Materials and methods

1.2.1 Standard preparation of gelatin nanoparticles via two step desolvation technique

GNPs were prepared according to the original preparation method by dissolving 1.25 g of gelatin type A (Bloom 175, Sigma, Taufkirchen, Germany) in 23.75 g of 0.2 µm filtered (Acrodisc, Pall, Dreieich, Germany) high purified water (HPW) under gentle heating to 50 °C. Constant stirring (500 rpm) was maintained during the whole preparation procedure. A first desolvation step was initiated by the addition of 25 ml of acetone. After sedimentation of the precipitated gelatin fractions for about 20 s, the supernatant consisting of dispersed as well as dissolved gelatin was discarded. Then, the sediment was weighed and redissolved by the addition of 0.2 µ m filtered HPW up to 25.0 g under heating to 50 °C. The pH (pH Meter MP 220, Mettler Toledo, Greifensee, Switzerland) was further adjusted to 2.5 with 2 M HCl. GNPs were formed in situ during a second desolvation step by drop wise addition of about 90 ml of acetone (3-5 ml/min) by a burette. After ten minutes, 175 µl of glutaraldehyde (25 %) were added to the reaction vessel, an Erlenmeyer flask, to crosslink the nanoparticles. Finally, after overnight stirring in an extractor hood, the particles were purified by two-fold centrifugation (19000 g for 18 min; Sigma Laborzentrifugen, Osterode,

Germany), redispersion in 0.2 µm filtered HPW and filtration through a 5 µm 0.2 µm filtered HPW rinsed filter (VWR, Fontenay sous Bois, France). The purified nanoparticles were stored as dispersion in HPW at 8 °C1.

1.2.2 Scale-up of the gelatin nanoparticle standard preparation method

In order to scale up the GNP standard preparation method, the batch size was first doubled. Consequently, 2.50 g of gelatin type A (Bloom 175) were dissolved in 47.50 g of 0.2 µm filtered HPW. Accordingly, the volume of acetone added for the first desolvation step, the mass of HPW added to redissolve the sediment and the amount of glutaraldehyde finally added to crosslink the nanoparticles were duplicated whereas temperature, stirring speed, pH, speed of acetone addition, centrifugation speed and centrifugation time were adopted from the standard preparation protocol. The sedimentation time and the volume of acetone added for the second desolvation step were each adapted to the batch size by visual judging of sediment formation and grade of turbidity, respectively. Turbidity was additionally measured by a nephelometer (Nephla, Dr. Lange, Berlin, Germany). In further experiments the batch size was increased three-, five- and ten-fold.

1.2.3 Modifications of the upscaled gelatin nanoparticle preparation method: Equipment changes.

The glass burette (Brand, Wertheim, Germany) used for the addition of acetone during the second desolvation step (see 1.2.1.) was first replaced by a peristaltic pump (4.9 ml/min; Sotax, Basel, Switzerland) employing acetone-proofed flexible tubes (Ismatec, Wertheim, Germany). Further, the previously used Erlenmeyer flask was replaced by a round bottom flask of an adequate volume.

For batch sizes greater or equal than three-fold an evaporation step was introduced to exclude residual acetone. Therefore, the non-purified GNP dispersion was transferred into a round bottom flask (1 l) and acetone was removed by a rotary evaporator (Heidolph Instruments, Schwabach, Germany) at a temperature of

40°C under low vacuum. The particles were then purified as described in the standard preparation protocol.

Moreover, the amount of glutaraldehyde (25 % V/V), added after the second desolvation step to crosslink the nanoparticles, was halved and the stability of the resulting particles was monitored.

1.2.4 Characterization of gelatin nanoparticles

1.2.4.1 Determination of concentration

About 20 µl of the aqueous GNP dispersion were dropped in a little aluminum vessel. That way, three samples were prepared. They were then dried in a drying chamber heated to 60 °C until their masses reached a constant level. In order to calculate the concentration of the dispersion (m/m), the aluminum vessels had to be weighed in empty, full and completely dried status (microbalance, Mettler Toledo, Greifensee, Switzerland).

1.2.4.2 Determination of particle size

An expendable cuvette was twice rinsed with 0.2 µm filtered HPW, before it was filled up to a height of about 1 cm with 0.2 µm filtered HPW again. Then, 15 µl of the aqueous GNP dispersion were added and mixed. Particle size and polydispersity index (PDI) were finally measured with a Zetasizer Nano ZS employing photon correlation spectroscopy (PCS; Malvern Instruments, Worcestershire, United Kingdom). Each indicated value was the mean of at least 15 subruns.

1.2.4.3 Determination of zeta potential

A zeta cuvette comprising two electrodes was once rinsed with 0.2 µm filtered HPW, before 65 µl of the aqueous GNP dispersion (see 2.2.) were filled in. Then, 650 µl of phosphate buffered saline (PBS) or 10 mM NaCl were added. Zeta potential was finally measured with a Zetasizer Nano ZS at a measuring voltage of 40 mV employing the standard Smoluchowski model. Each indicated value was the mean of 50 subruns.

1.2.5 Cationization of upscaled GNPs

First, standard conditions as established for single size batches (Zwiorek et al. 2008) were applied on a threefold up-scaled batch to include a reaction volume of 20 ml at room temperature with an incubation time of 30 minutes and an effective concentration of 0.50 mg of each cationization reagent (Cholamin and EDC) In parallel, the pH value was adjusted to values of 3.5, 4.0, 4.25, 4.5, 4.75, 5.0 and 5.5 for samples subjected to the above mentioned conditions. Furthermore, reaction volume was adjusted to adjust the starting GNP concentration (1.0 and 2.0 mg/ml) and cationization reagent concentrations (1.0, 2.0 and 3.0 mg/ml for cholamin and 1.1, 2.2 and 3.3 mg/ml for EDC). Monitored quality parameters included visible particles, GNP size and size distribution as well as the zeta potential.