Egg microneedles for transdermal vaccination of inactivated influenza virus.

The use of dissolving microneedles (DMNs) is a drug delivery technique in which drug dissolution occurs once it is administered into the skin. The skin is a remarkable site for vaccination due to its significant immunologic properties. Compared to the traditional hypodermic intramuscular (IM) injection, vaccination via DMN does not require cold chains and allows for minimal invasive drug delivery. On account of the significance of skin vaccination, preceding studies have been conducted to elucidate the importance of the DMN technology in vaccination. Most of these studies focused on formulations that maintain the activity of the vaccine, so formulations designed to be specific to the mechanical properties of the microneedle could not be used together independently. In this study, we have developed influenza vaccine loaded egg microneedles (EMN) and characterized the specificity of layer-specific functions of EMN by distinguishing between formulations that can maintain the activity of the vaccine and have the mechanical strength. By the use of in vitro tests such as ELISA and SRID assays, we quantitively evaluated the antigen activity of the formulation candidates to be 87% and 91%, respectively. In vivo tests were also conducted as mouse groups were inoculated with the formulation constructed into egg microneedles (FLU-EMN) to determine the protective efficacy against infection. The results demonstrated that FLU-EMN with functionalized formulations successfully enabled protective immune response even with a fractional dose compared to IM injection.

Lidocaine-loaded dissolving microneedle for safe local anesthesia on oral mucosa for dental procedure.

BACKGROUND: Lidocaine has been widely used as a short-acting local anesthetic agent to reduce the pain caused by needle insertion. Dissolving microneedles (DMNs), which are minimally invasive, can effectively deliver drugs by overcoming the oral mucosal barrier and relieving patient discomfort. METHODS: Lidocaine solution prepared by mixing lidocaine-HCl and hyaluronic acid was used to fabricate oral lidocaine HCl-encapsulated DMNs (oral Li-DMNs) via centrifugal lithography. The dissolution, penetration ability, and local transmucosal drug delivery of oral Li-DMNs into the oral mucosa were evaluated in porcine jaws. Pharmacokinetic analysis and safety assessment were performed using rabbits. RESULTS: The insertion depth of the oral Li-DMNs satisfies the safety standard. The oral Li-DMNs were completely dissolved after 3 min of application. The local transmucosal drug delivery, pharmacokinetic, and safety evaluations showed that the oral Li-DMNs can obtain a local anesthesia effect at a relatively lower dose, and there was no oral mucosal irritation in rabbits. CONCLUSIONS: A novel and safe oral Li-DMNs have potential applications in large animals and clinical trials and would possibly enter the anesthesia market.

Fabrication of liraglutide-encapsulated triple layer hyaluronic acid microneedles (TLMs) for the treatment of obesity.

Obesity is a chronic metabolic disease that is prevalent worldwide, causing complications that affect the quality of life and longevity of humans. Currently, the low bioavailability upon subcutaneous injection of an appetite suppressant, liraglutide, and health problems in the locally injected region remain to be overcome. In this study, we developed a novel hyaluronic acid-based liraglutide-encapsulated triple-layer microneedle (TLM) as a painless and patient-friendly long-term drug delivery system. In contrast to previous anti-obesity microneedle approaches, this TLM is composed of three layers for complete skin insertion, protecting the encapsulated liraglutide from environmental stresses. Daily topical application of the liraglutide-loaded TLM significantly reduced body weight and improved body composition in a mouse model of high-fat diet-induced obesity. Additionally, it ameliorated diet-induced hepatic steatosis in obese mice. This novel TLM could promote a glucagon-like peptide-1 drug release system for long-term daily administration with relatively higher patient compliance compared to subcutaneous injection.

Egg Microneedle for Transdermal Delivery of Active Liraglutide.

Liraglutide, a human glucagon-like peptide-1 (GLP-1) analog, is promising for safely treating type 2 diabetes mellitus (T2DM), compared to insulin, by significantly reducing the risk of glucose-dependent hypoglycemia. Concerns related to injection prevent T2DM patients from taking liraglutide regularly, even though once-a-day subcutaneous (SC) injections. Dissolving microneedles (DMNs) are promising substitutes for SC injection and for improving patient convenience. However, there are two fundamental limitations: the low drug delivery due to incomplete insertion and loss of drug activity during DMN fabrication. Here, it is shown that an egg microneedle (EMN) designed with three functional layered structures can maintain the maximum activity of the loaded compound during DMN fabrication and deliver it completely into the skin, with the base layer allowing the complete delivery of liraglutide, and the shell layer maintaining the drug activity by mimicking the role of albumin in eggs. In a diabetic mouse model, liraglutide administration via EMN exhibited similar effect when compared to that of injection. Therefore, EMN-mediated liraglutide administration is a good potential option for replacing liraglutide injections in T2DM treatment.

Shape of dissolving microneedles determines skin penetration ability and efficacy of drug delivery.

Dissolving microneedles (DMNs) are used for minimally invasive transdermal drug delivery. Dissolution of drugs is achieved in the body after skin penetration by DMNs. Unlike injections, the insertion depth of the DMN is an important issue because the amount of dissolved DMN in the skin determines the amount of drug delivered. Therefore, the inaccurate drug delivery due to the incomplete insertion is one of the limitations of the DMN. Thus, many insertion and penetration tests have been essentially conducted in DMN studies, yet only incomplete insertion is known and the exact standard for how much it is not inserted is still unknown. Moreover, there are various shapes have been introduced in the microneedle field, there have been only few studies that have compared and evaluated the insertion depth of the shapes. Here, we present an intensive approach for DMN insertion based on DMN shape among various insertion deciding factors. We numerically analyzed the volumetric distribution of three types of DMN shapes: conical-shaped DMN, funnel-shaped DMN, and candlelit-shaped DMN, and introduced a new insertion evaluation criterion while covering previous insertion evaluations. Using optical coherence tomography, the images of DMNs embedded in the skin were analyzed in rea l-time, and the amount of drug delivered was analyzed at sectioned depth with a cryotome. The in vitro data confirmed that the insertion depth differed based on shape, and the resulting drug delivery depended on the volume assigned to the insertion depth. Insulin-loaded DMNs were applied to C57BL/6 mice, and the results of pharmacokinetic and pharmacodynamic analyses supported the results of the in vitro analysis. Our approach, which considers the correlation between DMN shape and insertion depth, will contribute to establishing criteria for various DMN design and maximizing drug delivery.

Enhanced Micro-Channeling System via Dissolving Microneedle to Improve Transdermal Serum Delivery for Various Clinical Skincare Treatments.

Topical liquid formulations, dissolving microneedles (DMNs), and microscale needles composed of biodegradable materials have been widely used for the transdermal delivery of active compounds for skincare. However, transdermal active compound delivery by topical liquid formulation application is inhibited by skin barriers, and the skincare efficacy of DMNs is restricted by the low encapsulation capacity and incomplete insertion. In this study, topical serum application via a dissolvable micro-channeling system (DMCS) was used to enhance serum delivery through micro-channels embedded with DMNs. Transdermal serum delivery was evaluated after the topical-serum-only application and combinatorial serum application by assessing the intensity of allophycocyanin (APC) loaded with the serum in the porcine skin. APC intensity was significantly higher in the skin layer at a depth of 120-270 µm upon combinatorial serum application as compared to topical-serum-only application. In addition, the combinatorial serum application showed significantly improved efficacy in the clinical assessment of skin hydration, depigmentation, improvement of wrinkles, elasticity, dermal density, skin pores, and skin soothing without any safety issues compared to the serum-only application. The results indicate that combinatorial serum application with DMCS is a promising candidate for improving skincare treatments with optimal transdermal delivery of active compounds.

Development of Clinical Weekly-Dose Teriparatide Acetate Encapsulated Dissolving Microneedle Patch for Efficient Treatment of Osteoporosis.

Teriparatide acetate (TA), which directly promotes bone formation, is subcutaneously injected to treat osteoporosis. In this study, TA with a once-weekly administration regimen was loaded on dissolving microneedles (DMNs) to effectively deliver it to the systemic circulation via the transdermal route. TA activity reduction during the drying process of various TA polymer solutions formulated with hyaluronic acid and trehalose was monitored and homogeneities were assessed. TA-DMN patches fabricated using centrifugal lithography in a two-layered structure with dried pure hyaluronic acid on the base layer and dried TA polymer solution on the top layer were evaluated for their physical properties. Rhodamine-B-loaded TA-DMNs were found to form perforations when inserted into porcine skin using a shooting device. In addition, 87.6% of TA was delivered to the porcine skin after a 5-min TA-DMN patch application. The relative bioavailability of TA via subcutaneous injection was 66.9% in rats treated with TA-DMN patches. The maximal TA concentration in rat plasma was proportional to the number of patches used. Therefore, the TA-DMN patch fabricated in this study may aid in the effective delivery of TA in a patient-friendly manner and enhance medical efficacy in osteoporosis treatment.

Film-trigger applicator (FTA) for improved skin penetration of microneedle using punching force of carboxymethyl cellulose film acting as a microneedle applicator.

BACKGROUND: Dissolving microneedle (DMN) is a transdermal drug delivery system that creates pore in the skin and directly deliver drug through the pore channel. DMN is considered as one of the promising system alternatives to injection because it is minimally invasive and free from needle-related issues. However, traditional DMN patch system has limitations of incomplete insertion and need of complex external devices. Here, we designed film-trigger applicator (FTA) system that successfully delivered DMN inside the skin layers using fracture energy of carboxymethyl cellulose (CMC) film via micropillars. We highlighted advantages of FTA system in DMN delivery compared with DMN patch, including that the film itself can act as DMN applicator. METHODS: FTA system consists of DMNs fabricated on the CMC film, DMN array holder having holes aligned to DMN array, and micropillars prepared using general purpose polystyrene. We analyzed punching force on the film by micropillars until the film puncture point at different CMC film concentrations and micropillar diameters. We also compared drug delivery efficiency using rhodamine B fluorescence diffusion and skin penetration using optical coherence tomography (OCT) of FTA with those of conventional DMN patch. In vivo experiments were conducted to evaluate DMN delivery efficiency using C57BL/6 mice and insulin as a model drug. RESULTS: FTA system showed enhanced delivery efficiency compared with that of the existing DMN patch system. We concluded CMC film as a successful DMN applicator as it showed enhanced DMN penetration in OCT and rhodamine B diffusion studies. Further, we applied FTA on shaved mouse dorsal skin and observed successful skin penetration. The FTA group showed higher level of plasma insulin in vivo than that of the DMN patch group. CONCLUSIONS: FTA system consisting of simple polymer film and micropillars showed enhanced DMN delivery than that of the existing DMN patch system. Because FTA works with simple finger force without sticky patch and external devices, FTA is a novel and promising platform to overcome the limitations of conventional microneedle patch delivery system; we suggest FTA as a next generation applicator for microneedle application in the future.

Optimization of Layered Dissolving Microneedle for Sustained Drug Delivery Using Heat-Melted Poly(Lactic-Co-glycolic Acid).

Dissolving microneedles (DMNs) have been used as an alternative drug delivery system to deliver therapeutics across the skin barrier in a painless manner. In this study, we propose a novel heat-melting method for the fabrication of hydrophobic poly(lactic-co-glycolic acid) (PLGA) DMNs, without the use of potentially harmful organic solvents. The drug-loaded PLGA mixture, which consisted of a middle layer of the DMN, was optimized and successfully implanted into ex vivo porcine skin. Implanted HMP-DMNs separated from the patch within 10 min, enhancing user compliance, and the encapsulated molecules were released for nearly 4 weeks thereafter. In conclusion, the geometry of HMP-DMNs was successfully optimized for safe and effective transdermal sustained drug delivery without the use of organic solvents. This study provides a strategy for the innovative utilization of PLGA as a material for transdermal drug delivery systems.

Development of Lidocaine-Loaded Dissolving Microneedle for Rapid and Efficient Local Anesthesia.

Lidocaine is a local anesthetic agent used in the form of injection and topical cream. However, these formulation types have limitations of being either painful or slow-acting, thereby hindering effective and complete clinical performance of lidocaine. Dissolving microneedles (DMNs) are used to overcome these limitations owing to their fast onset time and minimally invasive administration methods. Using hyaluronic acid and lidocaine to produce the drug solution, a lidocaine HCl encapsulated DMN (Li-DMN) was fabricated by centrifugal lithography. The drug delivery rate and local anesthetic quality of Li-DMNs were evaluated using the pig cadaver insertion test and Von Frey behavior test. Results showed that Li-DMNs could deliver sufficient lidocaine for anesthesia that is required to be utilized for clinical level. Results from the von Frey test showed that the anesthetic effect of Li-DMNs was observed within 10 min after administration, thus confirming fast onset time. A toxicity test for appropriate clinical application standard was conducted with a microbial limit test and an animal skin irritation test, showing absence of skin irritation and irritation-related microorganisms. Overall, Li-DMN is a possible alternative drug delivery method for local anesthesia, meeting the requirements for clinical conditions and overcoming the drawbacks of other conventional lidocaine administration methods.

Tissue Interlocking Dissolving Microneedles for Accurate and Efficient Transdermal Delivery of Biomolecules.

The interest in safe and efficient transdermal drug delivery systems has been increasing in recent decades. In light of that, polymeric dissolving microneedles (DMNs) were developed as an ideal platform capable of delivering micro- and macro-biomolecules across the skin in a minimally invasive manner. A vast majority of studies, however, suggest that the shape of DMNs, as well as the elastic properties of skin, affects the delivery efficiency of materials encapsulated within DMNs. Likewise, in dynamic tissues, DMNs would easily distend from the skin, leading to inefficient delivery of encapsulated agents. Thus, herein, to improve delivery efficiency of DMN encapsulated agents, a novel hyaluronic acid backbone-based tissue interlocking DMN (TI-DMN) is developed. TI-DMN is simple to fabricate and significantly improves the transdermal delivery efficiency of encapsulated materials compared with traditional DMNs. The enhanced tissue interlocking feature of TI-DMN is achieved through its sharp tip, wide body, and narrow neck geometry. This paper demonstrates that TI-DMN would serve as an attractive transdermal delivery platform to enhance penetration and delivery efficiency of a wide range of biomolecules into the body.

Two-phase delivery using a horse oil and adenosine-loaded dissolving microneedle patch for skin barrier restoration, moisturization, and wrinkle improvement.

BACKGROUND: Dissolving microneedles (DMNs) have been used for skin restoration and wrinkle improvement. Although lipophilic compounds, for example, natural oils or ceramides, enrich the skin barrier, their delivery via DMNs is challenging because of DMN fabrication difficulties. OBJECTIVES: In the present study, we combined a topical formulation and a DMN patch to perform two-phase delivery comprising a lipophilic formulation and hydrophilic compound-loaded DMNs to improve skin barrier status and the efficacy of drug delivery. METHODS: Horse oil-spread and adenosine-loaded DMN arrays were developed in a single patch (HOS-Ad-DMN patch). In vitro analysis was conducted to confirm the successful delivery of the compositions. Clinical assessments were conducted on the lateral canthus of 20 women to compare the efficacy of HOS-Ad-DMN patches with that of adenosine-loaded DMN patches (Ad-DMN patches). RESULTS: Adenosine was delivered via the DMNs after skin penetration and horse oil was delivered successfully into the skin through the microchannels created by the Ad-DMNs. Compared with Ad-DMN patches, HOS-Ad-DMN patches significantly improved skin elasticity, hydration, dermal density, and wrinkles. No adverse events were observed. CONCLUSION: HOS-Ad-DMN patches are a safe and efficient system for skin restoration and wrinkle improvement.

Combinatorial application of dissolving microneedle patch and cream for improvement of skin wrinkles, dermal density, elasticity, and hydration.

BACKGROUND: Dissolving microneedles (DMNs), microscale needles with a biodegradable polymer matrix, have been widely investigated for transdermal drug delivery. However, the restricted drug loading space of DMNs limited the delivery of the desired quantity of active compounds. In this study, we developed novel combinatorial therapies involving sequential application of adenosine-loaded DMN (Ad-DMN) patches and a topical adenosine-loaded cream (Ad-cream). The application of DMNs created skin channels, which delivered encapsulated drugs from both the DMNs and cream. The use of combinatorial therapies can maximize drug delivery. METHODS: To compare the efficacy of combinatorial therapies and Ad-cream application, a double-blind clinical test was conducted over 10 weeks on 21 females with wrinkles around their eyes, and the skin parameters such as wrinkles, dermal density, elasticity, and hydration were analyzed. The skin irritation test was assessed by expert interviewers to elucidate undesirable side effects. RESULTS: The combinatorial therapies showed statistically significant efficacy for the improvement of average depth of wrinkles, dermal density, elasticity, and hydration after an 8-week application (P < 0.001). Adverse effects on the skin were not observed in any subject during the test period. CONCLUSION: The efficacy and safety results showed that the combinatorial therapies were a safe and outstanding innovation for the optimization of transdermal therapy.

Comparative Study of Two Droplet-Based Dissolving Microneedle Fabrication Methods for Skin Vaccination.

Dissolving microneedles (DMNs) have been widely studied in medical applications due to their pain-free administration, superior efficiency, and safe drug delivery. In skin vaccination, preserving the activity of the encapsulated antigen is an important consideration, as antigen activity is lost during DMN fabrication because of various stress factors. These stress factors vary between fabrication methods and each method affects the antigen's activity to different degrees. In this study, the activity of encapsulated antigens delivered by DMNs is compared between two recently developed DMN fabrication methods; droplet-born air blowing (DAB) and centrifugal lithography (CL) for a model scrub typhus vaccine antigen, ScaA. Although the in vitro analysis of ScaA-loaded DMNs (ScaA-DMNs) does not show any differences in physical properties depending on the fabrication methods, the immunogenicity of the CL-produced ScaA-DMN is significantly higher based on cytokine measurement and humoral immunity. DAB and CL differ in their solidification conditions, suggesting that solidification factors critically affect the encapsulated antigen's activity. ScaA-DMNs may also be stably stored for 4 weeks at room temperature. In conclusion, CL is a superior DMN fabrication method compared with DAB, and this study proves that DMN is feasible and practical for skin vaccination.

Transcutaneous implantation of valproic acid-encapsulated dissolving microneedles induces hair regrowth.

The interest in alternative material systems and delivery methods for treatment of androgenetic alopecia has been increasing in the recent decades. Topical application of valproic acid (VPA), an FDA-approved anticonvulsant drug, has been shown to effectively stimulate hair follicle (HF) regrowth by upregulating Wnt/ß-catenin, a key pathway involved in initiation of HF development. Moreover, a majority of studies have suggested that cutaneous wound re-epithelialization is capable of inducing HF through Wnt/ß-catenin pathway. Here, we report fabrication and evaluation of a novel VPA-encapsulating dissolving microneedle (DMN-VPA) that creates minimally invasive dermal micro-wounds upon application, significantly improving the VPA delivery efficiency. DMN-VPA not only delivers encapsulated VPA with higher accuracy than topical application, it also stimulates wound re-epithelialization signals involved in HF regrowth. Through a series of in vivo studies, we show that micro-wounding-mediated implantation of DMN-VPA upregulates expression of Wnt/ß-catenin pathway, alkaline phosphatase, proliferating cell nuclear antigen, loricrin and HF stem cell markers, including keratin 15, and CD34 more effectively than topical application.

Effects of two droplet-based dissolving microneedle manufacturing methods on the activity of encapsulated epidermal growth factor and ascorbic acid.

Dissolving microneedle (DMN) is an attractive, minimally invasive transdermal drug delivery technology. The drugs encapsulated in the DMNs are exposed to a series of thermal, chemical, and physical stresses during the fabrication process, decreasing their therapeutic activity. Current DMN fabrication methods, such as micro-molding, drawing lithography, droplet-born air blowing, and centrifugal lithography, undergo different manufacturing processes involving differing stress conditions. Among the methods, we compared the effects of two droplet-based methods, droplet-born air blowing and centrifugal lithography, on the activity of encapsulated drugs using epidermal growth factor and ascorbic acid as model drugs. Although the appearance and physical properties of DMNs fabricated by the two methods were similar, the immunoreactivity of encapsulated epidermal growth factor in centrifugal lithography and droplet-born air blowing was 92.08±2.86% and 80.67±8.00%, respectively, at baseline, and decreased to 75.32±19.40% and 41.75±16.17%, respectively, 24h after drug-loading. The free-radical scavenging activity of ascorbic acid was maintained at 88.24±0.78% in DMNs fabricated by centrifugal lithography, but decreased over time to 67.02±1.11% in DMNs fabricated by droplet-born air blowing. These findings indicate that the manufacturing conditions of centrifugal lithography exert less stress on the drug-loaded DMNs, minimizing activity loss over time, and therefore that centrifugal lithography is suitable for fabricating DMNs loaded with fragile biological drugs.

Physicochemical study of ascorbic acid 2-glucoside loaded hyaluronic acid dissolving microneedles irradiated by electron beam and gamma ray.

A dissolving microneedle (DMN) patch encapsulated with ascorbic acid 2-glucoside (AA2G) in a needle-shaped hyaluronic acid (HA) backbone was fabricated and sterilized by electron beam (e-beam, 5-40kGy) and gamma ray (γ-ray, 5-30kGy). DMN structures maintained their morphologies and fracture force regardless of e-beam and γ-ray irradiation doses. Both e-beam (40kGy) and γ-ray (20 and 30kGy) met the product sterility requirements for cosmetics and vaccines; however, γ-ray irradiation significantly degraded the encapsulated AA2G, while e-beam maintained AA2G activity. Thus, an e-beam dose of 40kGy, which satisfied the sterility requirements without loss of AA2G, is suitable for terminal sterilization of DMNs. Moreover, we confirmed that the optimized irradiation (e-beam, 40kGy) did not affect dissolution rate and drug release profile of DMNs. Further, we confirmed that HA, the backbone polymer of DMNs, could be utilized as a stabilizer that inhibits degradation of encapsulated AA2G by irradiation. This detailed analysis can be developed further to optimize various biological drugs in transdermal drug delivery systems.

Centrifugal Lithography: Self-Shaping of Polymer Microstructures Encapsulating Biopharmaceutics by Centrifuging Polymer Drops.

Polymeric microstructures encapsulating biopharmaceutics must be fabricated in a controlled environment to preserve the biological activity. There is increasing demand for simple methods designed to preserve the biological activity by utilizing the natural properties of polymers. Here, the paper shows that centrifugal lithography (CL) can be used for the fabrication of such microstructures in a single centrifugation, by engineering the self-shaping properties of hyaluronic acid (HA). In this method, HA drops are self-shaped into hourglass-microstructures to produce two dissolving microneedles (DMN), which facilitate transdermal delivery via implantation on the skin. In addition, tuberculin purified protein derivatives are encapsulated into HA DMNs under refrigerated conditions (4 °C) during CL. Therefore, the tuberculin skin test (TST) with the DMNs indicates minimal damage, as opposed to the case of TST with traditional hypodermic needles. These findings on the fabrication of polymeric microstructures with biopharmaceutics may trigger the development of various biomedical devices and therapies.

Enhanced Transdermal Delivery by Combined Application of Dissolving Microneedle Patch on Serum-Treated Skin.

Dissolving microneedle (DMN), a transdermal drug delivery system in which drugs are encapsulated in a biodegradable polymeric microstructure, is designed to dissolve after skin penetration and release the encapsulated drugs into the body. However, because of limited loading capacity of drugs within microsized structures, only a small dosage can be delivered, which is often insufficient for patients. We propose a novel DMN application that combines topical and DMN application simultaneously to improve skin permeation efficiency. Drugs in pretreated topical formulation and encapsulated drugs in DMN patch are delivered into the skin through microchannels created by DMN application, thus greatly increasing the delivered dose. We used 4-n-butylresorcinol to treat human hyperpigmentation and found that sequential application of serum formulation and DMNs was successful. In skin distribution experiments using Alexa Fluor 488 and 568 dyes as model drugs, we confirmed that the pretreated serum formulation was delivered into the skin through microchannels created by the DMNs. In vitro skin permeation and retention experiments confirmed that this novel combined application delivered more 4-n-butylresorcinol into the skin than traditional DMN-only and serum-only applications. Moreover, this combined application showed a higher efficacy in reducing patients' melanin index and hyperpigmented regions compared with the serum-only application. As combined application of DMNs on serum-treated skin can overcome both dose limitations and safety concerns, this novel approach can advance developments in transdermal drug delivery.