The immunological effects of intradermal particle-based vaccine delivery using a novel microinjection needle studied in a human skin explant model.

For intradermal (ID) immunisation, novel needle-based delivery systems have been proposed as a better alternative to the Mantoux method. However, the penetration depth of needles in the human skin and its effect on immune cells residing in the different layers of the skin has not been analyzed. A novel and user-friendly silicon microinjection needle (Bella-muTM) has been developed, which allows for a perpendicular injection due to its short needle length (1.4-1.8 mm) and ultrashort bevel. We aimed to characterize the performance of this microinjection needle in the context of the delivery of a particle-based outer membrane vesicle (OMV) vaccine using an ex vivo human skin explant model. We compared the needles of 1.4 and 1.8 mm with the conventional Mantoux method to investigate the depth of vaccine injection and the capacity of the skin antigen-presenting cell (APC) to phagocytose the OMVs. The 1.4 mm needle deposited the antigen closer to the epidermis than the 1.8 mm needle or the Mantoux method. Consequently, activation of epidermal Langerhans cells was significantly higher as determined by dendrite shortening. We found that five different subsets of dermal APCs are able to phagocytose the OMV vaccine, irrespective of the device or injection method. ID delivery using the 1.4 mm needle of a OMV-based vaccine allowed epidermal and dermal APC targeting, with superior activation of Langerhans cells. This study indicates that the use of a microinjection needle improves the delivery of vaccines in the human skin.

Three-phase vaporization theory for laser-activated microcapsules.

Precision control of vaporization, both in space and time, is critical for numerous applications, including medical imaging and therapy, catalysis and energy conversion, and it can be greatly improved through the use of micro- or nano-sized light absorbers. Ultimately, optimization of these applications also requires a fundamental understanding of the vaporization process. Upon laser irradiation, polymeric microcapsules containing a dye can vaporize, leading to the growth of a vapor bubble that emits a strong acoustic signature. Here, we compare laser-activated capsules containing either a volatile or a non-volatile oil core. We theoretically explore the vaporization of the capsules based on a three-phase thermodynamics model, that accounts for the partial vaporization of both the surrounding fluid and the oil core as well as for the interaction between heat transfer and microbubble growth. The model is compared to ultra-high-speed imaging experiments, where we record the cavitation events. Theory and experiments are in convincing agreement.

Laser-activated microparticles for multimodal imaging: ultrasound and photoacoustics.

The increasing personalization of medical treatment demands refined imaging and increased monitoring capabilities, as well as an improved efficacy through targeted drug delivery. Such a transition in health care can be facilitated by the use of multimodal contrast agents. In this paper, we present a novel type of multimodal contrast agents, that enhances contrast both in ultrasound and in photoacoustic imaging, while at the same time being capable of triggered drug delivery. Upon pulsed laser irradiation, polymeric microparticles-containing a dye and an oil core-can create a cavitation bubble that subsequently emits a strong acoustic wave. We investigated different formulations of these particles, by changing the oil content, dye concentration and probing conditions using a combination of pulsed laser excitation and an ultrasound chirp. We demonstrated that capsules with a core containing a low boiling point oil give the highest photoacoustic and acoustic response. The laser activation threshold for this system is high in the visible range, but within the near infrared medical limits. The same system also produces a stable bubble. US scattering by these stable bubbles results in medically relevant frequencies, making the particles of interest for biomedical and pre-clinical imaging. Finally, the system has potential to carry a functional drug-load, and a route to these applications is discussed.

Laser-Activated Polymeric Microcapsules for Ultrasound Imaging and Therapy: In Vitro Feasibility.

Polymeric microcapsules with a light-absorbing dye incorporated in their shell can generate vapor microbubbles that can be spatiotemporally controlled by pulsed laser irradiation. These contrast agents of 6-8 µm in diameter can circulate through the vasculature, offering possibilities for ultrasound (molecular) imaging and targeted therapies. Here, we study the impact of such vapor bubbles on human endothelial cells in terms of cell poration and cell viability to establish the imaging and therapeutic windows. Two capsule formulations were used: the first one consisted of a high boiling point oil (hexadecane), whereas the second was loaded with a low boiling point oil (perfluoropentane). Poration probability was already 40% for the smallest bubbles that were formed (<7.5 µm diameter), and reached 100% for the larger bubbles. The hexadecane-loaded capsules also produced bubbles while their shell remained intact. These encapsulated bubbles could therefore be used for noninvasive ultrasound imaging after laser activation without inducing any cell damage. The controlled and localized cell destruction achieved by activation of both capsule formulations may provide an innovative approach for specifically inducing cell death in vivo, e.g., for cancer therapy.

Microsphere-Based Rapamycin Delivery, Systemic Versus Local Administration in a Rat Model of Renal Ischemia/Reperfusion Injury.

PURPOSE: The increasing prevalence and treatment costs of kidney diseases call for innovative therapeutic strategies that prevent disease progression at an early stage. We studied a novel method of subcapsular injection of monodisperse microspheres, to use as a local delivery system of drugs to the kidney. METHODS: We generated placebo- and rapamycin monodisperse microspheres to investigate subcapsular delivery of drugs. Using a rat model of acute kidney injury, subcapsular injection of placebo and rapamycin monodisperse microspheres (monospheres) was compared to subcutaneous injection, mimicking systemic administration. RESULTS: We did not find any adverse effects related to the delivery method. Irrespective of the injection site, a similar low dose of rapamycin was present in the circulation. However, only local intrarenal delivery of rapamycin from monospheres led to decreased macrophage infiltration and a significantly lower amount of myofibroblasts in the kidney, where systemic administration did not. Local delivery of rapamycin did cause a transient increase in the deposition of collagen I, but not of collagen III. CONCLUSIONS: We conclude that therapeutic effects can be increased when rapamycin is delivered subcapsularly by monospheres, which, combined with low systemic concentrations, may lead to an effective intrarenal delivery method.

Mucus permeating carriers: formulation and characterization of highly densely charged nanoparticles.

The GI mucus layer represents a significant block to drug carriers absorption. Taking an example from nature, virus-mimicking nanoparticles (NPs) with highly densely charged surface were designed with the aim to improve their mucus permeation ability. NPs were formulated by combining chitosan with chondroitin sulfate and were characterized by particle size, ζ-potential and hydrophobicity. The interaction occurring between NPs and diluted porcine intestinal mucus was investigated by a new method. Furthermore, the rotating tube technique was exploited to evaluate the NPs permeation ability in fresh undiluted porcine intestinal mucus. NPs (400-500 nm) presenting a slightly positive (4.02 mV) and slightly negative (-3.55 mV) ζ-potential resulted to be hydrophobic and hydrophilic, respectively. On the one hand the hydrophobic NPs undergo physico-chemical changes when incubated with mucus, namely the size increased and the ζ-potential decreased. On the other hand, the hydrophilic NPs did not significantly change size and net charge during incubation with mucus. Both types of NPs showed a 3-fold higher diffusion ability compared to the reference 50/50 DL-lactide/glycolide copolymer NPs (136 nm, -23 mV, hydrophilic). Based on these results, this work gives valuable information for the further design of mucus-penetrating NPs.

Local therapeutic efficacy with reduced systemic side effects by rapamycin-loaded subcapsular microspheres.

Kidney injury triggers fibrosis, the final common pathway of chronic kidney disease (CKD). The increase of CKD prevalence worldwide urgently calls for new therapies. Available systemic treatment such as rapamycin are associated with serious side effects. To study the potential of local antifibrotic therapy, we administered rapamycin-loaded microspheres under the kidney capsule of ureter-obstructed rats and assessed the local antifibrotic effects and systemic side effects of rapamycin. After 7 days, microsphere depots were easily identifiable under the kidney capsule. Both systemic and local rapamycin treatment reduced intrarenal mTOR activity, myofibroblast accumulation, expression of fibrotic genes, and T-lymphocyte infiltration. Upon local treatment, inhibition of mTOR activity and reduction of myofibroblast accumulation were limited to the immediate vicinity of the subcapsular pocket, while reduction of T-cell infiltration was widespread. In contrast to systemically administered rapamycin, local treatment did not induce off target effects such as weight loss. Thus subcapsular delivery of rapamycin-loaded microspheres successfully inhibited local fibrotic response in UUO with less systemic effects. Therapeutic effect of released rapamycin was most prominent in close vicinity to the implanted microspheres.

Computer modeling assisted design of monodisperse PLGA microspheres with controlled porosity affords zero order release of an encapsulated macromolecule for 3 months.

PURPOSE: The aim of this study was the development of poly(D,L-lactide-co-glycolide) (PLGA) microspheres with controlled porosity, to obtain microspheres that afford continuous release of a macromolecular model compound (blue dextran). METHODS: PLGA microspheres with a size of around 40 µm and narrow size distribution (span value of 0.3) were prepared with a double emulsion membrane emulsification method. Gene expression programming (GEP) analysis was applied to design and formulate a batch of microspheres with controlled porosity that shows continuous release of blue dextran. RESULTS: Low porous microspheres with a high loading efficiency were formed at high polymer concentrations (30% w/w in the oil phase) and were characterized with a burst release <10% and a three-phasic release profile of blue dextran. Increasing porosity (10% w/w polymer concentrations), a sustained release of blue dextran was obtained albeit with up to 40% of burst release. The desired formulation, calculated by GEP, resulted in microspheres with 72% loading efficiency and intermediate porosity. Blue dextran was indeed released continuously in almost a zero order manner over a period of 3 months after an initial small burst release of 9%. CONCLUSIONS: By fine-tuning the porosity, the release profile of PLGA microspheres for macromolecules can be predicted and changed from a three-phasic to a continuous release.

Three different strategies to obtain porous calcium phosphate cements: comparison of performance in a rat skull bone augmentation model.

Preprosthetic surgery has become a routine procedure to obtain sufficient bone quantity and quality for dental implant installation in patients with an initial inadequate bone volume. Although autologous bone onlay or inlay grafting is still the preferred bone augmentation technique, a broad range of synthetic bone substitutes have been developed, for example, calcium phosphate cement (CPC). The introduction of porosity within CPC can be used to increase CPC degradation and bone ingrowth. Therefore, three different strategies to obtain porous CPCs were evaluated in this preclinical study. Instantaneously porous CPC (CPC-IP) was compared with delayed porous CPC in vitro and in vivo. CPC-IP was obtained by the creation of CO2 bubbles during setting, whereas delayed porous CPC was obtained after the degradation of incorporated poly(lactic-co-glycolic acid) (PLGA) microspheres. As an additional aspect, delayed porous CPC was created by the incorporation of either hollow or dense degradable PLGA microspheres (CPC-hPLGA and CPC-dPLGA). All CPC compositions showed appropriate clinical handling properties and an interconnected porous structure with a final porosity above 70% (v/v). In vitro degradation studies showed the gradual formation of pores and further CPC-matrix dissolution for CPCs containing PLGA microspheres (dPLGA microspheres > hPLGA microspheres). For in vivo evaluation of the CPCs, an augmentation model was used, allowing a CPC injection into a rigidly immobilized Teflon ring on the rat skull. Histological evaluation after 12 weeks of implantation showed bone formation using all three CPCs. Bone apposition reached volumetric amounts of up to 10% of the augmentation area and a maximum augmentation height of ∼1 mm. CPC-IP showed significantly more bone formation and resulted in a superior bone apposition height compared with both CPCs containing PLGA microspheres. No differences in biological performance were observed between the CPCs containing hPLGA and those containing dPLGA microspheres. Further research is necessary to enhance the bone appositional speed and amount of CPCs for bone augmentation procedures before them being used in a potential clinical setting.

The intracellular uptake of CD95 modified paclitaxel-loaded poly(lactic-co-glycolic acid) microparticles.

The CD95/CD95L receptor-ligand system is mainly recognised in the induction of apoptosis. However, it has also been shown that CD95L is over-expressed in many cancer types where it modulates immune-evasion and together with its receptor CD95 promotes tumour growth. Here, we show that CD95 surface modification of relatively large microparticles >0.5 µm in diameter, including those made from biodegradable polylactic-co-glycolic acid (PLGA), enhances intracellular uptake by a range of CD95L expressing cells in a process akin to phagocytosis. Using this approach we describe the intracellular uptake of microparticles and agent delivery in neurons, medulloblastoma, breast and ovarian cancer cells in vitro. CD95 modified paclitaxel-loaded PLGA microparticles are shown to be significantly more effective compared to conventional paclitaxel therapy (Taxol) at the same dose in subcutaneous medulloblastoma (∗∗∗P < 0.0001) and orthotopic ovarian cancer xenograft models where a >65-fold reduction in tumour bioluminescence was measured after treatment (∗P = 0.012). This drug delivery platform represents a new way of manipulating the normally advantageous tumour CD95L over-expression towards a therapeutic strategy. CD95 functionalised drug carriers could contribute to the improved function of cytotoxics in cancer, potentially increasing drug targeting and efficacy whilst reducing toxicity.

Parallelized edge-based droplet generation (EDGE) devices.

We here report on three parallelized designs of the new edge-based droplet generation mechanism, which, unlike existing mechanisms, produces many equally sized droplets simultaneously at a single droplet formation unit. Operation of the scaled-out systems is straight forward; only the oil inlet pressure has to be controlled to let all the units produce oil droplets, given certain basic design constraints. For systems with a typical nozzle depth of 1.2 microm, the mean droplet diameter is 7.5 microm and the coefficient of variation is below 10%. The number of droplets that is formed per unit can easily be increased by increasing the length of the unit. The stable pressure range in which monodisperse droplets are formed can be extended by small adjustments to the design. Overall, the EDGE devices are simple in design and robust in use, making them suitable for massive outscaling.