Loading and release of porous silicon nanoparticles with Flightless I neutralizing antibodies to aid wound healing

Abstract

Event Abstract Back to Event Loading and release of porous silicon nanoparticles with Flightless I neutralizing antibodies to aid wound healing Steven Mcinnes1*, Christopher T. Turner1*, Allison J. Cowin1* and Nicolas H. Voelcker1* 1 University of South Australia,, Mawson Institute, Australia Introduction: Flightless I (Flii) is upregulated in acute and chronic wounds and is a negative regulator of wound healing[1]. Flii neutralizing antibodies (FnAbs) are able to improve the rate of wound healing and decrease scarring[2]. However, retaining FnAb activity in a hostile environment, such as a diabetic wound, is challenging. The aim of this work is to incorporate FnAb into biocompatible and biodegradable porous silicon[3] nanoparticles (pSiNPs) to protect them from the proteolytic degradation in chronic wounds. We investigated the ability of the pSiNP materials to effectively deliver FnAb to diabetic mouse wounds. Materials and Methods: A pSi membrane was fabricated by anodization of p-type Si wafers in an 18 cm2 etching cell in 3:1 HF:ethanol (v/v) solution using a square waveform (50 mA/cm2 for 7.3 s and 400 mA/cm2 for 0.3 s, 474 repeats) followed by electropolishing in 1:20 HF:ethanol at 4 mA/cm2 for 250 s. The freestanding pSi membrane was fractured into porous silicon nanoparticles (pSiNPs) by ultra-sonication for 16 h in DMSO. The resulting NPs solution was spun at 10,000 × g then filtered through a 220 nm PTFE syrine filter before centrifugation at 22000 × g. The pSiNPs were loaded with FnAb by incubating in 1 mg/mL solutions in PBS for 1 hour at 4 oC. UV-Vis based loading quantification was performed on the supernatant, after centrifugation at 16000 x g for 1 minute. Results and Discussion: pSiNPs loaded with FnAb, typically at loadings of up to 724.39 ± 34.79 μg of FnAb per mg of pSi, were incubated with keratinocytes. We observed an increase in cell proliferation and wound closure similar to that seen with FnAb alone, confirming that the released FnAb had not undergone any structural alteration or lost functionality (Fig 1A). To confirm a protective effect, IgG loaded pSiNP were incubated with proteases, rinsed, and then allowed to release the IgG at 25 oC for 7 days. The supernatant was collected and showed intact IgG for up to 7 days post-treatment, suggesting the pSiNPs were protecting the antibodies from the proteolytic environment (Fig 1B). Using a streptozotocin-induced murine model of diabetic wound healing, the effect of the FnAb-pSiNPs was assessed on healing. FnAb loaded pSiNPs were intradermally injected into excisional wounds at day 0 with healing assessed at day 7. A significant improvement in healing was observed compared to controls. Additionally, healing was more effective with FnAb-pSiNPs than with FnAb alone (Fig 2). Figure 1: A) Electric cell impedance sensing. Confluent HaCaTs were treated with FnAb-NPs (t = 0), and wounded (2,500 µA, 48,000 Hz) for 30 seconds (t = 1.5 hrs). Cells recovery was then monitored (24,000 Hz) for 10 hrs. Cells were treated with FnAb-NPs (25 ug/well NP; red, 12.5 ug/well NP; blue) and unloaded NPs (black). n = 3, with data presented as mean +/- 1 SD. B) Effect of proteases on IgG-loaded pSi. IgG loaded pSi was incubated with proteases, washed and then incubated for 7 days at 25 oC. Daily supernatants were run on coomassie-stained SDS-PAGE gels. Figure 2: Excisional wound trial. 6mm excisional wounds were treated with FnAb-NPs intradermally injected at the wound margins. All treatments contained 50 ug of the FnAb or IgG control. Mice were humanely killed at day 7, with wounds examined macroscopically (a, b). Data in (a) is wound area at day 7 as % of original size. * P<0.05. ** P<0.005. FnAb-NP = FnAb loaded pSiNPs, FnAb + NP = Separate FnAb and pSiNPs injections. Conclusions: pSiNPs are able to protect antibody based payloads from a proteolytic environment as well as provide a novel and effective vehicle for delivering antibodies to wound environments. These materials may also be fabricated into bandages to help develop novel non-invasive therapeutic approaches for wound healing. This research was in part conducted and funded by the Australian Research Council Centre of Excellence in Convergent Bio-Nano Science and Technology (project number CE140100036).