UWE (UML based Web engineering)

First, the influence of equipment changes on GNP properties were determined. Therefore, GNPs in one- to three-fold batch sizes were prepared employing an Erlenmeyer flask and burette, Erlenmeyer flask and peristaltic pump and finally round bottom flask and peristaltic pump as combinations of laboratory equipment. GNPs that were manufactured that way were examined by PCS to determine particle size and PDI. Further, drying loss experiments were run to determine the concentration of the GNP dispersion and the particle yield, respectively.

1.3.1.1 Effects on particle size and polydispersity index

In general, particle size is an important parameter for the characterization of nanoparticles, especially when they are intended for clinical use e.g. in cancer applications. In this case, particle size should preferably not exceed a target value of 200 nm when administrated i.v. to benefit from the enhanced permeability and retention effect (EPR) which is characteristic for tumor vasculature (Torchilin 2007). Larger particles also increase the risk for thrombosis and should consequently be avoided. However, for s.c. delivery, particles up to 350 nm can be suitable when a local presentation of drugload is indended (Zwiorek 2006).

Fig. 1 displays the particle size resulting from the different employed preparation methods that were each used for one-, two- and three-fold batch size. Fortunately, all the manufactured particles do not exceed a size of 200 nm.

The columns on the left side (Erlenmeyer flask, burette) indicate that an upscaling of the standard preparation method is generally possible. Although the particle size increases with the batch size, the GNPs seem to be of high quality. So, the PDI values of two- and three-fold batch size average 0.030 and 0.027, respectively whereas the one of the standard batch size averages 0.096. Values below 0.1-0.15 can be regarded as strong indication for monomodal particle distribution (Pishvaei et al. 2006).

0 50 100 150 200 250

Erle nme ye r flask, bure tte

Erle nme ye r flask, pe ristaltic pump

round bottom flask, pe ristaltic pump p a rt ic le s iz e [ n m ]

one-fold batch size two-fold batch size three-fold batch size

Fig. 1. Particle size resulting from three different GNP preparation

methods, each employed for one-, two- and three-fold batch size. Each data point displays the mean of three single batches (n=3, ± S.D., altogether nine individual measurements).

In order to semi automate the GNP production process, the burette was replaced by a peristaltic pump. That way, the speed of acetone addition was exactly adjusted to 4.9 ml/min and maintained during the whole second desolvation step. As a consequence, slightly smaller particle size standard deviations of the upscaled batches than by using the burette were observed as it can be see columns in the middle). Furthermore, the related mean PdI values are still in a range

indicating high GNP quality. Once again, the particle size increases with the batch size. Although this seems to be characteristic for the scale-up in Erlenmeyer flasks, we could confute this hypothesis in further experiments including five- and ten-fold batch sizes.

Finally, the Erlenmeyer flask was replaced by a round bottom flask of an adequate volume. So, we could further improve the particle size standard deviations (see columns on the right side) while the mean PdI values remained nearly constant. Nevertheless, the production of nanoparticles in round bottom flasks is not as easy to handle as in Erlenmeyer flasks during the desolvation process as they need additional fixation for safe positioning. On the other hand, employment of round bottom flasks allows the removal of organic solvents without the need of intermediate vessel change.

Summarizing, each investigated method is appropriate for an upscaling of the GNP standard preparation method and leads to particles of high quality.

1.3.1.2 Effects on particle yield

One of the main objectives of an upscaling process is to increase the yield of the manufactured product while its high quality is maintained. For the scale-up of the GNP production process the latter was already demonstrated so that the GNP yield can exclusively be focused in the following.

Fig. 2 shows the GNP yield resulting from the different preparation methods described in 3.1. that were each employed for one-, two- and three-fold batch size. Referring to the initially employed gelatin mass, the yield was invariably specified in %. Thus, the resulting normalized values of the different batches couldn be checked against each other. They generally varied between 1.5 % and 13.0 %. The black columns which display the GNP yield of the standard-sized batches indicate that the preparation process could be optimized, first by replacing the burette by a peristaltic pump, then by replacing the Erlenmeyer flask by a round bottom flask. That way, the GNP yield almost decupled. Unfortunately, this trend did not proceed in two- and three-fold batch sizes.

The columns on the left side (Fig. 2) show that the applied method employing Erlenmeyer flask and burette led to mean GNP yields smaller than 5 % and

relatively high standard deviations, which were both inapplicable for further scale-up experiments.

Regrettably, the GNP yields of the upscaled batch sizes were not optimized by replacing the burette by a peristaltic pump (see columns in the middle). Quite the opposite, the particle yields further declined. However, the standard deviations could clearly be reduced.

When the Erlenmeyer flask was finally replaced by a round bottom flask (see columns on the right side), the GNP yield of the two-fold batch size markedly increased by 5.4% up to 9.4%. Nevertheless, it did not measure up to the mean yield of the standard batch size (13.0%). Its standard deviation also worsened again.

0 5 10 15

Erle nm eye r flask , burette Erle nm eye r flask , pe ristaltic pum p

round bottom flask , pe ristaltic pum p G N P y ie ld r e la ti n g t o g e la ti n m a s s [% ]

one-fold batch size two-fold batch size three-fold batch size

Fig. 2. GNP yield relating to the initially employed gelatin mass, which resulted

from three different GNP preparation methods, each employed for one-, two- and three-fold batch size. Each data point displays the mean of three single batches (n=3, ± S.D., altogether nine individual measurements).

With the exception of the first dark column, the particle yield decreased with an increasing batch size meaning that the efficiency of the GNP production process declines. This was probably due to the sedimentation time starting after the first desolvation step, which was adapted to the batch size by visual judging of the sediment formation. Presumably, with respect to larger total volumes and therefore longer particle sedimentation distances this time needed to be prolonged in future upscaling experiments. Additionally, both the centrifugation speed and time greatly influenced the GNP yield. Consequently, they should not be handled

as constant process parameters but dynamically be adapted to the requirements of each batch. However, a “one fits all” procedure would be more desirable to avoid time consuming pre-studies to optimize each single batch.

Summarizing, each investigated upscaled GNP preparation method employing a peristaltic pump provided heretofore non-satisfactory yields in comparison to those of the standard-sized preparation methods. Although the GNP preparation in round bottom flasks seemed to be more effective than the preparation in Erlenmeyer flasks, we continued our experiments using Erlenmeyer flasks because of an easier and hence time-saving handling. However, a future use of round bottom flaks should not be excluded due to the ease of subsequent organic solvent evaporation whithout the need of in between vessel change.