Hybrid microneedle arrays for antibiotic and near-IR photothermal synergistic antimicrobial effect against Methicillin-Resistant Staphylococcus aureus

Abstract

The rise of antibiotic-resistant skin and soft tissue infections (SSTIs) necessitates the development of novel treatments to improve the efficiency and delivery of antibiotics. The incorporation of photothermal agents such as plasmonic nanoparticles (NPs) improves the antibacterial efficiency of antibiotics through synergism with elevated temperatures. Hybrid microneedle (MN) arrays are promising local delivery platforms that enable co-therapy with therapeutic and photothermal agents. However, to-date, the majority of hybrid MNs have focused on the potential treatment of skin cancers, while suffering from the shortcoming of the intradermal release of photothermal agents. Here, we developed hybrid, two-layered MN arrays consisting of an outer water-soluble layer loaded with vancomycin (VAN) and an inner water-insoluble near-IR photothermal core. The photothermal core consists of flame-made plasmonic Au/SiO₂ nanoaggregates and polymethylmethacrylate (PMMA). We analyzed the effect of the outer layer polymer, polyvinyl alcohol (PVA) and polyvinylpyrrolidone (PVP), on MN morphology and performance. Hybrid MNs produced with 30 wt% PVA contain a highly drug-loaded outer shell allowing for the incorporation of VAN concentrations up to 100 mg g^-1 and temperature increases up to 60 °C under near-IR irradiation while showing sufficient mechanical strength for skin insertion. Furthermore, we studied the combinatorial effect of VAN and heat on the growth inhibition of methicillin-resistant Staphylococcus aureus (MRSA) showing synergistic inhibition between VAN and heat above 55 °C for 10 min. Finally, we show that treatment with hybrid MN arrays can inhibit the growth of MRSA due to the synergistic interaction of heat with VAN reducing the bacterial survival by up to 80%. This proof-of-concept study demonstrates the potential of hybrid, two-layered MN arrays as a novel treatment option for MRSA-associated skin infections.

Keywords: Combination treatment; Gold nanoparticles; Local delivery; MRSA; Skin patch; Thermotherapy.

Fig. 1. Schematic illustration of the fabrication process of two-layered hybrid MN arrays.

(a) Illustration of the aerosol synthesis of photothermal Au/SiO₂ (4 wt% SiO₂) nanoaggregates. A liquid precursor containing Au and Si is combusted in a methane/oxygen supported flame and the nanoaggregate-containing aerosol is filtered downstream via vacuum-aided filtration. The nanoaggregate filter cake is collected, and the powder is used for further fabrication of the hybrid MN arrays. The insert shows a TEM image of the Au/SiO₂ aggregates, the SiO₂ layer is highlighted with the red arrow. (b) Schematic of the fabrication steps for production of two-layered hybrid MN arrays using either PVA or PVP for the drug-containing, water-soluble outer layer and PMMA for the nanoaggregate-containing, water-insoluble core. (c) Final synergistic antibacterial action of hybrid MN arrays by (i) release of the antibiotic VAN through dissolution of the water-soluble outer layer followed by (ii) photothermal heating under near-IR laser radiation. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 2. Microscopy images of two-layered hybrid MN arrays loaded with the dye sulforhodamine for two different water-soluble polymers (PVA and PVP) and two polymeric weight concentrations (15 and 30 wt%).

(a) MNs are shown imaged with bright field microscopy to visualize the pink dye and the dark Au/SiO₂ (4 wt% SiO₂) nanoaggregates. (b) Fluorescence microscopy images are shown to visualize the water-soluble layer via the fluorescence signal from the dye (in white). (c) The MN arrays were also imaged via SEM to highlight the nanoaggregate-loaded layer (in white). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 3

Drug loading of VAN in hybrid MN arrays. SEM images of hybrid MN arrays (a) before and (b) after dissolution in PBS for different polymers and weight percentages. (c) Quantification via HPLC of VAN released into PBS from hybrid MN arrays produced with 10 mg g⁻¹ VAN in different polymers for different weight percentages, n = 3. Insert shows schematic illustration of the influence of increasing weight percentages of PVP or PVA, respectively, on the formation of the VAN-loaded layer in the hybrid MN arrays. (d) VAN release over time from hybrid MN arrays produced with 30 wt% PVA, n = 3.

Fig. 4. Photothermal heating of hybrid MN arrays (prepared with 10 mg g⁻¹ VAN and 20 mg g⁻¹ Au/SiO₂ NPs) at 808 nm laser radiation in air.

(a) Temperature increase of hybrid MN arrays prepared with (a) 15 or 30 wt% of PVP, or (b) 15, 20, 30, and 40 wt% PVA, n = 3. (c-f) Thermal images obtained during laser irradiation of hybrid MN array prepared with 30 wt% PVA at 0, 80, 160, or 240 s, respectively.

Fig. 5. In vitro synergistic effect between heat and VAN on bacterial growth inhibition of MRSA.

(a) Growth inhibition of MRSA 24 h after exposure to different temperatures (50–60 °C), for several treatment durations (5–15 min) and in presence of VAN at varying concentrations (0–0.75 μg mL⁻¹). The color indicates the average growth inhibition for three replicates. (b) Calculation of combination effect of heat exposure for 10 min and VAN on the bacterial growth of MRSA after 24 h according to Loewe additivity. Three replicates were analyzed using the software tool Combenefit.

Fig. 6. In vitro antibacterial performance of hybrid MN arrays against MRSA.

(a) Schematic illustration of experimental set-up for photothermal heating of hybrid MNs arrays in liquid MRSA culture under NIR irradiation at 808 nm and 1 W cm⁻². Indication of location of temperature measurement points (1, triangle) and (2, diamond) at the center or edge of the sample well, respectively. (b) Thermal images obtained after 5 and 15 min depicting the measurement points. (c) Temperature profile over 30 min of measurement points in the bacterial solution for NIR irradiation of 15 min. (d,e) Dot spots of ten-fold dilutions of MRSA culture when exposed to hybrid MNs loaded with different concentrations of VAN (0, 0.5, 1, 10 mg g⁻¹) and additionally exposed to NIR irradiation (+). Dot spots were prepared after (d) 4 h or (e) 24 h of incubation of samples after treatment end. (f) Quantification of CFU from bacterial samples after treatment with hybrid MN arrays with NIR laser irradiation for 15 min.