Chitosan microneedle patches for sustained transdermal delivery of macromolecules.

This paper introduces a chitosan microneedle patch for efficient and sustained transdermal delivery of hydrophilic macromolecules. Chitosan microneedles have sufficient mechanical strength to be inserted in vitro into porcine skin at approximately 250 µm in depth and in vivo into rat skin at approximately 200 µm in depth. Bovine serum albumin (BSA, MW=66.5 kDa) was used as a model protein to explore the potential use of chitosan microneedles as a transdermal delivery device for protein drugs. In vitro drug release showed that chitosan microneedles can provide a sustained release of BSA for at least 8 days (approximately 95% of drugs released in 8 days). When the Alexa Fluor 488-labeled BSA (Alexa 488-BSA)-loaded microneedles were applied to the rat skin in vivo, confocal microscopic images showed that BSA can gradually diffuse from the puncture sites to the dermal layer and the fluorescence of Alexa 488-BSA can be observed at the depth of 300 µm. In addition, encapsulation of BSA within the microneedle matrix did not alter the secondary structure of BSA, indicating that the gentle nature of the fabrication process allowed for encapsulation of fragile biomolecules. These results suggested that the developed chitosan microneedles may serve as a promising device for transdermal delivery of macromolecules in a sustained manner.

Implantable microneedles with an immune-boosting function for effective intradermal influenza vaccination.

This study details effective influenza vaccination via sustained intradermal (ID) release of vaccines using implantable and patch-free chitosan microneedles (MNs). The microneedle (MN) patch is composed of vaccine-loaded chitosan MNs with a dissolvable supporting array that gives extra length for complete insertion of MNs and is dissolved within the skin during insertion. Chitosan MNs can be quickly and entirely implanted into the dermis to function as a depot and an immune-boosting agent for the extended release of vaccines and simultaneous activation of the immune system. We found the influenza virus-specific antibody levels induced by chitosan MN vaccination were significantly higher than those elicited by intramuscular (IM) immunization with influenza vaccine alone. The MN induced immune-enhancing effect was obvious 4 week after the vaccination and lasted for at least 16 weeks. Most importantly, MN-immunized mice were completely protected from H1N1 viral challenge without major weight loss, whereas mice receiving IM injection at the same dose had a mortality rate of 60% and experienced notable weight loss after challenge. Our results suggest that the chitosan MNs cannot only be a viable tool for precise ID vaccine delivery but also exert strong adjuvanticity to enhance vaccine potency and induce protective immunity against influenza virus infections. STATEMENT OF SIGNIFICANCE: There is an urgent need for generating a new vaccination strategy to address the threat of global pandemic influenza. This study presents implantable chitosan microneedles (MNs) with immune-boosting function for effective influenza vaccination. We demonstrate that the chitosan MN can not only be an efficient tool for sustained intradermal delivery but also serve as an immunological adjuvant to boost vaccine efficacy. Continuous antigen exposure and immune stimulation provided by the implanted MNs may enhance the immunogenicity of influenza vaccines and evoke long-lasting immune responses to completely protect mice from lethal influenza challenge. The proposed MN system has great potential to be used as a new adjuvanted vaccine formulation and make influenza vaccination more effective and more accessible.

Enhancing immunogenicity of antigens through sustained intradermal delivery using chitosan microneedles with a patch-dissolvable design.

Reducing the dosage required for vaccination is highly desirable, particularly in cases of epidemic emergencies. This study evaluated the potential of a chitosan microneedle (MN) system with a patch-dissolvable design for low-dose immunization. This system comprises antigen-loaded chitosan MNs and a hydrophilic polyvinyl alcohol/polyvinyl pyrrolidone supporting array patch, which provides extra strength to achieve complete MN insertion and then quickly dissolves in the skin to reduce patch-induced skin irritation. After insertion, MNs could be directly implanted in the dermal layer as an intradermal (ID) depot to allow a sustained release of the model antigen ovalbumin (OVA) for up to 28 days. We found that rats immunized with MNs containing low-dose OVA (approximately 200 µg) had persistently high antibody levels for 18 weeks, which were significantly higher than those observed after an intramuscular injection of full-dose OVA (approximately 500 µg), demonstrating at least 2.5-fold dose sparing. Moreover, OVA-encapsulated chitosan MNs had superior immunogenicity to OVA plus chitosan solution, indicating that MN-based delivery and prolonged skin exposure can further enhance chitosan's adjuvanticity. Therefore, this patch-dissolvable MN system offers a needle-free, accurate, and reliable ID delivery of antigens and has potential as a sustained ID delivery device to improve vaccine efficacy and facilitate dose sparing with existing vaccines. STATEMENT OF SIGNIFICANCE: This study developed implantable chitosan microneedles (MNs) with a patch-dissolvable design for the sustained intradermal (ID) delivery of antigens and demonstrated their antigen dose-sparing potential. We found that rats immunized with chitosan MNs containing low-dose OVA had persistently high antibody levels for 18 weeks, which were significantly higher than those observed after an intramuscular injection of full-dose OVA, demonstrating at least 2.5-fold dose sparing. Our results indicate that chitosan MNs can not only serve as an efficient vaccine delivery system but also exert their promising adjuvant activity by forming an ID depot for prolonged antigen exposure and activating dendritic cells for promoting immune responses.

New diterpenes leojaponins G-L from Leonurus japonicus.

Six new diterpenes, leojaponins G-L (1-6) along with 19 known compounds (7-25) were isolated from Leonurus japonicus. Their structures were elucidated by NMR, MS, IR, UV, and ECD spectroscopic data. Anti-melanogenesis assay indicated that 7 could safely and dose-dependently decrease melanin production in B16F10 melanoma cell with an IC50 value of 59.1 µM, but moderately inhibit tyrosinase activity. Without cytotoxicities at 20 µM, compounds 11, 14, 15, and 17-21 showed significant melanogenesis stimulation activities at the percentages of 7.7-48.2. Antioxidants 19 and 24 could notably inhibit ROS production in a dose-dependent manner with percentages of 24.7-42.2 and 27.9-40.2, respectively among the concentrations of 0.16 to 100 µM. Our results demonstrated L. japonicus and its constituents could be potential botanical resources of cosmeceutical development for treatment and prevention of skin disorders.

Fully embeddable chitosan microneedles as a sustained release depot for intradermal vaccination.

This study introduces a microneedle transdermal delivery system, composed of embeddable chitosan microneedles and a poly(L-lactide-co-D,L-lactide) (PLA) supporting array, for complete and sustained delivery of encapsulated antigens to the skin. Chitosan microneedles were mounted to the top of a strong PLA supporting array, providing mechanical strength to fully insert the microneedles into the skin. When inserted into rat skin in vivo, chitosan microneedles successfully separated from the supporting array and were left within the skin for sustained drug delivery without requiring a transdermal patch. The microneedle penetration depth was approximately 600 µm (i.e. the total length of the microneedle), which is beneficial for targeted delivery of antigens to antigen-presenting cells in the epidermis and dermis. To evaluate the utility of chitosan microneedles for intradermal vaccination, ovalbumin (OVA; MW = 44.3 kDa) was used as a model antigen. When the OVA-loaded microneedles were embedded in rat skin in vivo, histological examination showed that the microneedles gradually degraded and prolonged OVA exposure at the insertion sites for up to 14 days. Compared to traditional intramuscular immunization, rats immunized by a single microneedle dose of OVA showed a significantly higher OVA-specific antibody response which lasted for at least 6 weeks. These results suggest that embeddable chitosan microneedles are a promising depot for extended delivery of encapsulated antigens to provide sustained immune stimulation and improve immunogenicity.