Glucose regulation by modified boronic acid-sulfobetaine zwitterionic nanogels – a non-hormonal strategy for the potential treatment of hyperglycemia

Amin GhavamiNejad a, Brian Lu a, Adria Giacca b and Xiao Yu Wu *a
aAdvanced Pharmaceutics and Drug Delivery Laboratory, Leslie L. Dan Faculty of Pharmacy, University of Toronto, Toronto, Canada. E-mail: sxy.wu@utoronto.ca
bDepartment of Physiology, Faculty of Medicine, University of Toronto, Toronto, Canada

Received 24th February 2019 , Accepted 14th May 2019

First published on 15th May 2019


We have introduced a non-hormonal hyperglycemia treatment strategy by using an injectable glucose-responsive boronic acid- zwitterionic nanogel. The synthesized system, similar to an artificial liver, is capable of storing/releasing glucose at high/low blood glucose concentrations. In vivo performance revealed that the injection of the nanogels can effectively regulate blood glucose in type 1 diabetic rats for at least 6 hours.


Insulin therapy is the only treatment for those with type 1 diabetes (T1D).1 However, insulin therapy was found to be relatively ineffective in yielding tight blood glucose control for a considerable number of patients with diabetes mellitus, particularly among those with HIV and obesity due to a reduced insulin sensitivity in the peripheral tissues of these patients.2–4 Therefore, other forms of diabetic treatment (i.e., non-insulin therapy) may be needed for diabetic patients, especially for those with severe insulin resistance. To date, extensive research efforts have been directed toward developing smart materials that release various hormones in a glucose-responsive manner, but encountered challenges of hormone instability.5,6 To this end, hydrogels that exhibit reversible glucose-binding properties could serve as a non-hormonal glucose-regulation system.7,8 Of various glucose-responsive hydrogels, phenylboronic acid-containing polymers are more suitable for this application owing to their non-immunogenicity compared to sugar-binding protein concanavalin A.9 To obtain phenylboronic acid-based hydrogels with glucose-responsiveness at physiological pH, varying comonomers are introduced, including those carrying amine or glyco-groups.10–14 However, to the best of our knowledge, there is no report on boronic acid-containing sulfobetaine zwitterionic polymer in a nanogel form. Zwitterionic hydrogels are considered one of the most attractive types of hydrogels for use in biomedical applications due to their biocompatibility,15 superhydrophilicity,16 antifouling property17 and long circulation lifetime with minimal immunogenic response in comparison to the FDA-approved polyethylene glycol (PEG).18 Zwitterionic micro/nanogels have been shown to be well protected from aggregation in biological media, which makes them suitable for injection.16,19 Here, we introduce a non-hormonal antidiabetic treatment strategy by using an injectable glucose-responsive boronic acid zwitterionic core–shell nanogel. The synthesized glucose-responsive nanogels can store or release glucose at different glucose concentrations due to the dynamic interactions between glucose molecules and the boronic acid functional groups. As shown in Fig. 1, at high glucose concentrations, glucose will form 1[thin space (1/6-em)]:[thin space (1/6-em)]1 monocomplexes with available boronic acid groups residing on different polymer chains, leading to an expansion of the nanogel volume and storage of the glucose in its network. At low glucose concentrations, boronic acid groups will form 2[thin space (1/6-em)]:[thin space (1/6-em)]1 biscomplexes with glucose acting as secondary crosslinks, which leads to shrinkage of the hydrogel matrix and thus, facilitate rapid release of the stored glucose. In vivo evaluation of the synthesized glucose-responsive nanogels demonstrated an excellent antidiabetic effect, comparable to that of regular inulin, but with longer effective duration than that of insulin in streptozotocin (STZ)-induced T1D rats. Our study demonstrates a novel idea for the treatment of diabetes mellitus by using a glucose-responsive polymeric system for non-hormonal blood glucose regulation.
image file: c9nr01687b-f1.tif
Fig. 1 A schematic illustration of the swelling/shrinkage of nanogels in presence of high/low glucose concentrations.

Experimental

Synthesis of 4-acrylamido-3-fluorophenylboronic acid (AFBA)

4-Amino-3-fluorophenylboronic acid (1.0 mmol) and NaHCO3 (2.0 mmol) were dissolved in a mixture of water and THF (2/1 v/v). To this solution, acryloyl chloride (2.0 mmol) was added at 0 °C, and the mixture was stirred for 1 h. At this time, a slurry-like mixture had formed and the resulting solid in the mixture was vacuum filtered and the obtained aqueous solution was extracted with 30 mL of ethyl acetate. The extracted layer in the ethyl acetate was dried over MgSO4 and the volume was reduced to 10 mL with a rotary evaporator. The obtained solution was added dropwise to 100 mL of hexane with vigorous stirring to precipitate a yellowish solid. To purify, the obtained yellowish solid was dissolved in 10 mL of ethyl acetate, precipitated in 100 mL of hexane, and then the formed suspension was refrigerated to maximize the size of crystal formation. The final solid powder was dried in a vacuum overnight with a yield of 47%. The composition of the monomer was confirmed by 1H-NMR (Fig. SI1).

Synthesis of glucose-responsive nanogels

Copolymeric nanogels composed of sulfobetaine methacrylate (SBMA) and AFBA were synthesized using dispersion polymerization. Various monomer feed ratios (Table 1) of AIBN as an initiator (1 mol%), PVP as a stabilizer (1 mol%), and 30 g of water/ethanol (30/70 w/w) as medium, were all mixed, filtered and then added to a 100 mL flask. The mixture was deoxygenated for 20 min with nitrogen and then transferred to an oil bath set at 65 °C to start the polymerization. After the reaction started, the produced oligomers precipitated from the medium forming nuclei for particle growth. After the solution became turbid, a solution of 1.5 mol% crosslinker (MBA) in 10 mL of water/ethanol (30/70 w/w) was added dropwise by a syringe pump at a rate of 5 mL h−1. The delay in the addition of crosslinker was due to the higher reactivity of the crosslinker that may prevent the initial nucleation step.20 The mixture was stirred at 200 rpm overnight. The obtained polymeric particles were cooled, washed twice with ethanol and dialyzed for 2 days in 1 L of distilled water to remove free stabilizer chains and unreacted monomers. The final polymeric nanogels were lyophilized to obtain white solid particles. Table 1 presents the experimental conditions for the synthesis of the nanogels. It should be mentioned that the nanogel composition containing 40 wt% of AFBA (SB60-BA40) was chosen to carry out the experiments in this work because it contains the highest boronic acid functional groups with an acceptable polydispersity index (PDI) and yield of polymerization.
Table 1 Chemical composition of the synthesized nanogels
Sample name SBMA (wt%) feed AFBA (wt%) feed AFBA (wt%) product Yield (%) PDI
MBA = 1.5 mol% with respect to monomers; AIBN = 1 mol% with respect to monomers; stabilizer = 1 mol% with respect to monomers.
SB50-BA50 50 50 32 0.521
SB60-BA40 60 40 45 73 0.119
SB70-BA30 70 30 33 78 0.174
SB80-BA20 85 15 17 74 0.210


Characterization of nanogels

The effect of glucose concentration on nanogel size and zeta potential was characterized using dynamic light scattering (DLS) and zeta sizer (Malvern Zetasizer Nano-ZS) in phosphate-buffered saline (PBS, pH 7.4, ionic strength: 154 mM). The stability of the nanogels was studied by incubating the nanogels in PBS with different glucose concentrations at 37 °C over 72 h. The size of the nanogels were measured as a function of time by DLS. Dimensions and surface morphology of freeze-dried nanogels were examined by scanning electron microscopy/energy dispersive X-ray spectroscopy (SEM/EDX) at an acceleration voltage of 20 kV. The final percentages of AFBA incorporated to the synthesized nanogels were determined by ICP-AES (Optima 7300 DV ICP) and calculated according to the known concentration of nanogel in the suspension. Transmission electron microscopy (TEM) images were performed on a Hitachi H-7500 transmission electron microscope operated at 80 kV. The bonding configurations of the samples were characterized by means of their FTIR spectra using a Paragon 1000 spectrometer (PerkinElmer).

In vivo studies on a type 1 diabetic rat model

All conducted in vivo studies strictly adhered to the ethical and legal requirements of the Ontario Animals for Research Act and the Federal Canadian Council on Animal Care guidelines, and were approved by the Animal Care Committee of the University of Toronto. Male Sprague Dawley rats (Charles River) were treated with 65 mg kg−1 STZ to induce T1D. The rats receiving STZ were carefully monitored for 1 week during which the blood sugar was measured in 2 day intervals using a glucose meter (OneTouch® Ultra®, LifeScan, Inc., USA). Diabetic rats with blood sugar stabilized above 17 mmol L−1 were selected for the study. All rats were fasted for 4 hours prior to the experiment to eliminate the possible confounding effect of food.21 The diabetic rats were divided into four groups (n = 5 per group): one group was treated with a subcutaneous (s.c.) injection of glucose-responsive nanogels (83 mg kg−1), two groups were treated with a s.c. injection of human recombinant insulin (0.5 or 1.0 IU per kg doses), and one group without treatment was used as fasting control. Non-diabetic rats were also treated with glucose-responsive nanogels (83 mg kg−1) as the healthy control. The blood glucose levels were measured every 15 min after treatment and stopped whenever they were above or below the euglycemic window.

Results and discussion

First, a novel boronic acid-containing monomer, AFBA, was synthesized to provide the necessary glucose sensitivity at physiological pH by the incorporation of an electron withdrawing group, fluorine, on the phenyl aromatic ring. Then copolymeric nanogels of zwitterionic SBMA and AFBA were synthesized via a one-pot radical precipitation/dispersion polymerization. The SEM and TEM images depict that the synthesized nanogels exhibit uniform morphology with symmetrical core–shell structure and average particle sizes of around 400 nm (Fig. 2A–C). The observed core–shell structure may be attributed to three possible reasons: (1) the concentration gradient of the crosslinker in the nanogel,22,23 (2) the faster polymerization rate of AFBA than SMBA, which is evidenced by higher concentration of AFBA in the nanogel than in the feed24,25 (Table 1), and (3) higher hydrophobicity of AFBA due to the presence of the phenyl group that reduces the global polarity of the monomer, which favors the polymerization of AFBA at the early stage of particle nucleation, resulting in a AFBA-rich core of the nanogel.
image file: c9nr01687b-f2.tif
Fig. 2 Characterization of glucose-responsive nanogels. (A) SEM; (B, C) TEM images; and (D) EDX mapping of sulfur elements of the nanogels.

The surface elemental composition of the nanogels was determined by EDX-mapping. The EDX analysis identified a high percentage of sulfur element on the surface of the nanogels (Fig. 2D), suggesting that most of the boron were present in the core and surrounded by the zwitterionic shell. Existence of antifouling and biocompatible zwitterionic polymer as the shell imparts several beneficial properties to the particles: (1) preventing nanogel aggregation in biological media,16 and (2) facilitating glucose diffusion into the nanogel owing to the highly hydrated SMBA-rich shell.26 The chemical composition of the nanogel was further confirmed by FTIR (Fig. SI2). The existence of sulfobetaine methacrylate segments in the nanogels were confirmed by the presence of ester carbonyl groups and sulfonate groups observed from the bands corresponding to –SO3 stretching at 1033 cm−1 and O–C[double bond, length as m-dash]O stretching at 1727 cm−1, respectively.15 Furthermore, the FTIR band at 1183 cm−1 can be attributed to the S[double bond, length as m-dash]O group, and the absorption band at 1480 cm−1 was mainly due to the C–H stretching of the quaternary ammonium. FTIR spectrum of substituted fluorine derivatives of fluorophenylboronic acid can also be assigned to the wavenumber 1254 cm−1 due to the C–F stretching mode.27 Bright-field (transmitted light) microscopy and dynamic light scattering (DLS) were used to record the changes of nanogel size at different glucose concentrations.

Fig. 3(A–C) shows that the synthesized nanogels exhibited significant changes in size at different glucose concentrations corresponding to hypoglycemia (70 mg dL−1) and hyperglycemia (200 mg dL−1). Binding of glucose molecules to AFBA moieties of nanogels affects the size of nanogels in two ways.28 At high glucose concentrations (hyperglycemia), AFBA groups form 1[thin space (1/6-em)]:[thin space (1/6-em)]1 monocomplexation with glucose which leads to an expansion of the hydrogel volume. When glucose concentration is reduced, AFBA groups form a 2[thin space (1/6-em)]:[thin space (1/6-em)]1 biscomplexation with glucose, leading to additional (secondary) crosslinking and thus, shrinkage of the hydrogel matrix and release of the stored glucose. It is noted that due to the incorporation of an electron withdrawing group on the aromatic ring of boronic acid functional groups, the AFBA units are in the tetrahedral (ionized) state at the physiological pH. Thus, the nanogels were in a swollen state in the absence of glucose (Fig. SI3). Furthermore, zeta potential measurements showed that the surface charge of the nanogel did not significantly change after swelling (−1.82 ± 0.6 mV to −2.17 ± 1.1 mV), suggesting that the ionization equilibrium associated with AFBA groups occurred in the core of the nanogel. Repeated nanogel swelling/shrinking cycles in response to varying glucose levels were observed when the nanogels were immersed in solutions of different glucose concentrations (Fig. 3D). The results suggest that the nanogel is a stable and reversible glucose-responsive system. The drastic reduction in particle size when glucose level was changed from hyperglycemia (200 mg dL−1) to hypoglycemia (70 mg dL−1) is consistent with the proposed glucose storing/releasing mechanism. The nanogels were stable in buffer solutions of different glucose concentrations with no noticeable change in particle size and polydispersity (PDI < 0.2) over 72 hours at 37 °C (Fig. SI4). Cytotoxicity of the nanogels was examined on NIH-3T3 fibroblast cells. The MTT assay showed little change in cell viability when incubated with the nanogels (Fig. SI3) attributable to the low cytotoxicity of the zwitterionic polymer. In vivo evaluation of the efficacy of the glucose-responsive nanogels were performed using an STZ-induced T1D rat model. In this experiment, we compared the glucose-regulating effect of s.c. injected nanogels with human recombinant insulin (a fast-acting insulin form) at different doses. As shown in Fig. 4, the blood glucose levels in the nanogel- and insulin-treated diabetic rats rapidly decreased in contrast to the un-treated diabetic rats; whereas the glucose level of the non-diabetic (healthy) rats treated with the nanogels remained in the euglycemic range during the experimental period. The blood glucose level in rats treated with 0.5 IU per kg insulin decreased to euglycemia and then rebounded to hyperglycemia after 3 hours, while the blood glucose level of the rats treated with 1 IU per kg insulin rapidly fell to euglycemia but continued to drop below the hypoglycemic threshold (<3.9 mmol L−1).


image file: c9nr01687b-f3.tif
Fig. 3 Bright-field (transmitted light) microscopy image of nanogels at (A) 70 mg dL−1; (B) 200 mg dL−1 glucose concentration; (C) representative DLS result of nanogels at the above-mentioned glucose concentrations; (D) changes of the hydrodynamic diameters of nanogels when the glucose concentrations were alternated between 70 and 200 mg dL−1.

image file: c9nr01687b-f4.tif
Fig. 4 Antidiabetic tests of glucose-responsive nanogel vs. 0.5 or 1.0 IU per kg dose of insulin, and control groups. The dashed-line box indicates the euglycemic window.

In contrast, rats treated with the glucose-responsive nanogels reached euglycemia at 60 minutes post-injection (albeit slightly slower than insulin treated rats) and continued to maintain euglycemia for at least another 6 hours without falling to hypoglycemia. In other words, the glucose-responsive nanogels exhibited superior capability of regulating glucose levels within the euglycemic window for extended periods in diabetic rats as compared to insulin.

In conclusion, a novel glucose-responsive polymeric nanogel containing zwitterionic and modified boronic acid moieties were developed. The boronic acid moieties, which can directly interact with glucose molecules, were rationally tuned to enable glucose storage or release at high or low blood glucose concentrations, respectively. The synthesized nanogels demonstrated excellent reversible glucose-responsive volume change and exhibited a desirable profile for glucose regulation. At high glucose concentrations (hyperglycemia), the nanogels absorb glucose molecules, resulting in the swelling of the nanogels. In contrast, the nanogels shrink at low glucose concentrations (hypoglycemia) to release stored glucose. Finally, in vivo experiments demonstrated an excellent glucose-regulating effect of the nanogels in T1D rats for a duration of at least 6 hours upon a single dose of nanogel injection. The results from this work warrant further development of a novel non-hormonal polymeric glucose regulation system as a potential solution for those with severe insulin resistance.

Conflicts of interest

There are no conflicts to declare.

Acknowledgements

This work was supported by a grant from JDRF (2-SRA-2016-268-A-N, XYW, AG), BBDC fellowship (AGN), QEII-GSST scholarship (BL), and NSERC Discovery ((RGPIN 170460-13) and Equipment grants (EQPEQ 374799-09; EQPEQ 440689-13) to XYW). The authors would like to thank the staff of ANALEST and DCM at UofT, for helping with analytical and animal experiments. For animal studies. All institutional and national guidelines for the care and use of laboratory animals were followed.

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Footnotes

Electronic supplementary information (ESI) available. See DOI: 10.1039/c9nr01687b
These authors contributed equally to this work.

This journal is © The Royal Society of Chemistry 2019