Modeling ischemic stroke in a triculture neurovascular unit on-a-chip.

BACKGROUND: In ischemic stroke, the function of the cerebral vasculature is impaired. This vascular structure is formed by the so-called neurovascular unit (NVU). A better understanding of the mechanisms involved in NVU dysfunction and recovery may lead to new insights for the development of highly sought therapeutic approaches. To date, there remains an unmet need for complex human in vitro models of the NVU to study ischemic events seen in the human brain. METHODS: We here describe the development of a human NVU on-a-chip model using a platform that allows culture of 40 chips in parallel. The model comprises a perfused vessel of primary human brain endothelial cells in co-culture with induced pluripotent stem cell derived astrocytes and neurons. Ischemic stroke was mimicked using a threefold approach that combines chemical hypoxia, hypoglycemia, and halted perfusion. RESULTS: Immunofluorescent staining confirmed expression of endothelial adherens and tight junction proteins, as well as astrocytic and neuronal markers. In addition, the model expresses relevant brain endothelial transporters and shows spontaneous neuronal firing. The NVU on-a-chip model demonstrates tight barrier function, evidenced by retention of small molecule sodium fluorescein in its lumen. Exposure to the toxic compound staurosporine disrupted the endothelial barrier, causing reduced transepithelial electrical resistance and increased permeability to sodium fluorescein. Under stroke mimicking conditions, brain endothelial cells showed strongly reduced barrier function (35-fold higher apparent permeability) and 7.3-fold decreased mitochondrial potential. Furthermore, levels of adenosine triphosphate were significantly reduced on both the blood- and the brain side of the model (4.8-fold and 11.7-fold reduction, respectively). CONCLUSIONS: The NVU on-a-chip model presented here can be used for fundamental studies of NVU function in stroke and other neurological diseases and for investigation of potential restorative therapies to fight neurological disorders. Due to the platform's relatively high throughput and compatibility with automation, the model holds potential for drug compound screening.

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.

Hollow microneedle-mediated micro-injections of a liposomal HPV E743-63 synthetic long peptide vaccine for efficient induction of cytotoxic and T-helper responses.

Recent studies have shown that intradermal vaccination has great potential for T cell-mediated cancer immunotherapy. However, classical intradermal immunization with a hypodermic needle and syringe has several drawbacks. Therefore, in the present study a digitally controlled hollow microneedle injection system (DC-hMN-iSystem) with an ultra-low dead volume was developed to perform micro-injections (0.25-10µL) into skin in an automated manner. A synthetic long peptide derived from human papilloma virus formulated in cationic liposomes, which was used as a therapeutic cancer vaccine, was administered intradermally by using the DC-hMN-iSystem. Fused silica hollow microneedles with an inner diameter of 50µm and a bevel length of 66±26µm were successfully fabricated via hydrofluoric acid etching. Upon piercing these microneedles into the skin using a protrusion length of 400µm, microneedles were inserted at a depth of 350±55µm. Micro-injections of 1-10µL had an accuracy between 97 and 113% with a relative standard deviation (RSD) of 9%, and lower volumes (0.25 and 0.5µL) had an accuracy of 86-103% with a RSD of 29% in ex vivo human skin. Intradermal administration of the therapeutic cancer vaccine via micro-injections induced strong functional cytotoxic and T-helper responses in mice, while requiring much lower volumes as compared to classical intradermal immunization. In conclusion, by using the newly developed DC-hMN-iSystem, very low vaccine volumes can be precisely injected into skin in an automated manner. Thereby, this system shows potential for minimally-invasive and potentially pain-free therapeutic cancer vaccination.