CONTEXTO CONFLICTOS VISIÓN DEL PERSONAJE INFANTIL

Finally, the influence of total drug load on FITC dextran 70 release was investigated. Numerous studies on various delivery systems have shown that this parameter may substantially influence the release behavior, e.g. for collagen minipellets (Sano et al.; 2003), collagen discs (Gilbert; 1988b), poly(orthoester) discs (Sparer et al.; 1984), EVAc matrices (Saltzman; 2001) and tristearin implants (Mohl; 2004).

Maeda et al. investigated the incorporation of HSA into minipellets (Maeda et al.; 1999). HSA was found as fine particulated clusters inside the matrices which were homogeneously distributed throughout the complete collagen extrudate. After water contact, these clusters dissolve and the drug could be released. As a result, small cavities remained inside the matrix at the areas where HSA was located before dissolution (Maeda et al.; 1999; Sano et al.; 2003). Although Gilbert et al. postulated that drug loads higher than 10% inulin were necessary to create additional cavities (Gilbert et al.; 1990), Maeda et al. could demonstrate that even 1% HSA was sufficient to observe small pores in the surface of collagen extrudates during release (Maeda et al.; 1999). Since the structure of our minirods resembled more Maeda’s extrudates and because model drugs with higher molecular weights were used, it was assumed that new pores could be built during FITC dextran release as well. A 2% FITC dextran 70 concentration was used as highest model drug concentration. FITC dextran solutions with higher concentrations show markedly increased viscosity which may lead to inhomogeneous drug distribution. Furthermore, the intense fluorescence may cause trouble in fluorescent investigations. As was also observed for HSA, interferon alpha and G-CSF release from collagen minipellets (Sano et al.; 2003), an increase in drug concentration resulted in a relative increase in drug liberation (for FITC dextran 70 see Figure 4-47a), because more cavities could be built. The density of the FITC dextran 70 loaded

devices (see Table 4-1; 1%: 1.109g/cm³, 2%: 0.829g/cm³) and consequently the thickness of the diffusion barrier decreased and a faster release took place (Sano et al.; 2003). This observation was made with highly water soluble compounds which are readily released by diffusion. Besides “normal” diffusion through water filled pores, diffusion through the swollen matrix itself could take place. This was observed for the release of hGH from air dried 2% collagen films (Maeda et al.; 2001). A 3% drug load resulted in Fickian diffusion, whereas release of 30% hGH was accelerated and became dependent on diffusion through water filled pores as well as the swollen matrix itself. If the drug is less soluble under physiological conditions or if interactions between drug and collagen occur, drug loading and diffusion controlled release becomes less important. This effect was observed for rh-BMP 2 (recombinant human bone morphogenetic protein 2) release from minipellets (Maeda et al.; 2004b). For rh- BMP 2, diffusion controlled delivery could be amplified if glutamic acid was added which lowers the pH value inside the collagen matrix and increases the solubility of collagen and rh-BMP 2 (Maeda et al.; 2004a).

0 96 192 288 384 0 10 20 30 40 50 60 70 80 90 100 110 releas ed F ITC dex tran 70 [%] time [h] 1% 2% a 0 96 192 288 384 0 20 40 60 80 100 b degraded collagen [%] time [h] 1% 2%

Figure 4-47 Effect of the FITC dextran 70 concentration on drug release (a) and collagen degradation (b) of 10mm equine non cross-linked minirods (0.1µg/ml enzyme was added after 0.25h; average ± SD; n=3)

Since degradation of 1% and 2% minirods was identically the assumption of pore formation and additional diffusion controlled release through these new

cavities could be accepted for our minirods. This resulted in a higher initial release for the 2% devices (see Figure 4-47).

4.3.6 Summary

The objective of this chapter was to investigate the in vitro release of FITC dextrans from collagen minirods. Since drug release from collagen devices is mainly governed by diffusion dependent liberation (Radu et al.; 2002), diffusion equations had to be implemented into the mathematical model (see 4.5). Therefore, the diffusion coefficients in water of FITC dextran 20, 70 and 150 were determined by FCS. According to their molecular weights, the diffusion coefficient decreased from 3*10-3_{cm²/h for FITC dextran 20 to 1.8*10}-3_{cm²/h for}

FITC dextran 150. The observed diffusion coefficient of FITC dextran 70 (2.4*10-3cm²/h) corresponded well with the diffusion coefficient of collagenase, described in the literature, and was consequently used for the enzyme in the mathematical model as well.

Drug release was investigated with respect to the used collagen matrix quality, the incorporated drug and the added enzyme. If higher amounts of collagenase were added, FITC dextran 70 release and collagen degradation became faster (24h release: 0µg/ml collagenase: 35%, 0.1µg/ml collagenase: 50%, 6.7µg/ml collagenase: 85%, 24h degradation: 0.1µg/ml collagenase: 15%, 6.7µg/ml collagenase: 55%) and less drug was entrapped inside the collagen matrix, suggesting that a distinct drug portion could only be released by matrix erosion. It was demonstrated that the addition of collagenase to a fully swollen matrix resulted in faster collagen digestion, because more enzyme binding and cleavage sites became accessible.

Using different collagen materials for minirod preparation gave a tool to control drug release. A fast release (and degradation) was observed for pulverized equine non cross-linked collagen and bovine tendon non cross-linked minirods. Delivery could be delayed by using lyophilized equine material. Further retardation in drug liberation was achieved by cross-linking. DHT treatment resulted in lower enzymatic resistance than EDC cross-linking (Pieper et al.; 1999) and in consequence release was not as suppressed as from chemically

cross-linked matrices. Different ratios of EDC cross-linking were examined. Both investigated types of collagen, equine and bovine corium, showed the most promising results for EDC 1 cross-linked materials.

With changing the dimensions of the extrudates, drug release could also be controlled. Increasing the length or diameter of the cylindrical rods resulted in a decrease of FITC dextran 70 delivery. This was attributed on the one hand to an increase of the smooth lateral surface area, if minirods were elongated, which exhibit a more hindered release pattern than the porous cross-section. On the other hand, if extrudates were thickened, diffusion ways increased and the specific surface area decreased which resulted in a lower particle release by diffusion.

The last parameters which can influence drug release derive from the incorporated drug itself. In general, it is assumed that increasing the molecular weight lead to slower release by diffusion. However, the theoretical order FITC dextran 20 - 70 - 150 could only be detected for the diffusion based release from equine EDC 1 cross-linked minirods. The other collagen matrices met another order: FITC dextran 70 was always released slowest followed by FITC dextran 150 and 20. This change in sequence was attributed to the higher apparent density of FITC dextran 70 extrudates resulting in a lower degradation behavior. Since the other two FITC dextran loads exhibited almost identical matrix densities, the liberation of FITC dextran 150 was delayed due to more pronounced interactions with the collagen mesh. This indicated that the influence of the matrix structure on the release and degradation pattern was more pronounced than the effect of the molecular size of the incorporated drugs. If the incorporated drug portion was increased, faster release occurred. It was assumed that the faster release from 2% matrices derive from diffusion controlled release through newly formed cavities built at positions formerly filled by drug, because 1% and 2% minirods were degraded identically.