Fabrication of boronic acid-functionalized nanoparticles via boronic acid–diol complexation for drug delivery

Abstract

We presented here a facile strategy for fabricating boronic acid-functionalized nanoparticles based on the complexation of phenylboronic acids in poly(3-methacrylamido phenylboronic acid) (PMAPBA) and glucose moieties in dextran via boronic acid–diol interactions. The formation of boronate crosslinked nanoparticles was confirmed by Fourier transform infrared spectrometry, thermal analysis, transmission electron micrographs, dynamic light scattering and UV spectrometry. The nanoparticles were well dispersed as individual, spherically shaped particles with an average size of 100 nm. The glucose-sensitivity was revealed by the swelling behavior of the nanoparticles at different glucose concentrations. Furthermore, insulin was encapsulated in the nanoparticles with a loading capacity up to 22%, and the structure of insulin had not been distorted in the loading procedure. The insulin release increased with the enhancement of the glucose level in the medium. More importantly, the nanoparticles had good cytocompatibility, as demonstrated by in vitro experiments. The facility of this strategy together with the high loading capacity, glucose-sensitivity and cytocompatibility of the produced nanoparticles should greatly boost their application in drug delivery.

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1. Introduction

Polymeric nanoparticles have been widely reported in areas such as drug delivery formulations, biomaterials, surfactants, and membrane and separation technology.^1–4 They can be prepared by the partial hydrophobic modifications of hydrophilic polymers that result in amphiphilic character and consequently induce the self-assembly of polymers into nanoparticles.^5,6 Nevertheless, the covalent modification processes are usually quite complicated and time consuming and thus it is virtually inconvenient to control the properties of nanoparticles through tuning the modification parameters. Non-covalent interactions, such as hydrogen bonding, acid–base interactions, and oppositely charged ionic interactions, can also be used to prepare self-assembled nanoparticles.^7–15 However, the stability of the formed nanoparticles is generally limited, especially in ionic, acidic and basic environments.^16,17 In contrast, the unique chemical properties of boronic acids make boronic acid-related reactions ideal candidates for constructing nanoparticles. The boronic acids can react rapidly and reversibly with diol groups.¹⁸ The hydrophobic molecules containing boronic acid groups can be attached to hydrophilic compounds with diol moieties rapidly in aqueous media without any aid from catalysts. Accordingly, the preparative process for nanoparticles would be simplified and more easily controlled by introducing boronic acid-related reactions.^19,20

Recently, there has been a great deal of interest in studying the interactions between boronic acids and diol-containing compounds.^21–23 Carbohydrate polymers are excellent reactants for the formation of complex nanoparticles with boronic acids, thanks to their abundant hydroxyl group. In pharmaceutics area, natural carbohydrate polymers are often preferred over synthetic polymers due to their non-toxic, low cost, ease of availability and biodegradability characteristics. Among the many natural carbohydrate polymers, dextran (a polysaccharide consisting of glucose molecules coupled into long branched chains, mainly through 1,6- and some through 1,3-glucosidic linkages) is very attractive because of its colloidal biocompatibility and because it is already widely used in the food and pharmaceutical industries.^24,25 Dextran is chemically similar to glycosaminoglycans that are important constituent of extracellular matrix and hence, it does not affect cell viability. Moreover, dextran can be degraded by the dextranase enzyme, which is present in colon.²⁶

In the current study, we report a facile strategy for designing boronic acid-functionalized nanoparticles based on the complexation of phenylboronic acids in PMAPBA and glucose moiety in dextran via a boronic acid–diol reaction. We evaluated the formation, glucose-sensitivity and cytocompatibility of the boronate crosslinked nanoparticles, and studied the encapsulation behavior and in vitro release of the nanoparticles.

2. Experimental

2.1 Materials

Dextran (M_w 40 kDa) was purchased from ACROS Company (Beijing, China). Pure crystalline porcine insulin (with a nominal activity of 28 IU mg⁻¹) was obtained from the Xuzhou Wanbang Biochemical Co., Ltd. (Jiangsu, China), and was used without further purification. The dialysis membrance (3.5 and 100 kDa cutoff) was purchased from Shanghai Green Bird Science and Technology Co., m Ltd. (Shanghai, China). Poly(3-methacrylamido phenylboronic acid) (PMAPBA) was prepared by reversible addition fragmentation chain transfer polymerization according to the previous method.^21,27 3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) was purchased from the J & K China Chemical Ltd. (Beijing, China). All other agents used were of analytical grades.

2.2 Preparation of nanoparticles

The PMAPBA homopolymer was dissolved in methanol, and dextran was dissolved in water. The 2.0 mL of dextran solution (0, 0.10, 0.20, 0.30, 0.40, 0.50, 0.60, 0.75, and 1.0 mg mL⁻¹) was added slowly into 0.10 mL of PMAPBA solution (10 mg mL⁻¹) under vigorous stirring. The resulting suspension was stirred for an additional 30 min and was then dialyzed against water in a dialysis bag with a molecular weight cutoff of 100 kDa to remove methanol and free dextran. The suspension was analyzed as is or freeze-dried for further characterization. For simplicity, the nanoparticles were marked as PMAPBA_a/dextran_b, in which letters “a” and “b” represented the weight (mg) of PMAPBA and dextran, respectively. As a result, the nanoparticles were marked as PMAPBA₁/dextran₀, PMAPBA₁/dextran_0.2, PMAPBA₁/dextran_0.4, PMAPBA₁/dextran_0.6, PMAPBA₁/dextran_0.8, PMAPBA₁/dextran₁, PMAPBA₁/dextran_1.2, PMAPBA₁/dextran_1.5 and PMAPBA₁/dextran₂.

2.3 Characterization of the nanoparticles

FT-IR spectra of dry dextran and PMAPBA/dextran nanoparticles were recorded on a Fourier Transform Infrared Spectrometer (FTS-6000, Bio-Rad Co.) with a KBr tablet containing the powders of above samples at a resolution of 8 cm⁻¹. The thermogravimetric analysis of the dextran, PMAPBA and PMAPBA/dextran nanoparticles, using 3–5 mg per sample, was conducted in nitrogen, at a heating rate of 10 °C min⁻¹, using a thermogravimetric analyzer (TGA; TG 209, NETZSCH). The hydrodynamic diameter (D_H), and polydispersity (PDI) of the nanoparticles were determined using dynamic light scattering (DLS) (Malvern, Nano ZS90/ZEN3690) at 25 °C. Furthermore, the D_H and PDI of the nanoparticles, before and after the treatment with 1 and 3 mg mL⁻¹ glucose, were also determined by DLS. The measurement was carried out at 25 °C in pH 7.4 phosphate buffer solution (PBS). The morphology of the nanoparticles was measured by transmission electron micrographs (TEM) (Philips, EM400ST). The turbidity measurement was conducted using a UV spectrometer (Shimadzu, UV-2550) at 500 nm.

2.4 Insulin loading capacity

To assess the potential use of these nanoparticles as drug delivery system, insulin was chosen to study the encapsulation behavior of the nanoparticles. PMAPBA was dissolved in methanol. Insulin and dextran was dissolved in distilled water, and were slowly dropped into PMAPBA solution under stirring. The insulin-loaded PMAPBA₁/dextran_0.2, PMAPBA₁/dextran_0.8 and PMAPBA₁/dextran_1.5 nanoparticle solutions were prepared as described for the blank nanoparticles. Finally, the insulin-loaded nanoparticle suspension was dialyzed against water in a dialysis bag with a molecular weight cutoff of 3.5 kDa to remove methanol and were then obtained by centrifugation for 30 min at 11 000 rpm. The amount of insulin in the supernatant was measured by the Bradford method, using a UV spectrometer (Shimadzu, UV-2550) at 595 nm. The insulin loading capacity (LC) and entrapment efficiency (EE) of the nanoparticles were determined using the following equations:

LC% = (total insulin − free insulin)/nanoparticle weight × 100%

EE% = (total insulin − free insulin)/total insulin × 100%

All measurements were performed in triplicate and averaged.

2.5 In vitro release studies

Insulin release from insulin-loaded PMAPBA/dextran nanoparticles was analyzed by incubating insulin-loaded nanoparticles at 37 °C in PBS (pH 7.4) with different glucose concentrations (0, 1 and 3 mg mL⁻¹) while shaking (100 rpm). At predetermined time points, 100 μL supernatant was withdrawn and fresh solution was replenished. The amount of free insulin was determined by the Bradford assay and a calibration curve was made using blank nanoparticles in order to correct for the intrinsic absorption of the polymer. In each experiment, the samples were analyzed in triplicate and the error bars in the plot represented the standard deviation.

2.6 Circular dichroism (CD) measurements

The insulin-loaded nanoparticles were destroyed, and the insulin was released from the nanoparticles. The stability of insulin was determined by circular dichroism (CD) spectroscopy. Solution of the standard insulin and the released insulin was diluted to 0.2 mg mL⁻¹. CD measurements were performed on a Jasco J-715 CD spectropolarimeter at 25 °C with a cell length of 1 mm. Samples were scanned from 190 to 260 nm and accumulated 10 times.

2.7 Cell viability

Cell viability was evaluated by using NIH3T3 cells. The cell line was cultured in Dulbecco's modified Eagle's medium (DMEM) in 5% CO₂, 95% O₂. The cells were seeded on to 96-well plates at 10 000 cells per well. Cells were allowed to grow until cell monolayers were obtained. The nanoparticle solution (PMAPBA₁/dextran₀, PMAPBA₁/dextran_0.8 and PMAPBA₁/dextran_1.5) was diluted with culture medium to give a final range of concentrations from 60 to 500 μg mL⁻¹. The medium from each well was replaced with 100 μL of the nanoparticle suspension. The plates were incubated at 37 °C in 5% CO₂ for 48 h. Each sample was plated in five replicates per plate. After 48 h, the culture medium and 10 μL of MTT were used to replace the mixture in each well. The cells were incubated for another 4 h in 5% CO₂ at 37 °C prior to removal of the culture medium and MTT. Isopropanol (100 μL) was added to each well to dissolve the formazan crystals that formed in response to MTT exposure. Plates were incubated in 5% CO₂ at 37 °C for 10 min and at 6 °C for 15 min prior to determination of optical density using a microplate reader at 570 nm. Relative cell proliferation rate was determined as a percentage of the positive control; untreated cells were used as the positive control and their proliferation rate was set to 100%.

3. Results and discussion

3.1 Preparation of the nanoparticles

Phenylboronic acid compounds were in equilibrium between an uncharged and a charged form, and they had unique reversible covalent interaction with diol moiety in sugars to form cyclic boronate moieties.^28,29 As shown in Scheme 1, the phenylboronic acids in PMAPBA could form stable complexes with diol moieties in dextran, when dextran solution was added slowly into PMAPBA solution. Due to the hydrophobic and hydrophilic moieties of the PMAPBA/dextran complex, it readily self-assembled into nanoparticles.

Scheme 1 Proposed formation process of PMAPBA/dextran nanoparticles.

3.2 Characterization of the nanoparticles

3.2.1 FT-IR spectra. Fig. 1 shows the FT-IR spectra of dextran, PMAPBA and PMAPBA₁/dextran_0.2 nanoparticles. For PMAPBA₁/dextran_0.2 nanoparticles, the dextran concentration was 0.2 mg mL⁻¹. After dialysis to remove free dextran, the nanoparticle suspension was freeze-dried for FT-IR characterization. In the range of 3695 and 3081, the dextran displayed absorption bands, which were attributed to the free (3550 cm⁻¹) and associated (3285 cm⁻¹) hydroxyl stretching. Compared to the peaks of dextran, the nanoparticles showed a wider peak in this range which was caused by the decrease of the free hydroxyl groups resulted from the interactions between the hydroxyl groups of dextran and boronic acid groups of PMAPBA.³⁰ The nanoparticles showed the absorption peaks in the range of 3000 and 2841 cm⁻¹, which were assigned to saturated hydrocarbon stretching vibration in the dextran (at 2939 and 2905 cm⁻¹) and PMAPBA (at 2990 and 2930 cm⁻¹). Except for the clear signals of phenyl groups at 1605, 1584, 1529, and 1484 cm⁻¹, which reflected the appearance of PMAPBA in nanoparticles, the peaks at 795, 765, and 707 cm⁻¹ were assigned to C–H bending vibration of meta-substituted benzene rings in PMAPBA.^20,30 The absorption peaks of the nanoparticles in the region 1461–1300 cm⁻¹ were ascribed to C–H bending vibration of saturated hydrocarbon in dextran (at 1424 and 1353 cm⁻¹) and PMAPBA (at 1425 and 1342 cm⁻¹). Additionally, the nanoparticles displayed the absorption bands in the range of 1300 and 1006 cm⁻¹, which was attributed to the C–O stretching vibration of alkoxy bonds in dextran.²⁰ These results confirmed that the nanoparticles were fabricated by the complexation of PMAPBA and dextran.

Fig. 1 FT-IR spectra of dextran, PMAPBA, and PMAPBA₁/dextran_0.2 nanoparticles.

3.2.2 Thermal analysis. The traces of thermogravimetric (TG) and derivative thermogravimetric (DTG) for dextran, PMAPBA and PMAPBA₁/dextran_0.2 nanoparticles were shown in Fig. 2. For PMAPBA₁/dextran_0.2 nanoparticles, the dextran concentration was 0.2 mg mL⁻¹. After dialysis to remove free dextran, the nanoparticle suspension was freeze-dried for thermal analysis. As shown in Fig. 2b, it was obvious that dextran (green curve) had low thermal stability with its major degradation temperature occurring at 280 °C, while PMAPBA (red curve) had major degradation at 340 °C. On the other hand, the nanoparticles (blue curve) showed two-stage degradation. Due to the hydroscopic dextran moieties, the nanoparticles showed a minor weight loss (7%) under 105 °C due to moisture. A 20% weight loss of nanoparticles in the first degradation from 225 to 331 °C corresponded to the thermal decomposition of dextran.³¹ The last stage of thermal degradation of nanoparticles, occurring between 331 and 480 °C, was assigned to the thermal degradation of PMAPBA.^32,33 At this stage, the weight loss was approximately 37%. Additionally, the degradation rate of the nanoparticles in the range of 420–800 °C was slower than that of the PMAPBA, this was because that the complexation between the hydroxyl groups in dextran and phenylboronic acids in PMAPBA increased the thermal stability of the nanoparticles. The changes in nanoparticle weight were calculated from the thermogravimetric curves displayed in Fig. 2a.

Fig. 2 Thermal analysis of dextran, PMAPBA, and PMAPBA₁/dextran_0.2 nanoparticles: (a) TG; (b) DTG.

3.2.3 Size and transmittance of nanoparticles. The 2.0 mL of dextran solution (0, 0.10, 0.20, 0.30, 0.40, 0.50, 0.60, 0.75, and 1.0 mg mL⁻¹) was added slowly into 0.10 mL of PMAPBA solution (10 mg mL⁻¹) to form a series of nanoparticles. Fig. 3 shows the diameters and percent transmittance (%T) of the PMAPBA/dextran nanoparticles. The diameters of the nanoparticles were decreased with an increase in the dextran concentration in the range of 0–0.4 mg mL⁻¹. This can be attributed to the increase of dextran leading to a high cross-linking degree of the nanoparticles. As a result, the nanoparticles became smaller and more compact. At the dextran concentration of 0.4 mg mL⁻¹, the molar ratio of phenylboronic acid groups to glucose groups was calculated to be 1.0 and was very close to the stoichiometric ratio. In principle, all of the phenylboronic acid groups on the PMAPBA can bind the glucose groups on the dextran. So, the dextran concentration of 0.4 mg mL⁻¹ can be considered as a critical value at which the PMAPBA is cross-linked with the highest degree. However, above the dextran concentration of 0.4 mg mL⁻¹, the diameters of the nanoparticles increased with a further increase of the dextran concentration. When more dextran was added, some molecules may only have part of glucose groups bound with the phenylboronic acid groups. The unbound glucose groups could increase the nanoparticle diameter. Additionally, %T values increased first, and then decreased with increasing dextran concentration.

Fig. 3 Effect of dextran concentration (0, 0.10, 0.20, 0.30, 0.40, 0.50, 0.60, 0.75, and 1.0 mg mL⁻¹) on the size and percent transmittance (%T) of PMAPBA/dextran nanoparticles.

3.2.4 Polydispersity of nanoparticles. Fig. 4 shows the variation of the polydispersity of the PMAPBA/dextran nanoparticles with the dextran concentration. The polydispersity decreased with increasing dextran concentration in the range of 0–0.4 mg mL⁻¹. Above 0.4 mg mL⁻¹, the polydispersity was kept nearly constant, which was about 0.05. Additionally, the inset of Fig. 4 shows the hydrodynamic diameter distributions f(D_H) of the nanoparticles. Compared to nanoparticles without dextran, the nanoparticles with dextran concentration of 0.4 mg mL⁻¹ exhibited a narrower distribution. Therefore, based on the complexation of dextran and PMAPBA, the PMAPBA/dextran nanoparticles had a very narrow and monadal size distribution.

Fig. 4 Effect of dextran concentration (0, 0.10, 0.20, 0.30, 0.40, 0.50, 0.60, 0.75, and 1.0 mg mL⁻¹) on the polydispersity of PMAPBA/dextran nanoparticles. The inset shows the hydrodynamic diameter distributions of nanoparticles with dextran concentration of 0 (black line) and 0.4 (red line) mg mL⁻¹.

3.2.5 Morphology of nanoparticles. The morphology of the PMAPBA₁/dextran_0.8 nanoparticles was observed using TEM. The size of the PMAPBA₁/dextran_0.8 nanoparticles was approximately 100 nm, with a spherical shape, as shown in Fig. 5. The sizes of PMAPBA₁/dextran_0.8 nanoparticles in TEM were little smaller than those in DLS. This was because PMAPBA₁/dextran_0.8 nanoparticles were measured by TEM in dry state, while the sizes measured by DLS were in aqueous solution.

Fig. 5 TEM micrograph of PMAPBA₁/dextran_0.8 nanoparticles.

The FT-IR spectra, thermal analysis, DLS, UV spectrometer, and TEM clearly indicated the formation of spherical PMAPBA/dextran nanoparticles with a narrow size distribution via a boronic-diol reaction.

3.3 Glucose-sensitivity of nanoparticles

The introduction of phenylboronic acids in PMAPBA made the resulting nanoparticles glucose-sensitive.^34,35 Table 1 shows the D_H and PDI of PMAPBA/dextran nanoparticles in pH 7.4 PBS with different glucose concentrations measured at 25 °C. Even after glucose treatment, there was a narrow size distribution, with the PDI ranging from 0.040 to 0.145 for all samples analyzed. The D_H values of the nanoparticles after treatment with 1 mg mL⁻¹ glucose were slightly greater than that without glucose. After treatment with 3 mg mL⁻¹ glucose, the nanoparticle size expanded dramatically. In the absence of glucose, the D_H of PMAPBA₁/dextran_1.5 nanoparticles was only 174.9 nm. However, the nanoparticles swelled to nearly 45 nm in size when glucose concentration was 3 mg mL⁻¹. The swelling phenomenon indicated that the glucose exposure influenced the morphology of the nanoparticles and PMAPBA/dextran nanoparticles were responsive to glucose. This was due to the boronic ester linkages between the boronic acid-containing polymers with diols being dynamic-covalent, and the dynamic-covalent nature of the boronic ester allowed the linkages to reconfigure their structure in the presence of other diols that competed for bonding with the boronic acids. Thus, the linkages could be induced to dissociate via competitive exchange reactions, which resulted in the swelling phenomenon of nanoparticles. Furthermore, the increase of the D_H value was more in 3 mg mL⁻¹ glucose than that in 1 mg mL⁻¹, which was attributed to more dissociation of nanoparticles in higher glucose concentration.

Table 1 D_H and PDI of the nanoparticles at various glucose concentrations measured by DLS

Sample	Glucose concentration
	0 mg mL^−1	1 mg mL^−1	3 mg mL^−1
	DH (nm)/PDI	DH (nm)/PDI	DH (nm)/PDI
PMAPBA1/dextran0.2	190.6 ± 2.7/0.053	199.2 ± 2.9/0.114	223.1 ± 4.3/0.145
PMAPBA1/dextran0.8	150.3 ± 0.8/0.040	159.1 ± 0.9/0.054	183.2 ± 1.7/0.116
PMAPBA1/dextran1.5	174.9 ± 2.8/0.053	182.3 ± 3.6/0.094	219.1 ± 1.2/0.131

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3.4 Insulin-loading of nanoparticles

Insulin with random sequenced hydrophilic and hydrophobic amino acid in the linear chain can self-assemble with PMAPBA and dextran due to hydrophilic and hydrophobic interaction. As shown in Table 2, the mean size of PMAPBA/dextran nanoparticles became greater after the drug loading. The PMAPBA₁/dextran_0.8 particle size increased from 150.3 to 215.3 nm as the insulin concentration was increased from 0 to 0.5 mg mL⁻¹. This implied that the encapsulated insulin contributed to the enlargement of the particles. Additionally, all of the insulin-loaded nanoparticles also had good dispersity.

Table 2 The physicochemical properties of PMAPBA/dextran nanoparticles

Sample	Insulin concentration (mg mL^−1)	DH (nm)/PDI	Zeta potential (mV)	EE (%)	LC (%)
PMAPBA1/dextran0.2	0	190.6 ± 2.7/0.053	−30.5 ± 0.53	—	—
PMAPBA1/dextran0.2	0.25	212.5 ± 3.9/0.133	−37.7 ± 0.62	62.2 ± 0.53	15.9 ± 0.49
PMAPBA1/dextran0.8	0	150.3 ± 0.8/0.040	−26.3 ± 0.64	—	—
PMAPBA1/dextran0.8	0.10	162.3 ± 1.2/0.120	−30.9 ± 0.32	80.8 ± 0.52	8.9 ± 0.45
PMAPBA1/dextran0.8	0.25	184.2 ± 1.9/0.100	−35.4 ± 0.89	70.1 ± 0.93	17.7 ± 0.93
PMAPBA1/dextran0.8	0.50	215.3 ± 1.0/0.140	−46.4 ± 1.73	55.3 ± 0.75	22.1 ± 0.68
PMAPBA1/dextran1.5	0	174.9 ± 2.8/0.053	−21.5 ± 0.42	—	—
PMAPBA1/dextran1.5	0.25	201.7 ± 4.7/0.153	−29.8 ± 0.64	85.7 ± 0.73	20.1 ± 0.58

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The zeta potential is an important parameter in evaluating the stability of nanoparticles in solution. The zeta potential of the PMAPBA/dextran nanoparticles was about −26 mV (Table 2), which was due to the charged phenylboronic in the PMAPBA. Furthermore, the net negative zeta potential of the nanoparticles decreased with increasing dextran content in PMAPBA/dextran nanoparticles. Additionally, the surface charge of insulin-loaded PMAPBA/dextran nanoparticles was more negative than that of blank nanoparticles. This was attributed to the net negative charge of insulin (pI 5.3) in pH 7.0 aqueous solution.

LC and EE of the PMAPBA/dextran nanoparticles are also shown in Table 2. The LC and EE of the nanoparticles were as high as reach 22% and 85% respectively, and increased with increasing hydrophilic dextran content in PMAPBA/dextran nanoparticles. Furthermore, the LC and EE of the nanoparticles were affected by initial concentration of the insulin used. The LC increased with increasing initial concentration of insulin, while the EE decreased under the same conditions.

3.5 In vitro release of insulin

Fig. 6a shows the cumulative release profiles of insulin in response to different concentrations of glucose at pH 7.4. The results indicated a burst release phase for all glucose concentrations was examined within 2 h. After the first couple of hours, insulin was gradually released from the PMAPBA₁/dextran_1.5 nanoparticles. The burst release without glucose corresponded to the diffusion of insulin located on the nanoparticle surfaces. In the medium containing glucose, the burst of insulin was much higher, with 37% and 56% of the insulin being released following treatment with 1 and 3 mg mL⁻¹ of glucose, respectively, compared to 25% insulin release in nanoparticles incubated in glucose-free medium. The amounts of insulin released after 7 h in 0, 1 and 3 mg mL⁻¹ glucose concentration were 42%, 60%, and 95%, respectively. These results suggested that the nanoparticles were responsive to glucose and released insulin rapidly upon exposure to glucose.

Fig. 6 In vivo release profiles of insulin. (a) In vitro release profiles from PMAPBA₁/dextran_1.5 nanoparticles at various glucose concentrations; (b) in vitro release profiles of insulin from PMAPBA/dextran nanoparticles at a glucose concentration of 3 mg mL⁻¹.

Fig. 6b shows the release profile of insulin from three insulin-loaded nanoparticles in PBS with 3 mg mL⁻¹ of glucose. Insulin was released from the nanoparticles in the following order: PMAPBA₁/dextran_1.5 > PMAPBA₁/dextran_0.8 > PMAPBA₁/dextran_0.2, and the cumulative amounts of insulin released after 7 h for each nanoparticles was 95%, 79% and 63%, respectively. The insulin-loaded nanoparticles became swollen after glucose exposure, which resulted in a rapid release of insulin. Furthermore, the more the hydrophilic dextran content in PMAPBA/dextran nanoparticles, the easier it was for the glucose molecular to access and penetrate the nanoparticles and react with phenylboronic acids in PMAPBA.

3.6 Circular dichroism spectra

Fig. 7 shows the CD spectra of standard insulin and encapsulated insulin. Two bands at 209 and 220 nm were found in the CD spectrum of standard insulin and encapsulated insulin, which suggested the existence of α-helix and β-structure.^36,37 The ratio between both bands ([Φ]₂₀₉/[Φ]₂₂₀) can be used to generate a qualitative measure of the overall conformational structure of insulin. In our study, the [Φ]₂₀₉/[Φ]₂₂₀ ratio for standard insulin was 1.16. The ratio for encapsulated insulin was about 1.10, which was similar with that of the standard insulin. The strong double minima at 220 and 208 nm occurred without significant difference from those of the native insulin. The result indicated the structure of insulin had not been distorted in the loading procedure.

Fig. 7 CD spectra of standard insulin and encapsulated insulin.

3.7 Cell viability

Most of the boronated moieties and their derivatives have cytotoxic activity.³⁸ Therefore, it was important to verify the harmless nature of the nanoparticles. To evaluate the potential toxicity of these nanoparticles, in vitro cytotoxicity assays, using the PMAPBA/dextran nanoparticles and the NIH3T3 cell line, were performed and analyzed by the MTT method (Fig. 8). The cells were exposed to various concentrations of the nanoparticle solutions and incubated for 48 h. It was found that the cell viability increased obviously as the dextran moiety in PMAPBA/dextran nanopaticles increased (PMAPBA₁/dextran_1.5 > PMAPBA₁/dextran_0.8 > PMAPBA₁/dextran₀). The cell viability was lower than 80% for all of the PMAPBA₁/dextran₀ nanoparticle concentration tested. After 48 h treatment at 500 μg mL⁻¹ PMAPBA₁/dextran₀ nanoparticle concentration, the cell viability was measured to be only 50%. However, the cell viability of NIH3T3 cells was maintained over 80% after incubation with PMAPBA₁/dextran_0.8 and PMAPBA₁/dextran_1.5 nanoparticles (500 μg mL⁻¹) for 48 h. The reduction of the cytotoxicity of PMAPBA was mainly attributed to the introduction of the dextran moieties, which is in agreement with the results reported previously by our group.³⁹ A possible mechanism for these is that hydrophilic dextran containing hydroxyl groups covered the phenylboronic acid, thus reducing the interaction between phenylboronic acid and the cells. The results of cytotoxicity tests suggest that the boronic acid-functionalized nanoparticles can be used as a drug delivery vehicle without serious cytotoxicity.

Fig. 8 Cytotoxicity of PMAPBA₁/dextran₀, PMAPBA₁/dextran_0.8 and PMAPBA₁/dextran_1.5 nanoparticles against NIH3T3 cells for 48 h.

4. Conclusion

In this study, we had developed a facile approach for preparing PMAPBA/dextran nanoparticles via the boronic acid–diol reaction. The resulted nanoparticles were spherically shaped particles with a narrow size distribution. The particle size increased in correlation with the glucose concentration. Additionally, the nanoparticles had high loading capacity with the insulin as model drug. Insulin release profiles indicated that the release of insulin was in correlation with the increasing glucose concentration. The cell viability tests revealed that the nanoparticles had good cytocompatibility. These demonstrate that the PMAPBA/dextran nanoparticles therefore have potential applications in drug delivery.

Acknowledgements

This work was supported by the Fundamental Research Funds for the Central Universities (Grant no. 3142014006) and the Youth Foundation of Hebei Educational Committee (Grant no. QN2014301).

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