Inulin vinyl sulfone derivative cross-linked with bis-amino PEG: new materials for biomedical applications

J. DRUG DEL. SCI. TECH., 19 (6) 419-423 2009

Inulin vinyl sulfone derivative cross-linked with bis-amino PEG: new materials for biomedical applications G. Pitarresi1*, G. Tripodo1, D. Triolo1, C. Fiorica1, G. Giammona1, 2 Lab. of Biocompatible Polymers, Dipartimento di Chimica e Tecnologie Farmaceutiche, Università degli Studi di Palermo, Via Archirafi 32, 90123 Palermo, Italy 2 IBF-CNR, Via Ugo La Malfa 153, 90143 Palermo, Italy *Correspondence: [email protected]

1

In this work new hydrogels based on biocompatible polymers such as inulin (INU) and O,O’-bis(2-aminoethyl)polyethyleneglycol (PEGBa) have been prepared and charaterized. In particular, INU has been derivatized with divinyl sulfone (DV) thus obtaining the INUDV derivative, a copolymer bearing double bonds highly reactive towards the conjugate addition by nucleophilic molecules. INUDV has been characterized by FT-IR, 1H-NMR and SEC analyses that have confirmed the success of the derivatization reaction. With the aim to obtain novel hydrogel systems, INUDV derivative has been cross-linked with PEGBa in phosphate buffer solution pH 7.4. The reaction has been carried out for 4 h at room temperature and various samples have been obtained by changing the amount of PEGBa. These hydrogels resulted to be homogeneous, transparent, colorless and odorless and they have been characterized by spectroscopic analysis, swelling, chemical and enzymatic degradation studies. Their cell compatibility has been evaluated in vitro by direct and indirect assays on CaCo-2 cells. Key words: Inulin – Divinyl sulfone – Polyethylene glycol – Chemical cross-linking – Hydrogels.

In the last years several efforts have been made to develop new materials, like hydrogels, based on biocompatible polymers to be used as drug delivery systems, contact lens or scaffolds for tissue engineering [1-9]. Various techniques can be employed to obtain a hydrogel, but the principal feature that a biomaterial must have, is the absence of secondary products that could affect the biocompatibility of the final material. The preparation of such a system is possible, for example, by employing a clean chemistry. In this context, a chemical cross-linking, in a biocompatible medium, in mild reaction conditions and by using non toxic materials could be a promising approach. In the literature there are various examples of hydrogels prepared by cross-linking between thiol- or amine- containing polymers and vinyl sulfone groups [10, 11], but there are not examples of hydrogels starting from inulin sulfone derivative and amine-containing polyethylene glycol. In this work, inulin has been chosen because it is a biocompatible, biodegradable, FDA approved polymer, already employed in many fields, from food to pharmaceutical to cosmetic industry and here used to prepare a divinyl sulfone (INUDV) derivative able to react with O,O’-bis(2-aminoethyl)polyethyleneglycol (PEGBa). PEGBa has been chosen as a biocompatible cross-linking agent and the reaction between INUDV and PEGBa has been performed in phosphate buffer solution pH 7.4 at room temperature for 4 h. Three hydrogels have been obtained by changing the amount of PEGBa that reacts with INUDV derivative and studies concerning their swelling behaviour, chemical and enzymatic degradation and in vitro cell compatibility have been reported here.

2. Apparatus

Molecular weight of INUDV was determined by a SEC system equipped with a pump and a 410 differential refractometer (DRI) as a concentration detector, all from Waters. A Ultrahydrogel 1000 (size exclusion range 10,000-500,000) and a Ultrahydrogel 250 (size exclusion range 1,000-50,000), both from Waters, were used as columns. 0.05 M phosphate buffer solution (PBS) pH 7.2 was used as a mobile phase at 35°C with a flow of 0.6 mL/min. Pullulan (Mw range 300150,000 Da) was used as a standard. 1 H-NMR (D2O) spectra were obtained with a Bruker AvanceII 300 MHz. FT-IR spectra were recorded as pellets in KBr in the range 4,000400 cm-1 using a Perkin-Elmer 1720 Fourier Transform Spectrophotometer with a resolution of 1 cm-1; each spectrum was recorded after 100 scans. Centrifugations were performed with a Beckman Coulter Allegra X-22R equipped with a fixed-angle rotor F0850 and refrigeration system. Degradation studies were performed in a Benchtop 80°C Incubator Orbital Shaker model 420. AThermo Labsystems Multiskan Ex 96-well microplate photometer was used to evaluate cell viability after MTS test.

3. Synthesis of INUDV derivative

Before use inulin (INU) was dried for 24 h at 70°C, then 1 g was dissolved in 20 mL of anhydrous DMF under argon for at least 3 h. After complete dissolution, always under stirring and argon, a suitable amount of divinyl sulfone (DV) was added according to X = 5, where X is the moles of DV/moles of INU repeating units. After 3 min, a suitable amount of triethylamine (TEA) was added according to Y = 5, where Y is the moles of TEA/moles of INU repeating units. The reaction mixture was placed into a thermostatic bath and stirred at 60°C under argon for 24 h. After this time, the obtained product was precipitated in 400 mL of ether and centrifuged for 15 min at 10,000 rpm and 4°C. The product was washed with ether (5 × 50 mL) and then dried under vacuum. The final product, named INUDV, was obtained with a yield of 92% w/w based on the starting inulin, then it was characterized by FT-IR, 1H-NMR and SEC analyses.

I. EXPERIMENTAL 1. Materials

Inulin (INU) from Dahlia Tubers Mw ≈ 5000 Da, diethyl ether, divinyl sulfone ≥98.0 % (DV), O,O’-bis(2-aminoethyl)polyethyleneglycol (PEGBa) Mw ≈ 2000 Da, inulinase from Aspergillus niger and triethylamine (TEA) were obtained from Fluka (Italy). Anhydrous N,N-dimethylformamide 99.9 % (DMF) and acetone were purchased from Aldrich Chemical Co. (Italy). Pullulan GPC standards were obtained from Polymer Laboratories (Germany). Caco-2 cells were purchased from Istituto Zooprofilattico Sperimentale della Lombardia e dell’Emilia Romagna “Bruno Umbertini” (Italy). 419

J. DRUG DEL. SCI. TECH., 19 (6) 419-423 2009

Inulin vinyl sulfone derivative cross-linked witn bis-amino PEG: new materials for biomedical applications G. Pitarresi, G. Tripodo, D. Triolo, C. Fiorica, G. Giammona

4. Characterization of INUDV derivative

8. Enzymatic degradation studies with inulinase

FT-IR (KBr) spectrum showed a broad band centred at 3400 cm-1 (νas OH); bands at 1311 (νas O=S=O), 1294 (scissoring -C=CH2), 1127 cm-1 (νs O=S=O) and 761 (wagging –C=CH2) cm-1. 1 H-NMR (D2O) showed: δ 3.20-4.0 (5H, m: -CH2-OH; CH-CH2OH; -CH2-CH2-O-), 4.14 (1H, t: CH-OH), 4.25 (1H, d: CH-OH), 6.21 (2H, m: CH2=) and 6.77 (1H, m: =CH-). The degree of derivatization (DD %) in DV was determined by 1H-NMR by comparing the integrals of peaks at δ 6.21 and 6.77 (3H, 2m: CH2=CH-) relative to DV double bond protons, with the peaks between δ 3.20-4.25 relative to inulin fructose unit protons (7 H). The value of DD % in DV resulted to be 25 ± 1 % mol/mol.

Aliquots (30 mg) of INUDV/PEGBa-A, INUDV/PEGBa-B or INUDV/PEGBa-C hydrogels were incubated with 10 mL of phosphate buffer solution pH 4.7 in the absence or in the presence of inulinase (final enzyme concentration 10 U/mL), under continuous stirring (100 rpm) at 37 ± 0.1°C for 24 h. Enzyme solution was prepared immediately before the experiment. The degradation of the samples was evaluated by using the anthrone method following a procedure elsewhere reported [12]. Each experiment was performed in triplicate.

9. Cell culture

Caco-2 cells were maintained in MEM containing 10 vol.-% fetal calf serum (FCS), 2 mM L-glutamine, 100 U/mL penicillin, 100 μg/mL streptomycin, 2.5 μg/mL amphotericin B (Sigma Aldrich, Italy), under standardized conditions (95% relative humidity, 5% CO2, 37°C).

5. Preparation of INUDV/PEGBa hydrogels

INUDV/PEGBa hydrogels were prepared by varying the amount of PEGBa, according to Z = 0.5, 0.8 or 1.0, where Z is the moles of PEGBa/moles of INUDV repeating units bearing DV groups, considering a DD in DV of 25 % mol/mol respect to inulin. In particular, 100 mg of INUDV derivative were dissolved in 400 μL of phosphate buffer solution pH 7.4, at room temperature and degassed under vacuum. Separately, 132, 211 or 264 mg of PEGBa were dissolved in 600 μL of PBS pH 7.4, at room temperature and degassed under vacuum. Then, 400 μL of INUDV solution were added to each PEGBa solution and the cross-linking reaction was left 4 h at room temperature. After this time, the obtained hydrogels, named INUDV/PEGBa-A, INUDV/PEGBa-B and INUDV/PEGBa-C for Z = 0.5, 0.8 or 1.0, respectively, were recovered, washed with distilled water (5 × 50 mL) and freeze dried.

In vitro cell compatibility studies Cell compatibility of INUDV/PEGBa-A, INUDV/PEGBa-B and INUDV/PEGBa-C hydrogels was evaluated in vitro, by both “direct” and “indirect” method, using Caco-2 cells with a viability of 98 ± 1%, as revealed by the trypan blue exclusion assay. Before analysis, each hydrogel was sterilized by washing with 96% (v/v) ethanol for 30 min and dried at room temperature under sterile hood. Direct method: Caco-2 cells in complete MEM were seeded in a 96-well plate at 1·105 cells/mL (0.1 mL per well) and incubated at 37°C and 5% CO2 until they reached a confluent monolayer. Then, the medium was replaced with fresh complete MEM and each freezedried hydrogel (2.5 mg ) was added to every well. After 24 or 48 h of incubation, the medium and the hydrogel were removed from each well and replaced with fresh medium, then 20 µL per well of MTS reagent were added. After 2 h of incubation, the absorbance at 492 nm was recorded and cell viability data were calculated. Relative cell viability (in percentage) was expressed as (Abs492 treated cells/Abs492 control cells) ×100. Cells incubated in MEM in the absence of the hydrogel were used as a control. Each experiment was performed in triplicate. Indirect method: the viability of Caco-2 cells cultured in a medium where each hydrogel was suspended and swelled (indicated as “conditioned medium”) was evaluated by means of MTS assay. In particular, each hydrogel (2.5 mg/mL of medium) was incubated in MEM without FCS at 37 ± 0.1°C for 5 days under orbital stirring at 120 rpm. After incubation, the medium (“conditioned medium”) was centrifuged at 11,800 rpm, 4°C for 30 min, then filtered to remove the hydrogel. Caco-2 cells in complete MEM were seeded in a 96-well plate at 1·105 cells/mL (0.1 mL per well) and incubated at 37°C and 5% CO2 until they reached a confluent monolayer. Subsequently, the culture medium was replaced with the “conditioned” medium supplemented with 10% v/v FCS. After 24 or 48 h of incubation, cell viability was assayed by MTS assay, as described above. Cells incubated in MEM were used as a control. Each experiment was performed in triplicate.

6. Swelling studies

Aliquots (30 mg) of INUDV/PEGBa-A, INUDV/PEGBa-B or INUDV/PEGBa-C hydrogels were placed in tared 5-mL sintered glass filters (ø 10 mm; porosity, G3) and left to swell at 37 ± 0.1°C until 24 h by immersing the filters plus supports in beakers containing twice-distilled water. At established times, the weight of each swollen hydrogel was evaluated after percolation under atmospheric pressure and centrifugation at 3,000 rpm for 5 min. Swelling was also evaluated in HCl solution pH 1.0 (simulated gastric fluid) for 2 h and then, in phosphate buffer solution pH 6.8 (simulated intestinal fluid) until 24 h. The filter tare was determined after centrifugation with water alone. The weight swelling ratio (q) was calculated according to: q = Ws/Wd where Ws and Wd are the weights of the swollen and dry sample, respectively. Each experiment was performed in triplicate.

7. Chemical hydrolysis studies in simulated gastric fluid

Aliquots (30 mg) of INUDV/PEGBa-A, INUDV/PEGBa-B or INUDV/PEGBa-C hydrogels were incubated with 6 mL of HCl 0.1 N under continuous stirring (100 rpm) at 37 ± 0.1°C for 2 h. After this time, each sample was recovered after centrifugation (15 min, 10,000 rpm, 4°C) and washed with distilled water (5 × 50 mL) under continuous stirring for 30 min, in order to extract soluble polymer degradation products entrapped within the network. Samples were freeze-dried and weighed. Degradation was expressed as:

II. RESULTS AND DISCUSSION

In order to obtain an inulin (INU) derivative able to react with nucleophilic molecules, for the production of chemical hydrogels, the polysaccharide has been derivatized with divinyl sulfone (DV). In particular, the derivatization of INU with DV has been performed in organic solvent by using TEA as a catalyst and INUDV derivative thus obtained has been characterized by 1H-NMR, FT-IR and SEC analyses. 1 H-NMR (D2O) showed two peaks at: δ 6.21 (2H, m: CH2=) and 6.77 (1H, m: =CH-) relative to vinyl sulfone linked to inulin (Figure 1). The molar degree of derivatization (DD %) in DV was determined by 1H-NMR, as reported in the experimental part, and resulted to be 25 ± 1% mol/mol.

Degradation % = [(weight of starting hydrogel - weight of hydrogel recovered after degradation)/weight of starting hydrogel] × 100 Each experiment was performed in triplicate.

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Inulin vinyl sulfone derivative cross-linked witn bis-amino PEG: new materials for biomedical applications G. Pitarresi, G. Tripodo, D. Triolo, C. Fiorica, G. Giammona

J. DRUG DEL. SCI. TECH., 19 (6) 419-423 2009

FT-IR (KBr) spectrum showed peaks (see experimental) that confirmed the formation of INUDV derivative. SEC studies showed a mean molecular weight of INUDV derivative of 7.5 kDa (polydispersity index 1.1), higher than that of native inulin (5.0 kDa, polydispersity index 1.2), thus confirming that no degradation occurred on inulin backbone during its reaction with divinyl sulfone. The cross-linking of INUDV with O,O’-bis(2-aminoethyl) polyethylene (PEGBa) was carried out at room temperature and in phosphate buffer solution pH 7.4 by varying the molar amount of PEGBa respect to INUDV. No chemical initiator was employed and within 4 h a complete cross-linking occurred. Three different amounts of PEGBa were used according to Z = 0.5, 0.8 or 1 where Z is the moles of PEGBa/moles of INUDV repeating units bearing DV groups, taking into consideration a molar degree of derivatization of INUDV derivative equal to 25% mol/mol as calculated by 1H-NMR. The obtained hydrogels, named INUDV/PEGBa-A, INUDV/

PEGBa-B and INUDV/PEGBa-C for Z = 0.5, 0.8 or 1, respectively, were characterized by FT-IR analysis, and studies of swelling in various media, chemical and enzymatic hydrolysis. Figure  2 reports the formation of a INUDV-PEGBa hydrogel and Figure 3 shows as an example, hydrogel INUDV/PEGBa-B that appears homogeneous, transparent and colorless. FT-IR spectra of INUDV/PEGBa-A, INUDV/PEGBa-B and INUDV/PEGBa-C hydrogels showed the disappearance of the peak at 761 cm-1 belonging to wagging of double bonds in INUDV derivative, thus indicating that the cross-linking reaction involves the opening of these bonds. Another important peak, useful to demonstrate the chemical cross-linking, was the signal at 1294 cm-1 (scissoring -C=CH2); in this case it is possible to evidence a remarkable reduction in the intensity, but because of the overlapping of PEG peak at 1290 cm-1, it was not possible to observe the complete disappearance of such a peak after cross-linking. Another evidence of the cross-linking was the appearance, in the spectra of the hydrogels, of the strong peak at 1107 cm-1 relative to PEG (C-O) stretching (Figure 4). The yield of obtained hydrogels was equal to 65, 80 and 90 wt % for INUDV/PEGBa-A, INUDV/PEGBa-B and INUDV/PEGBa-C, respectively, therefore, as expected, the yield increases by increasing the amount of PEGBa. It is known that the swelling ability of a hydrogel is very important since it affects many properties, like interaction with biological surfaces, mechanical properties, absorption and diffusion of solutes, etc.

Figure 1 - 1H-NMR spectrum of INUDV derivative. OH HO HO

O OH

O

O

HO

O

n

NH2

+

O

HO

O HO

PEGBa

O

O

Figure 3 - Photograph of INUDV/PEGBa-B hydrogel.

O

S

HO

O

OH

CH2 O

INUDV

OH

HO O

HN

O n

S

NH

O S

O

O

O

OH

HO O

O

CH2

OH O

O

CH2

OH

O

H2N

O

O

S

O

S O

N H

O n

N H

OH

S O

O

HO

INUDV/PEGBa hydrogel Figure 2 - Schematic representation of formation of INUDV/PEGBa hydrogel.

Figure 4 - FT-IR spectra of INUDV derivative, PEGBa and INUDV/ PEGBa-B hydrogel. 421

J. DRUG DEL. SCI. TECH., 19 (6) 419-423 2009

Inulin vinyl sulfone derivative cross-linked witn bis-amino PEG: new materials for biomedical applications G. Pitarresi, G. Tripodo, D. Triolo, C. Fiorica, G. Giammona

For these reasons swelling studies were performed in twice-distilled water for 24 h, in HCl solution pH 1.0 (simulated gastric fluid) for 2 h and then in phosphate buffer solution pH 6.8 (simulated intestinal fluid) until 24 h; results are showed in Figure 5. All investigated hydrogels showed a high affinity towards the aqueous medium and no significant difference was observed by changing the pH value from 1.0 to 6.8 according to the absence of ionizable groups in the samples. However, the following trend in the swelling ability was observed: INUDV/PEGBa-C>INUDV/PEGBa-B>INUDV/PEGBa-A thus confirming that, due to the hydrophilic character of the cross-linking agent (PEGBa), swelling increased by increasing its amount in the hydrogel. Since the prepared hydrogels are proposed for oral administration, in order to evaluate their stability in simulated gastric conditions, samples were incubated for 2 h with HCl solution pH 1.0. Results reported in Table I show that a very low chemical degradation occurred due to the absence of easily hydrolytically degradable bonds, independently of the amount of PEGBa present in the hydrogel. Therefore it is reasonable to suppose that these hydrogels could pass through the stomach without undergoing an appreciable degradation. In the intestinal fluid they could undergo a degradation by intestinal enzymes, such as inulinase. For this reason, these hydrogels were incubated for 24 h in the presence of inulinase in phosphate buffer solution pH 4.7 (or in the absence of enzyme as a negative control). As reported in Table I, a negligible degradation occurred when inulinase was absent in the medium, in contrast, in the presence of enzyme, all hydrogels underwent a pronounced degradation that increased by increasing the amount of PEGBa present in the hydrogel according with the increase in swelling degree that could promote enzyme permeation into the network. Therefore, enzymatic degradation data confirm that

inulin, even if derivatized with divinyl sulfone and cross-linked with PEGBa, can be degraded by inulinase. Test of cell compatibility is a good first step to provide a predictive evidence of a material biocompatibility. In particular, through rapid, standardized, sensitive and inexpensive means, it is possible to determine if a material causes acute effects to isolated cells. Therefore, cell compatibility of INUDV/PEGBa hydrogels was evaluated on Caco-2 cells, chosen as a model line of intestinal cells. These studies were performed by using two different methods: the direct test and the indirect test. In particular, in the direct test, INUDV/PEGBa hydrogels were directly incubated for 24 or 48 h with Caco-2 cells, whereas the indirect test was performed to evaluate if cells are compatible after an incubation of 24 or 48 h, with a culture medium where INUDV/ PEGBa hydrogels have been previously incubated for 5 days. Cells viability was evaluated by MTS test (see experimental part) and results are showed in Figure 6. It is evident that all the investigated hydrogels do not cause significant variation in cell viability after their direct or indirect contact with the cells, i.e. these materials are free (or contain not significant amounts) of biologically harmful extractable. * In this work novel hydrogels have been obtained by chemical cross-linking between an inulin-divinyl sulfone (INUDV) derivative and O,O’-bis(2-aminoethyl)polyethyleneglycol (PEGBa). The crosslinking has been performed in phosphate buffer pH 7.4 in the absence of

90

C ell Viability %

13

11

9

7

80 70 60

6

8

10

12

14

16

18

20

22

24

l ro nt Co

EG /P DV

U

24 h

IN

4

IN

2

Ba

-B EG

/P U

DV U 0

IN

1

Ba

-A Ba EG /P

3

-C

50

5

DV

q

A

100

48 h

Time (h)

Table I - Values of degradation % in HCl solution pH 1.0 and in phosphate buffer solution pH 4.7 in the absence or in the presence of inulinase (10 U/mL) for INUDV/PEGBa-A, INUDV/PEGBa-B and INUDV/ PEGBa-C hydrogels.

INUDV/PEGBa-A INUDV/PEGBa-B INUDV/PEGBa-C

90

C ell V iability %

Figure 5 - Dynamic swelling studies for INUDV/PEGBa-A, INUDV/ PEGBa-B and INUDV/PEGBa-C hydrogels in twice-distilled water for 24 h, HCl 0.1 N (pH 1.0) (simulated gastric fluid) for 2 h and then in phosphate buffer solution (PBS) pH 6.8 (simulated intestinal fluid) for 24 h.

80 70 60 l ro nt

DV

/P

Co

EG

EG /P DV

22.4 ± 2.0 29.6 ± 1.5 36.5 ± 1.8

U

3.1 ± 0.1 2.4 ± 0.2 2.9 ± 0.1

IN

3.3 ± 0.3 2.7 ± 0.2 2.5 ± 0.3

/P

at pH 4.7 with inulinase (10 U/mL)

DV

at pH 4.7 without inulinase

U

at pH 1.0

Ba

Ba EG

Degradation %

-C

-

50

IN

Hydrogel

B

100

-B

INUDV/PEGbA-C Water

Ba

INUDV/PEGbA-B pH 1.0-pH 6.8

INUDV/PEGbA-C pH 1.0-pH 6.8

U

NUDV/PEGbA-A Water

INUDV/PEGbA-B Water

IN

INUDV/PEGbA-A pH 1.0-pH 6.8

Figure 6 - Cell viability % evaluated by MTS test after 24 or 48 h of incubation of Caco-2 cells with INUDV/PEGBa hydrogels: A) direct test; B) indirect test. 422

Inulin vinyl sulfone derivative cross-linked witn bis-amino PEG: new materials for biomedical applications G. Pitarresi, G. Tripodo, D. Triolo, C. Fiorica, G. Giammona

J. DRUG DEL. SCI. TECH., 19 (6) 419-423 2009

initiators and at room temperature. INUDV/PEGa hydrogels, obtained with a high yield, are transparent, colorless, odorless and resistant to chemical hydrolysis, but degraded by inulinase. They show a good affinity towards aqueous media and a high cell compatibility as demonstrated by in vitro tests on Caco-2 cells. Taking into account that the cross-linking procedure between INUDV and PEGBa could be useful to load bioactive substances that are sensible to drastic cross-linking conditions such as high temperature, acid or basic medium or organic solvents, without need a further purification, the results of this paper encourage to study drug incorporation ability and release properties of these hydrogels.

6.

7. 8.

9.

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Manuscript Received 18 February 2009, accepted for publication 18 August 2009.

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