EP4673178A1 - Engineered nanocomplexes - Google Patents

Engineered nanocomplexes

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Publication number
EP4673178A1
EP4673178A1 EP24762825.8A EP24762825A EP4673178A1 EP 4673178 A1 EP4673178 A1 EP 4673178A1 EP 24762825 A EP24762825 A EP 24762825A EP 4673178 A1 EP4673178 A1 EP 4673178A1
Authority
EP
European Patent Office
Prior art keywords
insulin
fpba
nanocomplex
ncs
ins
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
EP24762825.8A
Other languages
German (de)
French (fr)
Inventor
Christoph Hagemeyer
Rong Xu
Sukhvir Kaur BHANGU
Francesca Cavalieri
Frank Caruso
Mark E COOPER
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
University of Melbourne
Monash University
Royal Melbourne Institute of Technology
Melbourne Institute of Technology
Original Assignee
Royal Melbourne Institute of Technology Ltd
University of Melbourne
Monash University
Melbourne Institute of Technology
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from AU2023900518A external-priority patent/AU2023900518A0/en
Application filed by Royal Melbourne Institute of Technology Ltd, University of Melbourne, Monash University, Melbourne Institute of Technology filed Critical Royal Melbourne Institute of Technology Ltd
Publication of EP4673178A1 publication Critical patent/EP4673178A1/en
Pending legal-status Critical Current

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Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6921—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere
    • A61K47/6927—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores
    • A61K47/6929—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle
    • A61K47/6931—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle the material constituting the nanoparticle being a polymer
    • A61K47/6939—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle the material constituting the nanoparticle being a polymer the polymer being a polysaccharide, e.g. starch, chitosan, chitin, cellulose or pectin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/61—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule the organic macromolecular compound being a polysaccharide or a derivative thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/22—Hormones
    • A61K38/26—Glucagons
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/22—Hormones
    • A61K38/28—Insulins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • A61K9/5107—Excipients; Inactive ingredients
    • A61K9/5123—Organic compounds, e.g. fats, sugars
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • A61K9/5107—Excipients; Inactive ingredients
    • A61K9/513—Organic macromolecular compounds; Dendrimers
    • A61K9/5161—Polysaccharides, e.g. alginate, chitosan, cellulose derivatives; Cyclodextrin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • A61K9/5107—Excipients; Inactive ingredients
    • A61K9/513—Organic macromolecular compounds; Dendrimers
    • A61K9/5169—Proteins, e.g. albumin, gelatin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • C07K14/605—Glucagons
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • C07K14/62—Insulins

Definitions

  • NCs nanocomplexes
  • NP polysaccharide nanoparticle
  • Glucagon a hormone selected from insulin, glucagon, or glucagon-like protein-1
  • NCPs nanocomplexes
  • NP polysaccharide nanoparticle
  • Glucagon-like protein-1 a hormone selected from insulin, glucagon, or glucagon-like protein-1
  • SC subcutaneous
  • exogenous insulin formulations rapid, short, intermediate, or long acting
  • blood glucose level monitoring by finger-prick tests or in-arm continuous blood glucose monitoring sensors remain the main strategy for blood glucose management and treatment of type 1 diabetes as well as some type 2 diabetes.
  • SC administration of insulin is often associated with hypoglycemia, which can be life threatening, and lead to glucose fluctuations and other adherence issues.
  • a glucose-responsive insulin delivery system that can act as an insulin reservoir, after a single daily SC injection, is desirable. Such systems would rapidly deploy insulin to counteract spikes in blood glucose levels and ensure a sustained release of insulin to maintain extended normoglycemia.
  • the ideal insulin delivery system would display sufficient insulin-loading capacity, colloidal stability for storage and administration together with a long shelf life and be cost effective.
  • a simple, reproducible, and scalable manufacturing process as well as biodegradability with limited toxicity and immunogenicity are also highly desirable.
  • formulations that dynamically regulate insulin release in response to blood glucose are not clinically available.
  • Various glucose-responsive materials for insulin delivery have been preclinically investigated for the controlled delivery of insulin in diabetic animal models, including glucose oxidase enzyme (GOx) based hydrogels (Gu Z, et al. ACS Nano 2013, 7(8): 6758-6766), GOx loaded nanoparticles (NPs) (Volpatti LR, et al.
  • the present invention provides a nanocomplex comprising a polysaccharide nanoparticle selected from the group consisting of glycogen, bovine glycogen, phytoglycogen or a combination thereof reversibly complexed with a hormone selected from the group consisting of insulin, glucagon and glucagon-like protein- 1.
  • the polysaccharide nanoparticle is covalently coupled to amine moieties.
  • the polysaccharide nanoparticle is covalently coupled to boronic acid moieties.
  • the present invention provides a method for reducing the blood glucose level of a subject in need thereof, the method comprising administering to the subject an effective amount of a nanocomplex according to the invention.
  • the invention provides a method for treating diabetes mellitus in a subject in need thereof, the method comprising administering to the subject an effective amount of a nanocomplex according to the invention.
  • a pharmaceutical composition comprising the nanocomplex according to the invention.
  • Figure 1 illustrates characterization of phytoglycogen nanoparticles (PG NPs), phytoglycogen-EDA nanoparticles (PGEDA NPs), and phytoglycogen-EDA-FPBA nanoparticles (PGEDA-FPBA NPs).
  • PG NPs phytoglycogen nanoparticles
  • PGEDA NPs phytoglycogen-EDA nanoparticles
  • PGEDA-FPBA NPs phytoglycogen-EDA-FPBA nanoparticles
  • FIG. 1 illustrates the determination of degree of substitution by 1 H-NMR spectroscopy.
  • Figure 3 illustrates the deconvolution of the peaks integrating for H-2,3,7,8 of the functionalized moieties of PG EDA .
  • Figure 4 illustrates size characterization of PGEDA-FPBA by 13 C-NMR spectroscopy 13 C-NMR.
  • FIG. 1 Solid state spectra of PG (A), PGEDA-FPBA (B).
  • Figure 5 illustrates the characterization of PG EDA-FPBA by 1 H-NMR spectroscopy.
  • Figure 6 illustrates the determination of degree of substitution by UV–vis spectroscopy.
  • Figure 7 illustrates size characterization of PGEDA-FPBA NPs.
  • B Size distribution of PGEDA-FPBA NPs.
  • Figure 8 illustrates the stability of PG EDA-FPBA NPs assessed by an ⁇ -and ⁇ - amylase degradation assay.
  • Figure 9 illustrates characterization of Ins-PGEDA-FPBA NC by TEM.
  • Figure 10 illustrates fluorescence emission spectra of insulin (top) and supernatant recovered from the complexes prepared at different ratios (w/w) of PGEDA- FPBA /insulin. The spectra were acquired at an excitation wavelength of 275 nm.
  • Figure 11 illustrates (A) the release kinetics of insulin from Ins-PGEDA-FPBA NCs in PBS and at different glucose concentrations. B) Release kinetics of insulin from Ins- PG EDA NCs in PBS and at different glucose concentrations.
  • FIG. 13 illustrates the release kinetics of insulin from Ins-PG EDA-FPBA NCs in presence of human serum albumin (0.11 mg/mL) at 400 mg/dl glucose concentrations and in PBS.
  • Figure 14 illustrates the UV–vis (A) and fluorescence (B) spectra of TA, PG EDA- FPBA NP and TA coated PGEDA-FPBA NP.
  • Figure 15 illustrates the release kinetics of insulin from Ins-TA-PGEDA-FPBA NCs in presence of 100 mg/dl glucose, 400 mg/dl glucose concentrations and in PBS.
  • Figure 16 illustrates the determination of degree of substitution by 1 H-NMR spectroscopy. 1 H-NMR spectra of unfunctionalized BG EDA (A) and BG EDA-FPBA (B).
  • Figure 17 illustrates glucose responsive insulin release capability of Ins-BGEDA- FPBA NCs (a) Western-blot analysis of p-AKT in starved HepG2 cells after 15 mins of exposure to Ins-BGEDA-FPBA NCs, Vehicle (Milli-Q water) and free insulin.
  • (b) Cumulative insulin release profiles in different glucose concentrations (400 mg/dl and 100 mg/dl) and PBS at different time points (0, 1, 4, 6 and 24 h), n 3 per group.
  • Statistical analyses used one-way analysis of variance (ANOVA) with a Tukey post-hoc, or the Student’s t-test.
  • Figure 18 illustrates the effective conjugation of PG EDA-FPBA NPs with a cyclic peptide for targeting pancreas by fluorescence (A) and UV-vis spectra (B) acquired before and after purification of samples.
  • Figure 19 illustrates the effective pancreas cell targeting of PGEDA-FPBANPs- cyclic peptide conjugates by Amnis ® Image Flow Cytometry (left) of ⁇ -cell lines, MIN6.
  • FIG 20 illustrates in vivo evaluation of Ins-PGEDA-FPBA NCs injection in an Akita spontaneous type 1 diabetic mouse model.
  • B) Duration of normoglycemic conditions maintained in Akita diabetic mice treated with subcutaneously injected naked insulin or Ins-PGEDA-FPBA NCs (insulin dose 80 IU/kg) (n 5).
  • ANOVA analysis of variance
  • Figure 21 illustrates in vivo evaluation of Ins-PG EDA-FPBA NCs injection in an STZ-induced diabetic mouse model.
  • A) Blood glucose concentration in STZ-induced diabetic mice (n 3–4).
  • B) Duration of normoglycemic conditions maintained in STZ- induced diabetic mice treated with subcutaneously injected naked insulin (insulin dose 16 IU/kg) or Ins-PG EDA-FPBA NCs (insulin dose 80 IU/kg) (n 3–4).
  • D) IPGTT results of STZ-induced diabetic mice at 4 h after treatment with Ins-PGEDA-FPBA NCs (insulin dose 80 IU/kg) or naked insulin (insulin dose 16 IU/kg). The glucose dose was set to 1.5 g/kg; n 3–4.
  • E) AUC of IPGTT response at 120 min, with the baseline set at the 0– min blood glucose reading (n 3–4).
  • FIG. 23 illustrates blood glucose regulation by Ins-BGEDA-FPBA NCs in the Akita T1D mouse model.
  • (a) Blood glucose level from Akita T1D mice treated with INS- Small Nano Sugar and insulin; n 2.
  • (b) Duration of normoglycemic conditions maintained in Akita diabetic mice; n 2.
  • (c) IPGTT results in Akita diabetic mice at 4 h after treatments. glucose dose: 1.5 g kg -1 ; n 2.
  • Figure 25 illustrates hepatobiliary excretion of Ins-PG EDA-FPBA NCs.
  • B) Ins-PG EDA-FPBA NCs in the faces (n 3).
  • Figure 26 illustrates the in vivo evaluation of Ins-PG NCs injection in an Akita mice spontaneous type 1 diabetic mouse model.
  • FIG. 27 illustrates kidney elimination of Ins-BGEDA-FPBA NCs.
  • (a) Representative near-infrared fluorescence scans of organs from STZ-induced T1D mice collected at 8 h, 24 h and 1-week post-subcutaneous injection of Cy5.5-labelled ⁇ INS-Small Nano Sugar’ (108 mg kg -1 ) or control (free insulin; INS) (n 2).
  • nanocomplexes according to the invention effectively deliver a rapid and sustained release of a hormone selected from insulin, glucagon, or glucagon-like protein-1 in a therapeutically relevant dose and time scale.
  • a single subcutaneous injection of the nanocomplexes according to the invention has been shown to provide a rapid and efficient response to a glucose challenge in two distinct diabetic mouse models, resulting in optimal blood glucose levels (below 200 mg/dL) for up to 13 h.
  • the morphology of the nanocomplexes was found to be key to controlling rapid and extended glucose-regulated insulin delivery in vivo.
  • the inventors have engineered nanocomplexes comprising a biodegradable and a charge-switchable polysaccharide nanoparticle capable of releasing a therapeutically effective amount of a hormone selected from insulin, glucagon, or glucagon-like protein-1.
  • the polysaccharide nanoparticle in accordance with the invention is comprised of polymerised monomer residues.
  • the polysaccharide nanoparticle may be a homopolymer in which the monomer residues are the same or may be a copolymer comprising two or more distinct monomer units.
  • the polysaccharide nanoparticle comprises glycogen.
  • the polysaccharide nanoparticle comprises bovine glycogen.
  • the polysaccharide nanoparticle comprises phytoglycogen.
  • the polysaccharide nanoparticle comprises two or more of glycogen, bovine glycogen or phytoglycogen.
  • the polysaccharide nanoparticle according to the invention will generally have diameter ranging from about 1 to about 110 nm, preferably 30 to 100 nm.
  • the polysaccharide nanoparticle is covalently coupled to amine moieties.
  • amine moieties to the polysaccharide nanoparticle increases the positive surface charge of the polysaccharide nanoparticle, enabling the polysaccharide nanoparticle to form stable nanocomplexes with a negatively charged hormone selected from insulin, glucagon, or glucagon-like protein-1 via electrostatic interactions.
  • the amine moieties are of the formula -N(R 1 ) 2 , wherein each R 1 is independently selected from H and -C1-C4alkyl-N(R 2 )2; and each R 2 is independently selected from H and C 1 -C 4 alkyl; or two R 1 groups, when taken together with the nitrogen atom to which they are attached, form a heterocyclic ring.
  • R 1 is independently selected from H and -C1-C4alkyl-N(R 2 )2
  • each R 2 is independently selected from H and C 1 -C 4 alkyl; or two R 1 groups, when taken together with the nitrogen atom to which they are attached, form a heterocyclic ring.
  • alkyl used either alone or in compound words, denotes straight chain or branched alkyl. Prefixes such as "C1-C4" are used to denote the number of carbon atoms within the alkyl group (from 1 to 4 in this case).
  • the amine moieties are selected from the group consisting of methylenediamine, ethylenediamine (EDA), 1,1-dimethylethylenediamine, 1,2-dimethylenediamine, 1,3-diaminopropane, and putrescine.
  • the amino moieties are ethylenediamine (EDA).
  • the polysaccharide nanoparticle is covalently coupled to boronic acid moieties via the amine moieties.
  • the present invention provides a nanoparticle comprising a polysaccharide selected from the group consisting of glycogen, bovine glycogen, phytoglycogen or a combination thereof, having covalently coupled to the polysaccharide, amine moieties and pendant boronic acid moieties of Formula (I): wherein Ring A is selected from C4-C8aryl or a C4-C10heteroaryl comprising at least one heteroatom selected from N, O and S, wherein the aryl or heteroaryl is optionally substituted with one or more halo, amino, C1-C4alkyl, -C1-C4alkyloxy, -NO2, or -N(R 3 )2; and each R 3 is independently selected from H and C1-C4alkyl.
  • a polysaccharide selected from the group consisting of glycogen, bovine glycogen, phytoglycogen or a combination thereof, having covalently coupled to the polysaccharide, amine moieties and pendant
  • the amine moieties are ethylenediamine (EDA) and the pendent boronic acid moieties are 4-carboxy-3-fluorophenylboronic acid (FPBA).
  • EDA ethylenediamine
  • FPBA 4-carboxy-3-fluorophenylboronic acid
  • the present invention provides a nanocomplex comprising a polysaccharide nanoparticle selected from the group consisting of glycogen, bovine glycogen, phytoglycogen or a combination thereof, reversibly complexed with a hormone selected from the group consisting of insulin, glucagon, or glucagon-like protein- 1, wherein the polysaccharide nanoparticle is covalently coupled to amine moieties, and the amine moieties are covalently coupled with boronic acid moieties.
  • the amine moieties are ethylenediamine (EDA) and the boronic acid moieties are 4-carboxy-3-fluorophenylboronic acid (FPBA).
  • the polysaccharide nanoparticle comprises glycogen.
  • the polysaccharide nanoparticle comprises bovine glycogen.
  • the polysaccharide nanoparticle comprises phytoglycogen.
  • the polysaccharide nanoparticle comprises two or more of glycogen, bovine glycogen or phytoglycogen.
  • the polysaccharide nanoparticle is bioconjugated with a biomolecule selected from a peptide.
  • Bioconjugation of the polysaccharides nanoparticle according to the invention enables targeting of specific organs.
  • bioconjugation of the polysaccharide nanoparticle with the pancreas-targeting cyclic peptide enables targeting of the pancreas.
  • the polysaccharide nanoparticle according to the invention further comprises a coating.
  • the coating is selected from albumin or tannic acid.
  • the addition of a coating to the polysaccharide nanoparticle, such as an albumin coating or a tannic acid coating may improve loading of the hormone to the polysaccharide nanoparticle and enables the release kinetics of the hormone to be tuned.
  • the nanocomplexes can therefore readily and passively diffuse through the extracellular matrix network after subcutaneous injection to access lymph nodes and navigate the lymphatic system and enter the blood stream.
  • the nanocomplexes are able to distribute to different organs, accumulate in the liver and are cleared by bile-hepatic extraction.
  • the nanocomplexes according to the invention are reversibly complexed with a hormone selected from the group consisting of insulin, glucagon and glucagon-like protein 1.
  • the hormone is insulin.
  • the insulin is fast-acting insulin.
  • the insulin is intermediate-acting insulin.
  • the insulin is long-acting insulin.
  • the insulin is a combination of two or more of fast-acting insulin, intermediate-acting insulin and long- acting insulin.
  • the present invention provides a method for reducing the blood glucose level of a subject in need thereof, the method comprising administering to the subject an effective amount of the nanocomplex according to the invention where the hormone is insulin.
  • the present invention provides a method for treating diabetes mellitus in a subject in need thereof, the method comprising administering to the subject an effective amount of the nanocomplex according to the invention where the hormone is insulin.
  • the subject has diabetes mellitus selected from type 1 diabetes mellitus or type 2 diabetes mellitus.
  • the present invention provides a method for treating hypoglycaemia in a subject in need thereof, the method comprising administering to the subject an effective amount of the nanocomplex according to the invention where the hormone is glucagon or glucagon-like protein-1.
  • the term “subject” refers to an animal, such as a bird or a mammal. Specific animals include rat, mouse, dog, cat, cow, sheep, horse, pig or primate. A subject may be a human, alternatively referred to as a patient. A subject may further be a rodent, such as a mouse or a rat.
  • the pharmaceutical composition comprises nanocomplexes comprising bovine glycogen nanoparticles. In a further embodiment, the pharmaceutical composition comprises nanocomplexes comprising phytoglycogen nanoparticles. In yet another embodiment, the pharmaceutical composition comprises two or more nanocomplexes of glycogen, bovine glycogen or phytoglycogen nanoparticles. In one embodiment, the pharmaceutical composition comprises nanocomplexes of bovine glycogen nanoparticles and nanocomplexes of phytoglycogen nanoparticles. [0078] As will be readily appreciated by those skilled in the art, the route of administration and the nature of the pharmaceutically acceptable carrier will depend on the nature of the condition and the mammal to be treated.
  • nanocomplexes according to the invention will be administered to a subject subcutaneously. It is believed that the choice of a particular carrier or delivery system and route of administration could be readily determined by a person skilled in the art. In the preparation of any formulation containing the nanocomplex according to the invention care should be taken to ensure that the activity of the nanocomplex is not destroyed in the process and that the nanocomplex is able to reach its site of action without being destroyed. Similarly, the route of administration chosen should be such that the nanocomplex reaches its site of action. [0079] Those skilled in the art may readily determine appropriate formulations for the nanocomplexes of the present invention using conventional approaches. Identification of preferred pH ranges and suitable excipients, for example antioxidants, is routine in the art.
  • the solvent or dispersion medium for the injectable solution or dispersion may contain any of the conventional solvent or carrier systems for the active compound, and may contain, for example, water, ethanol, polyol (for example, glycerol, propylene glycol and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils.
  • the proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
  • the prevention of the action of microorganisms can be brought about where necessary by the inclusion of various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal and the like. In many cases, it will be preferable to include agents to adjust osmolarity, for example, sugars or sodium chloride.
  • the formulation for injection will be isotonic with blood.
  • Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminium monostearate and gelatin.
  • Pharmaceutical forms suitable for injectable use may be delivered by any appropriate route including intravenous, intramuscular, intracerebral, intrathecal, epidural injection or infusion.
  • Sterile injectable solutions are prepared by incorporating the aqueous liquids of the invention in the required amount in the appropriate solvent with various of the other ingredients such as those enumerated above, as required, followed by filtered sterilization.
  • dispersions are prepared by incorporating the various sterilised active ingredient into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above.
  • Pharmaceutically acceptable vehicles and/or diluents include any and all solvents, dispersion media, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like. The use of such media and agents for pharmaceutical active substances is well known in the art.
  • Unit dosage form refers to physically discrete units suited as unitary dosages for the subjects to be diagnosed; each unit containing a predetermined quantity of the nanocomplex calculated to produce the desired efficacy in association with the required pharmaceutically acceptable vehicle.
  • the specification for the novel unit dosage forms of the invention are dictated by and directly dependent on (a) the unique characteristics of the nanocomplex and the particular outcome to be achieved, and (b) the limitations inherent in the art of compounding the nanocomplexes of the invention in living subjects having a diseased condition in which bodily health is impaired.
  • the nanocomplexes may be compounded for convenient and effective administration in effective amounts with a suitable pharmaceutically acceptable vehicle in unit dosage form.
  • a unit dosage form can, for example, contain the nanocomplexes in amounts ranging from 0.25 ⁇ g to about 2000 mg. Expressed in proportions, the nanocomplexes may be present in from about 0.25 ⁇ g to about 2000 mg/mL of carrier.
  • compositions containing supplementary active ingredients are determined by reference to the usual dose and manner of administration of the said ingredients.
  • word “comprise”, and variations such as “comprises” or “comprising”, will be understood to imply the inclusion of a stated integer or group of integers or steps but not the exclusion of any other integer or group of integers.
  • the reference in this specification to any prior publication (or information derived from it), or to any matter which is known, is not, and should not be taken as an acknowledgment or admission or any form of suggestion that that prior publication (or information derived from it) or known matter forms part of the common general knowledge in the field of endeavour to which this specification relates.
  • insulin (2 mg/mL) in NaHCO3 (100 mM) was incubated overnight with AF488- NHS (35 ⁇ L, 1 mg/mL) and AF647-NHS (8 ⁇ L, 1 mg/mL) dyes and purified via dialysis against water (dialysis tubing size was 10 kDa).
  • PGEDA-FPBANPs (1.5 mg/mL) were likewise incubated with AF647-NHS (12 ⁇ L) and AF555-NHS (20 ⁇ L) dyes, and the mixture was stirred overnight, and excess dye was removed using NAP-10 column and freeze-dried.
  • a 1 cm quartz cuvette was used to analyze a solution of PG EDA-FPBA NPs (1 mg/ml, pH 7) and a calibration curve for FPBA was generated. The scattering of the PG NPs at the same concentration was subtracted from the spectra.
  • Degradation of PG EDA-FPBA NPs by ⁇ -amylase and ⁇ -amylase The rate of degradation of PG NPs and PGEDA-FPBANPs by ⁇ -amylase and ⁇ -amylase was determined using the Somogyi–Nelson assay.
  • copper-carbonate-tartrate reagent which is composed of stock I (sodium potassium tartrate tetrahydrate (1.2 g), sodium carbonate (2.4 g), sodium bicarbonate (1.6 g), and sodium sulfate (14.4 g) in 80 mL of Milli-Q water) and stock II (copper sulfate pentahydrate (0.4 g) and sodium sulfate (3.6 g) dissolved in 20 mL of Milli-Q water), was prepared. The working reagent was then prepared by mixing 4 parts of stock I with one part of stock II.
  • the arsenomolybdate color reagent was prepared by dissolving ammonium molybdate (2.5 g) in water which was then mixed with concentrated sulfuric acid (2.1 mL). This solution was mixed with a solution of sodium arsenate dibasic pentahydrate (0.3 g in 2.5 mL of Milli-Q water). Aliquots (45 ⁇ L) of PG or PGEDA-FPBA NPs solution, in triplicate, before and after treatment with ⁇ -amylase were added to a 96-well microplate (Costar 3596, Corning, MA, USA). The working reagent (45 ⁇ L) was added and the plate was covered in aluminium foil and heated at 90 °C for 20 min.
  • Cell viability was measured using an alamarBlue assay. 3T3 Fibroblasts and raw cells were plated on a 96-well plate at a seeding density of 7000 cells per well in Dulbecco’s modified Eagle medium (DMEM; 100 ⁇ L) supplemented with 10% fetal bovine serum. After 24 h, cells were incubated with different concentrations of PG EDA- FPBA NPs for 96 h.
  • DMEM Dulbecco’s modified Eagle medium
  • the complexes were purified using a 100 kDa spin column at 10,000 rpm for 5 min, and the supernatant was analyzed by fluorescence spectroscopy (emission at 310 nm at excitation wavelength 275 nm) and high-performance liquid chromatography to estimate the amount of loading.
  • the size and charge of the complexes were measured by DLS and electrophoretic mobility, respectively, and the morphology was studied by STORM.
  • the complexes were also prepared in the presence of PBS (20 mM, pH ⁇ 7), where PG EDA-FPBA NPs were dissolved in PBS and the pH of the insulin solution was gradually increased to ⁇ 6.5.
  • TA complex with PG EDA-FPBA was estimated using DLS.
  • the complex at ratio 1: 0.016 (PG EDA-FPBA :TA) was purfied overnight by dialyses (tube size 14 kDa) against Milli-Q (water changes every 2 h in a day for 4 times). The complex was then characterized using absorption and fluorescence spectroscopy.
  • STORM images were acquired on a Nikon N-STORM system equipped with a Nikon 100 ⁇ 1.4 NA oil immersion objective. The focus and total internal reflection fluorescence imaging angle were adjusted to obtain a high signal-to-noise ratio. Lasers (647, 561, and 488 nm) were used for the excitation of the fluorophores. All time lapses were recorded within a 256 ⁇ 256 pixels region using an EMCCD camera. For each image, 4000 frames were acquired sequentially using full laser power. STORM images were first processed with the STORM module of the NIS Elements Nikon software, where drift correction was performed, and a list of particle localizations was obtained by Gaussian fitting of the fluorescence spots of blinking dyes.
  • Circular dichroism (CD) spectroscopy Ins-PG EDA-FPBA NCs (ratio 1:13) and naked insulin samples were prepared as described above and then diluted 8 times with Milli- Q water. Each sample was split into two aliquots, the first aliquot was treated at 60 °C for 1 h with shaking at 300 rpm and the second aliquot was kept at RT for 1 h.
  • pancreas cell targeting effect of PG-EDA-FPBA-cyclic peptide conjugates Pancreas cell line MIN6 cultured with DMEM supplemented with 10% FCS and L-glutamine and Penicillin and Streptomycin (Pen/Strep). MIN6 cells (200,000 per 100 ⁇ l) incubated with blocking solution (5% FCS in PBS) for an hour on ice. Centrifuged at 500 g for 5 minutes, and remove supernatant, incubate cells with Cy7.5 labelled PG- EDA- FPBA -cyclic-peptides conjugates for an hour on ice (cyclic peptides target EPHA4, which enriched on the surface of pancreas cell line MIN6).
  • mice were subcutaneously administered either labeled Ins- PG EDA-FPBA-Cy5.5 NCs, naked insulin as control, dual-labeled Ins -Cy7.5 -PG EDA-FPBA-Cy5.5 NCs, naked Ins-Cy7.5, or PGEDA-FPBA-Cy5.5 NP in Akita mice.
  • mice were humanely killed, and vital organs (heart, liver, spleen, lung, kidney, and brain), skin, and muscle were isolated for ex vivo fluorescence imaging to determine sample biodistribution.
  • Representative Odyssey ⁇ images of Akita diabetic mice and STZ-induced diabetic mice showed a prolonged retention (24 h post-injection) of Ins-PGEDA-FPBA-Cy5.5 NCs in the proximity of the injection site (i.e., under the skin).
  • Ins-PGEDA-FPBA-Cy5.5 NCs can likely cross the organ’s fenestrated endothelium, which permits the passage of NPs of up to 100 nm in diameter (Garnett MC, Kallinteri P. Occup Med (Lond) 2006, 56(5): 307-311). Ins-PGEDA- FPBA-Cy5.5 NCs are then taken up by hepatocytes and enter the intrahepatic system of bile ducts.
  • kidney tissue confirmed that the small nanoparticle is rapidly filtered by the glomeruli (8 h post-administration) and appears in the urine (Figure 27b), with evidence that glomeruli uptake is localised to podocytes ( Figure 27c & d).
  • the nanoparticle material is quickly re-absorbed by proximal tubule cells (Figure 27c), then fully degraded, and excreted within one week (Figure 27a).
  • an insulin delivery system utilising Ins-BGEDA-FPBA NCs has the potential to provide hypoglycaemia-free glucose control over multiple days with high biocompatibility via renal clearance after injection.
  • this technology may provide a direct route for drug delivery to the kidney to protect this vital organ from diabetic damage when loaded with suitable protective agents.

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Abstract

The present invention relates to nanocomplexes (NCs) comprising a polysaccharide nanoparticle (NP) and a hormone selected from insulin, glucagon, or glucagon-like protein-1, and uses thereof for reducing the blood glucose level, in particular, for the treatment of diabetes.

Description

Engineered nanocomplexes Field of the invention [0001] The present invention relates to nanocomplexes (NCs) comprising a polysaccharide nanoparticle (NP) and a hormone selected from insulin, glucagon, or glucagon-like protein-1, and uses thereof for reducing the blood glucose level, in particular, for the treatment of diabetes. Background of the invention [0002] Diabetes mellitus (type 1 and type 2) is a group of metabolic disorders associated with persistent hyperglycemia (high blood glucose levels) that currently affect more than 463 million people worldwide and an estimated 700 million people by 2045. Frequent, subcutaneous (SC) administration of exogenous insulin formulations (rapid, short, intermediate, or long acting) combined with blood glucose level monitoring by finger-prick tests or in-arm continuous blood glucose monitoring sensors remain the main strategy for blood glucose management and treatment of type 1 diabetes as well as some type 2 diabetes. However, SC administration of insulin is often associated with hypoglycemia, which can be life threatening, and lead to glucose fluctuations and other adherence issues. To improve diabetes therapy, a glucose-responsive insulin delivery system that can act as an insulin reservoir, after a single daily SC injection, is desirable. Such systems would rapidly deploy insulin to counteract spikes in blood glucose levels and ensure a sustained release of insulin to maintain extended normoglycemia. In addition, the ideal insulin delivery system would display sufficient insulin-loading capacity, colloidal stability for storage and administration together with a long shelf life and be cost effective. A simple, reproducible, and scalable manufacturing process as well as biodegradability with limited toxicity and immunogenicity are also highly desirable. [0003] To date, formulations that dynamically regulate insulin release in response to blood glucose are not clinically available. Various glucose-responsive materials for insulin delivery have been preclinically investigated for the controlled delivery of insulin in diabetic animal models, including glucose oxidase enzyme (GOx) based hydrogels (Gu Z, et al. ACS Nano 2013, 7(8): 6758-6766), GOx loaded nanoparticles (NPs) (Volpatti LR, et al. ACS Nano 2020, 14(1): 488-497) or concanavalin A (ConA) based microparticles (Yin R, et al. Colloids Surf B Biointerfaces 2010, 76(2): 483-488), and phenyl boronic acid (PBA) based –microspheres (Wu JZ, et al. Drug Deliv 2017, 24(1): 1513-1525), -complexes (Li C, et al. Langmuir 2018, 34(40): 12116-12125), -liposomes (Yu JC ZY, et al. Nano Research 2019, 12: 1539-1545), -microneedles (Yu J, et al. Nat Biomed Eng 2020, 4(5): 499-506), -cells (Wang C, et al. Adv Mater 2017, 29(18)) and -insulin analogues (Chou DH, et al. Proc Natl Acad Sci U S A 2015, 112(8): 2401-2406). [0004] Among other glucose-responsive materials that have been investigated, a glucose-responsive, charge-switchable complex based on non-biodegradable cationic boronated polyacrylamide and water-insoluble micrometer-sized poly-L-lysine (PLL) have been investigated (Wang J, et al. Sci Adv 2019, 5(7): eaaw4357; Wang J, et al. ACS Nano 2021, 15(3): 4294-4304), displaying robust glucose-triggered insulin release in diabetic pig and mouse models. However, those systems face long-term biocompatibility issues due to the non-biodegradable acrylic polymer chains and variability in the insulin release profile that is affected by rapid and uncontrolled aggregation of the PLL-boronate-insulin mixture. Notably, variability in the effect of subcutaneous absorption of insulin can give rise to an unpredictable therapeutic response, resulting in inadequate glycemic control or increased risk of hypoglycaemia. [0005] Accordingly, there exists a need to provide an improved stable and cost-effective insulin delivery system that is biodegradable with limited toxicity and immunogenicity. Summary of the invention [0006] New nanocomplexes and methods are provided for reducing blood glucose levels and treating diabetes. [0007] Accordingly, in one aspect the present invention provides a nanocomplex comprising a polysaccharide nanoparticle selected from the group consisting of glycogen, bovine glycogen, phytoglycogen or a combination thereof reversibly complexed with a hormone selected from the group consisting of insulin, glucagon and glucagon-like protein- 1. [0008] In one embodiment, the polysaccharide nanoparticle is covalently coupled to amine moieties. [0009] In another embodiment, the polysaccharide nanoparticle is covalently coupled to boronic acid moieties. [0010] In another aspect, the present invention provides a method for reducing the blood glucose level of a subject in need thereof, the method comprising administering to the subject an effective amount of a nanocomplex according to the invention. [0011] In a further aspect, the invention provides a method for treating diabetes mellitus in a subject in need thereof, the method comprising administering to the subject an effective amount of a nanocomplex according to the invention. [0012] In yet another aspect of the invention there is provided a pharmaceutical composition comprising the nanocomplex according to the invention. [0013] These and other aspects of the present invention will become more apparent to the skilled addressee upon reading the following detailed description in connection with the accompanying examples and claims. Brief Description of the Drawings [0014] The invention will herein be described by way of example only with reference to the following non-limiting Figures in which: [0015] Figure 1 illustrates characterization of phytoglycogen nanoparticles (PG NPs), phytoglycogen-EDA nanoparticles (PGEDA NPs), and phytoglycogen-EDA-FPBA nanoparticles (PGEDA-FPBA NPs). A) Cryo-TEM image of PG NPs. B-C), Size (B) and size distribution (C) of PG, PGEDA, and PGEDA-FPBA NPs measured by DLS. D) ζ-Potential of PG, PGEDA, and PGEDA-FPBA NPs. Conjugation of boronate moieties with the amine group reduces the surface charge of PGEDA NPs. [0016] Figure 2 illustrates the determination of degree of substitution by 1H-NMR spectroscopy. 1H-NMR spectra of unfunctionalized PG (A) and PGEDA (B); HSQC NMR spectra of unfunctionalized PG (C), and PGEDA (D). The assignment of the numbered peaks are provided in Table 1. [0017] Figure 3 illustrates the deconvolution of the peaks integrating for H-2,3,7,8 of the functionalized moieties of PGEDA. [0018] Figure 4 illustrates size characterization of PGEDA-FPBA by 13C-NMR spectroscopy 13C-NMR. Solid state spectra of PG (A), PGEDA-FPBA (B). [0019] Figure 5 illustrates the characterization of PGEDA-FPBA by 1H-NMR spectroscopy. [0020] Figure 6 illustrates the determination of degree of substitution by UV–vis spectroscopy. UV–vis spectra of PGEDA NPs (A), FPBA (B), and PGEDA-FPBA NPs (C). [0021] Figure 7 illustrates size characterization of PGEDA-FPBA NPs. A) Representative TEM image of PGEDA-FPBA NPs. B) Size distribution of PGEDA-FPBA NPs. [0022] Figure 8 illustrates the stability of PGEDA-FPBA NPs assessed by an α-and ^- amylase degradation assay. A) Diffusion coefficients of PGEDA-FPBA NPs were measured by FCS during incubation with α-amylase showing that the NPs remain intact. The data show that unlike native PG NPs, PGEDA-FPBA NP are likely stable when injected in the blood stream or in the gastrointestinal tract where α-amylase is abundant. B) ^-Amylase enzymatic degradation assay analysis of PG NPs, and PGEDAFPBA NPs at different time points (0, 1, and 3 h). [0023] Figure 9 illustrates characterization of Ins-PGEDA-FPBA NC by TEM. [0024] Figure 10 illustrates fluorescence emission spectra of insulin (top) and supernatant recovered from the complexes prepared at different ratios (w/w) of PGEDA- FPBA/insulin. The spectra were acquired at an excitation wavelength of 275 nm. [0025] Figure 11 illustrates (A) the release kinetics of insulin from Ins-PGEDA-FPBA NCs in PBS and at different glucose concentrations. B) Release kinetics of insulin from Ins- PGEDA NCs in PBS and at different glucose concentrations. The complexes were prepared in 10 mM PBS at pH 6.5 and FITC-labeled insulin was used. C) Pulse insulin release from Ins-PGEDA-FPBA NCs by alternate addition of glucose at 100 and 400 mg dL-1. The complex was incubated in each solution for 5 min. D) Quantification of blood insulin in human normal and diabetic PPP via ELISA. Percentage of insulin released from Ins-PGEDA-FPBA NCs in normal and diabetic PPP (ex vivo) measured over 2 h. [0026] Figure 12 illustrates the characterization of in vitro cellular toxicity of PGEDA and PGEDA-FPBA NP. Cell viability of (A) 3T3 cells and (B) raw cells when treated with PGEDA and PGEDA-FPBA NP after 96 h. [0027] Figure 13 illustrates the release kinetics of insulin from Ins-PGEDA-FPBA NCs in presence of human serum albumin (0.11 mg/mL) at 400 mg/dl glucose concentrations and in PBS. [0028] Figure 14 illustrates the UV–vis (A) and fluorescence (B) spectra of TA, PGEDA- FPBA NP and TA coated PGEDA-FPBA NP. [0029] Figure 15 illustrates the release kinetics of insulin from Ins-TA-PGEDA-FPBA NCs in presence of 100 mg/dl glucose, 400 mg/dl glucose concentrations and in PBS. [0030] Figure 16 illustrates the determination of degree of substitution by 1H-NMR spectroscopy. 1H-NMR spectra of unfunctionalized BGEDA (A) and BGEDA-FPBA (B). [0031] Figure 17 illustrates glucose responsive insulin release capability of Ins-BGEDA- FPBA NCs (a) Western-blot analysis of p-AKT in starved HepG2 cells after 15 mins of exposure to Ins-BGEDA-FPBA NCs, Vehicle (Milli-Q water) and free insulin. Total AKT was used as the loading control and the ratio of p-AKT over total AKT was quantified n=3 per group. (b) Cumulative insulin release profiles in different glucose concentrations (400 mg/dl and 100 mg/dl) and PBS at different time points (0, 1, 4, 6 and 24 h), n=3 per group. (c) Percentage of insulin released into supernatant from Ins-BGEDA-FPBA NCs in different plasma glucose concentrations, 400 mg dL-1 and 100 mg dL-1 at different time points (0.5, 1, 2, 4, and 24 h). Statistical analyses used one-way analysis of variance (ANOVA) with a Tukey post-hoc, or the Student’s t-test. *p < 0.05, **p < 0.01, ***p < 0.001. ****p < 0.0001. Error bars represent the mean ± standard deviation (SD). [0032] Figure 18 illustrates the effective conjugation of PGEDA-FPBANPs with a cyclic peptide for targeting pancreas by fluorescence (A) and UV-vis spectra (B) acquired before and after purification of samples. [0033] Figure 19 illustrates the effective pancreas cell targeting of PGEDA-FPBANPs- cyclic peptide conjugates by Amnis® Image Flow Cytometry (left) of β-cell lines, MIN6. [0034] Figure 20 illustrates in vivo evaluation of Ins-PGEDA-FPBA NCs injection in an Akita spontaneous type 1 diabetic mouse model. A) Blood glucose level (BGL) in Akita type 1 diabetic mice after treatment with naked insulin (insulin dose 16 IU/kg, n = 5), Ins- PGEDA-FPBA NCs (insulin dose 80 IU/kg, n = 5)), or PGEDA-FPBA NPs (36 mg/kg, n = 1). Data are mean ^ standard deviation (SD). B) Duration of normoglycemic conditions maintained in Akita diabetic mice treated with subcutaneously injected naked insulin or Ins-PGEDA-FPBA NCs (insulin dose 80 IU/kg) (n = 5). C) Concentration of plasma insulin (quantified by insulin ELISA kit) in Akita diabetic mice treated with naked insulin (insulin dose 16 IU/kg, n =3) or Ins-PGEDA-FPBA NCs (insulin dose 80 IU/kg, n =3). Inset: magnified view of data at 14 h post-treatment. p ^ 0.01. D) IPGTT results of Akita diabetic mice at 4 h after treatment with Ins-PGEDA-FPBA NCs (insulin 80 IU/kg) or naked insulin (insulin dose 16 IU/kg). The glucose dose was set to 1.5 g/kg; n = 3–4. E) AUC of IPGTT response at 120 min, with the baseline set at the 0–min blood glucose reading (n = 3–4). F) In vivo glucose-responsive insulin release triggered by intraperitoneal glucose injection at 4 h after treatment with naked insulin (insulin dose 16 IU/kg) or Ins-PGEDA-FPBA NCs (insulin dose 80 IU/kg), as quantified using an insulin ELISA kit; the glucose dose was set to 1.5 g/kg (n = 3–4). All statistical analyses were performed by one-way analysis of variance (ANOVA) with a Tukey post hoc test or Student’s t-test. *p < 0.05, **p < 0.01, ***p < 0.001 [0035] Figure 21 illustrates in vivo evaluation of Ins-PGEDA-FPBA NCs injection in an STZ-induced diabetic mouse model. A) Blood glucose concentration in STZ-induced diabetic mice (n = 3–4). B) Duration of normoglycemic conditions maintained in STZ- induced diabetic mice treated with subcutaneously injected naked insulin (insulin dose 16 IU/kg) or Ins-PGEDA-FPBA NCs (insulin dose 80 IU/kg) (n = 3–4). C) Concentration of plasma insulin (n =3) in STZ-induced diabetic mice after treatment with naked insulin (insulin dose 16 IU/kg) or Ins-PGEDA-FPBA NCs (insulin dose 80 IU/kg). Data are mean ^ SD (n = 3). D) IPGTT results of STZ-induced diabetic mice at 4 h after treatment with Ins-PGEDA-FPBA NCs (insulin dose 80 IU/kg) or naked insulin (insulin dose 16 IU/kg). The glucose dose was set to 1.5 g/kg; n = 3–4. E) AUC of IPGTT response at 120 min, with the baseline set at the 0– min blood glucose reading (n = 3–4). F) In vivo glucose-responsive insulin release triggered by intraperitoneal glucose injection at 4 h after treatment with naked insulin (insulin dose 16 IU/kg) or Ins-PGEDA-FPBA NCs (insulin dose 80 IU/kg), as quantified using an insulin ELISA kit; the glucose dose was set to 1.5 g/kg (n = 3–4). All statistical analyses were performed by ANOVA with a Tukey post hoc test or Student’s t-test. *p < 0.05, **p < 0.01, ***p < 0.001. [0036] Figure 22 illustrates blood glucose regulation by Ins-BGEDA-FPBA NC in the STZ T1D mouse model. (a) BGL from STZ-induced T1D mice treated subcutaneously with free insulin (16 IU kg-1, n=4), Ins-BGEDA-FPBA NCs (insulin dose 108 IU kg-1, n=4) and BGEDA- FPBA NPs alone (n =1) as a control. (b) Duration of normal blood levels with each treatment group in the STZ-induced T1D mouse model. (c) Intraperitoneal glucose tolerance test (IPGTT) in STZ-induced T1D mice at 4 h after treatment with Ins-BGEDA-FPBA NCs or free insulin. Glucose dose: 1.5 g kg-1; n = 3. (d), AUC of IPGTT response at 120 min, with the baseline set at the 0–min blood glucose reading (n = 3). (e) In vivo glucose-responsive insulin release triggered by intraperitoneal glucose injection at 4 h after treatment with insulin (insulin dose 16 IU kg-1) or Ins-BGEDA-FPBA NCs (insulin dose 108 IU kg-1), as quantified using an insulin ELISA kit; the glucose dose was set to 1.5 g kg-1 (n = 3). Statistical analyses used ANOVA with a Tukey post-hoc, or the Student’s t-test. *p < 0.05, **p < 0.01, ***p < 0.001. ****p < 0.0001. Error bars represent the mean ± standard SD. [0037] Figure 23 illustrates blood glucose regulation by Ins-BGEDA-FPBA NCs in the Akita T1D mouse model. (a) Blood glucose level from Akita T1D mice treated with INS- Small Nano Sugar and insulin; n=2. (b) Duration of normoglycemic conditions maintained in Akita diabetic mice; n = 2. (c) IPGTT results in Akita diabetic mice at 4 h after treatments. glucose dose: 1.5 g kg-1; n = 2. (d), AUC of IPGTT response at 120 min, with the baseline set at the 0–min blood glucose reading (n = 2). Statistical analyses used ANOVA with a Tukey post-hoc, or the Student’s t-test. *p < 0.05, **p < 0.01, ****p < 0.0001. Error bars represent the mean ± SD. [0038] Figure 24 illustrates blood clearance of PGEDA-FPBA NPs monitored over 24 h in WT C57bL/6 (n =3) mice. [0039] Figure 25 illustrates hepatobiliary excretion of Ins-PGEDA-FPBA NCs. A) Ins- PGEDA-FPBA NCs in the bile. B) Ins-PGEDA-FPBA NCs in the faces (n =3). [0040] Figure 26 illustrates the in vivo evaluation of Ins-PG NCs injection in an Akita mice spontaneous type 1 diabetic mouse model. Blood glucose level (BGK) in Akita type 1 diabetic mice after treatment with Ins-PG NCs (insulin dose 90 IU/kg, n =3). Data are mean Data are mean ^ standard deviation (SD). [0041] Figure 27 illustrates kidney elimination of Ins-BGEDA-FPBA NCs. (a) Representative near-infrared fluorescence scans of organs from STZ-induced T1D mice collected at 8 h, 24 h and 1-week post-subcutaneous injection of Cy5.5-labelled `INS-Small Nano Sugar’ (108 mg kg-1) or control (free insulin; INS) (n =2). (b) Representative curves of urine insulin levels in Akita T1D mice. (c, d) Representative deconvoluted images of perfused kidney sections co-immunostained for INS-Small Nano Sugar (pink) and proximal tubule cells or podocytes 8 h after subcutaneous injection of Cy5.5-labelled nanoparticles into STZ-induced T1D mouse. [0042] Figure 28 illustrates insulin release profiles of Ins-BGEDA-FPBA and PGEDA-FPBA in a co-formulation (1:9 ratio). Cumulative insulin release profiles in different glucose concentrations and PBS at different time points. The error bars represent mean ± SEM (n=3 per group). All statistical analyses were performed with Two-way ANOVA with a Tukey post hoc test. **** p<0.0001, ***p<0.001, **p<0.01, *p<0.05. Detailed description of the invention [0043] The present inventors have surprisingly found that nanocomplexes according to the invention effectively deliver a rapid and sustained release of a hormone selected from insulin, glucagon, or glucagon-like protein-1 in a therapeutically relevant dose and time scale. [0044] A single subcutaneous injection of the nanocomplexes according to the invention has been shown to provide a rapid and efficient response to a glucose challenge in two distinct diabetic mouse models, resulting in optimal blood glucose levels (below 200 mg/dL) for up to 13 h. The morphology of the nanocomplexes was found to be key to controlling rapid and extended glucose-regulated insulin delivery in vivo. The studies revealed that the injected nanocomplexes enabled efficient insulin release, with optimal bioavailability, pharmacokinetics, and safety profiles. [0045] The inventors have engineered nanocomplexes comprising a biodegradable and a charge-switchable polysaccharide nanoparticle capable of releasing a therapeutically effective amount of a hormone selected from insulin, glucagon, or glucagon-like protein-1. [0046] The polysaccharide nanoparticle in accordance with the invention is comprised of polymerised monomer residues. The polysaccharide nanoparticle may be a homopolymer in which the monomer residues are the same or may be a copolymer comprising two or more distinct monomer units. [0047] In one embodiment, the polysaccharide nanoparticle comprises glycogen. In another embodiment, the polysaccharide nanoparticle comprises bovine glycogen. In a further embodiment, the polysaccharide nanoparticle comprises phytoglycogen. In yet another embodiment, the polysaccharide nanoparticle comprises two or more of glycogen, bovine glycogen or phytoglycogen. [0048] The polysaccharide nanoparticle according to the invention will generally have diameter ranging from about 1 to about 110 nm, preferably 30 to 100 nm. [0049] In one embodiment, the polysaccharide nanoparticle is covalently coupled to amine moieties. [0050] The present inventors have found that the addition of amine moieties to the polysaccharide nanoparticle increases the positive surface charge of the polysaccharide nanoparticle, enabling the polysaccharide nanoparticle to form stable nanocomplexes with a negatively charged hormone selected from insulin, glucagon, or glucagon-like protein-1 via electrostatic interactions. [0051] In one embodiment, the amine moieties are of the formula -N(R1)2, wherein each R1 is independently selected from H and -C1-C4alkyl-N(R2)2; and each R2 is independently selected from H and C1-C4alkyl; or two R1 groups, when taken together with the nitrogen atom to which they are attached, form a heterocyclic ring. [0052] As used herein, the term "alkyl", used either alone or in compound words, denotes straight chain or branched alkyl. Prefixes such as "C1-C4" are used to denote the number of carbon atoms within the alkyl group (from 1 to 4 in this case). Examples of straight chain and branched alkyl include methyl, ethyl, n-propyl, isopropyl, n-butyl, sec- butyl and t-butyl. [0053] In another embodiment, the amine moieties are selected from the group consisting of methylenediamine, ethylenediamine (EDA), 1,1-dimethylethylenediamine, 1,2-dimethylenediamine, 1,3-diaminopropane, and putrescine. In a preferred embodiment, the amino moieties are ethylenediamine (EDA). [0054] In one embodiment, the polysaccharide nanoparticle is covalently coupled to boronic acid moieties via the amine moieties. Esterification of the polysaccharide nanoparticle with boronic acid moieties enables glucose to bind to the polysaccharide nanoparticle. The binding of glucose to the polysaccharide nanoparticle reduces the overall positive charge of the polysaccharide nanoparticle, thereby weakening the electrostatic attraction between the polysaccharide nanoparticle and the hormone to stimulate release of the hormone. [0055] In one embodiment, the present invention provides a nanoparticle comprising a polysaccharide selected from the group consisting of glycogen, bovine glycogen, phytoglycogen or a combination thereof, having covalently coupled to the polysaccharide, amine moieties and pendant boronic acid moieties of Formula (I): wherein Ring A is selected from C4-C8aryl or a C4-C10heteroaryl comprising at least one heteroatom selected from N, O and S, wherein the aryl or heteroaryl is optionally substituted with one or more halo, amino, C1-C4alkyl, -C1-C4alkyloxy, -NO2, or -N(R3)2; and each R3 is independently selected from H and C1-C4alkyl. [0056] In one embodiment with reference to Formula (I), the pendant amine moieties are of the formula -N(R1)2, wherein each R1 is independently selected from H and -C1-C4alkyl-N(R2)2; and each R2 is independently selected from H and C1-C4alkyl; or two R1 groups, when taken together with the nitrogen atom to which they are attached, form a heterocyclic ring. [0057] In another embodiment, Ring A is selected from optionally substituted phenyl and an optionally substituted 5-membered heteroaryl. [0058] In one embodiment, the boronic acid moieties are 4-carboxy-3- fluorophenylboronic acid (FPBA) moieties: . [0059] In a further embodiment, the amine moieties are ethylenediamine (EDA) and the pendent boronic acid moieties are 4-carboxy-3-fluorophenylboronic acid (FPBA). [0060] Accordingly, in one embodiment the present invention provides a nanocomplex comprising a polysaccharide nanoparticle selected from the group consisting of glycogen, bovine glycogen, phytoglycogen or a combination thereof, reversibly complexed with a hormone selected from the group consisting of insulin, glucagon, or glucagon-like protein- 1, wherein the polysaccharide nanoparticle is covalently coupled to amine moieties, and the amine moieties are covalently coupled with boronic acid moieties. In a preferred embodiment, the amine moieties are ethylenediamine (EDA) and the boronic acid moieties are 4-carboxy-3-fluorophenylboronic acid (FPBA). [0061] In one embodiment with reference to Formula (I), the polysaccharide nanoparticle comprises glycogen. In another embodiment, the polysaccharide nanoparticle comprises bovine glycogen. In a further embodiment, the polysaccharide nanoparticle comprises phytoglycogen. In yet another embodiment, the polysaccharide nanoparticle comprises two or more of glycogen, bovine glycogen or phytoglycogen. [0062] In one embodiment, the polysaccharide nanoparticle is bioconjugated with a biomolecule selected from a peptide. Bioconjugation of the polysaccharides nanoparticle according to the invention enables targeting of specific organs. As an example, bioconjugation of the polysaccharide nanoparticle with the pancreas-targeting cyclic peptide: enables targeting of the pancreas. [0063] In another embodiment, the polysaccharide nanoparticle according to the invention further comprises a coating. In one embodiment, the coating is selected from albumin or tannic acid. The addition of a coating to the polysaccharide nanoparticle, such as an albumin coating or a tannic acid coating, may improve loading of the hormone to the polysaccharide nanoparticle and enables the release kinetics of the hormone to be tuned. [0064] Innovative technologies for diabetes treatment which act like an artificial pancreas in vivo are in great demand but are difficult to attain (Goyal SN, et al. Chem Biol Interact 2016, 244: 49-63). The clinical translation of biomaterials formulated to gain glucose responsive insulin delivery is typically hampered by long-term biocompatibility and bioavailability issues, variability in the insulin release profile and risk of hypoglycaemia. The present inventors have engineered a glucose-responsive nanosugar that acts as a highly biocompatible glucose-responsive insulin delivery platform for diabetes treatment using two distinct insulin-deficient diabetic mouse models. It has been demonstrated that insulin was delivered by the engineered nanocomplexes according to the invention after a single subcutaneous (SC) injection in mice, in a therapeutically relevant dose and time scale resulting in optimal blood glucose levels (below 200 mg/dL) for up to 13 h. [0065] These findings suggest that a rapid glucose-responsive hormone release is mediated by nanocomplexes according to the invention, which are capable of promptly sensing high glucose levels and a fast in situ insulin release by a surface charge switch mechanism. The polysaccharide nanoparticle according to the invention have unique intrinsic size, hydrophilicity, biodegradability and tunable morphology. The nanocomplexes can therefore readily and passively diffuse through the extracellular matrix network after subcutaneous injection to access lymph nodes and navigate the lymphatic system and enter the blood stream. The nanocomplexes are able to distribute to different organs, accumulate in the liver and are cleared by bile-hepatic extraction. [0066] The nanocomplexes according to the invention are reversibly complexed with a hormone selected from the group consisting of insulin, glucagon and glucagon-like protein 1. In one embodiment, the hormone is insulin. In one embodiment, the insulin is fast-acting insulin. In another embodiment, the insulin is intermediate-acting insulin. In a further embodiment, the insulin is long-acting insulin. In yet another embodiment, the insulin is a combination of two or more of fast-acting insulin, intermediate-acting insulin and long- acting insulin. [0067] In one embodiment, the present invention provides a method for reducing the blood glucose level of a subject in need thereof, the method comprising administering to the subject an effective amount of the nanocomplex according to the invention where the hormone is insulin. [0068] In another embodiment, the present invention provides a method for treating diabetes mellitus in a subject in need thereof, the method comprising administering to the subject an effective amount of the nanocomplex according to the invention where the hormone is insulin. [0069] In one embodiment, the subject has diabetes mellitus selected from type 1 diabetes mellitus or type 2 diabetes mellitus. In one embodiment, the subject has type 2 diabetes mellitus. [0070] In another embodiment, the hormone reversibly complexed to the polysaccharide nanoparticle is glucagon. [0071] In a further embodiment, the hormone reversibly complexed to the polysaccharide nanoparticle is glucagon-like protein-1. [0072] In one embodiment, the present invention provides a method for increasing the blood glucose level of a subject in need thereof, the method comprising administering to the subject an effective amount of the nanocomplex according to the invention where the hormone is glucagon or glucagon-like protein-1. [0073] In yet another embodiment, the present invention provides a method for treating hypoglycaemia in a subject in need thereof, the method comprising administering to the subject an effective amount of the nanocomplex according to the invention where the hormone is glucagon or glucagon-like protein-1. [0074] As used herein, the term “subject” refers to an animal, such as a bird or a mammal. Specific animals include rat, mouse, dog, cat, cow, sheep, horse, pig or primate. A subject may be a human, alternatively referred to as a patient. A subject may further be a rodent, such as a mouse or a rat. [0075] The present invention also provides a pharmaceutical composition comprising the nanocomplex according to the invention, together with at least one pharmaceutically acceptable carrier or diluent. [0076] Accordingly, in one embodiment, the present invention provides a pharmaceutical composition comprising the nanocomplex according to the invention, wherein the nanocomplex comprises a polysaccharide selected from the group consisting of glycogen, bovine glycogen, phytoglycogen or a combination thereof, reversibly complexed with a hormone selected from the group consisting of insulin, glucagon and glucagon-like protein-1. [0077] In one embodiment, the pharmaceutical composition comprises nanocomplexes comprising glycogen nanoparticles. In another embodiment, the pharmaceutical composition comprises nanocomplexes comprising bovine glycogen nanoparticles. In a further embodiment, the pharmaceutical composition comprises nanocomplexes comprising phytoglycogen nanoparticles. In yet another embodiment, the pharmaceutical composition comprises two or more nanocomplexes of glycogen, bovine glycogen or phytoglycogen nanoparticles. In one embodiment, the pharmaceutical composition comprises nanocomplexes of bovine glycogen nanoparticles and nanocomplexes of phytoglycogen nanoparticles. [0078] As will be readily appreciated by those skilled in the art, the route of administration and the nature of the pharmaceutically acceptable carrier will depend on the nature of the condition and the mammal to be treated. It is envisaged that nanocomplexes according to the invention will be administered to a subject subcutaneously. It is believed that the choice of a particular carrier or delivery system and route of administration could be readily determined by a person skilled in the art. In the preparation of any formulation containing the nanocomplex according to the invention care should be taken to ensure that the activity of the nanocomplex is not destroyed in the process and that the nanocomplex is able to reach its site of action without being destroyed. Similarly, the route of administration chosen should be such that the nanocomplex reaches its site of action. [0079] Those skilled in the art may readily determine appropriate formulations for the nanocomplexes of the present invention using conventional approaches. Identification of preferred pH ranges and suitable excipients, for example antioxidants, is routine in the art. Buffer systems are routinely used to provide pH values of a desired range and include carboxylic acid buffers for example acetate, citrate, lactate and succinate. A variety of antioxidants are available for such formulations including phenolic compounds such as BHT or vitamin E, reducing agents such as methionine or sulphite, and metal chelators such as EDTA. [0080] It is envisaged that the nanocomplexes according to the invention will be prepared in parenteral dosage forms, including those suitable for subcutaneous, intravenous, intrathecal, and intracerebral or epidural delivery. The pharmaceutical forms suitable for injectable use include sterile injectable solutions or dispersions, and sterile powders for the extemporaneous preparation of sterile injectable solutions. They should be stable under the conditions of manufacture and storage and may be preserved against reduction or oxidation and the contaminating action of microorganisms such as bacteria or fungi. [0081] The solvent or dispersion medium for the injectable solution or dispersion may contain any of the conventional solvent or carrier systems for the active compound, and may contain, for example, water, ethanol, polyol (for example, glycerol, propylene glycol and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. The prevention of the action of microorganisms can be brought about where necessary by the inclusion of various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal and the like. In many cases, it will be preferable to include agents to adjust osmolarity, for example, sugars or sodium chloride. Preferably, the formulation for injection will be isotonic with blood. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminium monostearate and gelatin. Pharmaceutical forms suitable for injectable use may be delivered by any appropriate route including intravenous, intramuscular, intracerebral, intrathecal, epidural injection or infusion. [0082] Sterile injectable solutions are prepared by incorporating the aqueous liquids of the invention in the required amount in the appropriate solvent with various of the other ingredients such as those enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilised active ingredient into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. [0083] Pharmaceutically acceptable vehicles and/or diluents include any and all solvents, dispersion media, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like. The use of such media and agents for pharmaceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, use thereof in the therapeutic compositions is contemplated. Supplementary active ingredients can also be incorporated into the compositions. [0084] It is especially advantageous to formulate the compositions in unit dosage form for ease of administration and uniformity of dosage. Unit dosage form as used herein refers to physically discrete units suited as unitary dosages for the subjects to be diagnosed; each unit containing a predetermined quantity of the nanocomplex calculated to produce the desired efficacy in association with the required pharmaceutically acceptable vehicle. The specification for the novel unit dosage forms of the invention are dictated by and directly dependent on (a) the unique characteristics of the nanocomplex and the particular outcome to be achieved, and (b) the limitations inherent in the art of compounding the nanocomplexes of the invention in living subjects having a diseased condition in which bodily health is impaired. [0085] As mentioned above, the nanocomplexes may be compounded for convenient and effective administration in effective amounts with a suitable pharmaceutically acceptable vehicle in unit dosage form. A unit dosage form can, for example, contain the nanocomplexes in amounts ranging from 0.25 μg to about 2000 mg. Expressed in proportions, the nanocomplexes may be present in from about 0.25 μg to about 2000 mg/mL of carrier. In the case of compositions containing supplementary active ingredients, the dosages are determined by reference to the usual dose and manner of administration of the said ingredients. [0086] Throughout this specification and claims which follow, unless the context requires otherwise, the word “comprise”, and variations such as “comprises” or “comprising”, will be understood to imply the inclusion of a stated integer or group of integers or steps but not the exclusion of any other integer or group of integers. [0087] The reference in this specification to any prior publication (or information derived from it), or to any matter which is known, is not, and should not be taken as an acknowledgment or admission or any form of suggestion that that prior publication (or information derived from it) or known matter forms part of the common general knowledge in the field of endeavour to which this specification relates. [0088] The invention will now be described with reference to the following non-limiting examples: MATERIALS AND METHODS [0089] Synthesis of phytoglycogen-ethylenediamine nanoparticles (PGEDA NPs): Phytoglycogen (200 mg, equivalent to 1.2 mmol of glucose monomers) was dissolved in acetic buffer (5 mL, 0.6 M, pH 5.5) with stirring. To this solution, sodium periodate (42 mg, 0.24 mmol) was added in the dark for oxidation of the 1,2 diols. After 2 h, EDA (72 mg, 1.2 mmol) was added followed by the addition of sodium cyanoborohydride (10 eq.) and the mixture was stirred overnight. The product (PGEDA NPs) was purified by dialysis (molecular weight cutoff (MWCO) 14 kDa) against Milli-Q water for 3 days (the medium was changed 6 times) and freeze-dried. The yield of the reaction was ~90%. [0090] Synthesis of phytoglycogen-ethylenediamine-4-carboxy-3- fluorophenylboronic acid nanoparticles (PGEDA-FPBA NPs): FPBA (40 mL; 110 mg, 0.6 mmol) was added to a mixture of N-hydroxysuccinimide (NHS; 140 mg, 1.2 mmol) and N- (3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC•HCl; 120 mg, 0.6 mmol) and stirred for 30 min. Next, PGEDA NPs (100 mg) were added to the reaction mixture and stirred overnight in dark. The product (PGEDA-FPBA NPs) was purified by dialysis (MWCO cutoff 14 kDa) against NaCl (0.1 M) for 1 day and Milli-Q water for 3 days (6 times changed) and freeze dried. The yield of the reaction was ~87%. The NPs were characterized by 1H-NMR spectroscopy, absorption spectroscopy, and DLS. [0091] Labeling of insulin and PGEDA-FPBANPs for STORM and FCS analyses: PGEDA-FPBA NPs (5 mg/mL) were dissolved in sodium bicarbonate buffer pH = 8 (100 mM) and mixed with AF488-NHS, AF647-NHS, Cy5.5, or Cy7.5 (20 µL, 1 mg/mL) dye. The mixture was stirred overnight, and excess dye was removed using NAP-10 column and freeze-dried. Similarly, for studying the co-localization of insulin and PGEDA-FPBA NPs with STORM, insulin (2 mg/mL) in NaHCO3 (100 mM) was incubated overnight with AF488- NHS (35 µL, 1 mg/mL) and AF647-NHS (8 µL, 1 mg/mL) dyes and purified via dialysis against water (dialysis tubing size was 10 kDa). PGEDA-FPBANPs (1.5 mg/mL) were likewise incubated with AF647-NHS (12 µL) and AF555-NHS (20 µL) dyes, and the mixture was stirred overnight, and excess dye was removed using NAP-10 column and freeze-dried. [0092] Characterization of engineered NPs and NCs by NMR spectroscopy and UV– vis spectroscopy: 1H-NMR spectra of PG NPs, PGEDA NPs, and PGEDA-FPBA NPs were recorded on a 600 MHz Bruker Avance III at 40 °C in H2O and 10% D2O using the sequence (zgesgp) and the following parameters: 128 scans, 2 s relaxation delay, and spectral width of 9.6 kHz. 1H-NMR spectra of insulin, Ins-PGEDA-FPBA NCs, Ins-PGEDA NCs, and Ins-PG NCs were recorded on a 700 MHz Bruker Avance IIIHD at 25 °C in H2O and 10% D2O using the sequence (zgesgp) and the following parameters : 64 scans, 2 s relaxation delay, and spectral width of 11.2 kHz. The spectra were processed using Topspin 4.1.4, and 1H chemical shifts were referenced to d4-trimethylsilylpropanoate at 0 ppm. UV–vis spectroscopy was performed using SPECORD 250 PLUS. A 1 cm quartz cuvette was used to analyze a solution of PGEDA-FPBA NPs (1 mg/ml, pH 7) and a calibration curve for FPBA was generated. The scattering of the PG NPs at the same concentration was subtracted from the spectra. [0093] Degradation of PGEDA-FPBANPs by α-amylase and β-amylase: The rate of degradation of PG NPs and PGEDA-FPBANPs by α-amylase and β-amylase was determined using the Somogyi–Nelson assay. The PG or PGEDA-FPBA NPs (200 µL, 1 mg/mL) were dissolved in 10 mM PBS (pH = 7.4) and incubated for 1, 2, and 3 h with enzyme solutions, where the final concentration of the enzyme was 1 U/mL. Following incubation, the samples were analysed using the Somogyi–Nelson assay. For the assay, copper-carbonate-tartrate reagent, which is composed of stock I (sodium potassium tartrate tetrahydrate (1.2 g), sodium carbonate (2.4 g), sodium bicarbonate (1.6 g), and sodium sulfate (14.4 g) in 80 mL of Milli-Q water) and stock II (copper sulfate pentahydrate (0.4 g) and sodium sulfate (3.6 g) dissolved in 20 mL of Milli-Q water), was prepared. The working reagent was then prepared by mixing 4 parts of stock I with one part of stock II. The arsenomolybdate color reagent was prepared by dissolving ammonium molybdate (2.5 g) in water which was then mixed with concentrated sulfuric acid (2.1 mL). This solution was mixed with a solution of sodium arsenate dibasic pentahydrate (0.3 g in 2.5 mL of Milli-Q water). Aliquots (45 µL) of PG or PGEDA-FPBA NPs solution, in triplicate, before and after treatment with α-amylase were added to a 96-well microplate (Costar 3596, Corning, MA, USA). The working reagent (45 µL) was added and the plate was covered in aluminium foil and heated at 90 °C for 20 min. The plate was cooled to room temperature and arsenomolybdate color reagent (45 µL) was added to each well, and after 15–20 min, the absorbance was recorded at 600 nm using an Infinite M200 microplate reader (Tecan, Switzerland). [0094] Cell viability: Cell viability was measured using an alamarBlue assay. 3T3 Fibroblasts and raw cells were plated on a 96-well plate at a seeding density of 7000 cells per well in Dulbecco’s modified Eagle medium (DMEM; 100 μL) supplemented with 10% fetal bovine serum. After 24 h, cells were incubated with different concentrations of PGEDA- FPBA NPs for 96 h. Cell viability was estimated after incubating the cells with alamarBlue reagent for 2 h using an Infinite M200 microplate reader. [0095] Complexation between insulin and PGEDA-FPBA NPs to form Ins-PGEDA-FPBA NCs: Both PGEDA-FPBA NPs (20 mg/mL in Milli-Q water) and insulin were dissolved (1 mg, 28.8 U, in 0.01 N HCl). The complexes were then prepared with different weight-to-weight (w/w) ratio of insulin and PGEDA-FPBA NPs in Milli-Q water (pH 6) and PBS (50 mM, pH 7). Then, the complexes were purified using a 100 kDa spin column at 10,000 rpm for 5 min, and the supernatant was analyzed by fluorescence spectroscopy (emission at 310 nm at excitation wavelength 275 nm) and high-performance liquid chromatography to estimate the amount of loading. The size and charge of the complexes were measured by DLS and electrophoretic mobility, respectively, and the morphology was studied by STORM. The complexes were also prepared in the presence of PBS (20 mM, pH ~7), where PGEDA-FPBA NPs were dissolved in PBS and the pH of the insulin solution was gradually increased to ~6.5. (20 mg/mL, 5 mM PBS pH 6.5) were added a solution of tannic acid (0.2 mg/mL dissolved in MQ water) at different wt/wt ratio. The size of TA complex with PGEDA-FPBA was estimated using DLS. The complex at ratio 1: 0.016 (PGEDA-FPBA:TA) was purfied overnight by dialyses (tube size 14 kDa) against Milli-Q (water changes every 2 h in a day for 4 times). The complex was then characterized using absorption and fluorescence spectroscopy. [0098] This formulation was then incubated with various amount of insulin and the complexes were then purified using 100 kDa spin column at 10,000 rpm for 5 min and supernatant was analysed using fluorescncee spectroscopy (emission at 310 nm at excitation wavelength 275 nm). The size and charge of the complexes was measured using DLS. In the presence of PBS (20 mM, pH ~7), PGEDA-FPBA NPs were complexed with insulin at an insulin-to-PGEDA-FPBA NPs weight ratio of 1:13. The complexes were then added into Human serum albumin or bovine Albumin (final concentration of albumin ̴50 %) at pH 7.4 in centrifuge tubes. Release of insulin was estimated at different concentrations of glucose (100 and 400 mg dL-1). [0099] Conjugation of PGEDA-FPBANPs with targeting peptide: PGEDA-FPBA NPs (10 mg, equivalent to 0.06 mmol of glucose monomers) were dissolved in 5 mL of PBS pH=6.8 with stirring. To this, sodium periodate (0.8 mg, ~ 0.004 mmol) was added and reacted in the dark. After 2 h, 0.8 mg of targeting cyclic peptide (0.0006 mmol) was added followed by 10 eq. of sodium cyanoborohydride to sodium periodate (0.04 mmol), and the mixture was stirred overnight. Product was purified by dialysis (tube size 14 kDa) against Milli-Q water for 3 days and freeze dried. [0100] Characterization of Ins-PGEDA-FPBA NCs by STORM, FCS and TEM: For STORM analysis, FITC-Ins-PGEDA-FPBA NCs were prepared as described above at varying insulin-to-PGEDA-FPBA NPs weight ratios of 1:0, 1:1.5, 1:3.5, 1:10, 1:13. Ins-FITC -PGEDA-FPBA NCs were deposited on a glass slide, and after 30 min of incubation at 25 °C, unbound molecules and NPs were washed away with freshly prepared, standard imaging buffer with cysteamine (MEA). STORM images were acquired on a Nikon N-STORM system equipped with a Nikon 100× 1.4 NA oil immersion objective. The focus and total internal reflection fluorescence imaging angle were adjusted to obtain a high signal-to-noise ratio. Lasers (647, 561, and 488 nm) were used for the excitation of the fluorophores. All time lapses were recorded within a 256 × 256 pixels region using an EMCCD camera. For each image, 4000 frames were acquired sequentially using full laser power. STORM images were first processed with the STORM module of the NIS Elements Nikon software, where drift correction was performed, and a list of particle localizations was obtained by Gaussian fitting of the fluorescence spots of blinking dyes. Blinking events that were detected in ≤5 consecutive frames were counted as single molecules, whereas events detected in >5 consecutive frames were discarded (max trace length 5). The list of localizations was exported as a .txt file and analyzed using an in-house-built clustering analysis script in Phyton, where the localizations were clustered using a kernel density estimation with a bandwidth of 50 nm. An ellipse was fitted to the obtained clusters with a minimum of 10 localizations and a maximum elongation factor of 1.5 (ratio of long and short axes of the ellipse). Then, the circles containing 90% of detected spots in the cluster were fitted to determine the NP size distribution and number of localizations. For the multicolour STORM analysis, insulin was dual labelled with AF488/AF647 (green) and the PGEDA-FPBA NPs were dual labelled with AF555/AF647 (red). FCS experiments on AF647 PGEDA-FPBA NPs incubated with enzymes were performed on a Nikon A1R confocal microscope combined with a MicroTime PicoQuant system with 40×/1.1 NA water immersion objective and a 647 nm laser for illumination. The confocal volume (Veff) was calibrated by the measurements of AF 647 dye with the known DAF647 = 3.3 ± 0.1 × 10−6 cm2 s−1 at the beginning of each experiment. The measurements were carried out for 30 s and repeated at least 20 times in different positions. The generated autocorrelation function (ACF) curves were analysed using the SymPhoTime 64 software. For TEM imaging, a drop (10 µL) of a sample dispersion (10 mg/mL) was deposited on a copper grid for 10 min. Excess sample was then removed and the grid was washed with Milli-Q water twice. Subsequently, uranyl acetate (10 µL) was added to the grid for 1 min and the grid was washed twice with water. The grid was then air-dried and imaged on a JEOL JEM-1010 transmission electron microscope. Cryo-TEM images were obtained using a TECNAI F30 microscope equipped with a high- angle annular dark-field scanning transmission electron microscopy detector, a Gatan quantum 965 energy filter, and an upper CETA 4k × 4k CMOS camera. [0101] Circular dichroism (CD) spectroscopy: Ins-PGEDA-FPBA NCs (ratio 1:13) and naked insulin samples were prepared as described above and then diluted 8 times with Milli- Q water. Each sample was split into two aliquots, the first aliquot was treated at 60 °C for 1 h with shaking at 300 rpm and the second aliquot was kept at RT for 1 h. CD spectra were acquired on a Chirascan spectropolarimeter (Applied Photophysics Ltd., UK) between 190 and 260 nm using a 0.1 mm path length cylindrical quartz cell (Starna Scientific Ltd, Hainault, UK). Spectra were acquired with 0.5 nm data intervals, 1 s integration time and 3 scans accumulation. Signal was recorded as millidegrees at 25 °C. Spectra were zeroed at 260 nm, the background from the solvent or PGEDA-FPBA NPs was subtracted, and normalized to give units of mean-residue ellipticity (MRE) according to [θ]MRE = θ/(c × l × Nr), where θ is the recorded ellipticity (mdeg), c is the peptide concentration (dmol/L), l is the cell path length (cm), and Nr is the number of residues. [0102] Effect of glucose on PGEDA-FPBA NPs: The effect of glucose concentration on the ζ-potential of PGEDA-FPBA NPs (10 mg/mL) was determined after the addition of different amounts of glucose (100 and 400 mg/dL) below and above the pKa of FPBA. The binding of glucose to PGEDA-FPBA NPs was quantified as follows. Glucose (100 or 400 mg/dL) was added to 300 µL of 8 mg/mL PGEDA NP or PGEDA-FPBA NP solution in PBS (100 mM, pH 7.4), and the mixture was incubated at 37 °C. After various time points, the supernatant was collected using a spin column and the glucose concentration was estimated using an Accu- Chek Performa glucose monitor. [0103] In vitro insulin release study: Briefly, the PGEDA and PGEDA-FPBA NCs with 0.3 mg insulin were incubated with PBS and with different concentrations of glucose (100 and 400 mg/dL) at pH 7.4 in centrifuge tubes. The centrifuge tubes were incubated at 37 °C and stirred at 500 rpm. The supernatant was then recovered after various time points using a 100 kDa spin column at 10,000 rpm for 5 min and analyzed by fluorescence spectroscopy (emission at 310 nm at excitation wavelength 275 nm) to estimate the amount of loading. When FITC-conjugated insulin was used, the fluorescence emission was recorded at 515 nm at an excitation wavelength of 480 nm. [0104] In vitro assessment of insulin bioactivity of Ins-PGEDA-FPBA NCs: HepG2 cells (0.5 × 106; human liver hepatocellular carcinoma, American Type Culture Collection, HB- 8065) were seeded over 48 h with 10% FCS/DMEM (1× penicillin-streptomycin) and starved in serum-free DMEM media overnight. Insulin (equivalent concentration of 100 nM) was then loaded into the PGEDA-FPBA NPs at an Insulin-to-PGEDA-FPBA NPs ratio of 1:13. An aliquot (2 µL) of each sample was added to the HepG2 cells and incubated for 15 min. The medium was then discarded and the cells were washed with PBS twice, and RIPA buffer with a phospho-inhibitor cocktail and protease inhibitor cocktail was added for cell lysis. The supernatant was collected after centrifugation at 16,000 g for 30 min, and the protein amount was quantified using the BCA kit before the supernatant was added to 6× sodium dodecyl sulfate sample buffer and heated at 95 ^C for 10 min. For immunoblotting, each cell lysate (60 µg) was loaded into 12% acrylamide gel for electrophoresis, as previously described (Xu R, et al. Proteomics 2019, 19(8): e1700453). Transferred membranes were probed with primary antibodies (rabbit p-AKT Ser473, Pan-AKT, 1:1000 dilution). Incubation transferred membrane with secondary antibody (goat anti-rabbit HRP, 1:3000 dilution). Membranes were washed thrice with PBS and the chemiluminescent Western Blot reagent was added (Thermo Fisher Scientific). Images were obtained using a ChemiDoc machine (BioRad) and quantified using Image Lab 6.1 (BioRad). [0105] In vitro pancreas cell targeting effect of PG-EDA-FPBA-cyclic peptide conjugates: Pancreas cell line MIN6 cultured with DMEM supplemented with 10% FCS and L-glutamine and Penicillin and Streptomycin (Pen/Strep). MIN6 cells (200,000 per 100 μl) incubated with blocking solution (5% FCS in PBS) for an hour on ice. Centrifuged at 500 g for 5 minutes, and remove supernatant, incubate cells with Cy7.5 labelled PG-EDA- FPBA-cyclic-peptides conjugates for an hour on ice (cyclic peptides target EPHA4, which enriched on the surface of pancreas cell line MIN6). Centrifuged at 500 g for 5 minutes, remove supernatant, added 250 μl PBS and resuspend gentle for the Amnis® Image Flow Cytometry. PG-EDA-FPBA-pancreas targeting cyclic-peptide targets primary pancreas cell line MIN6. [0106] Ex vivo plasma insulin release study: Blood samples were collected from healthy donors, with informed consent, at the Australian Centre for Blood Diseases. PPP was obtained after centrifugation at 2000 g for 10 min twice. Samples (25 µL) were prepared that were either normal PPP glucose (100 mg/dL) or diabetic PPP glucose (600 mg/dL, additional glucose was added to normal PPP). All samples had an equivalent insulin concentration of 1.33 IU/mL and were incubated at 37 ^C while shaking at 500 rpm. The supernatant was then recovered after various time points using a 100 kDa spin column at 10,000 g for 5 min and analyzed for the free insulin amount via ELISA (ALPCO 80- INSMSU-E01 kit). The supernatant was diluted 3000× using a Zero standard solution from the ELISA kit. Briefly, aliquots (5 µL) of each of the standard curve samples, insulin controls, and final diluted samples were loaded into a 96-well microplate. A working conjugate buffer (75 µL) was added, and the microplate was sealed and placed on an Eppendorf Thermomixer machine for 2 h and mixed at 800 rpm. Wells were washed 6 times with working strength wash buffer (350 µL) before tetramethylbenzidine (TMB) Substrate (100 µL) was added. The microplate was incubated for a further 30 min at room temperature while shaking at 800 rpm. The Stop Solution (100 µL) was added to each well and the absorbance was measured using a FLUOstar OPTIMA machine at 450 nm. [0107] In vivo blood glucose regulation studies in Akita and STZ-induced type 1 diabetic mice models: In vivo studies were conducted using 8–12-week-old heterozygous Akita type 1 diabetic male mice (C57BL/6-Ins2-Akita/J) purchased from the Alfred Research Alliance Precinct Animal Centre (Australia). All animal procedures were approved by the Alfred Research Alliance Animal Ethics Committees (ARAAEC), Monash University (AEC number E/1695/2016/M and E/P8220/2022). STZ induction was conducted by continuous intraperitoneal injection of STZ at low dose (55 mg/kg) over 5 days, and blood glucose was monitored over 4–5 weeks. The mice were considered diabetic when the BGL was over 25 mg/dL. Akita mice (n = 5) and STZ-induced diabetic mice (n =3–4) were distributed into groups treated with naked insulin (insulin dose 16 IU/kg) and NCs (e.g. insulin dose 80 IU/kg). The blood glucose was monitored before and after treatment until the blood glucose returned to initial levels. The blood samples were taken from the tail tip and plasma glucose concentration was measured by a glucose meter (ACCU- CHEK). Blood samples (2.5 µL) of the Akita mice (n =3) and STZ-induced diabetic mice (n = 3) treated with native insulin (insulin dose 16 IU/kg) and NCs (insulin dose 80 IU/kg) were extracted and transferred into Eppendorf tubes and mixed with 2.5 µL 10% sodium citrate (3.2%) PBS solution. The obtained blood was centrifuged (2000 g, 15mins, twice) and the plasma was stored at −20 °C until measurement. The plasma insulin level was quantified via ELISA. [0108] IPGTT in Akita and STZ-induced type 1 diabetic mice: Akita (n = 3–4) and STZ-induced type 1 diabetic mice (n = 3–4) were randomly assigned to be treated with insulin NCs (80 IU/kg) and naked insulin (16 IU/kg). At 4 h post-treatment, the Akita and STZ-induced diabetic mice were intraperitoneally injected with glucose (1.5 g/kg). Blood samples were taken from the tail tip and the plasma glucose concentration was measured by a glucose meter (ACCU-CHEK). [0109] Intraperitoneal glucose injection-induced insulin release study in Akita and STZ-induced type 1 diabetic mice: Akita (n = 3–4) and STZ-induced type 1 diabetic mice (n =3) were randomly assigned to be treated with various insulin NCs (insulin dose 80 IU/kg). At 4 h post-treatment, the Akita mice were intraperitoneally injected with glucose (1.5 g/kg). Blood samples (2.5 µL) were extracted and transferred into Eppendorf tubes and mixed with 2.5 µL 10% sodium citrate (3.2%) PBS solution. The obtained blood was centrifuged and the plasma was stored at −20 °C until measurement. The plasma insulin level was quantified via ELISA. [0110] Biodistribution studies in Akita, STZ-induced diabetic, and WT C57BL/6 mice: Mice were housed on a 12 h light/dark cycle with ad libitum access to food and water. Biodistribution studies were conducted using Akita male mice (8–12 weeks old), STZ- induced C57Bl/6 male mice (12–13 weeks old), and C57BL/6 male mice (8–12 weeks old) bred in house. All animal experiments were approved by the ARAAEC Monash University (AEC number Akita mice E/1695/2016/M and C57Bl/6 mice E/1625/2016/M). To evaluate the biodistribution of Ins-PGEDA-FPBA NCs and PGEDA-FPBA NPs, the mice were subcutaneously administered Ins-PGEDA-FPBA-Cy5.5 NCs (insulin dose 80 IU/kg) and naked insulin (control, insulin dose 16 IU/kg), Ins-Cy7.5-PGEDA-FPBA-Cy5.5 NCs (insulin dose 80 IU/kg), naked Ins-Cy7.5 (insulin dose 16 IU/kg), or PGEDA-FPBA-Cy5.5 NPs (36 mg/kg) (Akita mice (n =3) and STZ-induced mice (n =3)). In WT C57BL/6 mice, PGEDA-FPBA-Cy5.5 NPs (36 mg/kg), Ins-PGEDA-FPBA-Cy5.5 NCs (insulin dose 24 IU/kg, NPs 36 mg/kg), Ins-PGEDA-FPBA- Cy5.5 NCs (insulin dose 12 IU/kg, NPs 36 mg/kg), or Ins-PGEDA-FPBA-Cy5.5 NCs (insulin dose 8 IU/kg, NPs 36 mg/kg) were administrated subcutaneously. Blood samples were then collected from the mouse tail and blood withdrawn (2.5 µL) using a pipette at the indicated time points post-injection. Collected blood was diluted in 10% sodium citrate (3.2%) PBS solution (total volume 50 µL) and those diluted blood samples (50 µL) were measured on an Odyssey CLx scanner (LI-COR Biosciences, NE, USA), with the fluorescence detected using the 800 nm (Cy7.5 dye) or 700 nm (Cy5.5) channel, accordingly. [0111] At indicated time points, the mice were euthanized by an overdose intraperitoneal injection of ketamine (300 mg/kg) and xylazine (30 mg/kg) and subsequently subjected to transcardial perfusion with PBS (20 mL) before the brain, heart, lung, liver, spleen, kidney, muscle, and skin were harvested. The organs were briefly washed and stored in PBS at 4 °C and in the dark until fluorescence imaging was performed. The scans were recorded on an Odyssey CLx scanner, with the fluorescence detected using the 800 nm (Cy7.5 dye) or 700 nm (Cy5.5 dye) channel. The mean fluorescence intensity value (a.u.) was determined for each sample, as well as the total fluorescence of each injected dose, calculated from a standard curve of stock volume. The percentage of the remaining material for blood clearance, and the percentage of injected dose per gram tissue for biodistribution, were calculated (Yu H, et al. ACS Appl Mater Interfaces 2022, 14(3): 3740-3751). [0112] Immunohistochemistry and immunofluorescence: Tissues were fixed in 10% neutral buffered formalin (NBF), paraffin embedded, sectioned at 4 microns, and float sections into superfrost slides. One series was subjected to H&E staining, and an Olympus BX51 fluorescence microscope was used to image the H&E-stained tissue slides. The waxed tissue slides were placed on a heating block at 60 °C for 2 h and then dewaxed with incubation with xylene (5 min, 3 times), 100% ethanol (20 dips, 3 times), and PBS (5 min, 2 times). Antigen retrieval was achieved via incubation of tissue slides with sodium citrate buffer at 90 °C for 20 min with 30 min cooling, followed by PBS washes (5 min, 2 times). Permeabilization was performed with 0.5% Triton X/PBS for 30 min at room temperature. Tissue slides were blocked with 5% serum at room temperature for 1 h. Tissue slides were incubated with primary antibody (Insulin, mouse monoclonal Antibody, eBioscience ^) and Wheat Germ Agglutinin Alexa Fluor 488 conjugate (WGA-AF488, Thermo Fisher Scientific for plasma membrane staining) overnight at 4 °C and washed with PBS three times, then incubated with secondary antibody (Donkey anti-mouse AF568, Thermo Fisher Scientific) for one hour at RT and Hoechst staining at RT for 15 min. Confocal microscopy was conducted on a Nikon A1 microscope, sequentially acquiring using Plan Fluor 20× MImm NA 0.75 objective (FITC was used for plasma membrane staining, AF568 was used for insulin staining, and channel 647 nm was used for Cy5.5 dye (PGEDA-FPBA NPs), Hoechst was used for nuclear staining). [0113] Complexation between insulin and PG NPs to form Ins-PG NCs and in vivo studies: Insulin was loaded into unmodified porous PG nanoparticles by a soaking method. The FITC labeled insulin was dissolved at concentration 1 mg/mL. Next, to the 100 µL insulin solution was added different amounts of unmodified PG in centrifuge tubes. After overnight incubation, the complexes were purified using 100 k spin column at 10,000 rpm for 5 min and the supernatant was analysed using fluorescence spectroscopy (emission at 515 nm at excitation wavelength 480 nm) to estimate the insulin loading efficiency (80 µg insulin/mg PG). [0114] Akita mice (n = 3) were treated with Ins-PG NCs (e.g. insulin dose 80 IU kg-1). The blood glucose was monitored before and after treatment until the blood glucose returned to initial levels. The blood samples were taken from the tail tip and plasma glucose concentration was measured by a glucose meter (ACCU-CHEK). [0115] Statistical Analysis: One way ANOVA with Tukey post-hoc tests and two-way ANOVA were used to carry out multiple comparisons. RESULTS Engineering charge-switchable polysaccharide nanoparticles for glucose-responsive insulin delivery [0116] Phytoglycogen (PG) is a highly branched polysaccharide nanoparticle (NP) derived from sweet corn with a dendrimer-like molecular structure and a multilobular morphology, as shown by the cryo-transmission electron microscopy (cryo-TEM) images in Figure 1A. The PG NPs displayed a hydrodynamic diameter of approximately 80 ± 30 nm (Figure 1B, C) and a ζ-potential of –5 ± 2 mV (Figure 1D), as determined by dynamic light scattering (DLS) and electrophoretic mobility measurements, respectively. The PG NPs were endowed with a positive surface charge and glucose sensitive moieties upon chemical derivatization with EDA and FPBA groups in aqueous solvents as illustrated in Scheme 1 below to obtain PGEDA NPs and PGEDA-FPBA NPs respectively. The degrees of substitution of the PG NPs by EDA (~19%) and FPBA (10%) were determined by 1H nuclear magnetic resonance (NMR) spectroscopy (Figures 2-5, Table 1) and UV–visible (UV–vis) spectroscopy (Figure 6). Table 1. Peak assignment illustrated in Figure 2 and 5 [ppm] Assignment 7.909-7- 257 H-9,10,11 5.583- 4.987 H-1 from functionalized moieties 5.348 H-1 4.963 H-1 from 1,6 branching 4.158- 3.490 H-2,3,5,6,6’ 3.422 H-4 chain end 3.024 H-2,2’,3,3',7,7' from functionalized moieties 2.826 H-8,8' from functionalized moieties Scheme 1. Reaction scheme for the reductive amination of PG NP to form PGEDA NPs. Step 1, oxidative cleavage of the C2–C3 bond to form two aldehyde groups. Step 2, imines (Schiff bases) formation in equilibrium; Step 3, reduction of the imine to amine groups. Possible chemical modifications of the glycoside units in the polysaccharide chains, glycoside unit inside the chain can bear one EDA moiety (A), two EDA moieties (B), two alcohol groups (C). The glycoside unit at the chain ends can also bear one or two EDA moieties (D). [0117] The hydrodynamic diameters of PGEDA NPs and PGEDA-FPBA NPs (Figure 1C) increased by ~25% relative to that of the unmodified PG NPs. Subsequent conjugation of the FPBA groups (pKa 7.2) led to a significant reduction of the positive charge density on PGEDA-FPBANPs from 40 mV (PGEDA NPs) to 25 mV (Figure 1D). TEM images revealed a significant size shrinkage of the PGEDA-FPBA NPs upon drying to approximately 34 ± 10 nm (Figure 7), indicating that the PGEDA-FPBA NPs were highly hydrated. The binding of the PGEDA-FPBA NPs to glucose by esterification with the FPBA moieties was evaluated as a function of time after the addition of glucose at clinically relevant glucose concentrations of 100 mg/dL (5.6 mmol/L) and 400 mg/dl (22.2 mmol/L) in phosphate-buffered saline (PBS) pH 7.4. The concentration of glucose in the supernatants of the original glucose solutions of 100 and 400 mg/dL decreased by approximately 50 and 120 mg/dL, respectively, after 24 h, reflecting the effective binding of glucose to the PGEDA-FPBA NPs. [0118] In agreement with these results, the ζ-potential of the PGEDA-FPBA NPs decreased from 25 mV in the absence of glucose to approximately 15 mV after 24 h incubation with 400 mg/dL glucose. The binding of glucose to FPBA groups has been reported to induce a decrease in the pKa (~6.4) of boronic acid (Yu J, et al. Nat Biomed Eng 2020, 4(5): 499-506; Shao Y, Lin AH. Food Chem 2018, 240: 898-903) and therefore a shift of the equilibrium toward the boronate form. Consequently, the negative charges arising from the boronate moieties reduced the positive charge density on the PGEDA-FPBA NPs. The biodegradability of the PGEDA-FPBA NPs by α-amylase, which is typically present in plasma, was measured in vitro. Compared to the unmodified PG NPs, PGEDA-FPBA NPs exhibited minimal degradation (8–10%) during incubation with α-amylase for 3 h, as determined using the Somogyi–Nelson amylolytic enzyme activity assay. The stability of the PGEDA-FPBA NPs against α-amylase was also confirmed by fluorescence correlation spectroscopy (FCS) and STORM imaging where the diffusion coefficient and corresponding radius of the Alexa Fluor (AF) 555 (AF555)-labeled PGEDA-FPBA NPs showed minimal changes in the presence of the hydrolytic enzyme (Figure 8A). [0119] However, the PGEDA-FPBA NPs were partially degradable (40%) when treated for 3 h with ^-amylase, mimicking lysosomal enzymes (Figure 8B). This result suggests that upon cellular uptake and intracellular trafficking, the lysosomal exo-amylases can break down the PGEDA-FPBA NPs. Overall, those results indicate that the engineered PGEDA-FPBA NPs exhibit affinity for glucose under the relevant blood glucose concentration observed in diabetes (400 mg/dL) and likely maintain their structural integrity in the extracellular environment when injected in the bloodstream or subcutaneously. [0120] Next, the electrostatic interactions between the positively charged PGEDA-FPBA NPs and negatively charged insulin (isoelectric point of 5.3) were exploited to obtain glucose-responsive Ins-PGEDA-FPBA NCs. As the binding of glucose to the FPBA moieties causes a decrease in the positive charge density on the PGEDA-FPBA NPs, it was hypothesized that this condition can weaken the attraction between insulin and the PGEDA-FPBA NPs to stimulate insulin release. To gain a deeper insight into the morphology of individual NCs at a molecular level, the complexation process was investigated by single molecules localization microscopy STORM. The Ins-PGEDA-FPBA NCs were prepared using fluorescein isothiocyanate (FITC)-labeled insulin at different FITC-labeled insulin-to-PGEDA-FPBA NPs weight ratios (1:0, 1:1.5, 1:3.5, 1:10, and 1:13), where the concentration of insulin was fixed at 1 mg/mL. Naked insulin molecules (free insulin without association with NPs) or Ins- PGEDA-FPBA NCs were deposited on a glass coverslip for STORM-total internal reflection fluorescence imaging. The list of localizations was processed using a clustering analysis script to quantitatively estimate the number of localizations inside a single NC and the size of the NCs. Notably, the number of localizations is proportional to the number of insulin molecules. At low ratios (1:1.5, 1:3.5), free insulin molecules were imaged, while with increasing weight ratios up to 1:13, the free insulin molecules originally visualized in the background gradually disappeared and only round NCs of 160 ± 40 nm in size were detected. This result indicates that all insulin molecules have been complexed with the PGEDA-FPBA NPs. [0121] Results indicate that the number of localizations detected within a single NC obtained at the ratio of 1:13 increases with the diameter of the NC. A limited number of localizations, ranging from 15 to 50, were detected on the small NCs (50–100 nm), whereas heterogeneous clusters of molecules ranging from 80 to 600 were observed in the larger NCs (100–300 nm). Hence, STORM imaging analysis allows the elucidation of the morphologies of the NCs, suggesting that the insulin molecules can either deposit on the brushed charged surface of individual Ins-PGEDA-FPBA NCs to form smaller NCs or remain embedded between multiple Ins-PGEDA-FPBA NCs to form larger NCs; this is also evident by TEM analysis (Figure 9). [0122] Multicolor STORM imaging was employed to verify the co-localization between insulin molecules and the PGEDA-FPBA NPs and confirmed the efficient loading of insulin molecules onto the PGEDA-FPBA NPs. The maximum insulin loading capacity (95%) of the PGEDA-FPBA NPs was calculated by fluorescence analysis of the Ins-PGEDA-FPBA NCs after spin filtration of the unbound insulin at increasing PGEDA-FPBA NPs-to-insulin weight ratios (Figure 10). The charge of the Ins-PGEDA-FPBA NCs was reduced from 25 to 15 mV at the insulin-to-PGEDA-FPBANPs weight ratios 1:13. This reduction in surface charge indicates that complexation is primarily mediated by electrostatic interactions and likely by an increase in entropy due to the release of counterions and coordinated water molecules. The increase in size of the PGEDA-FPBA NPs to 160 ± 70 upon binding to insulin was also confirmed by DLS measurements. The Ins-PGEDA-FPBA NCs maintained their colloidal stability and size after storage for 6 months. In vitro and ex vivo glucose-responsive and pulsatile release of insulin from Ins-PGEDA- FPBA NCs [0123] The kinetics of insulin release from the glucose-responsive Ins-PGEDA-FPBA NCs was investigated in vitro under normal (100 mg/dL) and elevated blood glucose (400 mg/dL) concentrations. Ins-PGEDA-FPBA NCs loaded with 2 IU insulin showed rapid insulin release at elevated glucose levels with approximately 70% of the payload released after 4 h (Figure 11A). In contrast, under no glucose (PBS alone) and normal blood glucose concentration conditions (100 mg/dL), only 20% and 30% of the loaded insulin was released, respectively (Figure 11A). The disassembly of the Ins-PGEDA-FPBA NCs upon glucose incubation was confirmed by a shift in the size distribution of the NCs. For comparison, when the insulin release was monitored under the same experimental conditions from a non-glucose- responsive NCs, obtained by the electrostatic association of insulin with PGEDA NPs (Ins- PGEDA NCs), a very slow and glucose-insensitive insulin release was observed (Figure 11B). In addition, Ins-PGEDA-FPBA NCs further enabled pulsatile insulin release in vitro for up to three cycles by switching the glucose concentrations between 100 and 400 mg/dL (Figuire 11C). [0124] Moreover, the released insulin phosphorylated AKT (p-AKT) in HepG2 liver cells, confirming its bioactivity in vitro. Cytotoxicity assays on fibroblasts (3T3) and macrophages, representing the primary cells types that reside in the SC connective tissues25, found no toxicity following exposure to the PGEDA-FPBA NPs for up to 96 h (Figure 12). Overall, these results suggest that the engineered Ins-PGEDA-FPBA NCs are non-toxic and have the potential to rapidly respond to dynamic glucose changes in vivo in the SC tissue by deploying fully bioactive insulin. [0125] Ex vivo, insulin release from Ins-PGEDA-FPBA NCs incubated in normal plasma for 2 h was approximately 18% of the total insulin loaded (Figure 11D), in agreement with the in vitro results. However, the percentage of insulin released in ‘diabetic plasma’ (addition of glucose to normal plasma) after 2h incubation was lower than that observed in vitro (25% versus 55%). This suggests that the adsorption of plasma proteins onto the Ins- PGEDA-FPBA NCs (i.e., a protein corona) may hinder and slow the release of insulin in vivo. This was confirmed by insulin release studies from Ins-PGEDA-FPBA NCs in the presence of human serum albumin (Figure 13). Whole blood analysis following SC injection of fluorescently labeled PGEDA-FPBA NPs in Wild-type (WT) C57BL/6 mice demonstrated that most of the PGEDA-FPBA NPs were associated with the peripheral blood mononuclear cells (PBMCs), with a fraction of the PGEDA-FPBANPs still visible in the plasma. The thermal stability of insulin, either ‘naked’ or embedded into Ins-PGEDA-FPBA NCs, was then studied by monitoring the heat-induced aggregation and changes in the secondary structure of insulin. The monomeric insulin band disappeared after incubation for 24 h at 60 °C due to insulin aggregation, whereas Ins-PGEDA-FPBA NCs preserved insulin in its active monomeric state. Furthermore, a change in the secondary structure was observed by circular dichroism (CD) analysis for naked insulin after heat treatment for 1 h at 60 °C, whereas the structure of insulin attached to the NCs (Ins-PGEDA-FPBA NCs) remained unchanged. Thus, the Ins- PGEDA-FPBA NCs formulation could potentially improve diabetes care in resource-poor settings where there is a reduced capacity to avoid insulin exposure to high temperatures (Kaufmann B, et al. PLoS One 2021, 16(2): e0245372). [0126] The association of tannic acid to PGEDA-FPBA NPs was demonstrated by UV and fluorescence spectroscopy. The new absorption band at 320 nm (Figure 14A) and enhancement in fluorescence emission at 400 nm at excitation wavelength of 320 nm (Figure 14B), suggest the binding of TA to PGEDA-FPBA NPs likely by hydrogen bonding. [0127] Insulin was loaded onto tannic acid coated PGEDA-FPBA NPs to form a nanocomplex where the adhesive tannic acid enhanced the uptake and interaction strength with insulin. The release of insulin kinetic was studied. The release kinetics of insulin from PGEDA-FPBA NPs TA: Insulin nanocomplex at different glucose concentrations and in PBS (Figure 15) shows that the presence of tannic acid enables a sustained release of insulin. In vitro and ex vivo glucose-glucose-triggered insulin release pattern of Ins-BGEDA-FPBA NCs [0128] Smaller nanoparticles were examined by replacing phytoglycogen with bovine glycogen to prepare bovine glycogen-ethylenediamine-4-carboxy-3-fluorophenylboronic acid nanoparticles (BGEDA-FPBA NPs) following the methodology outlined above. As can be seen from Table 2, use of bovine glycogen resulted in smaller nanoparticles around 25 nm in size with a zeta potential value of +25 mV and bearing 11 % and 7 % boronate. The degree of substitution determined by 1H-NMR spectroscopy is shown in Figure 16. Table 2. Size, zeta potential and degree of functionalization for BGEDA-FPBA NPs Sample Size (nm) Zeta potential (mV) Functionalization (%) BG 23 -11±6 NA BGEDA 25±1 40±3 18 BGEDA-FPBA 39±3 29 11 boronate 14 amine [0129] Insulin was complexed with the BGEDA-FPBA NPs as described above to form Ins- BGEDA-FPBA NCs. Briefly, complexes of insulin (0.46 mg mL-1) and BGEDA-FPBA NPs (6 mg mL-1) (ratio was 1:13) were prepared with 0.2 x PBS (pH 7.4). [0130] Western-blot analysis demonstrates that the normalised percentage ratio of P- AKT to total AKT of Ins-BGEDA-FPBA NCs exhibited statistical significance when compared to the vehicle control (Figure 17a), showing a three-fold difference in phosphorylation levels between the Ins- BGEDA-FPBA NCs and the vehicle control. The concentration of insulin is the same across all groups (insulin equivalent of 100 nM). This confirms that the bioactivity of the insulin was preserved following complexation with BGEDA-FPBA NPs. [0131] Subsequently, the insulin release profiles of Ins-BGEDA-FPBA NCs were evaluated by quantifying the free insulin levels in the supernatant upon incubation at clinically relevant glucose concentrations 100 mg dL-1 (normal blood glucose level (BGL)) and 400 mg dL-1 (diabetic BGL) and PBS (control). The glucose responsive release of insulin was determined by monitoring the amount of insulin released as a function of time and glucose levels. As depicted in Figure 17b, the Ins-BGEDA-FPBA NCs exhibited a rapid release profile with 80 % of the payload released at 400 mg dL-1 glucose concentration within 1 h. A slower kinetic release was observed at 100 mg dL-1 glucose concentration with only 50 % of the payload released after 1 h. In the absence of glucose (PBS), the cumulative release of insulin over time levelled off at the values of 20 % after 1 h (Figure 17b). Additionally, Ins-BGEDA- FPBA NCs displayed a much extended but still highly glucose-responsive insulin release pattern with the potential to release insulin for over 24 hours when incubated in diabetic plasma ex vivo (Figure 17c). Conjugation of PGEDA-FPBANPs with targeting peptide and in vitro activity: [0132] PGEDA-FPBA NPs (10 mg, equivalent to 0.06 mmol of glucose monomers) were dissolved in 5 mL of PBS pH=6.8 with stirring. To this, sodium periodate (0.8 mg, ~ 0.004 mmol) was added and reacted in the dark. After 2 h, 0.8 mg of targeting cyclic peptide (0.0006 mmol) was added followed by 10 eq. of sodium cyanoborohydride to sodium periodate (0.04 mmol), and the mixture was stirred overnight. Product was purified by dialysis (tube size 14 kDa) against Milli-Q water for 3 days and freeze dried. [0133] The effective conjugation of PGEDA-FPBANPs with a cyclic peptide for targeting pancreas was demonstrated by fluorescence (Figure 18A) and UV- absorbance (Figure 18B) spectra where the tryptophan absorption at 280 nm and emission at 340 nm were acquired and evaluated after dialysis. [0134] The effective pancreas cell targeting of PGEDA-FPBA NPs-cyclic peptide conjugates was validated by Amnis® Image Flow Cytometry were indicated in Ch06 (Cy7.5) and high intensity. Ch01/Brightfield clearly showed images of each MIN6 with morphological and structural detail (Figure 19). In vivo blood glucose regulation in two distinct diabetic mouse models [0135] To evaluate the therapeutic properties of the nanocomplexes in vivo, two distinct insulin-deficient mouse models were employed: (1) the spontaneously type 1 diabetic Akita mouse model (C57BL/6-Ins2-Akita/J), in which a mutation in the proinsulin gene causes insulin misfolding and beta cell degeneration (Yoshioka M, Kayo T, Ikeda T, Koizumi A. Diabetes 1997, 46(5): 887-894), and (2) the widely used streptozotocin (STZ)-induced diabetic mouse model (Yu J, et al. Nat Biomed Eng 2020, 4(5): 499-506). [0136] For Ins-PGEDA-FPBA NCs, Akita mice and STZ-induced diabetic mice were randomly grouped and subcutaneously injected with either Ins-PGEDA-FPBA NCs (insulin dose 80 IU/kg) or naked insulin (16 IU/kg) as appropriate control. [0137] Both naked insulin and Ins-PGEDA-FPBA NCs triggered a rapid decrease of the elevated blood glucose level (BGL) (500–600 mg/dL) in the treated diabetic mice (both Akita and STZ-induced diabetic mice) to reach normoglycemia (<200 mg/dL) within 30–40 min (Figure 20A and 21A). These results indicate that the injected Ins-PGEDA-FPBA NCs can rapidly sense and reduce the high concentration of interstitial glucose in these diabetic mice, as the NCs can promptly deploy bioactive insulin in the SC injection site that directly enters the blood stream. When PGEDA-FPBA NPs (without loaded insulin) were injected as a control, a slight decrease in BGL was induced in the diabetic Akita mice (Figure 20A), possibly a result of glucose binding to the boronate moieties. Ins-PGEDA-FPBA NCs maintained normoglycemia for 13.5 h, whereas naked insulin maintained the blood glucose at a normal level for 3 h only in both the Akita and STZ-induced diabetic mice (Figure 20B and 21B). Additionally, plasma insulin analysis by ELISA revealed a rapid insulin release, with a peak plasma insulin level of approximately 700 and 950 µU/mL at 1 h post- Ins-PGEDA-FPBA NCs administration to the Akita and STZ-induced diabetic mice, respectively (Figure 20C and 21C); this result is consistent and correlated with the rapid decrease in BGL observed in those diabetic mice (Figure 20A and 21A). [0138] Subsequently, the plasma insulin level decreased considerably to 39 µU/mL within 4 h in the Akita diabetic mice due to the pharmacological elimination of insulin. A sustained insulin release was observed for up to 13 h when Ins-PGEDA-FPBA NCs were injected in both the Akita and STZ-induced diabetic mice. In contrast, both Akita and STZ-induced mice treated with naked insulin showed a spike in plasma insulin level at 1 h after injection (1500 and 1600 µU/mL, respectively), followed by a steep fall to undetectable levels of insulin within 4 h (Figure 20C and 21C). [0139] To further verify the in vivo efficacy of Ins-PGEDA-FPBA NCs in responding to a glucose challenge mimicking a meal, an intraperitoneal glucose tolerance test was performed (IPGTT, 1.5 g/kg glucose) in both the Akita and STZ-induced diabetic mice 4 h after treatment with naked insulin or Ins-PGEDA-FPBA NCs. In response to the IPGTT, the mice receiving naked insulin rapidly returned to their initial hyperglycemic state (Figure 20D and 20D). Conversely, mice treated with Ins-PGEDA-FPBA NCs, an initial increase of BGL to 240 mg/dL was robustly controlled to a BGL to normal range (<200 mg/dL) up to 2 h after the IPGTT (Figure 20D, E and Figure 21D, E). The area under the curve (AUC) shown in Figures 20E and 21E was used to compare the responsiveness to IPGTT after administration of naked insulin or Ins-PGEDA-FPBA NCs in the Akita and STZ-induced diabetic mice. The glycemic control in mice treated with Ins-PGEDA-FPBA NCs was associated with a significant spike in plasma insulin from 60 to 220 µU/mL in the Akita mice and from 30 to 320 µU/mL in the STZ-induced diabetic mice, respectively (Figure 20F and 21F). This is while mice treated with naked insulin showed no significant change in the plasma insulin level upon IPGTT, as expected (Figure 20F and 21F). [0140] Overall, data in the two insulin-deficient diabetic mouse models show that SC injection of Ins-PGEDA-FPBA NCs in diabetic mice enabled a rapid insulin release under the hyperglycemic conditions at high glucose levels at the local injection site and a sustained insulin release under normal low glucose levels. Normoglycemic conditions were maintained by Ins-PGEDA-FPBA NCs up to 13 h post-administration through an early-stage rapid insulin release and a later-stage basal insulin release kinetics profile, while avoiding both severe hypoglycemia and transient hyperglycemia. In addition, Ins-PGEDA-FPBA NCs showed a rapid and efficient response to a glucose challenge, providing extended glycemic control in vivo when compared to naked insulin. [0141] For Ins-BGEDA-FPBA NCs, the diabetic mice were randomly assigned to be treated with either Ins-BGEDA-FPBA NCs (insulin dose 108 IU kg-1), insulin (16 IU kg-1) or BGEDA- FPBA NP control. [0142] Both insulin and Ins-BGEDA-FPBA NCs triggered a rapid decrease in the BGL from the hyperglycemic state (600 mg dL-1) to reach a normoglycemic BGL (<200 mg dL-1) in both T1D mice models within 60 minutes (Figure 22a and 23a). The BGL was not maintained under normoglycemic levels for longer periods in insulin injected mice, and increased back to hyperglycemic conditions (>200 mg dL-1) after 4 hours. In contrast, Ins- BGEDA-FPBA NCs were able to maintain normal BGL (70-200 mg dL-1) in STZ-induced and Akita T1D mice models, up to 70.7h and 71.5h, respectively. (Figure 22a and 23a). Taken together, the initial in vivo data illustrates that this novel nanocomplex (insulin dose 108 IU kg-1) maintains normoglycemia for at least 3 days in the two distinct T1D mouse models after a single subcutaneous injection, without inducing hypoglycaemia (Figure 22a, b, Figure 23a, b). Injecting BGEDA-FPBA NPs as a control resulted in a slight decrease in BGL, but overall, the BGL remained very high (>400 mg dL-1) throughout the 84 hours in STZ- induced T1D mice (Figure 22a). Intriguingly, there was a rapid blood glucose lowering response when an intraperitoneal glucose tolerance test (IPGTT) was performed to mimic meals in both STZ-induced and Akita T1D mice, (Figure 22c, d & 23c, d). The glucose- triggered insulin release was reported in the STZ-induced T1D mice model (Figure 22e). Thus, smaller INS-BGEDA-FPBA NCs are a promising means of alleviating the burden of multiple insulin injections and achieving a long-term control of blood glucose. The mechanism of insulin release and the fate of Ins-PGEDA-FPBA NCs [0143] To shed light on the underlying mechanism of insulin release from Ins-PGEDA- FPBANCs and the fate of PGEDA-FPBANCs in vivo, pharmacokinetic studies of the glucose- responsive Ins-PGEDA-FPBA NCs (lymphatic absorption and trafficking, systemic circulation and blood clearance, tissue distribution, extravasation, and excretion) were performed in both the Akita mice and STZ-induced diabetic mice after SC injection of the NCs. The in vivo biosafety of the engineered PGEDA-FPBA NPs, administrated SC, was first assessed and showed a survival rate of 100%, with no significant weight loss or any signs of illness for 7 days post-delivery. Next, the mice were subcutaneously administered either labeled Ins- PGEDA-FPBA-Cy5.5NCs, naked insulin as control, dual-labeled Ins-Cy7.5-PGEDA-FPBA-Cy5.5 NCs, naked Ins-Cy7.5, or PGEDA-FPBA-Cy5.5 NP in Akita mice. Super-resolution microscopy imaging combined with an Odyssey ^ near infrared (NIRF) scanning was used to monitor the presence of Ins-PGEDA-FPBA NCs and insulin in the organs and blood. At 24 h post-injection, mice were humanely killed, and vital organs (heart, liver, spleen, lung, kidney, and brain), skin, and muscle were isolated for ex vivo fluorescence imaging to determine sample biodistribution. Representative Odyssey ^ images of Akita diabetic mice and STZ-induced diabetic mice showed a prolonged retention (24 h post-injection) of Ins-PGEDA-FPBA-Cy5.5 NCs in the proximity of the injection site (i.e., under the skin). Significant accumulation of Ins- PGEDA-FPBA-Cy5.5 NCs in the liver and kidneys was observed 24 h post-injection, with only limited retention at 96 h and no uptake into the spleen, lung, heart, or brain. A similar biodistribution of the dual-labeled sample InsCy7.5-PGEDA-FPBA-Cy5.5 NCs was demonstrated as well as a comparable biodistribution of the PGEDA-FPBA-Cy5.5 NPs was observed in Akita mice and WT C57BL/b mice. Importantly, the analysis of all organs 16 weeks post-injection in WT C57BL/6 mice revealed complete elimination of the injected PGEDA-FPBA-Cy5.5 NPs from the liver and kidneys with a low signal still present at the injection site. [0144] The blood clearance of Ins-PGEDA-FPBA-Cy5.5 NCs or PGEDA-FPBA-Cy5.5 NPs was then monitored in Akita mice and healthy C57BL/6 mice (Figure 24). Fluorescence signal spikes of Ins-PGEDA-FPBA-Cy5.5 NCs or PGEDA-FPBA-Cy5.5 NPs, corresponding to 1% of injected dose, were observed in the blood 2 h post-injection of. A second peak corresponding to 1.5% of the injected dose was observed around 24 h post-injection, followed by a progressive decrease in the signal up to 96 h (where 0.5% of injected dose remained). The initial spikes at 2 h post injection can be attributed to the onset of vascular endothelial dysfunction that is typically observed in Akita mice28. This was confirmed by the contrasting results obtained in healthy C57BL/6 mice, where a gradual increase in circulating Ins-PGEDA-FPBA-Cy5.5 NPs was detected in the first 2 h post-injection (Figure 24). These data reveal that both Ins- PGEDA-FPBA NCs and PGEDA-FPBA NPs readily enter the blood stream upon SC administration. Yet, unlike insulin monomers and dimers that are rapidly absorbed by blood capillaries, Ins- PGEDA-FPBA NCs or PGEDA-FPBA NPs are too large to cross the tight endothelial junctions in blood capillaries21,29 (Gradel AKJ, et al. J Diabetes Res 2018, 2018: 1205121; Cai S, et al. Adv Drug Deliv Rev 2011, 63(10-11): 901-908). [0145] It was therefore hypothesized that Ins-PGEDA-FPBA NCs can enter the systemic circulation through the lymphatic system as it is reported that lymphatic capillaries enable the uptake of high molecular weight biomolecules and NPs (Cai S, et al. Adv Drug Deliv Rev 2011, 63(10-11): 901-908). To verify this hypothesis, several major lymph nodes (LNs) were examined at different time points after SC injection of Ins-PGEDA-FPBA-Cy5.5 NCs and control (unlabeled naked insulin), and PGEDA-FPBA-Cy5.5 NPs, naked Ins-Cy7.5, and dual-labeled Ins-Cy7.5-PGEDA-FPBA-Cy5.5 NCs in Akita mice and STZ-induced diabetic mice. In healthy C57BL/6 mice, the PGEDA-FPBA-Cy5.5 NPs first gained access to the ipsilateral inguinal LNs (iLNs) via the lymphatic vessels and then promptly (within 1 h) reached the contralateral iLN through the wide opening of the specialized gaps (also known as junctions, up to ~100 nm) between lymphatic endothelial cells in the lymphatic capillaries. The lymphatic vessels form a one-way trafficking pathway for lymph and leukocytes that specifically encompass tight “zipper-like” junctions for prevention of lymph leakage during lymphatic drainage (Alitalo K. Nat Med 2011, 17(11): 1371-1380; McCright J, et al. Front Pharmacol 2022, 13: 887402). Thus, both the ipsilateral left and contralateral iLNs, axillary LNs (AxLNs), brachial LNs (BrLNs), and mesenteric LNs (MLNs) were also examined 24 h after SC injection of Ins-PGEDA-FPBA-Cy5.5 NCs or Ins-Cy7.5-PGEDA-FPBA-Cy5.5 NCs in the Akita mice and STZ-induced diabetic mice. Ins-Cy7.5-PGEDA-FPBA-Cy5.5 NC was detected in the iLNs and associated lymphatic vessels 24 h post-SC injection in the Akita mice and STZ-induced diabetic mice. A similar uptake of Ins-PGEDA-FPBA-Cy5.5 NCs into LNs was observed in WT C57BL/6 mice. Furthermore, the distribution of Ins-PGEDA-FPBA NCs into different areas of iLNs in the Akita mice was visualized using confocal microscopy imaging. Super-resolution microscopy images with a nanoscale resolution, acquired from blood 24 h post-injection revealed the presence of intact circulating Ins-PGEDA-FPBA NCs, with insulin molecules appear still bound to the nanoparticle. [0146] As the literature reports, the presence of surface hydrophilicity, and optimal particle size and flexibility are critical for lymphatic transport, for example, chain-like dextran with high flexibility is superior to rigid polystyrene spheres (McCright J, et al. Front Pharmacol 2022, 13: 887402; Rohner NA, Thomas SN. ACS Biomater Sci Eng 2017, 3(2): 153-159). The excellent lymphatic transport properties displayed by the Ins-PGEDA-FPBA NCs may likely be due to the intrinsic size, hydrophilicity and flexibility of the multilobular nanosugar component. Further studies may be conducted to determine whether lymphatic transport of the nanosugars (Ins-PGEDA-FPBA NCs) occurs via passive transport, active cell- mediated trafficking, or mixed mechanisms. These results indicate that both the Ins-PGEDA- FPBA NCs and PGEDA-FPBA NPs navigate the lymphatic system upon injection into the SC tissue by entry into lymphatic capillaries, migrating to primary and ipsilateral LNs and subsequently entering the contralateral LNs or thoracic duct and right lymphatic trunk to access the systemic circulation and accumulate in the liver (McCright J, et al. Front Pharmacol 2022, 13: 887402). Indeed, analysis of the fixed liver tissue by H&E staining and confocal microscopy revealed that the Ins-PGEDA-FPBA-Cy5.5 NCs can likely cross the organ’s fenestrated endothelium, which permits the passage of NPs of up to 100 nm in diameter (Garnett MC, Kallinteri P. Occup Med (Lond) 2006, 56(5): 307-311). Ins-PGEDA- FPBA-Cy5.5 NCs are then taken up by hepatocytes and enter the intrahepatic system of bile ducts. STORM super resolution analysis of the fixed liver tissues further demonstrated the presence of intact ins-PGEDA-FPBA NCs, whereas a widespread signal was detected in the kidney, confirming that Ins-PGEDA-FPBA NCs are amenable to degradation in vivo. The Ins- PGEDA-FPBA NCs were also identified in the bile and faeces (Figure 25), indicating that Ins- PGEDA-FPBA NCs enter the hepatobiliary excretion pathway (Garnett MC, Kallinteri P. Occup Med (Lond) 2006, 56(5): 307-311). These data infer that Ins-PGEDA-FPBA NCs entering the hepatic portal vein are taken up and processed by liver hepatocytes and finally undergo either intracellular hepatic degradation or hepatobiliary excretion via the bile duct. Notably, histology of liver tissue and the skin injection site indicated no liver damage or inflammation after administration of Ins-PGEDA-FPBA NCs in Akita mice. [0147] After subcutaneous injection in Akita mice, the PG-insulin NC (e.q 2 U insulin), slowed down the absorption of insulin and extended the insulin therapeutic effect up to 8hr, while avoiding severe hyperglycemia (Figure 26). Biocompatibility of INS-BGEDA-FPBA NCs [0148] Developing biocompatible nanoparticles with excellent pharmacokinetic properties and minimal long-term toxicity is crucial for the effective treatment of lifelong diseases such as T1D (Besford, Q.A., Cavalieri F., Caruso, F. Adv Mater, 2020, 32 e1904625). Unlike endogenous insulin secreted by the pancreas, which enters the portal vein and is primarily removed by the liver during first-pass transit, exogenous insulin administered by subcutaneous injection escapes first-pass hepatic clearance and is instead removed via kidney filtration (Tokarz, V.L., MacDonald, P.E., Klip, A. J Cell Biol, 2018, 217 2273; Reutens, A.T., et al. Contemp. Clin. Trials, 2020, 90 105892). Accordingly, kidney filtration is important for insulin removal and metabolism in T1D patients. [0149] Interestingly, unlike the elimination of Ins-PGEDA-FPBA NCs via hepatic metabolism and biliary excretion, BGEDA-FPBA NPs (20 nm) were found to be eliminated from the body via the kidney-urine renal excretion pathway (Figure 27a & b). Indeed, histological examination of kidney tissue confirmed that the small nanoparticle is rapidly filtered by the glomeruli (8 h post-administration) and appears in the urine (Figure 27b), with evidence that glomeruli uptake is localised to podocytes (Figure 27c & d). Once filtered, the nanoparticle material is quickly re-absorbed by proximal tubule cells (Figure 27c), then fully degraded, and excreted within one week (Figure 27a). It was also found that lysosomal α-glucosidase in renal tubule cells is responsible for degradation of the BGEDA-FPBA NPs, as well as metabolism of endogenous and exogenous insulin to end their actions (Wojnilowicz, M., et al. ACS Nano, 2019, 13187; Tokarz, V.L., MacDonald, P.E., Klip, A. J Cell Biol, 2018, 2172273). Taken together, these data indicate that the Ins-BGEDA-FPBA NCs are highly biodegradable, nontoxic, and biocompatible. [0150] Overall, an insulin delivery system utilising Ins-BGEDA-FPBA NCs has the potential to provide hypoglycaemia-free glucose control over multiple days with high biocompatibility via renal clearance after injection. As an additional benefit, this technology may provide a direct route for drug delivery to the kidney to protect this vital organ from diabetic damage when loaded with suitable protective agents. Co-formulations of Ins-BGEDA-FPBA NCs and Ins-PGEDA-FPBA NCs [0151] It was hypothesised that insulin release kinetics could be optimised with a co- formulation of Ins-BGEDA-FPBA NCs and Ins-PGEDA-FPBA NCs to obtain a longer normoglycemia duration such that the formulation need only be injected to a subject in need thereof once per week or once per month. Several formulations were prepared and assessed in vitro for their glucose-responsive performance. [0152] After optimisation, a 1:9 volume-to-volume ratio of Ins-BGEDA-FPBA NCs and Ins-PGEDA-FPBA NCs was selected for further assessment. Both a 9:1 volume-to-volume ratio of Ins-BGEDA-FPBA NCs and Ins-PGEDA-FPBA NCs and a 5:5 volume-to-volume ratio of Ins- BGEDA-FPBA NCs and Ins-PGEDA-FPBA NCs exhibited similar insulin release kinetics to Ins- BGEDA-FPBA NCs. At a glucose concentration of 400 mg dL-1, the 1:9 formulation exhibited a delayed response, releasing only about ~15% of insulin within the first 30 minutes (Figure 27). Interestingly, the formulation released only ~48% of the encapsulated insulin in comparison to Ins-BGEDA-FPBA NCs, which released approximately 86% of insulin within 24 hours (Figure 17b). To assess the ability of the co-formulations to release insulin beyond 24 hours, one of the co-formulation replicates was incubated in all three glucose conditions for 48 hours and insulin release was quantified. Even after 48 hours of incubation, the formulation was able to release insulin, amounting to approximately 63% of the loaded insulin. This suggests the potential for the co-formulation to release insulin beyond 48 hours (Figure 28).

Claims

THE CLAIMS DEFINING THE INVENTION ARE AS FOLLOWS: 1. A nanocomplex comprising a polysaccharide nanoparticle selected from the group consisting of glycogen, bovine glycogen, phytoglycogen or a combination thereof reversibly complexed with a hormone selected from the group consisting of insulin, glucagon and glucagon-like protein-1.
2. The nanocomplex of claim 1, wherein the polysaccharide nanoparticle is covalently coupled to amine moieties.
3. The nanocomplex of claim 2, wherein the amine moieties are of the formula - N(R3)2, wherein each R3 is independently selected from H and -C1-C4alkyl-N(R4)2; and each R4 is independently selected from H and C1-C4alkyl; or two R3 groups, when taken together with the nitrogen atom to which they are attached, form a heterocyclic ring.
4. The nanocomplex of claim 2 or 3, wherein the amine moiety is ethylenediamine (EDA).
5. The nanocomplex of any one of claims 2 to 4, wherein the polysaccharide nanoparticle is covalently coupled to boronic acid moieties.
6. The nanocomplex of claim 5, wherein the boronic acid moieties are 4-carboxy- 3-fluorophenylboronic acid (FPBA).
7. The nanocomplex of any one of claims 1 to 6, wherein the polysaccharide nanoparticle is bioconjugated with a peptide.
8. The nanocomplex of any one of claims 1 to 7, further comprising a coating on the polysaccharide nanoparticle.
9. The nanocomplex of claim 8, wherein the coating comprises albumin or tannic acid (TA).
10. The nanocomplex of any one of claims 5 to 9, wherein charge density is tuned by controlling the ratio of amine and boronic acid moieties.
11. The nanocomplex of any one of claims 1 to 10, wherein the insulin is selected from fast-acting insulin, intermediate-acting insulin, long-acting insulin or a combination thereof.
12. The nanocomplex of any one of claims 1 to 11, wherein the hormone is complexed with the polysaccharide nanoparticle by a reversible electrostatic interaction.
13. The nanocomplex of any one of claims 1 to 12, wherein the hormone is complexed with the polysaccharide nanoparticle at around 80% to 99% loading capacity.
14. The nanocomplex of any one of claims 1 to 13, wherein the hormone is complexed with the polysaccharide nanoparticle at around 90% to 95% loading capacity.
15. The nanocomplex of any one of claims 1 to 14, wherein the hormone is released from the polysaccharide nanoparticle upon binding of glucose to the polysaccharide nanoparticle.
16. The nanocomplex of any one of claims 1 to 14, wherein the hormone is insulin.
17. A pharmaceutical composition comprising the nanocomplex of any one of claims 1 to 16 and at least one pharmaceutically acceptable carrier or diluent.
18. The pharmaceutical composition of claim 17, comprising nanocomplexes comprising a bovine glycogen nanoparticle and nanocomplexes comprising a phytoglycogen nanoparticle.
19. A method for reducing the blood glucose level of a subject in need thereof, the method comprising administering to the subject an effective amount of the nanocomplex of claim 16.
20. A method for treating diabetes mellitus in a subject in need thereof, the method comprising administering to the subject an effective amount of the nanocomplex of claim 16.
21. The method of claim 19 or 20, wherein the nanocomplex is administered to the subject by subcutaneous injection.
22. The method of any one of claims 20 or 21, wherein the subject has type 1 diabetes mellitus.
EP24762825.8A 2023-02-28 2024-02-28 Engineered nanocomplexes Pending EP4673178A1 (en)

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