Dissolving microneedle arrays (dMNAs) can be used to deliver vaccines via the intradermal route. Fabrication of dMNAs using centrifugation is the most common preparation method of dMNAs, but it... Show moreDissolving microneedle arrays (dMNAs) can be used to deliver vaccines via the intradermal route. Fabrication of dMNAs using centrifugation is the most common preparation method of dMNAs, but it results in a substantial loss of antigens. In order to solve the issue of antigen waste, we engineered an automatic dispensing system for dMNA preparation. Here, we report on the fabrication of influenza whole inactivated virus (WIV) vaccine-loaded dMNAs (WIV dMNAs) by using the automatic dispensing system. Prior to the dispensing process, polydimethylsiloxane (PDMS) moulds were treated with oxygen plasma to increase surface hydrophilicity. WIV dMNAs were prepared with 1% (w/v) trehalose and pullulan (50 : 50 weight ratio). During the dispensing process, reduced pressure was applied to the PDMS mould via a vacuum chamber to make microneedle cavities airless. After producing dMNAs, WIV was quantified and 1.9 μg of WIV was loaded per dMNA, of which 1.3 μg was in the microneedle tips. Compared to the centrifugation method, this automatic dispensing system resulted in a 95% reduction of antigen waste. A hemagglutination assay confirmed that WIV dMNA maintained the stability of the antigen for at least four weeks of storage, even at room temperature or at 37 °C. The WIV dMNAs displayed 100% penetration efficiency in human skin, and 83% of the microneedle volume was dissolved in the skin within 10 minutes. In a vaccination study, mice immunised with WIV dMNAs showed similar IgG levels to those that received WIV intramuscularly. In conclusion, using the automatic dispensing system for dMNA production strongly reduced antigen waste and yielded dMNAs with excellent physical, mechanical, and immunological properties. Show less
The induction of a potent T cell response is essential for successful tumor immunotherapy and protection against many infectious diseases. In the past few years, mRNA vaccines have emerged as... Show moreThe induction of a potent T cell response is essential for successful tumor immunotherapy and protection against many infectious diseases. In the past few years, mRNA vaccines have emerged as potent immune activators and inducers of a robust T cell immune response. The recent approval of the Moderna and the Pfizer/BioNTech vaccines based on lipid nanoparticles (LNP) encapsulating antigen-encoding mRNA has revolutionized the field of vaccines. The advantages of LNPs are their ease of design and formulation resulting in potent, effective, and safe vaccines. However, there is still plenty of room for improvement with respect to LNP efficacy, for instance, by optimizing the lipid composition and tuning LNP for specific purposes. mRNA delivery is known to be strongly dependent on the lipid composition of LNPs and the efficiency is mainly determined by the ionizable lipids. Besides that, cholesterol and helper lipids also play important roles in mRNA transfection potency. Here, a panel of LNP formulations was studied by keeping the ionizable lipids constant, replacing cholesterol with β-sitosterol, and changing the fusogenic helper lipid DOPE content. We studied the ability of this LNP library to induce antigen presentation and T cell proliferation to identify superior LNP candidates eliciting potent T cell immune responses. We hypothesize that using β-sitosterol and increasing DOPE content would boost the mRNA transfection on immune cells and result in enhanced immune responses. Transfection of immortal immune cell lines and bone marrow dendritic cells (BMDCs) with LNPs was studied. Delivery of mRNA coding for the model antigen ovalbumin (OVA-mRNA) to BMDCs with a number of LNP formulations, resulted in a high level of activation, as evidenced by the upregulation of the co-stimulatory receptors (CD40 and CD86) and IL-12 in BMDCs. The enhancement of BMDC activation and T cell proliferation induced by the introduction of β-sitosterol and fusogenic DOPE lipids were cell dependent. Four LNP formulations (C12-200-cho-10%DOPE, C12-200-sito-10%DOPE, cKK-E12-cho-10%DOPE and cKK-E12-sito-30%DOPE) were identified that induced robust T cell proliferation and enhanced IFN-γ, TNF-α, IL-2 expression. These results demonstrate that T cell proliferation is strongly dependent on LNP composition and promising LNP-mRNA vaccine formulations were identified. Show less
Aim: Pre-targeting is a proven strategy for in vivo delivery of a diagnostic or therapeutic payload. The pre-targeting concept can be realized through various conjugation strategies, one of which... Show moreAim: Pre-targeting is a proven strategy for in vivo delivery of a diagnostic or therapeutic payload. The pre-targeting concept can be realized through various conjugation strategies, one of which is based on copper-free "click" chemistry. Copper-free click reactions have shown in vivo potential for imaging and radionuclide therapy, but this conjugation strategy has not yet been explored in combination with microspheres or unicellular organisms. This study aims to evaluate the in vivo efficacy of strain-promoted azide-alkyne cycloaddition (SPAAC) reactions to achieve imaging and targeting of azide-functionalized macro-aggregated albumin (MAA) microspheres and Staphylococcus aureus bacteria. Methods: MAA microspheres (diameter 10-90 mu m) were functionalized with a biorthogonal Cy5 fluorophore, bearing an azide functionality (N-3), to generate MAA-Cy5-N-3. S. aureus (diameter similar to 1 mu m) were functionalized with Tc-99m-UBI29-41-Cy5-N-3, generating S. aureus-Tc-99m-UBI29-41-Cy5-N-3. In situ and in vitro click conjugation on the -N-3 moieties was studied for 20 h using a radioactivity-based assay and fluorescence microscopy. For in vivo validation, both primary entities, radiolabeled with Tc-99m, were deposited into the microvasculature of the liver via intrasplenic injections. Secondary targeting was realized following the intravenous administration of indium-111-radiolabeled diethylenetriaminepentaacetic acid-dibenzocyclooctyne (In-111-DTPA-DBCO). To assess click reaction efficiency in vivo, Tc-99m and In-111-biodistributions were measured (SPECT and %ID g(-1)). Use of In-111-DTPA-DBCO in mice without MAA deposits or mice infected with non-functionalized S. aureus served as controls. Ex vivo confocal fluorescence imaging was carried out in excised tissues to confirm the presence of functionalized MAA and bacteria. Results: In vitro data confirmed effective click reactions on both the MAA particles and the bacterial membrane. SPECT imaging and biodistribution studies revealed significantly (p < 0.05) increased accumulation of In-111-DTPA-DBCO at the sites where MAA-Cy5-N-3 (7.5 +/- 1.5%ID g(-1)vs. 3.5 +/- 0.5%ID g(-1) in control mice) and S. aureus-Tc-99m-UBI29-41-Cy5-N-3 (9.3 +/- 1.3%ID g(-1)vs. 6.0 +/- 0.5%ID g(-1) in control mice) resided. Ex vivo fluorescence imaging confirmed the presence of either functionalized MAA or S. aureus in excised spleens and livers of mice. Conclusion: Copper-free click chemistry between a DBCO moiety and Cy5-N-3-functionalized microspheres or bacterial entities in the liver can be used to realize in vivo imaging and targeting. Show less
Layer by layer (LBL) assembly has garnered considerable interest due to its ability to generate multifunctional films with high tunability and versatility in terms of substrates and... Show moreLayer by layer (LBL) assembly has garnered considerable interest due to its ability to generate multifunctional films with high tunability and versatility in terms of substrates and polyelectrolytes, allowing the option to use complex devices and drugs. Polyelectrolytes, such as growth factors (GFs), are essential, but costly, delicate, biological molecules that have been used in various tissue regeneration applications. For this reason, the controlled drug delivery of efficiently loaded GFs via LBL assembly (GF-LBL) can contribute to the establishment of cost-effective biologically triggered biomedical applications. We have developed an LBL method to load GFs (specifically, transforming growth factor beta 1, platelet-derived growth factor beta beta, and insulin growth factor 1), with up to 90% efficiency approximately, by gas plasma surface activation and tuning the pH to increase the ionic strength of polyelectrolytes. Poly(styrenesulfonate) (PSS) and poly(ethyleneimine) (PEI) have been used to provide the initial necessary charge for multilayer build-up. Heparin and dextran sulphate have been investigated as counter polyelectrolytes to enhance the activity of GFs by protecting their ligands, where heparin resulted in the highest achievable loading efficiency for all GFs. Oxygen gas plasma and acidic pH levels also resulted in a significant increase in GF loading efficiency. The three GFs were released by diffusion and erosion in a controlled manner over lengthy time scales and the bioactivity was maintained for up to 14 days. When tested as implants in vitro, GF-LBL constructs increased fibroblast proliferation, influenced cell morphology and migration, and enhanced myofibroblast differentiation, indicating that the biological functionalities of the GFs were preserved. In conclusion, this developed LBL assembly method can provide a simple drug delivery system, which may yield more effective applications for tissue regeneration as well as biomedical sciences at large. Show less
Electrospinning provides a simple robust method to manufacture scaffolds for tissue engineering applications. Though varieties of materials can be used, optimization and biocompatibility tests are... Show moreElectrospinning provides a simple robust method to manufacture scaffolds for tissue engineering applications. Though varieties of materials can be used, optimization and biocompatibility tests are required to provide functional tissue regeneration. Moreover, many studies are limited to 2D electrospun constructs rather than 3D templates due to the production of high density packed fibres, which result in poor cell infiltration. Here, we optimised electrospinning parameters for three different polymers: poly(epsilon-caprolactone) (PCL), polylactic acid (PLA) and poly(ethylene oxide terephthalate)/poly(butylene terephthalate) (PA) copolymers. Human mesenchymal stromal cells (hMSCs) were cultured on scaffolds for 14 days to study the scaffolds' biocompatibility and their multi-lineage differentiation potential or maintenance of stemness in the absence of chemical stimuli. For all scaffolds, a high and stable metabolic activity was measured throughout the culture time with a high proliferation rate compared to day 1 (PCL 5.8-, PLA 4-, PA 4.9-fold). The metabolism of hMSCs was also measured through glucose and lactate concentrations, showing no cytotoxic levels up to 14 days. Total glycosaminoglycan (GAG) production was the highest in PA electrospun scaffolds. When normalized to DNA, GAG production was the highest in PLA and PA scaffolds. All scaffolds were prone to differentiate to an osteogenic lineage, with PCL providing the highest alkaline phosphatase and collagen type Ia gene upregulation. As PA had the most stable fibre formation, it was chosen as a template to further incorporate stromal cell-derived factor-1 (SDF-1) and granulocyte colony-stimulating factor (G-CSF), and stimulate higher hMSC infiltration. These scaffolds provided significantly higher hMSC infiltration than normal PA scaffolds. In conclusion, our optimized biocompatible electrospun scaffolds have shown promising regulation of hMSC fate. When combined with migratory stimulating cytokines, these scaffolds may overcome the known challenges of poor cellular infiltration typical of micro- and nano-fibrillary random meshes. Show less
