Preparation and application of pH-responsive composite hydrogel beads as potential delivery carrier candidates for controlled release of berberine hydrochloride

For improving the effective concentration of berberine hydrochloride (BH) in the gastrointestinal tract, a series of pH-responsive hydrogel beads were prepared based on carboxymethylstarch-g-poly (acrylic acid)/palygorskite/starch/sodium alginate (CMS-g-PAA/PGS/ST/SA) in the present work. The developed hydrogel beads were characterized by Fourier transform infrared spectroscopy (FTIR), X-ray diffraction (XRD) and thermogravimetric analysis (TG). Effect of palygorskite (PGS) content on the swelling properties of hydrogel beads and BH cumulative release were discussed. The pH responsiveness of hydrogel beads was also investigated in different media. Results illustrated that swelling of hydrogel beads and BH cumulative release from hydrogel beads were obviously affected by PGS content. The swelling ratio and BH cumulative release of composite hydrogel beads remarkably slowed down with PGS content increasing in the range from 10 to 40 wt%. The composite hydrogel beads were pH-responsive. At pH 7.4, the swelling ratio and BH cumulative release from composite hydrogel beads were the fastest among the dissolution media of pH 1.2, pH 6.8 and pH 7.4. The BH cumulative release from hydrogel beads was related to the swelling and relaxation of composite hydrogel beads and could be fitted better by the Higuchi model. The obtained composite hydrogel beads could be potentially used for the development of BH pharmaceutical dosage forms.


JG, 0000-0002-0913-1643
For improving the effective concentration of berberine hydrochloride (BH) in the gastrointestinal tract, a series of pH-responsive hydrogel beads were prepared based on carboxymethylstarch-g-poly (acrylic acid)/palygorskite/starch/ sodium alginate (CMS-g-PAA/PGS/ST/SA) in the present work. The developed hydrogel beads were characterized by Fourier transform infrared spectroscopy (FTIR), X-ray diffraction (XRD) and thermogravimetric analysis (TG). Effect of palygorskite (PGS) content on the swelling properties of hydrogel beads and BH cumulative release were discussed. The pH responsiveness of hydrogel beads was also investigated in different media. Results illustrated that swelling of hydrogel beads and BH cumulative release from hydrogel beads were obviously affected by PGS content. The swelling ratio and BH cumulative release of composite hydrogel beads remarkably slowed down with PGS content increasing in the range from 10 to 40 wt%. The composite hydrogel beads were pH-responsive. At pH 7.4, the swelling ratio and BH cumulative release from composite hydrogel beads were the fastest among the dissolution media of pH 1.2, pH 6.8 and pH 7.4. The BH cumulative release from hydrogel beads was related to the swelling and relaxation of composite hydrogel beads and could be fitted better by the Higuchi model. The obtained composite hydrogel beads could be potentially used for the development of BH pharmaceutical dosage forms.

Introduction
Berberine hydrochloride (BH), an isoquinoline alkaloid, can be extracted from a variety of Chinese medicines such as Hydrastis canadensis, Berberis aristata, Phellodendron amurense and Tinospora cordifolia [1]. Traditionally, BH is used as a non-prescription drug to treat gastroenteritis, dysentery and abdominal pain for many years in China [2,3]. In recent years, a multitude of biological effects of BH, including anti-inflammatory [4], anti-tumour [5] and anti-hyperglycaemia [6], have also been demonstrated by numerous researches. Moreover, BH could be proven to exert the effects on mycotic infection, heart and cardiovascular diseases, and diabetic renopathy [7]. These novel bioactivities have evoked a strong desire on the potential use of BH. However, the further clinical application of BH is seriously limited due to its poor water-solubility and high polarity as an alkaloid [8,9], which resulted in low gastrointestinal absorption and bioavailability after oral administration [10]. To effectively increase the concentration of BH in the gastrointestinal tract and absorption, a new strategy for improving BH-release involves the utilization of drug carriers.
Drug carriers are an essential part for preparing new medicines in the field of pharmaceutics, which are receiving increasing attention owing to the advantage of responding to the pH value of the external environment changes, improving patient compliance, increasing drug-residence time and ensuring drug released at the desired site [11][12][13][14]. For example, Youssef et al. [15] prepared nanostructured lipid carriers loaded in an in situ gel system, which was able to prolong the residence time on the ocular surface after topical administration. Massoumi's group obtained novel pH-responsive PEGylated hollow nanocapsules, which had excellent potential for cancer chemotherapy [16]. The drug carriers are mainly from natural or synthetic polymeric hydrogels. During the past few decades, lots of natural or synthetic hydrogels such as xylan [17], chitosan [18], alginate [19], κ-carrageenan [20] and poly(N-isopropylacrylamide) [21], have great potential as drug carriers. However, using single polymer hydrogel as drug carrier could endow hydrogel with worse drug loading ability, biodegradability, mechanical property and water-solubility, causing the instability of drug release [22,23]. Currently, polymer/clay composite hydrogels as drug carrier exhibited encouraging results due to the synergistic effects of hydrogels and clay in biomedical and pharmaceutical applications [24,25]. For the polymer/clay composite hydrogels, literature precedents suggest that the introduction of clays could not only regulate the swelling ratio, increase the mechanical properties of the polymers but also give composite hydrogels better drug loading capacity and enhance long-term stable release [26][27][28][29]. Palygorskite (PGS), as a silicate clay with layered chain structure, has been widely used in the biological medicine industry because of its unique physical and chemical properties and low toxicity [30,31]. For example, Yahia et al. [32] report the comprehensive performance of palygorskite/chitosan beads could be greatly enhanced due to the synergistic effects of the palygorskite and chitosan.
Carboxymethyl starch (CMS) attracts increasing attention as an excipient in drug delivery systems due to its low price, good compaction for tablet preparation and pH-responsive properties, but pure CMS is apt to solubility causing a high burst effect of drug [33][34][35]. Based on the above description, the main objective of this paper is to fabricate a hydrogel bead with pH-responsive, good mechanical properties and high drug loading ability to be used as BH carrier. For this purpose, the composite materials carboxymethylstarch-g-poly (acrylic acid)/palygorskite (CMS-g-PAA/PGS) were prepared firstly. Afterwards, the resultant composite CMS-g-PAA/PGS was combined with soluble starch (ST) and sodium alginate (SA) by cross-linking SA with Ca 2+ to prepare the CMS-g-PAA/PGS/ST/SA composites hydrogels beads. The schematic of CMS-g-PAA/PGS/ST/SA composite hydrogel beads is given in scheme 1. The effects of PGS content and pH on swelling ratio, drug loading ability and drug release of the composite hydrogel beads were investigated. Meanwhile, the BH-release behaviour from the hydrogel beads was also explored for improving the effective concentration of BH in the gastrointestinal tract.

Preparation of CMS-g-PAA/PGS composites
CMS-g-PAA/PGS composites with different PGS contents were prepared as our previously reported method [36,37]. CMS (2.0 g) and distilled water (25 ml) were put in a 250 ml three-necked flask equipped with a condenser, a thermometer, a nitrogen line and a mechanical stirrer. The mixture was heated to 70°C stirring for 30 min. The initiator KPS (0.01 g) was then added to the mixture solution. Under nitrogen atmosphere stirring for 15 min later, AA with a 70% neutralization degree, cross-linker MBA and PGS were added. The reaction proceeded at 70°C and was kept for 1.5 h. Subsequently, the obtained composites were washed with ethanol to remove residual reactants and homopolymer. Finally, the CMS-g-PAA/PGS composites were dried in an oven at 60°C to constant weights. The dried composites were milled and screened, and the sizes of the obtained particles were 100-120 mesh. The preparation procedure of CMS-g-PAA was similar to that of the CMS-g-PAA/PGS, except without PGS being added.

Preparation of CMS-g-PAA/PGS/ST/SA composite hydrogel beads
The CMS-g-PAA/PGS/ST/SA hydrogel beads were prepared as follows. Firstly, BH (0.10 g) and distilled water (100 ml) were put in a 500 ml beaker equipped with a mechanical stirrer and dissolved. CMS-g-PAA/PGS (1.0 g) microparticles were then added into the mixture solution and stirred for 3 h. After that, SA (2.0 g) and ST (5.0 g) were added and stirred for 4 h at 1000 r.p.m. Subsequently, using a 1 ml syringe the slurry was dropped into a 5 wt% CaCl 2 solution to form CMS-g-PAA/PGS/ST/SA composite hydrogel beads (SA could be cross-linked with Ca 2+ in the CaCl 2 solution immediately). The obtained hydrogel bead products were screened and washed with distilled water several times to remove unreacted the CaCl 2 on surface, and then dried at 60°C in an oven to obtain the final product.

Evaluation of properties 2.4.1. Evaluation of BH loading
The BH-loaded CMS-g-PAA/PGS/ST/SA hydrogel beads were soaked in pH 6.8 phosphate buffer solution (PBS, 10 ml) for 12 h. Afterwards, the swollen beads were crushed and transferred into a beaker. Subsequently, the crushed hydrogel beads were soaked in fresh PBS (20 ml) again and sonicated for 30 min to extract BH from hydrogel beads. The BH solution was centrifuged at 5000 r.p.m. for 20 min to remove the polymeric debris. UV spectrophotometer was used to analyse the BH content. The drug loading (%) was calculated using the following equation: where W 0 represents the weight of BH in hydrogel beads, W 1 indicates the weights of BH-loaded composite hydrogel beads.

Evaluation of swelling properties
BH-loaded CMS-g-PAA/PGS/ST/SA hydrogel beads swelling properties were studied in pH 6.8 PBS. The 0.20 g of hydrogel beads were put into the baskets of intelligent disintegration instrument (ZRS-1C, Tianjing University Precision Instrument Factory, China) at 37 ± 0.5°C. At the set time intervals, the hydrogel beads were taken out from the swelling medium of pH 6.8 and weighed after removing residual liquid on the surface of hydrogel beads. The swelling ratio is calculated using the equation given below.
where M 0 and M t are the weight of hydrogel beads before and after soaking in the swelling medium of pH 6.8 at time t, respectively. The swelling ratio under various pH conditions was tested by the same procedure. The various buffer solutions were made by combining NaH 2 PO 4 , Na 2 HPO 4 , HCl and NaOH solutions. And pH meter (PHS-3E) was used to determine pH values. All swelling experiments were carried out thrice carefully under the same conditions and the average values were reported.

Evaluation of in vitro release of BH
In vitro release study of BH from BH-loaded CMS-g-PAA/PGS/ST/SA hydrogel beads was carried out as follows: 0.20 g of dried BH-loaded CMS-g-PAA/PGS/SA hydrogel beads were placed in 100 ml of the release medium, and incubated at 37 ± 0.5°C under 100 r.p.m. The release medium (pH 1.2, pH 6.8 or pH 7.4) was made by combining HCl, KH 2 PO 4 and NaOH solutions properly referring to the Chinese Pharmacopoeia 2015. At predetermined time intervals, 5 ml of the release medium was withdrawn and replaced by an equal amount of fresh release medium to keep a constant volume. The BH concentration in the release medium was assayed by UV spectrophotometer. For release medium pH 1.2, the BH concentration was assayed at 343 nm, and for release medium pH 6.8 and pH 7.4, the BH concentration was assayed at 224 nm. The BH cumulative release per cent was obtained using equation (2.3). All cumulative release results were done in triplicate.
where M and M t represent the BH initial amount and cumulative release amount of at time t, respectively. All release experiments were carried out thrice carefully under the same conditions and the average values were reported.

Analysis of in vitro release kinetics
The data of BH release kinetics from hydrogel beads was fitted using zero-order model, Higuchi model and Korsmeyer-Peppas model. Zero-order model: Higuchi model: and Korsmeyer-Peppas model: where M t /M ∞ represents the fraction of drug released in time t, n is the release exponent characterizing release mechanism and K is a constant. If n ≤ 0. 43

Characterization
Samples of FTIR spectra were taken in KBr pellets using a FTIR-FTS3000 spectrophotometer; an Ultra Plus scanning electron microscope (SEM) instrument (Carl Zeiss AG) was used to analyse micrographs; X-ray diffraction (XRD) was carried out on a Rigaku D/Max-2400 diffractometer in the 2θ range of 3-80°at a scan speed of 5°min −1 ; an America TA Company Instruments (TGA-Q100) was used for the thermogravimetric (TG) analysis of samples.

FTIR spectral analysis
The royalsocietypublishing.org/journal/rsos R. Soc. Open Sci. 7: 200676 smooth surfaces. After they were dried, the beads showed a rough surface and the size was approximate 2.5 mm (figure 2b). Additionally, CMS-g-PAA composite displayed a flat and tight surface (figure 2c), while CMS-g-PAA/10% PGS exhibited a relatively loose, undulant and coarse surface (figure 2d ). Moreover, some pores could also be seen in the surface of CMS-g-PAA/10% PGS. These results indicated that the introduction of PGS could affect the specific surface area of composites, and might eventually cause the changes of swelling behaviour and drug release property [41].

X-ray diffraction analysis
The XRD patterns of BH, CMS-g-PAA/10% PGS/ST/SA and BH-loaded CMS-g-PAA/10% PGS/ST/SA are shown in figure 3. As could be observed, some BH crystalline structure was absent in the XRD pattern of the BH-loaded CMS-g-PAA/10% PGS/ST/SA, which indicated BH crystalline structure converted into amorphous. Moreover, the XRD profile of CMS-g-PAA/10% PGS/ST/SA showed typical characteristic peaks at 2θ = 31.52°and 45.3°. While in the XRD profile of BH-loaded CMS-g-PAA/10% PGS/ST/SA, the two characteristic peaks were weakened. It could be concluded that BH crystal

Thermal stability analysis
The thermal degradation behaviour of BH, CMS-g-PAA/10% PGS/ST/SA and BH-loaded CMS-g-PAA/ 10% PGS/ST/SA was studied under a nitrogen atmosphere between 34 and 790°C. The results are shown in figure 4. It was evident all curves showed a decreasing trend with increasing temperature. The TG curve of BH underwent four decomposition phases. Initial weight loss of BH started at 109°C with 8.3% of weight loss, which corresponded to the loss of moisture [42]. The second step between 109 and 188°C, with 1.3% of weight loss, signified the melting temperature of the BH. While the third step showed 20.6% of weight loss at 250°C, revealing decomposition of the BH [43]. The fourth step in the 250-790°C range was ascribed to the destruction of the BH skeleton structure. However, for BHloaded CMS-g-PAA/10% PGS/ST/SA hydrogel beads, the maximum weight loss occurred at about 265°C with 18.3% of weight loss (with 19.4% of weight loss for CMS-g-PAA/10% PGS/ST/SA at the same temperature). This meant that the thermal stability of BH in hydrogel beads suffered from minimal weight loss. The CMS-g-PAA/10% PGS/ST/SA hydrogel might improve the thermal stability through hydrogen bonding and electrostatic attraction interactions [44].

Effect of the PGS content on BH loading
As shown in figure 5, BH loading increased in the BH-loaded CMS-g-PAA/10% PGS/ST/SA hydrogel beads with the increase of PGS content in CMS-g-PAA/PGS composite materials from 0 to 10 wt%. The BH loading amount in the hydrogel beads decreased with further increase of PGS content in CMS-g-PAA/PGS composite materials from 10 to 40 wt%. The variation of the curve could be ascribed to the following reasons. The introduction of moderate PGS in CMS-g-PAA/PGS could relieve the entanglement of the polymeric chains, which could be beneficial to decrease the physical cross-linking degree and facilitate the entrapment of drug in CMS-g-PAA/PGS. Consequently, the higher drug loading was observed in the hydrogel beads [45]. However, the additional PGS could also react with AA and CMS [46], which led to more cross-linking points in CMS-g-PAA/PGS and restricted the entrapment of BH in CMS-g-PAA/PGS, and then caused the decrease of the BH loading.
3.6. Swelling properties 3.6.1. Effect of the PGS content on swelling Clay content has a very important effect on the swelling of polymer/clay composites because clay could affect the network structure of polymer/clay composites [47]. Thus, the effect of PGS content on swelling performance of the CMS-g-PAA/PGS/ST/SA hydrogel beads in pH 6.8 PBS was studied. As shown in figure 6, when 10 wt% of PGS was introduced in the CMS-g-PAA/PGS composite material, the swelling ratio of the CMS-g-PAA/10 wt% PGS/ST/SA hydrogel beads reached maximum. Compared with CMSg-PAA/ST/SA hydrogel beads, the equilibrium swelling rate of CMS-g-PAA/10 wt% PGS/ST/SA hydrogel beads significantly increased. However, when PGS content ranged from 10 to 40 wt% in the CMS-g-PAA/PGS composites, the swelling ratio of CMS-g-PAA/PGS/ST/SA hydrogel beads decreased with increasing PGS content. According to previous reports [48,49], the reason might be ascribed to the fact that moderate PGS was able to impair the hydrogen-bonding interaction and the entanglement of the polymeric chains, which improved the swelling ratio of hydrogel beads. Nevertheless, excess PGS took up a position of cross-linking points in hydrogel beads by large amounts of -OH groups on the surface of PGS. Meanwhile, the excessive PGS may enhance the cross-linking density of composite and plugging network voids and minimize the swelling ratio.

Effect of pH on the release of BH
The effect of pH on BH release from BH-loaded CMS-g-PAA/10% PGS/ST/SA hydrogel beads is shown in figure 9. It could be observed that for the composite hydrogel beads in the medium of pH 1.2, the BH cumulative release ratio from the BH-loaded CMS-g-PAA/10 wt% PGS/ST/SA composite hydrogel beads was 3.68% for 1 h, and 6.77% for 8 h, while in pH 6.8 PBS, the BH cumulative release ratio was

Model fitting of BH release mechanism
The results of model fitting are shown in table 1. It could be found the BH release behaviour from the composite hydrogel beads fitted the Higuchi equation (R 2 = 0.9216-0.9433) better than the zero-order equation (R 2 = 0.8467-0.9332) in pH 6.8 PBS according to the respective correlation coefficients (R). For the relationship between BH cumulative release from hydrogel beads and the relaxation as well as erosion of hydrogel beads, the Korsmeyer-Peppas model showed all the n values were between 0.9451 and 1.397, which means that BH release mechanism was mainly related to the swelling and relaxation of composite hydrogel beads.

Conclusion
In this paper, a series of new pH-responsive composite hydrogel beads were successfully developed in order to improve the effective concentration of BH in the gastrointestinal tract. The incorporation of PGS could influence the amount of BH loading in hydrogel beads. The swelling ratio of hydrogel beads and BH cumulative release decreased with the increase of PGS content in hydrogel beads. The swelling ratio of hydrogel beads and BH cumulative release from hydrogel beads at pH 7.4 were faster than that at pH 6.8. BH cumulative release from hydrogel beads was related to the swelling ratio and relaxation of composite hydrogel beads and could be described better by the Higuchi model. These obtained results showed that the incorporation of PGS in composite hydrogel beads could regulate drug load and release, which would facilitate the preparation of novel drug carriers. The composite hydrogel beads is useful for improving the concentration of BH in the gastrointestinal tract. The method of preparation for hydrogel beads was also proved to be simple.
Data accessibility. The datasets supporting this article have been uploaded as part of the electronic supplementary material.