Emulsion Cross-linking Method Research Articles

Chitosan Nanoparticles: A Promising System in Novel Drug Delivery

The ability of nanoparticles to manipulate the molecules and their structures has revolutionized the conventional drug delivery system. The chitosan nanoparticles, because of their biodegradability, biocompatibility, better stability, low toxicity, simple and mild preparation methods, offer a valuable tool to novel drug delivery systems in the present scenario. Besides ionotropic gelation method, other methods such as microemulsion method, emulsification solvent diffusion method, polyelectrolyte complex method, emulsification cross-linking method, complex coacervation method and solvent evaporation method are also in use. The chitosan nanoparticles have also been reported to have key applications in parentral drug delivery, per-oral administration of drugs, in non-viral gene delivery, in vaccine delivery, in ocular drug delivery, in electrodeposition, in brain targeting drug delivery, in stability improvement, in mucosal drug delivery in controlled drug delivery of drugs, in tissue engineering and in the effective delivery of insulin. The present review describes origin and properties of chitosan and its nanoparticles along with the different methods of its preparation and the various areas of novel drug delivery where it has got its application.

Preparation and Characterization of 5-Fluorouracil Loaded Chitosan Microspheres by a Two-Step Solidification Method

Chitosan, as a natural biopolymer, has potential applications in controlling the release of drugs due to its unique natural and physiochemical properties. Conventionally, an emulsion crosslinking method was used to prepare chitosan microspheres. In this study, a novel method of preparing 5-fluorouracil-loaded chitosan microspheres was established by combining the emulsification technique and a two-step solidification method using both glutaraldehyde (GTA) and tripolyphosphate (TPP) as crosslinking agents. The microspheres were characterized by surface morphology, encapsulation efficiency, yield of microspheres and drug release profile in vitro. The optimal chitosan microspheres were achieved by crosslinking chitosan with both TPP (0.1%, pH 4.0) and GTA (1.0%, v/v) with span-80 additive under a crosslinking time of 1 h with TPP and GTA, respectively.

Comparative delivery of Diltiazem hydrochloride through synthesized polymer: Hydrogel and hydrogel microspheres

AbstractIn this study, interpenetrating polymer network (IPN) hydrogel based on polyvinyl alcohol (PVA) networking with polyacrylic acid (PAA) were prepared by a non‐conventional emulsion method without any added crosslinker, using benzoyl peroxide as initiator and sodium chloride (NaCl) as additive. The IPN hydrogel was characterized by using Fourier transformed infrared (FTIR) spectrophotometry, Thermo gravimetric analysis (TGA), and Scanning electron microscopy (SEM). (PVA‐co‐PAA)/NaCl normal IPN hydrogel (H) were fabricated into hydrogel microspheres (HM) by modified emulsion crosslinking method using glutaraldehyde‐saturated toluene as crosslinker and were loaded with Diltiazem hydrochloride (DL). The IPN hydrogel showed more swelling in simulated intestinal fluid (SIF). (PVA‐co‐PAA)/NaCl HM formulation A1 showed comparatively higher DL entrapment (79%) and better control over DL release up to 24 h. By comparing antihypertensive activity of DL loaded two formulations in normotensive rats, HM formulation A1 found more effective in reducing blood pressure to 40.1%. The experimental results demonstrated that (PVA‐co‐PAA)/NaCl HM had the greater potential than normal hydrogel to be used as a drug carrier. A single use of the prepared hydrogel microsphere system of DL can effectively control hypertension in rats. The system holds promise for clinical studies. © 2009 Wiley Periodicals, Inc. J Appl Polym Sci, 2010

The biological properties of decelluarized valve scaffolds modified by polyethylene glycol microspheres loading transforming growth factor-β1

Objective To prepare decelluarized valve scaffolds modified by polyethylence glycol (PEG) microspheres loading transforming growth factor-β1 (TGF-β1),and observe its biological proper-ties. Methods PEG microspheres were produced by an emulsion-crosslinking method,and scanning elec-tron microscopic observation was performed to examine the average diameter. Decelluarized valve scaffolds were modified with PEG microspheres, then TGF-β1 was loaded into them by adsorption. Controlled release of TGF-β1 was measured by ELISA, and histomorphology observation and molecular biological detection were performed. The myofibroblasts harvested from rats were seeded on the scaffolds. After culture for one week in vitro, histomorphology observation and molecular biological detection were performed. Results The average diameter of PEG microspheres was (42.72±3.48) nm. The percentage of cumulative release was 67.22% within 7 days,and the encapsulation efficiency was 82.01%. Compared with the simple de-celluarized valve scaffolds, the modified scaffolds showed that cells contacted closely and extracellular ma-trix were more bloomy,and DNA and hydroxyproline contents were also increased. Conclusion Decelluar-ized valve scaffolds modified by PEG microspheres loading TGF-β1 are beneficial to cell proliferation, se-cretion of collagen, and the construction of TEHV. Key words: Valve; Tissue engineering; Polyethylene glycol; Transforming growth factor-β1

Novel carboxymethyl chitin nanoparticles for cancer drug delivery applications

Carboxymethyl chitin (CMC) nanoparticles were prepared by cross-linking of CMC solution with CaCl 2 and FeCl 3. The cytotoxicity of the CMC nanoparticles was evaluated using MTT assay and they were found to be non-toxic into L929 mouse cells. The antibacterial and magnetic properties of the CMC nanoparticles were also studied. The prepared CMC nanoparticles were characterized using SEM and FTIR. The model hydrophobic anticancer drug 5-fluorouracil (5-Fu) was loaded into CMC nanoparticles via emulsion cross-linking method. The encapsulation efficiency and in-vitro drug release behaviour of drug-loaded nanoparticles were studied by UV spectrophotometer. Drug release studies showed that the CMC nanoparticles showed controlled and sustained drug release at pH 6.8. Moreover, the prepared nanoparticles were also found to be antibacterial and their magnetic properties reveals for their potential use in drug tracking. These results indicated that CMC nanoparticles are a promising carrier system for controlled drug delivery.

Formulation and in vitro–in vivo evaluation of ketoprofen-loaded albumin microspheres for intramuscular administration

The objective of this work was to prepare and evaluate ketoprofen-loaded albumin microspheres for intramuscular administration. Microspheres were prepared by emulsion cross-linking method using a 23 factorial design and the effect of different factors on entrapment efficiency was determined. Microspheres were evaluated for entrapment efficiency, percentage yield, particle size and release behaviour. Selected formulations were then tested by differential scanning calorimetry, X-ray diffraction and scanning electron microscopy. Further they were analysed for residual solvents, syringeability and stability. Microspheres were then sterilized and bioavailability studies were carried out in New Zealand white rabbits. The physical characteristics of microspheres showed that they were suitable for IM administration. The sterilization technique adopted was adequate to maintain sterility. In vivo studies showed increase in Cmax, AUC, t1/2 and MRT (p < 0.05) administered in the form of microspheres. MRT of ketoprofen was almost 3.2-times in the form of microspheres. From these results it was concluded that the developed albumin microspheres of ketoprofen is a potential delivery system for once-a-day intramuscular administration.

Anti-tumor activity of paclitaxel-loaded chitosan nanoparticles: An in vitro study

Chitosan nanoparticles containing the anticancer drug paclitaxel were prepared by a solvent evaporation and emulsification crosslinking method. The physicochemical properties of the nanoparticles were characterized by various techniques, and uniform nanoparticles with an average particle size of 116 ± 15 nm with high encapsulation efficiencies (EE) were obtained. Additionally, a sustained release of paclitaxel from paclitaxel-loaded chitosan nanoparticles was successful. Using different ratios of paclitaxel-to-chitosan, the EE ranged from 32.2 ± 8.21% to 94.0 ± 16.73 %. The drug release rates of paclitaxel from the nanoparticles were approximately, 26.55 ± 2.11% and 93.44 ± 10.96% after 1 day and 13 days, respectively, suggesting the potential of the chitosan nanoparticles as a sustained drug delivery system. Cytotoxicity tests showed that the paclitaxel-loaded chitosan had higher cell toxicity than the individual paclitaxel and confocal microscopy analysis confirmed excellent cellular uptake efficiency. TEM images showed the ultrastructure changes of A2780 cells incubated with paclitaxel-loaded nanoparticles. Flow cytometric analysis revealed two subdiploid peaks for the cells in the paclitaxel-loaded nanoparticles and paclitaxel treated groups, respectively, with the intensity of the former higher than that of the latter. Moreover, the cell cycle was arrested in the G 2-M phase, which was consistent with the action mechanism of the direct administration of paclitaxel. These results indicate that chitosan nanoparticles have potential uses as anticancer drug carriers and also have an enhanced anticancer effect.

Controlled release of theophylline through semi-interpenetrating network microspheres of chitosan-(dextran-g-acrylamide)

Semi-interpenetrating network microspheres of chitosan-(dextran-g-acrylamide) were prepared by emulsion-crosslinking method using glutaraldehyde (GA) as a crosslinking agent. Graft copolymerization of dextran with acrylamide (Dx-g-AAm) was carried out by aqueous free-radical polymerization using ceric ammonium nitrate (CAN) as initiator. The grafting efficiency was found to be 92%. Theophylline (TH), antiasthmatic drug, was successfully encapsulated into semi-INP microspheres by varying the ratio of Dx-g-AAm and amount of GA. The laser light scattering technique shows that the particles size increased with increasing amount of graft copolymer and decrease with increasing amount of GA. The % encapsulation efficiency was found to vary between 50 and 78. MPs were characterized by FTIR spectroscopy and differential scanning calorimetry (DSC) techniques to confirm the graft copolymer, formation of semi-IPN structure of MPs and molecular distribution of the drug molecules in the polymer matrix. In vitro release studies of TH from these matrices have been investigated at Ph 1.2 and 7.4 media and the slow release were extended up to 18 h at 37 degrees C. The release rates were fitted to an empirical equation to estimate the diffusion exponent n, which indicated that the release from the MPs follows non-Fickian type.

Preparation and evaluation of biodegradable microspheres of methotrexate

The objective of the present study is to prepare sustained-release methotrexate microspheres of bovine serum albumin in different ratios by the emulsion cross-linking method. The prepared microspheres were subjected to various physicochemical evaluation and in vitro release studies. The drug release from microspheres of 1:6 ratio is the most constant and prolonged drug release is diffusion followed by erosion. The characteristics of the prepared microspheres are conducive to the formulation of the sustained release drug delivery system.

Biodegradable Microspheres Based on Gelatin-Porcine Mucin Admixtures: in Vitro and in Vivo Delivery Studies

Soluble mucus glycoprotein (S-mucin) processed from the small intestines (ileal region) of freshly slaughtered pigs via homogenization, dialysis, centrifugation and lyophilization and their admixtures with type B gelatin were used to prepare cefaclor-loaded microspheres by the emulsification-crosslinking method. The microspheres were evaluated for the in vitro delivery of cefaclor in both simulated intestinal fluid (SIF) without pancreatin (pH 7.4) and simulated gastric fluid (SGF) without pepsin (pH 1.2). Results obtained indicated that the microspheres formulated were highly mucoadhesive and that release of cefaclor in both release media was non-Fickian and was much higher and more rapid in SGF than in SIF and from microspheres based on gelatin alone when compared to those based on gelatin-procine mucin admixtures. The mean area under the plasma level versus time curves (AUC) was shown to be dependent on the formulation with values of 172.3 mug.h/ml for the control, 278.5 mug.h/ml for microspheres based on gelatin only and 353.0 mug.h/ml for microspheres formulated with equal parts of gelatin and mucin indicating that the rectal route may provide a therapeutically viable alternative to the oral route for the delivery of cefaclor. Further indications also emerged of a possibility of site-specific delivery of cefaclor to the small intestine through a careful selection of gelatin type and porcine mucin admixtures prior to formulation of the microspheres. On the whole, the inclusion of S-mucin in the composition of the microspheres had an enhancer effect on the release and rectal bioavailability of cefaclor which may be exploited in the design of a rectal delivery system of the drug.

Physical properties and loading capacity of starch-based microparticles crosslinked with trisodium trimetaphosphate

Starch-based microparticles were prepared by emulsion crosslinking method, using trisodium trimetaphosphate (TSTP) as crosslinking agent. The prepared TSTP-crosslinked starch microparticles (TSMs) were characterized by scanning electron microscopy (SEM), X-ray diffraction (XRD) and Fourier transform infrared (FTIR) technique. SEM pictures showed smooth face and spherical shape for the TSMs. XRD patterns revealed that TSMs were of amorphous structure mostly. This result was confirmed by FTIR spectra. Influence of crosslinking density on particle size, swelling degree and loading capacity of TSMs was studied by varying TSTP concentration. It was found that with increasing TSTP concentration from 0.1 to 0.4 g/g, the particle size and loading capacity increased, but swelling degrees showed a bell-shaped dependence on TSTP concentration with maximum values at the concentration of 0.2 g/g in different media. However, the changes of these three aspects became inconspicuous when TSTP concentration increased to 0.4 g/g. It is estimated that the crosslinking density had nearly reached its full extent at such concentration. The present investigated TSMs could potentially be applied in dry powder form for food ingredients delivery.

Three-step experimental design to determine the effect of process parameters on the size of chitosan microspheres

Investigation was carried out to elucidate the influence of process parameters on the mean particle size of chitosan microspheres produced by water-in-oil (w/o) emulsion crosslinking method. The aqueous chitosan solution was dispersed in the oil phase composed of 40% (v/v) sunflower oil and 60% (v/v) n-hexadecane in the presence of Tween 80 surfactant. The droplets were solidified and hardened by glutaraldehyde crosslinking agent. The particle size of the obtained microspheres was influenced by four independent process variables, such as stirring rate, and the concentrations of chitosan, Tween 80 and glutaraldehyde. To study these effects, 4-factorial 3-level Box-Behnken type experimental design and statistical analysis of the experimental results were carried out in three consecutive steps. First, the 4 independent factors at 3 levels were taken into consideration without repetitions, resulting in 27 scheduled experiments. Second, the design was completed by further experiments to check the effects of some of the variables at five levels both inside and outside the original factor intervals used in the first step. In addition, five repeated experiments at the central points of the four factors were also involved in the second step to determine the pure error. In order to elucidate the effects of process variables on the mean particle size, non-linear multidimensional regression was determined. Third, the less significant effects were neglected resulting in a simplified model equation that proved to be suitable for the prediction of the mean particle size as a function of the studied process parameters.

Fabrication of starch-based microparticles by an emulsification-crosslinking method

In this study, starch-based microparticles (MPs) fabricated by a water-in-water (w/w) emulsification-crosslinking method could be used as a controlled release delivery vehicle for food bioactives. Due to the processing route without the use of toxic organic solvents, it is expected that these microparticles can be used as delivery vehicles for controlled release of food bioactives. Octenyl succinic anhydride (OSA) starch was used as raw material. Optical microscopy showed OSA starch-based microparticles (OSA-MPs) had a good dispersibility. Scanning electron microscopy (SEM) showed OSA-MPs had a solid structure and spherical shape. X-ray diffraction (XRD) patterns revealed that OSA-MPs were of amorphous structure. A Plackett–Burman screening design methodology was employed to evaluate the effects of the process and formulation parameters on the particle size of OSA-MPs. Considering the statistical analysis of the results, it appeared that the OSA starch concentration ( P = 0.0146), poly(ethylene glycol) (PEG) molecular weight ( P = 0.0155), volume ratio of dispersed phase/continuous phase ( P = 0.0204) and PEG concentration ( P = 0.0230) had significant effect on particle size.

Preparation and antibacterial activity of chitosan microshperes in a solid dispersing system

In this study, we investigated the interface contacting inhibition behaviors of chitosan against bacterial in the dispersing state. For that purpose, chitosan microspheres (CMs) in the dispersing state was prepared by the emulsification cross-linking method. The CMs had smooth surface and spherical shape with the diameter of about 124 µm. They were stable after sterilization at 121°C and 150 kPa for 20 min. The CMs had similar antibacterial activity to that of chitosan in the solution form. Their antibacterial activities increased with the increase of the CM concentration, while decreased with the increase of pH of the system. It was found that the CMs with the degree of deacetylation (DD) of 63.6% exhibited the highest antibacterial activity, while the CMs with the DD of 83.7% exerted the lowest antibacterial activity among the three tested samples.

Effect of cross-linking agent on the characteristics of celecoxib loaded chitosan microspheres

The purpose of the present investigation was to compare the characteristics of the microspheres of chitosan prepared using two different cross-linking agents: viz. formaldehyde and glutaraldehyde and by simple heat treatment. Chitosan microspheres were prepared by emulsification cross-linking method. Microspheres were characterized for entrapment efficiency, particle size, in-vitro drug release and surface morphology was studied by scanning electron microscopy. The entrapment efficiency of the glutaraldehyde and formaldehyde cross-linked microspheres was significantly higher (P<0.05) than the heat-cross-linked microspheres. In-vitro drug release studies indicated that the microspheres cross-linked using glutaraldehyde showed slower release rate than those cross-linked with formaldehyde while the heat cross-linked microspheres showed the fastest release.

Beta-galactosidase immobilization on chitosan microspheres

Application of solid-phase biocatalysts has become important during the last decades. Enzymes could be immobilized on various supports and by different methods. The properties of immobilized biocatalysts are influenced by the characteristics of enzyme, support material and the immobilization method. A suitable support should have high affinity to proteins, reactive functional groups, hydrophilicity, mechanical stability and rigidity. Chitosan is a natural polyaminosaccharide which offers the above characteristics, therefore it is often used for enzyme immobilization (Martino et al., 1996; Sheu et al., 1998). Chitosan microspheres suitable for enzyme immobilization were prepared by emulsion cross-linking method (Agnihotri et al., 2004). Aqueous chitosan solution was added dropwise into an oil phase consisted of sunflower oil and n-hexadecane. Cross-linking was performed with glutaraldehyde which make possible the covalent attachment of enzymes. �-Galactosidase, which is applied in dairy industry for lactose hydrolysis, was immobilized onto the microspheres. The solid-phase biocatalyst was characterized and the storage and operational stabilities were investigated.

Preparation and Characterization of Biodegradable β-TCP-Based Composite Microspheres as Bone Tissue Engineering Scaffolds

Since a small globular particle was first used as support for three-dimensional (3D) growth of anchorage-dependent cells in suspended cultures, a variety of microspheres as tissue engineering scaffolds have been developed. In this paper, β-TCP and chitosan were selected as the components of microspheres due to their biodegradability and osteogenic properties. The biodegradable β-TCP/chitosan composite microspheres were prepared by a solid-in-water-in-oil (s/w/o) emulsion cross-linking method in this paper. The size distribution, surface morphology, and microstructure of the microspheres were evaluated. Scanning electron microscopy revealed that the size of the microspheres with good spherical morphology was distributed in the range of 50~200μm. In vitro immersion experiments were carried out to evaluate the degradability of the microspheres, and the results demonstrated that the chitosan/β-TCP composite microspheres were potential materials as tissue engineering scaffolds for bone repair.

Formulation and evaluation of ketorolac tromethamine-loaded albumin microspheres for potential intramuscular administration

The objective of this work was to prepare and evaluate ketorolac tromethamine-loaded albumin microspheres using a factorial design. Albumin microspheres were prepared by emulsion cross-linking method. Selected formulations were characterized for their entrapment efficiency, particle size, surface morphology, and release behavior. Analysis of variance (ANOVA) for entrapment efficiency indicated that entrapment efficiency is best fitted to a response surface linear model. From the statistical analysis it was observed that as the drug:polymer (D:P) ratio and volume of glutaraldehyde increased, there was a significant increase in the encapsulation efficiency. Scanning electron microscopy of the microspheres revealed a spherical, nonporous and uniform appearance, with a smooth surface. Based on the entrapment efficiency and physical appearance, 9 formulations were selected for release study. The maximum particle size observed was below 40 microm. The release pattern was biphasic, characterized by an initial burst effect followed by a slow release. All selected microspheres, except those having less polymer proportion (D:P ratio is 1:1), exhibited a prolonged release for almost 24 hours. On comparing r (2) values for Higuchi and Peppas kinetic models, different batches of microspheres showed Fickian, non-Fickian, and diffusion kinetics. The release mechanism was regulated by D:P ratio and amount of cross-linking agent. From the experimental data obtained with respect to particle size and extent of drug release, it could be concluded that the prepared microspheres are useful for once-a-day intramuscular administration of ketorolac tromethamine.

Preparation and characterization of Pingyangmycin-loaded bovine serum albumin microspheres for embolization therapy

Bovine serum albumin microspheres (BSA-MSs) containing Pingyangmycin hydrochloride (PYM) were prepared for the interventional embolization by an emulsification-crosslinking method. The average diameter of the MSs was 83.6 μm with 80% ranging from 35 to 200 μm. The MSs showed a rather high entrapment efficiency (EE%) of 87.6 ± 5.7% and the drug loading efficacy (DL%) was 20.2 ± 1.3%. The drug release behaviors were evaluated both in vitro and in vivo, using UV-spectroscopy and HPLC, respectively. The in vitro results showed a significantly delayed release of drug for 10 days. The central auricular artery of rabbit was chosen as an embolization sites to study the in vivo drug release and the pharmacokinetics of the MSs compared with PYM injections. Experiments performed by artery perfusion and embolization in rabbits central auricular artery revealed that the PYM loaded BSA-MSs (PYM-BSA-MSs) could obviously prolong in vivo drug release, extend the mean residence time (MRT) and had equal bioavailability compared with plain PYM injections. These results demonstrated that by embolization of the central auricular artery with PYM-BSA-MSs, the local drug concentration could maintain at a relative high level for a longer time, thus achieve the aim of tumor targeting therapy. PYM-BSA-MSs are excellent potential alternatives of interventional embolization materials for the treatment of maxillofacial region tumors.

Biodegradable chitosan scaffolds containing microspheres as carriers for controlled transforming growth factor-β1 delivery for cartilage tissue engineering

Natural articular cartilage has a limited capacity for spontaneous regeneration. Controlled release of transforming growth factor-beta1 (TGF-beta1) to cartilage defects can enhance chondrogenesis. In this study, we assessed the feasibility of using biodegradable chitosan microspheres as carriers for controlled TGF-beta1 delivery and the effect of released TGF-beta1 on the chondrogenic potential of chondrocytes. Chitosan scaffolds and chitosan microspheres loaded with TGF-beta1 were prepared by the freeze-drying and the emulsion-crosslinking method respectively. In vitro drug release kinetics, as measured by enzyme-linked immunosorbent assay, was monitored for 7 days. Lysozyme degradation was performed for 4 weeks to detect in vitro degradability of the scaffolds and the microspheres. Rabbit chondrocytes were seeded on the scaffolds containing TGF-beta1 microspheres and incubated in vitro for 3 weeks. Histological examination and type II collagen immunohistochemical staining was performed to evaluate the effects of released TGF-beta1 on cell adhesivity, proliferation and synthesis of the extracellular matrix. TGF-beta1 was encapsulated into chitosan microspheres and the encapsulation efficiency of TGF-beta1 was high (90.1%). During 4 weeks of incubation in lysozyme solution for in vitro degradation, the mass of both the scaffolds and the microspheres decreased continuously and significant morphological changes was noticed. From the release experiments, it was found that TGF-beta1 could be released from the microspheres in a multiphasic fashion including an initial burst phase, a slow linear release phase and a plateau phase. The release amount of TGF-beta1 was 37.4%, 50.7%, 61.3%, and 63.5% for 1, 3, 5, and 7 days respectively. At 21 days after cultivation, type II collagen immunohistochemical staining was performed. The mean percentage of positive cells for collagen type II in control group (32.7% +/- 10.4%) was significantly lower than that in the controlled TGF-beta1 release group (92.4% +/- 4.8%, P < 0.05). Both the proliferation rate and production of collagen type II in the transforming growth factor-beta1 microsphere incorporated scaffolds were significantly higher than those in the scaffolds without microspheres, indicating that the activity of TGF-beta1 was retained during microsphere fabrication and after growth factor release. Chitosan microspheres can serve as delivery vehicles for controlled release of TGF-beta1, and the released growth factor can augment chondrocytes proliferation and synthesis of extracellular matrix. Chitosan scaffolds incorporated with chitosan microspheres loaded with TGF-beta1 possess a promising potential to be applied for controlled cytokine delivery and cartilage tissue engineering.