PREPARATION

BACKGROUND OF THE INVENTION

[001] The synthesis of microcapsules and their applications have attracted growing interest in recent years. Different methods to prepare microcapsules were reported. These include the chemical deposition of polymer or hydrogel coatings on substrate-loaded cores, followed by the etching of the core template. For example, CaC0 ₃ core templates were coated by a layer-by-layer deposition processes of oppositely-charged polyelectrolytes, or the use of interlayer biorecognition complexes, e.g., lectin/saccharide complexes or the use of covalent bonds, such as disulfides, and the etching of the core templates, e.g., by EDTA resulted in the substrate-loaded microcapsules. Alternatively, microcapsules loaded with substrates were prepared in oil-in-water or water-in-oil microemulsions.

[002] Different applications of microcapsules were suggested including their use as drug carriers for slow release, sensors, microreactors for chemical transformations food and cosmetic additives. A subclass of functional microcapsules includes stimuli-responsive capsules that are unlocked, in the presence of appropriate biomarkers or environmental conditions, to stimulate the selective and programmed release of the loads. For example, pH, light, heat, gases, salts, chemical reducing agents, carbohydrates, enzymes, magnetic field, and ultrasonic or microwave agitation were used to release the embedded substrate loads. Specific applications of stimuli-responsive microcapsules include the triggered release of encapsulated drugs by specific biomarkers or appropriate cellular/tissues environmental conditions. [003] The base sequence of nucleic acids dictates their structural reconfiguration functions in the presence of auxiliary triggers. For example, the pH-induced formation of i-motif or triplex structures, the K+-ion stabilized of G-quadruplex and their separation by crown ethers, and the cooperative stabilization of duplex nucleic acid by metal-ion bridge mismatched bases (e.g. T-Hg2+-T or C-Ag+-C).

[004] In addition, sequence-specific recognition of ligands (aptamers) or sequence dictated catalytic properties of nucleic acids (DNAzymes) provide important motifs for controlling the structure and chemical function of oligonucleotides. Nucleic acids thus provide a useful “tool-box” to synthesize stimuli-responsive nucleic acid drug-loaded micro/nano carriers.

[005] All-DNA-stabilized drug loaded microcapsules were reported and their unlocking by light, pH, and the formation of aptamer-ligand (ATP or VEGF) complexes were demonstrated. In addition, drug-loaded nucleic-acid-modified hydrogel-stabilized microcapsules were prepared, and the triggered reversible reconfiguration of the oligonucleotide units was used to control the stiffness of the hydrogel coating and to switch the reversible ON/OFF release of the encapsulated drugs. Cell experiments demonstrated the selective cytotoxicity of chemotherapeutic drug-loaded microcapsules toward cancer cells.

[006] Further enhancement of the complexities of stimuli-responsive microcapsules would involve the challenging assembly of microcapsule-in-microcapsule systems where two compartments are separated and stabilized by two different stimuli-responsive coating layers. Such systems could be applied for the programmed release of one of two drugs or the parallel release of two drugs (or prodrug and activator) and could act as an organized compartmentalized containment for chemical reactions. While microcapsule-in- microcapsule systems were fabricated in microemulsion, the inter-connected microcapsules were prepared using microfluidic devices, the design of stimuli-responsive switchable microcapsule-in-microcapsule systems and the application of these structures are basically unexplored.

[007] The inventors of the present invention provide a synthesis and characterization of polymer (such as for example hydrogel) stimuli-responsive microcapsule-in-microcapsule systems that includes, within the core of each microcapsule, different loads in separated aqueous compartments comprising the structures. This is demonstrated in the presence of one or two triggers the selective release of one load (core), or the two loads from the carrier proceeds. Also, in the presence of an appropriate trigger, loads embedded in the two aqueous compartments of the carrier can be mixed. In addition, the microcapsule-in microcapsule system of the invention provides a functional unit that operates as a closed- loop device that senses, for example, glucose and releases insulin, acting as a model of an “artificial pancreas”.

SUMMARY OF THE INVENTION

[008] Thus, the present invention provides a system comprising at least one first microcapsule having a first core and first shell layer structure; wherein said at least one first shell layer comprise at least one first polymer and at least one first stimuli-responsive nucleic acid sequence; said first shell layer encapsulating within its first core at least one first active agent and at least one second microcapsule having a second core and second shell layer structure; wherein said at least one second shell layer comprise at least one second polymer and at least one second stimuli-responsive nucleic acid sequence; said second shell layer encapsulating within its second core at least one second active agent. [009] A system of the invention may be referred to also as “microcapsule-in microcapsule” structure, which should be understood to relate to a system wherein at least one first microcapsule comprises within its core at least one second microcapsule as defined herein above and below.

[0010] When referring to a “stimuli-responsive nucleic acid sequence” it should be understood to relate to a nucleic acid sequence that is capable of changing at least one of its physical properties upon association with at least one stimulus. Such stimulus may be an association of said sequence with a biomarker and/or biological molecule. Examples may include but are not limited to a salt, a metal ion, a chemical reducing agent, a carbohydrate, a competing/complementary nucleic acid sequence, an enzyme, and any combinations thereof.

[0011] Other stimuli might be a cellular environment parameter. Examples may include but are not limited to pH, radiation, light, temperature, heat, gas, magnetic field, ultrasonic or microwave agitation and any changes and combinations thereof.

[0012] The integration of stimuli-responsive oligonucleotides (nucleic acid sequence) with at least one polymer (forming said shell layer of a microcapsule disclosed herein, may be achieved by any type of bond such as a chemical bond, an electronic association, a hydrogen bond, a metal bond and so forth. In some embodiments said at least one stimuli- responsive sequence is associated to said at least one polymer via cross-linking bonds, thus being integrated in the framework of said at least one polymer. In some other embodiments, said at least one polymer of said shell layer is modified with at least one tethering nucleic acid sequence. In some embodiments, said at least one tethering nucleic acid sequence anchors said at least one stimuli-responsive nucleic acid sequence. [0013] Such integration of stimuli-responsive oligonucleotides (nucleic acid sequence) with at least one polymer provides means to exploit the functional information encoded in the nucleic acid sequences to yield stimuli-responsive polymer exhibiting switchable physical, structural, and chemical properties. In such embodiments, said integration of said at least one stimuli-responsive nucleic acid sequence with said at least one polymer provides the ability to change the physical state of said shell layer upon association of said at least one stimuli-responsive sequence with said at least one stimulus. Thus, such an association with said stimulus may, for example, change the stiffness, the thickness, the permeability, the cross-linking degree, of said polymer and thus of said shell layer.

[0014] Therefore, in some embodiments, upon the association of said at least one stimuli- responsive sequence with at least one stimulus, said shell layer changes its properties to allow the core content of said microcapsule to be released from said core.

[0015] In other embodiments, upon the association of said at least one stimuli-responsive sequence with at least one stimulus, said shell layer changes its properties to allow components present at the close environment of said microcapsule to permeate into the core of said microcapsule.

[0016] In some other embodiments, said change in the properties of said shell layer, upon association with said at least one stimulus, is reversible, thus, upon the termination of said at least one stimulus, the properties of said shell layer are restored.

[0017] In some embodiments, said at least one first polymer of said at least one first shell layer is selected from a hydrophobic or a hydrophilic polymer.

[0018] In some embodiments, said hydrophilic polymer is a hydrogel polymer.

[0019] In some embodiments, said at least one first polymer of said at least one first shell layer is modified with at least one first tethering nucleic acid sequence. [0020] In some embodiments, said at least one first tethering nucleic acid sequence anchors said at least one first stimuli-responsive nucleic acid sequence.

[0021] In some embodiments, said at least one second polymer of said at least one second shell layer is selected from a hydrophobic or a hydrophilic polymer.

[0022] In some embodiments, said hydrophilic polymer is a hydrogel polymer.

[0023] In some embodiments, said at least one second polymer of said at least one second shell layer is modified with at least one second tethering nucleic acid sequence.

[0024] In some embodiments, said at least one second tethering nucleic acid sequence anchors said at least one second stimuli-responsive nucleic acid sequence.

[0025] In some embodiments, said at least one first polymer and said at least one second polymer are the same or different.

In some embodiments, said at least one first stimuli-responsive sequence is selected from a hairpin strand, a pH-responsive strands, a DNAzyme, a G-quadruplex, a stem-loop strand, an aptamer, a pathogen-specific sequence and any combinations thereof.

[0026] In some embodiments, said at least one second stimuli-responsive sequence is selected from a hairpin strand, a pH-responsive strands, a DNAzyme, a G-quadruplex, a stem-loop strand, an aptamer, a pathogen-specific sequence and any combinations thereof. [0027] In some embodiments, said at least one first microcapsule further comprises at least one third microcapsule having a third core and third shell layer structure; wherein said at least one third shell layer comprise at least one third polymer and at least one third stimuli-responsive nucleic acid sequence; said third shell layer encapsulating within its third core at least one third active agent.

[0028] In some embodiments, said at least one second microcapsule further comprises at least one third microcapsule having a third core and third shell layer structure; wherein said at least one third shell layer comprise at least one third polymer and at least one third stimuli-responsive nucleic acid sequence; said third shell layer encapsulating within its third core at least one third active agent.

[0029] In some embodiments, said at least one third polymer of said at least one third shell layer is selected from a hydrophobic or a hydrophilic polymer.

[0030] In some embodiments, said hydrophilic polymer is a hydrogel polymer.

[0031] In some embodiments, said at least one third polymer of said at least one third shell layer is modified with at least one third tethering nucleic acid sequence.

[0032] In some embodiments, said at least one third tethering nucleic acid sequence anchors said at least one third stimuli-responsive nucleic acid sequence.

[0033] In some embodiments, said at least one first active agent is released from said first core upon the association of said at least one first stimuli-responsive nucleic acid sequence with at least one stimulus.

[0034] In some embodiments, said stimuli is selected from at least one biomarker and/or at least one cellular environmental condition.

[0035] In some embodiments, said stimuli is selected from pH, radiation, light, heat, gas, salt, chemical reducing agents, carbohydrate, enzyme, magnetic field, ultrasonic or microwave agitation and any combinations thereof.

[0036] In some embodiments, said at least one second active agent is released from said second core upon the association of said at least one second stimuli-responsive nucleic acid sequence with at least one stimulus.

[0037] In some embodiments, said stimuli is selected from at least one biomarker and/or at least one cellular environmental condition. [0038] In some embodiments, said stimuli is selected from pH, radiation, light, heat, gas, salt, chemical reducing agents, carbohydrate, enzyme, magnetic field, ultrasonic or microwave agitation and any combinations thereof.

[0039] In some embodiments, said at least one third active agent is released from said third core upon the association of said at least one third stimuli-responsive nucleic acid sequence with at least one stimulus.

[0040] In some embodiments, stimulus is selected from at least one biomarker and/or at least one cellular environmental condition.

[0041] In some embodiments, said stimuli is endogenous. In some other embodiments, said stimuli is exogenous. In some further embodiments, said stimuli is selected from pH, radiation, light, heat, gas, salt, chemical reducing agent, carbohydrate, enzyme, magnetic field, ultrasonic or microwave agitation, pathogen, and any combinations thereof.

[0042] In some embodiments, each of said first, second and third active agents are each selected from a drug, a pro-drug, a labeling agent, a hormone, a reactive compound capable of forming at least one stimulus, a nucleic acid sequence, a steroid, a sensor, a transistor, a radioactive agent, and any combinations thereof.

[0043] In some embodiments, said at least one first active agent is glucose oxidase (thus encapsulated within the core of said first microcapsule) and at least one second active agent is insulin (thus encapsulated within the core of said second microcapsule, which is encapsulated within said first microcapsule). Under such embodiments, a system of the invention is used in the treatment of a subject needing to regulate their glucose levels. Under such embodiments, a system of the invention is used in the treatment of diabetes in a subject in need thereof. [0044] In some embodiments, said at least one first active agent is insulin (thus encapsulated within the core of said first microcapsule) and at least one second active agent is glucose oxidase (thus encapsulated within the core of said second microcapsule, which is encapsulated within said first microcapsule). Under such embodiments, a system of the invention is used in the treatment of a subject needing to regulate their glucose levels. Under such embodiments, a system of the invention is used in the treatment of diabetes in a subject in need thereof.

[0045] In some embodiments, the concentration of said insulin in the core of said first or second microcapsule is at least 20mg/dL. In some embodiments, the concentration of said insulin in the core of said first or second microcapsule is at least 30mg/dL. In some embodiments, the concentration of said insulin in the core of said first or second microcapsule is at least 40mg/dL. In some embodiments, the concentration of said insulin in the core of said first or second microcapsule is at least 50mg/dL. In some embodiments, the concentration of said insulin in the core of said first or second microcapsule is at least 60mg/dL. In some embodiments, the concentration of said insulin in the core of said first or second microcapsule is at least 70mg/dL. In some embodiments, the concentration of said insulin in the core of said first or second microcapsule is at least 80mg/dL. In some embodiments, the concentration of said insulin in the core of said first or second microcapsule is at least 90mg/dL. In some embodiments, the concentration of said insulin in the core of said first or second microcapsule is at least lOOmg/dL. In some embodiments, the concentration of said insulin in the core of said first or second microcapsule is between about 10 to 100 mg/dL.

[0046] In some embodiments, said system of the invention comprising insulin as active agent, is capable of releasing said insulin when glucose concentration of said subject is <100mg/dL. In some other embodiments, said system of the invention comprising insulin as active agent, is capable of terminating/halting/holding the release of said insulin when glucose concentration of said subject is >100mg/dL.

[0047] The invention further provides a composition comprising a system as disclosed herein above and below. In some embodiments, said composition is a pharmaceutical composition.

[0048] The invention provides a method to assemble stimuli-responsive nucleic acid- based hydrogel-stabilized microcapsule-in-microcapsule systems. An inner compartment stabilized by stimuli-responsive hydrogel layer (ca. 150 nm) provides the inner microcapsule (diameter ca. 2.5 pm). The inner microcapsule is separated from the outer compartment stabilized by an outer stimuli-responsive hydrogel layer (thickness of ca. 150 nm) that yields a microcapsule-in-microcapsule system. The hydrogel layers exist in a higher stiffness state that prevents inter-reservoir or leakage of the loads from the respective compartments. Subjecting the inner hydrogel layer to a stimulus, and the outer hydrogel layer to another stimuli, leads to the triggered separation of the bridging units associated with the respective hydrogel films. This results in 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 compartment, or the release of the loads from the two compartments.

[0049] In another option of a system of the invention 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 glucose oxidase 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 concentration range of diabetic concern 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.

[0050] A process for the preparation of a system of claim 1, said process comprising the steps of: (a) encapsulating at least one second active agent within at least one second shell layer comprising at least one second polymer and at least one second stimuli-responsive nucleic acid sequence; thereby forming at least one second microcapsule having a second shell layer and a second core; (b) encapsulating said at least one second microcapsule and at least one first active agent within at least at least one first shell layer comprising at least one first polymer and at least one first stimuli-responsive nucleic acid sequence; thereby forming a first microcapsule having a first shell layer and a first core; thereby forming said system.

BRIEF DESCRIPTION OF THE DRAWINGS

[0051] The subject matter regarded as the invention is particularly pointed out and distinctly claimed in the concluding portion of the specification. The invention, however, both as to organization and method of operation, together with objects, features, and advantages thereof, may best be understood by reference to the following detailed description when read with the accompanying drawings in which:

[0052] Figures 1A-1C. Figure 1A shows the synthesis of carboxymethyl cellulose (CMC)- stimuli-responsive nucleic acid-based hydrogel microcapsule-in-microcapsule loaded with two different fluorophores and the unlocking of the system by two different triggers: Zn2+-ion and pH, which results in the release of the loads from the different aqueous compartments. Figure IB shows a detailed outline of the substrate (3) and Zn2+- ion-dependent DNAzyme (4) associated with the inner hydrogel layer. Figure 1C shows a detailed outline of the outer hydrogel layer comprising the pH-responsive reconfiguration of the (5)/(6) supramolecular duplexes into the separated i-motif structure (at pH = 5.0, lower stiffness) and the reverse assembly of the (5)/(6) duplex-bridged hydrogel (at pH = 7.0, higher stiffness).

[0053] Figures 2A-2E. Figures 2A-2C show SEM images corresponding to Panel I (2A) - The CaC03 microparticles before modification. Panel P (2B) - The microparticles coated with the two-films of hydrogel (prior to etching off the CaC03 component). Panel III (2C) - An example of a broken microparticle consisting of two-layers of deposited hydrogel films. Figures 2D-2E show the focus-ion beam (FIB) images of: Panel I (2D) - The microparticle core coated with the first hydrogel film. Panel II (2E)- The microparticle consisting of core-shell/core-shell CaC03/hydrogel films.

[0054] Figures 3A-3K. Figures 3A - 3D show confocal microscopy images corresponding to the two hydrogel coated CaC03 microparticles that includes: (3A) CdSe/ZnS QDs in the outer CaC03 core (Zex=464nm; Zem=482nm) (3B) TMR-D in the inner CaC03 core (lec =546nm ; Zem=580nm) (3C) Overlay of the channel-separate fluorescence images shown in (3A) and (3B). (3D) Bright-field image of the bi-layer hydrogel functionalized microparticles. Figures 3E-3K show the orthogonal projections of the overlaid confocal microscopy images of the bi-layer-hydrogel-functionalized microparticles that include the CdSe/ZnS QDs in the outer CaC03 core and TMR-D in the inner CaC03 core. [0055] Figures 4A - 4B. Triggered time-dependent release of the loads from the bi- compartmentalized aqueous compartments of the microcapsule-in-microcapsule system loaded with TMR-D in the inner compartment and CdSe/ZnS QDs in the outer compartment. The compartments are separated by Zn2+-ion-dependent DNAzyme hydrogel (inner compartment). The outer compartment and the bulk solution are separated by pH-responsive hydrogel. Figure 4A show the release of: (a) CdSe/ZnS QDs and (b) TMR-D, upon subjecting the microcapsule-in-microcapsule system to pH = 5.5. Inset: Switchable “ON” and “OFF” release of the CdSe/ZnS QDs from the microcapsule-in microcapsule system upon the reversible treatment at pH = 5.5 and pH = 7.0. Figure 4B show the release of: (a) CdSe/ZnS QDs and (b) TMR-D upon treatment of the microcapsule-in-microcapsule system with Zn2+-ions, 20 mM and pH = 5.5.

[0056] Figure 5. Confocal microscopy images and bright-field images of the microcapsule-in-microcapsule system loaded with CdSe/ZnS QDs and TMR-D upon triggering the system with Zn2+-ions, 20 mM, at different time intervals (a) The single channel QDs fluorescence (green) (b) The single channel TMR-D fluorescence (red) (c) The overlaid fluorescence of (a) and (b). (d) The bright-field image. Panel I: t=0 minutes; Panel II: t=5 minutes. Panel III: t=10 minutes. Panel IV: t=25 minutes. Note that after 25 minutes an overlaid yellow image is observed confirming the mixture of the fluorophores in the two aqueous compartments.

[0057] Figures 6A-6C. Figure 6A shows a schematic synthesis of the microcapsule-in microcapsule system composed of glucose oxidase (GOx) entrapped in the inner aqueous compartment, stabilized by a supramolecular duplex nucleic acid-bridged hydrogel, and the fluorophore-labeled insulin loaded in the outer aqueous compartment, stabilized by the pH responsive hydrogel layer. Scheme depicts the switchable pH-stimulated release of the insulin by the reconfiguration of the duplex units, bridging the outer hydrogel layer into the i-motif structure. Inset: Schematic biocatalytic reaction of GOx entrapped in the inner aqueous reservoir. Note that the release of insulin is reversibly controlled by the concentration of glucose and the accompanying GOx-stimulated pH changes. Figure 6B shows a detailed outline of the nucleic acid bridging elements associated with the inner hydrogel layer. Figure 6C shows a detailed outline of the nucleic acid bridging units comprising the outer pH-responsive reconfiguration of the (5)/(6) supramolecular duplexes into the separated i-motif structure (at pH = 5.5, lower stiffness) and the reverse assembly of the (5)/(6) duplex-bridged hydrogel (at pH = 7.0, higher stiffness) associated with the outer hydrogel layer.

[0058] Figures 7A-7B. Figure 7 A is a time-dependent fluorescence changes corresponding to the release of the Fluorescein-labeled insulin from the microcapsule-in microcapsule systems subjected to different concentrations of glucose: (a) 0 mM (b) 5 mM (c) 10 mM (d) 15 mM (e) 20 mM (f) 30 mM. Figure 7B shows the switchable “ON” and “OFF” release of the insulin upon subjecting the microcapsule-in-microcapsule system to “elevated”/” normal” glucose levels. The capsules are initially introduced to a glucose solution, 10 mM, and this results in the release of insulin for 50 minutes and the leveling off of the insulin release (where glucometer evaluated values of the glucose in the bulk solution corresponds to 4 mM). At point (a) the capsules are subjected to an additional added concentration of glucose, 10 mM. This results in the reactivation of the release of insulin and the leveling off of the release process after ca. 60 minutes.

[0059] Figures 8A-8B. Determination of the ratio between nucleic acid units and CMC polymer units on the HI/ (2) (5AmMC6 modified) conjugated copolymer PI. Figure 8A shows the absorbance spectra corresponding to different concentrations of CMC in the presence of constant concentration of the amino-modified nucleic acid (2) and HI, in total concentration of 0.5mM at a 2:1 ratio respectively. Ratio CMC/DNA was as followed: (a) 0:1 , (b) 10:1, (c) 20:l, (d) 50:1, (e) 100:1, (f) 150:1 (g) 200:1 (h) 250:1 (i) 300:1 (j) 350:1 (k) 400:1 (1) 500:1 (m) 600:1 (n) 700:1. Figure 8B shows the calibration curve between the molar ratio of CMC/H1(2) (5AmMC6 modified) vs. the ratio of absorbance at wavelength of 205 nm and 260 nm. The absorbance of the modified polymer was measured and the ratio of CMC to nucleic acid strand was calculated to be 60: 1.

[0060] Figures 9A-9B. Determination of the ratio between nucleic acid units and CMC polymer units on the (x)/ (2) (5AmMC6 modified) conjugated copolymer P2 (without H2). Figure 9A shows the absorbance spectra corresponding to a different concentration of CMC in the presence of constant concentration of the amino-modified nucleic acid (2) and (x) in total concentration of 0.5mM at a 2:1 ratio respectively. Ratio CMC/DNA was as followed: (a) 0:1 , (b) 10:1, (c) 20:1, (d) 50:1, (e) 100:1, (f) 150:1 (g) 200:1 (h) 250:1 (i) 300:1 (j) 350:1 (k) 400:1 (1) 500:1 (m) 600:1 (n) 700:1. Figure 9B shows the calibration curve between the molar ratio of CMC/(x) (2) (5AmMC6 modified) vs. the ratio of absorbance at wavelength of 205 nm and 260 nm. The absorbance of the modified polymer was measured and the ratio of CMC to nucleic acid strand was calculated to be 60:1.

[0061] Figures 10A-10B. Figure 10A shows the lH-NMR spectrum of CMC polymer PI, CMC/DNA. Figure 10B shows the DOSY-NMR spectrum of CMC polymer PI.

[0062] Figures 11A-11B. Figure 11A shows the lH-NMR spectrum of CMC polymer P2, CMC/DNA. Figure 1 IB shows the DOSY-NMR spectrum of CMC polymer P2. [0063] Figures 12A-12B. Determination of the ratio between nucleic acid units and acrylamide units on the HI/ (2) (5Acryd modified) conjugated copolymer PI. Figure 12A shows the absorbance spectra corresponding to a different concentration of acrylamide in the presence of constant concentration of the acrydite-modified nucleic acid (2) and HI, in total concentration of 0.5mM at a 2:1 ratio respectively. Ratio acrylamide units /DNA was as followed: (a) 1:1 , (b) 5:1, (c) 10:1, (d) 25:1, (e) 50:1, (f) 75:1 (g) 100:1 (h) 125:1 (i) 150:1 (j) 175:1 (k) 200:1 (1) 250:1. Figure 12B shows the calibration curve between the molar ratio of acrylamide/Hl(2) (5Acryd modified) vs. the ratio of absorbance at wavelength of 205 nm and 260 nm. The absorbance of the modified polymer was measured and the ratio of acrylamide units to nucleic acid strand was calculated to be 30:1.

[0064] Figures 13A-13B. Determination of the ratio between nucleic acid units and acrylamide units on the (x)/( 2) (5Acryd modified) conjugated copolymer P2 (without H2). Figure 13 A shows the absorbance spectra corresponding to a different concentration of acrylamide in the presence of constant concentration of the acrydite-modified nucleic acid (2) and (x), in total concentration of 0.5mM at a 2:1 ratio respectively. Ratio acrylamide units /DNA was as followed: (a) 1:1 , (b) 5:1, (c) 10:1, (d) 25:1, (e) 50:1, (f) 75:1 (g) 100:1 (h) 125:1 (i) 150:1 (j) 175:1 (k) 200:1 (1) 250:1. Figure 13B shows the calibration curve between the molar ratio of acrylamide/(x)(2) (5Acryd modified) vs. the ratio of absorbance at wavelength of 205 nm and 260 nm. The absorbance of the modified polymer was measured and the ratio of acrylamide units to nucleic acid strand was calculated to be 30: 1.

[0065] Figures 14A-14B. Figure 14A is the 1H-NMR spectrum of acrylamide polymer PI, polyacrylamide /DNA. Figure 14B shows the DOSY-NMR spectrum of acrylamide polymer PI. [0066] Figures 15A-15B. Figure 15 A shows the 1H-NMR spectrum of acrylamide polymer P2, polyacrylamide/DNA. Figure 15B shows the DOSY-NMR spectrum of acrylamide polymer P2.

[0067] Figure 16. Calibration curve corresponding to the diffusion coefficients of a series fof CMC polymers of known average molecular weights (Mw= 90 kDa, 250 kDa and 700 kDa). The diffusion coefficients of the respective polymers were derived by recording the DOSY spectrum of each of the polymers. (500 KDa for PI, and 200 KDa for P2).

[0068] Figure 17. Calibration curve of the diffusion coefficients against known molecular weights of polyacrylamide (549.4 kDa for PI, and 390.63 kDa for P2). The diffusion coefficients of the respective polymers were derived by recording the DOSY spectrum of each of the polymers. Squares represent the known polymers utilized to form standard line, and squares represent the polymers measured for experimental studies.

[0069] Figure 18. Time-dependent release of the loads from the bi-compartmentalized aqueous compartments of the microcapsule-in-microcapsule system at pH =7.0, in the absence of Zn2+. No release of the CdSe/ZnS QDs, curve (a), (pH-responsive hydrogel, outer compartment) or TMR-D, curve (b), (Zn2+-ion-dependent DNAzyme hydrogel, inner compartment) was detected.

[0070] Figure 19. Triggered time-dependent release of CdSe/ZnS QDs upon subjecting the polyacrylamide bi-compartmentalized microcapsule-in-microcapsule to pH = 5.5. [0071] Figure 20. Triggered time-dependent release of CdSe/ZnS QDs upon subjecting the polyacrylamide bi-compartmentalized microcapsule-in-microcapsule to pH = 7.

[0072] Figure 21. Switchable “ON” and “OFF” release of the CdSe/ZnS QDs from the polyacrylamide microcapsule-in-microcapsule system upon the reversible treatment at pH = 5.5 and pH = 7.0. [0073] Figure 22. Triggered time-dependent release of TMR-D upon subjecting the polyacrylamide bi-compartmentalized microcapsule-in-microcapsule to different concentrations of Zn2+ ions at pH = 5.5: (a) 20 mM, (b) 0 mM.

[0074] Figure 23. Triggered time-dependent release of the loads (QD and TMR) upon subjecting the polyacrylamide bi-compartmentalized microcapsule-in-microcapsule to Zn2+ ions, 20 mM, and different pH values (a) Release of TMR-D at pH 7, (b) Release of QDs at pH 7, (c) Release of QDs at pH 5.5, (d) Release of TMR-D at pH 5.5.

[0075] Figure 24. Triggered time-dependent release of TMR-D from the inner compartment of the microcapsule-in-microcapsule system, at pH=5.5 in the presence of different concentrations of Zn2+-ions (a) OMm, (b)10Mm, (c) 20mM, (d) 30Mm.

[0076] It will be appreciated that for simplicity and clarity of illustration, elements shown in the figures have not necessarily been drawn to scale. For example, the dimensions of some of the elements may be exaggerated relative to other elements for clarity. Further, where considered appropriate, reference numerals may be repeated among the figures to indicate corresponding or analogous elements.

DETAILED DESCRIPTION OF THE PRESENT INVENTION

[0077] In the following detailed description, numerous specific details are set forth in order to provide a thorough understanding of the invention. However, it will be understood by those skilled in the art that the present invention may be practiced without these specific details. In other instances, well-known methods, procedures, and components have not been described in detail so as not to obscure the present invention. [0078] The synthesis of dually-triggered, stimuli-responsive, nucleic acid-based hydrogel- microcapsule-in-microcapsule microstructures carrying two different loads (e.g., tetramethyl rhodamine-dextran, TMR-D, and CdSe/ZnS quantum dots, QDs) is schematically outlined in Figure 1. In addition, the mechanism of unlocking the capsules, and the dictated release of the loads by two different triggers, e.g., the Zn2+ and/or pH are schematically exemplified in Figure 1. CaC03 microparticles loaded with TMR-D were coated with a polyallylamine hydrochloride, PAH, layer followed by the electrostatic adsorption of the DNA strand (1) on the coated particles. The strand (1) acts as a promoter strand for inducing the hybridization chain reaction (HCR).

[0079] The TMR-D loaded (l)-functionalized microparticles were reacted with two carboxym ethyl cellulose (CMC) polymers, PI and P2, that were modified with hairpins HI and H2, respectively, and with the nucleic acid (2) that acts as anchoring site for the stimuli responsive units (vide infra). Note that hairpin H2 was conjugated to P2 through tether x linked to the polymer. This modification is essential to retain the appropriate directionality of the hairpins for the HCR process. The stimuli-responsive unlocking unit of the first hydrogel layer in this specific example is Zn2+-ions-dependent DNAzyme (4). Accordingly, the strand (3) includes the sequence of the Zn2+-ions-dependent DNAzyme substrate extended at its 5’- and 3’ -ends by sequences that are complementary to the anchoring tethers, (2), associated with the polymer chains, P1/P2. The strand (4) includes the sequence of the Zn2+-ions-dependent DNAzyme (it includes the loop domain for binding Zn2+-ions, and two extended arms that bind to the substrate (3)) as shown in Figure 1(B). The loading of the nucleic acid units on the CMC chains were evaluated spectroscopically and corresponded to 1:60 for PI and 1:60 for P2 (nucleic acid: CMC unit). For experimental details describing the evaluation of the average molecular weight and nucleic acid loadings on the different chains, see Figures si -17 and experimental section. Treatment of the (l)-functionalized microparticles with the polymer chains PI and P2 in the presence of the substrate (3) and the DNAzyme sequence (4) initiates the HCR process, where the promoter (1) hybridizes with hairpin HI associated with PI and the "toehold" strand associated with the open HI hybridizes with H2 and the "toehold" single strand of the opened H2 reopens HI and vice versa. This HCR process leads to the formation of a CMC hydrogel film, crosslinked by duplexes formed upon the counter opening of hairpins H1/H2, on the microparticle core. As the HCR process is stimulated in the presence of (3) and (4), the free anchoring tether (2) hybridizes with the substrate chain (3) and the single-stranded domain of (3) hybridizes with the arms of (4) that include in the loop domain the Zn2+-ion-dependent DNAzyme sequence. This yields the supramolecular structure that in the presence of the Zn2+-ions generates the activation of the Zn2+-i on-dependent DNAzyme. Figure 1(B) shows the domains of the duplex crosslinking units generated by the HCR process and the supramolecular structure of the (3)/(4) units. The core microparticles coated with the first Zn2+-ions stimuli-responsive hydrogel were then reacted with mixture of CaC12, CdSe/ZnS QDs (modified units carboxylic acid functionalized) and Na2C03. The in-situ generated QD-impregnated CaC03 particles were then deposited on the CMC hydrogel coating film using Ca2+-ions as "glue" that bind the CaC03 layer to the CMC hydrogel. Subsequently, the CaC03 layer was first modified with PAH and then with the promoter strand (1). The resulting (1)- functionalized particles were interacted with the polymer chains PI and P2 in the presence of the strands (5) and (6). Strand (5) includes at its 3’-end the (q’)-sequence extended by the sequence z that is capable to form under acidic conditions i-motif structure. The strand (6), includes at its 5’ -end the sequence z’ (complementary to z), extended by the sequence q’ (complementary to q), Figure 1(C). Under these conditions, the (l)-stimulated HCR process leads to the formation of the second hydrogel film consisting of the crosslinking duplexes generated between HI and H2, via the HCR process, and cooperatively stabilized by the supramolecular stmcture consisting of the (5)/(6) duplex anchored through the (q’) toeholds to the (2)-tethers associated with the polymer PI and P2 (note that the supramolecular Zn2+-ion-dependent DNAzyme (3)/(4), Figure 1(B), and the duplex structure (5)/(6) are crosslinked to the polymer chains by identical (q)/(q') duplex anchoring site, where q corresponds to strand (2)).

[0080] The resulting two hydrogel films, coating the core and interlayer of CaC03,were then etched with EDTA, yielding the microcapsule-in-microcapsule structure consisting of two aqueous compartments separated by two stimuli -responsive hydrogel layers. The inner aqueous reservoir, protected by the Zn2+-dependent stimuli-responsive hydrogel, includes the TMR-D load, and the outer aqueous compartment includes the CdSe/ZnS QDs, protected by the outer pH-responsive hydrogel layer. Figure 1(A) describes the triggered release of the loads associated with the two aqueous compartments of the microcapsule-in-microcapsule system.

[0081] Treatment of the microcapsule-in-microcapsule system with Zn2+-ions cleaves the substrate (3) and leads to a lower degree of crosslinking and to a consequently inner hydrogel layer of lower stiffness, resulting in the mixture of the loads present in the two aqueous compartments. Subjecting the microcapsule-in-microcapsule system to pH = 5.5 results in the reconfiguration of the bridging strand (6) into i-motif structure and in the separation of the (5)/(6) duplex bridges (Figure 1C). This process leads to an outer- hydrogel layer of lower stiffness that allows the release of the load from the outer aqueous compartment, while the load in the inner reservoir stays intact.

[0082] Treatment of the microcapsule-in-microcapsule system with Zn2+-ions and pH = 5.5 leads to the triggering of the inner and outer hydrogel layers into lower stiffness matrices. This allows the release of the loads from the inner and outer reservoirs into the bulk aqueous solution.

[0083] The versatility of the method to assemble and trigger the microcapsule-in microcapsule system should be emphasized: (i) the production of CMC -microcapsule-in microcapsule systems is shown. This concept can be, however, adapted to other stimuli- responsive hydrogel materials. For example, Figure 1 and accompanying discussion describe the synthesis of stimuli-responsive polyacrylamide-based microcapsule-in microcapsule hydrogel systems using an identical concept (ii) the loading of the aqueous compartments with TMR-D and QD was shown. The method can be applied, however, to load the different aqueous compartments with any other dyes, particles, proteins or nucleic acids MW > 5KDa (vide infra) (iii) the Zn2+-ion-dependent DNAzyme and the pH- stimulated formation of the i-motif structures as unlocking motifs for releasing the loads. Nonetheless the unlocking mechanisms are not limited to these hydrogel uncaging principles. Other methods to reconfigure the bilayer microcapsule-in-microcapsule hydrogel system may be envisaged including the application of other DNAzymes or the use of other stimuli-responsive DNA-reconfiguration principles, e.g., K+-ion-stabilized G- quadruplexes (vide infra) (iv) the bi-lay ered microcapsule-in-microcapsule microstructure reveals in/out permeation of low molecular-weight substrates or products. Nonetheless loads exhibiting molecular weights > 5KDa are confined to the respective aqueous compartments, with no leakage through the hydrogel layers of the respective compartment, provided that the hydrogels exist in their locked higher stiffness configurations.

[0084] In the first stage the bi-layer microcapsule-in-microcapsule structures were characterized. Figure 2(A), panel I, shows the SEM image of the bare inner CaC03 core microparticles. Figure 2(A), panel P shows the SEM image of the CaC03 microparticle after the stepwise deposition of the two Zn2+-ion-dependent DNAzyme and/pH responsive hydrogel films. A rough, porous coating is observed, consistent with the formation of hydrogel coated particles. The formation of a bi-layer hydrogel coating on the CaC03 microparticles is supported by identifying “defective”, broken bi-layer-coated particles, Figure 2(A), panel PI. The yield of intact bi-layer hydrogel coated microparticles is very high ca. 99% yet ca. 1% consists of defective structures, such as displayed in panel III. The SEM image clearly demonstrate the bi-layer coating of the particles, and eventually shows “broken-off’ pieces of microparticle coated by hydrogel. The formation of the bi-layer hydrogel films on the CaC03 microparticles is further supported by focused ion-beam (FIB) imaging of the hydrogel coated microparticles, Figure 2(B). Panel I show FIB image of a cut particle coated only with the first hydrogel film.

[0085] The thickness of the hydrogel film is ca. 150 nm. Panel II shows a FIB image of the two-hydrogel coating films, separated by the outer CaC03 template film (thickness 200-150 nm). The two hydrogel films reveal similar thickness (ca. 150 nm).

[0086] The formation of the bi-layer microcapsules after etching the CaC03 template units was supported by confocal fluorescence microscopy imaging experiments. Figure 3(A) shows the confocal fluorescence microscopy images of the TMR-D (red) and CdSe/ZnS QDs (green) loaded bi-layer microstructures, and the respective bright-field images before etching the CaC03 templates, Panel I, and after the EDTA-stimulated removal of the templates, Panel II. The overlaid fluorescence images indicate the formation of two separated fluorophore-containing compartments before and after etching (outside compartment green, inside compartment red). The single-channel microstructures generated after etching-off the CaC03 template show a void internal volume that implies the formation of the two-aqueous compartment microcapsule-in-microcapsule Panel II a and b. The overlay of a and b shows the location of the two different fluorophore containing compartments. In addition, Figure 3(B) shows the orthogonal projections of the bi-layer microparticles. Distinct and separated compartments are visible at the ZX projection and ZY projection.

[0087] In the next step, the triggered release of the loads from the bi-layer microcapsules was examined. Treatment of the microcapsules at pH =5.5 resulted in the release of the CdSe/ZnS QDs, Figure 4(A), curve (a). Under these conditions the TMR-D load, entrapped in the inner compartment, is not released, Figure 4(A), curve (b). In addition, at pH = 7.0 no release of the QDs was observed, Figure 18 curve (a). This allowed the switchable pH stimulated release of the QDs from the outer compartment of the microcapsules, Figure 4(A), inset. Figure 4(B) depicts the time-dependent release of the CdSe/ZnS QDs, curve (a), and of the TMR-D (from the inner compartment), curve (b), upon subjecting the microcapsules to pH = 5.5 and Zn2+-ions (20 mM). Under these conditions the two loads are released from the bi-layer compartments. Control experiments revealed that at pH =7.0, and in the absence of Zn2+, no release of the QDs (curve (a)) or TMR-D (curve (b)) was detected, Figure 18. (The triggered release of the loads from the polyacrylamide bi-layer microcapsules was also examined, Figures 19-23). [0088] Furthermore, the release at pH=5.5 of the TMR-D from the microcapsules was controlled by the concentration of Zn2+-ions, Figure 24. As the concentration of Zn2+-ion increases, the release of TMR-D is enhanced, consistent with the improved unlocking of the inner compartment by the Zn2+-ion-dependent crosslinking DNAzyme. Treatment of the microcapsules with Zn2+-ions at pH = 7.0 did not lead to the release of TMR-D or QDs to the bulk solution. Nonetheless, under these conditions, the inner compartment was unlocked by the Zn2+-ion-dependent DNAzyme and this allowed the mixing of the fluorophores between the two compartments.

[0089] The selective unlocking of the inner-compartment of the microcapsules by the Zn2+-ions dependent DNAzyme crosslinked and the mixture of the loads between the two compartments was confirmed by confocal fluorescence microscopy imaging. Figure 5 shows the confocal fluorescence microscopy images and bright-field images of the microcapsule-in-microcapsule fluorophore-labeled microstructures before the addition of Zn2+-ions to the system, Panel I, and after treatment of the microcapsules with Zn2+-ions, 20 mM, for different time intervals, Panels P - Panel IV. The non-treated microcapsule- in-microcapsule microstructures show the specific green fluorescence of the QDs and the red fluorescence of TMR-D upon the single-channel excitation of the fluorophores, images (a) and (b), respectively. Image (c) depicts the overlay of the two-channel fluorescence images of the microcapsule-in-microcapsule microstructures. An inner red fluorescence separated from an outer green fluorescence is observed, indicating that the fluorophores are confined in the two separated compartments. Treatment of the microcapsule-in-microcapsule microstructures with Zn2+-ions results in a yellow boundary between the inner red compartment and the outer green compartment, and this expands with time to a fully overlaid yellow fluorescence image, indicating the complete mixing of the two fluorophores as a result of the DNAzyme-catalyzed unlocking of the inner compartment.

[0090] The concept of synthesizing stimuli-responsive microcapsule-in-microcapsule microstructures and the triggered release of the loads from the bi-layer assemblies were expanded to include another switchable trigger. In an embodiment a microcapsule-in microcapsule microstructure system of the invention comprises an outer hydrogel layer crosslinked by K+-ion-stabilized G-quadmplex, and an inner hydrogel layer composed of the Zn2+-ions-dependent DNAzyme as crosslinkers. As before, the outer aqueous compartment was loaded with the CdSe/ZnS QDs, and the inner aqueous compartment was loaded with the TMR-D fluorophore. Subjecting the microcapsule-in-microcapsule system to 18-crown-6-ether (CE) separated the G-quadruplex crosslinking bridges, resulting in a hydrogel layer of lower stiffness that led to the release of the CdSe/ZnS QDs. Under these conditions the TMR-D, confined to the inner compartment, was not released from the microstructures, curve (b). The gated unlocking of the outer hydrogel layer could be switched between “ON” and “OFF” states by the cyclic treatment of the microstructures with CE and K+-ions. Treatment of the microcapsule-in-microcapsule system with CE and Zn2+-ions resulted in the triggered unlocking of the two hydrogel layers leading to the release of the loads from the two aqueous compartments. In addition, subjecting the microcapsule-in-microcapsule system to Zn2+-ions only resulted in the mixing of the fluorophores in the two aqueous compartments, with no release of the loads to the bulk solution.

[0091] The development of a versatile method to prepare two-reservoir stimuli-responsive microcapsule-in-microcapsule systems turns these ensembles as ideal drug carriers for controlled switchable release. Indeed, as a proof-of-concept, the inventors have applied these systems to tailor a glucose-regulated insulin release microcapsule-in-microcapsule system. Diabetes mellitus is a major public health problem across the world, accompanied by the constant increase of diabetes patients. Besides oral administration of drugs to regulate the glucose level in blood, the injection of insulin is a frequent practice to control glucose levels in blood. Nevertheless, poor control over glucose levels is often experienced, and complications such as hypoglycemia are often encountered. Indeed, major efforts were directed in the past decades to develop autonomous glucose responsive materials for the controlled release of insulin. Insulin-loaded polymer nanoparticles responding to pH changes generated by the GOx-catalyzed oxidation of glucose and accompanied by the generation of gluconic acid or glucose-induced swelling of boronic acid-functionalized polymers acted as useful glucose triggered insulin release matrices. In addition, pH-responsive polysaccharide particles loaded with insulin, glucose oxidase and catalase were used as functional carriers for the pH-stimulated release of insulin and the concomitant catalase-induced degradation of accompanying glucose-oxidase generated H202 that could lead to harmful ROS species. As well, a close-loop insulin delivery system consisting of an insulin-loaded silicon reservoir gated by enzyme-loaded pH- responsive hydrogel nanoparticles were used for the release of insulin.

[0092] Although substantial progress in designing close-loop insulin release systems was demonstrated, the development of other carriers revealing increased insulin loading, fast, selective and reversible insulin release functions under high/normal levels of glucose, injectability of the carrier (e.g., subcutaneous injectability with microneedle assays), and elimination of immunogenic effects of undesired leakage of proteins such as glucose oxidase are desired.

[0093] In contrast to the reported carriers that include the insulin- and stimuli-release element in one compartment, the two-reservoir, hydrogel-based microcapsule-in microcapsule system might introduce several advantages: (i) The thin hydrogel layers separating the two compartments are anticipated to allow effective switchable and selective release of insulin while protecting glucose oxidase in a confined inner compartment against leakage (ii) The availability of an aqueous compartment for the solubilization of insulin might allow enhanced loading of the carrier with the drug (iii) The hydrogel-based microcapsule-in-microcapsule carriers are suspendable in water/buffer solutions, and thus their injectability could be of useful practice. Accordingly, the bi-hydrogel layer microcapsule-in-microcapsule was applied to tailor a model system acting as an artificial pancreas. Figure 6 depicts the assembly of the bi-layer microcapsules carrying glucose oxidase (GOx) and insulin (In) in the distinct compartments comprising the microcapsules. The synthesis of GOx/insulin-loaded bi-layer microcapsules system followed the same protocol used in the previous systems, where GOx is confined to the inner aqueous compartment and insulin is confined to the outer aqueous compartment. It should be noted that in this microcapsule system the inner layer consists of the (2)/(3)/(2) bridged crosslinking units in the absence of the added DNAzyme sequence (4). Thus, these bridges are stable under all external conditions.

[0094] GOx was loaded on the core CaC03 microparticles, and these were coated with PAH and further functionalized with the promoter strand (1). The (l)-modified particles were subjected to the polymer chains PI and P2, were PI was modified with hairpin HI and polymer P2 was modified with hairpin H2. The two polymer chains included identical nucleic acid tether (2). The (l)-stimulated HCR process, in the presence of strand (3), resulted in the first hydrogel film, Figure 6(B). Subsequently, CaC03 was deposited on the first hydrogel film while loading the CaC03 with Insulin. The deposited CaC03 was then modified with PAH and the promoter strand (l).The (l)-functionalized surface was used to activate the HCR process in the presence of PI, P2 and the strands (5) and (6) to yield the second (outer) hydrogel film that is cooperatively stabilized by the duplex HI open/ H2 open and the superstructure bridge (2)/(5)/(6)/ (2), Figure 6(C). After etching the CaC03 template with EDTA, the microcapsule-in-microcapsule system is formed, where GOx is solubilized in the inner aqueous compartment and the insulin is solubilized in the outer aqueous compartment. The inner hydrogel layer is stabilized by the duplex HI open/ H2 open and the (2)/(3)/(2) bridges. The outer hydrogel layer is composed of the duplex HI open/ H2 open and the pH-responsive complex (2)/(5)/(6)/ (2). (cf. Figure 6(B) and Figure 6(C)).

[0095] The strand (5) associated with the bridging unit is cytosine rich and at pH 5.5 it reconfigures into the i-motif structure. This results in the separation of the bridging units and the unlocking of the hydrogel toward the release of insulin. The further neutralization of the pH unlocked the structure by dissociates the i-motif units and regenerates the gated higher stiffness hydrogel that prohibits the release of insulin. Thus, by the cyclic control of the pH at the hydrogel boundary the switchable ON/OFF release of insulin proceeds.

[0096] Under acidic conditions, the separation of the duplex (5)/(6) leads to a hydrogel of lower stiffness that provides the key properties for the operation of the “artificial pancreas”. Note that the bi-layer hydrogel microcapsule is freely permeable to low- molecular weight substrates, while the lower stiffness hydrogel is permeable to proteins, <5KDa, while the higher stiffness hydrogel is non-permeable to proteins. These features of the bi-layer microcapsule boundaries enable the operation of the “artificial pancreas”. The permeation of glucose across the two layers of the microcapsule-in-microcapsule boundaries leads to the GOx-catalyzed aerobic oxidation of glucose to gluconic acid and H202. The formation of gluconic acid acidifies the inner aqueous compartment and protons are permeating to the outer compartment and acidifying it. The acidic pH induces the reconfiguration of the duplex (5)/(6) associated with the outer hydrogel layer into the i- motif structure, a process which leads to the formation of a lower stiffness hydrogel that allows the release of insulin, to the bulk solution. Note that the pH-changes stimulated by GOx and, thus, the stiffness changes and release efficiency by the outer hydrogel layer are controlled by the concentrations of glucose. Furthermore, the control over the release of insulin by the concentrations of glucose is anticipated to yield a switchable dose release mechanism as required for an artificial pancreas-mimicking device. Figure 7(A) shows the time-dependent release of fluorescein-labeled insulin from the microcapsule system in the presence of different concentrations of glucose. In the absence of glucose, no release of Insulin is observed, curve (a). At a glucose concentration of 5 mM (normal levels of glucose in human blood) inefficient release of Insulin is observed, curve (b). At higher concentrations of glucose, > 10 mM, effective release of Insulin is observed, and the release of Insulin is enhanced as the concentration of glucose increased curves (c)-(f). At the high glucose concentration, 30mM, the release of insulin reaches a saturation value after ca. 35 minutes, curve (f). Further increase in the concentration of glucose do not affect the insulin release profile, and the release curve overlap curve (f) depicted in figure 7(A).

[0097] That is the saturated release of insulin corresponds to the release of the entire insulin loaded in the microcapsule-in-microcapsule system. Using an appropriate calibration curve for the fluorophore-labeled insulin, and knowing the concentration of the carrier, the loading of insulin in the bi-layer microcapsule was evaluated to be 4x10-15 mol/capsule, see supporting information. Figure 7(B) shows the switchable glucose triggered release of insulin from the microcapsules system. In this experiment, the insulin- loaded microcapsules were subjected to glucose, lOmM, and after a time-interval of ca. 50 minutes the release of insulin reaches a saturation value. By applying the appropriate calibration curve, it is estimated that a ca. 20% of the insulin that was loaded in the carriers was released. Parallel measurement of the glucose concentration in the solution, using a glucometer, resulted in a decrease in the glucose concentration from ca. 200 mg/dl to 75 mg/dl (lOmM to 4mM). The saturation value observed after 50 minutes is consistent with the inefficient release of insulin at glucose concentrations lower than <5mM. At point (a), marked with an arrow, the microcapsules were subjected to an addition increase in the glucose concentration (lOmM or 200mg/dl). This switches-on the release of the fluorophore-labeled insulin, resulting in a saturation value after ca. 60 minutes. In this second step additional 40% of the loaded insulin were released. Using the respective calibration curve, it was estimated that ca. 140 nM of insulin was within this step. Note that even though the additional concentration of glucose in the second step was lOmM a significantly higher insulin release is observed as compared to the release of insulin in the first steep. The glucometer-measured values of glucose outside the microcapsules indicated that even after a time-interval of 100 minutes, where the immediate release reach saturation, the outside concentration of glucose is ca. 4mM. Presumably, the flux of glucose penetration into the microcapsules decrease upon completion of the first insulin release cycle. The residual concentration of "inactive" glucose in the bulk solution increases the glucose concentration in the solution, to ca. 15mM (ca. 4mM from the first cycle that were not consumed in the addition to lOmM of the second cycle). This results in enhanced pH changes, cf. Figure 7(A), and increased concentrations of the released insulin.

[0098] The GOx bio-catalyzed oxidation of glucose in the core of bi-compartment microcapsule system is, however, accompanied by the generation of H202. This product might be harmful as it acts as precursor for reactive oxygen species (ROS). To eliminate this disturbing effect, the enzyme catalase, that disproportionate H202 into H20and 02, was added to the GOx-loaded insulin-modified microcapsule-in-microcapsule system Using the respective calibration curves, it is estimated that the loading of the catalase in the inner core corresponded to ca. 120 nM and the loading of GOx in the same inner confinement corresponded to ca 15 mM.

[0099] The system of the invention was compared with a couple of previous reports on the design of "artificial pancreas" systems, in Table 1 below.

Table 1 - Comparison of glucose controlling systems

System Size of Insulin Glucose Stability Remarks

Carrier Loading Concentration

Releasing Insulin

Glucose-Sensitive 5 pm Not lOOmM 2 weeks No

Microcapsules mentioned (equivalent to switching

2000mg/dL) not reported

Gu, Z. et al. ACS relevant to H202 not Nano 2013, 7(5), diabetic control, degraded 4194-4201 release at relevant glucose concentration is missing

Polysaccharide 100mm ² 7.9 to lOOmg/dL no 4 weeks Carrier is

Hydrogel patch 11.4 wt% significant release degraded,

Network Particles per possible particle 400mg/dL release immune

Qi, W. et al. proceed (very response Biomacromolecules high glucose 2009, 10(5), 1212— concentration)

1216 release at relevant glucose concentration 80 - 300mg/dL is not reported

Bi-compartmental 2.6±0.3pm 40mg/dL Release of insulin 2 - 3 Switchable Triggered Glucose complies with the months (8 ON/OFF oxidase/insulin of glucose range - 12 as long as the present diabetics weeks) loaded invention controlling 80 - lOOmg/dL - no release

In glucose concentration

<100mg/dL - insulin is released efficiently [00100] The invention provides a versatile method to assemble stimuli-responsive hydrogel -based microcapsule-in-microcapsule systems. The resulting microcapsule systems included two aqueous reservoirs, loaded with different loads and separated by stimuli-responsive hydrogel layers. It was demonstrated that the microcapsule-in microcapsule systems can be based on nucleic acid-functionalized CMC or nucleic acid modified polyacrylamide scaffolds as hydrogel -building scaffolds. The nucleic acids embedded in the hydrogel layer provide crosslinking duplex units and, most important, stimuli-responsive nucleic acids that upon reconfiguration control the stiffness of the hydrogel layers and their permeability towards loads. Specifically, the Zn2+-ions were used as irreversible trigger to unlock one of the hydrogel layer and pH or K+-ions/crown ether triggers were successfully applied to reversibly control the stiffness/permeability of the hydrogel through the switchable reconfiguration of i-motif or G-quadruplex structures, respectively.

[00101] These results suggest that other cofactor-dependent DNAzymes or irreversible light-induced cleavage of DNA duplex nucleic acid could lead to irreversible unlocking of the hydrogel layers. In addition, the reversible control over the stiffness of the hydrogel by means of metal-ions/ligands (e.g., T-Hg2+-T bridges/cysteine) or light (cis/trans isomerization of azobenzene intercalators) could be used to control the reversible permeabilities of the hydrogel layers.

[00102] It was found that the specific hydrogel layers used in the present study revealed a substrate permeability cut-off of < M.W. 5KDa, lower molecular-weight of substrates will permeate through the hydrogel layers. Substrates of higher molecular weight were non-permeable at higher stiffness of the hydrogel (no leakage phenomena were detected on a time-scale of ten days). These features of the systems are dictated by the degree of crosslinking of the hydrogel by the respective nucleic acid bridges. Further tuning the loading, and the return of nucleic acid bridges could further control the permeabilities of the hydrogels. Finally, to adapt the controlled release of the loads to the triggered stiffness properties of the hydrogels, luminescent quantum dots and drug functionalized polymers were applied as loads. One may, however, apply other nanoparticles or macromolecular nanostructures as loads. Besides the controlled release of the loads associated with the two aqueous reservoirs, in the presence of the appropriate triggers, an interesting enzyme- stimulated release of a drug (insulin) from the bi-compartmentalized microcapsule was demonstrated, thereby providing a microencapsulated system acting as an “artificial pancreas”. That is, the enzyme glucose oxidase was encapsulated in the inner aqueous compartment of the microcapsule, and insulin was loaded in the outer aqueous compartment. The freely permeating glucose led to the glucose GOx-catalyzed aerobic oxidation of glucose to gluconic acid. The acidification of the microcapsule-in microcapsule microenvironment, and the accompanying release of insulin were then guided by the concentration of glucose. That is, an autonomous microcarrier for the release of insulin in response to up-regulated contents of glucose is demonstrated. In principle, other enzymes altering the pH of the microcarriers may be loaded in the microcapsules for releasing other therapeutics. For example, acetylcholine esterase could be loaded in the microcapsules. Changes driven upon the hydrolysis of acetylcholine upon over-activation or inhibition of the enzyme may then release drugs that perturb the neural system. Furthermore, the triggered intercommunication (mixing) of the two aqueous compartments might lead to signal -dictated reactions in microreactors. The diversity of structural stimuli-responsive microcapsule-in-microcapsule systems and the variability of loads in the bi-compartmentalized microcapsule system provide different versatile applications of such carriers.

[00103] Experimental Section

[00104] Reagents and materials: Magnesium chloride, sodium chloride, 4- carboxyphenylboronic acid, 4-(2- hydroxyethyl)piperazine-l-ethanesulfonic acid sodium salt (HEPES base), 4-(2- hydroxyethyl)piperazine-l-ethanesulfonic acid (HEPES acid), N- (3 -dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC),

Nhydroxysulfosuccinimide sodium salt (sulfo-NHS), ammonium persulfate (APS), N,N,N',N'-tetramethylethylenediamine (TEMED), acrylamide solution (40%), poly(allylamine hydrochloride) (PAH, MW = 58 kE ⁾ a), ethyl enediaminetetraacetic acid disodium salt dihydrate (EDTA), glucose oxidase, insulin, catalase, zinc chloride, carboxymethyl cellulose (CMC, medium viscosity, D.S 0.9), rhodamine B isothiocyanate, fluorescein isothiocyanate isomer I (FITC), 7-hydroxycoumarin-3- carboxylic acid N- succinimidyl ester were purchased from Sigma- Aldrich. Tetramethylrhodamine-dextran (TMR-D, MW = 70 kDa) was purchased from Life Technologies Corporation (EISA). CdSe/ ZnS (2.25nm) QDs purchased from Evident technologies. All oligonucleotides were synthesized, purified by standard desalting, and freeze-dried by Integrated DNA Technologies, Inc. (Table 2). Ultrapure water from a NANOpure Diamond (Bamstead International, Dubuque, IA) source was used in all experiments. A Magellan XHR 400L scanning electron microscope (SEM), FV-1000 confocal microscope (Olympus, Japan) and Focused Ion Beam (FIB)- Helios NanoLab™ 460F1 DualBeam™ were employed to characterize the microparticles.

[00105] Synthesis of acrydite-modified oligo/ acrylamide copolymers: 100 pL of a solution consisting of 0.75 mM acrydite-modified oligonucleotide ((2), HI or (x)) and 1.5% acrylamide was bubbled with nitrogen for 3 min, followed by the addition of 7.5 pL of an initiator mixture (prepared as 10 mg APS in 5 pL TEMED and 95 pL H20). The solutions were subjected to an additional 5 min of nitrogen bubbling, followed by incubation at 4 °C for 12 h to form the copolymer chains. Polymers PA (2) was purified and separated from the unreacted compounds using a centrifugal filter device (Amicon, 30k MWCO), whereas polymers PB (3) was filtered using a 10k MWCO Amicon filter. After being washed with water three times, the copolymer solutions were dried under a gentle flow of nitrogen gas and redispersed in buffer (25 mM HEPES, pH 7.2, containing 25 mM MgC12 and 10 mM NaCl). 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.

[00106] Synthesis of 5' -Amino Modifier C6-modified oligo/Carboxymethyl cellulose (CMC) copolymers: A MES buffer solution (2 ml, 10 mM, pH = 5.5), , containing CMC, 20 mg, N-(3- dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride, 20 mg, was incubated for 5 minutes and then sulfo-N-hydroxysuccinimide, 26 mg, was added and the solution was incubated for additional 10 minutes. 2 ml of HEPES buffer (50 mM, pH = 7.0) were added, containing the amine-functionalized nucleic acid, (HI or (x)), 300 pM and 600 pM of (2). The mixture was allowed to react for 2 h in room temperature on gentle shaking. The modified polymer was separated using a MWCO 10K spin filter. After being washed with water three times, the copolymer solutions were dried and redispersed in buffer (25 mM HEPES, pH 7.2, containing 25 mM MgC12 and 10 mM NaCl). 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. [00107] Preparation of MPA-capped CdSe/ZnS QDs were prepared as it was described in Wang, X.; Zhang, M.; Xing, F.; Han, N. Effect of a Healing Agent on the Curing Reaction Kinetics and Its Mechanism in a Self-Healing System. Appl. Sci. 2018, 8 (11). [00108] Preparation of Insulin/GOx/Catalase-Fluorophore Conjugates were prepared as it was described as previously by G. Sitta Sittampalam et.al.

[00109] Preparation of CaC03 microparticles with different loads CaC03 particles were prepared by a precipitation reaction between equal amounts of CaC12 and Na2C03 under magnetic stirring at RT. CaC03 particles loaded with TMR-D, were obtained through coprecipitation by mixing of CaC12 (307 pL, 0.33 M) and Na2C03 (307 pL, 0.33 M) solutions, in the presence of TMR-D; the ratio of TMR-D and deionized water was adjusted to obtain a total volume of 1020 pL and 0.2 mg 3 mL-1 of TMR-D as final concentration. After magnetic stirring for 110s, the suspension was left for 70s at RT to settle down. The particles were centrifuged at 100 ref for 20s, followed by the removal of the supernatant solution, and the subsequent resuspension of the particles in water. This washing procedure was repeated for another two times, to remove byproducts resulting from the precipitation reaction.

[00110] The GOx contained core and the GOx -catalase contained core were prepared as described above.

[00111] Synthesis of DNA acrylamide/CMC hydrogel microcapsule-in-microcapsule: CaC03 microparticles (6.0 mg) were suspended in 300 pL of a 1 mg/mL solution of PAH (10 mM HEPES, pH 7.2, containing 500 mM NaCl and 50 mM MgC12) and kept under continuous shaking for an adsorption time interval of 30 min. The PAH-coated particles were washed twice with buffer (25 mM HEPES, pH 7.2, containing 25 mM MgC12 and 10 mM NaCl), followed by centrifugation at 100 ref for 20 s. Subsequently, the PAH- coated microparticles were incubated with 300 pL of the promoter nucleic acids (1) (the final concentration of the promoter nucleic acids was 10 pM) and kept under continuous shaking in room temperature for an adsorption time interval of 30 min. After being washed twice with buffer (25 mM HEPES, pH 7.2, containing 25 mM MgC12 and 10 mM NaCl), followed by centrifugation at 100 ref for 20 s, the DNA hydrogel particles were prepared by mixing each of the polymer sets (PA/PB) immediately before adding the solution to the CaC03 particles coated with the promoter. The final concentration of each hairpin was 10 pM. The particles were incubated at room temperature under continuous shaking overnight (approximately 12h), followed by centrifugation at 100 ref for 20 s to remove non-adsorbed polymers, and were washed twice with buffer (25 mM HEPES, pH 7.2, containing 25 mM MgC12 and 10 mM NaCl). The DNA-acrylamide/CMC hydrogel one-layer particles that formed were suspended in buffer solution (480 pL, 25 mM HEPES, pH 7.2, containing 25 mM MgC12 and 10 mM NaCl), and mixed with a solution of 307 pi CaC12 0.33M and 307 pi Na2C03 0.33M, in the presence of CdSe/ZnS QD in order to create the second core of CaC03; the ratio of CdSe/ZnS QD and deionized water was adjusted to obtain a total volume of 1020 pL and 0.2 mg 3 mL-1 of QD as final concentration. After magnetic stirring for 110 s, the suspension was left for 70 s at room temperature to settle down.

[00112] The particles were centrifuged at 100 ref 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, to remove byproducts resulting from the precipitation reaction. Then, the outer hydrogel layer was prepared with the same procedure as the inner layer. The microparticles were suspended in 300 pL of a 1 mg/mL solution of PAH (10 mM HEPES, pH 7.2, containing 500 mM NaCl and 50 mM MgC12) and kept under continuous shaking for an adsorption time interval of 30 min.

[00113] The PAH-coated particles were washed twice with a buffer (25 mM HEPES, pH 7.2, containing 25 mM MgC12 and 10 mM NaCl), followed by centrifugation at 100 ref for 20 s. Subsequently, the PAH-coated microparticles were incubated with 300 pL of the promoter nucleic acids (1) (the final concentration of the promoter nucleic acids was 10 pM) and kept under continuous shaking in RT for an adsorption time interval of 30 min. After being washed twice with a buffer (25 mM HEPES, pH 7.2, containing 25 mM MgC12 and 10 mM NaCl), followed by centrifugation at 100 ref for 20 s, the DNA hydrogel particles second-layer were prepared by mixing each of the polymer sets (PA/PB) immediately before adding the solution to the CaC03 particles coated with the promoter. The final concentration of each hairpin was 10 pM. The particles were incubated at room RT under continuous shaking overnight (approximately 12h), followed by centrifugation at 100 ref for 20 s to remove non-adsorbed polymers and were washed twice more with a buffer (25 mM HEPES, pH 7.2, containing 25 mM MgC12 and 10 mM NaCl). The DNA-acrylamide/CMC hydrogel bi-layer particles that were formed were suspended in a buffer solution (480 pL, 25 mM HEPES, pH 7.2, containing 25 mM MgC12 and 10 mM NaCl). The microcapsule-in-microcapsule system triggered by Zn2+ ions and K+ ions was prepared as described above using the G-quadruplexes sequence instead i-motif (see Table 2 of the sequences used). Table 2 - G-quadruplexes sequences used in microcapsule-in-microcapsule system of the invention triggered by Zn2+ ions and K+ ions

[00114] Similarly, the microcapsule-in-microcapsule system containing fluorophore modified- GOx (60 pL of 1 mg/mL solution) or fluorophore modified-GOx/catalase (100 pL of 24 mg/mL solution) in the inner core and fluorophore modified insulin (30 pL of 1 mg/mL solution) in the outer core was prepared as described above excluding the Zn2+- ion dependent DNAyzme (4) in the inner hydrogel layer and exchanged the dyes for the respective enzymes.

[00115] Dissolution of the Core: 120 pL of a 0.5 M solution of EDTA (pH 7.5) was added into the solution of the particles and incubated for 1 h to dissolve the CaC03 cores. When the suspension became clear, the resulting capsules were washed with a buffer (25 mM HEPES, pH 7.2, containing 25 mM MgC12 and 10 mM NaCl) using slow centrifugation (50 ref, 20 min) three times.

[00116] Stimuli-induced unlocking of the hydrogel microcapsules and release of the encapsulated loads Zn2+ ions and pH responsive system: Samples of 9 pL of HC1 (1 M) and/or ZnC12 (1 M) were added to 120 pL of the DNA- aery 1 ami de\CMC hydrogel bi layer responsive microcapsule-in-microcapsule, containing 1700 capsule per pi. Then the release of the respective dyes was measured through fluorescence (TMR-D (lec =546nm ; Zem = 580nm ), CdSe/ZnS (2.25nm) QD (lec =464nm ; Zem = 482nm )). Zn2+ ions, K+ ions responsive system Samples of 9 pL of CE (1.5 M) and/or ZnC12 (1M) were added to 120 pL of the DNA- acrylamide\CMC hydrogel bi-layer responsive microcapsule-in microcapsule, containing 1700 capsule per pi. Then the release of the respective dyes was measured through fluorescence (TMR-D (lec =546nm ; Zem = 580nm ), CdSe/ZnS (2.25nm) QD (lec = 464nm ; Zem = 482nm )).

[00117] While certain features of the invention have been illustrated and described herein, many modifications, substitutions, changes, and equivalents will now occur to those of ordinary skill in the art. It is, therefore, to be understood that the appended claims are intended to cover all such modifications and changes as fall within the true spirit of the invention.