Two different strategies can be applied in order to overcome the limited physical and chemical stability of suspensions during storage. On the one hand, stabilizers are used to avoid a loss in physical stability due to steric stabilization and increased electrostatic repulsion [42]. On the other hand, pharmaceutical drying techniques are employed to withdraw the water from aqueous suspensions. For that purpose, freeze-drying is the most commonly used drying technique [23]. As one advantage of freeze-drying is the excellent stabilization of sensitive drugs like therapeutic proteins [45], freeze-drying was applied to the development of stable protein loaded eADF4(C16) particle formulations. Previous knowledge of other groups, which already reported on freeze-drying of nanosuspensions, gives indications concerning the choice of possible excipients (lyo- and cryoprotectants) and formulation parameters [23, 24, 46, 47]. An overview of all investigated formulations and parameters in this work is given in Table 3.3.
Table 3.3. Freeze-drying of empty and lysozyme-loaded eADF4(C16) particles using different types of excipients, excipient-to-eADF4(C16) particles ratios [w/w] and reconstitution volumes after lyophilization
Excipient Filling volume [mL] Excipient-to-eADF4(C16) particles weight ratio Reconstitution volume [mL] empty eADF4(C16) particles
sucrose 1.0 0 / 5 / 10 / 30 / 50 0.1 / 0.2 / 1.0 sucrose trehalose mannitol 0.5 0.5 0.5 2.5 / 5 / 20 / 50 2.5 / 5 / 20 / 50 2.5 / 5 / 20 / 50 0.5 0.5 0.5 lysozyme-loaded eADF4(C16) particles
sucrose trehalose mannitol 0.5 0.5 0.5 30 30 30 0.5 0.5 0.5
3.5.1 F
EASIBILITY STUDYThe focus was to test the feasibility of freeze-drying eADF4(C16) nanosupensions. Therefore, a conventional lyophilization cycle could be employed because spider silk particles had shown no influence on Tg’ compared to placebo formulations (data not shown). Results from this freeze-drying cycle with empty eADF4(C16) particles and different concentrations of sucrose as freeze-drying excipient are displayed in Figure 3.19. Compared to particle sizes and polydispersity indices of the suspensions before lyophilization, none of the freeze-dried formulations containing 10:1 [w/w] or more sucrose showed increased values after reconstitution with the original filling volume of 1.0 mL (dashed bars). In contrast, freeze-drying of formulations lacking sucrose or comprising sucrose at a w/w-ratio of 5:1
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resulted in agglomerated particles represented by the increase in particle size and PI. Therefore, a minimum excipient-to-eADF4(C16) particle ratio of 10:1 [w/w] is considered to be required for a sufficient stabilization of spiders silk particles during freeze-drying.
Moreover, the effect of reduced reconstitution volumes was investigated according to Zillies et al. [25] as an opportunity to concentrate the formulations after freeze-drying. For instance, this technique may be necessary to obtain a required nanoparticle concentration and the corresponding drug load for a potential in vitro or in vivo study. An impact of reduced reconstitution volumes was only observed at an excipient-to-eADF4(C16) particles ratio of 5:1 [w/w]. Herein, reduced volumes led to an increase in particle size and polydispersity index, indicating that reconstitution with low volumes creates agglomerated spider silk particles. At excipient-to-eADF4(C16) particles ratios of 10:1 [w/w] or above, the lower reconstitution volumes of 1/5th and 1/10th of the original volume led to only minor changes in
particle size and PI. Therefore, the impact of the reconstitution volume can be neglected above the determined threshold and an increase of the nanoparticle concentration after lyophilization is possible without modification of the spider silk particles’ colloidal properties.
Figure 3.19. Particle size of reconstituted empty eADF4(C16) particles right after freeze-drying using sucrose as freeze-drying excipient at different sucrose-to-eADF4(C16) particles ratios [w/w] and using different reconstitution volumes from 1.0 to 0.1 mL
3.5.2 F
ORMULATION DEVELOPMENTIn a second set of freeze-drying experiments, two commonly used disaccharides, namely sucrose and trehalose as amorphous lyo- and cryoprotectants, and mannitol as bulking agent were compared regarding their ability to stabilize the physical properties of eADF4(C16) suspensions during freeze-drying (see Figure 3.20). As it can be concluded from the increased particle size after reconstitution, none of the three excipients was able to sufficiently prevent eADF4(C16) particles from agglomeration at an excipient-to-eADF4(C16) particles ratio of 5:1 [w/w] or less. The results above the determined threshold of a w/w-ratio of 10:1 were different. All formulations comprising either sucrose or trehalose showed no significant changes in eADF4(C16) particle size and no difference between the employed excipients. It is commonly known that freeze-dried formulations containing mannitol exhibit a different nature compared to sucrose or trehalose as mannitol readily crystallizes during freeze-drying. Hence, decreased protein drug recovery was detected after freeze-drying of different proteins with mannitol due to the prevention of requisite hydrogen bonding [48]. Nevertheless, it was described that in the case of freeze-drying particulate formulations mannitol was able to maintain nanoparticles’ integrity [25]. The authors explained their results by the so-called particle isolation theory which describes that almost any excipient disregarded of its behavior during freeze-drying is able to separate particles in the unfrozen fraction if sufficient amounts of the excipients are used. Our results confirm this hypothesis as an excipient-to-eADF4(C16) particle ratio of 10:1 is the threshold for sufficient stabilization of eADF4(C16) particles independent from the excipient used in the different formulations.
Figure 3.20. Particle size of empty eADF4(C16) particles after freeze-drying with sucrose, trehalose or mannitol at different excipient-to-eADF4(C16) particles ratios
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Nevertheless, the three employed excipients exhibit different properties regarding their stabilizing mechanism during freeze-drying and further storage of dried formulations. Sucrose and trehalose act as amorphous and glassy matrix formers which can be distinguished by their glass transition temperature. As shown in Figure 3.21, freeze-dried trehalose formulations possess a significantly higher Tg than sucrose formulations at almost
equal residual moisture contents of around 1%. A higher Tg is known to be superior during
storage stability of dried protein formulations so that trehalose containing formulations are recommended in the case of freeze-drying eADF4(C16) nanosuspensions. Mannitol containing formulations exhibited a typical crystalline structure represented by the melting point detected at 170°C. Therefore, mannitol does not possess any lyoprotectant activity regarding the stabilization of protein molecules [49]. The macroscopic appearance of sucrose containing formulations of the second freeze-drying experiment is exemplarily shown in Figure 3.21. The porosity of the resulting cake increased with decreasing solid content, but all investigated formulations exhibited an acceptable appearance after freeze-drying.
50:1 [w/w] formulation excipient moisture [%] residual Tg
[°C] [°C] mp
sucrose 1.11 46.1 -
trehalose 0.86 120.6 -
mannitol 0.32 - 170.1
Figure 3.21. Residual moisture and glass transition or melting temperature of freeze-dried formulations comprising a 50:1 [w/w]-ratio of excipient to eADF4(C16) particles. On the right hand side the macroscopic appearance of sucrose containing formulations is illustrated.
In addition to empty eADF4(C16) particles, 15% [w/w] lysozyme-loaded eADF4(C16) particles (via remote loading) were freeze-dried either with sucrose, trehalose or mannitol at an excipient-to-particles weight ratio of 30:1 using the same freeze-drying cycle as before. All investigated formulations exhibited the same particle size and PI after lyophilization and reconstitution (see Figure 3.22).
Therefore, two conclusions can be drawn from the set of freeze-drying studies. At first, freeze-drying of empty as well as lysozyme-loaded eADF4(C16) particles with an excipient- to-eADF4(C16) particles weight ratio of at least 10:1 is possible without losing the physical properties of the original eADF4(C16) particle nanosuspensions. Secondly, trehalose at a mass ratio of 30:1 or 50:1 is the recommended excipient concerning the storage stability of lyophilized eADF4(C16) formulations as the highest Tg was obtained and the colloidal
Figure 3.22. Particle size and polydispersity indices of reconstituted lysozyme-loaded eADF4(C16) particles using sucrose, mannitol or trehalose as freeze-drying excipients at a w/w-ratio of 30:1
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