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Intracellular delivery of membrane-impermeable cargo offers unique opportunities for biological research and the development of cell-based therapies. Despite the breadth of available intracellular delivery tools, existing protocols are often suboptimal and alternative approaches that merge delivery efficiency with both biocompatibility, as well as applicability, remain highly sought after. Here, a comprehensive platform is presented that exploits the unique property of cationic hydrogel nanoparticles to transiently disrupt the plasma membrane of cells, allowing direct cytosolic delivery of uncomplexed membrane-impermeable cargo. Using this platform, which is termed Hydrogel-enabled nanoPoration or HyPore, the delivery of fluorescein isothiocyanate (FITC)-dextran macromolecules in various cancer cell lines and primary bovine corneal epithelial cells is convincingly demonstrated. Of note, HyPore demonstrates efficient FITC-dextran delivery in primary human T cells, outperforming state-of-the-art electroporation-mediated delivery. Moreover, the HyPore platform enables cytosolic delivery of functional proteins, including a histone-binding nanobody as well as the enzymes granzyme A and Cre-recombinase. Finally, HyPore-mediated delivery of the MRI contrast agent gadobutrol in primary human T cells significantly improves their T1 -weighted MRI signal intensities compared to electroporation. Taken together, HyPore is proposed as a straightforward, highly versatile, and cost-effective technique for high-throughput, ex vivo manipulation of primary cells and cell lines.

Drug delivery systems which can generate multiple drug pulses after a single injection can be of major importance for e.g. the delivery of vaccines which often require multiple booster injections to generate sufficient immunity. In this paper, we report self-exploding microcapsules which can release their content in a pulsed fashion after a certain incubation time at physiological conditions. The system is based on a biodegradable microgel core surrounded by a bio-polyelectrolyte membrane. When the microgel core degrades, the swelling pressure increases and finally ruptures the membrane leading to release of the encapsulated materials. We show that the explosion time and subsequent release of 50 nm, latex nanoparticles (used as a model), can be tailored by varying the cross-link density of the microgels. Further we also address the biocompatibility in vivo after subcutaneous injection of the microcapsules; in mice. (c) 2009 Elsevier B.V. All rights reserved.

This work describes the tailorability of the network properties of self-assembling hydrogels, based on ionic crosslinking between dextran microspheres. Copolymerization of hydroxyethyl methacrylate-derivatized dextran (dex-HEMA), emulsified in an aqueous poly(ethylene glycol) (PEG) solution, with methacrylic acid (MAA) or dimethylaminoethyl methacrylate (DMAEMA) resulted in negatively or positively charged microspheres, respectively, at physiological pH. The monomer/HEMA ratio ranged between 6 and 57, resulting in microspheres with zeta (zeta)-potentials from -6 to -34mV and +3 to +23mV, for the monomers MAA and DMAEMA, respectively. By altering the emulsification procedure, microsphere batches with various sizes and size distributions were obtained. The aim of the research was to assess the effect of particle size (distribution) and charge on the network properties of the macroscopic hydrogels. The ability to tailor the mechanical properties such as strength and elasticity increases the potential of the hydrogels to be used in a variety of pharmaceutical applications. Additionally, the injectability of these self-assembling hydrogels was investigated. Injectability is an important feature of drug delivery systems, since it allows avoiding surgery. Rheological analysis showed that an increasing surface charge of the microspheres led to stronger hydrogels. Relatively small microspheres (7microm) with a narrow size distribution (99% smaller than 14microm) gave rise to stronger hydrogels when compared to larger microspheres of 20microm with a broad distribution (99% smaller than 50microm). When small microspheres were combined with large microspheres of opposite charge, it was found that the strongest gels were obtained with 75% small and 25% large microspheres, instead of equal amounts (50/50) of positively and negatively charged microspheres. Computer modeling confirmed these findings and showed that the most favorable composition, related to the lowest potential energy, comprised of 75% small microspheres. Taking both charge and size effects into account, the storage moduli (G') of the almost fully elastic hydrogels could be tailored from 400 to 30,000Pa. Injectability tests showed that hydrogels (G' up to 4000Pa) composed of equal amounts of oppositely charged microspheres (-7 and +6mV, average particle size 7microm) could be injected through 25G needles using a static load of 15N, an ISO accepted value. In conclusion, a variety of options to control the network properties of macroscopic hydrogels are provided, related to the charge and particle size of the composing dextran microspheres. Furthermore, it is shown that the hydrogels are injectable, making them attractive candidates for a diversity of pharmaceutical applications.