Fluorophenylboronic acid substituted chitosan for insulin loading and release

Highlights

• CS-FPBA could be responsive to glucose better than normal CS-PBA in physiological environment.

• CS-FPBA could effectively load with insulin with approximately 50% (wt%) encapsulation efficiency in 0.5 mg/mL insulin.

• Insulin release speed could be regulated by choosing different molecular weights.

• The zeta potential of CS-FPBA was 6.7~7.1, lower than physiological pH (7.4).

• CS-FPBA could appear various morphologies in different substituted degree.

Abstract

Phenylboronic acid (PBA) can form dynamical reversible chemical bonds with sugars, and has potential in glucose response and controlling insulin delivery. In this paper, 4-fluorophenylboronic acid (FPBA) was employed which could work better than normal PBA in physiological environment. Herein, FPBA-substituted chitosans (CS-FPBA) with three different molecular weights were prepared, and affecting factors on preparation, including solvent, pH, raw materials ratio, activation time and reaction time, were discussed. The glucose responsiveness and controlling insulin release properties of CS-FPBA were also determined. Results showed that the substitution of FPBA onto CS could decrease the zeta potential of CS to negative values, which was favorable for CS-FPBA to react with hydroxyl groups in biological environments. They could effectively load with insulin with approximately 50% (wt%) encapsulation efficiency in 0.5 mg/mL insulin. Also, CS-FPBA particles had significant binding efficiency to glucose, which was dramatically higher by over 6 folds compared to chitosan. The maximum releasing amount of insulin was >90% in 9 h in 3 mg/mL of glucose solution when the molecular weight of CS was 50 kDa. In summary, CS-FPBA has potential to control the release of insulin, and can regulate the release rate by controlling the molecular weight of CS.

Introduction

Insulin is currently an indispensable drug for diabetes treatment, but long-term injection of insulin can cause adverse reactions such as allergy, neuritis, insulin resistance, lipoatrophy, and so forth [1]. Therefore, designing a glucose responsive carrier which can release insulin with blood glucose changes would have practical significance in improving insulin compliance [[2], [3], [4]]. There are three major glucose- responsive systems at present, including glucose oxidase (GOD), concanavalin A (Con A) and phenylboronic acid (PBA). GOD could oxidize glucose by specific recognition to produce gluconic acid and hydrogen peroxide. The GOD-containing polymers would be responsive to the oxidation products and release insulin by swelling or disassembly [[5], [6], [7]]. Con A is a plant lectin extracted from bean seeds, which could specifically bind to sugar units through non-covalent bonds [[8], [9], [10]]. The binding to glucose would cause morphological changes of the polymer, and finally the release of insulin. Compared with the two proteinaceous systems, PBA system exhibits responsive activities in a wider range of pH and ionic strength due to its better chemical stability, and is superior in biomedical applications [[11], [12], [13]].

PBA is a type of Lewis acid which can combine 1,2- or 1,3- hydroxyl groups to form a dynamically reversible borate structure [14,15]. The stability and dynamic exchange feature of borate depends on the structure of boronic acid in different acidity, which is determined by the pKa of PBA (Scheme 1). When pH > pKa, boric acid would exhibit a negatively charged tetrahedral structure, and can easily react with hydroxyl groups; while pH < pKa, boric acid would turn into an uncharged planar structure which could hardly react with hydroxyl groups. Since the pKa (7.8–8.6) of PBA and some of its derivatives is too high to be fully ionized under physiological environments (~7.4) [[16], [17], [18]], the glucose borate would become unstable and ready to be hydrolyzed, and would cause a low glucose responsiveness [19]. The effective way to decrease the pKa of PBA includes the introduction of electrophilic groups (such as carboxyl groups, halogens, nitryls); or using amino or hydroxyl groups to coordinate with boron atom [20,21]. By using these methods, several phenylboronic acid-substituted polymeric materials with glucose-responsiveness and insulin-releasing properties under physiological conditions have been prepared [11,22,23]. Based on above methods, 3-carboxyl-4-fluorophenylboronic acid was employed in this paper.

Chitosan (CS) is the deacetylated product of chitin, with glucosamine and acetylglucosamine linked by β-1,4-glycosidic bond, and can be used as wound dressing, drug carrier and tissue engineering scaffold due to its good biodegradability, biocompatibility [[24], [25], [26], [27]]. Some PBA-substituted chitosans (CS-PBA) have been prepared [[28], [29], [30], [31], [32]], however, a study on fluorinated phenylboronic acid-substituted CS has rarely been reported. Theoretically, due to the high polarity of fluorine, the FPBA-substituted CS would exhibit lower pKa and be more negatively-charged under physiological conditions, which would be benefit to the combination of carbohydrates. Hence, CS-FPBA has potential to exhibit better drug-loading and release performance compared with CS-PBA under human body fluid environments.

In this study (Scheme 2), CS-FPBA was prepared by amide condensation reaction. 3-carboxy-4-fluorophenylboronic acid (FPBA) was firstly activated by 1-ethyl-3-(3-dimethylaminopropyl) carbodiimine (EDC) and N-hydroxyl succinimide (NHS), and then reacted with amine groups of CS to form CS-FPBA. The effects of solvent type, pH value, molar ratio of raw materials, molecular weights of CS, activation time and reaction time on the substitution reaction were studied. The products were characterized by acid-base titration, Fourier transform infrared spectroscopy (FT-IR), hydrogen nuclear magnetic spectroscopy (¹H NMR), zeta potential determination, and scanning electronic microscope (SEM). The insulin loading and release behavior of CS-FPBA in vitro were evaluated, and the relationship between insulin release and glucose concentration was determined.