Epoxide-derived mixed-mode chromatographic stationary phases for separation of active substances in fixed-dose combination drugs.

A method for the preparation of novel mixed-mode reversed-phase/strong cation exchange stationary phase for the separation of fixed-dose combination drugs has been developed. An epoxysilane bonded silica prepared by vapor phase deposition was used as a starting material to produce diol, octadecyl, sulfonate, and mixed octadecyl/sulfonate groups bonded silica phases. The chemical structure and surface coverage of the functional groups on these synthesized phases were confirmed by fourier-transform infrared and solid-state 13 C NMR spectroscopy and elemental analysis. Alkylbenzene homologs, basic drugs, nucleobases and alkylaniline homologs were used as probes to demonstrate the reversed-phase, ion exchange, hydrophilic interaction and mixed-mode retention behaviors of these stationary phases. The octadecyl/sulfonate bonded silica exhibits pronounced mixed-mode retention behavior and superior retentivity and selectivity for alkylaniline homologs. The mixed-mode retention is affected by either ionic or solvent strength in the mobile phase, permiting optimization of a separation by fine tuning these parameters. The mixed-mode stationary phase was applied to separate two fixed-dose combination drugs: compound reserpine tablets and compound methoxyphenamine capsules. The results show that simultaneous separation of multiple substances in the compound dosage can be achieved on the mixed-mode phase, which makes multi-cycles of analysis for multiple components obsolete.

Deformability- and size-based microcapsule sorting.

Biomedical applications often require to sort cells according to their physical properties, such as size, density or deformability. In recent years, microfluidics has provided a variety of tools to sort micro-objects. We present here a simple microfluidic device consisting of a channel containing a semi-cylindrical obstacle against which capsules are squeezed by the flow, followed by a diverging chamber where streamlines separate. We demonstrate that this basic system is capable of sorting elastic microcapsules according to their size at low flow strength, and according to the stiffness of their membrane at high flow strength. Contrary to most existing sorting devices, we show that the present one is very sensitive and capable of discriminating between capsules with differences in membrane elasticity of order unity.

Template-free synthesis of phosphate-based spheres via modified supersaturated phosphate buffer solutions.

Modified supersaturated phosphate buffer solutions were used to synthesize phosphate-based spheres, including calcium phosphate (CaP), strontium phosphate (SrP) and barium phosphate (BaP). A series of ions concentrations in the modified phosphate buffer solutions were investigated in order to study their effects in precipitates morphologies. During synthesis, it was found that magnesium was the key factor in sphere formation. The morphologies of calcium phosphate, strontium phosphate and barium phosphate precipitates varied as the concentration of magnesium ions varied. When sufficient magnesium was provided, the precipitates appeared spherical, and the diameter was in range of 0.5-2 µm. The morphologies, compositions and structure of spheres were characterized by x-ray diffraction (XRD), scanning electron microscopy (SEM), transmission electron microscopy (TEM) and N2 adsorption analysis. Moreover, the application of magnesium substituted calcium phosphate spheres in dentin tubules occlusion was investigated.

Rapid one-step purification of single-cells encapsulated in alginate microcapsules from oil to aqueous phase using a hydrophobic filter paper: implications for single-cell experiments.

By virtue of the biocompatibility and physical properties of hydrogel, picoliter-sized hydrogel microcapsules have been considered to be a biometric signature containing several features similar to that of encapsulated single cells, including phenotype, viability, and intracellular content. To maximize the experimental potential of encapsulating cells in hydrogel microcapsules, a method that enables efficient hydrogel microcapsule purification from oil is necessary. Current methods based on centrifugation for the conventional stepwise rinsing of oil, are slow and laborious and decrease the monodispersity and yield of the recovered hydrogel microcapsules. To remedy these shortcomings we have developed a simple one-step method to purify alginate microcapsules, containing a single live cell, from oil to aqueous phase. This method employs oil impregnation using a commercially available hydrophobic filter paper without multistep centrifugal purification and complicated microchannel networks. The oil-suspended alginate microcapsules encapsulating single cells from mammalian cancer cell lines (MCF-7, HepG2, and U937) and microorganisms (Chlorella vulgaris) were successfully exchanged to cell culture media by quick (~10 min) depletion of the surrounding oil phase without coalescence of neighboring microcapsules. Cell proliferation and high integrity of the microcapsules were also demonstrated by long-term incubation of microcapsules containing a single live cell. We expect that this method for the simple and rapid purification of encapsulated single-cell microcapsules will attain widespread adoption, assisting cell biologists and clinicians in the development of single-cell experiments.

Separation of empty microcapsules after microencapsulation of porcine neonatal islets.

Pancreatic islet transplantation is used to treat diabetes mellitus that has minimal complications and avoids hypoglycemic shock. Conformal microencapsulation of pancreatic islets improves their function by blocking immunogenic molecules while protecting fragile islets. However, production of empty alginate capsules during microencapsulation causes enlargement of the transplantation volume of the encapsulated islets and interferes with efficient transfer of nutrients and insulin. In this study, empty alginate capsules were separated after microencapsulation of neonatal porcine islet-like cell clusters (NPCC) using density-gradient centrifugation. Densities of NPCC and alginate capsules were determined using Percoll. Encapsulation products following alginate removal were 97 % of products, with less than 10 % of the capsules remaining empty. The viability of this process compared with manually-selected encapsulated islets indicates the separation process does not harm islets.

Preparation and properties of electrophoretic microcapsules for electronic paper.

This paper shows two types of microcapsules used for electrophoretic display. One is prepared by in-situ polymerization which is based on urea, melamine and formaldehyde and another by complex coacervation, which is composed of gelatin and gum Arabic. Microcapsules attract interests of many research groups for longer lifetime of electrophoretic display by reducing agglomerization or lateral movements of nanoparticles. The gelatin microcapsules were more attractive in providing more uniform microcapsule coverage on electrodes due to their flexibility as compared to the melamine-urea microcapsules. The properties of microcapsules were characterized by FTIR, OM, SEM and TGA. Migration of nanoparticles in the two types of microcapsules was also observed when an electric field was applied.

Preparation of microcapsules with self-microemulsifying core by a vibrating nozzle method.

Incorporation of drugs in self-microemulsifying systems (SMES) offers several advantages for their delivery, the main one being faster drug dissolution and absorption. Formulation of SMES in solid dosage forms can be difficult and, to date, most SMES are applied in liquid dosage form or soft gelatin capsules. This study has explored the incorporation of SMES in microcapsules, which could then be used for formulation of solid dosage forms. An Inotech IE-50 R encapsulator equipped with a concentric nozzle was used to produce alginate microcapsules with a self-microemulsifying core. Retention of the core phase was improved by optimization of encapsulator parameters and modification of the shell forming phase and hardening solution. The mean encapsulation efficiency of final batches was more than 87%, which resulted in 0.07% drug loading. It was demonstrated that production of microcapsules with a self-microemulsifying core is possible and that the process is stable and reproducible.

In vitro degradation and dissolution behaviours of microspheres prepared by three low molecular weight polyesters.

Three low-molecular weight polyesters, poly(L-lactic acid) (PLA), copoly(lactic acid/glycolic acid) (PLGA) and poly(delta-valerolactone) (PV), were used to prepare water-soluble sodium diclofenac-loaded microspheres by using the oil-in-oil (o/o) emulsification-solvent evaporation method. Their micromeritic and physicochemical properties, and degradation and dissolution behaviours were determined in vitro. The results indicate that high encapsulation efficiency and better monodispersity might be achieved by the o/o emulsification-solvent evaporation method, depending on the amount of drug loading used. The slower evaporation of organic solvent from the system during microencapsulation seemed to modify the crystallinity of drug and polyester in the microspheres, determined by powder x-ray diffractometry and differential scanning calorimetry. The in vitro degradation rate of all the microspheres in pH7.4 phosphate buffer solution showed first-order kinetics and ranked in the order of PLGA > PLA > PV microspheres. Furthermore, the first-order release rate was also found in all the microspheres after an initial drug burst and ranked in the order of PLGA> PLA > PV microspheres, too. The relationship between degradation and dissolution behaviours of these microspheres is discussed.

Branched oligoester microspheres fabricated by a rapid emulsion solvent extraction method.

Methyl formate was used as the solvent of biodegradable oligoesters for the fabrication of microspheres with encapsulated bovine serum albumin (BSA). The procedure of dispersion of the double emulsion of the w/o/w type and its dilution and solvent extraction is very rapid, taking only several minutes. A higher yield and better encapsulation efficiency were obtained with copolymers of DL-lactic acid with mannitol than with pure linear poly DL-lactic acid. The procedure was accelerated, and yields and encapsulation efficacy were enhanced by the addition of 5% methyl formate to the external water phase. The microspheres were smaller than 100 microm. No benefits were obtained from the addition of wetting agents or other additives to the intermediate (oligoesteric) phase. Further development should concentrate particularly on hydrodynamic conditions and optimization of the composition of the external phase.

Modelling of the solvent evaporation method for the preparation of controlled release acrylic microspheres using neural networks.

The purpose of the present study was to model the solvent evaporation procedure for the preparation of acrylic microspheres by using artificial neural networks (ANNs) to obtain an understanding of the selected preparative variables. Three preparative variables, the concentration of the dispersing agent (sucrose stearate), the stirring rate of emulsion system, and the ratio of polymers (Eudragit RS-L) were studied, each at different levels, as input variables. The response (output) variables examined to characterize microspheres and drug release were the size of the microspheres and T63.2%, the time at which 63.2% of drug is released. The results were also analysed by the multiple linear regression (MLR) to provide a comparison with the ANN methodology. Although both ANN and MLR methods were found to be similar in characterizing the process studied, the results showed that an ANN method gave a better prediction than the MLR method. For the size values of the microspheres, the predictability of the ANN model was quite high (R2 = 0.9602) based on the input variables. A relationship between these variables and size values of microspheres was also obtained by the MLR model (R2 = 0.9050). The performances of both models for the release data from microspheres based on the same input variables were at the level of 53%. According to the results, the ANN methodology can provide an alternative to the traditional regression methods, as a flexible and accurate method to study process and formulation factors.

Biodegradable nanoparticles as a delivery system for cyclosporine: preparation and characterization.

Cyclosporine (CyA) was incorporated into polycaprolactone nanoparticles (PCL-NP) in order to increase its oral bioavailability and to control drug distribution, thereby potentially reducing its toxicity. Prior to in vivo studies, the carrier was optimized and characterized by using different techniques. Light scattering (LS) and transmission and scanning electron microscopy (TEM and SEM) indicated the NP were spherical in shape with a mean size of approximately 100 nm. The influence of the solvent evaporation conditions and the polymer and drug amounts on CyA incorporation was established in order to optimize drug loading. When acetone and excess water were removed at constant temperature, no aggregation phenomena were observed. A value of 180 mg PCL was the minimum polymer amount necessary to encapsulate 95% of the drug initially added to the preparation. Under these conditions, HPLC analysis revealed that approximately 130 microg CyA per mg PCL were incorporated for a total CyA concentration of 2.5 mg/ml, being part of the drug adsorbed onto the particle surface. No structural changes or instability of the components during NP preparation were detected by gel permeation chromatography (GPC) and differential scanning calorimetry (DSC). However, GPC studies showed a competition between poloxamer and CyA for adsorption onto the carrier. In addition, DSC results suggested that at least part of the drug associated to NP remained in its crystal form. Therefore, CyA-loaded NP were easily manufactured and characterized and allow for the administration of therapeutic drug doses to experimental animals.

The preparation and drug-release behaviour of CTA/EC and PMS/EC composite microcapsules.

A cellulose triacetate (CTA) and three different molecular weights of poly(alpha-methyl styrene) (PMS) were used as co-wall materials to prepare composite microcapsules with ethylcellulose (EC). A non-solvent-addition phase-separation method was used. The core material was theophylline (TH) and the solvent-non-solvent pair was dichloromethane-n-hexane, and the drug-release rates of the microcapsules prepared from these two types of co-wall materials were compared. The effects of their phase-separation range on the properties of the microcapsules, such as particle size, release rate and the morphology of the microcapsules are also discussed. The release rate of microcapsules was also affected by the compatibility of the co-wall materials and the EC. The dissolution studies indicated that the drug-release time of CTA/EC and PMS/EC composite microcapsules was sustained to 10 and 3.5 times, respectively, in comparison with that for pure EC microcapsules.

Improved preparation and physical studies of polybutylcyanoacrylate nanocapsules.

An improved method for the preparation of alkylcyanoacrylate nanocapsules is proposed that involves the intermediate synthesis of a well defined adduct of a single monomer unit to an ethanol molecule. It leads to thinner capsule walls and, generally, to a more reproducible capsule structure. The chemical composition of the intermediate organic phase has been studied by nuclear magnetic resonance spectroscopy. The morphology and size of resulting structures is analysed, applying analytical ultracentrifugation and light microscopic particle tracking. The sizes of capsules prepared in the described manner depend on the concentrations of the oil and the monomer components, as is shown by the results of a set of experiments following a simple factorial design.

Influence of various technological parameters on the preparation of spray-dried poly(epsilon-caprolactone) microparticles containing a model antigen.

This work evaluates the efficacy of the spray-drying technique to prepare poly(epsilon-caprolactone) (PCL) microparticles containing an entrapped model antigen (bovine albumin, BSA). The presence of a stabiliser was found to be an important parameter when preparing PCL microparticles containing a hydrophilic antigen. The effect of various technological parameters (concentration of the polymer and protein solutions, organic/aqueous phases ratio, nature of solvents and emulsion parameters such as duration and speed of agitation) on microparticle morphology and size, BSA entrapment and encapsulation efficiency was studied. Microparticles were characterized by a mean size from 9.56+/-0.25 to 24.31+/-2.87 microm and a BSA entrapment from 0.80+/-0.02 to 24.21+/-0.23% (w/w). SDS-PAGE electrophoresis and isoelectric focusing (IEF) confirmed the conservation of the physicochemical characteristics of the BSA entrapped within PCL microparticles produced by spray-drying. Together, these results showed that spray-drying is an efficient technique to overcome the key obstacle that represents the scaling-up of the manufacturing process to produce sufficient quantities of vaccine for clinical trials and, ultimately, commercialization.

Long-term stability of PBCA nanoparticle suspensions.

In this study, the stability of poly(butyl cyanoacrylate) (PBCA) nanoparticle suspensions was examined for up to 1 year by measuring the nanoparticle sizes. The nanoparticles were prepared with different stabilizers (dextran 70.000, poloxamer 188, or polysorbate 85), and the particle size was determined before and after purification by centrifugation and after dilution with different solutions (0.1 N HCl, 0.01 N HCl, H2O, and PBS). The most constant sizes were with the untreated acidic nanoparticle suspensions. In all other cases, agglomeration of the particles occurred: the extent of this agglomeration and the time at which the agglomeration occurred depended on the experimental conditions. Nanoparticle polymer degradation, as indicated by size decrease, was not observed. Thus, PBCA nanoparticles can be stored as suspensions, making the lyophilization and the sometimes problematic resuspension by ultrasonication, unnecessary, which is advantageous for clinical applications.

Preparation of biodegradable microcapsules containing zidovudine (AZT) using solvent evaporation technique.

Sustained release biodegradable microcapsules of AZT were prepared using different concentrations of copolymer of poly(lactic/glycolic) acid (PLGA 50:50 and PLGA 90:10). Solid microcapsules were collected following the complete evaporation of the solvent. The yield of microcapsules was increased two fold with a two-fold increase of the polymer concentration. The efficiency of encapsulation of AZT was also increased with the increase of the polymer concentration. These microcapsules were characterized using scanning electron microscopy. The dissolution of AZT from the microcapsules of PLGA (50: 50) was higher than the microcapsules of PLGA (90:10); the PLGA (50:50) microcapsules containing 1:10 drug/polymer ratio showed higher dissolution than the microcapsules containing 1:20 drug/polymer ratio. The PLGA (90:10) microcapsules containing 1:6 drug/polymer ratio showed higher dissolution than the microcapsules containing 1:10 drug/polymer ratio. In conclusion, the dissolution of AZT was dependent on the type of the copolymer used and the relative concentrations of the drug and the copolymer.

Preparation and characterization of a complex microencapsulated diet for striped bass Morone saxatilis larvae.

The nutritional requirements of marine fish larvae are not well understood, primarily due to the lack of biochemically defined diets acceptable by the larvae. This study describes the methodology for the preparation and the characterization of a complex protein-walled microcapsule (CPWC) containing lipid-walled capsules (LWC). The CPWC were prepared by atomizing a mixture of casein, LWC and other dietary materials into a cyclohexane solution containing 1% (v/v) cross-linking reagent, adipoyl chloride. The primary purpose of complex microencapsulation was to allow the gradual release of low molecular weight phagostimulants and nutrients from the cross-linked protein wall. To determine the release kinetics of low molecular weight compounds from the complex diet, a solution of the amino acid, lysine, was encapsulated in LWC and its leaching rate from CPWC was assessed. A batch of conventional protein-walled capsules (PWC) were also prepared by mixing lysine with casein. The release of lysine from CPWC was significantly lower than that measured from conventional PWC (p < 0.05). Nearly 45% of the total lysine leached out of freeze-dried PWC within 5 min of hydration, reaching 100% after 2 h. CPWC, on the other hand, efficiently retained encapsulated lysine, releasing only 25% after 2 h. In-vitro experiments indicated that CPWC were readily digested by the crude enzyme extract from striped bass larvae and purified porcine pepsin and trypsin.

Poloxamer-coated three-ply-walled microcapsules for controlled delivery of diclofenac sodium.

The three-ply-walled-based system for controlled delivery of diclofenac was developed. The preparation of three-ply-walled microcapsules is essentially based on the technique of multiple-emulsion formation polymer at the interface followed by rigidization of the wall on evaporation of solvent. The protective colloids with surface-active properties were selected for the present study, viz. acacia gelatin, polyvinyl alcohol and sodium alginate. Ethyl cellulose was taken as hydrophobic polymer. The acacia/ethylcellulose/acacia-based three-ply-walled microcapsule system was selected for further studies. The three-ply-walled microcapsule were subsequently coated with poloxamer 188. The non-ionic hydrophilic surfactant coating retards uptake into the reticuloendothelial system. The coated and uncoated microcapsules were characterized for in vitro and in vivo performance. The microcapsules were noted to provide sustained release of the contained diclofenac. The plasma level observed indicated that poloxamer coating results in prolonged release of the drug. Organ distribution demonstrated a different distribution pattern when compared with uncoated microcapsules.

An experimental investigation of enzyme release from poly(vinyl alcohol) crosslinked microspheres.

Crosslinked poly(vinyl alcohol) particles were prepared by the addition of glutaraldehyde into a PVA methanol/water solution in the presence of 0.2 N sulphuric acid. The polymer solution was dispersed in mineral oil in a jacketed vessel, with the aid of a six-blade impeller. Spherical crosslinked particles in the size range 30-80 microns were obtained by varying the degree of agitation or/and the amount of suspending agent. The crosslinked particles, after washing and drying, were placed into a protease enzyme solution for loading. The enzyme-containing water-swollen particles were subsequently removed from the solution and the enzyme release kinetics determined by a UV spectrophotometer. The influence of the degree of crosslinking, ionic strength, pH, particle size, and degree of hydrophilicity of the polymer on the enzyme activity was retained during the adsorption-desorption studies. The release behaviour of enzymes from crosslinked PVA particles exhibited a biphasic kinetic model, with an initial fast release followed by a much slower release rate.