Table of Contents:
Within the scope of the present doctoral thesis, novel drug carrier systems based on naturally occurring and synthetic bolaamphiphiles for antimicrobial photodynamic therapy (aPDT) against bacteria and fungi were generated, characterized and first
in vitro tests were carried out. The aim of this work was the development of stable carrier systems, which allow adequate therapy while being skin and mucous membrane compatible.
Chapter 1 introduced the topic with an explanation of the problem of increasing antibiotic resistance in chronic wounds and mucosal infections caused by yeast, while simultaneously an adequate therapy and wound management is not available. Photodynamic therapy (PDT) was presented as a possible solution, highlighting the advantage of drug delivery systems made from bolaamphiphiles. The first chapter was followed by a summary and explanation of the manufacturing and characterization methods used in this work (chapter 2).
Chapter 3 addressed the production and characterization of both drug delivery systems. First, the natural bolaamphiphilic lipids (tetraether lipids - TEL) were successfully extracted from the freeze-dried biomass of Sulfolobus acidocaldarius. By adding the phospholipid DSPC in various proportions, stable vesicular phospholipid gels (VPGs) were developed. These were characterized by means of scanning electron microscopy (SEM), cryo-transmission electron microscopy (cryo-TEM) and rotational viscosimetry. SEM images showed differences between the native carrier system and those with incorporated methylene blue (MB). VPG produced with DSPC:TEL (50:50) without drug showed a phase separation of the system, which could be confirmed by cryo-TEM examinations. Cryo-TEM pictures were carried out with the VPGs diluted to 2 mg/mL and, as expected, showed the structures phospholipids and TEL form spontaneously in aqueous media. An increase in the vesicle size with increasing proportions of added TEL was observed. In contrast to the other systems, DSPC:TEL (50:50)-VPG did not show uniform vesicles, but rather micellar and vesicular structures occurring simultaneously. Viscosity characterization using rotational viscometer revealed differences between the carrier systems with and without MB for DSPC and DSPC:TEL (50:50) formulations, which could already been suspected from the SEM images. In contrast to that DSPC:TEL (80:20) and DSPC:TEL (70:30) VPGs showed nearly the same rheological behavior. During the rheological investigation of the hydrogels obtained from PC-C32-PC and
Me2PE-C32-Me2PE the stability of the self-organized hydrogels decreased with minimal shear forces. This fact indicates an inadequate stability at the application site using these hydrogels. With the help of sublimation in a conventional freeze-drier it was possible to create stable aerogels which could be transformed into hydrogels in situ by addition of water. The fully hydrated aerogels showed only slightly different behavior in the rheogram compared to original hydrogels. Morphological characterization of the aerogels was realized using SEM. Both aerogels showed uniform pores within the carrier system. This is comparable to freeze-dried hydrogels, which are already used as intelligent wound dressings in the therapy of deep wounds.
Chapter 4 investigated the drug release behavior of the drug delivery systems. All of the gels tested were able to release MB: The vesicular phospholipid gels were able to release MB in a sustained manner over several days. In particular, the VPGs made from DSPC:TEL (80:20) and DSPC:TEL (70:30) with a release time of seven days are very interesting candidates for long-term therapy of chronic wound infections. In contrast the release of PC-C32-PC and Me2PE-C32-Me2PE aerogels took only six and eight hours, respectively, due to the burst-release of the active ingredient at the beginning and the delayed gel formation. This fact hampers the use of native aerogels as intelligent drug delivery systems for long-term therapy. In contrast, the hydrated aerogels (hydrogels) show a clearly delayed release, which was superior to HEC 300 hydrogels used as reference. Combining the advantages of both systems appears to be more effective. Aerogels have long-term stability and are an easy-to-use system for in situ hydrogel formation.
The investigation of the photodynamic effect of the drug carrier systems on the gram-positive germs Staphylococcus saprophyticus subsp. bovis (DSM 18669) and Staphylococcus aureus subsp. aureus (ATCC 25923) and the gram-negative bacteria Escherichia coli (DH5α) was discussed in Chapter 5. To the gram-positive germ a bacteriostatic effect under the MB-mediated treatment with the carrier systems even without exposure to radiation could be observed due to the bacteriostatic effect of the MB itself. After irradiation an increased bacteriostatic effect was found for both germs. The bactericidal effect occurred under the aPDT in both germs but could only be quantified in S. saprophyticus subsp. bovis (DSM 18669), as the measurement data from the Šumperk Hospital in the Czech Republic, where the experiments were carried out, was not available. E. coli (DH5α) showed no sensitivity to the active substance carrier systems loaded with MB without applied aPDT. After irradiation both bacteriostatic and bactericidal effect took place. However, the effect was less distinctive compared to gram-positive bacteria.
Chapter 6 dealt with the characterization of the aPDT sensitivity of yeasts under therapy with the carrier systems made from natural and synthetic bolaamphiphiles. Saccharomyces cerevisiae was chosen as model germ. Similar to the gram-positive cocci, the microorganisms exhibited a slight fungistatic and occasional fungicidal effect even without irradiation. Using aPDT, both effects were considerably increased in all drug delivery systems. Additionally, with exception of the PC-C32-PC aerogel, all systems were superior compared to the control group.
Mucous membranes and the wound bed represent application sites which have special requirements on the drug delivery systems. For this reason, Chapter 7 examined the compatibility of gels made from DSPC and DSPC:TEL with blood components. All VPGs with and without MB were tested for their hemolytic potential. Drug delivery systems containing DSPC and DSPC:TEL (80:20) showed no hemolytic value. Based on the use of higher amount of GLE with included impurities VPGs made by DSPC:TEL (70:30) and DSPC:TEL 850:50) demonstrated moderate hemolytic potential. In summary, it was found that only the DSPC- and DSPC:TEL (80:20)-system fulfill all requirements of good tolerability with blood components. Due to the small sample volume, this experiment could not be carried out with the aerogels made by PC-C32-PC and Me2PE-C32-Me2PE.
Long-term biocompatibility of the aerogels, the mucosal tolerance and the tolerance of the applied aPDT of all drug delivery systems made from bolaamphiphils were finally evaluated in Chapter 8 using the chorioallantoic membrane (CAM). The mucosal compatibility of the gels of DSPC, DSPC: TEL, PC-C32-PC and Me2PE-C32-Me2PE was compared with a hydroxyethyl cellulose 300 (HEC 300) hydrogel, suitable for vaginal application, using the “henn´s egg test on the chorioallantoic membrane“ (HET-CAM. Biocompatibility of the bolaamphiphilic drug delivery systems matched that of
the HEC 300 hydrogel. In order to investigate the long-term biocompatibility of the
PC-C32-PC and Me2PE-C32-Me2PE aerogels daily observations starting on egg development day 9 (EDD 9) of the blood vessels on CAM surface were conducted till EDD 14. A different hydration behavior of both aerogels was noticed which, however, did not affect the tolerability. Both systems were inert to the growing blood vessel system and no defense reactions were visible.
Side effects of PDT can include eradication of the microorganisms on the skin, mucous membrane or in the wound bed, as well as damage to healthy tissue. An examination of the biocompatibility of the therapy on the CAM was therefore carried out in Chapter 8 also. In order to ensure that neither the acting radiation nor the drug delivery system without active ingredient influenced the integrity of the blood vessels, these parameters were excluded in advance. The LED-mediated aPDT with MB using the DSPC and DSPC: TEL VPGs as well as the PC-C32-PC and Me2PE-C32-Me2PE aerogels took place in comparison to aqueous MB solution of the same concentration. Compared to the MB solution, the damage after irradiation is significantly less for all drug delivery systems. In particular, 24 and 48 hours after the treatment, there are serious differences in terms of vascular integrity within the CAM. While the considerable damage during the treatment with the reference system caused a structural change in the CAM, which manifested itself in a whitish-necrotic appearance, no such changes could be observed for the aPDT with all the bolaamphiphile-drug delivery systems. This fact proves the superior compatibility of the gels compared to the reference solution of the same active substance concentration.
In conclusion it can therefore be noted that stable formulations have been successfully developed from both natural and synthetic bolaamphiphiles. They are suitable for antimicrobial photodynamic therapy and at the same time are well tolerated. These characteristics qualify them as potential delivery systems for clinical application on skin, mucous membrane and wound infections.