homogenisation or stirring rate, molecular weight and concentration of polymers as well as other processes involved in obtaining final products (e.g. centrifugation, freeze-drying). In-vitro evaluations in cultured cells have revealed that the derivative PLGA-PEI nanoparticles are capable of transfecting mammalian cells with siRNA better than the parent compound, PEI. In addition to that, chitosan nanoparticles simply prepared by ionic gelation have been shown to be more competent as siRNA carriers compared to other types of chitosan-based nanoparticles investigated in this study, either chitosan- siRNA complexes or PLGA-chitosan nanoparticles. Although type and molecular weight of chitosan are important in delineating characteristics of particle size and surface charge (the two factors normally important in determining capability of particulate systems to transfect cells), it appears that the type and molecular weight of chitosan have not shown any obvious correlation with the level of the targeted gene knockdown by siRNA. TAT-peptide siRNA complexes were shown to be capable of successfully delivering siRNA into cells without the need of chemical conjugation and the effect could be enhanced by the addition of calcium into the particle suspensions before transfection. Further investigations using chitosan nanoparticles prepared by ionic gelation, have been used to deliver MAPK-14 siRNA in the macrophage cell line, J774A.1 have shown that the system has the ability to transfect cells and subsequently allow the delivered siRNA to knockdown the targeted endogenous gene, MAPK p38α with a sustained effect and a relatively low cytotoxicity. In conclusion, the ability of these polymers as a carrier for siRNA is highly dependent on the method of preparation and their physicochemical characteristics of each of these polymeric particles

Preparation of polyethyleneimine incorporated poly(D,L-lactide-co-glycolide) nanoparticles by spontaneous emulsion diffusion method for small interfering RNA delivery

Gene therapy based on small interfering RNA (siRNA) has emerged as an exciting new therapeutic approach. However, insufficient cellular uptake and poor stability have limited its usefulness. Polyethyleneimine (PEI) has been extensively studied as a vector for nucleic acids and incorporation of PEI into poly(D,L-lactide-co-glycolide) (PLGA) particles has been shown to be useful in the development of gene delivery. PEI was incorporated into the PLGA particles by spontaneous modified emulsification diffusion method. Incorporation of PEI into PLGA particles with the PLGA to PEI weight ratio 29:1 was found to produce spherical and positively charged nanoparticles where type of polymer, type and concentration of surfactant could affect their physical properties. Particle size of around 100 nm was obtained when 5% (m/v) PVA was used as a stabiliser. PLGA-PEI nanoparticles were able to completely bind siRNA at N/P ratio 20:1 and to provide protection for siRNA against nuclease degradation. In vitro cell culture studies subsequently revealed that PLGA-PEI nanoparticles with adsorbed siRNA could efficiently silence the targeted gene in mammalian cells, better than PEI alone, with acceptable cell viability. PLGA-PEI nanoparticles have been found to be superior to its cationising parent compound; PEI polymer. Crown Copyright (C) 2008 Published by Elsevier B.V. All rights reserved