Glucose-responsive glucagon-micelle for the prevention of hypoglycemia and methods of use

Abstract

Disclosed is a polymer comprising an ordered arrangement of a polyethylene glycol block, a copolymer block comprising A-isopropyl acrylamide (NIPAM) and either acrylamidophenylboronic acid (2-APBA) and / or acrylic acid (AAc), and an end-group selected. Methods of making and using the polymer is also provided.

Description

[0001]790482.00511 GLUCOSE-RESPONSIVE GLUCAGON-MICELLE FOR THE PREVENTION OF HYPOGLYCEMIA AND METHODS OF USE CROSS-REFERENCE TO RELATED APPLICATIONS The present application claims benefit of priority to U.S. Provisional Application Ser. No.63 / 637,818, filed April 23, 2024, the contents of which is incorporated by reference in its entirety. REFERENCE TO AN ELECTRONIC SEQUENCE LISTING The contents of the electronic sequence listing (79048200511.xml; Size: 2,727 bytes; and Date of Creation: April 18, 2025) is herein incorporated by reference in its entirety. BACKGROUND OF THE INVENTION Glucagon is a peptide hormone that interacts with glucagon receptors in the liver to trigger the conversion of glycogen into glucose, raising blood glucose levels. It is commonly used by hypoglycemic patients and to treat bradycardia resulting from beta-blocker overdose; however, high cost, limited availability, and primarily instability currently thwart its full clinical potential. Currently glucagon is used for emergency treatments in clinical use. Considering the number of hypoglycemic episodes experienced by diabetic patients, there is a great need to create formulations that respond to low glucose levels to reveal glucagon. BRIEF SUMMARY OF THE INVENTION Disclosed is a polymer comprising an ordered arrangement of a polyethylene glycol block, a copolymer block comprising N-isopropyl acrylamide (NIPAM) and either acrylamidophenylboronic acid (2-APBA) and / or acrylic acid (AAc), and an end-group. The end-group may be selected from a conjugated therapeutic agent, a conjugated reporter, a disulfide exchange functional group, an activated disulfide, a pyridyl disulfide, a nitrobenzoic acid, a disulfide reductant, a Michael acceptors, a maleimide, a maleimide derivative, a dihalomaleimide, a vinyl group, a vinyl sulfone, an acryloyl derivative, a haloacetyl, an alkyl halide derivative, an aziridine, an arylating agent, an isothiocyanate, an isocyanate, an acryl azide, an activated ester, a N-hydroxysuccinimide ester, a para-nitrophenyl ester, a sulfonyl chloride, an aldehyde, a glyoxal (with or without reductive amination), an epoxide (also called oxirane), a carbonate, an arylating agent, an imidoester, a carbodiimide, a anhydride, a primary amine, a secondary amine, a tertiary amine, a diazoalkane, a diazoacetyl, a carbonyldiimidazole, a carbonate, a chloroformate, an alkyl halogen, an aminooxy (hydroxylamine), a hydrazine, an alkyne, a chain transfer agent, a derivative thereof, or a combination thereof.. Methods of making and using the polymer is also provided. The QB\790482.00511\96013124.1 1 790482.00511 copolymer block may comprise comprising N-isopropyl acrylamide (NIPAM) and acrylamidophenylboronic acid (2-APBA). Suitably, the polymer comprises the conjugated therapeutic agent. The conjugated therapeutic agent is a conjugated glucagon or derivative thereof, such as a cysteine-substituted glucagon. Also disclosed are nanostructures comprising a plurality of polymers described herein. The nanostructure may be stable above a threshold glucose concentration and below a threshold glucose concentration the nanostructure undergoes disassembly, partial disassembly, or swelling at a physiologically-relevant temperature and / or stable above threshold glucose concentration and below threshold glucose concentration the nanostructure undergoes disassembly, partial disassembly, or swelling. Methods for treating or preventing a disease or condition a subject in need of a therapeutic agent are also provided. The method may comprise administering any of the pharmaceutical compositions described herein to the subject. Suitably, the subject may be diabetic or in need of insulin therapy. In some cases, the subject is administered insulin prior to or concurrently with the administered pharmaceutical composition. Alternatively, the subject is administered insulin after the pharmaceutical composition is administered. BRIEF DESCRIPTION OF THE DRAWINGS Non-limiting embodiments of the present invention will be described by way of example with reference to the accompanying figures, which are schematic and are not intended to be drawn to scale. In the figures, each identical or nearly identical component illustrated is typically represented by a single numeral. For purposes of clarity, not every component is labeled in every figure, nor is every component of each embodiment of the invention shown where illustration is not necessary to allow those of ordinary skill in the art to understand the invention. Figures 1. Illustrates a schematic of glucagon-polymer conjugate. Mode of action overview of PEG-b- P(NIPAM-stat-2-APBA)-GCG between micellar form in normoglycemic conditions and linear (unimer) format in hypoglycemic conditions. (a) Glucose-responsive self- assembly and disassembly of PEG-b-P(NIPAM-stat-2-APBA)-GCG conjugate. Binding of glucose with PBA group, increases the hydrophobicity of the P(NIPAM-stat-2-APBA) block so that at 37 °C, the polymer forms micelles. When glucose levels are lowered, the P(NIPAM- stat-2-APBA) core becomes more hydrophilic, and the micelle disassembles. (b) Exposure to normoglycemic conditions ([Glc]> 100 mg / dL), GCG- micelles retain their nanoparticle format. Under exposure to deep hypoglycemia ([Glc] < 60 mg / dL), GCG-micelles disassemble promoting the release of GCG and subsequent increase of blood glucose concentration. QB\790482.00511\96013124.1 2 790482.00511 Figures 2. (a) Synthetic route of PEG-b-P(NIPAM-stat-2-APBA)-GCG (P2-GCG) conjugate (AIBN = 2,2’- azobis(2-methylpropionitrile), DMSO = dimethyl sulfoxide, EDC = N-3-dimethylaminopropyl)-N’- ethylcarbodiimide hydrochloride, DMAP = 4- (dimethylamino)pyridine, MeOH = methanol, HCl = hydrochloric acid) (b) GCG-SH conjugation to PEG-b-P(NIPAM-stat-2-APBA)-PDS monitored by HPLC at λ = 224 nm. (c) LC-MS mass spectra of fresh GCG-SH and (d) GCG-SH released from the conjugate with TCEP (10 mM). (e) Normalized cloud point (10 mg / mL) of block copolymer candidate (P2) before and after GCG-conjugation measured by UV-vis spectroscopy between 25-65 ºC by determining absorbance at λ = 600 nm. (f) Intensity- weighted size distributions obtained by DLS for GCG-micelles at 37 ºC, showing no micelle formation in absence of glucose or at hypoglycemia, micelle formation at normoglycemia and their disruption when the media is diluted to hypoglycemic level. [Glc] = 0, 60, 150, and 60 mg / dL upon dilution. (g) TEM image of GCG-micelle at 25 ºC, presenting no micelles and (h) at 40 ºC, presenting defined micelles. (i) Dose response curves of native GCG, GCG-SH, P2, GCG-micelle and TCEP reduced P2 using commercial kit cAMP Hunter™ eXpress GCGR CHO-K1 GPCR assay. j) EC50 values of native GCG, GCG-SH, GCG-micelle and TCEP reduced P2 using commercial kit cAMP Hunter™ eXpress GCGR CHO-K1 GPCR assay. Data are shown as the mean ± SEM of five to six independent repeats. p < 0.05 (*), p < 0.01 (**), p < 0.001 (***), p < 0.0001 (****). Figures 3. Chronic toxicity with daily injection of micelles for 14 days. (a) The schematic representation of the study design. (b) Whole blood analysis (CBC) (n = 5) (i) RBC (ii) WBC (iii) Platelet count (iv) % monocytes (v) % Lymphocytes (vi) % Neutrophils (vii) % Eosinophils (viii) Hemoglobin (ix) % Hematocrit (x) % Reticulocyte (xi) % Red Cell Distribution Width (xii) Mean Corpuscular Hemoglobin (MCH). (c) Hepatic function parameters (i) ALT (ii) AST (iii) LDH (n = 8). (d) Kidney function parameters (i) Calcium (n = 4) (ii) Glucose (n = 10). (e) Hematoxylin and eosin staining of the liver, spleen, heart, lungs, and kidney (magnification 1.25x, 4x, 10x). p < 0.05 (*) Figures 4. (a) Quantification of the immune markers (i) IFN γ (ii) TNF α and (iii) IL2 (n = 8). (b) Immunochemistry to show the tissue-specific markers by using F8 / 40 antibody (i) lungs (ii) liver. Figures 5. μPET-μCT imaging showing the time course biodistribution and excretion of18F-FBEM- labeled-micelle and18F-SFB-labeled GCG (n = 4). a. Chemical structure of (i)18F-FBEM-labeled micelle and (ii)18F-SFB-labeled GCG. b. PET-CT images of (i)18F-FBEM- labeled micelle and (ii)18F- SFB-labeled GCG. The time course biodistribution in blood, liver, QB\790482.00511\96013124.1 3 790482.00511 left kidney, right kidney, bladder, muscle, left lung, right lung, gall bladder, GI, spleen, and blood half-life time. ID, injected dose. CC, cubic centimeters. Figures 6. (a) (i) Impact of micelles on glycemia in male C57Bl / 6J mice over 8 h. Data is represented as mean ± SEM, n = 5-6. (b) (i) Micelle-induced prevention of insulin-stimulated hypoglycemia in fasted C57Bl / 6J mice. (c) Micelle-reversal of insulin induced i) deep or ii) moderate hypoglycemia in fasted C57Bl / 6J mice. Dotted line represents approximate gluco- counterregulatory threshold. Data is represented as mean ± SEM, n = 5-6. p < 0.05 (*), p < 0.01 (**), p < 0.001 (***), p < 0.0001 (****). Figures 7. Circular dichroism (CD) spectra of native and thiolated glucagon (GCG) recorded at 0.125 mg / mL using a mixture of DPBS (pH = 7.4) / HCl (pH = 3) (1 / 1). Figures 8. Predicted 3D structure of native and thiolated glucagon by AlphaFold.(61, 62) Figures 9.SDS-PAGE. Lane 1: protein ladder; lane 2: GCG-SH, lane 3: PEG44-b-P(NIPAM- stat-2-APBA) (P2); lane 4: PEG44-b-P(NIPAM-stat-2-APBA) reduced withdithiothreitol (DTT);lane 5: crude conjugation reaction; lane 6: crude conjugation reactionreduced with DTT; lane 7:pure conjugate PEG44-b-P(NIPAM-stat-2-APBA)-GCG (P2-GCG); lane 8: pure conjugate PEG44-b-P(NIPAM-stat-2-APBA)-GCG (P2-GCG) reducedwith DTT. Figure 10. (a) Normalized cloud points of P1-P4 (10 mg / mL in DPBS) measured by UV-vis spectroscopy in the 26-65 ºC range by determining absorbance at 600 nm. (b) Intensity- weighted size distributions obtained by DLS of P2 and P3 at 37 ºC in the presence of [Glc] = 150 mg / dL indicating no formation of well-defined micelles. Figure 11. a) Normalized cloud point of P4 and P4-GCG (10 mg / mL in DPBS) measured by UV-Vis spectroscopy in the range of 26-62 ºC by determining absorbance at λ = 600 nm. b) Intensity-weighted size distributions obtained by DLS for P4 and P4-GCG at 37 ºC, without glucose addition. Figure 12. a) Normalized cloud point of P3 and P3-GCG (10 mg / mL in DPBS) measured by UV-vis spectroscopy in the 26-65 ºC range by determining absorbance at λ = 600 nm. b) Intensity-weighted DLS curves of P3-GCG at 37 ºC and P3 at 37 and 40 ºC. Figure 13. a) Acute toxicity analysis: a. Whole blood analysis (CBC results) (i) RBC (ii) WBC (iii) percentage of monocyte (iv) percentage of Lymphocyte (v) percentage of neutrophils (vi) percentage of eosinophils (vii) hemoglobin (viii) percentage of hematocrit. b. Total weight (grams) of different organs collected. QB\790482.00511\96013124.1 4 790482.00511 Figure 14. Chronic toxicity analysis: a. The body weight of the mice treated with saline (control) and empty micelle b. The weight of the different organs in grams (i) liver (ii) lungs (iii) heart (iv) spleen (v) kidney. Figure 15. µPET / µCT analysis of89Zr-labeled linear polymer and89Zr-labeled micelle.(a)Chemical structure of 89Zr-chelated PEG44-b-P(NIPAM-stat-2-APBA)-DFO. (b) Intensity-weighted size distributions obtained by DLS for89Zr-labeled linear polymer and89Zr-labeled micelle. (c) Time course biodistribution and excretion of fresh89Zr-labeled linear polymer and89Zr-labeled micelle from (c) gastrointestinal (GI), (d) spleen, (e) right lung, (f) left lung, (g) muscle, (h) bladder, (i) right kidney, (j) left kidney, (k) liver, (l) blood, (m) brain and (n) blood half time of eight C57BL / 6 male (n = 8). Note the scale of %ID / cc differs between each organ. ID, injected dose. CC, cubic centimeter. Figure 16. Co-registered μPET / μCT scans post89Zr-labeled micelle injection to four C57BL / 6 male mice. ID, injected dose. CC, cubic centimeter. Figure 17. Co-registered μPET / μCT scans post89Zr-labeled linear polymer injection to four C57BL / 6 male mice. ID, injected dose. CC, cubic centimeter. Figure 18. Quantification of μPET / µCT scans for89Zr-labeled linear polymer and89Zr- labeled micelle 192 h post-injection from the organs with appreciable signal. ID, injected dose. CC, cubic centimeter. Figure 19. Co-registered μPET and μCT scans post18F-SFB-labeled glucagon injection to four C57BL / 6 male mice. ID, injected dose. CC, cubic centimeter. Figure 20. Co-registered μPET and μCT scans post18F-FBEM-labeled micelle injection to four C57BL / 6 male mice. ID, injected dose. CC, cubic centimeter. Figure 21. H&E staining of kidney and liver tissue of C57Bl / 6J mice treated with GCG- micelle (P2-GCG) or PBS. Figure 22. Conjugation of non-radioactive19F-SFB prosthetic group to GCG. (a) After 5 minutes, GCG is almost entirely labeled with19F-SFB. (b) Conjugation after 10 minutes is complete with no further conversion up to 60 minutes. (c) After purification by centrifugal filtration (MWCO = 3 kDa),19F-SFB-labeled GCG (1 SFB) is the primary species in solution. A small percentage of GCG is unlabeled or labeled with 2 SFB consistent with the 2 lysine residues present in GCG sequence. (d) LC-MS confirms the presence of the19F-SFB-labeled GCG conjugate. QB\790482.00511\96013124.1 5 790482.00511 Figure 23.19F-NMR with proton decoupling recorded in D2O. Conjugation of 19F-FBEMprosthetic group to the micelle. The FBEM alone appears at δ = -108 ppm and shifts toδ = -75 ppm after conjugation to the micelle. Figure 24. Intensity-weighted size distributions by DLS of micelle at 37 ºC prior to labeling and after labeling with non-radioactive19F-FBEM. Figure 25. HPLC chromatogram of (a) purified18F-SFB and (b) purified18F-SFB co- injected with SFB reference standard. Column: Column: Phenomenex C185 µ Luna 250 x 4.6mm; Mobilephase: 50% v / v MeCN in H2O (0.1% TFA); flow rate: 1 mL / min.Figure 26. HPLC chromatogram of (a) purified18F-FBEM and (b) purified18F-FBEM co- injected with FBEM reference standard. Column: Column: Phenomenex C185 µ Luna 250 x 4.6 mm; Mobile phase: 20% v / v MeCN in H2O; flow rate: 1.5 mL / min. Figure 27. (a) NMR of poly(ethylene glycol)-b-poly(N-isopropylacrylamide-stat-2- acrylamidophenylboronic acid-stat-pyridyl disulfide methacrylate. (b) DLS showing the glucose responsiveness of GCG micelles at different glucose concentration. (c) TEM imagesshowing the glucose responsiveness of GCG micelles at different glucose concentration. (d)GCG-SH conjugation to poly(ethylene glycol)-b-poly(N-isopropylacrylamide-stat-2- acrylamidophenylboronic acid-stat-pyridyl disulfide methacrylate monitored by HPLC at λ = 224 nm. Figure 28.In vitro stability analysis of glucagon degradation and fibrillation. a.Degradation of glucagon in both its free and conjugated forms was assessed at room temperature and 4 °C using HPLC. b. Fibrillation was evaluated by the ThT assay at room temperature (r.t.23 °C) and fridge (4 °C) for glucagon in the micelle and compared to glucagon alone at room temperature and ThT alone. Figure 29. The in vivo efficacy and dose-dependent studies of the glucagon micelles from 500 μg / Kg to 50 μg / Kg in insulin-induced hypoglycemic C57Bl / 6J male mice. Data are represented as mean ± SEM, n = 4–6. p < 0.05 (*), p < 0.01 (**), p < 0.001 (***), p < 0.0001 (****). Statistical comparison of glucose levels between the two groups at the same time point during the ITT. Figure 30. Impact of micelles on euglycemia in male C57Bl / 6J mice over 8 h. Data is represented as mean ± SEM, n = 5-6. Figure 31. The in vivo efficacy studies determine whether the micelles remain effective when administered (a) 30, (b) 60, and (c) 90 minutes prior to insulin injection. Data are QB\790482.00511\96013124.1 6 790482.00511 represented as mean ± SEM, n = 5–6. p < 0.05 (*), p < 0.01 (**), p < 0.001 (***), p < 0.0001 (****). Figure 32. Chronic toxicity with daily injection of micelles for 14 days. (a) Total IgG and (b) Total IgM levels were measured using ELISA to assess the immunogenicity of the glucagon micelles. (c) Hematoxylin and eosin staining of the liver, spleen, heart, lungs, and kidney (magnification 4x, 10x). (d) Hepatic function parameters (i) ALT (ii) AST. (e) Immunochemistry to show the tissue-specific markers by using F8 / 40 antibody spleen, kidney, lungs and liver. Figure 33. (a) Schematic of in vivo studies to screen micelle polymer blood half-lives. (b) Conjugation of 5 nm GNPs to micelle polymer and TEM analysis showing 1-3 GNPs per 50 nm micelle. (c) In vivo experiment scheme and ICP-MS analysis of blood samples. (D) %ID / cc (% gold per blood volume) of micelle polymers tested. DETAILED DESCRIPTION OF THE INVENTION The disclosed technology provides for a glucose-responsive polymer-glucagon conjugate, compositions and methods for making the same, and uses of the glucose-response polymer-glucagon conjugate, such as for prevention and / or reversion of hypoglycemia. The compounds described herein include a polymer comprising an ordered arrangement of a polyethylene glycol block, a copolymer block comprising N-isopropyl acrylamide (NIPAM) and either acrylamidophenylboronic acid (2-APBA) or acrylic acid (AAc), and an end-group selected from a conjugated therapeutic agent, a conjugated reporter, a disulfide exchange functional group, a chain transfer agent, an activated disulfide, a pyridyl disulfide, a nitrobenzoic acid, a disulfide reductant, a Michael acceptors, a maleimide, a maleimide derivative, a dihalomaleimide, a vinyl group, a vinyl sulfone, an acryloyl derivative, a haloacetyl, an alkyl halide derivative, an aziridine, an arylating agent, an isothiocyanate, an isocyanate, an acryl azide, an activated ester, a N-hydroxysuccinimide ester, a para-nitrophenyl ester, a sulfonyl chloride, an aldehyde, a glyoxal (with or without reductive amination), an epoxide (also called oxirane), a carbonate, an arylating agent, an imidoester, a carbodiimide, a anhydride, a primary amine, a secondary amine, a tertiary amine, a diazoalkane, a diazoacetyl, a carbonyldiimidazole, a carbonate, a chloroformate, an alkyl halogen, an aminooxy (hydroxylamine), a hydrazine, an alkyne, a derivative thereof, or a combination thereof. In some embodiments, the end-group is selected from a conjugated therapeutic agent, a conjugated reporter, a disulfide exchange functional group, and a chain transfer agent. The QB\790482.00511\96013124.1 7 790482.00511 copolymer block may include a conjugated therapeutic agent or disulfide exchange functional group. The glucose-responsive polymer-glucagon conjugate comprises a copolymer block including N-isopropyl acrylamide (NIPAM) and acrylamidophenylboronic acid (2-APBA). Placing boronic acid in the ortho position to the amide as in 2-acrylamidophenylboronic acid (2-APBA) results in formation of an intramolecular B-O dative bond. When glucose is added to a polymer containing this functional group, the resulting material becomes more hydrophobic leading to polymers with a lower critical solution temperature (LCST). When nanostructures are formed of the glucose-responsive polymer-glucagon conjugate, the LCST can lead to disassembly of the nanostructure and unmasking of the glucagon conjugate. The Examples demonstrates that the behavior of glucose-responsive polymer-glucagon conjugates may be tuned for applications. For example, glucose- and thermo-responsiveness may be tuned by altering the composition of the glucose-responsive polymer-glucagon conjugate. The copolymer block may further comprise a conjugated therapeutic agent and / or a disulfide exchange functional group. In some embodiments, the polymer comprises a conjugated therapeutic agent. The conjugated therapeutic agent may be incorporated in the block copolymer and / or the end-group of the polymer. Therapeutic agents contemplated include proteins as well as other types of therapeutic agents that may elicit the desired effect. The conjugated therapeutic agent may be glucagon or a glucagon analog. The term "glucagon," as used herein, refers to a peptide hormone, produced by alpha cells of the pancreas. Glucagon works to raise the concentration of glucose and fat in the bloodstream. Glucagon may be considered to be the main catabolic hormone of the body. Glucagon may also be used as a medication to treat a number of health conditions. Glucagon's effect is opposite to that of insulin, which lowers the extracellular glucose. Glucagon's primary structure in humans is: HSQGTFTSDYSKYLDSRRAQDFVQWLMNT (SEQ ID NO: 2). The polypeptide of glucagon has a molecular weight of 3482.75 Daltons. The term “glucagon analog," "glucagon derivative,” or “glucagon conjugate” as used herein, refers to a substance that binds to a glucagon receptor and elicits the same biological activity as that of glucagon. The glucagon derivative may have an amino acid sequence which shares at least 80% homology with native glucagon, and may include a chemical substitution, deletion, or modification at some amino acid residues. The glucagon derivative suitable for the present invention may be selected from the group consisting of agonists, derivatives, fragments and variants of native glucagon, and a combination thereof. In further embodiments, the glucagon analog is a cysteine-containing QB\790482.00511\96013124.1 8 790482.00511 glucagon. The cysteine-containing glucagon analog may be covalently grafted to the polymer end-group. The cysteine-containing glucagon analog may alternatively be incorporated into the copolymer block. It is envisioned that when a protein or peptide like glucagon is conjugated (e.g., covalently bonded) with the polymers as described herein, the protein or peptide retain at least some of its native bioactivity compared to protein or peptide by itself. The conjugated therapeutic agent may be conjugated glucagon, cysteine-substituted glucagon, such as SEQ ID NO: 1. Exemplary glucose-responsive polymer-glucagon conjugate include PEG-b-P(NIPAM- stat-2-APBA)-GCG, as illustrated in Figure 1, and PEG-b-P(NIPAM-stat-2-APBA-stat- GCG). The term "protein" used herein refers to any compound of two or more individual amino acids (whether or not naturally occurring) linked via peptide bonds, as occur when the carboxyl carbon atom of the carboxylic acid group bonded to the a-carbon of one amino acid (or amino acid residue) becomes covalently bound to the amino nitrogen atom of the amino group bonded to the a-carbon of an adjacent amino acid. These peptide bond linkages, and the atoms comprising them (i.e., a-carbon atoms, carboxyl carbon atoms (and their substituent oxygen atoms), and amino nitrogen atoms (and their substituent hydrogen atoms)) form the "polypeptide backbone" of the protein. In addition, as used herein, the term "protein" is understood to include the terms "polypeptide" and "peptide." Similarly, protein fragments, analogs, derivatives, and variants may be referred to herein as "proteins," and shall be deemed to be a "protein" unless otherwise indicated. The term "fragment" of a protein refers to a polypeptide comprising fewer than all of the amino acid residues of the protein. As may be appreciated, a "fragment" of a protein may be a form of the protein truncated at the amino terminus, the carboxyl terminus, and / or internally (such as by natural splicing), and may also be variant and / or derivative. A "domain" of a protein is also a fragment and comprises the amino acid residues of the protein required to confer biochemical activity corresponding to naturally occurring protein. The term "protein" used herein also include "protein conjugate" which refers to a compound complex comprising a "protein" which is interlinked to one another molecule or subject. The term "complex" is used herein to mean those compounds comprising at least two components. The protein may be produced using DNA recombination or mutation techniques. The protein may be produced in vivo in a whole animal, or in a eukaryotic or prokaryotic cell. Alternatively, the protein may be generated using an in vitro method such as cell-free in vitro translation e.g. using E. coli lysate, wheat germ extract, or rabbit reticulocyte. Cell free in vitro translation methods can be employed following in vitro transcription, e.g. QB\790482.00511\96013124.1 9 790482.00511 following phage or ribosome display. In yet other methods, the protein may be synthetically produced, e.g., using solid-phase peptide synthesis. The presently disclosed nanostructure comprise a plurality of the presently disclosed glucose-responsive polymers as described herein. The term “nanostructure” used herein refers to any compound with a structure of intermediate size between microscopic and molecular structures. Nanostructures may be characterized by a length of about 10 to about 1000 nm. In some cases, the vesicles may be characterized by their diameter, such as less than about 100 nm in diameter, about 100 to 500 nm, or greater than 500 nm. Under certain conditions, the polymers described herein self-assemble into nanostructures. Self-assembly may be driven by non-covalent interactions between the polymers. Nanostructures may also be characterized by their morphology. The nanostructure may comprise a single layer of amphiphilic polymers, e.g., a micelle or a reverse micelle, having a hydrophilic surface and a hydrophobic surface or a bilayer comprising two layers of amphiphilic polymers having an inner-hydrophilic surface, an outer-hydrophilic surface, and a hydrophobic core disposed between the inner-hydrophilic surface and the outer-hydrophilic surface, e.g., a liposome or a lipid nanoparticle. In some embodiments, the nanostructure is a micelle. The nanostructure may be stable above a threshold glucose concentration and below a threshold glucose concentration the nanostructure undergoes disassembly, partial disassembly, or swelling at a physiologically-relevant temperature (e.g., from 35-40 ⁰C). When the glucose concentration is lowered below the threshold glucose concentration, the nanostructures may become unstable and disassemble. The disassembly results in an unmasking of the conjugated therapeutic agent. In some embodiments, the nanostructure is stable above a threshold glucose concentration and below a threshold glucose concentration the nanostructure undergoes disassembly, partial disassembly, or swelling in vivo. The threshold glucose concentration may be varied depending on the composition of the polymer. For example, the nanostructure may be stable above a threshold glucose concentration for normoglycemia (e.g., 100 mg / dL) and unstable below the threshold for normal glycemia, the nanostructure may be stable above a threshold glucose concentration for mild hypoglycemia (e.g., 70 mg / dL) and below the threshold for mild hypoglycemia, the nanostructure may be stable above a threshold glucose concentration for moderate hypoglycemia (e.g., 65 or 60 mg / dL) and unstable below the threshold for moderate hypoglycemia, or the nanostructure may be stable above a threshold glucose concentration for severe hypoglycemia (e.g., 55 mg / dL) and unstable below the threshold for severe hypoglycemia. QB\790482.00511\96013124.1 10 790482.00511 The Examples demonstrate a glucose-responsive polymeric nanosystem with a cysteine-containing glucagon analog covalently grafted to the polymer end-group and / or copolymer block. Under normoglycemic conditions, phenylboronic acid units in the polymer chain reversibly bind glucose, triggering the self-assembly of the conjugate into micelles due to transition-induced increased hydrophobicity. During hypoglycemia however, the micelle disassembles into its original, unimeric state, revealing the active glucagon conjugate. Complete disassembly is not required for activity. In some embodiments, the micelle may partially disassemble or swell in response to the hypoglycemic environment. These partially disassembled micelles can reveal the active glucagon conjugate and elicit a therapeutic effect. The designed formulation showed a five-fold increase in activity compared to native glucagon when tested in vitro, indicating that the polymer may assist glucagon receptor binding and / or stabilize the peptide. The in vivo acute and chronic toxicity analysis, along with µPET / µCT imaging, established the biosafety profile of this formulation and demonstrated no organ accumulation and full polymer clearance within two hours. Intraperitoneal boluses of glucagon-micelle were injected into mice experiencing insulin-induced hypoglycemia, showing successful regulation of glucose levels by reverting and, most importantly, preventing deep hypoglycemia within two hours. Release was only observed at or below the counterregulatory threshold, further demonstrating safety in mice. However, the compositions described herein may be modified for release at moderate or mild hypoglycemia conditions. These Examples demonstrate a stimuli-responsive glucagon delivery platform to control glycemia. Compounds and methods for preparing the glucose-responsive polymer-glucagon conjugates are also provided. In some embodiments, the polymer comprises a disulfide exchange functional group. Compounds containing a disulfide group are able to participate in disulfide exchange reactions with another thiol. The disulfide exchange (also called interchange) process involves attack of the thiol at the disulfide, breaking the -S–S- bond, with subsequent formation of a new mixed disulfide comprising a portion of the original disulfide compound. Crosslinking or modification reactions using disulfide exchange processes form disulfide linkages with sulfhydryl-containing molecules. These bonds are reversible using disulfide reducing agents. Thus, conjugates may be created and later released with disulfide reductants. In some embodiments, the exchange group is selected from maleimides, vinyl sulfone, alkyl iodides, or any electrophile. An exemplary disulfide exchange functional group is a disulfide pyridyl group. A thiolated glucagon analogue may be covalently grafted at the ω- polymer-end group via the disulfide exchange functional group. Exemplary compounds QB\790482.00511\96013124.1 11 790482.00511 include PEG-b-P(NIPAM-stat-2-APBA)-PDS as illustrated in Figure 2. Another way to incorporate the thiolated glucagon analogue may be through covalent conjugation to form the disulfide bond in the copolymer block via pyridyl disulfide exchange. Exemplary disulfide exchange groups in the copolymer block may include pyridyl disulfide methacylate (PDSMA). Exemplary polymers include PEG-b-P(NIPAM-stat-2-APBA-stat-PDSMA), as illustrated in Figure 27. In some embodiments, the thiolated glucagon analogue may be incorporated covalently grafted at the ω-polymer-end-group via the disulfide exchange functional and through covalent conjugation to form the disulfide bond onto the polymer via pyridyl disulfide exchange. In some embodiments, the polymer comprises a chain transfer agent. Chain transfer agents are moieties that react with a growing polymer radical, causing the growing chain to terminate while creating a new reactive species capable of initiating polymerization. Chain transfer agents are useful for controlling the molecular weights of polymers. In some embodiments, the chain transfer agent is selected from trithocarbonates, dithiobenzoates, dithicarbamates, or xanthates. An exemplary chain transfer agent comprises a trithiocarbonyl group, such as prepared from 2-[[(ethylthio)carbonothioyl]thio]-2- methylpropanoic acid. Aminolysis or reduction of the polymer’s terminal trithiocarbonyl group may be done for the installation of a disulfide exchange functional group, e.g., a thiol-reactive pyridyl disulfide (PDS) moiety at the ω-end group. Exemplary compounds include PEG-b-P(NIPAM-stat-2- APBA), PEG-b-P(NIPAM-stat-AAc), and PEG-b- P(NIPAM-stat-2-APBA-stat-PDSMA). In some embodiments, the polymer comprises a conjugated reporter. The conjugated reported may comprise a conjugated fluorophore, a conjugated radiolabel, or a metal. The term “fluorophore” used herein refers to any compound that that absorb and emit energy in a predictable fashion. They re-emit light when excited, making them useful tags for identifying and characterizing cells or molecules in a mixture, visualizing proteins, or quantifying tagged compounds. In some embodiments, the fluorophore is a protein. The term “radiolabel” used herein refers to any compound with a radioactive atom or substance. Suitable radiolabels include but are not limited to radiolabeled89Zr- labeled micelle (n = 4), radiolabeled89Zr- labeled linear polymer (n = 4),18F- FBEM micelle polymer (n = 4), radiolabeled18F-SFB glucagon non-micelle polymer (n = 4). Suitable exemplary metals include but are not limited to gold nanoparticles. In some embodiments, the polymer has a Mn between 1 and 50 kDa, e.g., between 1 and 5 kDa, between 5 and 10 kDa, between 10 and 15 kDa, between 15 and 20 kDa, between 20 and 25 kDa, between 25 and 30 kDa, between 30 and 35 kDa, between 35 and 40 kDa, QB\790482.00511\96013124.1 12 790482.00511 between 40 and 45 kDa, between 45 and 50 kDa, between 50 and 55 kDa, between 55 and 60 kDa, between 60 and 65 kDa, between 65 and 70 kDa, between 70 and 75 kDa, between 75 and 80 kDa, between 80 and 85 kDa, between 85 and 90 kDa, between 90 and 95 kDa, between 95 and 100 kDa, between 100 and 110 kDa, between 110 and 115 kDa, or between 115 and 120 kDa. Exemplary polymers may have a Mn of approximately 2 kDa, 10 kDa, 15 kDa, 23 kDa, 36 kDa, or 48 kDa as illustrated in the Examples. In some embodiments, the ratio of N-isopropyl acrylamide (NIPAM) to either acrylamidophenylboronic acid (2-APBA) or acrylic acid (AAc) is from 5:95 to 95:5, e.g., 5:95, 10:90, 15:85, 20:80, 25:75, 30:70, 35:65, 40:60, 45:55, 50:50, 55:45, 60:40, 65:35, 70:30, 75:25, 80:20, 85:15, 90:10, or 95:5. In some embodiments, the ratio of pyridyl disulfide methacrylate (PDSMA) and N- isopropyl acrylamide (NIPAM), acrylamidophenylboronic acid (2-APBA), or acrylic acid (AAc) is from 5:95 to 95:5, e.g., 5:95, 10:90, 15:85, 20:80, 25:75, 30:70, 35:65, 40:60, 45:55, 50:50, 55:45, 60:40, 65:35, 70:30, 75:25, 80:20, 85:15, 90:10, or 95:5. In some embodiments, the polyethylene glycol block comprises between 3 and 10000 ethylene glycol units, e.g., between 3 and 50 ethylene glycol units, between 25 and 100 ethylene glycol units, between 100 and 500 ethylene glycol units, or between 500 and 1100 ethylene glycol units, between 1100 and 2500 ethylene glycol units, between 2500 and 5000 ethylene glycol units, or between 5000 and 10,000 ethylene glycol units. This PEG block can be either linear or branched. A linear PEG consists of a single, unbroken chain of ethylene glycol units. Exemplary average numbers of ethylene glycol units used for linear PEG include 44, 110, 220, 440, and 880. A branched PEG features multiple unbroken chains of ethylene glycol units extending from the polymer backbone (or main chain). Exemplary numbers of unbroken linear ethylene glycol units within a branched PEG include 4.5, 454.5, and 1363.6 with the number of unbroken linear ethylene glycol units per branched PEG being between 6.5 and 130. An exemplary branched PEG is poly(ethylene glycol methacrylate) (PEGMA). Branched PEG structures can increase the stability of the micelle via increased half-life compared to the linear PEG, as shown in Figure 33. The compounds employed in the compositions and methods disclosed herein may be administered as pharmaceutical compositions and, therefore, pharmaceutical compositions incorporating the compounds are considered to be embodiments of the compositions disclosed herein. Such compositions may take any physical form which is pharmaceutically acceptable; illustratively, they can be orally administered pharmaceutical compositions. Such pharmaceutical compositions contain an effective amount of a disclosed compound, which QB\790482.00511\96013124.1 13 790482.00511 effective amount is related to the daily dose of the compound to be administered. Each dosage unit may contain the daily dose of a given compound or each dosage unit may contain a fraction of the daily dose, such as one-half or one-third of the dose. The amount of each compound to be contained in each dosage unit can depend, in part, on the identity of the particular compound chosen for the therapy and other factors, such as the indication for which it is given. The polymers or nanostructures disclosed herein may be formulated as pharmaceutical compositions that include: a therapeutically effective amount of one or more polymers and / or nanostructures as disclosed herein; and one or more pharmaceutically acceptable carriers, excipients, or diluents. It should be recognized that the polymers and nanostructures may exist in equilibrium under certain conditions, and the pharmaceutical compositions may comprise both. It is understood by those skilled in the art that dosage amount will vary with the activity of a particular polymer or nanostructure, disease state, route of administration, duration of treatment, and like factors well-known in the medical and pharmaceutical arts. In general, a suitable dose will be an amount which is the lowest dose effective to produce a therapeutic or prophylactic effect. If desired, an effective dose of such a compound, pharmaceutically acceptable salt thereof, or related composition may be administered in two or more sub-doses, administered separately over an appropriate period of time. Pharmaceutical compositions comprising the compounds may be adapted for administration by any appropriate route, for example by a parenteral (including subcutaneous, intramuscular, intravenous or intradermal), oral (including buccal or sublingual), rectal, nasal, topical (including buccal, sublingual or transdermal), or vaginal route. Such formulations may be prepared by any method known in the art of pharmacy, for example by bringing into association the active ingredient with the carrier(s) or excipient(s). Pharmaceutical compositions adapted for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain antioxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents. The formulations may be presented in unit-dose or multi-dose containers, for example sealed ampoules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example water for injections, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets. QB\790482.00511\96013124.1 14 790482.00511 Pharmaceutical compositions adapted for oral administration may be presented as discrete units such as capsules or tablets; powders or granules; solutions or suspensions in aqueous or non-aqueous liquids; edible foams or whips; or oil-in-water liquid emulsions or water-in-oil liquid emulsions. Optionally, the disclosed compounds or pharmaceutical compositions comprising the disclosed compounds may be administered with additional therapeutic agents, optionally in combination. In some embodiments of the disclosed methods, one or more additional therapeutic agents are administered with the disclosed compounds or with pharmaceutical compositions comprising the disclosed compounds, where the additional therapeutic agent is administered prior to, concurrently with, or after administering the disclosed compounds or the pharmaceutical compositions comprising the disclosed compounds. In some embodiments, the disclosed pharmaceutical composition is formulated to comprise the disclosed compounds and further to comprise one or more additional therapeutic agents, for example, one or more additional therapeutic agents for treating cell proliferative diseases and disorders. An exemplary additional therapeutic agent is insulin. Insulin deficiency and reduced beta-cell mass are hallmarks of type 1 and type 2 diabetes mellitus, which is often accompanied by dysfunction of glucagon-secreting alpha-cells. While glucose-responsive insulin delivery systems are in widespread clinical use to treat insulin insufficiency, the on-demand supplementation of glucagon for acute hypoglycemia treatment remains understudied. A self-regulated glucagon release module is highly desired to mitigate the potential risks of severe insulin-induced hypoglycemia may lead to coma or death. An aspect of the technology is a kit comprising a first pharmaceutical composition comprising the polymers or nanostructures described herein. The kit may further comprise a second pharmaceutical composition comprising an additional therapeutic agent. Suitably the additional therapeutic agent may be insulin. The first and optional second pharmaceutical compositions may be provided in separate containers or vessels, e.g., separate pill containers, vials, blister packs, or any combination thereof. In other instances, the first and optional second pharmaceutical composition are provided in the same container or vessel, e.g., separated in the same blister pack. The kit may further comprise instructions for dosing each of the first and optional second pharmaceutical compositions. The kit may further comprise one or more devices for administering the first and optional second pharmaceutical compositions. Exemplary devices for administering the pharmaceutical compositions include syringes for injection. QB\790482.00511\96013124.1 15 790482.00511 The disclosed compounds and pharmaceutical compositions comprising the disclosed compounds may be administered in methods of treating or preventing a disease, disorder, or condition in a subject in need thereof. The method may comprise administering any of the polymers, nanostructures, or pharmaceutical compositions described herein to the subject. The disclosed compounds and pharmaceutical compositions may be administered in methods of treatment. For example, the disclosed compounds and pharmaceutical compositions may be administered in methods of treating or preventing hypoglycemia. As used herein, the terms “treating” or “to treat” each mean to alleviate symptoms, eliminate the causation of resultant symptoms either on a temporary or permanent basis, and / or to prevent or slow the appearance or to reverse the progression or severity of resultant symptoms of the named disease or disorder. As such, the methods disclosed herein encompass both therapeutic and prophylactic administration. The Examples demonstrate both reversal and prevention of hypoglycemia in vivo. Because the micelles retain activity for a period of time in vivo, the micelles may be administered prior to insulin administration. For example, the micelles may be administered between 1 min and 10 days, between 1 and 7 days, between 3 and 5 days, between 48 and 72 hours, between 36 and 48 hours, between 24 and 36 hours, between 12 and 24 hours, between 6 and 12 hours, between 3 hours and 8 hours, between 4 and 6 hours, between 1 and 3 hours, between 1 and 90 mins, between 1 and 60 mins, or between 1 and 30 mins prior to onset of hypoglycemia. Onset of hypoglycemia may be marked by the time insulin is administered. A “subject in need thereof” as utilized herein refers to a subject in need of treatment for hypoglycemia, at risk for hypoglycemia, or a disease, disorder or condition associated the absence or reduced levels of glucagon in the subject. Those at risk for hypoglycemia may include diabetics, those in need of insulin therapy, or those receiving insulin therapy. For subjects receiving insulin therapy, the subject may be administered insulin prior to or concurrently with the polymers, nanostructures, or pharmaceutical compositions disclosed herein and / or the subject may be administered insulin after administration of the polymers, nanostructures, or composition disclosed herein. Alternatively, the subject is administered the pharmaceutical composition alone. The term “subject” may be used interchangeably with the terms “individual” and “patient” and includes human and non-human mammalian subjects. In some embodiments, the treated subject may be a mammalian subject. Although the methods disclosed herein are particularly intended for the treatment of proliferative disorders in humans, other mammals are included. By way of non-limiting examples, mammalian subjects include monkeys, equines, cattle, canines, felines, mice, rats and pigs. QB\790482.00511\96013124.1 16 790482.00511 As used herein, the term "disorder" refers to a condition in which there is a disturbance of normal functioning. A "disease" is any abnormal condition of the body or mind that causes discomfort, dysfunction, or distress to the person affected or those in contact with the person. Sometimes the term is used broadly to include injuries, disabilities, syndromes, symptoms, deviant behaviors, and atypical variations of structure and function, while in other contexts these may be considered distinguishable categories. It should be noted that the terms "disease", "disorder", "condition" and "illness", are equally used herein. As used herein the term “effective amount” refers to the amount or dose of the compound, upon single or multiple dose administration to the subject, which provides the desired effect in the subject under diagnosis or treatment. An effective amount can be readily determined by the attending diagnostician, as one skilled in the art, by the use of known techniques and by observing results obtained under analogous circumstances. In determining the effective amount or dose of compound administered, a number of factors can be considered by the attending diagnostician, such as: the species of the subject; its size, age, and general health; the degree of involvement or the severity of the disease or disorder involved; the response of the individual subject; the particular compound administered; the mode of administration; the bioavailability characteristics of the preparation administered; the dose regimen selected; the use of concomitant medication; and other relevant circumstances. Unless otherwise specified or indicated by context, the terms “a”, “an”, and “the” mean “one or more.” For example, “a molecule” should be interpreted to mean “one or more molecules.” As used herein, “about”, “approximately,” “substantially,” and “significantly” will be understood by persons of ordinary skill in the art and will vary to some extent on the context in which they are used. If there are uses of the term which are not clear to persons of ordinary skill in the art given the context in which it is used, “about” and “approximately” will mean plus or minus ≤10% of the particular term and “substantially” and “significantly” will mean plus or minus >10% of the particular term. As used herein, the terms “include” and “including” have the same meaning as the terms “comprise” and “comprising.” The terms “comprise” and “comprising” should be interpreted as being “open” transitional terms that permit the inclusion of additional components further to those components recited in the claims. The terms “consist” and “consisting of” should be interpreted as being “closed” transitional terms that do not permit the inclusion additional components other than the components recited in the claims. The term “consisting essentially QB\790482.00511\96013124.1 17 790482.00511 of” should be interpreted to be partially closed and allowing the inclusion only of additional components that do not fundamentally alter the nature of the claimed subject matter. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention. All references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein. Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are now described. All publications and patents specifically mentioned herein are incorporated by reference for all purposes including describing and disclosing the chemicals, instruments, statistical analysis and methodologies which are reported in the publications which might be used in connection with the invention. All references cited in this specification are to be taken as indicative of the level of skill in the art. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention. Preferred aspects of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of those preferred aspects may become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect a person having ordinary skill in the art to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context. EXAMPLES Diabetes is a metabolic disorder caused by a decline in the function of insulin- producing beta-cells in the pancreas (type 1, T1D) or by peripheral insulin resistance and also QB\790482.00511\96013124.1 18 790482.00511 arguably reduced beta-cell mass (type 2, T2D).(1, 2) Together T1D and T2D affects more than 500 million people worldwide and is projected to increase to 1.3 billion by 2050.(3–5) In healthy individuals, pancreatic cells respond to fluctuations in blood glucose dynamically by stimulating insulin secretion from the pancreatic beta-cells.su, 7) During hypoglycemia, insulin secretion is inhibited, restoring glycemic homeostasis via the release of glucagon from pancreatic alpha-cells (epinephrine and norepinephrine are also released) to stimulate the production of glucose predominantly by the liver, and kidney to a smaller extent.(8) Glucagon (GCG) is an endogenous peptide that raises blood glucose levels by activation of hepatic gluconeogenesis and glycogenolysis.(9) Because glucose regulation is disrupted in diabetic patients, treatment generally involves regular insulin replacement by injections to combat rising blood glucose. Although insulin treatment is extremely effective in lowering blood glucose from dangerous highs, episodes of moderate to severe hypoglycemia are a common clinical complication associated with relative hyperinsulinemia. In these instances, glucagon is administered as an emergency medication to treat severe, insulin-induced hypoglycemia, to quickly restore normoglycemia and prevent clinical symptoms of hypoglycemia including malaise, cognitive impairment, seizure, and coma.(10) The disclosed technology provides a shift in glucagon treatment strategy and strives for the development of a formulation that prevents hypoglycemia. This idea has already been examined by Haidar et al. in a randomized crossover-controlled trial, revealing that diabetic adults using a closed loop, dual-hormone pump had improved glucose control and reduced risk of hypoglycemia.(21) While there may be physiological concerns with frequent glucagon administration, recent studies demonstrated that repeated doses of glucagon do not cause hepatic glycogen depletion. Additionally, glucose responsiveness to glucagon administration was similar regardless of prior glucagon administration.(22–24) An ideal glucagon delivery system can sensitively recognize and accurately respond to decrements in glycemia, so as to restore normoglycemia. While many glucose-responsive approaches to insulin delivery have been undertaken, efforts towards responsive glucagon delivery are limited.(25) Webber and co-workers have successfully designed a hydrogel with encapsulated glucagon that is destabilized in the absence of glucose.(26) Notable work has been conducted using glucose- responsive microneedle patches to deliver both glucagon and insulin, primarily pioneered by Gu,(27–29) Wu,(30, 31) and co-workers. These systems leverage the properties of phenyl boronic acids (PBA), small molecules able to form dynamic covalent bonds with 1,2- diols, such as glucose, when in anionic tetrahedral state. (32, 33) Other than providing stability, their QB\790482.00511\96013124.1 19 790482.00511 glucose interaction can be fine-tuned by chemical modification and has been extensively used to prepare functional polymers and nanomaterials for insulin delivery.(34–37) Typically, meta- and para-substituted PBA are used in polymeric delivery systems, where addition of glucose stabilizes charged tetrahedral glucose boronate esters, increasing the ionization degree of the polymer and its hydrophilicity, resulting in the disassembly of the micelles.(38, 39) However, by placing the boronic acid in the ortho position to the amide as in 2-acrylamidophenylboronic acid (2-APBA), an intramolecular B-O dative bond is formed, favoring the charged state.(40) When glucose is added to a polymer containing this functional group, the resulting material becomes more hydrophobic. This feature has been exploited to alter the lower critical solution temperature (LCST) of a thermoresponsive N- isopropylacrylamide copolymer (P(NIPAM-stat-2-APBA)).(41) The LCST is the temperature at which a polymer undergoes a phase transition from its soluble, hydrated form to a shrunken, dehydrated phase. This transition heavily depends on the overall hydrophobicity of the chain (i.e. more hydrophobic polymers have lower LCSTs). Therefore, upon glucose addition to P(NIPAM-stat-2-APBA), the LCST decreases due to increased hydrophobicity (41) Wang et al. exploited this to prepare poly(ethylene glycol)-block-poly(N-isopropylacrylamide-stat-2- acrylamidophenylboronic acid), PEG-b-P(NIPAM-stat-2-APBA), that self-assembled into micelles upon glucose addition at 30 °C (42) This work was foundational towards using phenylboronic acids to drive the assembly of micelles in the presence of glucose. However, the glucose levels required to induce this transition were orders of magnitude higher ([Glc] =180 mg / dL) than what would be experienced in a physiological setting (<60 mg / dL, hypoglycemia to 100-150 mg / dL normoglycemia in rodents). Consequently, although this PEG-b-P(NIPAM- stat-2-APBA) micelle system was promising for hypoglycemia sensing, the polymer structure / molecular weight needed to be extensively refined to respond to physiologically- relevant glucose levels. Example 1 Herein, a glucose-responsive glucagon delivery platform using PEG-b- P(NIPAM-stat- 2-APBA) micelles that disassemble at glucose levels at or below the counter regulatory threshold is reported (Figure 1). A panel of polymers were synthesized via a chain-extension of a PEGylated chain transfer agent by reversible addition−fragmentation chain-transfer (RAFT) polymerization. A thiolated glucagon analogue was covalently grafted at the ω- polymer-end group via pyridyl disulfide exchange. At 37 °C in the presence of 150 mg / dL glucose (normoglycemia) the polymer-GCG conjugate candidate was micellar, and thus the glucagon was not active (Figure 1). However, at 60 mg / dL (hypoglycemia) the micelles QB\790482.00511\96013124.1 20 790482.00511 disassembled into linear / unimeric polymers revealing the active glucagon conjugate. The in vivo acute and chronic toxicity analysis and biodistribution determined through μPET / μCT imaging showed the safety of this formulation. Blood glucose dynamics upon administration of glucagon-micelle in healthy mice demonstrated successful regulation of glucose levels by reverting and preventing insulin-induced hypoglycemia. Synthesis and Characterization of Polymer-GCG Conjugate Library. Considering instability and degradability of glucagon in aqueous media, our initial hypothesis was that a glucagon-polymer conjugate would promote stabilization at physiological pH as evidenced by the work of Stigsnaes et al. on PEGylated glucagon.(43) Thus, a PEGylated corona was selected for the micelle formulation. The P(NIPAM-stat-2- APBA) block was selected for glucose and thermo-responsiveness. A block-copolymer library based on PEG-b-P(NIPAM-stat-2-APBA) with varying PEG to P(NIPAM-stat-2-APBA) block length ratios (while keeping the NIPAM:2-APBA molar ratio to 85:15 and PEG molecular weight at 2000 Da) was synthesized to fine-tune the glucose-responsiveness at 37°C, specifically to make the micelles disassemble at 60 mg / dL. RAFT polymerization in DMSO was employed to copolymerize N-isopropyl acrylamide (NIPAM) and acrylic acid (AAc) using a PEG modified at the omega-end with 2-(((ethylthio)carbonothioyl)thio)-2-methylpropanoic acid macromolecular chain transfer agent (PEG44 macro-CTA), yielding PEG-b-P(NIPAM- stat-AAc) (polymers P1-P5, Mn = 9.0-47.2 kDa, see below for detailed synthetic conditions, Table 1). EDC coupling was performed between 2-aminophenyl boronic acid and the carboxylic acid of PAAc repeat units (Table 2). Aminolysis of the polymer’s terminal trithiocarbonyl group allowed for the installation of a thiol-reactive pyridyl disulfide (PDS) moiety at the ω-end group. Figure 2a shows the overall synthesis scheme. A thiolated glucagon analogue bearing a cysteine residue (Q24C), GCG-SH, was used in this study and covalently conjugated onto the polymer end-group via pyridyl disulfide exchange and formation of a disulfide bond (Figure 2a). Circular dichroism studies on the native and thiolated glucagon and predicted 3D structures using AlphaFold indicated that the alpha-helix was fully retained (Figure 7, 8). PEG-b-(NIPAM-stat-2-APBA)-PDS block copolymers were reacted with GCG-SH in a 1.2 :1 mixture of DPBS (pH = 7.4): HCl 10 mM (pH = 2). The reaction was monitored by high-performance liquid chromatography (HPLC) (Figure 2b). Within 1 min of the addition of GCG-SH to the PDS-containing polymer, 57.4% of the peptide was consumed, reaching a maximum conversion of 83.0% within 4h. To confirm GCG-SH presence in the conjugate, PEG-b-P(NIPAM-stat-2-APBA)-GCG was subjected to disulfide reduction with tris(2-carboxyethyl)phosphine (TCEP) for 30 min to QB\790482.00511\96013124.1 21 790482.00511 release GCG-SH from the polymer. The amount released was quantified by HPLC and was consistent with the amount of GCG-SH consumed during the reaction. The integrity of released GCG-SH was also verified by LC-MS, indicating identical masses in excellent agreement with theoretical molecular weight (3457.76 Da). (Figure 2c, 2d). Lastly, the purity of PEG-b- P(NIPAM-stat-2-APBA)-GCG and the release of GCG-SH after reduction of the disulfide bond were confirmed and visualized by SDS-PAGE stained with Coomassie (Figure 9). Altogether, these results confirm the successful synthesis of the conjugate PEG-b-P(NIPAM- stat-2-APBA)-GCG. Following this strategy, a library of glucagon-polymer conjugates (P1 / P2 / P3 / P4-GCG) was prepared (Table 3). Conjugation conversion was consistent for all polymers. Solution Behavior of GCG-Polymer Conjugates: Tuning Glucose- and Thermo- Responsiveness With this library of block copolymers (P1-P5), an initial screening of the thermo- and glucose-responsive behavior of the unconjugated polymers was assessed by dynamic light scattering (DLS). In absence of glucose, polymer samples P2-P4 retained their linear form at 37 °C, but upon increasing temperature to 40 °C, formed uniform micelles. (Figure 10a, Table 2). In contrast, the largest molecular weight polymer, P5, formed micelles at 37 °C, confirming the trend of lower LCSTs with increased hydrophobic block molecular weight. As a next step, the glucose responsiveness of the polymers P2-P4 was examined by DLS due to their linearity at physiological temperature, speculating that glucose addition could possibly induce their self- assembly into micelles. Both P2 and P3 did not form well-defined micelles in the presence of [Glc] = 150 mg / dL (Figure 10b). However, P4 exhibited self-assembly behavior at normoglycemic conditions ([Glc] = 150 mg / dL), forming uniform micelles with similar size to those obtained at 40 °C. Our observations confirmed that glucose effectively shifted the LCST to lower temperatures. Variable-temperature ultraviolet−visible (UV−Vis) spectroscopy was also employed to evaluate the cloud point of P3 and P4 in different glucose concentrations ([Glc] = 0-1000 mg / dL). A difference of 2.4 and 3.8 °C between cloud points of 0 and 1000 mg / dL of glucose was reported for P3 and P4, respectively, confirming that glucose addition does shift LCST and micelle formation to lower temperatures. The solution behavior of P4-GCG conjugate was primarily investigated as P4 showed optimal glucose sensitivity at physiological temperature. However, when examined by DLS, it was demonstrated that it self-assembled into micelles at 37 ºC without any glucose addition, unlike its unconjugated version (Figure 11b). In addition, these micelles demonstrated a significantly higher hydrodynamic diameter of (Dh = 119.6 nm) compared to the diameter of QB\790482.00511\96013124.1 22 790482.00511 micelles obtained from polymer without glucagon (Dh = 46.75 nm). Variable-temperature UV−Vis spectroscopy also confirmed that the cloud point of P4-GCG was shifted by 2.5 ºC lower than P4 alone, confirming the assembly behavior observed by DLS (Figure 11a). Due to glucagon’s net charge of 0 at neutral pH, the peptide is thought to add hydrophobicity to the conjugate, therefore shifting PEG-b-(NIPAM-stat-2-APBA)-GCG to a lower LCST. This trend was confirmed for P2-GCG as well, exhibiting a cloud point of 50.6 ºC, 11.5 ºC lower than P2 alone (Figure 2e). Dry-state transmission electron microscopy (TEM) of P2- GCG also confirmed the thermo-responsiveness of the system, showing non-unform assemblies at 25 ºC (Figure 2g) while at 40 ºC uniform micelles were observed (Figure 2h). This effect is more marked with P2-GCG because of its lower molecular weight compared to P4-GCG, thus allowing glucagon to exert a more significant hydrophobic contribution. P3- GCG, however, behaved differently, showing an increase in cloud point by 9.5 ºC higher than P3 alone (Figure 12a). Both polymer and conjugate start eliciting a response to the rise in temperature around 45-46 ºC, but the P3 response is much sharper. This may be attributed to a higher hydration of the conjugate, but further investigation is beyond the scope of this work. Nonetheless, when investigated by DLS, P3-GCG showed the same thermo-responsive behavior as P4-GCG and self-assembled into micelles already at 37 ºC without glucose addition (Figure 12b). Conjugate P2-GCG showed the optimal self-assembly characteristics by DLS (Figure 2f). Specifically, at 37 ºC, P2-GCG did not self-assemble into micelles in the absence of glucose or at hypoglycemic glucose concentrations ([Glc] = 60 mg / dL). However, it formed well-defined micelles at normoglycemia ([Glc] = 150 mg / dL) with a hydrodynamic diameter of 50.40 nm (Table 3). To further investigate the sensitivity of this system towards changes in glucose concentration, P2-GCG micellar solution was diluted ([Glc] = 60 mg / dL). Within 10 mins of incubation, a second multimodal peak was noted by DLS of a larger diameter, which can be attributed to swelled micelles (Figure 2f). Considering these results, P2-(GCG) was selected as the candidate polymer for further in vivo toxicity, biodistribution, and efficacy evaluation. In vitro Evaluation of PEG-b-P(NIPAM-stat-2-APBA)-GCG Conjugate Activity A cAMP HunterTM eXpress GCGR CHO-K1 GPCR commercial test kit (Figure 2i, 2j) was used to evaluate the in vitro effectiveness of native GCG, GCG-SH, P2, P2-GCG, and TCEP reduced P2-GCG. The kit uses cells in which human glucagon receptor (GCGR) is overexpressed, allowing for quantification of receptor activation via increased levels of intracellular cAMP. Native GCG showed an EC50 of 4.49 ± 0.52 nM, comparable to literature- reported values.(44, 45) The EC50 of GCG-SH was 39.30 ± 11.60 nM; the increase in EC50 QB\790482.00511\96013124.1 23 790482.00511 is attributed to the chemical modification of GCG-SH, resulting in reduced receptor interaction. However, the value for GCG-SH is still in the reported range for glucagon activity and careful selection of the modification site in the peptide chain (Q24C) is believed to maintain significant bioactivity. Polymer P2 was used as a negative control showing no bioactivity as expected. The EC50 of the linear polymer conjugate P2-GCG (i.e. not in the micellar form) showed the lowest value (0.87 ± 0.12 nM). These results confirm that the glucagon is still active after conjugation to the polymer and that reduction of the conjugate disulfide to release GCG- SH is not a requisite for receptor activation. The assay confirmed that the critical step for the onset of activity is micelle disassembly to unmask the glucagon conjugate, rather than GCG- SH release. Notably, the EC50 of GCG-P2 is 5-fold lower than native glucagon, indicating that the polymer might promote the receptor interaction or simply enhance the stabilization of the peptide. To confirm this hypothesis, the conjugate's disulfide bond was reduced using a 10 mM TCEP solution (TCEP removed through centrifugal filtration, molecular weight cut-off (MWCO) = 3.5 kDa), leading to a mixture of GCG-SH and polymer, P2. The pre-reduced solution showed an EC50 of 4.66 ± 0.64, 5-fold higher than unreduced P2-GCG and on the same order of magnitude as native GCG. This activity loss upon reduction confirms that the conjugate is a more potent agonist than native GCG. In vivo Acute and Chronic Toxicity of Empty Micelles (P2) Prior to study of this glucagon conjugate, acute toxicity of the empty micelles (candidate polymer P2) was evaluated in C57Bl / 6J male mice at 0 h, 24 h and 120 h following injection. Mice were injected intraperitoneally (IP) with a single dose of empty micelles (2.322 mg / Kg, equivalent to 500 μg / kg of glucagon) and were euthanized at different intervals to determine the complete blood count (CBC) (n = 4 or 5) and organ weights (liver, kidney, spleen, heart and lungs) (n = 6). White blood cell (WBC), red blood cell (RBC), monocytes, lymphocytes, neutrophils, eosinophils, hemoglobin, and hematocrit were measured. (Figure 13a and Table 4). No significant difference in CBC counts was observed when compared to mice treated with empty micelles after 0 h, 24 h, and 120 h. All values were in accordance with reported literature.(46–48) Additionally, there were no significant differences between the treatment groups for organ weights (Figure 13b). Following acute studies, a chronic toxicity study was conducted in C57Bl / 6J male mice. The mice were injected IP with empty micelles every day for 14 days and the control group was injected with saline (Figure 3a). After 14 days of injections, mice were euthanized to determine body weight, CBC count, organ weight, histopathology, hepatic function parameters, kidney function parameters, immune markers, and immunohistochemistry of lung QB\790482.00511\96013124.1 24 790482.00511 and liver tissues. Total body weight as well as organ weights of saline control and empty micelle-treated mice (liver, lungs, spleen, heart, kidney) showed no significant differences (Figure 14). The CBC counts were all in accordance with published values (Figure 3b (i)- (xii), Table 5).(46–48) Hepatic function parameters including alanine aminotransferase (ALT), aspartate aminotransferase (AST) levels analyzed from serum (Figure 3c, Table 6) were the same for the micelles as the negative controls. For lactate dehydrogenase (LDH), the values for the empty micelles were slightly lower, statistically significant compared to the control. Kidney function was determined by blood glucose level and calcium count in urine and found not to be different for mice exposed to the micelle (Figure 3d, Table 6). Organ histopathology of liver, spleen, heart, lung, kidney was examined via Hematoxylin & Eosin (H&E) staining (Figure 3e). No microscopic morphological changes were observed in histological sections. Markers IFN γ, TNF α, and IL 2 were also analyzed to determine immune reaction and inflammation in response to micelle injection and no differences were detected between the groups (Figure 4a and Table 7). Histology slices of the liver and lung were stained with F4 / 80 antibody, a macrophage marker, and micelle treated animals were the same as the control (Figure 4b). Together, the data for the acute and chronic toxicity analyses demonstrate the safety profile of the polymeric material. In vivo μPET / μCT Imaging of PEG-b-P(NIPAM-stat-2-APBA) Micelle and GCG-SH µPET / µCT89Zr-labeled micelle / 89Zr -labeled linear polymer. Upon establishing a general safety profile of the micelle, μPET / μCT imaging was performed to further understand the in vivo biodistribution and pharmacokinetic profiles of the polymeric system. In vivo, the polymer should responsively shift between its micellar and linear form depending on glucose concentration. Examining the differences between both forms allows for an understanding of the complete picture of biodistribution and pharmacokinetic properties of the system. Therefore, two different polymer batches were synthesized: one always in the micelle state and another always in the linear state at 37 ºC regardless of glucose concentration. Both PEG-b-P(NIPAM-stat-2-APBA) polymer end groups were aminolyzed to a free thiol and conjugated with deferoxamine-maleimide (DFO- maleimide), a robust chelator of the radiometal zirconium-89 (89Zr).(49) DFO- conjugated polymers were chelated with [89Zr]Zr-oxalate at room temperature, and upon purification, DLS analysis was used to confirm the formation of the89Zr-labeled micelle and 89Zr-labeled linear polymer (Figure 15a,b). The µPET / µCT imaging analysis revealed that the89Zr-labeled micelle maintained a higher concentration in the blood. In contrast, renal clearance rate of the89Zr-labeled linear polymer was accelerated during the first hour post-injection (Figure 16, 17). Compared with the89Zr- QB\790482.00511\96013124.1 25 790482.00511 labeled linear polymer, the89Zr-labeled micelle showed higher uptake at 2 h post injection by liver, spleen, intestine (GI) and lungs (Figures 15c-n, Table 8). At the endpoint of the experiment (192 h), both forms accumulated in liver, spleen, and the kidney, with the89Zr- labeled micelle at a higher concentration than the89Zr-labeled linear polymer (Figure 17). The PET results were different than what was observed in the toxicity studies where it was confirmed the absence of inflammation or immune cell activation in response to polymer injection. The large molecular weight and relative hydrophobicity of the [89Zr]Zr-DFO label when conjugated at the polymer-end group could contribute to the observed biodistribution. To test this hypothesis, additional µPET / µCT imaging experiments were performed using18F- labeling via a prosthetic group (18F-FBEM or18F-SFB), which allowed labeling of the polymer directly without the use of a radiometal or chelating ligand. The small size of the radio- fluorinated prosthetic group, and its reduced hydrophobicity compared to [89Zr]Zr-DFO are expected to impact the polymer structure minimally, better representing the true biodistribution of the micelle. µPET / µCT18F-FBEM-labeled micelle / 18F-SFB-labeled glucagon. The µPET / µCT imaging was performed using an18F-FBEM-labeled micellar polymer and18F-SFB-labeled glucagon as a negative control. Conjugation conditions of the micelle and glucagon were initially optimized using19F-FBEM / SFB (See below for detailed experimental conditions on synthesis and conjugation of18 / 19F-FBEM / SFB, Figures 22-26), Figure 5a. The µPET / µCT imaging analysis of18F-SFB-labeled glucagon showed higher uptake in all organs compared to18F-FBEM-labeled micelle, particularly in the kidney and spleen during the first 4 h after probe injection (Figures 5b, 5c, 19, 20). The18F-FBEM-labeled micelle was eliminated by renal clearance more rapidly than glucagon, as shown by PET signal intensity in the bladder. In addition, the18F-FBEM-labeled micelle was also rapidly cleared from the gallbladder. After 8 h post-injection, organs showing PET activity (>1%ID / cc) were kidney (18F-FBEM-labeled micelle 1.17%ID / cc), gallbladder (18F-FBEM-labeled micelle 3.88%ID / cc,18F-SFB-labeled glucagon 3.61%ID / cc), and bladder (18F-FBEM-labeled micelle 1.44%ID / cc,18F-SFB-labeled glucagon 16.75%ID / cc). This data contrasts with what was observed when the micelle was labeled with89Zr via a DFO chelator. Therefore, the uptake and accumulation seen with the89Zr-radiolabel may have been directly caused by the radiometal and chelator. In vivo Safety Evaluation of PEG-b-P(NIPAM-stat-2-APBA)-GCG Micelle To further investigate how P2-GCG elevates glycemia in vivo, the conjugate was administered IP in healthy 6h fasted C57Bl / 6J mice (Figure 6a). The control group was QB\790482.00511\96013124.1 26 790482.00511 administered PBS only. Upon injection, blood glucose levels of mice were monitored over 8 hours at different time intervals. An initial small spike was observed in the first 15 min, with glycemia level rising from 160 to 190 mg / dL. This is not uncommon, as the stress of injection can lead to transient increase in blood glucose and is observed in both the glucagon-micelle and the PBS control. After 15 min, the glucose level steadily dropped to 125 mg / dL within 60 min of the injection and remained constant until the end of the experiment. Overall, glycemia remained in the normal range throughout the study. Following the 8h study period, animals were euthanized, kidneys and liver were harvested, and subsequent histopathological analyses were conducted via H&E staining (Figure 21). No microscopic morphological changes in histological sections were observed in micelle-injected mice vs. controls. These findings indicate that micelles are not activated under normoglycemia, nor do they induce histopathological outcomes in kidney or liver. Next, the in vivo efficacy of the GCG-polymer conjugate was evaluated. In vivo Activity of PEG-b-P(NIPAM-stat-2-APBA)-GCG Micelles Two independent in vivo experiments were conducted to evaluate the effectiveness of P2-GCG in reversing or preventing insulin-induced deep hypoglycemia. The efficacy of the GCG-micelle was evaluated in healthy C57Bl / 6J male mice. First, the mice were fasted for 12 hours prior to the administration of the insulin of dose 0.90 U / kg intraperitoneally to induce hypoglycemia. Glucose levels decreased from 100 to 65 mg / dL after 30 minutes of insulin administration and was approximately 60 mg / dL after 1 hour. At this point, GCG-micelle at a dose of 500 µg / Kg of glucagon was administered intraperitoneally. Blood glucose immediately increased, reaching 90 mg / dL in 25 minutes and 100 mg / dL within 40 minutes, restoring baseline glycemia. The GCG-micelle response showed a statistical difference from the response of the control group injected with the empty micelle (similar to GCG micelles after induction of hypoglycemia at 60min), which exhibited hypoglycemia <65 mg / dL until the end of the experiment. (Figure 6c(i)). To evaluate the responsiveness of the GCG-micelle above the estimated gluco-counterregulatory threshold, moderate hypoglycemia (80 mg / dL) was induced by administering 0.85 U / kg of insulin at time 0. The moderate reduction in glycemia level should not trigger the disassembly of micelles avoiding overresponse and induction of hyperglycemia. After 60 minutes, 500 µg / Kg of glucagon (GCG-micelle) was administered intraperitoneally resulting in a less pronounced response, restoring initial normoglycemia of 120 mg / dL in 105 minutes. More importantly, there was no statistical difference in glycemia at any time point during the insulin tolerance test (ITT) between GCG-micelle and the empty micelle injected control. These data suggest that micelle disassembly was not triggered unless QB\790482.00511\96013124.1 27 790482.00511 insulin induced a sufficient level of hypoglycemia at or below the estimated gluco- counterregulatory threshold (Figure 6c(ii)). Overall, the GCG-micelle demonstrated in vivo glucose responsiveness and was able to quickly reverse deep hypoglycemia. Next, the in vivo efficacy of the GCG micelle to prevent deep hypoglycemia was assessed by injecting insulin and the micelles simultaneously at time 0 of the experiment. Healthy C57Bl / 6J male mice were fasted for 12 hours before being administered with 0.90 U / kg of insulin and either 500 µg / Kg of glucagon (GCG-micelle) or an equivalent dose of empty micelle. The mice injected with the glucagon-conjugate maintained normoglycemia for the duration of the experiment, whereas glucose levels of mice injected with empty micelle (P2) reduced below the counterregulatory threshold (Figure 6b). Afterwards, the glycemia level increased and reached the starting level of 130 mg / dL within 2 hours. Conversely, the control group injected with empty micelle quickly dropped below 80 mg / dL in less than 30 minutes, reaching a glucose nadir of 70 mg / dL at 60 minutes. In the following hour, glycemia increased only to 88 mg / dL in control vs GCG. Overall, in vivo efficacy studies confirmed that the glucagon micelle can both safely prevent and reverse deep hypoglycemia. Discussion Glucose-responsive drug delivery systems have been extensively investigated for insulin delivery.(25) However, glucose-responsive glucagon delivery is understudied. PEG-b- P(NIPAM-stat-2-APBA) was used as the polymer of choice for its ability to form micelles in the presence of glucose.(42) The system was tuned to be responsive in physiological conditions, self-assembling and disassembling in a narrow glucose range at relevant temperatures. Through RAFT polymerization, a library of block copolymers was synthesized with a 3yl end- group that could be chemically modified to allow for site-specific conjugation of glucagon. The LCST and glucose-responsiveness were altered to be effective at physiological conditions; this was possible by varying weight ratios between the PEG hydrophilic block, which was kept constant at 2 kDa, and P(NIPAM-stat-2-APBA) block. Initially, P4 was found to present responsivity at 37 ºC and relevant glucose concentrations, forming well-defined, uniform micelles in normoglycemia; but when GCG-SH was conjugated, the polymer LCST decreased due to the hydrophobicity of the glucagon, forming micelles at temperatures lower than necessary. As a result, P2-GCG presented the optimal responsiveness, even though P2 alone did not. In early experiments, native glucagon encapsulation was attempted by physical entrapment, but poor encapsulation and release were achieved. A covalent design was therefore pursued instead. Typically, polymer conjugation to a protein or peptide results in a loss of QB\790482.00511\96013124.1 28 790482.00511 activity. To minimize this loss, researchers often employ site selective conjugation(50) to biomolecules. To best ensure site-selectivity during polymer conjugation to glucagon, a single cysteine unit was engineered into the native peptide. First, the substitution site for amino acid modification was selected based on the crystal structure of a glucagon analogue binding to the glucagon receptor.(51) Additionally, Chabenne et al. found that when substituting the existing glutamine residue of glucagon with alanine (Q24A), full potency of glucagon receptor binding and activity was retained.(16) Therefore the glutamine residue in position 24 was selected to modify to a cysteine (Q24C) so that the peptide could then be conjugated to a PDS- functionalized polymer. This allowed a simple, site-selective conjugation to the cysteine without affecting any of other reactive residues of glucagon. Biomolecules conjugated to polymers often display reduced activity due to either undesirable changes in the protein structure itself or due to polymer interference between the biomolecule and its receptor. Much to our surprise, the in vitro experiment demonstrated a higher activity for the conjugate itself compared to both native, thiolated, and released GCG-SH after the reduction of the disulfide bond. The polymer is hypothesized to help configure GCG in the correct confirmation for interaction with the receptor and the related G protein.(52) Alternatively, the polymer might stabilize GCG in solution, similar to our previously published trehalose nanogels.(20) Although unusual, other examples where site-specific protein-polymer conjugation increased protein bioactivity can be found in the literature.(53–56) Ultimately, the increased conjugate activity remains under investigation, yet is fortuitous. The successful in vitro results prompted us to evaluate the formulation efficacy in vivo. First, the in vivo acute and chronic toxicity was evaluated to understand the toxicity of the formulation in a healthy mouse model. The in vivo acute toxicity was determined by analyzing the CBC count and the organ weights. Empty micelles showed no significant difference in the CBC count or organ weights, indicating no toxic acute effects upon administration. Further, a detailed chronic toxicity analysis was performed for 14 days with multiple dosages of empty micelles, examining organ weight, CBC count, hepatic and kidney function, histology, and immune / inflammation markers. The acute and chronic toxicity evaluation studies show that the micelles have no in vivo toxic effect over the dosages and time course studied. The in vivo μPET / μCT imaging experiments were performed to further understand the pharmacokinetics of the polymer. The μPET / μCT imaging using89Zr-labeling revealed a small, but unexpected percentage of polymer accumulated in certain organs. After hypothesizing that the hydrophobicity and large molecular weight of the DFO molecule may have led to the observed results, μPET / μCT imaging experiments were repeated using an18F-labeled version QB\790482.00511\96013124.1 29 790482.00511 of the polymer to avoid the need for a bulky chelator ligand. The blood half-life of the polymer from the experiments with89Zr-labeled construct was also determined to be short enough to be compatible with the shorter half-life of F-18 (109.7 minutes). In these experiments, accumulation was not observed in key organs such as the liver, kidney, lungs and spleen. Adding a large, hydrophobic, radiometal-chelator probe to the polymer system may have altered the biodistribution or triggered the immune system, although further studies would need to be done to verify this. Regardless, the results were in accordance with the toxicity tests, demonstrating safety. Next, experiments were undertaken to verify that injection of GCG-conjugate (GCG- P2) at normal glucose levels did not induce hyperglycemia. The blood glucose levels were monitored for eight hours after injection of GCG micelle, and it remained above 125 mg / dL, well within the normoglycemia range. Surprisingly, glycemia levels of the mice treated with GCG micelle were lower than the PBS control by 30 mg / dL. This could be due to counter- regulation effects and the release of endogenous insulin. Future experiments using a diabetic model would be needed to elucidate this hypothesis. Yet, the above experiments did show that the empty micelle was not causing any in vivo toxicity, and the GCG micelle was not inducing hyperglycemic conditions, indicating that the formulation was safe for further exploration. In vivo efficacy studies were performed in the healthy murine model, avoiding various variables associated with diabetes mouse models. According to experimental results, the GCG micelle was able to reverse deep hypoglycemic conditions in less than 25 minutes after injection, indicating that the glucagon formulation could be employed as an emergency treatment for hypoglycemia. Interestingly, when mice with moderate hypoglycemia were injected with GCG micelles, the normoglycemia level was restored within 15 minutes of injection. Furthermore, there was no hyperglycemic condition following this injection, demonstrating the safety of the GCG micelle. Finally, when the GCG formulation was injected together with insulin, blood glucose levels were maintained in the normal range, whereas the control group experienced a drop in the glycemia below the gluco-counterregulatory threshold. This indicates that the GCG- micelle, acting in tandem with insulin, proved more effective at maintaining normoglycemia than the empty-micelle-insulin combination. In conclusion, our experimental results indicate that the GCG micelle could be used to avert the onset of insulin-induced deep hypoglycemia. These studies highlight the potential for a glucagon formulation to manage glycemia by pre- or co-administration of glucagon-micelles with insulin. Experiments are now being conducted to gain a comprehensive understanding of GCG pharmacokinetics, which is required to move forward with this formulation. QB\790482.00511\96013124.1 30 790482.00511 Herein, the design of a responsive polymer-GCG conjugate system for treating or preventing hypoglycemia is reported. Initial experiments suggest that it can studied for both an emergency treatment and as a preventive measure. Comprehensive toxicology studies and immunogenicity are needed to confirm the short- and long-term safety of this construct including lethal dose studies. Moreover, diabetic murine models and other preclinical animal models will be tested to move our candidate forward towards clinical testing. Future work will also explore higher molecular weight PEG corona and alternative coronas to increase circulation time, as a high percentage of severe hypoglycemic episodes occur during sleep (nocturnal hypoglycemia). Nonetheless, the Examples demonstrate that PEG-b-P(NIPAM- stat-2-APBA)-GCG is provides for the treatment of insulin-induced hypoglycemia. Materials and Methods Study design The objective of this study was to design and develop a glucose responsive delivery system for glucagon to combat severe hypoglycemia, either as an emergency medication or as a preventive measure. Two main goals were identified: I) to synthesize a polymer, fine tuning its physicochemical characteristics, that serve as a carrier for glucagon and that is able to self- assemble into micelle and disassemble at the desired glucose levels, and II) to verify such formulation biological activity by testing for its potency in vitro and for its capacity to prevent or reverse severe hypoglycemia without side effects in vivo. First, PEG-b- P(NIPAM-stat-2- APBA) was identified as a polymer presenting the desired characteristics, such as the ability to self-assemble into micelles based on temperature and glucose response. The polymer was modified to be responsive in physiological conditions by altering the ratio of hydrophilic PEG block to responsive hydrophobic NIPAM-stat-2-APBA block, as well as the percentage of phenyl boronic units present in the hydrophobic block. The polymer was modified to insert a disulfide to react with a thiolated glucagon, forming an active conjugate. Through careful fine tuning of the length and ratios of each polymer block, the conjugate was engineered to respond to changes in glycemia at physiological range. After the above optimization, the conjugate activity was tested in vitro using cells overexpressing the glucagon receptor, finding an increased potency of the conjugate compared to native glucagon. Finally, the conjugate safety and activity were assessed in vivo using healthy C57Bl / 6J male mice. To test safety, animals were injected with either the conjugate or PBS as a control, and changes in their glycemia were monitored for 8 h. At the end of the study, kidney and liver tissues were collected to perform histopathological analysis. Two experiments were performed to study the capacity of the conjugate to reverse or prevent deep hypoglycemia. In the first, animals were fasted for 12 h QB\790482.00511\96013124.1 31 790482.00511 and then injected with insulin to induce severe hypoglycemia. After, either the conjugate or the naked polymer as a control were administered monitoring changes in glycemia for 2 h. The second experiment was conducted similarly, but the conjugate or naked polymer were administered at the same time as insulin. All animal procedures performed in this study were reviewed and approved by the UCLA Animal Oversite Committee and supervised by A.L.H. or R.M.VD. Statistical analysis In vitro and in vivo experimental values are reported as the mean ± SD and mean ± SEM, respectively. Graph Pad Prism 8 (GraphPad Software, San Diego, USA) was used for the statistical analyses. Two-way analysis of variance (ANOVA) followed by Bonferroni’s multiple comparison test was employed to compare the means and determine the significance of the differences. Results were considered significantly different if p < 0.05 (*); results are also reported with p < 0.01 (**), p < 0.001 (***) and p < 0.0001 (****). Materials Solvents were purchased as ACS grade and used without any further purification and anhydrous solvents used were freshly distilled. 2-(((Ethylthio)carbonothioyl)thio)-2- methylpropanoic acid chain transfer agent was synthesized following a protocol from literature.(57) Sterile dulbecco's phosphate-buffered saline (DPBS), 2,2’-dithiodipyridine (98%, DTP), N,N′- dicyclohexylcarbodiimide (99%, DCC), 4-(dimethylamino)pyridine (≥99%, DMAP), polyethylene glycol methyl ether (PEG44, Mn ~ 2,000 g.mol-1), 2- aminophenylboronic acid hydrochloride (≥95%), N-(3-dimethylaminopropyl)-N′- ethylcarbodiimide hydrochloride (≥98%, EDC), 4-fluorobenzoic acid (98%), N- hydroxysuccinimide (98%), N-(2-aminoethyl)maleimide trifluoroacetate salt (≥98%), N,N- diisopropylethylamine (≥99%), 1,4-dithiothreitol (DTT, 97%), tris-(2- Carboxyethyl)phosphine hydrochloride (TCEP) (≥ 97%), and ethanolamine (98%) were purchased from Sigma Aldrich and Fisher Scientific and used without further purification. Chemicals of 2,2′-azobis(2-methylpropionitrile) (98%, AIBN), and N-isopropylacrylamide (97%, NIPAM) were purchased from Sigma Aldrich and recrystallized from acetone and hexane, respectively. Acrylic acid (99%, AAc) was also purchased from Sigma Aldrich and purified by distillation. Activity assay kits of aspartate aminotransferase (AST), alanine aminotransferase (ALT), lactate dehydrogenase (LDH) and calcium-detecting assay kit were also purchased from Sigma Aldrich: Thiolated glucagon (GCG-SH,sequence: HSQGTFTSDYSKYLDSRRAQDFVCWLMNT (SEQ ID NO: 1)) and native GCG (sequence: HSQGTFTSDYSKYLDSRRAQDFVQWLMNT (SEQ ID NO: 2)) were purchased from QB\790482.00511\96013124.1 32 790482.00511 Biomatik at >90% purity. For the glucagon activity assay, commercial kit cAMP Hunter™ eXpress GCGR CHO-K1 GPCR assay was purchased from Eurofins DiscoverX Products, LLC.89Zr-oxalate was obtained from 3D Imaging LLC. Contrast agent Fenestra HDVC CT was purchased from MediLumine. Vaporized isoflurane was obtained from McKesson. Characterization techniques NMR Spectroscopy.1H-NMR spectra were recorded at 400 MHz or 500 MHz on an Avance spectrometer respectively, with chloroform-d (CDCl3), DMSO-d6 ((CD3)2SO) and deuterium oxide (D2O) as the solvent. Chemical shifts of protons are reported as δ in parts per million (ppm) and are relative to tetramethyl silane (TMS) at δ = 0 ppm when using CDCl3 or solvent residual peak ((CD3)2SO), δ = 2.50 ppm / D2O, δ = 2.79 ppm). Dynamic Light Scattering. Hydrodynamic diameters (Dh) and size distributions of micellar formulations were determined by dynamic light scattering (DLS) at a polymer concentration of 10 mg / mL in DPBS supplemented with the adequate glucose concentration using a Malvern Zetasizer Nano ZS with a 4 mW He-Ne 633 nm laser module. Measurements were carried out at an angle of 173° (back scattering), and results were analyzed using Malvern DTS 7.03 software. All determinations were repeated 3 times with at least 10 measurements recorded for each run. Reverse-Phase High Performance Liquid Chromatography (HPLC). HPLCs of polymer-glucagon conjugates were carried out on an Agilent 1260 Infinity II HPLC system equipped with an autosampler and a UV detector using a Zorbax 300SB-C18 (analytical: 3.5 μm, 3.0 × 150 mm) with monitoring at λ = 220 nm and with a flow rate of 0.8 mL / min, using a gradient 25-95% of solvent B (A: H2O + 0.1% TFA; B: ACN + 0.1% TFA) over 17 min. Electrospray Ionization Mass Spectroscopy (ESI-MS). ESI-MS was performed using an Agilent 6530 ESI-Q-TOF in tandem with a 1260 Infinity LC. Turbidimetry. Turbidimetric analysis was performed on a SpectraMax iD3 spectrophotometer plate reader. Aqueous polymer solutions were prepared at 10 mg.mL-1in DPBS with changes in transmittance monitored at λ = 600 nm by heating each sample from 20 °C to 65 °C at a rate of 1 °C min-1. Lower critical solution temperature (LCST) for each thermal phase transition curve was set for abs = 0.5. Transmission Electron Microscopy (TEM). Dry-state-stained TEM imaging was performed on a FEI T12 instrument microscope operating at an acceleration voltage of 120 kV. All dry-state samples were deposited onto formvar-coated carbon grids. Grids were glow discharged for 15 seconds. Samples (5 mg / mL) were incubated either at 25 ºC or 40 ºC for 30 min. After roughly 5 min, excess sample was blotted from the grid and the grid was stained QB\790482.00511\96013124.1 33 790482.00511 with an aqueous 2 wt% uranyl acetate (UA) solution for 1 min prior to blotting, drying and microscopic analysis. SDS-PAGE. SDS-PAGE gels, samples were loaded using 2X Laemmli sample buffer and run on Mini-Protean TGX, Any kD gels (Bio-Rad) at 195V for 35 min using Tris / Glycine / SDS buffer (Bio-Rad). Gels were stained with Coomassie. SDS-PAGE protein standards were obtained from Bio-Rad (Precision Plus Protein Prestained Standards). Circular Dichroism. Circular dichroism (CD) analysis of thiolated and native glucagon samples was performed on a Jasco J-1715 CD spectropolarimeter, featuring a 150 W air-cooled Xe lamp and fitted with a temperature controlling system. The sample was prepared at 0.25 mg / mL concentration, in a mixture of DPBS (pH = 7.4) / HCl (pH = 3) (1 / 1) and contained in a 1mm pathlength quartz cuvette (Hellma, USA) with a spectral bandwidth of 1.0 nm. Synthetic Procedures Synthesis of PEG44-macromolecular chain transfer agent (mCTA). In a round bottom flask, the chain transfer agent 2-[[(ethylthio)carbonothioyl]thio]-2- methylpropanoic acid (168 mg, 0.750 mmol, 1.5 eq), DCC (206 mg, 1.00 mmol, 2 eq), and DMAP (122 mg, 1.00 mmol, 2 eq) were dissolved in 4 mL of dry DCM under Ar(g). Polyethylene glycol methyl ether (Mn ~2000 Da) (1000 mg, 0.500 mmol, 1 eq) was dissolved in 1 mL of dry DCM and added dropwise to the mixture, which was then allowed to stir for 72 h at 25 °C. The reaction mixture was then filtered to remove solid urea byproduct. The solvent was evaporated under reduced pressure and the product was purified by flash chromatography (SiO2, gradient DCM to DCM / MeOH 8 / 2 as eluent) to obtain of a yellow solid as the final product (0.782 g, yield= 70.3%).1H NMR (400 MHz, CDCl3) δ (ppm) = 4.27 – 4.22 (m, 2H), 3.70 – 3.57 (m, 178H), 3.37 (s,3H), 3.27 (q, J = 7.4 Hz, 2H), 1.67 (d, J = 9.8 Hz, 6H), 1.31 (t, J = 7.4 Hz, 3H). Representative synthesis of PEG44-b-P(NIPAM-stat-AAc). A concentrated solution of AIBN (1.02 mg, 0.006 mmol) in dry DMSO was prepared and the desired amount was added to a Schlenk flask containing PEG44 macro-CTA (54.9 mg, 0.025 mmol), AAc (33.7 mg, 0.468 mmol), and NIPAM (300 mg, 2.65 mmol). Theoretical feed ratio for NIPAM:AAc was 85:15 in every case. Total monomer to initiator ratios were varied to target different molecular weights. Dry DMSO was added to reach 3 mL and 3 freeze pump / thaw cycles were performed. The flask was filled with Ar(g) and stirred at 75 °C for 2h. Polymerization was quenched by exposing to air and an aliquot of the crude mixture was taken for1H NMR analysis. NIPAM conversion was calculated by comparing the areas under the peak of monomeric and polymeric amide peak at δ = 7.9 ppm and 6.9-7.6 ppm, respectively. AAc mol% was calculated by comparing the area under the peak of PAAc polymeric acidic peak at 11.8-12.1 ppm and QB\790482.00511\96013124.1 34 790482.00511 PNIPAM polymeric amide peak at 6.9-7.6 ppm. DPPNIPAM was calculated by comparing PEG glycolic peak at 3.4-3.5 ppm and NIPAM polymeric amide peak at 6.9-7.6 ppm. DPPAAc was calculated by comparing PEG glycolic peak at 3.4-3.5 ppm and AAc polymeric acidic peak at 11.8-12.1 ppm. Procedure was repeated for PEG / NIPAM / AAc ratios of 1 / 57 / 10 (p1), 1 / 106 / 19 (p2), 1 / 212 / 38 (p3), 1 / 283 / 50 (p4), 1 / 472 / 83 (p5) (see Table 1). Note: Multiple polymer batches of P2 were synthesized across this study with near identical molecular weights 15,00 ± 0,5 kDa. Mn,NMR was calculated according to the formula Mn,NMR = Mn, PEG44 mCTA + (DPPNIPAM × MW NIPAM) + (DPPAAc ×MW AAc). The crude mixture was directly used in the following reaction.1H NMR (400 MHz, DMSO-d6) δ (ppm) = 11.96 (s, 22H), 7.16 (s, 110H), 3.85 (m, 121H), 3.47 (s, 178H), 1.96 (m, 154H), 1.76 – 1.16 (m, 246H), 1.12 – 0.75 (m, 717H). Representative synthesis of PEG44-b-P(NIPAM-stat-2-APBA). Crude PEG44-b- P(NIPAM-stat-AAc) (AAc eq, 0.468 mmol) solution in DMSO, was added to 10 mL of MilliQ water at 0 °C in a round bottom flask and stirred for 1 h until polymer dissolution. EDC (360 mg, 1.88 mmol) and DMAP (36.5 mg, 0.234 mmol) were then added, and the solution was stirred at 25 °C for 20 min. 2-APBA.HCl (159 mg, 0.938 mmol) was added, and the mixture stirred for 16 h at 25 °C. Solution was dialyzed against DI water for 3 days (MWCO = 3.5 kDa) and lyophilized to obtain a white solid with quantitative conversion. 2-APBA mol% was calculated by comparing the area under the peak of 2-APBA aromatic peak at δ = 6.8-7.5 ppm and NIPAM tertiary isopropyl peak at δ = 3.6-3.9 ppm. DPPNIPAM was calculated by comparing PEG glycolic peak at δ = 3.5 ppm and NIPAM tertiary isopropyl peak at 3.6-3.9 ppm. DPP(2-APBA) was calculated by comparing PEG glycolic peak at δ =3.5 ppm and 2- APBA aromatic peaks at δ = 6.8-7.5 ppm. Mn, NMR was calculated according to the formula Mn, NMR = Mn, PEG44 mCTA + (DPPNIPAM ×MW, NIPAM) + (DPP2-APBA * MW, 2- APBA).1H NMR (400 MHz, D2O) δ (ppm) = 7.20 (t, 83H), 3.74 (s, 80H), 3.57 (s, 178H), 2.18 – 1.74 (m, 118H), 1.43 (s, 187H), 1.00 (s, 553H). End-group modification of PEG44-b-P(NIPAM-stat-2-APBA) with pyridyl disulfide (PDS). PEG44-b-P(NIPAM-stat-2-APBA) (Mn = 15.0 kDa, 100 mg, 0.0067 mmol) and DTP (29.0 mg, 0.133 mmol) were dissolved in 1 mL of dry methanol. The solution was sparged for 20 min with Ar(g). Ethanolamine (1.63 mg, 0.027 mmol) was added directly by syringe. The solution was stirred under Ar(g) for 3 h at 25 °C. The polymer was first dialyzed (MWCO = 3.5 kDa) against an acetone / water mixture (50 / 50 v / v %) for 16 h to remove the excess of DTP, and then dialyzed against water for another 2 days to remove acetone. Polymer solution was lyophilized to obtain a white solid. Functionalization was calculated by comparing the signal QB\790482.00511\96013124.1 35 790482.00511 from PDS group at δ = 8.2- 8.3 ppm with signal from PEG at δ = 3.5-3.6 ppm. All polymers showed quantitative functionalization. Conjugation of glucagon (GCG-SH) with PEG44-b-P(NIPAM-stat-2-APBA)-PDS. PEG44-b-P(NIPAM-stat-APBA)-PDS (Mn = 15 kDa, 5.1 mg, 0.343 µmol) was dissolved in 0.5 mL of DPBS (pH =7.4) and added to a solution of GCG-SH (1 mg, 0.286 µmol) in 0.5 mL HCl (10 mM, pH = 2). The solution was stirred for 4 h at 4 ºC. Crude, conjugated mixture was purified by centrifugal filtration (MWCO = 10 kDa, 3x HCl 10 mM, 3x DPBS pH = 7.4, 10 min, 13200 rpm, 4 °C). Reaction was monitored by analytical HPLC, showing maximum conversion of 83.0% within 4 h. End-group modification of PEG44-b-P(NIPAM-stat-2-APBA) (P2 / P3) with deferoxamine- maleimide (DFO-mal). PEG44-b-P(NIPAM-stat-2-APBA)-PDS (Mn = 15.0 kDa, 20 mg, 0.0013 mmol) was dissolved in 1 mL of a 10 mM tris(2-carboxyethyl)phosphine hydrochloride (TCEP) aqueous solution. The mixture was further purified by centrifugal filtration to remove free pyridyl disulfide (MWCO = 3 kDa, 3×TCEP, 10 min, 13200 rpm, 4 °C). Thiol-terminated polymer was recovered and diluted in 1 mL of MilliQ water. Triethylamine (TEA, 0.0013 mmol, 1 mg) and deferoxamine-maleimide (DFO-mal) (0.00421 mmol, 3 mg) were added and the mixture was stirred at room temperature for 16 h. Crude polymer was further purified by centrifugal filtration (MWCO =10 kDa, 10× DPBS, 10 min, 13200 rpm, 4 °C) and lyophilized to obtain a white solid.1H NMR analysis confirmed successful functionalization. 89Zr-radiolabeling of P2 and P3. 89Zr-radiolabeling of the P2 and P3 were adapted from Tavaré et al.(58) with slight modifications. Chemicals and materials were purchased from Sigma Aldrich (St. Louis, MO) except when indicated. [89Zr]Zr-oxalate was obtained from 3D Imaging LLC. Upon arrival, [89Zr]Zr-oxalate was diluted with 40% (v / v) 2 M Na2CO3 and allowed to incubate for 3 min. The activity was then diluted with 2.5× volume of 1 M HEPES (pH = 7.0). Final pH was checked with pHydrion plastic indicator strips (Micro Essential Laboratory) to confirm a pH of 7. The DFO-conjugated micelle and non-micelle was added to the buffered [89Zr]Zr-oxalate to give a molar activity of 180– 220 MBq / mg and mixed for 1 h at 37 °C using a thermomixer with shaking (600–800 rpm). Radiolabeling efficiency was measured by ITLC (Biodex Medical Systems) using 20 mM citrate buffer pH = 5.6 as the mobile phase using a Wizard 3″ 1480 Automatic Gamma Counter (Perkin- Elmer). Radiolabeled non-micelle and micelle were purified using a Centriprep filter with a 10 kDa MWCO. Radiochemical purity was assessed by ITLC as above. The counts at the origin (O) and at the solvent front (SF) were used to calculate % labeling efficiency or % radiochemical QB\790482.00511\96013124.1 36 790482.00511 purity by the equation: O / (O + SF) × 100%. For dose calibration measurements, a Capintec CRC-55tR (Capintec, Ramsey NJ) calibrated for Zr-89 was utilized (Calibration # 465). A final molar activity of 0.16–0.2 MBq / µg [89Zr]Zr-DFO-micelle / non-micelle was used in the experiments. 18F-FBEM labeling of micelle. The synthesis of18F-FBEM was adapted from Collins et al.(59) A purified sample containing 159 MBq of dried18F-FBEM dissolved in 50 µL of DMSO was used for labeling the micelle.1.5 mg of micelle (1 equivalent, 112 nmol) was first dissolved in 500 µL of MilliQ water in a dram vial. 0.16 mg of TCEP (5 equivalents, 560. nmol) was then added to the solution and allowed to stir for 10 min. Following the stirring, 500 µL of 1M borate buffer (pH = 8) was added and stirred for 2 min. The18F-FBEM in 50 µL of DMSO was then pipetted into the micelle solution and allowed to react for 25 min at room temperature with stirring. TCEP and unreacted18F-FBEM were removed using a Centriprep filter with a 3 kDa MWCO. A final molar activity of 141 MBq / µg was produced for the imaging experiments. 18F-SFB labeling of GCG-SH. The synthesis of18F-SFB was adapted from Lazari et al.(60) A purified sample containing 330 MBq of dried18F-SFB in 50 µL of DMSO was used for labeling the glucagon. 0.5 mg (1 equivalent, 140 nmol) of glucagon-SH was dissolved in 500 µL of 10 mM HCl in a LoBind Eppendorf tube. Then 500 µL of pH = 8 borate buffer (1M) was added to the solution. The reaction mixture was allowed to react 5-30 min. The18F-SFB in 50 µL of DMSO was then pipetted into the glucagon solution and allowed to react for 25 min at room temperature with stirring. The unreacted18F-SFB was removed using a Centriprep filter with a 3 kDa MWCO. A final molar activity of 141 MBq / µg was produced for the imaging experiments. Methods In vitro glucagon activity assay. Commercial kit cAMP Hunter™ eXpress GCGR CHO-K1 GPCR assay was purchased from Eurofins and used following manufacturer protocol. In brief, cells were plated according to manufacturer’s protocol for 96-well plate assay (1 vials of cells / 96-well plate, 30000 cells / well). Cells were incubated for 18 h at 37 ºC, and 5% CO2 atmosphere. Cell plating buffer was then removed and replaced with 30 µL of cell assay buffer. 15 µL of agonist solution was added to appropriate wells and plates were incubated at 37 ºC for 30 min. After, 15 μL of antibody Solution and 60 μL of cAMP working detection solution were added to the cells, and plates were incubated for 1 h at 25 ºC in the dark. Finally, 60 μL of cAMP solution A were added and plates were incubated for 24 h at 25 ºC in the dark, before reading chemiluminescent signal. A four-parameter logistic fit was applied to the results to QB\790482.00511\96013124.1 37 790482.00511 obtain EC50 values using GraphPad Prism 7.0. The mean and standard error of the mean of five to six independent repeats were used for calculations. In vivo acute and chronic toxicity of PEG44-b-P(NIPAM-stat-2-APBA) (P2). In vivo acute and chronic toxicity of P2 was evaluated in C57Bl / 6J mice (11 weeks, male). The acute toxicity was evaluated for 0 h, 24 h and 120 h. The mice were given a single dose of 2.322 mg / Kg of empty micelles (P2) injected intraperitoneally and were sacrificed at different intervals to determine the whole blood count (CBC) (n = 4 or 5) and organ weight (n = 6). To evaluate chronic toxicity, mice were administered with 2.32 mg / Kg of empty micelle (P2) by intraperitoneal injection continuously for 14 days. Mice administered with saline was the control group for the chronic toxicity. After 14 days of injection the mice were sacrificed to determine the body weight, CBC count, organ weight, histopathology, hepatic function parameters, kidney function parameters, immune markers, and immunohistochemistry. The blood was collected and stored in EDTA coated tubes for CBC analysis (n=5). Remaining blood was centrifuged, and the serum was collected and stored in - 80°C for further analysis of other parameters. Urine was collected and stored in -80°C. The CBC was done by IDEXX BioAnalytics. All organs were incubated in formalin for 18 h at 4 ºC, and then transferred / stored in 70% ethanol until taken for sectioning and staining with hematoxylin and eosin (H&E) (n = 5) by the UCLA Translational Pathology Core Laboratory. The hepatic function parameters kidney function parameters were evaluated from the blood serum using an aspartate aminotransferase activity assay kit (AST), alanine aminotransferase activity assay kit (ALT) and a lactate dehydrogenase activity assay kit (LDH) (n = 8). The kidney function parameters were evaluated using a calcium-detecting assay kit from urine (n = 4) and blood glucose (n = 10) levels using the Hemocue glucometer. The immune markers IFN γ, TNF α and IL 2 (n = 10) were analyzed using the Meso Scale Discovery (MSD) assay kits and MSD software. The immunohistochemistry for the lung and liver tissue was done using organ-specific immune marker F8 / 40 antibody at the UCLA Translational Pathology Core Laboratory. µPET / µCT89Zr-labeled micelle / 89Zr-labeled linear polymer. Eight C57BL / 6 mice (7 weeks, male, Jackson Laboratory) were anesthetized with 1.5% vaporized isoflurane and were injected via i.v. injections (tail vein) with 100uL CT contrast agent Fenestra HDVC agent (MediLumine), followed by another i.v. injection of 25-30 µCi radiolabeled89Zr- labeled micelle (n = 4) or radiolabeled89Zr-labeled linear polymer (n = 4). Each mouse was scanned immediately after injection for a 1-h dynamic µPET scan (energy window 350-650 keV) followed by a 1-min µCT scan (voltage 80kVP, current 150 µA, 720 projections, 200µm QB\790482.00511\96013124.1 38 790482.00511 resolution) on a GNEXT PET / CT scanner (Sofie Biosciences, Dulles, VA). Mice were imaged by µPET / µCT again at 2h, 6h, 24h, 48h, 96h, and 192h (static µPET, 10-60 minutes), post tracer injection. The µPET images were reconstructed using the 3D-OSEM / MAP algorithm (24 subsets and 3 iterates) with random, attenuation, and decay correction. The µCT images were reconstructed using a Modified Feldkamp Algorithm. Amide software was used to analyze co- registered µPET / µCT images. µPET / µCT18F-FBEM-labeled micelle / / 18F-SFB-labeled glucagon. Eight C57BL / 6 mice (7 weeks, male, Jackson Laboratory) were anesthetized with 1.5% vaporized isoflurane and were injected via i.v. injection (tail vein) with 100 µL CT contrast agent Fenestra HDVC agent (MediLumine), followed by another i.v. injection of 70-75 µCi radiolabeled18F- FBEM micelle polymer (n = 4) or radiolabeled18F-SFB glucagon non-micelle polymer (n = 4). Each mouse was scanned immediately after injection for a 1-h dynamic µPET scan (energy window 350-650 keV) followed by a 1-min µCT scan (voltage 80kVP, current 150 µA, 720 projections, 200µm resolution) on a GNEXT PET / CT scanner (Sofie Biosciences, Dulles, VA). Mice were imaged by µPET / µCT again at 2h, 4h, 6h, and 8h (static µPET, 10-90 min), post tracer injection. The µPET images were reconstructed using the 3D-OSEM / MAP algorithm (24 subsets and 3 iterates) with random, attenuation, and decay correction. The µCT images were reconstructed using a Modified Feldkamp Algorithm. Amide software was used to analyze co- registered µPET / µCT images. In vivo safety evaluation of PEG44-b-P(NIPAM-stat-2-APBA)-GCG. C57Bl / 6J mice (11 weeks, male, n = 5-6, Charles River Laboratories) were fasted for 4 h before being injected intraperitoneally with either 50 µL of a solution of P2-GCG in DPBS (pH = 7.4) at a dose of 500 µg / Kg or 50 µL of DPBS (pH = 7.4) alone. Blood glucose level was assessed at predetermined time points (0, 15, 30, 45, 60, 480 min) using an Hemocue glucometer. At the end of the study, mice were sacrificed, and liver and kidneys were harvested for histopathological analysis. Organs were fixed in formalin for 36 h at 25 ºC, and then transferred in ethanol 70% and kept at 4 °C until taken for sectioning and staining with hematoxylin and eosin (H&E) by the UCLA Translational Pathology Core Laboratory. In vivo hypoglycemia reversal. C57Bl / 6J mice (11 weeks, male, n = 5-6, Charles River Laboratories) were fasted for 12 h and then injected intraperitoneally with either 0.90 U / kg of insulin or 0.85 U / kg of insulin, to induce deep or moderate hypoglycemia, respectively. After 60 min, mice were injected with either 50 µL of a solution of P2-GCG in DPBS (pH = 7.4) at a dose of 500 µg / Kg or 50 µL of P2 in DPBS (pH = 7.4) at polymer equivalent dose. Blood glucose levels were assessed every 15 min for 2h using an Hemocue glucometer. QB\790482.00511\96013124.1 39 790482.00511 In vivo hypoglycemia prevention. C57Bl / 6J mice (11 weeks, male, n = 5-6, Charles River Laboratories) were fasted for 12 h and then injected intraperitoneally with 200 µL of a solution containing both 0.90 U / kg of insulin and either 50 µL of a solution of P2-GCG in DPBS at a dose of 500 µg / Kg or 50 µL of P2 in DPBS at polymer equivalent dose. Blood glucose level was assessed every 15 min for 2h using an Hemocue glucometer. Micro-Positron Emission Tomography (μPET) / Micro-Computed Tomography (µCT). μPET / µCT for89Zr-micelle polymer,89Zr-non-micelle polymer,18F-FBEM micelle polymer and 18F-SFB glucagon was performed on an GNEXT PET / CT scanner (Sofie Biosciences, Dulles, VA). Upon injection, an initial µPET scan (energy window 350-650 keV) was performed followed by a 1-min µCT scan (voltage 80kVP, current 150 µA, 720 projections, 200µm resolution) for all samples. Example 2 An alternative method was developed to synthesize the polymer-thiol-functionalized glucagon peptide (GCG-SH) conjugate. This strategy involved incorporating the pyridyl disulfide functionality directly into the monomer feed instead of modifying the polymer chain end with the same group. By maintaining the feed ratio of pyridyl disulfide methacrylate at one equivalent, each polymer chain statistically contains one pyridyl disulfide unit, effectively preserving the one-to-one stoichiometry of peptide conjugation seen with end-functionalized polymers (Figure 27a). Furthermore, this strategy allows for controlled tuning of glucagon conjugation amount by varying the molar percentage of pyridyl disulfide monomer units in the polymer composition. A representative synthesis of PEG44-b-P(NIPAM-stat-AAc-stat-PDSMA) includes, a concentrated solution of AIBN (1.02 mg, 0.006 mmol) in dry DMSO was prepared and the desired amount was added to a Schlenk flask containing PEG44 macro-CTA (54.9 mg, 0.025 mmol), AAc (33.7 mg, 0.468 mmol), PDSMA (12.1 mg, .0473 mmol), and NIPAM (300 mg, 2.65 mmol). Theoretical feed ratio for NIPAM:Aac:PDSMA was 83.5:15:1.5 in every case. Total monomer to initiator ratios were varied to target different molecular weights. Dry DMSO was added to reach 3 mL and 3 freeze pump / thaw cycles were performed. The flask was filled with Ar(g) and stirred at 75 °C for 2.5h. Polymerization was quenched by exposing to air and an aliquot of the crude mixture was taken for1H NMR analysis. NIPAM conversion was calculated by comparing the areas under the peak of monomeric and polymeric amide peak at δ = 7.9 ppm and 6.9-7.6 ppm, respectively. AAc mol% was calculated by comparing the area under the peak of PAAc polymeric acidic peak at 11.8-12.1 ppm and PNIPAM polymeric amide peak at 6.9-7.6 ppm. PDSMA mol% was calculated by comparing the area under the QB\790482.00511\96013124.1 40 790482.00511 peak of PPDSMA aryl proton at 8.4-8.5 ppm and PNIPAM polymeric amide peak at 6.9-7.6 ppm. DPPNIPAM was calculated by comparing PEG glycolic peak at 3.4-3.5 ppm and NIPAM polymeric amide peak at 6.9-7.6 ppm. DPPAAc was calculated by comparing PEG glycolic peak at 3.4-3.5 ppm and AAc polymeric acidic peak at 11.8-12.1 ppm. DPPDSMA was calculated by comparing PEG glycolic peak at 3.4-3.5 ppm and PDSMA polymeric aryl proton at 8.4-8.5 ppm. Procedure was repeated for all PEG / NIPAM / AAc / PDSMA polymers synthesized and for all alternatives to PEG. Multiple polymer batches were synthesized of near identical molecular weights. Mn,NMRwas calculated according to the formula Mn,NMR= Mn, PEG44 mCTA + (DPPNIPAM × MW NIPAM) + (DPPAAc ×MW AAc) + (DPPDSMA x MW PDSMA). The crude mixture was directly used identical to previously described boronic acid functionalization.]1H NMR (400 MHz, DMSO) δ 12.06 (s, 29H), 8.42 (dd, J = 1.8, 1.0 Hz, 2H), 7.69 – 6.85 (m, 92H), 3.47 (s, 178H), 2.20 – 1.77 (m, 126H), 1.71 – 1.16 (m, 175H), 1.02 – 0.92 (s, J = 6.6 Hz, 656H). A representative synthesis of PEG44-b-P(NIPAM-stat-2-APBA-stat-PDSMA) includes a crude PEG44-b-P(NIPAM-stat-AAc-stat-PDSMA) (AAc eq, 0.468 mmol) solution in DMSO, was added to 10 mL of MilliQ water at 0 °C in a round bottom flask and stirred for 1 h until polymer dissolution. EDC (360 mg, 1.88 mmol) and DMAP (36.5 mg, 0.234 mmol) were then added, and the solution was stirred at 25 °C for 20 min.2-APBA.HCl (159 mg, 0.938 mmol) was added, and the mixture stirred for 16 h at 25 °C. Solution was dialyzed against DI water for 3 days (MWCO = 3.5 kDa) and lyophilized to obtain a white solid with quantitative conversion. 2-APBA mol% was calculated by comparing the area under the peak of 2-APBA aromatic peak at δ = 6.8-7.5 ppm and NIPAM tertiary isopropyl peak at δ = 3.6-3.9 ppm. DPPNIPAM was calculated by comparing PEG glycolic peak at δ = 3.5 ppm and NIPAM tertiary isopropyl peak at 3.6-3.9 ppm. DPP(2-APBA) was calculated by comparing PEG glycolic peak at δ =3.5 ppm and 2-APBA aromatic peaks at δ = 6.8-7.5 ppm. Mn, NMR was calculated according to the formula Mn, NMR = Mn, PEG44 mCTA + (DPPNIPAM ×MW, NIPAM) + (DPP2-APBA * MW, 2-APBA) + (DPPDSMA * MW, PDSMA).1H NMR (400 MHz, D2O) δ (ppm) = 8.45 – 8.39 (dd, 1H), 7.20 (t, 56H), 3.74 (s, 76H), 3.57 (s, 178H), 2.18 – 1.74 (m, 109H), 1.43 (s, 174H), 1.00 (s, 452H). Thiolated glucagon analogue bearing a cysteine residue (Q24C), GCG-SH, was used in this study and covalently conjugated onto the polymer via pyridyl disulfide exchange and formation of a disulfide bond. The conjugation was analyzed by high-performance liquid chromatography (HPLC) (Figure 27d). Within 1 hour of the addition of GCG-SH to the PDS- containing polymer, >99 % of the peptide was consumed, reaching a maximum conversion QB\790482.00511\96013124.1 41 790482.00511 (Figure 27d(i)). The glucose-responsive behavior of the GCG conjugated polymer was evaluated using dynamic light scattering (DLS). In the presence of glucose, notably, under normoglycemic conditions ([Glc] = 150 mg / dL), DLS revealed the presence of well-defined micelles with a hydrodynamic diameter of approximately 60 nm, indicating stable self- assembly at physiological glucose levels (Figure 27b). In the absence of glucose, GCG- micelles formed linear polymer at 37 °C. The micelles exhibited responsive changes in size distribution, as determined by DLS (Figure 27b). Specifically, at 37 °C, the micellar solution contained a mixture of linear conjugates, intact micelles, and larger aggregates. These larger structures are likely the result of micelle swelling or disassembly under hypoglycemic conditions. It is important to note that DLS cannot accurately quantify the relative abundance of each population due to its inherent bias toward larger particles in intensity-based measurements. To further probe the glucose sensitivity of the system, micelle solutions were diluted to simulate hypoglycemia ([Glc] = 75 mg / dL), which again yielded a heterogeneous population of linear polymers, micelles, and larger aggregates (Figure 27b). Transmission electron microscopy (TEM) further corroborated these findings. No distinct micellar structures were observed in the absence of glucose (0 mg / dL), while micelles became visible at glucose concentrations of 1.5 mg / dL, confirming the glucose-triggered assembly of the GCG-micelles (Figure 27c). A concern of glucagon delivery is its stability. In vitro stability studies were thus conducted to evaluate the chemical degradation and fibrillation behavior of glucagon when encapsulated within the modified micelle formulation. High-performance liquid chromatography (HPLC) and Thioflavin T (ThT) fluorescence assays were employed to assess the chemical and physical stability of glucagon, respectively. Glucagon conjugated to the modified micelles exhibited remarkable stability, remaining intact for at least 22 days at room temperature and more than 14 days at 4 °C (longest periods tested for each condition, Figure 28a). In contrast, free glucagon underwent rapid degradation within 3 days at room temperature and within 10 days at 4 °C. ThT fluorescence data further confirmed the enhanced physical stability of glucagon in micelles, with minimal fibril formation observed for over 10 days at room temperature, whereas fibrillation of free glucagon was evident starting from day 3 (Figure 28b). These results demonstrate that glucagon conjugation to the micelles significantly improves both the chemical and physical stability of glucagon under storage conditions. In vivo efficacy of micelles prepared according to Example 2 The in vivo efficacy of the glucagon-conjugated micelles (GCG-micelles) was evaluated in healthy male C57Bl / 6J mice. To evaluate the glycemic responsiveness of the QB\790482.00511\96013124.1 42 790482.00511 formulation above the glucose counterregulatory threshold, mice were fasted for 12 hours, and severe hypoglycemia (45 ± 5 mg / dL) was induced via intraperitoneal injection of insulin (1 U / kg) at time 0. Once hypoglycemia was established (30–45 minutes post-insulin administration), 500 µg / kg of GCG-micelle was administered intraperitoneally. Control groups received empty micelles and thiolated free glucagon (GCG-SH) at an equivalent dose. In the GCG-micelle group, normoglycemia (110 mg / dL) was rapidly restored within 120 minutes (Figure 29a). In comparison, free glucagon achieved normoglycemia in approximately 135 minutes, while mice treated with empty micelles showed no increase in blood glucose levels. To determine the minimum effective dose, a dose–response study was conducted using GCG- micelle formulations containing 500, 250, 100, and 50 µg / kg of glucagon. Both the 250 µg / kg and 100 µg / kg doses restore normoglycemia within 120 minutes, with no significant difference in efficacy between the GCG-micelle and free glucagon at these concentrations (Figure 29b and 29c). However, doses below 100 µg / kg did not result in significant glycemic recovery, even with glucagon alone (Figure 29d). To investigate if GCG micelles elevate glycemia in vivo at normal glycemic conditions, the conjugate was administered IP in healthy 6 h fasted C57Bl / 6J mice. The control group was administered with empty micelles. Upon injection, the blood glucose levels of mice were monitored over 2 h at different time intervals. After 15 min, the glucose level steadily dropped to 125 mg / dL within 120 min of injection (Figure 30). Overall, glycemia remained in the normal range throughout the study indicating the glucose responsiveness. Hypoglycemia Prevention Furthermore, to assess the temporal efficacy and glucose responsiveness of the glucagon micelles, additional studies were conducted in which GCG-micelles were administered 30–90 minutes prior to insulin injection. In these experiments, mice received the GCG-micelle intraperitoneally, followed by insulin administration (1 U / kg) after a 30-, 60-, or 90-minute interval. When GCG-micelles were administered 30 minutes prior to insulin, blood glucose levels showed a slight initial decline following insulin injection, indicating the onset of hypoglycemia. However, within 15 minutes, glucose levels began to rise, demonstrating the ability of the micelles to counteract insulin-induced hypoglycemia and restore euglycemia (Figure 31a). In the 60-minute pre-administration group, glucose levels began to increase approximately 40 minutes after insulin injection, confirming sustained in vivo bioactivity and glucose responsiveness of GCG micelles (Figure 31b). In contrast, when GCG-micelles were administered 90 minutes prior to insulin, no recovery in blood glucose was observed, and mice progressed into hypoglycemia (Figure 31c). These results demonstrate that GCG-micelles QB\790482.00511\96013124.1 43 790482.00511 retain therapeutic efficacy when administered up to 60 minutes prior to insulin challenge, highlighting their potential as a responsive and preemptive strategy for managing hypoglycemia. Safety Studies The chronic toxicity and immunogenicity of the GCG-micelles were evaluated through a combination of biochemical and histological analyses. Total IgG and IgM levels were quantified using enzyme-linked immunosorbent assay (ELISA) to assess systemic immune responses, while immunohistochemistry and histological examinations were conducted to evaluate macrophage activation and organ morphology, respectively. These assessments were performed using blood plasma and tissue samples collected from mice following daily intraperitoneal administration of empty micelles, free glucagon, or GCG-micelles over a 14- day period. ELISA results revealed no significant differences in IgG or IgM levels among the treatment groups, indicating that GCG-micelles did not elicit a detectable humoral immune response (Figure 32a). Histopathological analysis of the liver, spleen, heart, lung, and kidney using hematoxylin and eosin (H&E) staining showed no observable morphological abnormalities (Figure 32b). Hepatic function parameters including alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels analyzed from serum (Figure 32d) were the same for the GCG micelles as the negative controls. Additionally, immunohistochemical staining of liver and lung tissues using the macrophage-specific marker F4 / 80 demonstrated no difference in macrophage presence or activation across treatment groups (Figure 32e). Collectively, these findings suggest that GCG-micelles are well-tolerated and exhibit a favorable safety profile with no evidence of chronic toxicity or immunogenicity under the conditions tested. Micelles Increase Half life In circulation, our assembled micelle should have its hydrophilic block surface exposed. Because the hydrophilic corona should interact directly with the blood environment, changes to the hydrophilic corona are proposed to make a large difference in blood half-life. The blood half-life of the micelle described above was determined to be ~1.4h and the PEG corona was 2,000 Da. Different PEG sizes ranging from the current formulation PEG2K to PEG20K (Figure 33a) were investigated. The PEGs were conjugated to a RAFT agent by N,N’- dicyclohexylcarbodiimide (DCC) coupling and used as macroRAFT agents to polymerize the micelle polymer. Branched PEGs (PEGMA) were also investigated; these were first polymerized using a commercial RAFT agent and then used as macroRAFT agents for chain extension. Because μPET / μCT imaging is expensive and can be timing consuming, a quicker QB\790482.00511\96013124.1 44 790482.00511 and cheaper method was developed to quickly screen different polymer micelles. This consisted of conjugating the micelle polymer at the pyridyl disulfide to 5 nm gold nanoparticles (GNPs), as seen by TEM (Figure 33b) (~50 nm, 1-3 GNPs per micelle). Micelle-GNPs were then injected into mice followed by blood collection over time. Blood samples were digested using 70% nitric acid and analyzed by inductively coupled mass spectrometry (ICP-MS) using gold concentration as a proxy for polymer concentration in the blood (Figure 33c). From the results, PEG20K increased the blood half-life to from 1.4 h to 4 h. Notably, the PEG2K half- life by this method is observed to be similar to the half-life previously determined through PET imaging, validating the method. The effects of different corona architectures are also of interest, like branched PEGs. A micelle synthesized with a P(PEGMA)300 block with a molecular weight of 2 kDa was evaluated, identical to that of the current micelle formulation except for the PEG architecture. The branched system observed does have a longer half-life compared to the linear system (Figure 33d). A branched version of PEG20K may further increase the blood half-life beyond 4h. TABLES Table 1. Block copolymer characteristics of PEG44-b-P(NIPAM-stat-AAc) library. NIPAM Experimental %aa Table 2. Block characteristics of PEG44-b-P(NIPAM-stat-2-APBA) library. SampleMn, NMR2-APBA mol%a Cloud point (°C)b Dh(nm)c PDIc QB\790482.00511\96013124.1 45 790482.00511 SampleMn, NMR2-APBA mol%a Cloud point (°C)b Dh(nm)c PDIc(kDa)a (from 3 repeatmeasurements) obtained by dynamic light scattering (DLS) at 40 ºC (P1-P4) or 37 ºC (P5). Table 3. PEG44-b-P(NIPAM-stat-APBA)-GCG conjugate library characteristics. Conjugated polymer Total Mw(kDa)a Cloud point (°C)b Dh(nm)c PDIcP1-GCG 135 nd 3845 0489 0 6 2 b hand PDI values (from 3 repeatmeasurements) obtained by dynamic light scattering(DLS) at 37 ºC with the addition of [Glc] = 150 mg / dL. Table 4. Numerical values of the CBC results illustrated in Figure 13. QB\790482.00511\96013124.1 46 790482.00511 Table 5. Numerical values of the CBC results illustrated in Figure 3. Counts (mU) SI. No Hepatic Function Parameters s 0 Counts (pg / mL) I N I fl t i k s QB\790482.00511\96013124.1 47 790482.00511 Table 8. Quantification of μPET / µCT scans for89Zr-labeled linear polymer and89Zr-labeled micelle 2h post-injection from the organs showing the highest uptake (illustrated in Figure 15), with appreciable signal. ID, injected dose. CC, cubic centimeter. %ID / cc Organ89Zr-labeled linear polymer89Zr-labeled micelle 1. G. Brawerman, V. Ntranos, P. J. Thompson, Alpha cell dysfunction in type 1 diabetes is independent of a senescence program. Front. Endocrinol. (Lausanne). 13 (2022), doi:10.3389 / fendo.2022.932516. 2. G. C. Weir, J. Gaglia, S. Bonner-Weir, Inadequate β-cell mass is essential for the pathogenesis of type 2 diabetes. Lancet Diabetes Endocrinol.8, 249–256 (2020). 3. J. E. B. Reusch, J. E. Manson, Management of Type 2 Diabetes in 2017: Getting to Goal. JAMA 317, 1015–1016 (2017). 4. O. Veiseh, B. C. Tang, K. A. Whitehead, D. G. Anderson, R. Langer, Managing diabetes with nanomedicine: challenges and opportunities. Nat. Rev. Drug Discov. 14, 45–57 (2015). 5. K. L. 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Claims

790482.00511 CLAIMS We claim:

1. A polymer comprising an ordered arrangement of a polyethylene glycol block, a copolymer block comprising N-isopropyl acrylamide (NIPAM) and either acrylamidophenylboronic acid (2-APBA) and / or acrylic acid (AAc), and an end-group, wherein the end-group is optionally selected from a conjugated therapeutic agent, a conjugated reporter, a disulfide exchange functional group, and a chain transfer agent.

2. The polymer of claim 1, wherein the copolymer block comprises N-isopropyl acrylamide (NIPAM) and acrylamidophenylboronic acid (2-APBA).

3. The polymer of claim 2, wherein the copolymer block further comprises a conjugated therapeutic agent and / or a disulfide exchange functional group.

4. The polymer of claim 3, wherein the disulfide exchange group is pyridyl disulfide methacrylate (PDSMA).

5. The polymer of claim 3, wherein the conjugated therapeutic agent is a conjugated glucagon.

6. The polymer of claim 3, wherein the conjugated therapeutic agent is a conjugated cysteine-substituted glucagon.

7. The polymer of claim 3, wherein the conjugated therapeutic agent is a conjugated glucagon of SEQ ID NO:

1.

8. The polymer of any of claims 1-7, wherein the end-group comprises the conjugated therapeutic agent.

9. The polymer of claim 8, wherein the conjugated therapeutic agent is a conjugated glucagon.

10. The polymer of claim 8, wherein the conjugated therapeutic agent is a conjugated cysteine-substituted glucagon.

11. The polymer of claim 8, wherein the conjugated therapeutic agent is a conjugated glucagon of SEQ ID NO:

1.

12. The polymer of any of claims 2-7, wherein the end-group comprises the conjugated reporter.

13. The polymer of claim 12, wherein the conjugated reporter comprises a conjugated fluorophore, a conjugated radiolabel, or a metal.

14. The polymer of any of claims 2-7, wherein the end-group comprises the disulfide exchange functional group or the chain transfer agent. QB\790482.00511\96013124.1 54790482.00511 15. The polymer of claim 14, wherein the disulfide exchange functional group comprises a disulfide pyridyl group or the chain transfer agent comprises a trithiocarbonyl group.

16. The polymer of claim 1, wherein the copolymer block comprises N-isopropyl acrylamide (NIPAM) and acrylic acid (AAc).

17. The polymer of claim 16, wherein the end-group comprises the chain transfer agent.

18. The polymer of claim 17, wherein the chain transfer agent comprises a trithiocarbonyl group.

19. The polymer of any one of claims 1-18, wherein the ratio of N-isopropyl acrylamide (NIPAM) to either acrylamidophenylboronic acid (2-APBA) or acrylic acid (AAc) is from 5:95 to 95:

5.

20. The polymer of any one of claims 1-19, wherein the polymer has a Mn between 1 and 120 kDa.

21. The polymer of any one of claims 1-20, wherein the polyethylene glycol block comprises between 3 and 10000 ethylene glycol units.

22. The polymer of any one of claims 1-21, wherein the polyethylene glycol block is linear.

23. The polymer of any one of claims 1-21, wherein the polyethylene glycol block is branched.

24. The polymer of claim 23, wherein the branched polyethylene glycol block comprises poly(ethylene glycol methacrylate) (PEGMA).

25. A nanostructure comprising a plurality of polymers according to claim 1.

26. The nanostructure of claim 25, wherein the nanostructure is a micelle.

27. The nanostructure of any one of claims 25-26, wherein the nanostructure is stable above threshold glucose concentration and disassembles below the threshold glucose concentration at a physiologically-relevant temperature.

28. The nanostructure of any one of claims 26-27, wherein the nanostructure is stable above threshold glucose concentration and disassembles below the threshold glucose concentration in vivo.

29. The nanostructure of any one of claims 26-27, wherein the nanostructure is stable above threshold glucose concentration and disassembles below the threshold glucose concentration at a physiologically-relevant temperature or in vivo, wherein the threshold glucose concentration is between 55 mg / dL and100 mg / dL of glucose.

30. The nanostructure of any one of claims 26-29, wherein the polymer is the polymer according to any one of claims 2-24. QB\790482.00511\96013124.1 55790482.00511 31. A pharmaceutical composition comprising the polymer according to any one of the preceding claims 1-24 or the nanostructure comprising a plurality of polymers according to any one of claims 25-30 and a pharmaceutically acceptable carrier, excipient, or diluent, wherein the polymer comprises a conjugated therapeutic agent.

32. The pharmaceutical composition of claim 31 further comprising insulin.

33. A kit comprising the pharmaceutical composition according to claim 31 and further comprising a second therapeutic agent or a device for administering the composition according to claim 31.

34. The kit of claim 33, wherein the second therapeutic agent is insulin.

35. A method for treating or preventing a disease or condition a subject in need of a therapeutic agent, the method comprising administering the pharmaceutical composition comprising the polymer according to claim 1 or the nanostructure comprising a plurality of polymers according to claim 1 and a pharmaceutically acceptable carrier, excipient, or diluent, wherein the polymer comprises a conjugated therapeutic agent.

36. The method of claim 35, wherein the subject is in need of a therapeutic agent for hypoglycemia and the polymer comprises a conjugated glucagon.

37. The method of claim 36, wherein the subject is diabetic or in need of insulin therapy.

38. The method of claim 36, wherein the subject is administered insulin prior to or concurrently with the administered pharmaceutical composition.

39. The method of claim 36, wherein the subject is administered insulin after the pharmaceutical composition is administered.

40. The method of any one of claims 35-39, wherein the pharmaceutical composition is the pharmaceutical composition according to claim 31. QB\790482.00511\96013124.1 56

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