4.6.1 Dialysis bag experiments
The dialysis bag test (see Figure 18) was conducted for the extremely lipophilic dialkylindo-carbocyanine DiI and the moderately lipophilic dye NR. Nanocapsules labelled with 70 µg NR or DiI/g were diluted 1:15 with 1/15 M phosphate buffer pH 6.8. After certain periods of time the migration of the dye out of the capsule core, through the capsule shell, water phase and dialysis membrane into the MCT phase (10 mL) was detected. Then, the MCT filled dialysis bag was taken out of the water phase, dried and its fluorescence intensity was captured.
Spectral fluorescence images in vitro were obtained using the MaestroTM In vivo Imaging System (CRi Inc., Woburn, MA, USA). A green pass filter was used for emission and excitation of NR and DiI. The tunable filter was stepped in 2 nm increments from 550 to 800 nm while the Cri MaestroTM Multispectral Imaging System
48 Experimental
captured images at each wavelength interval with constant exposure time (3 ms). Quantification during the experiment was not possible since the oil could not be measured purely (intensity shielded by the cellulose acetate membrane). Thus only the final concentration of each dye in MCT was accessible after opening the bag.
The fluorescence emission of the dialysis bag at 600 nm (both dyes) was plotted versus time.
Figure 18 Assembly of the dialysis bag experiment (dispersion heated up to 37 °C)
4.6.2 Modified in vitro release simulation into lipophilic medium
This in vitro experiment was conducted in order to simulate the in vivo release behaviour of the incorporated fluorescence dyes nile red, pTHPP and DiR from the nanocapsules and to investigate their rate of accumulation in MCT which served as a fat-tissue analogue medium. It was performed (i) by covering the sample dispersion with the MCT phase and stirring the dispersion on a heated stirring plate, and (ii) by covering the sample dispersion with the MCT phase and permanent mixing of the phases by means of a rotating temperature-controlled end-over-end apparatus. The procedures are schematically shown in Figure 19.
Figure 19 Determination of in vitro release behaviour A: on a heated stirring plate, B: in a rotation apparatus (MCT: medium chain triglycerides, NC: diluted nanocapsule dispersion or nanoemulsion)
4.6.2.1 In vitro release on a heated stirring plate
During experiments the release vessels were protected from light and kept at 37 °C. For a 1 by 10 dilution of the sample dispersion, 18 mL of a 0.0333 M acetate buffer pH 4.5 and 10 mL MCT were filled into 40 mL glass
Methods for the investigation of release behaviour 49
vessels and heated up to 37 °C. Into that two-phase mixture 2 mL of sample were added, each containing 10 µg NR or pTHPP per gram sample. The dispersion in the vessels was mixed with a magnetic stirrer with a speed of about 200 rpm for 24 hours (NR) or 7 weeks (pTHPP). After certain time intervals, 600 µL of the upper MCT phase were withdrawn for quantification of NR and pTHPP and replaced by fresh MCT. For the 1 by 100 dilution, 19.8 mL of the acetate buffer, 5 mL MCT and 200 µL of sample were filled into the glass vessels.
1200 µL MCT were withdrawn from the upper phase and replaced by fresh MCT.
4.6.2.2 In vitro release in a rotation apparatus
During experiments the interior of the rotation apparatus was protected from light and kept at 37 °C. 15 mL of a 0.0333 M acetate buffer pH 4.5 and 10 mL MCT were filled into 30 mL glass vessels and heated up to 37 °C.
Into that dispersion, 200 µL of sample were added, each containing 70 µg NR or DiR per gram. This displays a dilution factor of 1:76. The dispersion in the vessels was stirred at a speed of 30 rpm. After certain time intervals, the rotation was stopped, 200 µL of the upper MCT phase were withdrawn and replaced by fresh MCT.
The MCT release medium into which NR, DiR or pTHPP possibly had migrated and was thus removed from the vessels, were diluted down to the calibration range and their fluorescence intensity was measured on a Perkin Elmer MPF-44 fluorescence spectrometer (Perkin Elmer Instruments, Überlingen, Germany) (NR: excitation 535 nm, emission 585 nm; DiR: excitation 690 nm, emission 770 nm; pTHPP: excitation 428 nm, emission 660 nm). For each sample, the release test was performed in triplicate. The concentration was calculated by external calibration. The released portion of model drug was plotted versus time.
4.6.3 Electron paramagnetic resonance spectroscopy
4.6.3.1 Instrumental setup
An EPR spectrometer with a microwave frequency of about 9.5 GHz (X-Band, Miniscope MS 200; Magnetech, Berlin, Germany) was used with the following parameters: modulation frequency 100 kHz, microwave power 10-25 mW, Bo-field 336 mT, scan range 1 mT (acquisition of the 3rd peak only) or 5 mT (acquisition of all 3 peaks of the TB signal), scan time 10 s (3rd peak only) or 30 s (all 3 peaks), modulation amplitude 0.05 mT. Short scan times were necessary for detecting fast releasing behaviour. The superimposed 3rd spectral line was fitted by means of a homemade function for the 1st derivative of a Lorentzian line, implemented into Origin Pro 7.5 software (Origin Lab Corp., Northhampton, Maryland, USA) and plotted with this software. This allowed calculating the fractions of TB each located in the oily and aqueous phase and estimating the release velocity.
TB was incorporated in the oily nanocapsule core by dissolving it in the oil in a concentration of 20 mM before the preparation of the nanoemulsion template. In order to allow a comparison of the samples (nanoemulsion, multi-layered nanocapsules) regarding their release behaviour, they were adapted to an approximately constant content of MCT (1.11 % v/v) by initial dilution, and thereby to a uniform content of TB of 0.222 mM.
50 Experimental
4.6.3.2 Dilution assay
In this experiment the aim was to measure the release kinetics of incorporated TB from the nanocapsule core induced by dilution of the three standardized samples with double-distilled water in the dilution factors 1:1 (200 µL + 200 µL water), 1:2 (200 µL + 400 µL water), 1:3 (200 µL + 600 µL water), 1:4 (200 µL + 800 µL water) and 1:9 (200 µL + 1800 µL water). Immediately after mixing, the EPR spectra were recorded over a period of 5 minutes.
4.6.3.3 Reduction assay
In the ascorbic acid reduction assay, the ability of the capsule shell to protect the encapsulated TB from ascorbic acid located in the aqueous environment was investigated. It is based on the reduction of the TB radical to the EPR silent TB hydroxylamine. Rapid loss of the EPR signal intensity indicates a fast reduction and a fast release of TB through the polymer shell into the aqueous environment since it is accessible for the ascorbic acid only in the aqueous phase [182]. The samples were diluted 1:1 (v/v) with 1.6 mmol aqueous ascorbic acid salt solution. Immediately after mixing, the EPR spectra were recorded over a period of 10 minutes.
4.6.4 Ultrafiltration at low pressure
The release of salicylic acid from the nanoemulsion as well as differently layered nanocapsules was investigated by ultrafiltration (UF) at low pressure and subsequent fluorescence spectroscopy. Salicylic acid was used as a model drug because previously studied lipophilic and fluorescencent substances (pTHPP, NR, hydroxyethyl and methyl salicalyte) showed strong adsorption onto the studied membranes (polyether sulphone (PES), regenerated cellulose, PVDF). Due to poor permeation through the membrane, these substances could not be separated from the drug carrier dispersion after the release. Thus, in order to facilitate fluorescence analysis after UF anyway, salicylic acid was chosen as release drug as it could permeate through the membrane and could be converted into the fluorescent anion by addition of NaOH after UF. UF was operated in a heatable, home-made stirred cell (Figure 20) equipped with an UF membrane UP 150 made of PES (diameter 47 mm, MWCO 150.000 Dalton, Microdyn-Nadir, Wiesbaden, Germany).
The samples were prepared with a salicylic acid content of 55.9 µg/mL, an oil content of each 1.11 % MCT and a constant acetate buffer concentration of 0.0333 M. Prior to the experiment, 5 mL of the release medium (0.0333 M acetate buffer pH 4.5, or 1/15 M phosphate buffer pH 6.8) were pressed through the membrane using low pressure (< 1 bar) in order to soak it with aqueous liquid and enhance the following fluid flux during release experiments. For each release test a new membrane was used. Afterwards, 32 mL of the release medium (buffer solution) were filled into the stirred cell and heated up to 37 °C. Under stirring, 8 mL of the preheated sample were added (1:5 dilution). During release time the dispersion was stirred at 100 rpm.
After definite points of time, low pressure of < 1 bar was applied on the cell and 300 µL of the release medium (including the portion of model drug released) were filtered through the membrane and collected (the first five droplets were discarded).
Methods for the investigation of release behaviour 51
Figure 20 Stirred cell composed of:
a: heat-transferring steel plate; b: UF membrane; c: pressure inlet; d:
pressure control valve; e: sample inlet; f: filtration outlet; g: magnetic stirrer; h: insulating plastic cylinder and release vessel; i: filtration orifice below the membrane zone
For fluorescence quantification, the collected filtrate (300 µL) was mixed with 300 µL of the release medium and 600 µL 0.1 M sodium hydroxide solution in order to convert the analyte into the fluorescent salicylate anion. The salicylic acid concentration in the filtrate was determined by external calibration at a MPF-44 fluorescence spectrometer (Perkin Elmer Instruments, Überlingen, Germany) using the wavelengths 321 nm (excitation) and 400 nm (emission). The amounts of salicylic acid released were corrected by the adsorption rate of the drug onto the membrane which was determined by ultrafiltration of a pure drug solution (concentration 4 µg/mL) before.