Efficacy of Nanoencapsulated Daptomycin in an Experimental Methicillin-Resistant Staphylococcus aureus Bone and Joint Infection Model

ABSTRACT

Staphylococcus aureus bone infections remain a therapeutic challenge, leading to long and expensive hospitalizations. Systemic antibiotic treatments are inconsistently effective, due to insufficient penetration into the infectious site. In an osteomyelitis model, the single local administration of nanoparticle-encapsulated daptomycin allows sterilization of the infectious sites after 4 and 14 days of treatment, while daily systemic daptomycin treatment for 4 days was not effective. These results demonstrate the great potential of this local antibiotic treatment.

INTRODUCTION

Bone and joint infections (BJIs) are difficult to treat, usually involving several months of intravenous (i.v.) antibiotic therapy (1–4). The costs to society are high (5, 6). The low vascularization of the site partly explains the frequent failure of treatment, due to the poor diffusion of antibiotics (6). Therefore, increasing the local concentration of antibiotics within the infected bone could improve treatment efficacy. No clear recommendations exist regarding the local use of antibiotics. Despite several years of use in oncology, the usefulness of nanoparticle-encapsulated anti-infective treatments is still lacking. This new approach could simplify the treatment of BJIs, limiting systemic antibiotic therapy and its harmful consequences. Using a rabbit osteomyelitis model, we evaluated the activity of a gel loaded with daptomycin (DAP) encapsulated in lipid nanocapsules (LNC-DAP), compared with free i.v. DAP.

(This study was presented at the 26th European Congress of Clinical Microbiology and Infectious Diseases, Amsterdam, The Netherlands, 9 to 12 April 2016.)

The LNC-DAP gel was prepared using the so-called phase inversion temperature method (7, 8). The mean particle size and polydispersity index of the LNC-DAP were determined by dynamic light scattering analysis using a Zetasizer Nano DTS analyzer. The quantification of DAP was performed by high-performance liquid chromatography (HPLC)-UV analysis (9). Female New Zealand White rabbits (weight, 2.0 to 2.5 kg) were maintained under specific-pathogen-free conditions at the UTE-IRS2 Nantes Biotech Animal Facility (Nantes, France) following institutional guidelines (ethics approval for all experimental procedures, approval APAFIS 919120150056547296). The BJI rabbit model was induced as described previously (10). Briefly, a Jamshidi bone marrow biopsy needle was inserted, under general anesthesia, between the two femoral condyles to reach the medullar canal. A methicillin-resistant Staphylococcus aureus (MRSA) strain isolated from a blood culture (belonging to sequence type 8 [ST8] and clonal complex 1 [CC1]) with an MIC of 0.5 μg/ml for free DAP and LNC-DAP was used. One milliliter of a 10⁸ CFU/ml bacterial suspension was injected into the knee cavity. After 3 days, surgical debridement and an articular wash were performed to mimic a surgical procedure. Animals were randomly assigned to no treatment (controls), local administration of one ml of LNC-DAP at 200 mg/ml inserted using the bone defect, or an i.v. DAP regimen mimicking a human daily dose of 6 mg/kg for 4 days (11, 12). Bacterial counts in marrow and bone were determined at 3, 7, and 14 days postinoculation. The efficacy measurements were made by comparing the bacterial loads at the start and end of treatment. If no growth was seen on agar plates, then 50 μl of the tissue homogenate was seeded in brain heart infusion (BHI) medium and incubated at 37°C for 14 days. A sample was considered sterile if no growth was seen in BHI medium. Selection of resistant mutants was sought by plating tissue homogenates on agar plates containing DAP at 4× MIC. DAP concentrations in plasma and tissues were determined in three animals for each time point (0, 1, 2, 4, 10, and 14 days) after local administration of LNC-DAP, by a HPLC-tandem mass spectrometry assay adapted from a previously described method (13). The local toxicity and general toxicity of LNC-DAP were evaluated in a rat osteomyelitis model (male Sprague Dawley rats). Histological analyses of bones exposed to LNC-DAP or LNC without DAP (deposited in the induced condyle defects) were performed for 4 days, in comparison with a control group (untreated). Longitudinal sections were prepared and hematoxylin-eosin staining was performed on each section to allow the evaluation of tissue morphology. For each screened lesion, a scoring system was applied based on a scale from 0 to 5 (0, normal/no lesion; 5, severe lesions). A Student-Newman-Keuls test was performed after analysis of variance (GraphPad Prism software). P values of ≤0.05 were considered significant.

HPLC analyses of LNC-DAP revealed an efficiency of DAP entrapment in the gel of close to 100%, with a final DAP concentration of 213.2 ± 5.2 mg/g. LNC particles exhibited a mean diameter of 78.8 nm, with a polydispersity index of 0.15. The final product is composed of both free DAP and LNC-DAP (25% of DAP is encapsulated in LNCs inserted in the gel, whereas 75% of DAP is free in the gel). Before treatment initiation (day 3), the bacterial load was 8 to 9 log₁₀CFU/g of tissue for both bone marrow and bone. The load was stable between day 3 and day 7 in control (untreated) animals, leading to a Δlog₁₀CFU/g value close to 0. After a 4-day treatment, a single dose of LNC-DAP showed significant activity in the bone and bone marrow, in comparison with free i.v. DAP (P < 0.001) (Fig. 1A and B). The percentage of negative cultures was more than 75% 4 days after a single local administration of LNC-DAP (Fig. 1C and D). No negative cultures were observed with the i.v. DAP regimen. Furthermore, 14 days following a single LNC-DAP dose, the antibacterial activity was still significant in bone and bone marrow (Fig. 1A and B) and the negative culture rate was 100% (Fig. 1C and D). No variant resistant to DAP was detected in the group treated with DAP or LNC-DAP. LNC-DAP implanted in rat femoral condyle defects was well tolerated, in terms of weight, macroscopic observations of tissues, and bone histological analyses (no significant difference) 4 days after implantation.

FIG 1.

(A and B) In vivo antibacterial activity of once-daily i.v. administration of DAP (DAP IV) or a single local administration of encapsulated DAP (LNC-DAP), after 4 and 14 days of treatment, for MRSA-induced osteomyelitis. (A) Bacterial counts in bone marrow. (B) Bacterial counts in bone. Data represent two pooled independent experiments (n = 6 to 10 animals per group). ****, P < 0.001 versus the control and i.v. DAP groups. (C and D) Percentage of sterilization of bone marrow (C) and bone (D) tissues after once-daily i.v. administration of DAP (DAP IV) or a single local administration of encapsulated DAP (LNC-DAP), after 4 and 14 days of treatment, for MRSA-induced osteomyelitis. Data represent two pooled independent experiments (n = 4 to 10 animals per group).

The poor diffusion in infected tissues could explain the limited efficacy of systemic DAP therapy, as previously observed by others (14–16) and by our team using the BJI rabbit model (see Table S1 in the supplemental material). The potential of nanomaterials to increase antibiotic efficacy has already been mentioned (17–20), as has their harmlessness (21). The recent introduction of coronavirus disease 2019 (Covid-19) vaccines (mRNA combined with lipidic nanocapsules) exhibited no problems with local tolerability (22).

Our results confirm that inclusion of antibiotics into LNCs confers prolonged local release (see Fig. S1 in the supplemental material) and provides effective treatment of BJIs, as shown (23). Our data show that the antibacterial efficacy of LNC-DAP after a 14-day treatment contrasts with the lack of efficacy of free i.v. DAP. This is likely due to the maintenance of high local concentrations in the infected tissue area, with plasma concentrations being similar to or lower than those observed with i.v. treatments (see Fig. S1 in the supplemental material) (11, 24). We suggest that this formulation could be used to prevent most BJIs and thereby reduce both the duration of stays and the cost of treatment.