Charged dendrimers as antimicrobial coatings for biomaterials

According to a survey conducted in October 2012 a third of all dental implants is affected by peri-implantitis ten years after insertion. Unfortunately, the impact of surface properties, such as charge, wettability and coating agility on protein adsorption and subsequent biological events, like bacterial adhesion and biofilm formation, is still not fully unravelled. To date there are no bi-functional coatings that suppress the adhesion and growth of pathogenic organisms while simultaneously stimulating the integration of the implant into the surrounding tissue. A coating combining both properties is believed to reduce the incidence of peri-implantitis at the trans-gingival passage. Therefore, this thesis focuses on the preparation and characterization of covalent surface modifications terminated by functional groups, capable of generating charge in aqueous solution. The following functionalities were chosen for this purpose: the ammonium (-NH_3^+), the pyridinium (-N^+C_5H_5), the sulfonate (-SO_3^-), and the carboxylate (-COO^-) functionality. They were inserted in two diverse basic coatings. On the one hand as a terminal group in the well known "self-assembled monolayer" (SAM) system based on linear alkyl silanes on silicon dioxide surfaces and on the other hand at the periphery of immobilised polyamidoamine (PAMAM) dendrimers. Non-ionizable methyl terminated coatings, poly(ethylene glycol) functionalized surfaces and highly hydrophilic oxidized \ce{SiO2} surfaces served as references. Dendrimers were chosen since they are an efficient and omnipresent structural element in nature (see e.g. nerves). The functionalities dendritically arranged at a surface are believed to be of higher concentration and possess a higher degree of freedom in comparison to the functionalities anchored within the rigid SAM system. The immobilization of PAMAM onto the substrata was achieved by a thin siloxane coating terminated by carboxylic acid anhydride moieties. The first part of this thesis focuses on the preparation and physico-chemical characterization of the above mentioned coatings. Diffuse-reflectance infrared Fourier transformed spectroscopy (DRIFT), X-ray photoelectron spectroscopy (XPS) and a chemiluminescence based assay for the detection of terminal amino groups were performed to determine the preparation success of the surface modifications. The impact of the functional groups in their corresponding fixation on the electrokinetic potential was assessed by electrokinetic measurements. Furthermore, the wettability of the surfaces was determined by static water contact angle measurements. The dendrimer backbone resulted in a higher or comparable wettability of the surface in comparison to the fixation of the identical functionality via linear alkyl chains. At SAM-coatings a mainly pronounced negative electrokinetic potential (-30 mV to -120 mV) was detected in neutral aqueous solution, with the exception of the amino-group terminated SAM at which a value around zero was determined. In contrast, the novel coatings based upon dendrimers are rather moderately charged (-20 mV to 30 mV) at pH 7. The zeta-potential value in dependence of the pH of the surrounding solution at the SAM-coatings was observed to depend solely on the ionization behaviour of the surface confined functionalities, whereas the "effective" zeta-potential-pH profiles at the PAMAM-coatings could only be explained considering film swelling and structural rearrangements. That the dendrimer maintained its flexibility upon immobilization was also supported by pilot sum frequency spectroscopy (SFG) measurements at the coating/air interface. No unambiguous correlation was recognized between wettability and charge. The second part of this thesis investigates the influence of the surface modifications on protein adsorption from saliva and serum, as well as bacterial adhesion and viability of Streptococcus gordonii DL1 and Staphylococcus aureus in the presence and absence of a pre-coat from the former mentioned physiological solutions. In addition the impact of the surfaces coatings on human MG-63 osteoblast attachment was inspected. The PAMAM-coatings showed comparable or lower amounts of protein upon the surfaces after the exposure to human whole saliva in comparison to their linear analogues. Serum exposure resulted in an adverse effect, maintaining or supporting the adsorption of proteins on PAMAM-coatings in comparison to the alkyl chain based films. Both bacteria investigated preferred dendrimer based coatings terminated by cationic functionalities and avoided the settlement on surfaces functionalized by dendrimers with anionic or neutral periphery. PAMAM-COOH is as effective and PAMAM-CH_3 nearly as effective as the gold standard SAM-PEG in prohibiting bacterial attachment. This property was even slightly enforced after serum conditioning, whereas it was slightly reduced after saliva contact. In contrast all SAM coatings were colonized by both bacteria irrespective of the terminal functionality. Regarding the vitality of the adhering bacteria, S. gordonii, was only influenced by pyridinium coated samples, whereas the bactericidal effect was stronger on PAMAM-Py than at the SAM-Py coated surfaces. This effect was lost after saliva pre-conditioning. The vitality of S. aureus was also reduced on pyridinium terminated coatings, but the most pronounced effect was achieved with PAMAM-CH_3. After serum exposure this bactericidal property was maintained. The antimicrobial property of PAMAM-NH_2, reported in literature, could not be preserved after tethering the molecule to the surface. Cell adhesion studies with human MG-63 osteoblasts showed comparable cell numbers on the dendrimer based coatings in comparison to the alkyl chain based coatings terminated by the same functionality, but after serum exposure higher cell numbers could be detected on the dendrimer based coatings in comparison to their linear analogues bearing the same functionality. This study demonstrates, that a surface fixation of the terminal groups via a dendrimer possesses a higher potential of modulating the oral biological response, than the supplement of the same functional groups via linear alkyl chains. Since PAMAM-CH_3 revealed a gentle antimicrobial property towards S. aureus and reduced the settlement of bacteria, PAMAM-CH_3 might be promising in reducing peri-implantitis at the trans-gingival passage without minimizing the attachment of bone forming cells.