Clinical investigation of use of an antimicrobial peptide hydrogel wound dressing on intact skin.

A material with the ability to rapidly eradicate bacteria via a contact-killing mechanism has the benefit of a more localised treatment that is easy to implement when needed to prevent or treat a bacterial infection. Here, we present an antimicrobial material based on covalently attached antimicrobial peptides (AMPs) to a soft amphiphilic hydrogel. This results in a material that exhibits an antimicrobial effect based on contact-killing. In this study, the antimicrobial efficacy of the AMP-hydrogel was investigated by observing the changes in total bioburden on the intact skin of healthy human volunteers when the AMP-hydrogel dressing was placed on the forearm for three hours. The AMP-hydrogel significantly reduced the bioburden on the skin from a mean value of 1200CFU/cm2 for the untreated skin to 23CFU/cm2. Biocompatibility evaluations of the AMP-hydrogel showed no sign of cytotoxicity, acute systemic toxicity, irritation or sensitisation, demonstrating the safety of the AMP-hydrogel as a potential wound dressing. Leachability studies confirmed no release of AMPs and that the antimicrobial effect was localised to the surface of the hydrogels, demonstrating a pure contact-killing mode of action.

Cross-linked lyotropic liquid crystal particles functionalized with antimicrobial peptides.

Antimicrobial peptides (AMPs) are promising alternatives to traditional antibiotics for addressing bacterial infections - including life-threatening antibiotic resistant infections. AMPs have a broad spectrum of antimicrobial activity and show a low probability to induce resistance. However, the poor serum stability of AMPs has limited their usage in clinical treatment. To enable improved serum stability while maintaining high antibacterial effect of AMPs, this study describes a material wherein AMPs are covalently bonded to micro-sized particles of cross-linked lyotropic liquid crystals, formed by the self-assembly of the block copolymer Pluronic F-127. The liquid crystal particles were shown to have antibacterial effect corresponding to a 4 log reduction against Staphylococcus aureus. The particles were structurally and chemically analyzed by small angle X-ray scattering, Fourier transform infra-red spectroscopy and Raman spectroscopy, confirming that the liquid crystal structure was maintained within the particles with the AMPs covalently bonded. The bonding to the particles gave the AMPs improved stability in serum, as they retained almost all of the antibacterial potency for 2 days compared to free AMPs, which lost all of its antibacterial potency within a day. Furthermore, insight regarding mode of action was obtained by cryogenic transmission electron microscopy, which showed the antimicrobial particles interacting with the surface of bacteria.

Antimicrobial Peptide-Functionalized Mesoporous Hydrogels.

Antimicrobial peptides (AMPs) are seen as a promising replacement to conventional antibiotics for the prevention of skin wound infections. However, due to the short half-life of AMPs in biological environments, such as blood, their use in clinical applications has been limited. The covalent immobilization of AMPs onto suitable substrates is an effective solution to create contact-killing surfaces with increased long-term stability. In this work, an antimicrobial peptide, RRPRPRPRPWWWW-NH2 (RRP9W4N), was covalently attached to amphiphilic and ordered mesoporous Pluronic F127 hydrogels made of cross-linked lyotropic liquid crystals through 1-ethyl-3-(3-(dimethylamino)propyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS) chemistry. The AMP-hydrogels showed high antibacterial activity against Staphylococcus epidermidis, Staphylococcus aureus, Pseudomonas aeruginosa, methicillin-resistant S. aureus (MRSA), and multidrug-resistant Escherichia coli for up to 24 h. Furthermore, the AMP-hydrogels did not present any toxicity to human fibroblasts. The AMPs retained their antimicrobial activity up to 48 h in human blood serum, which is a significant increase in stability compared to when used in dissolved state. A pilot in vivo rat model showed 10-100× less viable counts of S. aureus on AMP-hydrogels compared with control hydrogels during the first 3 days of infection. Studies performed on human whole blood showed that blood coagulated more readily in the presence of AMP-hydrogels as compared to hydrogels without AMPs, indicating potential hemostatic activity. Overall, the results suggest that the combination of amphiphilic hydrogels with covalently bonded AMPs has potential to be used as antibacterial wound dressing material to reduce infections and promote hemostatic activity as an alternative to antibiotics or other antimicrobial agents, whose use should be restricted.

Osseointegration effects of local release of strontium ranelate from implant surfaces in rats.

BACKGROUND: Numerous studies have reported the beneficial effects of strontium on bone growth, particularly by stimulating osteoblast proliferation and differentiation. Thus, strontium release around implants has been suggested as one possible strategy to enhance implant osseointegration. AIM: This study aimed to evaluate whether the local release of strontium ranelate (Sr-ranelate) from implants coated with mesoporous titania could improve bone formation around implants in an animal model. MATERIALS AND METHODS: Mesoporous titania (MT) thin coatings were formed utilizing the evaporation induced self-assembly (EISA) method using Pluronic (P123) with or without the addition of poly propylene glycol (PPG) to create materials with two different pore sizes. The MT was deposited on disks and mini-screws, both made of cp Ti grade IV. Scanning electron microscopy (SEM) was performed to characterize the MT using a Leo Ultra55 FEG instrument (Zeiss, Oberkochen, Germany). The MT was loaded with Sr-ranelate using soaking and the drug uptake and release kinetics to and from the surfaces were evaluated using quartz crystal microbalance with dissipation monitoring (QCM-D) utilizing a Q-sense E4 instrument. For the in vivo experiment, 24 adult rats were analyzed at two time points of implant healing (2 and 6 weeks). Titanium implants shaped as mini screws were coated with MT films and divided into two groups; supplied with Sr-ranelate (test group) and without Sr-ranelate (control group). Four implants (both test and control) were inserted in the tibia of each rat. The in vivo study was evaluated using histomorphometric analyses of the implant/bone interphase using optical microscopy. RESULTS: SEM images showed the successful formation of evenly distributed MT films covering the entire surface with pore sizes of 6 and 7.2 nm, respectively. The QCM-D analysis revealed an absorption of 3300 ng/cm2 of Sr-ranelate on the 7.2 nm MT, which was about 3 times more than the observed amount on the 6 nm MT (1200 ng/cm2). Both groups showed sustained release of Sr-ranelate from MT coated disks. The histomorphometric analysis revealed no significant differences in bone implant contact (BIC) and bone area (BA) between the implants with Sr-ranelate and implants in the control groups after 2 and 6 weeks of healing (BIC with a p-value of 0.43 after 2 weeks and 0.172 after 6 weeks; BA with a p-value of 0.503 after 2 weeks, and 0.088 after 6 weeks). The mean BIC and BA values within the same group showed significant increase among all groups between 2 and 6 weeks. CONCLUSION: This study could not confirm any positive effects of Sr-ranelate on implant osseointegration.

Development of a photon induced drug-delivery implant coating.

A thin surface coating intended for medical devices such as implants where local drug release is enabled using near infrared light (NIR) as an external stimulus has been developed. The delivery system consists of a thin Poly (N-isopropylacrylamide)-co-acrylamide (PNIPAAm-AAm) polymer layer with incorporated gold nanorods (GNRs). The aspect ratio of the GNRs were chosen to absorb NIR light, thus fitting the biological window of low tissue absorption, to locally heat the polymeric layer to initiate a drug release. Hence, controlled drug delivery from a surface within tissue orchestrated from outside the body is achievable. Composition of the PNIPAAm-AAm co-polymer was systematically varied to find a suitable phase transition temperature for in vivo applications. Differential scanning calorimetry (DSC) analysis showed that PNIPAAm-AAm containing 10% acrylamide had an appropriate phase transition temperature of 42 °C. As visualized by scanning electron microscopy (SEM), the surface coating consisted of 200 nm uniform polymer layer. Quartz crystal microbalance with dissipation monitoring (QCM-D) analysis coupled with in situ NIR irradiation demonstrated a dramatic shift in frequency that was attributed to mass being released from the surface upon irradiation. This mass release correlated well with the drug release profile as determined using UV/VIS spectroscopy with phenol as a model drug. In addition, proof-of-concept of the drug-delivery system was demonstrated by releasing the antibiotic vancomycin to eradicate Staphylococcus epidermidis bacteria in culture.

Antibiofilm elastin-like polypeptide coatings: functionality, stability, and selectivity.

Antimicrobial peptides (AMPs) are currently receiving interest as an alternative to conventional antibiotics to treat biomaterial-associated infection. However, the inherent instability of such peptides often limits their efficacy in intended clinical applications. Covalent immobilization of AMPs to surfaces is one strategy to increase the long-term stability and minimize the toxicity. In this work, an antimicrobial peptide, RRPRPRPRPWWWW-NH2 (RRP9W4N), was used to modify elastin-like polypeptide (ELP) surface coatings containing cell-adhesive peptide domains (RGD) using covalent chemistry. The AMP retained its antibacterial activity against Staphylococcus epidermidis, Staphylococcus aureus, and Pseudomonas aeruginosa when covalently bonded to ELP surfaces. Simultaneously, the AMP functionalization had insignificant effect on the viability, function, and differentiation of human osteosarcoma MG63 cells and human mesenchymal stem cells (hMSCs). Furthermore, stability of the immobilized AMP in human blood serum was investigated, and the results suggested that the AMP preserved its antibacterial activity up to 24 h. Combined, the results show that covalently attached AMPs onto RGD-containing ELP are an excellent candidate as an antimicrobial coating for medical devices. STATEMENT OF SIGNIFICANCE: Biomaterial associated infection, caused by adherent biofilm, is usually difficult to treat. There is a high demand for new materials and treatments to decrease the infection rates, especially with increasing threats concerning resistant bacteria. Formation of biofilms on medical devices lowers the bacteria susceptibility towards traditional antibiotics and also circumvent our immune system often resulting in revisional surgery and extensive use of antibiotics. One promising strategy is to develop surfaces having low bacterial attractiveness or bacterial killing properties, but still retaining the main function of the device. In this study, we have developed an implant coating that demonstrates a high antimicrobial effect and at the same time showing no negative affect on human cells.

Modulation of the nanometer pore size improves magnesium adsorption into mesoporous titania coatings and promotes bone morphogenic protein 4 expression in adhering osteoblasts.

OBJECTIVE: Mesoporous (MP) titania films used as implant coatings have recently been considered as release systems for controlled administration of magnesium to enhance initial osteoblast proliferation in vitro. Tuning of the pore size in such titania films is aimed at increasing the osteogenic potential through effects on the total loading capacity and the release profile of magnesium. METHODS: In this study, evaporation-induced self-assembly (EISA) was used with different structure-directing agents to form three mesoporous films with average pore sizes of 2nm (MP1), 6nm (MP2) and 7nm (MP3). Mg adsorption and release was monitored using quartz crystal microbalance with dissipation (QCM-D). The film surfaces were characterized with atomic force microscopy (AFM), scanning electron microscopy (SEM) and X-ray photoelectron spectroscopy (XPS). The effect of different Mg release on osteogenesis was investigated in human fetal osteoblasts (hFOB) using pre-designed osteogenesis arrays and real-time polymerase chain reaction (RT-PCR). RESULTS: Results showed a sustained release from all the films investigated, with higher magnesium adsorption into MP1 and MP3 films. No significant differences were observed in the surface nanotopography of the films, either with or without the presence of magnesium. MP3 films (7nm pore size) had the greatest effect on osteogenesis, up-regulating 15 bone-related genes after 1 week of hFOB growth and significantly promoting bone morphogenic protein (BMP4) expression after 3 weeks of growth. SIGNIFICANCE: The findings indicate that the increase in pore width on the nano scale significantly enhanced the bioactivity of the mesoporous coating, thus accelerating osteogenesis without creating differences in surface roughness.

Stem cell homing using local delivery of plerixafor and stromal derived growth factor-1alpha for improved bone regeneration around Ti-implants.

Triggering of the early healing events, including the recruitment of progenitor cells, has been suggested to promote bone regeneration. In implantology, local drug release technologies could provide an attractive approach to promote tissue regeneration. In this study, we targeted the chemotactic SDF-1α/CXCR4 axis that is responsible e.g. for the homing of stem cells to trauma sites. This was achieved by local delivery of plerixafor, an antagonist to CXCR4, and/or SDF-1α, from titanium implants coated with mesoporous titania thin films with a pore size of 7.5 nm. In vitro drug delivery experiments demonstrated that the mesoporous coating provided a high drug loading capacity and controlled release. The subsequent in vivo study in rat tibia showed beneficial effects with respect to bone-implant anchorage and bone-formation along the surface of the implants when plerixafor and SDF-1α were delivered locally. The effect was most prominent by the finding that the combination of the drugs significantly improved the mechanical bone anchorage. These observations suggest that titanium implants with local delivery of drugs for enhanced local recruitment of progenitor cells have the ability to promote osseointegration. This approach may provide a potential strategy for the development of novel implant treatments. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 2466-2475, 2016.

Antimicrobial performance of mesoporous titania thin films: role of pore size, hydrophobicity, and antibiotic release.

Implant-associated infections are undesirable complications that might arise after implant surgery. If the infection is not prevented, it can lead to tremendous cost, trauma, and even life threatening conditions for the patient. Development of an implant coating loaded with antimicrobial substances would be an effective way to improve the success rate of implants. In this study, the in vitro efficacy of mesoporous titania thin films used as a novel antimicrobial release coating was evaluated. Mesoporous titania thin films with pore diameters of 4, 6, and 7 nm were synthesized using the evaporation-induced self-assembly method. The films were characterized and loaded with antimicrobial agents, including vancomycin, gentamicin, and daptomycin. Staphylococcus aureus and Pseudomonas aeruginosa were used to evaluate their effectiveness toward inhibiting bacterial colonization. Drug loading and delivery were studied using a quartz crystal microbalance with dissipation monitoring, which showed successful loading and release of the antibiotics from the surfaces. Results from counting bacterial colony-forming units showed reduced bacterial adhesion on the drug-loaded films. Interestingly, the presence of the pores alone had a desired effect on bacterial colonization, which can be attributed to the documented nanotopographical effect. In summary, this study provides significant promise for the use of mesoporous titania thin films for reducing implant infections.

Controlling drug delivery kinetics from mesoporous titania thin films by pore size and surface energy.

The osseointegration capacity of bone-anchoring implants can be improved by the use of drugs that are administrated by an inbuilt drug delivery system. However, to attain superior control of drug delivery and to have the ability to administer drugs of varying size, including proteins, further material development of drug carriers is needed. Mesoporous materials have shown great potential in drug delivery applications to provide and maintain a drug concentration within the therapeutic window for the desired period of time. Moreover, drug delivery from coatings consisting of mesoporous titania has shown to be promising to improve healing of bone-anchoring implants. Here we report on how the delivery of an osteoporosis drug, alendronate, can be controlled by altering pore size and surface energy of mesoporous titania thin films. The pore size was varied from 3.4 nm to 7.2 nm by the use of different structure-directing templates and addition of a swelling agent. The surface energy was also altered by grafting dimethylsilane to the pore walls. The drug uptake and release profiles were monitored in situ using quartz crystal microbalance with dissipation (QCM-D) and it was shown that both pore size and surface energy had a profound effect on both the adsorption and release kinetics of alendronate. The QCM-D data provided evidence that the drug delivery from mesoporous titania films is controlled by a binding-diffusion mechanism. The yielded knowledge of release kinetics is crucial in order to improve the in vivo tissue response associated to therapeutic treatments.