A Phosphoramidate Prodrug Platform: One-Pot Amine Functionalization of Kinase Inhibitors with Oligoethylene Glycol for Improved Water-Solubility.

Small molecular kinase inhibitors play a key role in modern cancer therapy. Protein kinases are essential mediators in the growth and progression of cancerous tumors, rendering involved kinases an increasingly important target for therapy. However, kinase inhibitors are almost insoluble in water because of their hydrophobic aromatic nature, often lowering their availability and pharmacological efficacy. Direct drug functionalization with polar groups represents a simple strategy to improve the drug solubility, availability, and performance. Here, we present a strategy to functionalize secondary amines with oligoethylene glycol (OEG) phosphate using a one-step synthesis in three exemplary kinase inhibiting drugs Ceritinib, Crizotinib, and Palbociclib. These OEG-prodrug conjugates demonstrate superior solubility in water compared to the native drugs, with the solubility increasing up to 190-fold. The kinase inhibition potential is only slightly decreased for the conjugates compared to the native drugs. We further show pH dependent hydrolysis of the OEG-prodrugs which releases the native drug. We observe a slow release at pH 3, while the conjugates remain stable over 96 h under physiological conditions (pH 7.4). Using confocal microscopy, we verify improved cell uptake of the drug-OEG conjugates into the cytoplasm of HeLa cells, further supporting our universal solubility approach.

Microgels as Platforms for Antibody-Mediated Cytokine Scavenging.

Therapeutic antibodies are the key treatment option for various cytokine-mediated diseases, such as rheumatoid arthritis, psoriasis, and inflammatory bowel disease. However, systemic injection of these antibodies can cause side effects and suppress the immune system. Moreover, clearance of therapeutic antibodies from the blood is limiting their efficacy. Here, water-swollen microgels are produced with a size of 25 µm using droplet-based microfluidics. The microgels are functionalized with TNFα antibodies to locally scavenge the pro-inflammatory cytokine TNFα. Homogeneous distribution of TNFα-antibodies is shown throughout the microgel network and demonstrates specific antibody-antigen binding using confocal microscopy and FLIM-FRET measurements. Due to the large internal accessibility of the microgel network, its capacity to bind TNFα is extremely high. At a TNFα concentration of 2.5 µg mL-1 , the microgels are able to scavenge 88% of the cytokine. Cell culture experiments reveal the therapeutic potential of these microgels by protecting HT29 colorectal adenocarcinoma cells from TNFα toxicity and resulting in a significant reduction of COX II and IL8 production of the cells. When the microgels are incubated with stimulated human macrophages, to mimic the in vivo situation of inflammatory bowel disease, the microgels scavenge almost all TNFα that is produced by the cells.

Croconaine-Based Polymer Particles as Contrast Agents for Photoacoustic Imaging.

In the development and optimization of imaging methods, photoacoustic imaging (PAI) has become a powerful tool for preclinical biomedical diagnosis and detection of cancer. PAI probes can improve contrast and help identify pathogenic tissue. Such contrast agents must meet several requirements: they need to be biocompatible, and absorb strongly in the near-infrared (NIR) range, while relaxing the photoexcited state thermally and not radiatively. In this work, polymer nanoparticles are produced with croconaine as a monomer unit. Small molecular croconaine dyes are known to act as efficient pigments, which do not show photoluminescence. Here, for the first time croconaine copolymer nanoparticles are produced from croconic acid and a range of aromatic diamines. Following a dispersion polymerization protocol, this approach yields monodisperse particles of adjustable size. All synthesized polymers exhibit broad absorption within the NIR spectrum and therefore represent suitable candidates as contrast agents for PAI. The optical properties of these polymer particles are discussed with respect to the relation between particle size and outstanding photoacoustic performance. Biocompatibility of the polymer particles is demonstrated in cell viability experiments.

Microgels Sopping Up Toxins-GM1a-Functionalized Microgels as Scavengers for Cholera Toxin.

Vibrio cholerae is a Gram-negative bacterium that causes secretory diarrhea and constitutes a major health threat in the industrialized world and even more in developing countries. Its main virulence factor is the cholera toxin, which is internalized by intestinal epithelial cells after binding to the glycosphingolipid receptor GM1a on their apical surface. A potential future solution to dampen complications of cholera infection is by scavenging the cholera toxin by presenting competitive binding motifs to diminish the in vivo toxicity of V. cholerae. Here, we generate GM1a-functionalized and biocompatible microgels with diameters of 20 µm using drop-based microfluidics. The microgels are designed to exhibit a mesoporous and widely meshed network structure, allowing diffusion of the toxin protein deep into the microgel scavengers. Flow cytometry demonstrates strong and multivalent binding at high capacity of these microgels to the binding domain of the cholera toxin. Cell culture-based assays reveal the ability of these microgels to scavenge and retain the cholera toxin in direct binding competition to colorectal cells. This ability is evidenced by suppressed cyclic adenosine monophosphate production as well as reduced vacuole formation in mucus-forming colorectal HT-29 cells. Therefore, glycan-functionalized microgels show great potential as a non-antibiotic treatment for toxin-mediated infectious disorders.

Cell Encapsulation in Soft, Anisometric Poly(ethylene) Glycol Microgels Using a Novel Radical-Free Microfluidic System.

Complex 3D artificial tissue constructs are extensively investigated for tissue regeneration. Frequently, materials and cells are delivered separately without benefitting from the synergistic effect of combined administration. Cell delivery inside a material construct provides the cells with a supportive environment by presenting biochemical, mechanical, and structural signals to direct cell behavior. Conversely, the cell/material interaction is poorly understood at the micron scale and new systems are required to investigate the effect of micron-scale features on cell functionality. Consequently, cells are encapsulated in microgels to avoid diffusion limitations of nutrients and waste and facilitate analysis techniques of single or collective cells. However, up to now, the production of soft cell-loaded microgels by microfluidics is limited to spherical microgels. Here, a novel method is presented to produce monodisperse, anisometric poly(ethylene) glycol microgels to study cells inside an anisometric architecture. These microgels can potentially direct cell growth and can be injected as rod-shaped mini-tissues that further assemble into organized macroscopic and macroporous structures post-injection. Their aspect ratios are adjusted with flow parameters, while mechanical and biochemical properties are altered by modifying the precursors. Encapsulated primary fibroblasts are viable and spread and migrate across the 3D microgel structure.

Bio-degradable highly fluorescent conjugated polymer nanoparticles for bio-medical imaging applications.

Conjugated polymer nanoparticles exhibit strong fluorescence and have been applied for biological fluorescence imaging in cell culture and in small animals. However, conjugated polymer particles are hydrophobic and often chemically inert materials with diameters ranging from below 50 nm to several microns. As such, conjugated polymer nanoparticles cannot be excreted through the renal system. This drawback has prevented their application for clinical bio-medical imaging. Here, we present fully conjugated polymer nanoparticles based on imidazole units. These nanoparticles can be bio-degraded by activated macrophages. Reactive oxygen species induce scission of the conjugated polymer backbone at the imidazole unit, leading to complete decomposition of the particles into soluble low molecular weight fragments. Furthermore, the nanoparticles can be surface functionalized for directed targeting. The approach opens a wide range of opportunities for conjugated polymer particles in the fields of medical imaging, drug-delivery, and theranostics.Conjugated polymer nanoparticles have been applied for biological fluorescence imaging in cell culture and in small animals, but cannot readily be excreted through the renal system. Here the authors show fully conjugated polymer nanoparticles based on imidazole units that can be bio-degraded by activated macrophages.

PBCA-based polymeric microbubbles for molecular imaging and drug delivery.

Microbubbles (MB) are routinely used as contrast agents for ultrasound (US) imaging. We describe different types of targeted and drug-loaded poly(n-butyl cyanoacrylate) (PBCA) MB, and demonstrate their suitability for multiple biomedical applications, including molecular US imaging and US-mediated drug delivery. Molecular imaging of angiogenic tumor blood vessels and inflamed atherosclerotic endothelium is performed by modifying the surface of PBCA MB with peptides and antibodies recognizing E-selectin and VCAM-1. Stable and inertial cavitation of PBCA MB enables sonoporation and permeabilization of blood vessels in tumors and in the brain, which can be employed for direct and indirect drug delivery. Direct drug delivery is based on US-induced release of (model) drug molecules from the MB shell. Indirect drug delivery refers to US- and MB-mediated enhancement of extravasation and penetration of co-administered drugs and drug delivery systems. These findings are in line with recently reported pioneering proof-of-principle studies showing the usefulness of (phospholipid) MB for molecular US imaging and sonoporation-enhanced drug delivery in patients. They aim to exemplify the potential and the broad applicability of combining MB with US to improve disease diagnosis and therapy.

Efficient gas-liquid contact using microfluidic membrane devices with staggered herringbone mixers.

We describe a novel membrane based gas-liquid-contacting device with increased mass transport and reduced pressure loss by combining a membrane with a staggered herringbone static mixer. Herringbone structures are imposed on the microfluidic channel geometry via soft lithography, acting as mixers which introduce secondary flows at the membrane interface. Such flows include Dean vortices and Taylor flows generating effective mixing while improving mass transport and preventing concentration polarization in microfluidic channels. Furthermore, our static herringbone mixer membranes effectively reduce pressure losses leading to devices with enhanced transfer properties for microfluidic gas-liquid contact. We investigate the red blood cell distribution to tailor our devices towards miniaturised extracorporeal membrane oxygenation and improved comfort of patients with lung insufficiencies.