Triggered Release of Loads from Microcapsule-in-Microcapsule Hydrogel Microcarriers: En-Route to an “Artificial Pancreas”

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 μm). 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.

100 μL of a solution consisting of 0.75 mM acrydite-modified oligonucleotides ((2) and H1 or (x) in a ratio of 2:1) and 1.5 % acrylamide was bubbled with nitrogen for 3 min, followed by the addition of 7.5 μL of initiator mixture (prepared by 10 mg APS in 5 μL TEMED and 95 μL H2O). The resulting solution was subjected to additional 5 min of nitrogen bubbling, followed by incubation at 4 °C for 12 h to form the copolymer chains P1 and P2. Polymer P1 ((2) and H1) was purified and separated from the unreacted compounds using a 30k MWCO Amicon filter, whereas for polymer P2 ( (2) and (x)) a 10k MWCO Amicon filter was used. After being washed with water three times, the copolymer solutions were dried under a gentle flow of nitrogen gas and redispersed in buffer (10 mM HEPES, pH 7.2, containing 25 mM MgCl2). To polymer P2, after the determination of the concentration of (x), hairpin H2 was added in a molar ratio of 1:1. The polymer solutions were incubated at 95 °C for 5 min, followed by incubation on ice for 30 min to ensure the efficient closing of the hairpins.
Synthesis of 5'-Amino Modifier C6-modified oligo/Carboxymethyl cellulose (CMC) copolymers 2 mL of a MES buffer solution (10 mM, pH 5.5), containing CMC, 20 mg, N-(3dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride, 20 mg, were incubated for 5 minutes and then sulfo-N-hydroxysuccinimide, 26 mg, was added and the solution was incubated for additional 10 minutes. To the resulting solution, 2 mL of HEPES buffer (50 mM, pH 7.2) containing the amine-functionalized nucleic acids (300 µM of H1 or (x) and 600 µM of (2)), were added. The mixture was gentle shacked for 2 h at room temperature. The modified polymers, P1 ((2) and H1) and P2 ( (2) and (x)), were purified and separated from the unreacted compounds using MWCO 10K Amicon spin filters. After being washed with water three times, the copolymer solutions were dried and re-dispersed in buffer (10 mM HEPES, pH 7.0, containing 25 mM MgCl2). To polymer P2, after the determination of the concentration of (x), hairpin H2 was added in a molar ratio of 1:1. The polymer solutions were incubated at 95 °C for 5 min, followed immediately by incubation on ice for 30 min to ensure the efficient closing of the hairpins.

Preparation of CaCO3 microparticles with different loads
CaCO3 particles were prepared by a precipitation reaction between equal amounts of CaCl2 and Na2CO3 under magnetic stirring at room temperature. CaCO3 particles loaded with TMR-D were obtained through coprecipitation by mixing CaCl2 (307 μL, 0.33 M) and Na2CO3 (307 μL, 0.33 M) solutions, in the presence of TMR-D (30 μL, 6.25 mg mL -1 ). The final volume was adjusted to 1020 μL by addition of deionized water. After magnetic stirring for 110s, the suspension was left for 70s at room temperature to settle down. The particles were centrifuged at 100 rcf for 20s, followed by the removal of the supernatant solution, and the subsequent resuspension of the particles in water. This washing procedure was repeated twice in order to remove the byproducts resulting from the precipitation reaction .

Synthesis of DNA-acrylamide/CMC hydrogel microcapsule-in-microcapsule
The CaCO3 microparticles were suspended in 600 μL of 1 mg mL -1 PAH solution (10 mM HEPES, pH 7.2, containing 25 mM MgCl2) and kept under continuous shaking for 30 min. The PAH-coated particles were washed twice with buffer (10 mM HEPES, pH 7.2, containing 25 mM MgCl2), followed by centrifugation at 100 rcf for 20 s. Subsequently, the PAH-coated microparticles were incubated with 600 μL of the promoter nucleic acid (1) (final concentration 10 μM) and kept under continuous shaking at room temperature for 30 min. After being washed twice with buffer (10 mM HEPES, pH 7.2, containing 25 mM MgCl2), followed by centrifugation at 100 rcf for 20 s, the DNA hydrogel particles were prepared by mixing the polymer sets (P1 and P2) and the nucleic acid sequences (3) and (4) (or only (3) for the microcapsule-inmicrocapsule with GOx or GOx and catalase) with the promoter-coated CaCO3 microparticles. The final concentration of each hairpin was 10 μM. The particles were incubated overnight (approximately 12h) at room temperature under continuous shaking, followed by centrifugation at 100 rcf for 20 s to remove non-adsorbed polymers and the subsequent resuspension in buffer (10 mM HEPES, pH 7.0, containing 25 mM MgCl2). This washing procedure was repeated twice.
In order to create the CaCO3 interlayer, the one-layer DNA-acrylamide/CMC hydrogel particles were suspended in buffer solution (37.5 μL, 10 mM HEPES, pH 7.2, containing 25 mM MgCl2) and 337.5 μL of water, and mixed with 307 µL of CaCl2 0.33M and 307 µL of Na2CO3 0.33M, in the presence of CdSe/ZnS QD (30 μL, 240 nM). After magnetic stirring for 110 s, the suspension was left for 70 s at room temperature to settle down. The particles were centrifuged at 100 rcf for 20 s, followed by the removal of the supernatant solution, and the subsequent resuspension of the particles in water. This washing procedure was repeated twice in order to remove byproducts resulting from the precipitation reaction. Then, the outer hydrogel layer was prepared using the procedure described above for the inner hydrogel layer, adding together with the set of polymers (P1 and P2) the nucleic acid sequences (5) and (6) for the i-motif system and (7) for the G-quadruplex system.
Similarly, the microcapsule-in-microcapsule system containing -GOx or GOx and catalase in the inner core and fluorophore-modified insulin (30 μL of 16 mM solution) in the outer core was prepared as described above excluding the Zn 2+ -ion-dependent DNAyzme (4) in the inner hydrogel layer and exchanging the dyes for the respective enzymes.
Dissolution of the Core 120 μL of a 0.1 M EDTA solution (pH 7.5) were added into 60 μL of microparticle solution containing 1700 microparticles/L and 60 μL of buffer solution (10 mM HEPES, pH 7.2, containing 25 mM MgCl2). The resulting solution was incubated for 1 h to dissolve the CaCO3 cores. When the suspension became clear, the capsules were washed with buffer (10 mM HEPES, pH 7.2, containing 25 mM MgCl2) using slow centrifugation (50 rcf, 20 min) three times.
For the switchable pH-stimulated release of CdSe/ZnS QDs from the outer layer, 120 μL of buffer (10 mM HEPES, pH 7.2, containing 25 mM MgCl2) containing 20 μL of DNA-acrylamide\CMC hydrogel bilayer responsive microcapsules-in-microcapsules (1700 microcapsule-in-microcapsules per µL) were added to a cuvette. After the microcapsule-in-microcapsule precipitated, 9 μL of HCl (1 M) were added to the solution and the release of the QDs was measured through fluorescence. After 10 min the buffer was exchanging by HEPES buffer (10 mM HEPES, pH 7.2, containing 25 mM MgCl2). This process was repeated 3 times.
For the Zn 2+ controlled release of TMR-D at pH 5.5 different samples consisting of 120 μL of buffer (10 mM HEPES, pH 7.2, containing 25 mM MgCl2), containing 20 μL of DNA-acrylamide\CMC hydrogel bilayer responsive microcapsules-in-microcapsules (1700 microcapsules-in-microcapsules per µL) were added to a cuvette. After the microcapsules-in-microcapsules precipitated, 9 μL of HCl (1 M) and different concentrations of ZnCl2 (final concentration; 0 mM, 10 mM, 20 mM,·30 mM) were added to each sample and the release of TMR-D was measured through fluorescence.
For the switchable K + -stimulated release of CdSe/ZnS QDs from the outer layer, 120 μL of buffer (10 mM HEPES, pH 7.2, containing 25 mM MgCl2) containing 20 μL of DNA-acrylamide\CMC hydrogel bilayer responsive microcapsules-in-microcapsules (1700 microcapsules-in-microcapsules per µL) were added to a cuvette. After the microcapsules-in-microcapsules precipitated, 9 μL of CE (1.5 M) were added to the solution and the release of the QDs was measured through fluorescence. After 10 min the buffer was replaced by new buffer containing 9 μL of KCl (1 M). This process was repeated 2 times.

Glucose regulated insulin release
Different samples consisting of 120 μL of buffer (10 mM HEPES, pH 7.2, containing 25 mM MgCl2) containing 20 μL of CMC hydrogel bilayer microcapsules-in-microcapsules (1700 microcapsules-in-microcapsules per µL) were added to a cuvette. After the microcapsules-in-microcapsules precipitated different concentration of glucose were added (final concentration; 0 mM, 5 mM, 10 mM, 15 mM) to each sample and the release of insulin was measured through fluorescence.
For the switchable release of insulin from the outer layer, 120 μL of buffer (10 mM HEPES, pH 7.2, containing 25 mM MgCl2) containing 20 μL of CMC hydrogel bilayer microcapsules-in-microcapsules (1700 microcapsules-in-microcapsules per µL) were added to a cuvette. After the microcapsules-in-microcapsules precipitated, 2.4 μL glucose (10 mM final concentration) were added to the solution and the release of insulin was measured through fluorescence. 50 min after reaching the release saturation value, 2.4 μL additional of glucose were added and the release of insulin was switched on again. This process was repeated 2 times (the same procedure was done for the switching with 7 mM glucose by the addition of 1.68 µL of 500mM stock solution. The switching was done for 3 cycles).
For the switchable release of insulin with washing step from the outer layer, 120 μL of buffer (10 mM HEPES, pH 7.2, containing 25 mM MgCl2) containing 20 μL of CMC hydrogel bilayer microcapsules-in-microcapsules (1700 microcapsules-inmicrocapsules per µL) were added to a cuvette. After the microcapsules-inmicrocapsules precipitated, 1.68 μL glucose (7 mM final concentration) were added to the solution and the release of insulin was measured through fluorescence. 35 minutes since the addition of 7 mM glucose, the upper solution was extract and buffer (10 mM HEPES, pH 7.2, containing 25 mM MgCl2) was added. This process was repeated 5 times.

Glucometer
Detection of the glucose level in the presence of GOx and catalase/insulin microcapsules-in-microcapsules.
100 L of microcapsules-in-microcapsule were added to 400 L of buffer (10 mM HEPES, pH 7.2, containing 25 mM MgCl2). The glucose concentration was measured after time intervals of 0 min, 2 min, 5 min, 10 min, 20 min, 30 min, 60 min and 100 min by a glucometer (Accutrend plus kit mg/dL with Accutrend Glucose II 25 STR strips (Roche)). Table S1. The sequences of nucleic acids used in this system.  Table S2 compares two reported "artificial pancreas" systems (that to the best of our knowledge represent relevant configurations to our microcapsules-in-microcapsules insulin carriers). It should be noted that several other reports discussed the insulin release or applied non-hydrogel polymers or particles as carriers, yet the glucose stimulated release of insulin from the carriers is irrelevant for controlling diabetes (abnormal high concentration of glucose).
Realizing that our microcapsule-in-microcapsule system includes 1700 microcapsules in 1 μL, and knowing the insulin loading per microcapsule we estimate that ca. 400 units of insulin are present in one milliliter of an aqueous HEPES buffer mixture, pH 7.2, of the microcapsules. Presuming that treatment of high glucose levels, 130-200 mg/dL, requires 5 units of insulin, the injection of 100 μL of the microcapsules will allow the autonomous control of the glucose levels for eight cycles. Saying that and assuming that high glucose levels raise twice a day, 100 μL of microcapsules could autonomously control the glucose levels for four days. Alternatively, if the injected volume is 200 μL, an autonomous control of the glucose levels for eight days could be achieved. If the deviation in glucose levels increases to four-times daily, the 200 μL microcapsule mixture could autonomously control the glucose levels for four days.
Needless to say, further optimization of the system could be accomplished by increasing the load of insulin as well as by the modification of the hydrogel coating comprising the capsules.   2) and (x)) (5AmMC6 modified) vs. the ratio of absorbance at 205 nm/260 nm. The absorbance of the modified polymer was measured and the ratio of CMC to nucleic acid strands was calculated to be 60:1.       2) and (x)) (5Acryd modified) vs. the ratio of absorbance at 205 nm/260 nm. The absorbance of the modified polymer was measured and the ratio of acrylamide units to nucleic acid strands was calculated to be 30:1.          Entry I-The CaCO3-loaded bi-compartment particle prior etching.
Entry II-The bi-compartmentalized microcapsule after etching with EDTA.
Colum (a)-Fluorescence image of the red channel (TMR-D).

Colum (b)-Fluorescence image of the green channel (CdSe/ZnS QDs).
Colum (c)-Fluorescence overlay of the red and green channels.
Colum (d)-Bright-field image of the structures.
Entry I-Reveals in (a) and (b) the distinct red fluorescence and green fluorescence of the TMR-D (red) and CdSe/ZnS QDs (green) loaded in the inner and outer compartments of the microparticles-in-microparticles before etching. In (c) the overlay of (a) and (b) shows distinct separated compartments of (a) and (b). Entry IIreveals separated red and green compartments corresponding to the bi-compartment microcapsule-in-microcapsule system after etching. The overlay (c) shows distinct separated compartments of the red/green fluorophores. The bright field image (d) shows the two-compartments microcapsules-in-microcapsules. Entry III-(a) and (b) show the fluorophore image of HCl (pH 5.5) treated microcapsules-in-microcapsules.
As the release of the CdSe/ZnS QDs from the outer layer increase, no green fluorescence is observed, and only the inner red fluorescence is detected. In (d) the bright field image confirms the bilayer structure of the microcapsule. In Entry IV-(a) and (b) shows the fluorescent image of the red/green channels upon treatment of the microcapsules with HCl (pH 5.5) and Zn 2+ -ions (20mM). No fluorescence is detected in the two channels or in the overlay channel (c). Nonetheless, the bright field image confirms the existence of the intact bilayer microcapsule-in-microcapsule structure. These results confirm that under these conditions the two loads, TMR-D (inner compartment) and CdSe/ZnS QDs (outer compartment) were release from the microcapsules.    The loading of the microcapsules with the different loads was evaluated by two methods: (i) The concentration of the loads on the microparticles-in-microparticles was evaluated spectroscopically by determination of the absorbance of the loads in solution after and before deposition on the CaCO3 microparticles-in-microparticles. This particles were used to assemble the microcapsules and the concentrations of the loads that were washed off upon the synthesis of the loaded microcapsules were added to the residual concentrations to yield a value of non-loaded substrates in the microcapsules. The difference between the initial added concentrations of the loads and the total-concentrations of non-capsules-associated loads was assumed to be the loading degree of each of the loads within the microcapsules. (ii) The release process of the loads TMR-D, QDs or the labeled insulin from the microcapsules were recorded by subjecting the microcapsules to pH 5.5 (30 minutes), Zn 2+ -ions, 30mM (90 minutes) and glucose, 50mM (90 minutes). The recorded release profile for all loads reached saturation. For example, Figure S23 demonstrates the release profile of the coumarinlabeled-insulin. Knowing the concentrations of the microcapsules in each of the systems and applying appropriate calibration curves, the concentrations of the respective loads at the saturation levels of the release profiles were evaluated. Assuming that, the saturation levels in the release profiles corresponds to the total concentrations of the respective load in the carrier, this method provide an additional tool to estimate the degree of loading of the respective substrates. Indeed, the evaluated loading derived by this method overlap with 90%-100% of the loading degrees evaluated by method (i).
For example, from the saturated fluorescence intensity of the coumarin-labeled-insulin and knowing the number of microcapsules we estimate the loading of insulin to be 1.3X10 -12 mole/ microcapsule. Similarly, the loading of TMR-D and QDs corresponds to 1.8X10 -15 and 3.7X10 -14 mole/microcapsule, respectively. Figure S24. Switchable release of insulin upon the treatment of the bilayer GOx/insulin loaded microcapsules-in-microcapsules with glucose, 7mM, followed by precipitation of the microcapsules, and their re-dispersion in a new buffer solution and switching on the release by re-added glucose, 7mM.   . Time-dependent fluorescence changes upon subjection the GOx and catalase/insulin bi-reservoir microcapsule-in-microcapsule system to glucose and analyze the GOx-generated H2O2 using the oxidation of amplex-red to the fluorescent resorufin as assay, curve (a). No fluorescence change is observed with the addition of catalase to the microcapsule-in-microcapsule system implying that the co-added catalase degraded any harmful peroxide. Time-dependent fluorescence change upon subjection the GOx/insulin bi-reservoir microcapsule-in-microcapsule system to glucose and analyze the GOx-generated H2O2 using the oxidation of amplexred/resorufin assay, curve (b).

Evaluation of the molecular-weight cutoff value for the permeation of loads across single layer hydrogel microcapsules prepared by the integration of different loads in the microcapsules generated in the presence of P1 and P2.
The microcapsules were loaded with a series of FAM-modified nucleic acids of variable molecular weights NA1-NA4 (see Table S3). The release of the cargoes was examined in the stiffer hydrogel coating states and in the DNAzyme triggered lower stiffness coating states.  The release process leads to the following conclusions: NA1-Cannot be trapped in the higher-or lower-stiffness microcapsule states and it is washed out during the synthesis of the microcapsules.
NA2-Reveals partial release from the higher-stiffness microcapsules and free-release from the lower-stiffness microcapsules (the capsules reveal a lower loading degree due to the partial release of the load from the higher stiffness microcapsules during the synthesis of the microcapsules).
NA3 and NA4-No release from the higher-stiffness microcapsules is observed. Release from the lower-stiffness microcapsules proceeds.