Nano Capsule Preparation Method

By: Pharma Tips | Views: 3951 | Date: 12-Jul-2011

Nanocapsules were prepared by the method described by Fessi et al.based oninterfacial deposition of preformed polymer after solvent displacement.

Nanocapsules were prepared by the method described by Fessi et al. [67] based on interfacial deposition of preformed polymer after solvent displacement. Briefly, in this process, polymer, oil, drug and optionally a lipophilic stabilizer are dissolved in a semi-polar water-miscible solvent (e.g. acetone or ethanol) which is poured or injected into an aqueous solution containing a hydrophilic stabilizer (e.g. Poloxamer 188 or poly(vinyl alcohol)). Nanocapsules are formed instantaneously by rapid diffusion of the solvent into the water phase. In a subsequent step the solvent is eliminated from the suspension under reduced pressure. PLA, PEG-PLA-10% and PEG-PLA-20% were used as coating polymers and Miglyol as the oily phase.

Polymer and oil, and if required for ESR measurements, the lipophilic spin probes TEMPOL-benzoate (TB), 2-Heptadecyl-2,3,4,5,5-pentamethylimidazolidine-1-oxyl or 2-Heptadecyl-2,4,5,5-tetramethyl-3-imidazoline-1-oxyl (HD-PMI) were dissolved in acetone.
10 ml of the acetone solution (containing the polymer, Miglyol and if required the spin probe) were injected into 20 ml of external aqueous phase, with or without Poloxamer 188 as a hydrophilic surfactant, under moderate agitation. The solvents were evaporated to 10 ml under reduced pressure. An overview of all investigated formulations is given in Table 2.1.

The    “interfacial deposition of preformed polymer after solvent displacement” technique [66,67] for nanocapsule preparation was used because it is known for its simplicity and robustness at small scale [72]. In the literature there are several reviews  and articles which discuss the spontaneous emulsification process that leads to nanoparticle formation using explanations such as Marangoni effect [67]. Only recently the group of Katz  provided new insights into the physical phenomenon behind this method. They named this spontaneous emulsification phenomenon Ouzo effect, derived from the aperitif, which is an ethanol-water extract of anis seeds containing the water-insoluble substance anethol. Upon dilution with water Ouzo becomes cloudy and remains so for a long time. This general phenomenon can occur upon mixing large amounts of water with almost any solution consisting of a small concentration of oil in a hydrophilic solvent over a small range of concentrations.

The solvent phase, containing the water-immiscible oil, is poured into the aqueous phase, which may contain a surfactant (Figure 2.2 a). Upon diffusion of water into the oil-containing solvent droplet (b) supersaturation of oil is induced and droplet nucleation (c) occurs. Droplet growth ends when the aqueous phase is no longer supersaturated with the oil (d).

These metastable dispersions can be generated when mixing occurs in a special region in the phase diagram of water, the organic solvent and oil. Upon diffusion of the organic solvent into the water phase, the polymer diffuses with the organic solvent and is stranded at the interface between oil and water forming an envelope around the oil droplet. An oil-to-preformed-polymer ratio of approximately 3:1  as chosen because preliminary tests with this ratio yielded the best results. Poloxamer  88 was used at the same concentration of the polymer or at half the concentration.  hereas the production of PEG-PLA nanocapsules in the absence of Poloxamer was  uccessful, though the polydispersity index was high compared to the presence of  oloxamer, this attempt failed for PLA nanocapsules.

The chosen compositions lack  ecithin as a stabilizer which is commonly used in nanoemulsion and nanocapsule  reparations [70]. This decision was made on two accounts. One reason was to avoid the coexistence of liposomes. Groves and colleagues  reported that liposomal structures are normally present in phospholipid-stabilized emulsions and Mosqueira and colleagues [70] identified liposomes in addition to nanocapsules on TEM micrographs.

Since the intention of this study was to get a deep understanding of nanocapsules by ESR and SANS, an easy model system was needed. The coexistence of other colloidal species should be avoided. A second aim was to achieve a higher stability of nanocapsules in physiological media by omitting lecithin, which is known to be sensitive for hydrolysis and thereby forms the haemolytic product lysolecithin.

Nanoemulsions composed of 2.5% (v/v) Miglyol, 2.5% (w/v) Poloxamer 188 and water served as reference systems to nanocapsules.
Nanoemulsions were prepared by high pressure homogenization. Initially TB (0.1mM referring to the TB concentration in the final sample volume) was dissolved in Miglyol and heated to 80ºC. The oily phase, which contained the spin probe, was added to the aqueous phase of the same temperature which contained 2.5% (w/v) of the hydrophilic stabilizer Poloxamer.  An emulsion was formed using a rotor-stator mixer (Ultra turrax®, IKA® T18 basic, IKA®-Works, NC) for 120 s at 22.000 rpm. The emulsion was passed through a high pressure homogenizer (nG7400.270 P, Standsted Fluid Power Ltd., UK-Stansted) three times at 80ºC and 500 bar.

Nanoemulsions were produced via high pressure homogenization at 80ºC to obtain a system very similar to the examined nanocapsules but omitting the polymer wall. The Poloxamer concentration had to be increased from 0.3% for nanocapsules to 2.5% for nanoemulsions to stabilize these systems. Nanoemulsions were not produced by the solvent displacement method which was used for nanocapsule production because with this method lecithin is required for stable nanoemulsions.

Dynamic light scattering (PCS/3D-DLS)

For photon correlation spectroscopy (PCS) measurements, the non-invasive backscattering (NIBS, Malvern) technology was used, which suppresses multiple scattering in turbid colloidal suspensions.

Photon correlation spectroscopy is based on dynamic light scattering caused by Brownian movement of particles . Diffusion of small isometric particles in  liquids is fast, causing faster fluctuations in the intensity of scattered light compared to big particles which diffuse more slowly. These intensity fluctuations are recorded in the PCS experiment. Autocorrelation analysis of the measured diffusion coefficients, under assumption of a spherical shape of the particles, yields a mean particle diameter which is expressed as z-average. The polydispersity index (PDI) provides information on the heterogeneity of the sample, whereas the PDI value can be connected with the polydispersity (in %) by the following equation:
 
Polydispersity(%) = 100 ∗  PDI

PCS measurements were performed at a scattering angle of 173° (Malvern HPPS, Malvern Instruments, UK). The nanocapsule dispersions, containing 2.5% (v/v) Miglyol, were measured without further dilution. Contributions from multiple scattering can be excluded because reference measurements of diluted samples were performed. Only SANS samples (sample 1, Table 2.1) were diluted to a Miglyol concentration of 1% (v/v) before measurements because that was the concentration for SANS measurements. The mean radius of the nanocapsules and size distribution of the colloidal systems were determined at 25°C. Measurements were done in triplicate. Data treatment was performed using the Malvern software.
 
For comparison particle size measurements of the SANS samples (sample 1) were also performed using a 3D-DLS (LS Instruments GmbH, Fribourg, Switzerland). Here a DLS technique using cross-correlation schemes suppresses contributions from multiple scattering . To get a better intercept in the DLS signal the sample was diluted to 0.33% (v/v) Miglyol, 0.08% (w/v) poly(D,L-lactide) and 0.12% (w/v) Poloxamer. Dynamic light scattering measurements with variable scattering angles from 20 -140º were carried out at 20°C. Cumulant analysis was applied to the cross correlation function in the same way as for the NIBS measurement to receive the hydrodynamic radius. Since the aqueous dispersion medium of the nanocapsules contained Poloxamer, the given viscosity values for water were replaced by the Poloxamer solution values in the data analysis software. The viscosity alteration of the Poloxamer solutions compared to pure water was considered for both methods. The viscosities for different Poloxamer solutions were determined by Ubbelohdeviscosimeter at 20 and 25 °C.

Zeta potential measurements

The  potential was measured with a Zetasizer Nano ZS and a ZetaSizer 3000HS (Malvern Instruments, UK). All samples were diluted (1:1) with a solution of KCl (0.002 mol/l). The samples were measured at 25°C and 30 zeta runs were performed per sample at the Zetasizer Nano ZS. When using the ZetaSizer 3000HS four measurements with automatic measurement duration were carried out per sample.

Transmission electron microscopy (TEM)

For transmission electron microscopy nanocapsules were freeze-fixed using a propane jet-freeze device JFD 030 (BAL-TEC, Balzers, Liechtenstein). Afterwards the samples were freeze-fractured and freeze-etched (90 s; -110 °C) with a freeze-etching system BAF 060 (BAL-TEC, Balzers, Liechtenstein). The surfaces were shadowed with platinum to produce good topographic contrast (2 nm layer, shadowing angle 45°) and subsequently with carbon to stabilize the ultra-thin metal film (20 nm layer, shadowing angle 90°). The replica were floated in sodium chloride (4 % NaCl; Roth, Karlsruhe, Germany) for 30 minutes, rinsed in distilled water (10 minutes), washed in 30% acetone (Roth, Karlsruhe, Germany) for 30 minutes and rinsed again in distilled water (10 minutes). Thereafter the replica were mounted on grids and observed with a transmission electron microscope  TEM 900, Carl Zeiss SMT, Oberkochen) operating at 80 kV. Pictures were taken with a Variospeed SSCCD camera SM-1k-120 (TRS, Moorenweis, Germany).


Electron spin resonance (ESR) spectroscopy

In vitro determination of spin probe distribution

For ESR measurements undiluted aqueous nanocapsule dispersions were used which contained 0.1 mM spin probe (referring to the final volume). An ESR spectrometer of 9.5 GHz (X-Band; Miniscope MS 200) from Magnettech (Berlin, Germany) was used, where the probe is examined inside a glass capillary. Measurements were conducted at room temperature with the following typical parameters: B0 field: 335.4 mT; sweep: 10 mT (precisely 9.800 or 9.893 mT); modulation frequency: 100 kHz; microwave power: 20 mW; scan time: 30 s; modulation amplitude: 0.1 mT.

Dilution assay

For dilution assays nanocapsule dispersions containing 0.1 mM spin probe were diluted with water in different ratios (1:1, 1:2, 1:3, 1:4). Immediately after dilution the samples were placed inside the ESR spectrometer and changes in the spectral shape were monitored for one hour.

Centrisat is a ready-made unit for the centrifugal ultrafiltration of volumes up to 2.5 ml. Ultrafiltration is carried out against the centrifugal force. Centrisat tubes are usually used for the separation of proteins from small molecules. 500 µl of  LA-NK (sample 1, Table 2.1) were diluted with 2000 µl of H2O. The 2500 µl of nanocapsule dilution were put into a Centrisart (Centrisart I, Sartorius, cut-off 300.000) tube and the diluted nanocapsule sample was concentrated to a volume of 500 µl. ESR measurements were performed from the undiluted nanocapsule dispersion, the diluted nanocapsule dispersion, the concentrated nanocapsule dispersion after centrifugation and from the filtrate.

External incorporation of spin probe to nanocapsules

To study whether the spin probe was capable of penetrating from the aqueous environment through the nanocapsule wall into the oily core of the nanocapsule, spin-probe-free nanocapsule dispersions were prepared. The appropriate volume of a TB stock solution in acetone was added to an empty test tube, acetone was evaporated and the TB-free nanocapsule dispersion was added to the test tube. The tube was warmed up to 80ºC for a few seconds. ESR spectra were recorded immediately.

Ascorbic acid reduction assay

A reduction assay of the spin probe (0.1mM) in the samples was conducted by time-dependent ESR measurements after mixing  (1:1  v/v)  with  1.6  mM aqueous ascorbic acid sodium salt solution [104]. The chosen ascorbic acid concentration guaranteed a reduction speed which lead to quantifiable ESR spectra at the given times. The decrease of ESR signal intensity was calculated down to 10% only, because at lower intensities no proper simulation of the spectrum was possible because of an interfering spectrum of the ascorbic acid radical.
Simulation of the ESR spectra was performed by means of Public ESR Software Tools (P.E.S.T.) from National Institutes of Health (National Institute of Environmental Health Sciences, Research Triangle Park, NC 27709) . The optimization method used was LMB1.

 
Nuclear magnetic resonance (NMR) spectroscopy of protons

1H-NMR experiments were performed on a Bruker DRX 500 spectrometer (Bruker AG, Karlsruhe, Germany) with 500 MHz resonance frequency for protons. The experiments    were    run    on    the    aqueous    nanocapsule dispersions. D2O (150mg/sample) was used as an internal spin lock substance. The nanocapsule dispersions contained different concentrations (0, 1, 2 or 3 mM) of the spin probe TB. T1 (spin-lattice relaxation) was measured using a saturation-recovery pulse sequence. T2 was measured with a CPMG (Car-Purcell-Gill-Meiboom) pulse.


Small angle neutron scattering (SANS)

Small angle neutron scattering (SANS) is a technique, where cold neutrons permeate materials and, when they hit upon nano-sized structures, they are scattered to small angles. From the scattering image the structures can be reconstructed. SANS allows the characterization of structures or objects in the nanometer scale, typically in the range between 1 nm and 200 nm. The information one can extract from SANS is primarily the average size, size distribution and spatial correlation of nanoscale structures, as well as shape and internal structure of particles (e.g. core-shell structure). All in all, SANS is a valuable technique, widely used in many fields, to characterize particles (in solution or in bulk), clusters, (macro-) molecules, voids and precipitates in the nanometer size range. SANS measurements were carried out at the SANS facility of SINQ at Paul Scherrer
                  
Institute, Switzerland, using a neutron wavelength of    λ=1.3 nm and two sample-  detector distances of 6 and 18 m. The range of momentum transfer 0.01 < Q (nm-1)< 0.5 was covered. The momentum transfer Q is defined in the usual way as Q = 4π/λ sin(θ/2), where θ is the scattering angle. All measurements were performed at 20°C. The intensity data were corrected for background and transmission and for non-uniform detector efficiency by referring to the incoherent scattering of a water sample. For SANS measurements two nanocapsule dispersions were produced, one sample with H2O and one with D2O. The nanocapsule dispersions, which were produced as described above (2.3.1), were diluted with H2O or D2O to a final concentration of 1% (v/v) Miglyol, 0.24% (w/v) poly(D,L-lactide) and 0.12% (w/v) Poloxamer. The H2O and the D2O samples were mixed to receive the desired scattering contrast.

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