Table 1
Details on the samples’ possible processing scenarios
| Processing, storage, and sample usage parameters | Options |
|---|---|
| Freezing conditions | Flash freezing (liquid nitrogen, −196°C) |
| Fast freezing (precooled freezer, −70°C) | |
| Slow freezing (precooled freezer, −22°C) | |
| Reconstitution strategy | Freeze-drying and redispersion |
| Thawing | |
| Stock storage period | Immediate reconstitution |
| 1-week storage | |
| 1-month storage | |
| Sample use period (after stock reconstitution) | Immediate measurement |
| Measurement after 1 week | |
| Measurement after 1 month |
Table 2
Detailed description of various processing steps applied to the samples
| Flash freezing | Aliquoted samples were immersed in liquid nitrogen for at least 3 min, until visual inspection of samples confirmed full freeze. For prolonged storage, stock samples were moved to the precooled freezer (−70°C). |
| Fast freezing | Aliquoted samples were placed in −70°C precooled freezer for at least 12 h. For prolonged storage, stock samples were kept in the −70°C precooled freezer. |
| Slow freezing | Aliquoted samples were placed in −22°C precooled freezer for at least 12 h. For prolonged storage, stock samples were kept in −22°C precooled freezer. |
| Thawing | Reconstitution was realized by placing the stock sample at 4°C for thawing. The reconstituted sample was stored at 4°C for the whole sample use period. |
| Freeze-drying and redispersion | All stock samples, frozen in different conditions, after overnight treatment, were placed together in precooled freeze-dryer, at high vacuum, with a condenser surface temperature of −50°C, for 120 h. Stock samples in the form of freeze-dried cakes in conical tubes were vacuum-sealed and stored at 4°C until reconstitution. Redispersion was realized by adding 10 mL of ultrapure water to the cake and overnight moderate stirring at 37°C. Redispersed sample was stored at 4°C for the whole sample use period. |
| Stock storage period | Is considered to start when the processing of the particulate sample is completed, e.g., in the case of freeze-dried samples for redispersion – after completed lyophilization, and in the case of frozen samples for thawing – after samples are placed in the freezer. |
Fig. 1
Freeze-dried cakes after completed lyophilization: (A) flash freezing; (B) fast freezing; (C) slow freezing.
Fig. 2
Influence of the processing method on the mean particle size. Data for samples frozen using different options indicated in the graph, and, directly afterwards, subsequently subjected either to thawing or freeze-drying followed by immediate redispersion in water. The straight line indicates the mean size of the control aliquot of NGs. Error bars indicate standard deviations of mean.
Fig. 3
Influence of the stock storage time on the mean particle size. Error bars indicate standard deviations of mean.
Fig. 4
Influence of the storage time on the mean particle size during prolonged use: (A) immediate reconstitution; (B) short-term storage (1 week); (C) long-term storage (1 month). Error bars indicate standard deviations of mean.
Fig. 5
Influence of the treatment scenario on the mean particle size 1 month after completed synthesis and purification. Error bars indicate standard deviations of mean.