PH- and ligand-induced release of loads from DNA-acrylamide hydrogel microcapsules.
ABSTRACT: Herein, a method to construct stimuli-responsive DNA-acrylamide-based hydrogel microcapsules has been presented. This method involves the use of polyacrylamide chains modified with predesigned nucleic acid hairpin units and optionally single-strand tethers that provide the required hybridization and recognition functions to yield substrate-loaded stimuli-responsive hydrogel-based microcapsules. The synthesis of the microcapsules involves the loading of CaCO3 microparticles with the respective load substrates and the functionalization of the CaCO3 template particles with nucleic acid promoter units. In the presence of the hairpin-modified acrylamide chains, the promoter units induce the hybridization chain reaction (HCR), which leads to the formation of a hydrogel coating, which, after the dissociation of the CaCO3 cores, yields substrate-loaded stimuli-responsive hydrogel microcapsules. One of the microcapsule systems includes, in the hairpin-modified acrylamide constructs, and in the subsequent HCR-generated hydrogel shells, the caged sequences of anti-ATP or anti-cocaine aptamers. In the presence of ATP or cocaine, the duplex-caged aptamer sequences are separated via the formation of ATP- or cocaine-aptamer complexes, which results in the partial separation of the microcapsules and the release of the loads. The second type of microcapsule is cooperatively stabilized by bridges generated by HCR and pH-sensitive duplex units. Under acidic conditions, the pH-sensitive bridges dissociate via the formation of i-motif structures, which results in an increase in the fluidity of the microcapsule shells and the release of the loads. Preliminary studies indicate that ATP- or pH-responsive microcapsules loaded with the anticancer drug, doxorubicin, have a selective cytotoxic effect on MDA-MB-231 cancer cells.
Project description:A method to assemble stimuli-responsive nucleic acid-based hydrogel-stabilized microcapsule-in-microcapsule systems is introduced. An inner aqueous compartment stabilized by a stimuli-responsive hydrogel-layer (?150 nm) provides the inner microcapsule (diameter ?2.5 ?m). The inner microcapsule is separated from an outer aqueous compartment stabilized by an outer stimuli-responsive hydrogel layer (thickness of ?150 nm) that yields the microcapsule-in-microcapsule system. Different loads, e.g., tetramethyl rhodamine-dextran (TMR-D) and CdSe/ZnS quantum dots (QDs), are loaded in the inner and outer aqueous compartments. The hydrogel layers exist in a higher stiffness state that prevents inter-reservoir or leakage of the loads from the respective aqueous compartments. Subjecting the inner hydrogel layer to Zn2+-ions and/or the outer hydrogel layer to acidic pH or crown ether leads to the triggered separation of the bridging units associated with the respective hydrogel layers. This results in the hydrogel layers of lower stiffness allowing either the mixing of the loads occupying the two aqueous compartments, the guided release of the load from the outer aqueous compartment, or the release of the loads from the two aqueous compartments. In addition, a pH-responsive microcapsule-in-microcapsule system is loaded with glucose oxidase (GOx) in the inner aqueous compartment and insulin in the outer aqueous compartment. Glucose permeates across the two hydrogel layers resulting in the GOx catalyzed aerobic oxidation of glucose to gluconic acid. The acidification of the microcapsule-in-microcapsule system leads to the triggered unlocking of the outer, pH-responsive hydrogel layer and to the release of insulin. The pH-stimulated release of insulin is controlled by the concentration of glucose. While at normal glucose levels, the release of insulin is practically prohibited, the dose-controlled release of insulin in the entire diabetic range is demonstrated. Also, switchable ON/OFF release of insulin is achieved highlighting an autonomous glucose-responsive microdevice operating as an "artificial pancreas" for the release of insulin.
Project description:Nano-in-micro (NIM) system is a promising approach to enhance the performance of devices for a wide range of applications in disease treatment and tissue regeneration. In this study, polymeric nanofibre-integrated alginate (PNA) hydrogel microcapsules were designed using NIM technology. Various ratios of cryo-ground poly (lactide-co-glycolide) (PLGA) nanofibres (CPN) were incorporated into PNA hydrogel microcapsule. Electrostatic encapsulation method was used to incorporate living cells into the PNA microcapsules (~500 µm diameter). Human liver carcinoma cells, HepG2, were encapsulated into the microcapsules and their physio-chemical properties were studied. Morphology, stability, and chemical composition of the PNA microcapsules were analysed by light microscopy, fluorescent microscopy, scanning electron microscopy (SEM), Fourier-Transform Infrared spectroscopy (FTIR), and thermogravimetric analysis (TGA). The incorporation of CPN caused no significant changes in the morphology, size, and chemical structure of PNA microcapsules in cell culture media. Among four PNA microcapsule products (PNA-0, PNA-10, PNA-30, and PNA-50 with size 489 ± 31 µm, 480 ± 40 µm, 473 ± 51 µm and 464 ± 35 µm, respectively), PNA-10 showed overall suitability for HepG2 growth with high cellular metabolic activity, indicating that the 3D PNA-10 microcapsule could be suitable to maintain better vitality and liver-specific metabolic functions. Overall, this novel design of PNA microcapsule and the one-step method of cell encapsulation can be a versatile 3D NIM system for spontaneous generation of organoids with in vivo like tissue architectures, and the system can be useful for numerous biomedical applications, especially for liver tissue engineering, cell preservation, and drug toxicity study.
Project description:New methods for the preparation of reversible pH-responsive DNA hydrogels based on Hoogsteen triplex structures are described. One system consists of a hydrogel composed of duplex DNA units that bridge acrylamide chains at pH = 7.4 and undergoes dissolution at pH = 5.0 through the reconfiguration of one of the duplex bridging units into a protonated CG·C+ triplex structure. The second system consists of a hydrogel consisting of acrylamide chains crosslinked in the presence of an auxiliary strand by Hoogsteen TA·T triplex interaction at pH = 7.0. The hydrogel transforms into a liquid phase at pH = 10.0 due to the separation of the triplex bridging units. The two hydrogel systems undergo reversible and cyclic hydrogel/solution transitions by subjecting the systems to appropriate pH values. The anti-cancer drug, coralyne, binds specifically to the TA·T triplex-crosslinked hydrogel thereby increasing its stiffness. The pH-controlled release of the coralyne from the hydrogel is demonstrated.
Project description:BACKGROUND:Miniaturization of biochemical reaction volumes within artificial microcompartments has been the key driver for directed evolution of several catalysts in the past two decades. Typically, single cells are co-compartmentalized within water-in-oil emulsion droplets with a fluorogenic substrate whose conversion allows identification of catalysts with improved performance. However, emulsion droplet-based technologies prevent cell proliferation to high density and preclude the feasibility of biochemical reactions that require the exchange of small molecule substrates. Here, we report on the development of a high-throughput screening method that addresses these shortcomings and that relies on a novel selective permeable polymer hydrogel microcapsule. RESULTS:Hollow-core polyelectrolyte-coated chitosan alginate microcapsules (HC-PCAMs) with selective permeability were successfully constructed by jet break-up and layer-by-layer (LBL) technology. We showed that HC-PCAMs serve as miniaturized vessels for single cell encapsulation, enabling cell growth to high density and cell lysis to generate monoclonal cell lysate compartments suitable for high-throughput analysis using a large particle sorter (COPAS). The feasibility of using HC-PCAMs as reaction compartments which exchange small molecule substrates was demonstrated using the transpeptidation reaction catalyzed by the bond-forming enzyme sortase F from P. acnes. The polyelectrolyte shell surrounding microcapsules allowed a fluorescently labelled peptide substrate to enter the microcapsule and take part in the transpeptidation reaction catalyzed by the intracellularly expressed sortase enzyme retained within the capsule upon cell lysis. The specific retention of fluorescent transpeptidation products inside microcapsules enabled the sortase activity to be linked with a fluorescent readout and allowed clear separation of microcapsules expressing the wild type SrtF from those expressing the inactive variant. CONCLUSION:A novel polymer hydrogel microcapsule-based method, which allows for high-throughput analysis based on encapsulation of single cells has been developed. The method has been validated for the transpeptidation activity of sortase enzymes and represents a powerful tool for screening of libraries of sortases, other bond-forming enzymes, as well as of binding affinities in directed evolution experiments. Moreover, selective permeable microcapsules encapsulating microcolonies provide a new and efficient means for preparing novel caged biocatalyst and biosensor agents.
Project description:Cell-laden hydrogel microcapsules enable the high-throughput production of cell aggregates, which are relevant for three-dimensional tissue engineering and drug screening applications. However, current microcapsule production strategies are limited by their throughput, multistep protocols, and limited amount of compatible biomaterials. We here present a single-step process for the controlled microfluidic production of single-core microcapsules using enzymatic outside-in cross-linking of tyramine-conjugated polymers. It was hypothesized that a physically, instead of the conventionally explored biochemically, controlled enzymatic cross-linking process would improve the reproducibility, operational window, and throughput of shell formation. Droplets were flown through a silicone delay line, which allowed for highly controlled diffusion of the enzymatic cross-linking initiator. The microcapsules' cross-linking density and shell thickness is strictly depended on the droplet's retention time in the delay line, which is predictably controlled by flow rate. The here presented hydrogel cross-linking method allows for facile and cytocompatible production of cell-laden microcapsules compatible with the formation and biorthogonal isolation of long-term viable cellular spheroids for tissue engineering and drug screening applications.
Project description:A novel coaxial electrospray technology is developed to generate microcapsules with a hydrogel shell of alginate and an aqueous liquid core of living cells using two aqueous fluids in one step. Approximately 50 murine embryonic stem (ES) cells encapsulated in the core with high viability (92.3 ± 2.9%) can proliferate to form a single ES cell aggregate of 128.9 ± 17.4 ?m in each microcapsule within 7 days. Quantitative analyses of gene and protein expression indicate that ES cells cultured in the miniaturized 3D liquid core of the core-shell microcapsules have significantly higher pluripotency on average than the cells cultured on the 2D substrate or in the conventional 3D alginate hydrogel microbeads without a core-shell architecture. The higher pluripotency is further suggested by their significantly higher capability of differentiation into beating cardiomyocytes and higher expression of cardiomyocyte specific gene markers on average after directed differentiation under the same conditions. Considering its wide availability, easiness to set up and operate, reusability, and high production rate, the novel coaxial electrospray technology together with the microcapsule system is of importance for mass production of ES cells with high pluripotency to facilitate translation of the emerging pluripotent stem cell-based regenerative medicine into the clinic.
Project description:Imaging of implanted hydrogel-based biosystems usually requires indirect labeling of the vehicle or cargo, adding complexity and potential risk of altering functionality. Here, for the first time, it is reported that incorporation of genipin into the design of immunoisolation devices can be harnessed for in vivo imaging. Using cell-compatible in situ cross-linking reactions, a fast, efficient and noncytotoxic procedure is shown to maximize fluorescence of microcapsules. Moreover, genipin is validated as a quantitative imaging probe by injecting increasing doses of microcapsules in the subcutaneous space of mice, obtaining strong, stable fluorescence with good linearity of signal to microcapsule dose over several weeks. This allows immediate assessment of the actual injected dose and monitoring of its position over time, thereby significantly enhancing the efficacy and biosafety of the therapy. These outcomes may facilitate clinical translation and optimize medical applications of multiple hydrogel-based biotechnologies.
Project description:The mechanically-enhanced urea-formaldehyde (UF) microcapsules are developed through a multi-step in situ polymerization method. Optical microscope (OM) and field emission scanning electron microscope (FE-SEM) prove that the microcapsules, 147.4 ?m in diameter with a shell thickness of 600 nm, are well-formed. From 1H-nuclear magnetic resonance (1H-NMR) analysis, we found that dicyclopentadiene (DCPD), a self-healing agent encapsulated by the microcapsules, occupies ca. 40.3 %(v/v) of the internal volume of a single capsule. These microcapsules are mixed with EPDM (ethylene-propylene-diene-monomer) and Grubbs' catalyst via a solution mixing method, and universal testing machine (UTM) tests show that the composites with mechanically-enhanced microcapsules has ca. 47% higher toughness than the composites with conventionally prepared UF microcapsules, which is attributed to the improved mechanical stability of the microcapsule. When the EPDM/microcapsule rubber composites are notched, Fourier-transform infrared (FT-IR) spectroscopy shows that DCPD leaks from the broken microcapsule to the damaged site and flows to fill the notched valley, and self-heals as it is cured by Grubbs' catalyst. The self-healing efficiency depends on the capsule concentration in the EPDM matrix. However, the self-healed EPDM/microcapsule rubber composite with over 15 wt% microcapsule shows an almost full recovery of the mechanical strength and 100% healing efficiency.
Project description:Microencapsulation offers a unique potential for high cell density, high productivity mammalian cell cultures. However, for successful exploitation there is the need for microcapsules of defined size, properties and mechanical stability. Four types of alginate/poly-L: -Lysine microcapsules, containing recombinant CHO cells, have been investigated: (a) 800 mum liquid core microcapsules, (b) 500 mum liquid core microcapsules, (c) 880 mum liquid core microcapsules with a double PLL membrane and (d) 740 mum semi-liquid core microcapsules. With encapsulated cells a reduced growth rate was observed, however this was accompanied by a 2-3 fold higher specific production rate of the recombinant protein. Interestingly, the maximal intracapsular cell concentration was only 8.7 x 10(7) cell mL(-1), corresponding to a colonization of 20% of the microcapsule volume. The low level of colonization is unlikely to be due to diffusional limitations since reduction of microcapsule size had no effect. Measurement of cell leaching and mechanical properties showed that liquid core microcapsules are not suitable for continuous long-term cultures (>1 month). By contrast semi-liquid core microcapsules were stable over long periods with a constant level of cell colonization (varphi = 3%). This indicates that the alginate in the core plays a predominant role in determining the level of microcapsule colonization. This was confirmed by experiments showing reduced growth rates of batch suspension cultures of CHO cells in medium containing dissolved alginate. Removal of this alginate would therefore be expected to increase microcapsule colonization.
Project description:A novel core-shell microcapsule system is developed in this study to mimic the miniaturized 3D architecture of pre-hatching embryos with an aqueous liquid-like core of embryonic cells and a hydrogel-shell of zona pellucida. This is done by microfabricating a non-planar microfluidic flow-focusing device that enables one-step generation of microcapsules with an alginate hydrogel shell and an aqueous liquid core of cells from two aqueous fluids. Mouse embryonic stem (ES) cells encapsulated in the liquid core are found to survive well (>92%). Moreover, ~20 ES cells in the core can proliferate to form a single ES cell aggregate in each microcapsule within 7 days while at least a few hundred cells are usually needed by the commonly used hanging-drop method to form an embryoid body (EB) in each hanging drop. Quantitative RT-PCR analyses show significantly higher expression of pluripotency marker genes in the 3D aggregated ES cells compared to the cells under 2D culture. The aggregated ES cells can be efficiently differentiated into beating cardiomyocytes using a small molecule (cardiogenol C) without complex combination of multiple growth factors. Taken together, the novel 3D microfluidic and pre-hatching embryo-like microcapsule systems are of importance to facilitate in vitro culture of pluripotent stem cells for their ever-increasing use in modern cell-based medicine.