Hyaluronan molecular weight: Effects on dissolution time of dissolving microneedles in the skin and on immunogenicity of antigen.

Biomaterials used as matrix for dissolving micro needles (dMNs) may affect the manufacturing process as well as the potency of the active pharmaceutical ingredient, e.g. the immunogenicity of incorporated vaccine antigens. The aim of this study was to investigate the effect of the molecular weight of hyaluronan, a polymer widely used in the fabrication of dMNs, ranging in molecular weight from 4.8 kDa to 1.8 MDa, on the dissolution of microneedles in the skin in time as well as the antibody response in mice and T-cell activation in vitro. Hyaluronan molecular weight (HA-MWs) did not affect antibody responses (when lower than 150 kDa) nor CD4+ T-cell responses against model antigen ovalbumin. However, the HA-MWs had an effect on the fabrication of dMNs. The 1.8 MDa HA was not suitable for the fabrication of dMNs. Similarly, the 4.8 kDa HA generated dMN arrays less robust compared to the other HA-MWs requiring optimization of the drying conditions. Finally, higher HA-MWs led to longer application time of dMN arrays for a complete dissolution of microneedles into the skin. Specifically, we identified 20 kDa HA as the optimal HA-MW for the fabrication of dMNs as with this MW the dMNs are robust and dissolve fast in the skin without affecting immunogenicity.

Hyaluronan-based dissolving microneedles with high antigen content for intradermal vaccination: Formulation, physicochemical characterization and immunogenicity assessment.

The purpose of this study was to optimize the manufacturing of dissolving microneedles (dMNs) and to increase the antigen loading in dMNs to investigate the effect on their physicochemical properties. To achieve this, a novel single-array wells polydimethylsiloxane mold was designed, minimizing antigen wastage during fabrication and achieving homogeneous antigen distribution among the dMN arrays. Using this mold, hyaluronan (HA)-based dMNs were fabricated and tested for maximal ovalbumin (OVA) content. dMNs could be fabricated with an OVA:HA ratio as high as 1:1 (w/w), without compromising their properties such as shape and penetration into the ex vivo human skin, even after storage at high humidity and temperature. High antigen loading did not induce protein aggregation during dMN fabrication as demonstrated by complementary analytical methods. However, the dissolution rate in ex vivo human skin decreased with increasing antigen loading. About 2.7 µg OVA could be delivered in mice by using a single array with an OVA:HA ratio of 1:3 (w/w). Intradermal vaccination with dMNs induced an immune response similar as subcutaneous injection and faster than after hollow microneedle injection. In conclusion, results suggest that (i) the polydimethylsiloxane mold design has an impact on the manufacturing of dMNs, (ii) the increase in antigen loading in dMNs affects the microneedle dissolution and (iii) dMNs are a valid alternative for vaccine administration over conventional injection.

Universal Applicator for Digitally-Controlled Pressing Force and Impact Velocity Insertion of Microneedles into Skin.

Microneedle technologies have been developed for dermal drug and vaccine delivery, including hollow-, solid-, coated-, and dissolving microneedles. Microneedles have been made in many different geometries and of many different materials, all of which may influence their skin-penetrating ability. To ensure reproducible and effective drug and vaccine delivery via microneedles, the optimal insertion parameters should be known. Therefore, a digitally-controlled microneedle applicator was developed to insert microneedles into the skin via impact insertion (velocity) or via pressing force insertion. Six microneedle arrays with different geometries and/or materials were applied onto ex vivo human skin with varying velocities or pressing forces. Penetration efficiency and delivered antigen dose into the skin after application of microneedles were determined. In general, microneedles pierced the skin more efficiently when applied by impact application as compared to application via pressing force. However, the angle of application of the applicator on the skin can affect the velocity of the impact, influencing the penetration efficiency of microneedles. Regarding the antigen delivery into the skin, the delivered dose was increasing by increasing the velocity or pressure, and thus, increasing the penetration efficiency. These data demonstrate that an applicator is an important tool to determine optimal application conditions with ex vivo human skin.

Coated and Hollow Microneedle-Mediated Intradermal Immunization in Mice with Diphtheria Toxoid Loaded Mesoporous Silica Nanoparticles.

PURPOSE: To examine the immunogenicity of diphtheria toxoid (DT) loaded mesoporous silica nanoparticles (MSNs) after coated and hollow microneedle-mediated intradermal immunization in mice. METHODS: DT was loaded into MSNs and the nanoparticle surface was coated with a lipid bilayer (LB-MSN-DT). To prepare coated microneedles, alternating layers of negatively charged LB-MSN-DT and positively charged N-trimethyl chitosan (TMC) were coated onto pH-sensitive microneedle arrays via a layer-by-layer approach. Microneedle arrays coated with 5 or 3 layers of LB-MSN-DT were used to immunize mice and the elicited antibody responses were compared with those induced by hollow microneedle-injected liquid formulation of LB-MSN-DT. Liquid DT formulation with and without TMC (DT/TMC) injected by a hollow microneedle were used as controls. RESULTS: LB-MSN-DT had an average size of about 670 nm and a zeta potential of -35 mV. The encapsulation efficiency of DT in the nanoparticles was 77%. The amount of nano-encapsulated DT coated onto the microneedle array increased linearly with increasing number of the coating layers. Nano-encapsulated DT induced stronger immune responses than DT solution when delivered intradermally via hollow microneedles, but not when delivered via coated microneedles. CONCLUSION: Both the nano-encapsulation of DT and the type of microneedles affect the immunogenicity of the antigen.

Immunogenicity of diphtheria toxoid and poly(I:C) loaded cationic liposomes after hollow microneedle-mediated intradermal injection in mice.

In this study, we aimed to investigate the immunogenicity of cationic liposomes loaded with diphtheria toxoid (DT) and poly(I:C) after hollow microneedle-mediated intradermal vaccination in mice. The following liposomal formulations were studied: DT loaded liposomes, a mixture of free DT and poly(I:C)-loaded liposomes, a mixture of DT-loaded liposomes and free poly(I:C), and liposomal formulations with DT and poly(I:C) either individually or co-encapsulated in the liposomes. Reference groups were DT solution adjuvanted with or without poly(I:C) (DT/poly(I:C)). The liposomal formulations were characterized in terms of particle size, zeta potential, loading and release of DT and poly(I:C). After intradermal injection of BALB/c mice with the formulations through a hollow microneedle, the immunogenicity was assessed by DT-specific ELISAs. All formulations induced similar total IgG and IgG1 titers. However, all the liposomal groups containing both DT and poly(I:C) showed enhanced IgG2a titers compared to DT/poly(I:C) solution, indicating that the immune response was skewed towards a Th1 direction. This enhancement was similar for all liposomal groups that contain both DT and poly(I:C) in the formulations. Our results reveal that a mixture of DT encapsulated liposomes and poly(I:C) encapsulated liposomes have a similar effect on the antibody responses as DT and poly(I:C) co-encapsulated liposomes. These findings may have implications for future design of liposomal vaccine delivery systems.

Development of PLGA nanoparticle loaded dissolving microneedles and comparison with hollow microneedles in intradermal vaccine delivery.

Skin is an attractive but also very challenging immunisation site for particulate subunit vaccines. The aim of this study was to develop hyaluronan (HA)-based dissolving microneedles (MNs) loaded with PLGA nanoparticles (NPs) co-encapsulating ovalbumin (OVA) and poly(I:C) for intradermal immunisation. The NP:HA ratio used for the preparation of dissolving MNs appeared to be critical for the quality of MNs and their dissolution in ex vivo human skin. Asymmetrical flow field-flow fractionation and dynamic light scattering were used to analyse the NPs released from the MNs in vitro. Successful release of the NPs depended on the drying conditions during MN preparation. The delivered antigen dose from dissolving MNs in mice was determined to be 1 µg OVA, in NPs or as free antigen, by using near-infrared fluorescence imaging. Finally, the immunogenicity of the NPs after administration of dissolving MNs (NP:HA weight ratio 1:4) was compared with that of hollow MN-delivered NPs in mice. Immunization with free antigen in dissolving MNs resulted in equally strong immune responses compared to delivery by hollow MNs. However, humoral and cellular immune responses evoked by NP-loaded dissolving MNs were inferior to those elicited by NPs delivered through a hollow MN. In conclusion, we identified several critical formulation parameters for the further development of NP-loaded dissolving MNs.