Preparation and characterization of crosslinked

REACTIVE & FUNCTIONAL POLYMERS

Reactive & Functional Polymers 66 (2006) 1711–1717

www.elsevier.com/locate/react

Preparation and characterization of crosslinked polysucrose microspheres Xin Hou a

a,*

, Jing Yang a, Jincheng Tang a, Xiaomin Chen b, Xiukui Wang a, Kangde Yao a

School of Materials Science and Engineering, Tianjin University, Tianjin 300072, PR China b Institute of Chemistry, Chinese Academy of Science, Beijing 100080, PR China Received 8 March 2006; received in revised form 8 June 2006; accepted 24 June 2006 Available online 22 August 2006

Abstract A series of novel crosslinked polysucrose (PS) microspheres were prepared by a two-stage polymerization. A soluble (linear and/or comb) polysucrose was firstly synthesized by solution polymerization of sucrose and epichlorohydrin (EP); then the crosslinked polysucrose microspheres were prepared by reversed suspension polymerization of soluble PS with EP in chlorobenzene containing Span 80. The characterization by OM and SEM indicates that these spherical beads in diameters with the range from 250 to 450 lm have smooth surface. The hydrated and dry densities, equilibrium water contents, and hydroxyl loadings of PS microspheres were investigated. The equilibrium water content values of these microspheres changed from 79.6% to 94.1%, and the hydroxyl contents were 15.5–19.0 m mol/g dependent on the crosslinking degrees. 2006 Elsevier B.V. All rights reserved. Keywords: Polysucrose; Microspheres; Reversed suspension polymerization; Epichlorohydrin

1. Introduction Sucrose, a disaccharide, ubiquitously presents in nature with over 124 million tons annually in the world production [1]. Natural sucrose has quite high purity and does not require time-consumingly and costly purification. Sucrose-based polymers have been extensively studied since 1950s [2].The sucrose polymers, such as Splenda, Olean, Ficoll 400, have been widely applied [3]. Their performance such as

*

Corresponding author. E-mail address: [email protected] (X. Hou).

biodegradability, low toxicity and good biocompatibility makes the sucrose polymers suitable for application in biomedical, pharmaceutical and related fields [4–6]. Sucrose is liable to be crosslinked because it has eight chemically active hydroxyl groups (three primary and five secondary). Furthermore, crosslinking reaction could take place in water due to its excellent solubility. Crosslinked polysucrose (PS) networks (sucrose hydrogels or sucrogels) are generally obtained by incorporating vinyl groups to sucrose and then polymerization. This has been accomplished in enzymatic way [7,8], in chemical way [9], and in chemoenzymatic way [10]. Superporous sucrose

1381-5148/$ - see front matter 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.reactfunctpolym.2006.06.009

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X. Hou et al. / Reactive & Functional Polymers 66 (2006) 1711–1717

hydrogels have also been prepared in similar way [11]. Generally these sucrose polymers are gels without fixed shape, thus hinder their application. Hydrogel beads based on agarose and dextran have been developed and extensively used as supporting materials for protein purification by affinity, ion exchange, and hydrophobic interaction chromatography [12]. In this presentation our attempt is to synthesize a novel kind of crosslinked polysucrose (PS) microspheres using sucrose as a raw material and epichlorohydrin (EP) as a crosslinking agent. The crosslinked PS microspheres were prepared by a two-stage polymerization way. At the first step a solution polymerization of sucrose with EP was adopted for preparation of soluble PS, then reversed suspension polymerization of aqueous soluble PS with EP in chlorobenzene was carried out to offer crosslinked PS microspheres. 2. Experimental 2.1. Materials Sucrose (edible) was purchased from Tianjin Development Area Lande Food Co., Ltd. (Tianjin, China). Epichlorohydrin (analar) was purchased from Tianjin No. 1 Chemical Reagent Factory (Tianjin, China). Chlorobenzene (analar) was purchased from Beijing Chemical Agents Company (Tianjin, China). Span80 (analar) was purchased from Tianjin No. 3 Chemical Reagent Factory (Tianjin, China). Sodium hydroxide, acetic anhydride (analar) and Pyridine (analar) were purchased from Tianjin Kewei Company. All other reagents were of analytical grade.

after pretreated with acidic ion exchange resins (001 · 7, 122), and basic anion exchange resins (201 · 7, 330) to remove NaCl completely. 2.3. Synthesis of crosslinked PS Microspheres In a typical procedure, to a mixture of 5 g of soluble PS, 20 g DDW, 2 mL of 50% aqueous NaOH (w/w) and 1.69 mL of EP in a 250 mL flask at room temperature was added 160 mL of chlorobenzene containing 3 g of Span 80 with mechanically stirring to make a W/O suspension system. Crosslinked PS microspheres were formed during this reversed suspension polymerization was carried out at 70 C for 2 h in a thermostated oil bath. Then the temperature was raised to 90 C. The polymerization was allowed to proceed for another 4 h. At the end of the reaction, the PS microspheres were filtered and washed with ethanol to extract oil from the beads, then washed by large amounts of DDW. Procedures for preparation of other crosslinked PS microspheres with different crosslinking degrees were very similar to the typical one, and involved altering the different ratios in the weights of soluble PS and EP. The five crosslinked microspheres were prepared and designated as PS-1, PS-2, PS-3, PS-4 and PS-5, respectively. 2.4. Densities of PS microspheres Hydrated and dry densities of PS microspheres were measured with a 6 mL pycnometer using DDW and heptane as steep solution, respectively. 2.5. Equilibrium water content of PS microspheres

2.2. Synthesis of soluble PS To a solution of sucrose (80 g), distilled, deionized water (DDW) (25 g) and epichlorohydrin (35 g) was added 50% aqueous NaOH (w/w) (30 g). The mixture was stirred at room temperature for 2 h and the system was maintained at pH = 13 as above-mentioned NaOH solution. Then, the polymerization was allowed to proceed for another 2 h at 60 C. A hydrochloric acid (HCl) standard solution was added dropwise to the mixture until pH = 7. The resulting products were fractioned by stepwise precipitation method [13]. The Mn of prepolymers was determined by gel permeation chromatography (GPC). The prepolymers with Mn of 3–4 · 105 were selected for further polymerization

Equilibrium water content (EWC) of crosslinked PS microspheres was determined by immersing the microspheres in DDW at 25 C for 24 h to complete equilibration. The excess surface-adhered liquid was removed by blotting and the swollen microspheres were weighed using an electronic microbalance. Then these PS microspheres were dried in a vacuum oven at 60 C for 10 h until constant weight. The EWC was calculated according to the following formula [14] EWCð%Þ ¼

WsWd 100% Wd

ð1Þ

where Ws and Wd denote the weight of swollen and dried microspheres, respectively.


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2.6. Hydroxyl content of PS microspheres The hydroxyl content of crosslinked PS microspheres was determined by non-aqueous titration [15].To the acetic anhydride solution (20 mL, 25%, v/v) in pyridine was added PS microspheres (0.30 g), and the acetylation was carried out at 100 C for 1 h, then 5 mL of DDW was added to the mixture and stood for 30 min. The methanol solution of NaOH (1.0 mol/L) was used to titrate the excess of acetic acid after acetylation using a phenolphthalein solution as the indicator. Blank titration was performed in the same way to avoid systematic errors. 2.7. Characterization of PS microspheres

2933

1643

C

B

1643

2890 2893

2886

2926 2939

3427 3418

A two-stage route for the preparation of crosslinked PS microspheres is shown in Fig. 1. In the first step, sucrose reacted with EP in aqueous solution under alkaline medium to form sucrose oligomers due to the multiple active hydroxyl groups of sucrose. Substitution might happen between these resultant molecular chains and produce the chain propagation yielding the branching oligomers. So the oligomers (soluble polysucrose) might contain linear and comb polymers. In the second stage, reversed suspension polymerization of soluble PS with EP under the alkaline condition in chlorobenzene containing Span 80 was carried out for preparation of crosslinked microspheres. Water phase (aqueous solution of soluble sucrose oligomers, EP and NaOH) was dispersed

A

3386

3.1. Preparation of crosslinked PS microspheres

into droplets enwrapped by oil (chlorobenzene) and Span 80 as dispersion agent. After temperature was raised above 70 C, crosslinking reaction between soluble PS and EP took place in these suspension droplets to offer the crosslinked PS microspheres. The FTIR spectra of sucrose, soluble PS oligomers and crosslinked PS microspheres were showed in Fig. 2, respectively. It was observed that the sharp peak at 3560 cm1, which is assigned to primary hydroxyl groups, exsists only in spectrum of sucrose, and disappeared in the spectra of soluble PS and crosslinked PS microspheres. It indicates that all of the primary hydroxyl groups were consumed in the polymerization. It revealed that the

3560

3. Results and discussion

Fig. 1. Synthesis route for the preparation of crosslinked PS microspheres.

Transmittance

Fourier transform infrared (FTIR) spectra were recorded on Bio-Rad FTS135 spectrometer (BIORAD, USA). The dry samples were powered and mixed with KBr and pressed into pellets. The morphologies of wet PS microspheres and dried PS microspheres were observed by Olympus BX51 optical microscope (Olympus, Japan) and Philips XL-30 scanning electron microscope (Philips, The Netherlands), respectively. Samples were sputter coated with a thin layer of gold to enhance the surface contrast and reduce surface charging. The particle size distribution of PS microspheres was determined using Mastersizer S particle size analyzer (Malvern Instrument, UK).

4000

3000

2000

1000

0

-1

Wavenumber (cm )

Fig. 2. FTIR spectra of sucrose, soluble polysucrose and crosslinked PS microspheres. A sucrose B soluble polysucrose C crosslinked PS microspheres.

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polymerization of sucrose with EP firstly took place at primary hydroxyl groups due to the fact that primary hydroxyl groups of sucrose have the most reactivity. The peak at 3418 cm1 of soluble PS, which is assigned to secondary hydroxyl groups [16], displays a decrease in its intensity in comparison to that of sucrose, and the peak of PS was much weaker. This indicated that some secondary hydroxyl groups were consumed during the reaction. The peaks at 2933 and 2886 cm1 in spectra of crosslinked PS microspheres, which are assigned to methylene groups [17], show an increase in their intensities in comparison to that of sucrose and soluble PS. It is attributed to more methylene groups introduced by EP. The chemical structure of crosslinked PS microspheres is similar to that of soluble PS, which is confirmed by their almost same FTIR spectra. 3.2. Physical properties of PS microspheres The crosslinked PS microspheres were found to be in good spherical shape and swellable in water. Five crosslinked PS microspheres were obtained by altering crosslinker amounts during the crosslinking reaction. Some properties of these five PS microspheres were listed in Table 1. It is shown that the size of beads could be smaller with increasing the crosslinking degrees. The increase of the amounts of EP could make the inner structure of PS microspheres more compact, which is proved by the increase of the dry density of the beads. Whereas the hydrated density of PS microspheres remained almost unchanged. 3.3. Morphology of PS microspheres Fig. 3 shows the morphology of crosslinked PS microspheres. The shape of the microspheres is spherical, which is observed with an optical micro-

Fig. 3. OM photograph of crosslinked PS microspheres (PS-1, 100·).

scope. The PS microspheres are translucent under swollen state, which is a typical feature of hydrogel. The spherical shape and the smooth surface of PS microspheres unchanged after dried under vacuum at 60 C, but the size diminished as shown in Fig. 4. 3.4. Equilibrium water content and crosslinking density Crosslinked polymeric networks can be characterized by the crosslinking density q[18], which is inversely related to the average weights of chain fragments between crosslinking points, Mc, according to the following formula 1

q ¼ ðtM c Þ

ð2Þ

where t denotes the specific volume of the polymer. The dry density of the crosslinked PS microspheres can be used for the calculation of the volume fraction of soluble PS in the swollen beads, Vs, and the volume fraction of soluble PS in solution before crosslinking, Vr [19]:

Table 1 Properties of crosslinked PS microspheres with different crosslinker amountsa Microspheres

EP/soluble PS weight ratio in feed

D(v, 0.5) (lm)

D[4,3] (lm)

Wet density (g/mL)

Dry density (g/mL)

PS-1 PS-2 PS-3 PS-4 PS-5

0.4:1 0.5:1 0.6:1 0.7:1 0.8:1

443.36 398.96 339.94 313.98 290.62

449.40 406.93 345.69 318.61 295.27

1.00 ± 0.04 1.01 ± 0.03 0.99 ± 0.03 1.00 ± 0.02 1.01 ± 0.01

0.91 ± 0.03 0.95 ± 0.02 1.11 ± 0.02 1.13 ± 0.02 1.22 ± 0.01

a

Preparation conditions: [soluble PS] = 20%; W/O ratio (v:v) = 1:5; temperature = 70 ± 2 C; stirring speed = 240 ± 10 rpm.


Fig. 4. SEM photograph of crosslinked PS microspheres (PS-1, 240·).

Vs ¼

ð1 EWCÞ=d EWC þ ð1 EWCÞ=d

Vr ¼

W p =d W p =d þ W s ð3Þ

where d is the dry density of the crosslinked PS microspheres, Wp is the weight of soluble PS added before crosslinking, Ws is the weight of total water in PS solution before crosslinking, and EWC is the equilibrium water content. The density of water is taken as 1 g/mL. Bray and Merrill [20] modified Flory’s equation to calculate 1/Mc when crosslinkages were introduced between polymer chains while the polymer already existed in solution: 2

t ½lnð1 V s Þ þ V s þ vðV s Þ 1 2 V ¼ l 1=3 Mc Mn V r ½ðV s Þ 1 ðV s Þ Vr

ð4Þ

2 Vr

where t denotes the specific volume of the polymer, Vl is the molar volume of the solvent (18 mL/mol) and Mn is average molecular weight of the polymer. The Flory polymer-solvent interaction parameter v takes a value of 0.494, while Mn of soluble PS is known. The results are given in Table 2. The low Mc values of below 2000 suggested that the crosslinking density is very high. Since the soluble PS

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consists of linear and comb polysucrose and many of hydroxyl groups of soluble PS have been consumed during crosslinking reaction, the parameters used in calculations are rather approximate, and the results obtained here are not precise. It is found that Mc dropped sharply for these five PS microspheres, but the EWC value did not decline much, which is shown in Fig. 5. Naturally, the increase of crosslinking density q, which is characterized by the decrease of Mc, will depress the EWC because the soluble PS molecular chains would hold together much more tightly. Although q should have a direct influence on EWC of the PS microspheres, EWC displayed a relatively mild change to q. It is concluded that the hydroxyl content of PS microspheres is more important in determining the EWC of the beads. Both free and bound water exist in most swellable colloids in water [21]. The amount of bound water in the PS microspheres is mainly depended on or even proportional to the number of free hydroxyl groups [22]. So the EWC value of 79.6% indicates that these crosslinked PS microspheres have a quite high hydroxyl content even though their crosslinking densities are rather high. And there is only a small difference among five PS microspheres in EWC because their hydroxyl contents are quite close to each other. 3.5. Hydroxyl content To determine the hydroxyl content of the crosslinked PS microspheres, the beads were acetylated with an excess of acetic anhydride. After acetylation the excess of acetic anhydride was then converted to acetic acid and titrated with a standardized base solution. The results are listed in Table 3, in which each value corresponds to an average of three assessments. The results indicated that the hydroxyl contents ranged from 15.5 to 19.0 m mol/g and decreased with the increase of EP, which is shown in Fig. 6. This could be expected that the numbers

Table 2 Equilibrium water contents and crosslinking densities of crosslinked PS microspheres Microspheres


EWC (%)

Vs

Vr

Mc

q · 104


0.4:1 0.5:1 0.6:1 0.7:1 0.8:1

94.13 ± 0.21 90.09 ± 0.18 86.35 ± 0.13 84.02 ± 0.38 79.61 ± 0.35

0.064 0.104 0.125 0.144 0.174

0.216 0.208 0.184 0.181 0.17

16747 4622 2390 1506 759

0.65 2.37 4.59 7.28 14.44

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Equilibrium water content Crosslinking density

14

95

12 10

90

8

85

6 80 4 2

70

-4

75

Crosslinking density (× 10 )

Equilibrium water content (%)

100

0 PS-1

PS-2

PS-3

PS-4

PS-5

Crosslinked PS microspheres with different degree of crosslinking Fig. 5. The effect of EP amounts in feeds on equilibrium water content and crosslinking density of crosslinked PS microspheres. EP/ soluble PS weight ratio in the feed: PS-1, 0.4:1; PS-2, 0.5:1; PS-3, 0.6:1; PS-4, 0.7:1; PS-5, 0.8:1.

Table 3 Hydroxyl contents of crosslinked PS microspheres Microspheres


Hydroxyl content (m mol/g)

Number of consumed hydroxyl groups per sucrose unita


0.4:1 0.5:1 0.6:1 0.7:1 0.8:1

19.04 ± 0.18 18.84 ± 0.15 17.90 ± 0.12 17.13 ± 0.11 15.48 ± 0.15

1.4 1.5 1.8 2.1 2.6

a

Calculated from hydroxyl content experimental value of sucrose (experimental value = 23.1 m mol/g; theory value = 23.4 m mol/g).

Hydroxyl content (mmol/g)

19.5 19.0 18.5 18.0 17.5 17.0 16.5 16.0 15.5 15.0 PS-1

PS-2

PS-3

PS-4

PS-5

Crosslinked PS microspheres with different degree of crosslinking Fig. 6. Hydroxyl contents of crosslinked PS microspheres. EP/soluble PS weight ratio in the feed: PS-1, 0.4:1; PS-2, 0.5:1; PS-3, 0.6:1; PS4, 0.7:1; PS-5, 0.8:1.


of hydroxyl groups on the crosslinked PS microspheres would happen somewhat change after the crosslinking reactions, because each EP molecule reacted with two hydroxyl groups from different sucrose/soluble polysucrose molecules, yielding one hydroxyl group. Therefore, the increase of amounts of used EP would result in decrease of the hydroxyl content in the crosslinked PS microspheres. However, there could be some side reactions in crosslinking reaction, for example, some EP molecules might only react with one hydroxyl group or react with unreacted pendant epoxy groups. Since it is difficult to quantify such side reactions, it is reasonable to assume that amount of hydroxyl groups from such side reactions is directly related to the amount of EP used in the crosslinking reaction. 4. Conclusion A series of crosslinked PS microspheres with different crosslinking degrees were prepared by a twostage route including synthesis of soluble PS using sucrose and EP, followed by a reversed suspension polymerization through soluble PS with EP as crosslinker. The chemical structure of the crosslinked PS microspheres was characterized by FTIR. The PS microspheres have smooth surface and good spherical shape by OM and SEM. It was found that the PS microspheres with higher crosslinking density results in smaller particle size, higher dry density and lower equilibrium water content. These crosslinked PS microspheres are easily swellable in water and their equilibrium water content values are as high as 94.13%. These microspheres possess quite high hydroxyl contents even though they have a relatively high crosslinking density, which makes them suitable for further chemical modification. The sharp increase of crosslinking densities of PS microspheres may cause somewhat decrease in hydroxyl content. These PS microspheres have a potential application for controlled drug releases and separation of proteins.

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Acknowledgements This work was supported by National Nature Science Foundation of China (Grant no. 50403017) and Nature Science Foundation of Tianjin (Grants 05yfgpgx06600). References [1] [2] [3] [4] [5] [6] [7]

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