Abstract: BACKGROUND: Zoledronic acid is an effective drug for treating bone resorption, but systemic use has relatively large side effects. Dissolvable microneedles is an efficient method of local transdermal drug delivery, which can effectively penetrate the mucosal barrier and deliver drugs locally to avoid drug side effects. 
OBJECTIVE: To prepare a dissolvable microneedle patch loaded with zoledronic acid, and characterize their mechanical strength, penetrability, and effects on osteoclast differentiation.
METHODS: Polyvinyl alcohol solution was used as the backing matrix, and hyaluronic acid solution or hyaluronic acid solutions containing varying concentrations of zoledronic acid (1, 5, and 10 mg/mL) as the microneedle tip matrix. Blank soluble microneedle patches and dissolvable microneedle patches loaded with zoledronic acid were prepared by a two-step casting method. The morphology and drug loading of the microneedles were characterized. Microneedles loaded with 10 mg/mL zoledronic acid were pressed into tinfoil or porcine gingival tissue to assess their penetration ability. Microneedles loaded with 10 mg/mL zoledronic acid were inserted into Parafilm M films, and the holes formed by the microneedles in each layer of Parafilm M were observed to assess their penetration depth. Microneedles loaded with 10 mg/mL zoledronic acid were inserted into porcine gingival tissue, and their dissolution was observed at different time points. Extracts of blank and 1 mg/mL zoledronic acid-loaded dissolvable microneedles were co-cultured with RAW264.7 cells. The biosafety of the microneedles was assessed using CCK-8 assay and live-dead staining. RAW264.7 cells were cultured in three groups: a blank group without any drug, a lipopolysaccharide group receiving 100 ng/mL lipopolysaccharide (to induce osteoclast differentiation), and a zoledronic acid microneedle group receiving both 100 ng/mL lipopolysaccharide and a 1 mg/mL zoledronic acid-loaded dissolvable microneedle extract. Osteoclast differentiation was analyzed by tartrate-resistant acid phosphatase staining and RT-qPCR.
RESULTS AND CONCLUSION: (1) Optical microscopy revealed that the zoledronic acid-loaded soluble microneedles exhibited uniform structure, sharp tips, and a well-defined pyramidal morphology. The tips adhered tightly to the backing without defects or bubbles. The tip height was approximately 595 µm, and the distance between adjacent tips was approximately 495 µm. The drug loading of 1, 5, and 10 mg/mL zoledronic acid-soluble microneedles was (24.07±1.33), (203.4±9.14), and (576.74±9.46) µg, respectively, indicating that the drug loading of the microneedles could be controlled by adjusting the drug concentration in the microneedle matrix solution. (2) Microneedles inserted into tinfoil or porcine gingival tissue formed effective drug delivery micropores on the tissue surface. Microneedles inserted into Parafilm M membrane had a penetration depth of approximately 252 µm, demonstrating that the microneedles possess sufficient mechanical strength and tissue penetration properties to penetrate the mucosal barrier, form effective micropores, and deliver drug deep into the gingival tissue. (3) CCK-8 assay and live-dead staining revealed that both blank and zoledronic acid-loaded soluble microneedles showed no significant cytotoxicity. Tartrate-resistant acid phosphatase staining and RT-qPCR assay showed that the number of osteoclasts and the mRNA expressions of tartrate-resistant acid phosphatase, c-Fos, matrix metalloproteinase-9, and cathepsin K were significantly higher in the lipopolysaccharide group than in the control group (P < 0.05). The number of osteoclasts and the mRNA expression levels of tartrate-resistant acid phosphatase, c-Fos, matrix metalloproteinase-9, and cathepsin K were significantly lower in the zoledronic acid microneedle group than in the lipopolysaccharide group (P < 0.05). This suggests that zoledronic acid-loaded dissolvable microneedles can effectively inhibit osteoclast differentiation under inflammatory conditions.

Key words: zoledronic acid, dissolvable microneedle, hyaluronic acid, lipopolysaccharide, osteoclast, macrophage, penetrability