Control of Klebsiella pneumoniae Infection in Mice by Using Dissolving Microarray Patches Containing Gentamicin

Using a murine model of Klebsiella pneumoniae bacterial infection, we demonstrate that gentamicin dissolving microarray patches, applied to murine ears, could control K. pneumoniae infection. Mice treated with microarray patches had reduced bacterial burden in the nasal-associated lymphoid tissue and lungs compared with their untreated counterparts.

Upon insertion into the skin, the needles create microscopic holes, bypassing the stratum corneum barrier and subsequently delivering drug contained in the MNs into the viable skin (reviewed in reference 13). MNs are typically fabricated such that they are short enough to avoid stimulation of dermal nerves, and therefore, they provide a simplified, painless method of drug delivery that is well accepted by human subjects (14,15). MNs offer the possibility for GEN delivery by less-experienced personnel and easier logistics for supplying to remote areas (16). As MNs dissolve upon insertion in the skin, they eliminate the requirement for sharps disposal and avoid transmission of blood-borne infections through needlestick injuries (17). Our previous work has demonstrated the successful transdermal delivery of therapeutically relevant concentrations of GEN using dissolving MNs in an in vivo model (7). However, the in vivo efficacy of antibiotics delivered transdermally via MN to control bacterial infection has yet to be demonstrated.
In this study, we aimed to test the therapeutic efficacy of antibiotics delivered via MNs. MNs containing GEN were prepared utilizing simplified manufacturing processes, as previously described ( Fig. 1) (12). MNs were formulated from aqueous blends containing 3.4% sodium hyaluronate, with molecular weight (MW) 250 to 400 kDa, in combination with 1% polyvinylpyrrolidone (PVP; 58 kDa; Sigma-Aldrich, Dorset, UK) and containing 10% GEN sulfate (Tokyo Chemical Industry UK Ltd., Oxford, UK). Upon insertion of MNs into skin, the drug content in the needles is delivered concurrently with MN dissolution. Some drug may also diffuse into the skin layers from the baseplate, thus, allowing for sustained drug release (18). GEN exhibits a concentrationdependent bactericidal effect with peak and trough serum concentrations that are therapeutically effective and nontoxic. Sustained delivery of GEN, with peak serum levels above 10 to 12 g/ml and trough serum levels above 2 g/ml, may be toxic (7,19). Accordingly, we fabricated MNs in two steps to localize the GEN content to needles and, thus, prevent toxicity associated with sustained GEN delivery. Thus, the baseplates contained no GEN and were formulated from 15% PVP (360 kDa). The resultant MNs had heights of approximately 500 m and base widths of approximately 300 m, as confirmed by microscopic analysis (Fig. 1).
We subsequently probed the capacity of GEN MNs to induce therapeutic effects in vivo. We tested the GEN MNs in a Klebsiella pneumoniae murine model of pneumonia. K. pneumoniae is one of the most important Gram-negative pathogens associated with a wide spectrum of infections, including pneumonia, intraabdominal infections and bloodstream infections (20,21). GEN is a clinically relevant antibiotic treatment against K. pneumoniae, and therefore, this bacteria was selected as a model pathogen (22)(23)(24). Mice were infected with a live culture of K. pneumoniae (ATCC 43816) delivered intranasally (10 5 CFU per mouse in 30 l of endotoxin-free phosphate-buffered saline [PBS]), and this results in dissemination 24 h postinfection. The inoculum was plated for confirmation of bacterial number/ load. Following this, mice (n ϭ 6 to 7/group) were treated as per the schematic in Fig. 2A. In short, 8 h post-K. pneumoniae infection, GEN MNs were applied to the dorsal surface of each murine ear and held in place using micropore tape for 24 h, after which they were removed and replaced with additional MNs. GEN delivered i.m. to the thigh muscle of the hind limb was included as control. At 48 h postinfection, mice were sacrificed and the organs were harvested for analysis of bacterial burden. Body weights were monitored over the course of infection; however, no significant differences between groups were observed 48 h postinfection (Fig. 2B). As demonstrated in Fig. 2C, mice which received GEN i.m. or GEN MNs exhibited a greater capability to control infection, as evidenced by reduced numbers of CFU in the nasal-associated lymphoid tissue (NALT) and lungs. A significant reduction in CFU was observed in the NALT (P ϭ 0.0053) and lungs (P ϭ 0.0006) of mice treated with MNs compared with their untreated counterparts, demonstrating the in vivo activity of GEN delivered via MN. Specifically, application of GEN MN resulted almost a two-log reduction in the number of CFUs in the NALT and a 3.4-log reduction in lung CFU. A one-way analysis of variance (ANOVA), followed by correction for false discovery rate, was used for determination of statistical significance. A greater spread of results was evident in mice receiving GEN MNs than that of mice receiving GEN i.m., and this is likely attributable to the variability in the MNs manufactured under small-scale laboratory conditions. All experiments were performed in accordance with the UK Home Office and approved by the Queens University Belfast Ethical Review Committee. The baseplates were then applied to the backs of the MN, and pressure was applied as previously described. The combined MNs were dried for 48 h before being carefully removed from the molds and microscopically analyzed to ensure complete formation.
In conclusion, the results presented herein collectively demonstrate that MNs containing GEN effectively control K. pneumoniae infection in mice. While further studies are warranted to demonstrate complete clearance, this is the first reported study utilizing MNs for the treatment of bacterial infection. MNs may be a potentially viable delivery platform for antibiotic delivery, offering the possibility to expand access to lifesaving antibiotic treatment in low-resource settings. As MNs dissolve upon insertion in the skin, they circumvent the generation of sharps waste and associated transmission of blood-borne pathogens. Our ongoing efforts entail the optimization of GEN MNs to increase bioavailability and develop a thorough understanding of the pharmacokinetics and pharmacodynamics of GEN delivered via this route compared with that delivered i.m. In the era of increasing antimicrobial resistance, novel approaches for empirical therapy are necessitated and MNs may offer an ideal solution. Importantly, as MNs bypass the gastrointestinal microbiota, they also offer an alternative delivery option for antibiotics that are currently delivered orally and could potentially prevent dysbiosis of the gut microbiota.

ACKNOWLEDGMENTS
This work was supported by Biotechnology and Biological Sciences Research Council (BBSRC) grants (BB/L007223/1 and BB/P006078/1) to J.B. and the United States Agency nasal-associated lymphoid tissue (NALT), (D) lungs, and (E) spleens. CFU counts for individual mice are shown with solid lines corresponding to mean values, n ϭ6-7 mice/group, and "ϩ" indicates an animal that died. Statistical significance was determined using Prism 7 (GraphPad) software using a one-way ANOVA, followed by correction for false discovery rate via the two-stage step-up method of Benjamini, Krieger, and Yekuutieli.