Purpose: Bisphosphonates such as alendronate (ALE) have been proven as a class of molecules with high affinity for the key bone material hydroxyapatite (HA) and have been used as the bone-targeting moiety when conjugated with proteins and compounds. In this study, we aimed at developing and characterizing bone-targeting lipid-polymer hybrid nanocarriers (BTN) and evaluating their antimicrobial ability for osteomyelitis treatment.
Methods: BTN were prepared based on the nanoprecipitation and self-assembly technique using a combination of biocompatible polymers, lipids and phospholipids including PLGA (poly(lactic-co-glycolic acid)) and DSPE-PEG (polyethylene glycoldistearoyl-glycero-phosphoethanolamine). To equip BTN with bone-targeting function, DSPE-PEG-NHS (N-hydroxysuccinimide) was conjugated with ALE by NHS ester crosslinking reaction. BTN were characterized for their size, zeta potential and dispersion stability using photon correlation spectroscopy. To test BTN’s affinity for HA, HA affinity test, in vitro and in vivo studies on rats and mice were conducted. These bone materials were treated with nanoparticles labeled with near-infrared dye under different conditions, and the bone-binding performance was evaluated using a LI-COR Odyssey Imaging System. Linezolid (LIN) was encapsulated as an antimicrobial agent for BTN. MRSA (Methicillin-resistant Staphylococcus aureus) were used for standard MIC (minimum inhibitory concentration) and MBEC (minimum biofilm eradication concentration) assays on LIN-BTN nanocarriers. Confocal microscopy was performed to study the cellular interactions and uptake of BTN into osteoblasts cell lines. Also, bioluminescent S. aureus were studied for the development of animal osteomyelitis model.
Results: BTN-LIN around 110 nm in diameter with polydispersity index less than 0.2 were prepared. This nanocarrier was stable in serum-enriched medium at 37 °C within 5 days with only modest aggregation observed. The BTN can also be made as a lyophilized powder and rehydrated with minor changes in physical characteristics. HA conjugation assay showed positive correlation between the DSPE-PEG-ALE density on BTN and BTN-HA affinity. Bone samples including porcine bone chips and rat leg bones exposed to infrared dye-labeled BTN ex vivo were shown to emit substantially stronger fluorescence signals than the controls even after rinsing, indicating desirable bone-binding properties of BTN. Local injections to rodent legs led to similar results, with high local levels of BTN and LIN detected in the bones near the injection sites. MIC and MBEC assays demonstrated that BTN had comparable or significantly superior antimicrobial activities against MRSA and its biofilms. Confocal microscope images demonstrated efficient uptake of BTN into human osteoblasts and indicated the potential of BTN to treat intracellular bacterial infections.
Conclusion: We have successfully developed a stable lipid-polymer hybrid nanocarrier system with strong affinity for bones under different conditions. With its abilities to efficiently carry antibiotic agent(s) and bind to the bones, BTN system shows excellent potential to achieve high local antibiotic concentration in the infected bone tissues. This not only can reduce the systemic drug toxicity, it may also increase the effectiveness to eradicate the bacteria harbored in the bones and lower the possibility of development of drug resistance by the bacteria that is frequently reported in the patients with bone infections.
Pengbo Guo– Temple University, Pennsylvania
Hui-Yi Xue– Temple University School of Pharmacy, Pennsylvania
Bettina Buttaro– Temple University School of Medicine, Pennsylvania
Ngoc Tran– Temple University School of Pharmacy, Pennsylvania