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Journal of Advanced Research logoLink to Journal of Advanced Research
. 2024 Jul 25;72:605–614. doi: 10.1016/j.jare.2024.07.020

A mosquito proboscis-inspired cambered microneedle patch for ophthalmic regional anaesthesia

Xuequan Liu a,b,1, Xuequan Sun c,d,1, Hongyu Zhu b, Rubing Yan e, Chang Xu c,d, Fangxing Zhu c,d, Ruijie Xu f, Jing Xia b, He Dong b,⁎, Bingcheng Yi a,⁎⁎, Qihui Zhou a,⁎⁎
PMCID: PMC12147604  PMID: 39067695

Graphical abstract

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Keywords: Cambered microneedles, Ophthalmic regional anaesthesia, Dissolubility, Lidocaine, Mosquito proboscis

Highlights

  • •

    A rapidly dissolving cambered microneedle (MN) patch was fabricated to load lidocaine.

  • •

    The MN patch demonstrated effective ophthalmic regional anesthesia within 30 min.

  • •

    The corneal pinhole channels can self-heal within 24 h after MN administration.

Abstract

Introduction

One of the methods for pain management involves the use of local anesthesia, which numbs sensations in specific body regions while maintaining consciousness.

Objectives

Considering the certain limitations (e.g., pain, the requirement of skilled professionals, or slow passive diffusion) of conventional delivery methods of local anesthetics, developing alternative strategies that offer minimally invasive yet therapeutically effective delivery systems is of great concern for ophthalmic regional anesthesia.

Methods and results

In this study, a rapidly dissolving cambered microneedle (MNs) patch, composed of poly(vinylpyrrolidone) (PVP) and hyaluronic acid (HA) and served as a delivery system for lidocaine (Lido) in local anesthesia, was developed taking inspiration from the mosquito proboscis’s ability to extract blood unnoticed. The lidocaine-containing MNs patch (MNs@Lido) consisted of 25 microneedles with a four-pronged cone structure (height: 500 μm, base width: 275 μm), arranged in a concentric circle pattern on the patch, and displays excellent dissolubility for effective drug delivery of Lido. After confirming good cytocompatibility, MNs@Lido was found to possess adequate rigidity to penetrate the cornea without causing any subsequent injury, and the created corneal pinhole channels completely self-healed within 24 h. Interestingly, MNs@Lido exhibited effective analgesic effects for local anesthesia on both heel skin and eyeball, with the sustained anesthetic effect lasting for at least 30 min.

Conclusions

These findings indicate that the mosquito proboscis-inspired cambered MNs patch provides rapid and painless local anesthesia, overcoming the limitations of conventional delivery methods of local anesthetics, thus opening up new possibilities in the treatment of ophthalmic diseases.

Introduction

Local anesthesia is commonly employed in ophthalmic surgeries to initiate the rapid depolarization of action potentials in electrically excitable cells, such as nerve and muscle cells. These anesthetics exert their effect by specifically targeting voltage-gated Na+ channels, ensuring optimal conditions for surgical procedures [1]. Traditional local anesthetics for ophthalmic surgeries involve needle block techniques, such as retrobulbar anesthesia and peribulbar anesthesia [2]. Retrobulbar injection, first described in 1884, provides reliable anesthesia and akinesia with a rapid onset [3]. On the other hand, peribulbar block, relying on anesthetic diffusion into the muscle cone via connective tissue, offers a safer alternative to retrobulbar block [4]. Over the years, surgical techniques have evolved, and extensive research has aimed at improving the safety and success of ophthalmic regional anesthesia. This includes optimization of eye positioning, selection of appropriate needles, and precise control of needle direction [5]. However, needle-based local anesthesia is associated with inherent risks, such as needle misplacement, inadvertent globe perforation and rupture, injury to extraocular muscles, as well as the subarachnoid spread of anesthetics [6]. Moreover, the appearance of long needles and the accompanying pain often cause patient anxiety deterring timely interventions [7]. In attempts to alleviate such discomfort, topical anesthesia (TA) has emerged as a painless and rapid method for administering regional ophthalmic anesthesia [8]. Although the administration of local anesthetic eye drops provides several advantages, including ease of use, cost-effectiveness, and the avoidance of injection-related hazards, the tear film and the flushing action of tears could impede the diffusion process of anesthetics in the transcorneal route, leading to prolonged anesthesia time and weakened local anesthetic effect. As such, multiple administrations of eye drops are often necessary for ophthalmic surgeries. However, this strategy poses challenges in achieving therapeutically relevant doses and increases the risk of complications related to anesthetic entering the nasolacrimal system [9]. Despite attempts to achieve rapid onset anesthesia by utilizing techniques such as phonophoresis, magnetophoresis, or iontophoresis, these methods are restricted by their complex application, issues with patient compliance, and safety concerns [10]. Therefore, given the importance of prioritizing patient safety and addressing these concerns, there is a growing interest in exploring alternative strategies that offer minimally invasive yet therapeutically effective delivery systems for local anesthesia.

In nature, female mosquitoes, blood-sucking insects, possess a strong proboscis that easily penetrates human skin to feed on blood for essential proteins and nutrients. Despite leaving a prominent mark upon biting, individuals typically do not immediately feel anything, experiencing swelling and itchiness later. Researchers have discovered that the effectiveness of mosquito blood-sucking lies in their saliva, which is injected into the skin through a sophisticated system of thin needles within their proboscis (Fig. 1A). This saliva acts as an anesthetic, numbing the bite area temporarily [11]. Inspired by this natural phenomenon and the rapid and effective local anesthesia achieved through syringe injections, the development of a mosquito proboscis-inspired biomimetic needle shows promise as an alternative to traditional syringe-based local anesthetic systems. It aims to overcome needle phobia, minimize associated pain and risks, and provide a practical solution. Microneedle patches (MNs), which have emerged as effective drug delivery systems, resemble mosquito proboscis with their arrays of microneedles. MNs not only enhance drug delivery efficiency but also retain the transcorneal drug delivery route used in topical anesthesia. MNs offer painless and minimally invasive administration, mitigating emotional trauma and injection risks compared to syringe injections, which has been confirmed by accumulating evidence [12], [13]. By precisely controlling the number and length of microneedle tips on the MNs array, the anesthetic delivery effect can be accurately regulated [14]. Considering the convenience and reliability of MNs administration, the application of dissolvable MNs as anesthetic delivery systems has gained attention because it does not suffer from the easy detachment from tissues and the requirement of long-term compression for administration completion [15]. Studies have shown that dissolvable MNs effectively enhance the delivery of large model molecules for ocular drug delivery by disrupting the cornea's barrier function [16]. Collectively, these evidences indicate that the development of dissolvable MNs that combine the biomimetic blood-sucking approach of mosquitoes with the drug delivery route of topical anesthesia holds promise for providing rapid and effective local anesthesia in a painless manner (Fig. 1B), without the limitations associated with topical applications or hypodermic injections of local anesthetics. Certainly, to ensure convenience in handling and improved fitting to the eye tissue, MNs should be fabricated with a concave morphology, biomimicking the curvature of eyeball.

Fig. 1.

Fig. 1

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Schematic diagrams of dissolvable microneedles design, inspired by mosquito proboscis, for local anesthesia in ophthalmic surgeries: (A) Mosquito blood-feeding mechanism utilizing a sophisticated system of slim needles and the release of saliva as an anesthetic; (B) Dissolvable MNs designed for drug delivery of Lido in ophthalmic surgeries; (C) Synthesis of cambered MNs@Lido.

Lidocaine (Lido), an amide-class local anesthetic, has extensive clinical use in inhibiting acute and chronic pain sensations [17]. Its antinociceptive activity stems from its ability to block voltage-gated sodium channels, resulting in reversible inhibition of action potential propagation. Additionally, lidocaine exhibits antiarrhythmic, anti-inflammatory, and antithrombotic effects through toll-like receptor (TLR) and nuclear factor kappa-β (NF-kβ) signaling pathways, as well as downstream cytokine effectors high mobility group box 1 (HMGB1) and tumor necrosis factor-α (TNF-α) [18]. The aim of this study is to explore the potential application of rapidly dissolving cambered MNs composed of poly(vinylpyrrolidone) (PVP) and hyaluronic acid (HA) as a delivery system for lidocaine in local anesthesia. The lidocaine-containing MNs, referred to as MNs@Lido, were fabricated using a micro-molding technique (Fig. 1C). Morphology, mechanical strength, dissolution behavior, and insertion depths of the MNs in corneal tissues were thoroughly characterized. In vitro cell viability studies using L929 fibroblast cells were conducted to assess the biocompatibility of MNs@Lido. Furthermore, in vivo investigations determined the ability of MNs@Lido to penetrate corneal tissue and examined the self-healing properties of the eye following MNs treatment. Finally, an analysis of the analgesic effect using the plantar incision model (PIM) and single blink reflex completion time was performed to evaluate the capacity of MNs@Lido in providing rapid and sustained pain relief, confirming the feasibility of MNs for local anesthesia.

Materials and methods

Fabrication of MNs@Lido

The fabrication of MNs@Lido involved two components: HA needles containing Lido and a backing layer composed of HA and PVP. A cambered polydimethylsiloxane (PDMS) mold, purchased from Xiamen Chipper Medical & Technology in China, with 25 needles arranged in a concentric circle, was employed for the micro-molding technique. Each needle exhibited a four-pronged cone shape. To create the needle layer, a 2 % Lido solution was prepared by dissolving lidocaine hydrochloride powder (Macklin, China) in double-distilled water. The mixture was stirred at room temperature for 0.5 h. Subsequently, 1 % HA (MW: 100,000–200,000 Da, Macklin, China) was added to the Lido solution and stirred for 1 h at room temperature. The resulting HA/Lido mixed solution was coated onto the PDMS template, and vacuumed for 30 min to ensure complete filling of the mold cavity. The excess solution was then removed. For the backing layer, a HA/PVP mixed solution was prepared by dissolving 0.7 g of HA and 0.3 g of PVP (PVP40000, Solarbio, China) in 10 mL of double-distilled water at 95 °C. This solution was added to cover the needles. After sealing the mold, the MNs@Lido were obtained by demolding and drying.

Characterizations of MNs@Lido

The stereomicroscope (SMZ745T, Japan) was employed to capture the macroscopic morphology of MNs@Lido. The loading capacity of lidocaine in MNs was determined at 262 nm using a UV–Vis spectrophotometer (Shanghai Metash Instruments, China). Briefly, a series of lidocaine solutions with concentrations of 12.5, 25, 50, 100, 200, 400, and 800 mg/mL was prepared in PBS buffer, and a standard curve correlating concentration and absorbance value was generated using the UV spectrophotometer. Subsequently, the MNs@Lido was dissolved in PBS buffer, and the absorbance was measured to determine the loading capacity of lidocaine. The scanning electron microscope (Regulus 8100, Japan) was used to observe the microscopic morphology of the needle tips in MNs@Lido. To investigate the distribution of drugs (e.g., lidocaine) within the needle tips, 7-(Diethylamino)-coumarin-3-carboxylic acid (Aladdin, China), a fluorescent compound detectable under UV exposure, was utilized as a drug model to fabricate the microneedles. The resulting microneedles were then observed using a confocal laser scanning microscope (TCS SP8, Germany).

The infrared spectra of MNs@Lido, Lido, and HA were measured using a Fourier transform infrared (FTIR) spectrometer (Thermo Fisher Scientific, USA). The measurements were performed in the wavelength range of 500–4000 cm−1. The mechanical properties of MNs@Lido were determined using a universal testing machine (CMT6103, China). The samples were positioned on a metal fixed station, and an axial force was applied to compress the samples using a mechanical sensor at a rate of 1.1 mm/min. The resulting force–displacement relationship was recorded.

To assess the dissolubility of MNs@Lido, the samples were manually applied onto the surface of living rabbit eyeballs. After predetermined intervals of 5, 10, 15, 20, and 25 s, the samples were removed, and the morphology of the needle tips was observed using a scanning electron microscope. For drug release analysis, the remaining MNs@Lido was dissolved in 5 mL of phosphate-buffered saline (PBS, meilunbio, China) solution. Subsequently, the absorbance of the resulting solution was measured at 262 nm using a UV–Vis spectrophotometer (Shanghai Metash Instruments, China). The concentration of Lido in the solution was then determined by referring to a standard curve. To evaluate drug delivery, 7-(Diethylamino)-coumarin-3-carboxylic acid was applied as a drug model for MNs preparation. The drug-loaded MNs were inserted into an ex vivo rabbit eyeball for 30 s. Subsequently, upon removal of the samples, the cornea was obtained and the distribution of the drug within the cornea was observed using a confocal laser scanning microscope.

Biocompatibility of MNs@Lido

L929 fibroblast cells (Shanghai YanJin Biotech, China) were utilized to assess the biocompatibility of MNs@Lido and pristine Lido drug. Initially, solutions with different concentrations (25, 50, 100, 200, 400, and 800 μg/mL) of Lido, PVP, HA, or MNs@Lido were prepared by dissolving the samples in Dulbecco's modified Eagle's medium (DMEM) containing 1 % penicillin–streptomycin and 10 % fetal bovine serum (meilunbio, China). Subsequently, cells were seeded in 96-well plates at a density of 1 × 104 cells/well and cultured with the prepared solutions in a constant temperature and humidity chamber (37 °C and 5 % CO2) for 24 h. For the Live-dead assay, the cells were rinsed with PBS and incubated with a solution containing 2.5 μM calcein-AM and 2.5 μM ethidium homodimer (Beyotime, China) for 15 min at 37 °C. The fluorescence staining of the cells was then observed using an inverted fluorescence microscope (Nikon A1 MP, Japan). To evaluate cell viability, 10 μL of Cell Counting Kit-8 (CCK-8, meilunbio, China) reagent was added to each well. Following a 1 h incubation at 37 °C, the optical density (OD) of the solution was measured at 450 nm using a microplate reader (Bio Tek, America).

Self-healing ability of eyeball after MNs@Lido administration

The animal experiment protocol was approved by the Medical Ethics Committee of the Affiliated Hospital of Qingdao University (contract grant QYFY WZLL 28146). The research project's content and design adhered to ethical norms and followed the guidelines outlined in the Laboratory Animal Care and Use Guide. Male adult Sprague Dawley (SD) rats (∼200 g) and New Zealand white rabbits (∼2 kg) were procured from the Jinan Pengyue Laboratory Animal Breeding Center (Jinan, China). The animals were provided with free access to food and water during the experimental period.

To assess the self-healing ability of the eyeball, MNs@Lido was administered by inserting them into the eyeball of New Zealand white rabbits. Initially, to confirm the successful insertion of microneedles, the rabbits were sacrificed, and the eyeballs were surgically removed. The eyeballs were then fixed in 4 % paraformaldehyde, dehydrated, and embedded in paraffin. Subsequently, 6–8 μm thick cornea sections were cut and subjected to routine hematoxylin and eosin (H&E) staining. Tissue images were captured using an optical microscope (Nikon, Japan), and the depth of needle insertion was analyzed using Image J software. To dynamically observe the self-healing changes, optical coherence tomography (OCT, CIRRUS HD-OCT 4000, Germany) was used to evaluate corneal changes at 1 min and 5 min after MNs@Lido treatment. Additionally, corneal confocal microscopy (HRT3-CM, Germany) was employed to examine changes in the stroma and endothelium after 1 h of MNs@Lido administration. To further verify the self-healing process, corneal samples were obtained from rabbits at 0, 1.5, 3, 6, 12, and 24 h post-treatment for HE staining. The healing status of the cornea was observed using an optical microscope.

Analysis of the analgesic effect of MNs@Lido in vivo

The analgesic effect of MNs@Lido on rat paw

Brennan's PIM was established in male SD rats to evaluate the analgesic effect of MNs@Lido. Briefly, anesthesia was induced and maintained using sevoflurane in a mixture of oxygen and spontaneous respiration (R500IE, China). The left hind paw was aseptically treated with a 10 % povidone-iodine solution (Livzon Pharmaceutical Group Inc., China). Subsequently, a 1 cm longitudinal surgical incision was made on the left hind paw, starting from 0.5 cm proximal to the heel's edge, using a number 11 blade, and then the toe muscle was exposed in the incision. The flexor digitorum brevis muscle was carefully raised and longitudinally split through blunt dissection, while keeping the muscle origin and insertion intact. Hemostasis was achieved, and the wound was sutured with two stitches using a 4-0 nylon thread on an FS-2 needle. Following the procedure, the rats were returned to their original cages and allowed to recover from anesthesia for approximately 15 min.

To evaluate the analgesic effect of MNs@Lido, male SD rats with PIM were randomly divided into three groups: blank (without any treatment), blank MNs (pristine MNs without drugs), and MNs@Lido. The mechanical nociceptive threshold in rats was assessed by measuring the mechanical Paw Withdrawal Threshold (PWT) using the von Frey test. Each rat was placed in individual plastic chambers (15 cm × 15 cm × 30 cm) with a mesh floor, suspended on a bracket. Restraints on rats were minimized to avoid interfering with the behavior tests. After a 30-min adaptation period, the PWT was measured using von Frey filaments with bending forces of 0.4, 0.6, 1, 2, 4, 6, 8, and 15 g. The filaments were applied successively from beneath the cage through the mesh floor to the plantar skin of the planned surgical area. Using von Frey filaments, an appropriate starting filament was placed perpendicular to the plantar surface of the hind paw for 2–3 s. Positive responses, such as sudden withdrawal of the paw, body tremors, or licking of the affected paw, were marked with “x”. If there was no response, it was recorded as “o”. The up-down method was employed, wherein a higher intensity stimulus was used for testing to determine the 50 % mechanical withdrawal threshold. If there was still no response at the bending force of 15 g, 15 g was designated as the cutoff value.

The analgesic effect of MNs@Lido on rabbit eyes

To directly assess the analgesic effect of MNs@Lido on rabbit eyes, New Zealand white rabbits were randomly divided into three groups: blank MNs (pristine MNs without drugs), Lido-loaded patch (without the tips) and MNs@Lido. Briefly, the samples were inserted into the eyeballs of rabbits for 25 s. Subsequently, a cotton swab was applied to stimulate the cornea at predetermined intervals, and the rabbits' reactions were recorded using a camera. The experiment involved using a microneedle patch to puncture the rabbit's eyeball and starting the timer immediately after. Videos were recorded to document the rabbit's response to the cotton swab stimulation at different time points. The cotton swab was applied three times consecutively at each time point. A video editing software (CapCut) was used to calculate the time from the start of each stimulation until the end of the rabbit's blink response. This time duration represented a complete reflex response, referred to as single blink reflex completion time. If the rabbit did not blink within 1 s after being stimulated, the single blink reflex completion time was recorded as 1 s.

Statistical analysis

The data are presented as mean values ± standard deviation. Statistical analysis was performed using GraphPad Prism 8 software. One-way analysis of variance (ANOVA) and two-way ANOVA were employed to determine the statistical significance, considering the data to be statistically significant at *p < 0.05, **p < 0.01, ***p < 0.001.

Results and discussion

The cambered MNs@Lido exhibits rapid dissolution capabilities for Lido delivery

In previous studies, non-water-soluble biomaterials were commonly utilized for encasing anesthetic drugs to fabricate microneedles, enabling the delivery of anesthetics to the eye through drug diffusion or material degradation [19], [20], [21]. Nevertheless, this strategy often encounters challenges related to a suitable anesthetic release rate due to the physical adsorption of the microneedles and slow degradation of the materials [21]. Consequently, the overall anesthetic effect is compromised. Conversely, the adoption of rapidly dissolving microneedles can facilitate rapid local release of anesthetics, leading to a swift anesthetic effect. To address these issues, a mixed MNs@Lido system comprising HA and PVP was selected in this study to load lidocaine, thereby preparing microneedle patches. HA is an essential constituent of ocular fluid and cornea, and it plays a crucial role in regulating the wound-healing process [22], [23]. On the other hand, PVP is a highly water-soluble biomaterial that has obtained approval from the US Food and Drug Administration (FDA) [24]. To accommodate the shape of the eyeball [13], the backing layer of MNs@Lido was designed with a cambered shape, featuring a diameter of 14.2 mm and a sagittal height of 3.85 mm (Fig. 2A).

Fig. 2.

Fig. 2

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Structure and chemical characterizations of MNs@Lido: (A) Schematic diagram of microneedle size; (B) Optical images; (C) SEM images; (D) Fluoroscopic image of single microneedle loading 7-(Diethylamino)-coumarin-3-carboxylic acid; (E) FTIR spectra of HA, Lido, and MNs@Lido; (F) Compressive mechanical properties; (G) The dissolution of microneedles after the administration into rabbit eyes for 25 s; (H) The cumulative release of Lido; (I) Confocal laser scanning microscopy images of the formed microhole on the cornea at varied depths after the insertion of 7-(Diethylamino)-coumarin-3-carboxylic acid-loaded microneedles.

For the release profile of Lido, following the penetration of MNs into the cornea, the rapid dissolution of HA is speculated to facilitate the swift release of Lido into the corneal tissue, typically within tens of seconds. Subsequently, the released Lido undergoes local diffusion into the nerve tissue, where it impedes the production and conduction of nerve impulses, thereby eliciting a rapid local anesthetic effect. Hence, to enhance the stiffness of the microneedles for better insertion into the eyeball, a total of 25 microneedles arranged in a concentric circle pattern on the patch were formed with a four-pronged cone structure (Fig. 2A). This design strategy is derived from the strong correlation between the needle structure and its rigidity [25]. The average corneal thickness in humans is typically less than 500 μm [26]. To enable the microneedles to penetrate the corneal layer effectively, each individual microneedle was designed with a height of 500 μm and a base width of 275 μm. As expected, the micro-molding technique was utilized to successfully fabricate the cambered microneedle patch with the microneedles uniformly distributed in the concentric circle of the patch (Fig. 2B), and the microneedles patch allows for loading of approximately 70 μg of Lido, which enable the effective pain relief with the minimized toxic side effect. Notably, each needle exhibited a distinct four-pronged cone structure, characterized by a height of 420 ± 20 μm and a base width of 250 ± 20 μm (Fig. 2C). Due to the absence of a fluorophore in Lido, direct observation of its distribution within microneedles is challenging. To overcome this limitation, 7-(Diethylamino)-Coumarin-3-carboxylic acid, which possesses a molecular structure similar to that of Lido, was utilized as a substitute for Lido to visualize the drug distribution within the microneedles. Swiss ADME software analysis determined that 7-(Diethylamino)-Coumarin-3-carboxylic acid exhibited lipid solubility similar to Lido. As illustrated in (Fig. 2D), the fluorescent dye was observed to be uniformly dispersed within the microneedle tips, suggesting a high likelihood of similar and uniform distribution of Lido within the needles. The FTIR spectra (Fig. 2E) revealed the presence of broad peaks at 1541 cm−1 and 1672 cm−1, indicating the C—N stretching in amine vibration and the C Created by potrace 1.16, written by Peter Selinger 2001-2019 C stretching in the aromatic nucleus vibration of Lido [27], [28], respectively. These findings serve as further confirmation of the successful loading of Lido in the MNs patch. However, the incorporation of Lido has been observed to compromise the mechanical strength of the microneedles (Fig. 2F), which can be attributed to that the incorporation of Lido disrupts the hydrogen bonding between HA molecules [29].

Eye drops, despite being a painless and rapid method for administering regional ophthalmic anesthesia, face hindrance from the tear film and the flushing action of tears, impeding the diffusion process of anesthetics in the transcorneal route and weakening the local anesthetic effect. Conversely, the MNs patch offers a minimally invasive transcorneal drug delivery route for local anesthesia, triumphing over the tear film barrier. As shown in Fig. 2G, when MNs@Lido were administered into rabbit eyeballs for a duration of 25 s, a notable dissolution of the microneedles was observed, accompanied by a cumulative release of Lido exceeding 75 % (Fig. 2H), which enables the rapid achievement of the anesthetic effect. Additionally, a uniform fluorescence pattern was observed in the surrounding area, with the center positioned on the micropore channel (Fig. 2I). These findings highlight the excellent dissolubility of HA for effective drug delivery of Lido, and the even distribution of MNs tips ensures the homogeneous diffusion of lidocaine into the corneal tissue.

Rapid dissolved MNs@Lido possesses acceptable cytocompatibility

Given the rapid dissolubility of microneedle patch in vivo, the culture media containing different concentrations of microneedle components (e.g., Lido, HA, PVP, and MNs@Lido) were performed to assess the biocompatibility of MNs@Lido. As shown in Fig. 3A, although Lido at a high concentration significantly reduces cell viability owing to the disturbance of the ionization equilibrium between intracellular and extracellular environments [30], indicative of certain cytotoxicity, low dose of Lido (<200 μg/mL) possesses no significant damage to cells. This was reconfirmed by the results of live/dead staining (Fig. 3B). Additionally, administration of high concentrations of Lido generally results in excessive blood intake, which brings in the risk of causing mild headaches, vision problems, muscle twitches, and even cardiac arrest [31]. Given that the intravenous dosage of Lido is typically administered at a rate of 1 mg/kg [32], the Lido (70 μg) released from MNs@Lido can be speculated to be biosafe for local anesthesia. Furthermore, in addition to HA that possesses a slight cytotoxicity to L929 fibroblast cells at high concentration (>800 μg/mL), both PVP and the dissolved MNs@Lido were observed to show satisfactory biocompatibility at the concentration range of 0–800 μg/mL. No significant difference was observed in the number of dead cells and the quantified cell viability from the live/dead staining images (Fig. 3C). Overall, these findings indicated that the presence of rapid dissolution of MNs@Lido showed no significant risk in damaging cells surrounding the tissue area of anesthesia, indicative of acceptable biocompatibility.

Fig. 3.

Fig. 3

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Biocompatibility of the dissolved MNs@Lido: (A) Relative cell viability of L929 fibroblast cells affected by the microneedle component at different concentrations of 0–800 μg/mL; (B) Live/dead staining of cells after the coincubation with Lido for 24 h and the quantified living cells (%); (C) Live/dead staining of cells after the coincubation with the dissolved MNs@Lido for 24 h and the quantified living cells (%).

MNs@Lido penetrates the cornea without causing any consequential injury sequelae

To verify the sufficient mechanical penetration of MNs@Lido in the cornea, local observation was conducted on the eyeball tissue surrounding the microneedle-treated area. As anticipated, distinct micropinhole channels were observed on the surface of the eyeball (Fig. 4A), with a calculated size of 175 μm width and 220 μm depth (Fig. 4B), matching the size of the microneedles. Meanwhile, optical coherence tomography was also performed on the cornea within 5 min of microneedle insertion. As depicted in Fig. 4C, a clear cavity (indicated by the yellow dashed box) was visible on the cornea's surface 1 min after insertion, corresponding to the backing layer of the microneedle. After 5 min, the cornea's surface cavity disappeared, and certain regions within the cornea exhibited distinct colors compared to the surrounding tissue (indicated by the white arrow), suggesting corneal injury due to microneedle insertion. These findings not only confirmed the ability of microneedles to penetrate the cornea but also demonstrated the rapid dissolvability of the microneedle patch. Importantly, no significant changes were observed in the stromal and endothelial cell layers of the cornea tissue 1 h after microneedle insertion into the eyeball (Fig. 4D), substantiating the biosafety of microneedles. Notably, the self-healing of the micropinhole channels was observed, as indicated by the HE images in Fig. 4E. Despite the limited trace of micropinhole channels induced by MNs@Lido insertion, the corneal pinhole channels gradually diminished and nearly self-healed completely within 24 h. This demonstrates that the application of microneedles does not cause significant damage to corneal tissue. Regarding the potential impact of MNs@Lido insertion on eyesight, given the reported applications of microneedle patches in the treatment of ocular diseases and the observation of positive self-repair of the micropore channels within 24 h following MNs@Lido administration, it is reasonable to speculate that MNs@Lido administration is unlikely to significantly affect vision.

Fig. 4.

Fig. 4

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Self-healing of the micropinhole channels induced by MNs@Lido insertion: (A) Optical image of eyeball after MNs@Lido insertion; (B) HE images of eyeball tissue after MNs@Lido insertion; (C) OCT images of corneal changes within 5 min of MNs@Lido insertion; (D) Corneal confocal microscopy images of changes in the stroma and endothelium after 1 h of MNs@Lido administration; (E) HE images of living eyeball tissue within 24 h of MNs@Lido insertion.

MNs@Lido exhibits an excellent analgesic effect for local anesthesia

The Brennan plantar incision model (PIM) is a well-established method for assessing acute postoperative pain [33]. It was applied to evaluate the local analgesic effect of MNs@Lido (Fig. 5A). In this model, rats exhibit specific behaviors such as foot shrinking, flight, or neighing when the stimulation intensity reaches a certain threshold during Von-Frey fiber stimulation of the plantar [34] (Fig. 5B). The PIM was constructed by making a lengthwise incision on the heel skin and fascia to expose the toe muscle, followed by lengthwise splitting of the short flexor of the toe (Fig. 5C). Mechanical claw retraction thresholds were measured one day after the operation (Fig. 5D). It was observed that the mechanical threshold of all rats in the PIM group decreased significantly to 16.38 % ± 7.8 % of the pre-PIM baseline, indicating successful modeling of PIM. Importantly, the MNs@Lido group demonstrated significantly improved thresholds at 15, 30, and 45 min after administration compared to the Blank group and MNs alone. This confirmed a substantial and sustained anesthetic effect lasting for 60 min. Additionally, the analgesic effect of MNs@Lido directly on the eyeball was conducted by assessing the closure response of rabbit eyes upon cotton swab contact with the cornea (Fig. 5E). As shown in Fig. 5F, the Lido-loaded patch demonstrated a rapid analgesic response similar to the MNs@Lido group. However, its analgesic effect was slightly lower than that of the MNs@Lido group. After 15 min, the analgesic effect subsided, and no significant difference was observed compared to the Blank MNs group. In contrast, the MNs@Lido group not only exhibited the highest analgesic effect but also prolonged anesthesia time for at least half an hour. This was attributed to the direct delivery of the local anesthetic to corneal tissue through the tear film, avoiding dilution and loss of the anesthetic. Besides, such anesthesia duration possesses the capacity to meet the anesthesia requirements for the majority of ophthalmic surgeries, and helps to reduce discomfort resulting from prolonged anesthesia and minimize adverse effects on ocular tissues, such as dryness of the ocular surface and corneal hypoxia.

Fig. 5.

Fig. 5

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Analgesic effect of MNs@Lido: (A) Schematic diagram of evaluating the acute postoperative pain using PIM; (B) Effect of Von-Frey fibers stimulation on rat behaviors; (C) PIM establishment; (D) Paw-withdrawal threshold measurement; (E) Rabbit's blink response; (F) Single blink reflex completion time.

Inspired by the mosquito proboscis's ability to extract blood unnoticed, the objective of this study was to develop a minimally invasive yet therapeutically effective delivery system for ophthalmic regional anesthesia using a rapidly dissolving cambered MNs patch composed of PVP and HA as a carrier for Lido in local anesthesia applications. While the general goal was achieved, several limitations were encountered. Specifically, the lack of suitable testing methods resulted in the detection of general, rather than precise, efficacy of ocular anesthesia in small animals, necessitating further research. Additionally, the precise anatomical locations within the eye where lidocaine delivered via the MN patch reaches and meets the requirements for surgical anesthesia were not investigated. Subsequent studies may employ techniques such as quantifying the concentration of lidocaine in the aqueous humor and ocular contents to elucidate the drug's diffusion pattern within the eye. Despite these limitations, the results of this study support the affirmation that the mosquito proboscis-inspired cambered MN patch provides rapid and painless local anesthesia, overcoming the constraints of traditional local anesthetic delivery methods, thus presenting new avenues for ophthalmic disease treatment.

Conclusion

In summary, a rapidly dissolving cambered MNs patch composed of PVP and HA was successfully fabricated using a micro-molding technique to serve as a delivery system for Lido in ophthalmic regional anesthesia. The MNs@Lido patch featured four-pronged cone-structured microneedles arranged in a concentric circle pattern, demonstrating sufficient rigidity for corneal penetration and effective drug delivery of lidocaine. The MNs@Lido patch exhibited excellent dissolubility and cytocompatibility, ensuring biosafety without notable sequelae of injury, and the corneal pinhole channels created by the MNs@Lido patch completely self-healed within 24 h of administration. Importantly, MNs@Lido demonstrated effective analgesic effects for ophthalmic regional anesthesia, with a sustained anesthetic effect lasting for a minimum of 30 min, thus offering a rapid and painless method of local anesthesia in the treatment of ophthalmic diseases.

Compliance with ethics requirements

Project profile

This project aims to develop dissolvable concave microneedles to solve the clinical problems of traditional anesthesia in ophthalmology. Lidocaine was loaded into a microneedle transdermal drug delivery system prepared with hyaluronic acid to form a biomaterial with anesthetic effect, so as to replace the traditional anesthesia for eye surgery. The physical and chemical properties of lidocaine were tested and the ratio was optimized. The biocompatibility of the microneedle transdermal drug delivery system was verified by in vitro and in vivo experiments. The safety and anesthetic effect of the transdermal drug delivery system of microneedles were verified by animal experiments in vivo. This project developed a microneedle transdermal drug delivery system for ophthalmic anesthesia, and carried out research from the aspects of material optimization and related performance detection, cell evaluation in vitro, and animal experiments. This project will use SD rats to construct plantar incision pain model and New Zealand rabbits to explore the anesthetic effect of microneedle transdermal drug delivery system. Guided by clinical needs, it provides new materials, new methods and ideas for ophthalmic anesthesia, aiming to solve complications such as retrobulbar hemorrhage and infection faced in traditional ophthalmic surgery anesthesia, and provide research basis and theoretical basis for realizing good clinical application value and transformation of dissolvable concave microneedles for ophthalmic anesthesia. The project team promises to abide by the Declaration of Helsinki (1996 edition) and China's research norms and regulations on cell experiments and animal experiments, and the animals involved are only used for the legitimate research of the project team.

Comments from the Medical Ethics Committee

In this project, the rights and interests of animals were fully protected, which met the requirements of the Medical Ethics Committee. Agree on the research protocol.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgments

The authors are very thankful for financial support by the Young Taishan Scholars Program of Shandong Province (Grant No. tsqn202306272), National Key Research and Development Project of China (Grant No. 2023YFFO715101), National Natural Science Foundation of China (Grant No. 82302388), Qingdao Natural Science Foundation (Grant No. 23-2-1-132-zyyd-jch), and the Leading Project of Science and Technology of Yantai Development Zone (Grant No. 2021RC016).

Contributor Information

He Dong, Email: dongh@qdu.edu.cn.

Bingcheng Yi, Email: yibingcheng@uor.edu.cn.

Qihui Zhou, Email: qihuizhou@uor.edu.cn.

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