Defined pH-sensitive nanogels as gene delivery platform for siRNA mediated in vitro gene silencing.

In the present study, a pH sensitive nanogel platform for gene delivery was developed. The cationic nanogels based on dendritic polyglycerol (dPG) and low molecular weight polyethylenimine units were able to encapsulate siRNA during the manufacturing process. The thiol-Michael nanoprecipitation method, which operates under mild conditions and did not require any catalyst or surfactant, was used to develop tailor-made nanogels in the sub-100 nm range. The incorporation of pH sensitive benzacetal-bonds inside the nanogel network enables the controlled intracellular release of the cargo. The functionality to transport therapeutic biomolecules was tested by an in vitro GFP-siRNA transfection assay. Encapsulated siRNA could silence GFP expressing HeLa cells (up to 71% silencing in GFP). Furthermore, significantly reduced toxicity of the nanogel platform compared to the non-degradable PEI was observed. These properties realize a new carrier platform in the field of gene therapy.

Synthesis and Validation of Functional Nanogels as pH-Sensors in the Hair Follicle.

In the present study, a pH responsive dendritic polyglycerol nanogel (dPG-NG) is developed to measure the pH values inside the hair follicle (HF) using an ex vivo porcine ear model. The macromolecular precursors are labeled with a pH sensitive indodicarbocyanine dye (pH-IDCC) and a control dye (indocarbocyanine dye: ICC) and crosslinked via a mild and surfactant-free Thiol-Michael reaction using an inverse nanoprecipitation method. With this method, it is possible to prepare tailor-made particles in the range of 100 nm to 1 µm with a narrow polydispersity. The dPG-NGs are characterized using dynamic light scattering, nanoparticle tracking analysis, and atomic force microscopy. Systematic analysis of confocal microscope images of histological sections of the skin enables accurate determination of the pH gradient inside the HF. The results show that these novel pH-nanosensors deeply penetrate the skin via the follicular pathway and the pH of the pig hair follicles increase from 6.5 at the surface of the skin to 7.4 in deeper areas of the HF. The pH-nanosensor shows no toxicity potentials.

Synthesis of pH-Cleavable dPG-Amines for Gene Delivery Application.

The development of effective nonviral vectors for gene therapy is still a challenge in research, due to the high toxicity of many existing polycationic nanocarriers. In this paper, the development of two pH-cleavable polyglycerol-amine-based nanocarriers is described. The benz-acetal bond represents the pH-sensitive cleavage site between dendritic polyglycerol (dPG) and glycerol-based 1,2-diamines that can complex genetic material. Due to the acid lability of the acetal moiety, the cleavable dPG-amines are less toxic in vitro. Cell-mediated degradation results in non-toxic dPG with low amine functionalization and low molecular weight cleavage products (cp). The genetic material is released because of the loss of multivalent amine groups. Interestingly, the release kinetics at the endosomal pH could be controlled by simple chemical modification of the acetals. In vitro experiments demonstrate the ability of the cleavable dPG-amine to transfect HeLa cells with GFP-DNA, which resulted in cell-compatible cleavage products.

Hybrid Microgels with Thermo-Tunable Elasticity for Controllable Cell Confinement.

Stimuli-responsive hydrogels are able to change their physical properties such as their elastic moduli in response to changes in their environment. If biocompatible polymers are used to prepare such materials and if living cells are encapsulated within these networks, their switchability allows the cell-matrix interactions to be investigated with unprecedented consistency. In this paper, thermo-responsive macro- and microscopic hydrogels are presented based on azide-functionalized copolymers of poly(N-(2-hydroxypropyl)-methacrylamide) and poly(hydroxyethyl methacrylate) grafted with poly(N-isopropylacrylamide) side chains. Crosslinking of these comb polymers is realized by bio-orthogonal strain-promoted azide-alkyne cycloaddition with cyclooctyne-functionalized poly(ethylene glycol). The resulting hybrid hydrogels exhibit thermo-tunable elasticity tailored by the polymer chain length and grafting density. This bio-orthogonal polymer crosslinking strategy is combined with droplet-based microfluidics to encapsulate living cells into stimuli-responsive microgels, proving them to be a suitable platform for future systematic stem-cell research.

Visualization of real-time degradation of pH-responsive polyglycerol nanogels via atomic force microscopy.

Polyglycerol nanogels (nPG) have a huge impact in biomedical applications as drug deliverer due to their high biocompability. For such nPG nanogels, particle degradation is widely used as drug delivery method. The knowledge of this degradation process is limited up to date. In this communication, a real time visualization of such a degradation process is presented for pH-responsive nPG nanogels via atomic force microscopy (AFM) under ambient and in liquid conditions. The particle height plays a major role in the degradation process and decays exponentially in the beginning of this process. The particle width increases during the process indicating a "decross-linking" step of the particles into their starting monomers. Measurements under ambient conditions confirm this assumption and provide further insight in the "decross-linking" step of the nanogels into individual dendritic particles. The present work gives a detailed insight in the particle degradation process, which is essential for further progress for the development of new drug delivery systems.

The effect of polyglycerol sulfate branching on inflammatory processes.

In this study, the extent to which the scaffold architecture of polyglycerol sulfates affects inflammatory processes and hemocompatibility is investigated. Competitive L-selectin binding assays, cellular uptake studies, and blood compatibility readouts are done to evaluate distinct biological properties. Fully glycerol based hyperbranched polyglycerol architectures are obtained by either homopolymerization of glycidol (60% branching) or a new copolymerization strategy of glycidol with ethoxyethyl glycidyl ether. Two polyglycerols with 24 and 42% degree of branching (DB) are synthesized by using different monomer feed ratios. A perfectly branched polyglycerol dendrimer is synthesized according to an iterative two-step protocol based on allylation of the alcohol and subsequent catalytic dihydroxylation. All the polyglycerol sulfates are synthesized with a comparable molecular weight and degree of sulfation. The DB make the different polymer conjugates perform different ways. The optimal DB is 60% in all biological assays.

A facile approach for dual-responsive prodrug nanogels based on dendritic polyglycerols with minimal leaching.

A novel pH and redox dual-responsive prodrug nanogel was prepared by an inverse nanoprecipitation method, which is mild and surfactant free, and based on a thiol-disulfide exchange reaction and thiol-Michael addition reaction. Highly biocompatible hyperbranched polyglycerol (hPG) was cross-linked with disulfide bonds, to obtain biodegradable nanogels, which could be degraded under intracellular reductive conditions. Doxorubicin (DOX) was conjugated to the biodegradable nanogel matrix via an acid-labile hydrazone linker. This is the first dual-responsive prodrug nanogel system that shows very low unspecific drug leaching, but efficient intracellular release of the payload triggered by the intracellular conditions. Two different prodrug nanogels were prepared with a size of approximately 150nm, which is big enough to take the advantage of the enhanced permeation and retention (EPR) effect in tumor tissue. Cell culture analysis by microscopy and flow cytometry revealed that the prodrug nanogels were efficiently internalized by tumor cells. Distinct release profiles of DOX were achieved by adjusting the nanogel architecture, and online detection of cytotoxicity showed that, unlike free DOX, the dual-responsive prodrug nanogels exhibited a delay in the onset of toxicity, indicating the different uptake mechanism and the need for prodrug activation to induce cell death.

A microgel construction kit for bioorthogonal encapsulation and pH-controlled release of living cells.

pH-Cleavable cell-laden microgels with excellent long-term viabilities were fabricated by combining bioorthogonal strain-promoted azide-alkyne cycloaddition (SPAAC) and droplet-based microfluidics. Poly(ethylene glycol)dicyclooctyne and dendritic poly(glycerol azide) served as bioinert hydrogel precursors. Azide conjugation was performed using different substituted acid-labile benzacetal linkers that allowed precise control of the microgel degradation kinetics in the interesting pH range between 4.5 and 7.4. By this means, a pH-controlled release of the encapsulated cells was achieved upon demand with no effect on cell viability and spreading. As a result, the microgel particles can be used for temporary cell encapsulation, allowing the cells to be studied and manipulated during the encapsulation and then be isolated and harvested by decomposition of the microgel scaffolds.

Surfactant free preparation of biodegradable dendritic polyglycerol nanogels by inverse nanoprecipitation for encapsulation and release of pharmaceutical biomacromolecules.

In this paper we report a novel approach to generate biodegradable polyglycerol nanogels on different length scales. We developed a mild, surfactant free inverse nanoprecipitation process to template hydrophilic polyglycerol nanoparticles. In situ crosslinking of the precipitated nanoparticles by bioorthogonal copper catalyzed click chemistry allows us to obtain size defined polyglycerol nanogels (100-1000nm). Biodegradability was achieved by the introduction of benzacetal bonds into the net points of the nanogel. Interestingly, the polyglycerol nanogels quickly degraded into low molecular weight fragments at acidic pH values, which are present in inflamed and tumor tissues as well as intracellular organelles, and they remained stable at physiological pH values for a long time. This mild approach to biodegradable polyglycerol nanogels allows us to encapsulate labile biomacromolecules such as proteins, including the therapeutic relevant enzyme asparaginase, into the protein resistant polyglycerol network. Enzymes were encapsulated with an efficacy of 100% and after drug release, full enzyme activity and structural integrity were retained. This new inverse nanoprecipitation procedure allows the efficient encapsulation and release of various biomacromolecules including proteins and could find many applications in polymer therapeutics and nanomedicine.

Multivalent, biodegradable polyglycerol hydrogels.

In this paper we describe the preparation of disulfide crosslinked polyglycerol hydrogels by the ring-opening crosslinking polymerization of glycerol and polyethylene glycol-based polyepoxides and Na2S2. Multivalent polyglycerol hydrogels were prepared by acid-catalyzed hydrolysis of remaining epoxide functionalities. Additionally, a near infrared fluorescent dye was encapsulated in the hydrogel network. Our hydrogels show complete degradation in reducing environments and a controlled release of the fluorescence dye was observed. These hydrogels are interesting scaffolds for bioactive substances, which can be released, triggered by the hydrogel degradation.

Hyperbranched polyglycerols on the nanometer and micrometer scale.

We report the preparation of polyglycerol particles on different length scales by extending the size of hyperbranched polyglycerols (3 nm) to nanogels (32 nm) and microgels (140 and 220 µm). We use miniemulsion templating for the preparation of nanogels and microfluidic templating for the preparation of microgels, which we obtain through a free-radical polymerization of hyperbranched polyglycerol decaacrylate and polyethylene glycol-diacrylate. The use of mild polymerization conditions allows yeast cells to be encapsulated into the resultant microgels with cell viabilities of approximately 30%.