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.Drug Delivery Technolog

Quantification of lipid and peptide content in antigenic peptide-loaded liposome formulations by reversed-phase UPLC using UV absorbance and evaporative light scattering detection

Antigenic peptide-loaded cationic liposomes have shown promise as cancer vaccines. Quantification of both peptides and lipids is critical for quality control of such vaccines for clinical translation. In this work we describe a reversed phase ultra-performance liquid chromatography (RP-UPLC) method that separates lipids (DOTAP, DOPC and their degradation products) and two physicochemically different peptides within 12 min. Samples were prepared by dilution in a 1:1 (v/v) mixture of methanol and water. Peptide quantifi-cation was done via UV detection and lipids were quantified by an evaporative light scattering detector (ELSD), both coupled to the RP-UPLC system, with high precision (RSD < 3.5%). We showed that the presence of lipids and peptides did not mutually influence their quantification. Limit of detection (LOD) and limit of quantification (LOQ), as determined in the ICH guidelines, were 6 and 20 ng for DOTAP, 12 ng and 40 ng for DOPC, 3.0 ng and 8.0 ng for peptide A and 2.4 ng and 7.2 ng for the more hydrophobic peptide B. Finally, lipid degradation of DOTAP and DOPC was monitored in peptide loaded DOTAP:DOPC liposomes upon storage at 4 degrees C and 40 degrees C.(c) 2022 The Authors. Published by Elsevier Inc. on behalf of American Pharmacists Association. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/)Drug Delivery Technolog

Diphtheria toxoid and N-trimethyl chitosan layer-by-layer coated pH-sensitive microneedles induce potent immune responses upon dermal vaccination in mice

Dermal immunization using antigen-coated microneedle arrays is a promising vaccination strategy. However, reduction of microneedle sharpness and the available surface area for antigen coating is a limiting factor. To overcome these obstacles, a layer-by-layer coating approach can be applied onto pH-sensitive microneedles. Following this approach, pH-sensitive microneedle arrays (positively charged at coating pH 5.8 and nearly uncharged at pH 7.4) were alternatingly coated with negatively charged diphtheria toxoid (DT) and N-trimethyl chitosan (TMC), a cationic adjuvant. First, the optimal DT dose for intradermal immunization was determined in a dose-response study, which revealed that low-dose intradermal immunization was more efficient than subcutaneous immunization and that the EC50 dose of DT upon intradermal immunization is 3-fold lower, as compared to subcutaneous immunization. In a subsequent immunization study, microneedle arrays coated with an increasing number (2, 5, and 10) of DT/TMC bilayers resulted in step-wise increasing DT-specific immune responses. Dermal immunization with microneedle arrays coated with 10 bilayers of DT/TMC (corresponding with ± 0.6 μg DT delivered intradermally) resulted in similar DT-specific immune responses as subcutaneous immunization with 5 μg of DT adjuvanted with aluminum phosphate (8-fold dose reduction). Summarizing, the layer-by-layer coating approach onto pH-sensitive microneedles is a versatile method to precisely control the amount of coated and dermally-delivered antigen that is highly suitable for dermal immunization.Drug Delivery Technolog

Reversible NaCl-induced aggregation of a monoclonal antibody at low pH: Characterization of aggregates and factors affecting aggregation

We investigated the influence of pH and sodium chloride concentration on aggregation kinetics of a monoclonal antibody. Aggregation was induced by sodium chloride addition at low pH. Protein conformation before and after salt addition was determined as well as the reversibility of aggregation. Aggregation was monitored at pH values between 2 and 7 with NaCl up to 1.5M by turbidity measurement and size-exclusion chromatography. Particle size distribution was assessed by using size-exclusion chromatography as well as nanoparticle tracking analysis and flow imaging microscopy. Structural changes were monitored by circular dichroism, Fourier transform infrared and fluorescence spectroscopy. Thermal stability was measured by differential scanning fluorimetry. Aggregation propensity was maximal at low pH and high ionic strength. While thermal stability decreased with pH, the secondary structure remained unchanged down to pH 3.5 and up to 1.5M NaCl. Precipitated protein could be largely reverted to monomers by dilution into salt-free buffer. The re-solubilized antibody was indistinguishable in structure, solubility and monodispersity from the unstressed protein. Also, binding to Protein A was steady. Aggregation could be reduced in the presence of trehalose. The results suggest a reversible aggregation mechanism characterized by a limited change in tertiary structure at low pH and a subsequent loss of colloidal stability resulting from electrostatic repulsion once salt is added to the sample. The experimental setup is robust and allows high-throughput quantification of the effect of additives on aggregation kinetics

Cationic Liposomes: A Flexible Vaccine Delivery System for Physicochemically Diverse Antigenic Peptides

PurposePersonalized peptide-based cancer vaccines will be composed of multiple patient specific synthetic long peptides (SLPs) which may have various physicochemical properties. To formulate such SLPs, a flexible vaccine delivery system is required. We studied whether cationic liposomes are suitable for this purpose.MethodsFifteen SIINFEKL T cell epitope-containing SLPs, widely differing in hydrophobicity and isoelectric point, were separately loaded in cationic liposomes via the dehydration-rehydration method. Particle size and polydispersity index (PDI) were measured via dynamic light scattering (DLS), and zeta potential with laser Doppler electrophoresis. Peptide loading was fluorescently determined and the immunogenicity of the formulated peptides was assessed in co-cultures of dendritic cells (DCs) and CD8(+) T-cells in vitro.ResultsAll SLPs were loaded in cationic liposomes by using three different loading method variants, depending on the SLP characteristics. The fifteen liposomal formulations had a comparable size (< 200nm), PDI (< 0.3) and zeta potential (22-30mV). Cationic liposomes efficiently delivered the SLPs to DCs that subsequently activated SIINFEKL-specific CD8(+) T-cells, indicating improved immunological activity of the SLPs.ConclusionCationic liposomes can accommodate a wide range of different SLPs and are therefore a potential delivery platform for personalized cancer vaccines

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

To examine the immunogenicity of diphtheria toxoid (DT) loaded mesoporous silica nanoparticles (MSNs) after coated and hollow microneedle-mediated intradermal immunization in mice. 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. 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. Both the nano-encapsulation of DT and the type of microneedles affect the immunogenicity of the antigen. PURPOSE METHODS RESULTS CONCLUSIONSupramolecular & Biomaterials Chemistr

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

To examine the immunogenicity of diphtheria toxoid (DT) loaded mesoporous silica nanoparticles (MSNs) after coated and hollow microneedle-mediated intradermal immunization in mice. 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. 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. Both the nano-encapsulation of DT and the type of microneedles affect the immunogenicity of the antigen. PURPOSE METHODS RESULTS CONCLUSIONSupramolecular & Biomaterials Chemistr