chitosan nanospheres

Accepted Manuscript Title: Preparation and Sustainable Release of Modified Konjac Glucomannan/Chitosan Nanospheres Author: Congjiao Shi Pei Zhu Na Chen Xiaozhou Ye Yun Wang Shaobo Xiao PII: DOI: Reference:

S0141-8130(16)30485-8 http://dx.doi.org/doi:10.1016/j.ijbiomac.2016.05.073 BIOMAC 6134

To appear in:

International Journal of Biological Macromolecules

Received date: Revised date: Accepted date:

18-3-2016 12-5-2016 18-5-2016

Please cite this article as: Congjiao Shi, Pei Zhu, Na Chen, Xiaozhou Ye, Yun Wang, Shaobo Xiao, Preparation and Sustainable Release of Modified Konjac Glucomannan/Chitosan Nanospheres, International Journal of Biological Macromolecules http://dx.doi.org/10.1016/j.ijbiomac.2016.05.073 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Preparation and Sustainable Release of Modified Konjac Glucomannan/Chitosan Nanospheres

Congjiao Shi†, Pei Zhu†, Na Chen, Xiaozhou Ye*, Yun Wang*, Shaobo Xiao

State Key Laboratory of Agricultural Microbiology, College of Science, Huazhong Agricultural University, Wuhan 430070, PRChina

*Corresponding author. Tel.: +86 27 87284018; fax: +86 27 87282133. E-mail address: [email protected] (Yun Wang), [email protected](XiaozhouYe). † Equal contribution by the first two authors.

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Highlights 1. A novel CKGM/HACC composite nanospheres were prepared. 2. The particle size and zeta potential of the nanospheres are tunable. 3. CKGM/HACC/OVA has good sustained release properties for OVA in vitro. 4. The raw materials are low toxicity, good biocompatibility and biodegradability. 5. The polyelectrolyte complexes method is organic solvent free, and convenient.

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Abstract Biodegradable and biocompatible polymer nanospheres are useful materials for controlled drug delivery. In the present study, novel composite nanospheres were prepared from carboxymethyl konjac glucomannan (CKGM) and 2-hydroxypropyl trimethyl ammonium chloride chitosan (HACC) as a vaccine delivery vehicle by electrostatic complexation in a neutral aqueous solution without the use of chemical crosslinkers. By altering the CKGM and HACC concentrations, the average CKGM/HACC particle size could be tuned from approximately 600 nm to 1460 nm and the zeta potential from 39 mV to 50 mV. Furthermore, using ovalbumin (OVA) as a model molecule for vaccines, various parameters were determined to affect the CKGM/HACC nanosphere encapsulation efficiency and in vitro controlled release properties. Under optimum conditions, the OVA encapsulation efficiency of CKGM/HACC nanospheres was 71.8 %, while sustained and continuous in vitro OVA release over a period of more than 24 hours was observed. Therefore, CKGM/HACC nanospheres are novel drug delivery carriers with great potential for medical applications.

Keywords Konjac glucomannan; Chitosan; Nanospheres; Ovalbumin; Sustained release

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1. Introduction Biodegradable nano-size polymers, especially nanoparticles and nanocapsules of polysaccharides, are a class of novel carriers for sustained drug release [1, 2]. Due to their good biocompatibility, ultra-fine particle size, low toxicity, and efficient utilization of drugs, they are ideal carriers for entrapping biologically active macromolecular drugs, such as polypeptides, proteins, nucleic acids, and vaccine vectors [3-6]. Oppositely charged polysaccharides can be mixed in aqueous solutions and form polyelectrolyte complexes (PECs) without the use of chemical covalent cross-linkers [7, 8]. Owing to their biodegradability, non-toxicity, and sensitivity to stimuli, polyelectrolyte complexes of polysaccharides have attracted wide attentions and have been investigated in regard to drug encapsulation and delivery [9, 10]. Konjac glucomannan (KGM), a major active ingredient in konjac tuber, is a natural polysaccharide macromolecule of D-glucose and D-mannose that is linked through a β-l,4-glycosidic bond [11, 12]. As a natural and renewable polymer resource with excellent biocompatibility, biodegradability and biological activity, KGM has good application prospects in the biomedical field [13, 14]. Carboxymethylation of konjac glucomannan produces a negatively charged polymer (CKGM) with increased water solubility, swelling rate and stability compared to KGM [15, 16]. Due to these improved properties in combination with its excellent biological activity, CKGM nanoparticles have been prepared and used as a drug delivery vehicle. For instance, Li et al. prepared cholesterol-modified CKGM amphiphilic nanomicelles that had a maximum etoposide encapsulation rate of 39.4 % and steady drug release for 23 h [17]. Zhang et al. immobilized asparaginase on nanospheres, and the immobilized enzyme retained its activity, while showing improved thermal stability and tolerance to acidic and alkaline environments [18]. Chitosan (CS) is a positively charged natural polysaccharide that contains a large number of amine groups. Due to its biodegradable and biocompatible properties, chitosan is considered a promising biomaterial for applications in biomedicine, food, health care, cosmetics, and others [10, 19]. In particular, chitosan has been used as a raw material for the preparation of nanosphere drug carriers that showed good drug loading and sustained release properties [20-22]. Positively-charged 2-hydroxypropyl trimethyl ammonium chloride 4

chitosan (HACC), a water-soluble chitosan derivative [23], may be superior to chitosan, as its quaternized cationic nature enables stronger electrostatic interactions with negatively charged tumour cells when used as a drug carrier for tumour therapy [24]. In addition, HACC can potential be applied in many fields, e.g., prevention of fungal skin infections [25, 26], orthopaedics [27], nanofiltration [28, 29] and drug delivery [24, 30], due to its biocompatibility, water-solubility, low cytotoxicity, permanent cationic charges, among others. Negatively charged CKGM and positively charged CS may form PECs with potential drug loading and delivery applications [31]. For instance, Du et al prepared CKGM/CS nanospheres by complex coacervation in an acidic solution that had a high encapsulation efficiency for bovine serum albumin (BSA) [32, 33] and the potential for colloidal drug delivery [34]. However, despite these promising results of CKGM/CS complex nanospheres in the field of drug delivery, there have been few published articles on this topic, while the use of CKGM/HACC composite nanospheres for drug delivery, especially that of vaccines, to our knowledge has not yet been reported. Herein, we report the facile preparation of CKGM/HACC complex nanospheres in a neutral aqueous solution without chemical crosslinkers. The effects of the concentrations of CKGM and HACC on the average particle size and zeta potential of CKGM/HACC composite nanospheres were also determined. To investigate the potential application of CKGM/HACC nanospheres for vaccine loading and delivery, we used ovalbumin (OVA) as a model drug and evaluated its encapsulation and sustained release.

2. Materials and methods 2.1. Materials KGM with a viscosity average molecular weight of approximately 100000 g/mol was purchased from Wuhan Shenshi Chemical Technology Co. (Wuhan, China). CS with a viscosity average molecular weight of approximately 8000 g/mol and a degree of deacetylation of 92 % was purchased from Sinopharm Chemical Reagent Co. Ltd. (Beijing, China). The reagent 2,3-epoxypropyl trimethyl ammonium chloride, with a degree of substitution of 61 %, was prepared in-house according to published methods [35]. OVA was 5

purchased from Aladdin Reagents Co. Ltd. (Shanghai, China). All reagents were of analytical grade.

2.2. Preparation of CKGM/HACC nanospheres 2.2.1. Degradation and carboxymethyl modification of KGM KGM (35 g) was hydrolysed with 250 mL of a HCl-ethanol solution (v/v, 70:180) in a 500-mL three-neck flask under mechanical stirring at room temperature for 2 h. The product was subsequently washed with a 70 wt.% aqueous ethanol solution, vacuum filtered, and vacuum dried at 30 C for 16 h to obtain acid-hydrolysed KGM (AHKGM) for the preparation of carboxymethyl-modified KGM. AHKGM (10 g) was mixed with 20 mL of a 50 wt.% aqueous methanol solution in a three-neck flask and mechanically stirred at room temperature for 30 min until the AHKGM had completely swollen. Subsequently, 50 mL of anhydrous methanol was added to the three-neck flask and the mixture was heated to 50 C. Then, 20 mL of a 30 wt.% aqueous NaOH solution was added to the mixture dropwise, which was followed by a 30 min reaction. After the addition of 7.5 g of monochloroacetic acid, the reaction was allowed to continue for 15 h at 50 C under mechanical stirring. Finally, the product solution was neutralized with HCl; washed several times with 70 wt.%, 80 wt.% and 90 wt.% aqueous methanol solutions to remove impurities, vacuum filtered; and vacuum dried at 50 C to produce CKGM. The reaction scheme was presented in Scheme 1a. The degree of carboxymethyl substitution of KGM was measured according to the literature to be 0.49 [36]. KGM, AHKGM, and CKGM compounds were swollen in 100 mL of water (1 g each), and their apparent viscosities, measured with a NDJ-79 rotary viscometer, were 9, 1.4, and 2.6 mPa·s, respectively.

2.2.2. Modification of cationic CS CS (8.0 g) was mixed with 72 mL of isopropanol in a three-neck flask, heated to 60 C, and mechanically stirred for 1 h. Subsequently, 80 mL of a 37 wt.% aqueous 2,3-epoxypropyl trimethyl ammonium chloride solution was added and the mixture heated to 80 C under mechanical stirring for 14 h. The resulting product was washed with 80 wt.% isopropanol by 6

suction filtration. The filter cake was dissolved in distilled water in a dialysis bag and dialysed for 48 h against distilled water. The dialysis solution was replaced every 4 h. The dialysed solution was then precipitated with a certain amount of acetone, suction filtered and vacuum dried at 50 C to produce HACC. The reaction scheme was presented in Scheme 1b.The degree of 2,3-epoxypropyl trimethyl ammonium chloride substitution of CS was measured according to the literature to be 0.61 [37]. 2.2.3. Preparation of blank nanospheres and drug loaded nanospheres Blank CKGM/HACC composite nanospheres and CKGM/HACC/OVA composite nanospheres were prepared by complex coacervation. Preparation of blank CKGM/HACC nanospheres. A series of CKGM and HACC solutions at different concentrations were prepared and mechanically stirred at room temperature. Five millilitres of a CKGM solution was added dropwise to 10 mL of a HACC solution in a conical flask and mechanically stirred for 15 min. The nanospheres were collected by ultracentrifugation at 12000 r/min for 30 min, washed with deionized water three times, freeze-dried, and stored at -18 C before use. Preparation of drug loaded CKGM/HACC/OVA nanospheres. Ten millilitres of a HACC solution was mixed with 4 mL of OVA under mechanical stirring at room temperature. Five millilitres of a CKGM solution was added to the HACC/OVA mixture dropwise and mechanically stirred for 15 min. The produced CKGM/HACC/OVA nanospheres were collected by ultracentrifugation at 12000 r/min, washed with deionized water three times, freeze-dried, and stored at -18 C before use.

2.3. Characterization of modified KGM and CS and their composite nanospheres 2.3.1. Fourier transform infrared (FT-IR)spectroscopy FT-IR spectra of KGM, CS, and their modified forms were obtained with KBr pellets on an AVATAR330 (ThermoNicolet, USA) in the range of 4000 to 500 cm-1. 2.3.2. Particle size distribution and zeta potential The

particle

size

distribution

and

zeta

potential

of

CKGM/HACC

and

CKGM/HACC/OVA nanospheres were measured in 0.1 % aqueous solutions on a laser particle size analyser (Zetasizer Nano ZS90, Malvern, England). 7

2.3.3. Transmission electron microscopy(TEM) CKGM/HACC and CKGM/HACC/OVA nanosphere aqueous solutions (0.1 %) were deposited onto copper grids, negatively stained with phosphotungstic acid, dried at room temperature, and imaged with a transmission electron microscope (TEENAI10, Philips, Holland). 2.3.4. In vitro release Certain amounts of CKGM/HACC/OVA nanospheres were dispersed in 5 mL of phosphate buffer saline (PBS) solutions (pH 7.4) in test tubes. The tubes were placed in a 37 C thermostable oscillator and rotated at 100 rpm. At specific time intervals, 500 µL of the supernatants were sampled and the tubes were supplemented with 500 μL of PBS to ensure a constant total volume. The sampled supernatants were diluted and analysed with a fluorescent spectrophotometer to determine OVA concentrations.

3. Results and discussion 3.1. FT-IR analysis Figure 1a shows the FT-IR spectra for KGM, AHKGM, and CKGM. For KGM, the absorption peak at 3450 cm-1 indicates the presence of -OH groups, the weak absorption peak at approximately 2920 cm-1 was ascribed to the stretching vibration of methylene groups (-CH2-), the absorption peak at approximately 1730 cm-1 was assigned to the asymmetric stretching vibration of carbonyl groups (C=O) that are characteristic of acetyl groups in the KGM chain, the absorption peak at 1650 cm-1 was attributed to the bending vibration of the -OH of absorbed water, and the absorption peaks at 890 cm-1 and 810 cm-1 were attributed to the β-glycosidic linkages and pyran rings, respectively. AHKGM possessed a similar FT-IR spectrum as KGM, indicating that only degradation of long-chain KGM to form short-chain KGM occurred during the acid modification step. In the FT-IR spectrum of CKGM, compared to that of KGM, the acetyl group absorption peak at 1730 cm-1 disappeared and the absorption peak of the -OH bending vibration shifted from 1646 cm-1 to 1616 cm-1, indicating that the structure of KGM had changed after carboxymethylation [3]. The degree of carboxymethyl substitution of KGM was measured according to the literature to be 0.49 [36]. 8

Figure 1b shows the FT-IR spectra for CS and HACC. In the spectrum for HACC, the absorption peak at 1597 cm-1 associated with the bending vibration of the amino group (-NH2) in CS was weakened , and a new absorption peak at 1482 cm-1 for the deformation vibration of the methyl group (-CH3) in trimethyl ammonium cations appeared [37]. This indicates that hydroxypropyl trimethyl ammonium chloride quaternary ammonium side chains were successfully bound to the amino group (-NH2) of HACC [38]. The degree of 2,3-epoxypropyl trimethyl ammonium chloride substitution of CS was measured according to the literature to be 0.61 [37].

3.2 The formation mechanism of CKGM/HACC nanospheres for OVA encapsulation In general, by reacting two oppositely charged polysaccharides, PECs can be formed due to electrostatic complex coacervation. When CKGM aqueous solutions are dropped into HACC solutions at room temperature, molecular electrostatic attractions occur between the cationic quaternary amine groups of HACC and anionic carboxyl groups of CKGM, as shown in Scheme 2. These intramolecular electrostatic attractions may have made the macromolecular chains of HACC and CKGM aggregate and curl up, which led to the formation of insoluble CKGM/HACC composite nanospheres. After the addition of OVA to the solution during the aggregation process of HACC and CKGM, the amount of OVA in the supernatant after centrifugation was significantly decreased, indicating successful encapsulation of OVA in the CKGM/HACC composite nanospheres. Further encapsulation efficiency results are discussed in 3.5.

3.3. Morphology of the composite nanospheres Figure 2 shows the TEM images of CKGM/HACC (a) and CKGM/HACC/OVA (b) nanospheres prepared with 1.5 mg/mL of HACC and 2.0 mg/mL of CKGM under optimum conditions. The nanospheres, negatively stained with phosphotungstic acid, showed up as a deep colour in the TEM images. The blank nanospheres were evenly distributed, were spherical particles of approximately 200 nm, and did not aggregate. Compared with the blank nanospheres, the drug loaded CKGM/HACC/OVA nanospheres exhibited a wider particle size distribution. 9

3.4. Effects of CKGM and HACC concentrations on the average particle size and zeta potential of blank nanospheres The particle size of CKGM/HACC nanoparticles, which were prepared by dropwise addition of CKGM to a HACC solution at different concentrations, was investigated. As shown in Table 1, when the concentration of HACC was fixed at 1.0 mg/mL, the average particle size of the CKGM/HACC nanospheres increased from 599 nm to 1068 nm as the CKGM concentration increased from 0.5 to 2.5 mg/mL. Similarly, the average particle size increased from 637 nm to 1463 nm as the CKGM concentration was fixed at 1.5 mg/mL and the HACC concentration increased from 0.5 to 2.5 mg/mL. These results indicate that by simply altering the CKGM and HACC concentrations, the particle size of CKGM/HACC nanospheres can be controlled over a large range, which is beneficial for in vivo drug delivery. The zeta potential of CKGM/HACC nanoparticles could also be altered by varying the CKGM and HACC concentrations, as shown in Table 1. As the CKGM concentration increased, the zeta potential of CKGM/HACC nanoparticles decreased from 47 mV to 39 mV; as the HACC concentration increased; however, the opposite effect of an increase in zeta potential occurred. This may be because HACC is positively charged and CKGM is negatively charged. Overall, by altering the concentrations of CKGM and HACC, the zeta potential of the prepared nanospheres could be controlled between 39 mV and 50 mV. Typically, higher absolute values for the zeta potential lead to improved particle stability and a more uniform particle size, due to stronger repellent interactions among the particles.

3.5. Optimization of the preparation conditions of CKGM/HACC/OVA nanospheres To obtain a high encapsulation efficiency, an orthogonal experiment L9 (34) of four factors and three levels was designed to optimize the preparation process (Table 2). The effects of the HACC, CKGM and OVA concentrations and the pH of the OVA solution on the preparation and properties of the drug loaded nanospheres were investigated. Table 3 shows the orthogonal experimental results. The OVA encapsulation efficiency was used to evaluate the preparation process of CKGM/HACC/OVA composite nanospheres. As shown, the effects of the four factors follow the order of B > C > D > A. As a result, Factor B, 10

the concentration of CKGM, had the most significant effect on the OVA encapsulation efficiency, while Factor A, the concentration of HACC, had the smallest effect. Under the optimized preparation conditions of A1B3C3D3, which were 1.5 mg/mL HACC, 2.0 mg/mL CKGM, 2.0 mg/mL OVA and an OVA solution of pH 9.0, the OVA encapsulation efficiency reached 71.8 %.

3.6. In vitro drug release property The effects of the CKGM and HACC concentrations for the preparation of CKGM/HACC/OVA nanospheres on its OVA release performance were investigated. 3.6.1. Effects of the CKGM dosage on the drug release performance of composite nanospheres Figure 3a shows the in vitro OVA release curves of the CKGM/HACC/OVA nanoparticles prepared with different CKGM concentrations. Three different types of CKGM/HACC/OVA composite nanoparticles were prepared by adding 1.0, 1.5, and 2.0 mg/mL of a CKGM solution to 1.5 mg/mL of a HACC solution. The resulting nanospheres showed OVA encapsulation efficiencies of 38.2, 56.7, and 65.7 %, respectively. The OVA release profile for the composite nanospheres exhibits three stages. In the first 30 min, with CKGM concentrations of 1.0, 1.5, and 2.0 mg/mL, approximately 47, 46, and 42 % of OVA was released. The reason for this burst release stage might be the rapid diffusion of physically adsorbed or weakly bound OVA on the surface of nanoparticles to the release medium. This burst release was followed by a second, slow release phase. During this stage, the drug needs to overcome resistance to its release from the interior of the nanospheres. Therefore, the release occurs slower and over a prolonged time, and the release rate is relatively stable. After 48 h, OVA release reaches a third, equilibrium stage, and the OVA concentration stabilizes. After 72 h, approximately 99, 97, and 90 % of OVA was released at CKGM concentration of 1.0, 1.5, and 2.0 mg/mL, respectively. As shown from the OVA release curves, the OVA release rate decreased with an increase in CKGM concentration. This can be explained by the increase in anions with an increase in CKGM concentration, leading to an increasingly linked internal structure and enhanced encapsulation of OVA and thus a reduced OVA release rate. Thus, CKGM/HACC composite 11

nanospheres with higher CKGM concentration would retard the OVA diffusion from the composites.

3.6.2. Effects of the HACC dosage on the drug release performance To investigate the effects of the HACC dosage on the drug release performance, CKGM/HACC/OVA nanospheres were prepared with 1.5, 2.0, and 2.5 mg/mL HACC at a fixed CKGM concentration under optimum conditions. The encapsulation efficiencies of the nanospheres were 65.7, 61.2, and 61.9 %, respectively. As shown in Figure 3b, the in vitro release curves of CKGM/HACC/OVA nanospheres are similar to those of the CKGM/HACC/OVA nanospheres discussed in 3.6.1. Similarly, the OVA release rate decreased with an increase in the HACC concentration, leading to a prolonged release process. In addition, an increase in the HACC concentration could effectively reduce the burst release of OVA. This can be explained by the increase in positive charges with an increase in positively-charged HACC, leading to a more compact internal structure. The positively charged nanospheres’ attraction to and encapsulation of OVA became stronger, and OVA therefore had to overcome stronger electrostatic interactions and internal resistance to diffuse into the PBS buffer. Therefore, higher HACC concentrations for the preparation of the composite nanospheres can reduce OVA burst release and extend OVA release.

4. Conclusion A novel CKGM/HACC composite nanosphere was prepared by the complex coacervation of chemically modified natural polymers of KGM and CS under mild neutral conditions. The size of the as-prepared CKGM/HACC nanospheres could be controlled from 600 to 1068 nm by simply adjusting the CKGM and HACC concentrations during fabrication. The zeta potential of the nanospheres was positive and also tunable. We also investigated the OVA encapsulation efficiency of the nanospheres by orthogonal experiments, and the CKGM concentration was shown to have the most significant effect. Finally, the as-prepared CKGM/HACC nanospheres showed excellent in vitro OVA release kinetics, and the results suggested that the higher CKGM and HACC concentrations during the preparation process can both retard drug release. In addition, the raw materials for the preparation of composite 12

nanospheres are the abundant natural polymers with high biodegradability and biocompatibility. Therefore, CKGM/HACC nanospheres have the potential to be used carriers biologically active macromolecular drugs, such as protein, vaccines, among others.

Acknowledgments The authors gratefully acknowledge the financial supports from the Fundamental Research Founds for the Central Universities (2013SC25, 2014PY051) and the open funds of the State Key Laboratory of Agricultural Microbiology (AMLKF201507).

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Figure 1

17

Figure 2

18

Figure 3

19

Scheme 1

20

Scheme 2

21

Table 1 Effects of the concentration of CKGM and HACC on the average particle size and zeta potential of composite nanospheres c(HACC)(mg/mL)

c(CKGM)(mg/mL)

Average size (nm)

1.0

0.5

598.7

47.15±1.90

675.2

44.00±2.60

1.0

1.0

Zeta potential (mV)

1.0

1.5

768.5

42.10±0.42

1.0

2.0

896.4

41.32±0.14

1.0

2.5

1068.1

38.90±2.47

0.5

1.5

637.0

40.00±1.70

1.0

1.5

768.5

42.10±0.42

1.5

1.5

884.2

44.10±0.99

2.0

1.5

944.8

46.65±0.64

2.5

1.5

1463.4

50.00±0.88

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Table 2 Design table of orthogonal experiment for OVA encapsulation Number

A

B

C

D

Factor

HACC

CKGM

OVA

The

(mg/mL)

(mg/mL)

(mg/mL)

OVA

Level 1

1.5

1.0

1.0

6.0

Level 2

2.0

1.5

1.5

7.4

Level 3

2.5

2.0

2.0

9.0

pH

of

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Table 3 Orthogonal experiment results for OVA encapsulation A

B

C

D

Number

Encapsulation HACC

CKGM

OVA

The pH of

(mg/mL)

(mg/mL)

(mg/mL)

OVA

Experiment 1

1

1

1

1

40.0

Experiment 2

1

2

2

2

65.6

Experiment 3

1

3

3

3

71.8

Experiment 4

2

1

2

3

51.6

Experiment 5

2

2

3

1

63.0

Experiment 6

2

3

1

2

56.9

Experiment 7

3

1

3

2

55.8

Experiment 8

3

2

1

3

61.3

Experiment 9

3

3

2

1

59.4

K1a

59.1

49.1

52.7

54.1

K2b

57.1

63.3

58.9

59.4

K3c

58.8

62.7

63.5

61.6

Rd

2.0

14.2

10.8

7.5

Factor

efficiency(%)

a The average value of encapsulation efficiency at level 1 b The average value of encapsulation efficiency at level 2 c The average value of encapsulation efficiency at level 3 d The pole difference of average value of encapsulation efficiency

24