Biodegradable and pH-Sensitive Hydrogels for Potential Colon

Dec 1, 2007 - Giovanna Pitarresi, via Archirafi, 32, 90123 Palermo, Italy., † ... Dipartimento di Chimica e Tecnologie Farmaceutiche, Università di...
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Biomacromolecules 2008, 9, 43–49

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Biodegradable and pH-Sensitive Hydrogels for Potential Colon-Specific Drug Delivery: Characterization and In Vitro Release Studies Maria Antonietta Casadei,† Giovanna Pitarresi,*,‡ Rossella Calabrese,‡ Patrizia Paolicelli,† and Gaetano Giammona‡ Dipartimento di Studi di Chimica e Tecnologia delle Sostanze Biologicamente Attive, Università “La Sapienza”, Piazzale Aldo Moro 5, 00185 Roma, Italy, and Dipartimento di Chimica e Tecnologie Farmaceutiche, Università di Palermo, via Archirafi 32, 90123, Palermo, Italy Received June 28, 2007; Revised Manuscript Received October 15, 2007

A novel pH-sensitive and biodegradable composite hydrogel, based on a methacrylated and succinic derivative of dextran, named Dex-MA-SA, and a methacrylated and succinic derivative of R,β-poly(N-2-hydroxyethyl)-DLaspartamide (PHEA), named PHM-SA, was produced by photocross-linking. The goal was to obtain a colonspecific drug delivery system, exploiting both the pH-sensitive behavior and the colon-specific degradability. The hydrogel prepared with a suitable ratio between the polysaccharide and the polyaminoacid was characterized regarding its swelling behavior in gastrointestinal simulated conditions, chemical and enzymatic degradability, interaction with mucin, and cell compatibility on CaCo-2 cells. Moreover, 2-methoxyestradiol was chosen as a model of anticancer drug and release studies, were performed in the absence or in the presence of dextranase and esterase. The obtained hydrogel, due to its pH-sensitive swelling and enzymatic degradability, together with mucoadhesion and cell compatibility, could be potentially useful as system for the oral treatment of colonic cancer.

1. Introduction Colon-specific drug release systems are useful for the treatment of pathological conditions such as Crohn and inflammatory diseases, ulcerative colitis, infections, and carcinomas. In fact, a selective release of bioactive substances in the colon allows not only to lower the dosage necessary to obtain the therapeutic effect but also to reduce the side effects that these drugs produce when released and absorbed by the upper gastrointestinal tract.1 In this context, pH-sensitive hydrogels, whose swelling depends on the environmental pH (e.g., in the gastrointestinal tract), are very effective as intestinal drug delivery systems,2–4 as well as hydrogel biodegradable by intestinal enzymes, such as dextran based networks.5 These latter were produced by chemical cross-linking of dextran in organic solvent, in the presence of bifunctionalized cross-linkers6 or by UV cross-linking of photoreactive derivatives as methacrylated dextran (Dex-MA), usually in the presence of photoinitiators.7,8 However, only for derivatives having a high derivatization molar degree (DD) in methacrylic groups (20 mol %), the photocross-linking takes place in aqueous solution and in the absence of photoinitiators.9 These hydrogels were unable to avoid drug release in simulated gastric fluid, therefore, in order to obtain networks suitable for intestinal drug release, pH-sensitive hydrogels, based on acidic derivatives of methacrylated dextran, have been prepared in aqueous solution and without photoinitiators.9 Nevertheless, these hy* Corresponding author. E-mail: [email protected]. Telephone: 0039 091 6236154. Fax: 0039 091 6236150. Postal address: Prof. Giovanna Pitarresi, via Archirafi, 32, 90123 Palermo, Italy. † Dipartimento di Studi di Chimica e Tecnologia delle Sostanze Biologicamente Attive, Università “La Sapienza”. ‡ Dipartimento di Chimica e Tecnologie Farmaceutiche, Università di Palermo.

drogels were not susceptible of degradation by microbial enzymes so that drug release was probably related to the pHsensitive behavior of the hydrogel. In fact, it is known that the degradation by dextranase of photocross-linked methacrylated dextran (Dex-MA) is strongly affected by the cross-linking density, which in turn, depends on the DD in photoreactive groups of the starting polymer.8 However, in a previous work, we demonstrated that the combination of methacrylated dextran (with a DD in methacrylic groups of 20 mol %) with a photosensitive polyaminoacid derivative, such as methacrylated R,β-poly(N-2-hydroxyethyl)-DL-aspartamide, named PHM, was a useful way to obtain hydrogels with degradability by dextranase.10 On the other hand, we reported also the synthesis of the succinic derivative of PHM, named PHM-SA to produce hydrogels with a pronounced pH-sensitive behavior.11 For these reasons, to obtain a new colon specific drug delivery system, we have performed the combination of pH-responsiveness and enzymatic biodegradability in a single material. In particular, in this work, we report the preparation of a novel polysaccharide/polyaminoacid composite hydrogel, obtained by UV irradiation, without radical initiators, of a mixture of a succinic derivative of Dex-MA (named Dex-MA-SA) and PHM-SA. The hydrogel was widely characterized; swelling behavior in gastrointestinal simulated conditions, mucoadhesion, hydrolytic resistance, enzymatic degradability in the presence of dextranase, and/or esterase and in vitro cell compatibility on CaCo-2 cells were studied. Finally, 2-methoxyestradiol (2-ME), an antiangiogenic drug proposed for the colon-rectal cancer, was chosen and the ability of the hydrogel to release 2-ME in simulated intestinal fluid, as a result of its pH-sensitive behavior and specific enzymatic degradation, was in vitro investigated.

10.1021/bm700716c CCC: $40.75  2008 American Chemical Society Published on Web 12/01/2007

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2. Materials and Methods 2.1. Chemicals. All reagents were of analytical grade, unless otherwise stated. DL-Aspartic acid, ethanolamine, N,N-dimethylformamide (DMF), anhydrous N,N-dimethylacetamide (DMA), dimethylsulfoxide (DMSO), methacrylic anhydride (MA), succinic anhydride (SA), triethylamine (TEA), and dextran from Leuconostoc spp. (Mw 40.0 kDa) were purchased from Fluka (Italy). Glycidyl methacrylate (GMA), 4-dimethylaminopyridine (4-DMAP), DOWEX 50WX4–50 ionexchange resin and deuterium oxide (D2O) were purchased from Aldrich (Italy). Dextranase from Penicillium spp. (400–800 U/mg protein), esterase from porcine liver (184 U/mg protein), mucin from porcine stomach type II, hyaluronic acid sodium salt from rooster comb, 2-methoxyestradiol (2-ME), Dulbecco’s phosphate buffered saline (DPBS), minimum essential medium Eagle (MEM), trypsin-EDTA solution, trypan blue solution, amphotericin B solution, penicillin– streptomycin solution, and fetal bovine serum (FBS) were purchased from Sigma (Italy). Acetic acid, acetone, acetonitrile, diethylether, ethanol, 2-propanol, and tetrahydrofuran were purchased from Merck (Germany). The 3-(4,5-dimethlthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium inner salt (MTS)/phenazine ethosulphate (PES) solution (CellTiter 96 AQuqeous one solution cell proliferation assay) was purchased from Promega (Italy). Caco-2 cells were purchased from Istituto Zooprofilattico Sperimentale della Lombardia e dell’Emilia Romagna “Bruno Umbertini” (Italy). R,β-Poly(N-2-hydroxyethyl)-DL-aspartamide (PHEA) was synthesized, purified, and characterized according to a procedure reported elsewhere.12 Spectroscopic data (FT-IR and 1H NMR) were in agreement with previous results.13 The batch of PHEA used in this study has a weight-average molecular weight of 47.0 kDa (Mw/Mn 1.9) based on PEO/PEG standards, determined by size exclusion chromatography (SEC) analysis.13 2.2. Dex-MA-SA Synthesis. Succinic methacrylated dextran (DexMA-SA) was synthesized from dextran as already reported.9 The molar derivatization degree (DD mol %) in methacrylic groups, calculated by means of 1H NMR analysis, was equal to 18 ( 1 mol %, whereas the DD mol% in succinic groups, calculated by means of 1H NMR analysis and confirmed by potentiometer titration, was equal to 21 ( 1 mol %. 2.3. PHM-SA Synthesis. The succinic methacrylated derivative of PHEA (named PHM-SA) was produced and characterized as already reported in a previous work.11 Spectroscopic data (FT-IR, 1H NMR, and 13C NMR) were in agreement with previous results.11 The molar derivatization degrees in methacrylic and succinic groups, determined by 1H NMR, resulted equal to 30 ( 1 mol % and 35 ( 1 mol %, respectively. 2.4. Preparation of Dex-MA-SA/PHM-SA Based Hydrogel. An aqueous solution of Dex-MA-SA and PHM-SA (7% w/v) with a weight ratio between Dex-MA-SA and PHM-SA equal to 1:4, was prepared. This solution was placed in a Pyrex tube (internal diameter 14 mm) equipped with an internal Pyrex piston (diameter 10 mm) in order to reduce the thickness of polymeric solution to about 2 mm, then irradiated under argon with a UV source for 90 min. UV irradiation was performed using a Rayonet-like photochemical reactor, hand built and equipped with a merry-go-round for 9 tubes (with a diameter of 15 cm) and 16 mercury lamps with an intensity of 8 W at medium pressure and an emission at 313 nm (supplied by Helios Italquartz s.r.l., Italy). The distance between the sample and the lamp was 3.5 cm. The cross-linked Dex-MA-SA/PHM-SA hydrogel was recovered and washed with twice-distilled water (6 × 40 mL), centrifuging every time for 20 min at 9800 rpm and 4 °C to recover the hydrogel. Centrifugation was performed with an International Equipment Company Centra MP4R equipped with an 854 rotor and temperature control. After freeze-drying, the hydrogel was recovered with a yield of 90 ( 1% (w/w), based on the starting amount of Dex-MA-SA and PHM-SA. Dex-MA-SA/PHM-SA hydrogel has been characterized by FT-IR analysis. Infrared spectra were recorded as pellets in KBr, in the range 4000–400 cm-1, using a Perkin-Elmer 1720 Fourier transform spec-

Casadei et al. trophotometer with a resolution of 1 cm-1; each spectrum was recorded after 100 scans. 2.5. Swelling Studies. The swelling behavior of Dex-MA-SA/PHMSA based hydrogel was evaluated after 2 h of incubation in 0.1 N HCl (simulated gastric fluid, SGF) and after 24 h of incubation in twicedistilled water or pH 6.8 Na2HPO4/KH2PO4 buffer (simulated intestinal fluid, SIF). Dynamic swelling was also evaluated, i.e., the sample was incubated in SGF for 2 h, then in SIF until 24 h, and swelling was determined at various times. Exactly weighed aliquots (30 mg) of hydrogel were immersed, through tared sintered glass filters (10 mm diameter, G3 porosity), in the swelling medium and incubated at 37 °C, under continuous orbital shaking (150 rpm), for the established times. The excess of liquid was removed by percolation under atmospheric pressure and the filters, after centrifugation at 3000 rpm for 5 min, were weighed. The weight swelling ratio (q) of the hydrogel was calculated as follows:

q ) Ws ⁄ Wd where Ws and Wd were the weights of the swollen and dry samples, respectively. Each experiment was performed in triplicate and results are reported as mean ( standard deviation. 2.6. Chemical Hydrolysis Studies. Chemical hydrolysis of DexMA-SA/PHM-SA based hydrogel was performed in SGF, in SIF, or in pH 7.4 phosphate buffer solution (PBS, NaCl/Na2HPO4/KH2PO4). In particular, aliquots exactly weighed of the hydrogel (30 mg) were dispersed in the hydrolysis medium (30 mL) and incubated at 37 ( 0.1 °C under continuous orbital shaking (150 rpm). At established times (2 h in SGF and 24 h in SIF or PBS), the samples (neutralized when necessary) were recovered after centrifugation (15 min, 9800 rpm, 4 °C) and washed under continuous stirring for 1 h with twice-distilled water (5 × 40 mL), in order to extract soluble degradation products and electrolytes entrapped within the network. The samples were freezedried, weighed, and characterized by swelling studies in twice-distilled water. Each experiment was performed in triplicate, and results are reported as mean ( standard deviation. 2.7. Enzymatic Hydrolysis Studies. Aliquots (30 mg) of the DexMA-SA/PHM-SA based hydrogel were incubated for 24 h under continuous stirring (100 rpm) at 37.0 ( 0.1 °C in 30 mL of SIF (pH 6.8) or PBS (pH 7.4) containing dextranase (final enzyme concentration 15 U/mL) and/or esterase (final enzyme concentration 125 U/mL). The enzyme solutions were prepared just before use. The activities of dextranase and esterase have been confirmed by performing the assays reported in the literature.14,15 After the treatment, the hydrogel was purified and characterized with the same procedure used for the sample recovered after the chemical hydrolysis. Each experiment was performed in triplicate and results are reported as mean ( standard deviation. 2.8. In Vitro Cell Compatibility Studies. Cell compatibility of DexMA-SA/PHM-SA based hydrogel was evaluated in vitro, by “direct” and “indirect” method, using Caco-2 cells with a viability of 98 ( 1%, as revealed by the trypan blue exclusion assay. These cells were routinely cultured in RPMI-1640 medium, supplemented with 10% (v/ v) of FBS, 1% (v/v) of penicillin-streptomycin solution, and 1% (v/ v) of amphotericin B solution, at 37 °C in a 5% CO2 incubator. Before analysis, the hydrogel was sterilized by washing with 96% (v/v) ethanol for 30 min and dried at room temperature under sterile hood. Direct Method. Confluent cell monolayers were incubated in contact with the hydrogel, in complete RPMI-1640 at 37 °C and 5% CO2. After incubation, the cell viability was evaluated by means of trypan blue exclusion assay and MTS assay. In particular, as far as the trypan blue exclusion assay is concerned, Caco-2 cells in complete RPMI-1640 were seeded at 100000 cells/mL (1 mL per well) in a 24-well plate and incubated for 48 h. Then, the medium was replaced with fresh complete medium, freeze-dried hydrogel was added to each well, and the plate was incubated for 24 h. After the incubation time, the medium and the hydrogel were removed from each well, and the cells, previously detached by a trypsin-EDTA/DPBS sterile solution, were resuspended in fresh complete RPMI-1640 (1 mL per well). Then, 100 µL of each cell suspension were gently mixed (1:1) with a trypan blue solution

Hydrogels for Potential Colon-Specific Drug Delivery (0.2% w/v) and transferred into the both sides of a Bürker chamber (5–10 µL); therefore, under a light microscope, the number of viable and not viable cells in each well was determined. Cell viability data were calculated as percentage ratio between viable cell number and total cell number. Cells incubated in RPMI-1640 in the absence of the hydrogel were used as a control. Each experiment was performed in triplicate, and results are reported as mean ( standard deviation. As far as the MTS assay is concerned, Caco-2 cells in complete RPMI-1640 were seeded in a 96-well plate at 100000 cells/ml (0.1 mL per well) and incubated at 37 °C and 5% CO2 for 48 h. Subsequently, the medium was replaced with fresh complete RPMI1640 and the freeze-dried hydrogel was added to each well. After 24 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 by using a Thermo Labsystems Multiskan Ex 96-well microplate photometer and cell viability data were calculated. Cells incubated in RPMI-1640 in the absence of the hydrogel, were used as a control. Each experiment was performed in triplicate and results are reported as mean ( standard deviation. Indirect Method. The viability of Caco-2 cells cultured in a medium where the hydrogel was previously suspended and swelled (indicated as “conditioned” medium) was evaluated. In particular, the hydrogel was incubated in RPMI-1640 without FBS at 37 ( 0.1 °C for 5 days under orbital stirring at 120 rpm. After incubation, the medium “conditioned” by the hydrogel, was centrifuged at 11800 rpm, 4 °C for 30 min, then filtered to remove the hydrogel. Caco-2 cells in complete RPMI-1640 were seeded at 100000 cells/ml in a 24-well plate (1 mL per well) or in a 96-well plate (0.1 mL per well), for trypan blue or MTS assay, respectively, and they were incubated at 37 °C and 5% CO2 for 48 h. Subsequently, the culture medium was replaced with the “conditioned” medium supplemented with 10% v/v FBS. After 24 h of incubation (37 °C, 5% CO2), cell viability was evaluated by the trypan blue exclusion assay and MTS assay, as described above. Cells incubated in RPMI-1640 were used as a control. Each experiment was performed in triplicate and results are reported as mean ( standard deviation. 2.9. Studies of Interaction with Mucin. Mucin (10 mg) were dispersed in 10 mL of SIF at 37 ( 0.1 °C for 24 h under orbital shaking (150 rpm). Then, adequate amounts of Dex-MA-SA, PHM-SA, DexMA-SA/PHM-SA mixture (1:4) or hyaluronic acid sodium salt (chosen as a positive control) were added to the mucin dispersion (mucin/ polymer weight ratio equal to 1), and each sample was incubated at 37 ( 0.1 °C under orbital shaking (150 rpm) for 2, 7, 24, 30, and 48 h. After each incubation time, the transmittance % of the samples was recorded at 500 nm by using a Shimadzu UV-2401 spectrophotometer. 2.10. Drug Loading by Soaking Procedure. A concentrated solution of 2-ME in ethanol was added to Dex-MA-SA/PHM-SA based hydrogel. The mixture was maintained at room temperature under stirring for 4 days. After this time, the solvent was removed by filtration and the sample was rapidly washed with ethanol in order to remove 2-ME adsorbed on the surface. The drug loaded sample was dried at atmospheric pressure. 2.11. Determination of Drug Amount Entrapped in Dex-MASA/PHM-SA Based Hydrogel. 50 mg of drug loaded Dex-MA-SA/ PHM-SA based hydrogel were extensively extracted at room temperature with 60 mL of ethanol. The liquids of extraction were collected and evaporated under vacuum at 40 °C. The obtained residue was dissolved in ethanol and assayed by UV analysis at 285 nm for the quantitative determination of 2-ME. The amount of entrapped drug was 10% w/w. 2.12. Drug Release Studies. Aliquots of drug loaded Dex-MA-SA/ PHM-SA based hydrogel were incubated at 37 ( 0.1 °C under orbital shaking (150 rpm) in SGF until 2 h, then in SIF from 2 until 48 h, in the absence or in the presence of dextranase (15 U/mL) and esterase (125 U/mL). Sink conditions were maintained during the experiments (drug concentration was always below 10% of drug solubility). Then,

Biomacromolecules, Vol. 9, No. 1, 2008 45 at fixed time intervals, each aliquot of hydrogel was filtered through a 0.45 µm Millipore filter and assayed by UV analysis at 285 nm. Each experiment was performed in triplicate and results are reported as mean ( standard deviation.

3. Results and Discussion In a previous work, Dex and PHEA have been derivatized with methacrylate groups, by reaction with GMA or MA, thus obtaining two photocross-linkable derivatives, named Dex-MA and PHM, respectively. These polymers have been used as starting materials to produce biodegradable hydrogels for colon drug delivery by means of UV cross-linking in aqueous solution and in the absence of photoinitiators.10 In addition, as previously reported, succinic derivatives of Dex-MA (Dex-MA-SA) and PHM (PHM-SA) have been prepared and successfully employed to produce pH-sensitive hydrogels.9,11 Now, in order to obtain a better drug targeting to the colon, we have prepared a hydrogel based on Dex-MA-SA and PHM-SA showing both colonic biodegradability and pH-sensitive behavior. 3.1. Preparation of the Hydrogel. The high reactivity of methacrylic groups in Dex-MA-SA and PHM-SA toward radical reactions activated by UV rays, allows one to produce hydrogels even in the absence of photoinitiators. It is well-known that these molecules, because of their high reactivity, are often very toxic; as a consequence, the possibility to produce hydrogels without the use of photoinitiators gives the advantage of a potential biocompatibility of the resulting biomaterial. Moreover, UV cross-linking is a very advantageous method if compared with the conventional chemical cross-linking, for its safety, ease, and low cost.16,17 Preliminary experiments were carried out in order to evaluate the optimal conditions to perform the photocross-linking reaction. The solutions, prepared by mixing aqueous solutions of each polymer at concentration 7% w/v, with a weight ratio PHM-SA /Dex-MA-SA equal to 4, were irradiated at 313 nm for 90 min (Scheme 1). It is evident that a chemical hydrogel is formed where all the groups belonging to the starting polymers, such as glucosidic, ester, and amide linkages, as well as free carboxylic groups, are present. Obviously, due to the nature of these groups, several hydrogen bonds are also present in the network, as showed in the Scheme 1. 3.2. FT-IR Analysis. The FT-IR spectrum of Dex-MA-SA/ PHM-SA based hydrogel is reported in Figure 1 and compared with those of uncross-linked Dex-MA-SA and PHM-SA. It shows the following principal features: a broadband centered at 3360 cm-1 (νOH of Dex-MA-SA and νOH + νNH of PHMSA); bands at 1718 (νCOO ester), 1660 (amide I of PHM-SA), 1540 (amide II of PHM-SA) cm-1. In the region between 1400 and 400 cm-1, there are bands belonging to Dex-MA-SA and PHM-SA. The disappearance of the peaks at 814 and 950 cm-1 (wagging -CdC-H) belonging to Dex-MA-SA and PHM-SA, respectively, and of the peak at 1300 cm-1 (scissoring -CdCH) belonging to both polymers, confirmed that the cross-linking reaction involves the opening of the double bonds of both polymers, probably through the formation of free radicals that lead to inter- and intrapolymeric carbon-carbon cross-linked bonds. 3.3. Swelling Studies. The swelling behavior is one of the most important properties for a hydrogel designed for biomedical purpose. The biocompatibility, the chemical or enzymatic degradability, and the permeability/diffusion of molecules (for example drugs) through the network strongly depend on this characteristic. For these reasons and in order to evaluate the

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Scheme 1. Schematic representation of UV cross-linking between Dex-MA-SA and PHM-SA

effect of the environmental pH on the swelling behavior of DexMA-SA/PHM-SA based hydrogel, swelling studies were performed in various aqueous media, such as twice-distilled water, SGF, and SIF. In Table 1, equilibrium swelling data, obtained after 2 h in SGF and 24 h in twice-distilled water or in SIF, are reported. These results show the good affinity of the hydrogel toward the aqueous media and a pronounced pH-sensitive behavior; in fact, when pH is 6.8 (SIF), the swelling is 2.5 times greater than the swelling showed at pH 1.0 (SGF). Moreover, the q value in SGF or SIF is lower than that measured in twicedistilled water, probably due to the ionic strength and osmotic pressure of these media.

Table 1. Values of Weight Swelling Ratio, q, of Dex-MA-SA/ PHM-SA Based Hydrogel Determined after 2 h of Contact with SGF and after 24 h of Contact with Twice-Distilled Water or SIFa weight swelling ratio, q

a

twice-distilled water

SGF

SIF

23.3 ( 0.5

4.6 ( 0.2

11.5 ( 0.3

Values are reported as mean ( standard deviation (n ) 3).

Figure 2. Dynamic weight swelling ratio, q, measured in simulated gastrointestinal conditions: the sample was incubated in SGF for 2 h and subsequently in the SIF until 24 h.

Figure 1. FT-IR spectra of Dex-MA-SA (a), PHM-SA (b), and crosslinked Dex-MA-SA/PHM-SA (c).

The pH-responsive swelling is also showed in Figure 2, which reports the dynamic swelling, i.e., data are determined in a continuous way in SGF until 2 h, then in SIF until 24 h, in order to mimic the gastrointestinal transit. As expected, there is a considerable increase in the q value in SIF respect to the value measured in SGF. This behavior is probably due to the pendant acid groups present in the network, which are undissociated at the low pH value in SGF, but they become mostly dissociated at pH 6.8 in SIF, thus causing an electrostatic repulsion between the polymeric chains and a consequent increase in the water uptake.

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Table 2. Values of Yield % (w/w) and Weight Swelling Ratio, q, for Dex-MA-SA/PHM-SA Based Hydrogel, Determined after Chemical Hydrolysis in SGF for 2 h, in SIF pH 6.8 or PBS pH 7.4 for 24 ha qb in twice-distilled water after chemical hydrolysis

yield % (w/w) after chemical hydrolysis

a

SGF

SIF

PBS

96.0 ( 2.0

93.0 ( 1.2

95.2 ( 1.0

Values are reported as mean ( standard deviation (n ) 3).

b

SGF

SIF

PBS

24.0 ( 0.3

26.0 ( 0.4

23.9 ( 0.6

The q values were measured after 24 h.

Table 3. Values of Yield % (w/w) and Weight Swelling Ratio, q, for Dex-MA-SA/PHM-SA Based Hydrogel Determined after Enzymatic Hydrolysis in SIF pH 6.8 or in PBS pH 7.4 for 24 h in the Presence of Dextranase and/or Esterasea qb in twice-distilled water after enzymatic hydrolysis

yield % (w/w) after enzymatic hydrolysis dextranase in SIF

esterase in PBS

dextranase/ esterase in SIF

dextranase/ esterase in PBS

dextranase in SIF

esterase in PBS

dextranase/ esterase in SIF

dextranase/ esterase in PBS

85.5 ( 2.0

87.8 ( 1.0

72.0 ( 1.0

75.0 ( 1.5

34.0 ( 0.3

32.2 ( 0.2

47.0 ( 0.1

41.0 ( 0.3

a

Values are reported as mean ( standard deviation (n ) 3).

b

The q values were measured after 24 h.

3.4. Chemical and Enzymatic Hydrolysis Studies. Taking into account the presence of potentially degradable linkage in the structure of Dex-MA-SA/PHM-SA based hydrogel, such as glucosidic, ester, and amide groups, chemical and enzymatic degradation studies were performed in simulated physiological conditions. In particular, the sample was incubated at 37 °C for 2 h in SGF (pH 1.0) and, subsequently, for 24 h in SIF (pH 6.8) or in PBS (pH 7.4), with or without the presence of dextranase (15 U/ml) and/or esterase (125 U/ml). The entity of degradation was evaluated by determining both the loss in weight and the increase in the weight swelling ratio q of the sample recovered after the treatment. The values reported in Table 2 show that the hydrogel undergoes a negligible chemical hydrolysis with a small decrease in the yield of recovered hydrogel and a slight increase in the q value. On the contrary, the hydrogel is partially degraded after 24 h of incubation with dextranase and esterase, as evidenced by the pronounced increase in the q value (Table 3). It is also evident that the mixture dextranse/esterase is more effective than each single enzyme. These results confirm that the combination of the polyaminoacid derivative with the dextran derivative is a useful method to allow the degradation by dextranase even when the derivatization molar degree of dextran in methacrylic groups is high (18 mol %). On the contrary, cross-linked Dex-MA based hydrogels are hardly degraded by enzymes if the DD in methacrylic groups is greater than 7 mol %.8 It is likely that the polyaminoacid chains confer to the composite hydrogel a permeability to dextranase greater than that of Dex-MA hydrogels, probably due to the formation of a less compact network. Furthermore, degradation of the hydrogel was measured by changes in a bulk property, such as the weight swelling ratio, q. Because a pronounced increase in the q value was observed after the incubation with dextranase and esterase, the degradation could be a bulk rather than a surface phenomenon. Therefore it is probable that, after an initial surface erosion, enzymes diffuse into the polymeric network, thus causing a degradation of the more internal sites with a consequent pronounced increase in the swelling. Similar considerations have been reported for the enzymatic degradation of other hydrogels.18 3.5. In Vitro Cell Compatibility Studies. The biocompatibility of the new hydrogel was evaluated in vitro on Caco-2 cells, chosen as a model of human intestinal cells by “direct” and “indirect” methods (see Experimental Section). In the direct method, hydrogel was kept in direct contact with a Caco-2 cell monolayer for 24 h (see Figure 3), then cell viability was evaluated by the trypan blue exclusion assay and MTS assay. Cell viability was expressed as percentage ratio

Figure 3. Optical microscopic images of Caco-2 cell monolayer (magnification 200×) after 24 h of incubation in complete RPMI-1640 in direct contact with Dex-MA-SA/PHM-SA based hydrogel (a), or in the absence of the hydrogel as a control (b).

between viable cell number and total cell number, and results were compared with cells control incubated in the growth medium without the presence of the hydrogel. In the indirect method, the growth medium was first “conditioned” for 5 days with the hydrogel and then used to incubate the cells. After 24 h of incubation, cell viability was determined by the trypan blue exclusion assay and MTS assay and expressed as a percentage ratio between viable cell number and total cell number. The results were compared with cells control incubated in the growth medium not “conditioned” by the hydrogel. Figure 4 shows the results obtained for direct and indirect method by using the trypan blue exclusion assay. It is evident that no significant difference was found between the sample and the control. Analogous results were obtained by performing

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Figure 4. Cell viability data evaluated by the trypan blue exclusion assay on Caco-2 cells after 24 h of incubation in direct contact with Dex-MA-SA/PHM-SA based hydrogel (direct method) or in RPMI1640 medium previously “conditioned” by the hydrogel (indirect method). Cell viability data are reported as percentage ratio between viable cell number and total cell number. Cells cultured in fresh complete RPMI-1640 medium were used as a control.

the MTS assay. This suggests that Dex-MA-SA/PHM-SA based hydrogel does not release, in the growth medium, substances that interfere with the cell viability, and it does not cause a decrease in the cell viability after direct contact with cells. 3.6. Studies of Interaction with Mucin. The ability of an oral drug delivery system to remain at the absorption site for a prolonged time is a very important objective for a local treatment as well as for a sustained systemic absorption. An effective strategy to obtain a hydrogel able to adhere to the intestinal mucosa is the use of starting polymers with mucoadhesive properties, i.e., able to interact with mucin present in the intestinal mucus.19 Therefore, with the aim to have a preliminary information about the potential bioadhesive properties of DexMA-SA and PHM-SA, the interaction between these polymers and mucin has been evaluated by a turbidimetric method (already used successfully by other authors20,21) employing hyaluronic acid sodium salt, a known bioadhesive polymer, as a positive control. In particular, Dex-MA-SA, PHM-SA, a DexMA-SA/PHM-SA 1:4 mixture or hyaluronic acid sodium salt were added to a 0.1% w/v mucin dispersion (obtaining a final weight ratio polymer/mucin equal to 1) and, at established times of incubation, the transmittance values of the polymer-mucin mixture were recorded at 500 nm. The greater the interaction between the polymer and mucin is, the lower the transmittance value of the investigated sample (greater UV–vis scattering) due to the formation of macro-aggregates is.20,21 Data reported in Figure 5 show that both the polymers alone and their mixture show a mucoadhesive behavior, comparable to the positive control. Moreover, it is evident that after 24 h of incubation with mucin, the transmittance value of each sample becomes constant, thus suggesting that the maximum interaction polymer-glycoprotein has been reached. It is reasonable to suppose that also Dex-MA-SA/PHM-SA based hydrogel could have a mucoadhesive behavior potentially useful for a sustained drug release. 3.7. Drug Release Studies. To investigate the suitability of Dex-MA-SA/PHM-SA based hydrogel as an oral drug delivery system, 2-methoxyestradiol (2-ME), an antiangiogenic agent proposed for the oral therapy of the colorectal cancer, was chosen as a model drug. The drug loading was carried out by soaking the hydrogel in a concentrated drug solution in ethanol. Because the hydrogel

Casadei et al.

Figure 5. Transmittance % values at 500 nm of mucin dispersions in the presence of Dex-MA-SA, PHM-SA, Dex-MA-SA/PHM-SA (D/P) 1:4 mixture or hyaluronic acid sodium salt (HA) chosen as a positive control.

Figure 6. 2-ME release profile from Dex-MA-SA/PHM-SA based hydrogel in gastrointestinal simulated fluids. The hydrogel was incubated in SGF for 2 h and then in SIF for 46 h in the presence (continuous line) or in the absence (broken line) of dextranase/ esterase mixture.

can also swell in ethanol, it is possible to obtain the diffusion of drug molecules into the polymeric network, thus obtaining an effective drug entrapment (drug loading 10% w/w). Figure 6 reports drug release data, expressed as percentage of delivered drug, related to the entrapped total dose, as a function of time in SGF for the first 2 and in SIF in the absence or in the presence of a mixture of dextranase (15 U/ml) and esterase (125 U/ml) from 2 to 48 h. This time was chosen taking into consideration the potential mucoadhesion of the hydrogel that could prolong the residence time in the intestinal tract. It is possible that the drug release is due to a combination of the effect of the hydrogel and the low aqueous solubility of the drug. However, by observing the release profile obtained for the investigated sample, it seems that the predominant effect is due to the nature of the hydrogel. In fact, the release profile is biphasic, being lower at acidic pH (about 6% of drug released after 2 h) and higher when pH is 6.8 (about 36% after 48 h) according to the pH-sensitivity of the hydrogel, i.e., the higher swelling of the hydrogel in SIF causes a greater drug release. Another evidence of the hydrogel influence is due to the effect of dextranase and esterase that increase the drug release (more than 87% after 48 h) as a consequence of the hydrogel degradation. On the contrary, when enzymes are absent, hydrogel does not release all the dose of the entrapped drug even after 46 h of contact with SIF because it is necessary, besides swelling, to degrade internal sites to allow an almost complete drug release.

Hydrogels for Potential Colon-Specific Drug Delivery

4. Conclusions A novel composite polysaccharide/polyaminoacid hydrogel has been produced by photocross-linking of a methacrylated succinic dextran in the presence of a methacrylated succinic derivative of R,β-poly(N-2-hydroxyethyl)-DL-aspartamide, named Dex-MA-SA and PHM-SA, respectively. Photocross-linking has been performed by irradiating an aqueous polymer solution at 313 nm for 90 min, in the absence of photoinitiators. The resulting hydrogel shows a pH-responsive swelling, a chemical resistance in simulated gastrointesinal fluids, but it undergoes a degradation by dextranase and esterase. In vitro cell compatibility studies on Caco-2 cells indicate the absence of toxic effects due to the hydrogel or its degradation products. In vitro drug release studies, performed using 2-methoxyestradiol as a model drug, show that Dex-MA-SA/PHM-SA based hydrogel is able to release the drug in simulated intestinal fluid, especially in the presence of dextranase and esterase. Furthermore, the potential mucoadhesive behavior of the hydrogel could promote drug release in the site of action for a prolonged time. The obtained results show that this polysaccharide/polyaminoacid hydrogel is potentially useful for the oral treatment of colonic cancer. Acknowledgment. This work has been financially supported by MIUR (Ministero dell’Istruzione, dell’Università e della Ricerca) grants.

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