Biocompatible and Mucoadhesive Bacterial Cellulose-g-Poly(acrylic

Article pubs.acs.org/molecularpharmaceutics

Biocompatible and Mucoadhesive Bacterial Cellulose-g-Poly(acrylic acid) Hydrogels for Oral Protein Delivery Naveed Ahmad,† Mohd Cairul Iqbal Mohd Amin,*,† Shalela Mohd Mahali,‡ Ismanizan Ismail,§,∥ and Victor Tuan Giam Chuang⊥ †

Centre for Drug Delivery Research, Faculty of Pharmacy, Universiti Kebangsaan Malaysia, 50300 Kuala Lumpur, Malaysia School of Informatics & Applied Mathematics, Universiti Malaysia Terengganu, 21030 Kuala Terengganu, Terengganu, Malaysia § Institute of Systems Biology (INBIOSIS), Universiti Kebangsaan Malaysia, 43600 Bangi, Selangor, Malaysia ∥ School of Biosciences and Biotechnology, Faculty of Science and Technology, Universiti Kebangsaan Malaysia, 43600 Bangi, Selangor, Malaysia ⊥ School of Pharmacy, Curtin Health Innovation Research Institute, Faculty of Health Sciences, Curtin University, Perth, Western Australia 6845, Australia ‡

ABSTRACT: Stimuli-responsive bacterial cellulose-g-poly(acrylic acid) hydrogels were investigated for their potential use as an oral delivery system for proteins. These hydrogels were synthesized using electron beam irradiation without any cross-linking agents, thereby eliminating any potential toxic effects associated with cross-linkers. Bovine serum albumin (BSA), a model protein drug, was loaded into the hydrogels, and the release profile in simulated gastrointestinal fluids was investigated. Cumulative release of less than 10% in simulated gastric fluid (SGF) demonstrated the potential of these hydrogels to protect BSA from the acidic environment of the stomach. Subsequent conformational stability analyses of released BSA by SDS-PAGE, circular dichroism, and an esterase activity assay indicated that the structural integrity and bioactivity of BSA was maintained and preserved by the hydrogels. Furthermore, an increase in BSA penetration across intestinal mucosa tissue was observed in an ex vivo penetration experiment. Our fabricated hydrogels exhibited excellent cytocompatibility and showed no sign of toxicity, indicating the safety of these hydrogels for in vivo applications. KEYWORDS: bacterial cellulose, electron beam, hydrogels, oral protein delivery, protein conformational stability, biocompatibility

1. INTRODUCTION The advent of hybridoma and recombinant DNA technologies has accelerated the large-scale manufacture of therapeutic proteins. Due to their superior safety profile, tolerance, and low immunogenicity, the role of these biotechnologically produced therapeutic proteins has grown significantly in almost every field of medicine. In addition, therapeutic proteins are capable of performing highly specific and complex functions that may not be achieved by other therapeutic moieties.1,2 The specificity and biofunctionality of therapeutic proteins is attributed to their delicate three-dimensional structure that is subject to proteolytic, chemical, and physical degradation, which may result in the loss of activity and elicit an immune response.3,4 Therefore, most therapeutic proteins are administered parenterally; however, the pain, trauma, and discomfort associated with frequent injections often leads to low patient compliance and restricts the application and acceptance of proteins.1 Patient compliance may best be obtained with safe and effective oral administration of the proteins, but oral administration of these drugs is challenged by chemical, © XXXX American Chemical Society

enzymatic, and absorbance barriers in the gastrointestinal tract (GIT).5−8 Numerous strategies have been adapted to circumvent these challenges, such as the use of absorption enhancers, enzyme inhibitors, particulate drug delivery systems, mucoadhesive biopolymers, and formulations to protect drugs from the harsh environment of the GIT. Most of these strategies offer long-term possibilities for oral protein delivery, but none of them has been proven to be successful for clinical application.7,9 Hence, drug delivery scientists are still presented with the challenge of developing safe and effective oral delivery methods for therapeutic proteins. Stimuli-responsive and mucoadhesive polymeric hydrogels have shown potential promise as candidate biomaterials for oral protein delivery.7,10,11 Hydrogels are three-dimensional polymeric networks that imbibe large amounts of water while Received: April 24, 2014 Revised: September 19, 2014 Accepted: September 24, 2014

A

dx.doi.org/10.1021/mp5003015 | Mol. Pharmaceutics XXXX, XXX, XXX−XXX

Molecular Pharmaceutics

Article

Table 1. Composition, Gel Fraction, and pH-Responsive Swelling Index of BC-g-P(AA) Hydrogelsa

a

hydrogel (code)

acrylic acid (% v/v)

1% bacterial cellulose (% v/v)

electron beam dose (kGy)

gel fraction (%)

responsive index (%

208035 307035 406035

20% 30% 40%

80% 70% 60%

35 35 35

60.2 ± 3.4# 86.7 ± 1.7* 90.6 ± 3.1*

243.1 ± 8.2# 199.4 ± 5.7* 175.9 ± 6.3*#

The asterisk (*) represents significant difference (*p < 0.05) from 208035 and hash (#) represents significant difference (#p< 0.05) from 307035.

2. EXPERIMENTAL SECTION 2.1. Materials. Nata de coco as source of BC was purchased from the Nuraim industries (Malaysia). AA, BSA, Bradford reagent, Laemmli sample buffer, 30% acrylamide/bis-acrylamide solution, fluorescein isothiocyanate-BSA conjugate (FITCBSA), 4-nitrophenyl acetate, p-nitrophenol, and propidium iodide (PI) were supplied by Sigma-Aldrich, Malaysia. Prestained protein marker, broad range (7−175 kDa) was purchased from New England BioLabs, U.K.. SGF and SIF without enzymes were prepared according to the procedure described by the United States Pharmacopoeia (2010). Human colorectal adenocarcinoma cells (Caco-2) were purchased from the American Type Culture Collection (ATCC, U.S.A.). Dulbecco’s modified Eagle medium (DMEM), fetal bovine serum (FBS), trypsin-ethylenediaminetetraacetic acid (EDTA), Alamar Blue, and LIVE/DEAD Cell viability/Cytotoxicity Kit was purchased from Life Technologies (U.S.A.). The in vivo acute oral toxicity studies were approved by the Animal Ethics Committee of the Universiti Kebangsaan Malaysia. Wistar rats weighing 250 ± 25 g were obtained from the Laboratory Animal Center of the Universiti Kebangsaan Malaysia and were used for oral acute toxicity tests. The animals were housed at a controlled temperature of 20−22 °C, relative humidity of 50− 60%, and under 12 h light−dark cycles. Free access to food and water was allowed. 2.2. Preparation of Microfine Bacterial Cellulose Powder. Microfine BC powder was prepared from nata de coco cubes, according to our previously reported method.21 Briefly, nata de coco was washed several times with distilled water then heated in a 1 N NaOH solution at 90 °C for 1 h to remove bacteria, debris, and culture media. Samples were subjected to a final wash and rewash with distilled water to ensure neutrality. The resultant purified BC was freeze-dried at −110 °C (Coolsafe-110, Scan Vac, Denmark) for 48 h and micronized in a variable-speed rotor mill (Pulverisette14; Fritsch, Germany) to obtain microfine BC powder of particle size ≤50 μm (determined by sieve analysis). 2.3. Hydrogel Synthesis. BC-g-P(AA) hydrogels were synthesized using the EB irradiation technique, as previously reported.20 Briefly, a 1% (w/v) dispersion of microfine BC was homogenized with AA solution at different ratios to produce homogeneous dispersion. The BC/AA dispersions were then poured into plastic molds and exposed to EB irradiation at 35 kGy using an EB accelerator (EPS-3000, Japan) at room temperature. The EB irradiation doses were induced at 5 kGy/ pass with an accelerator energy of 3 MeV and 1 mA beam current. The resultant BC-g-P(AA) hydrogels were then dried at 60 °C to a constant weight (Go) and extracted in distilled water for 7 days to remove unreacted AA monomers and BC. The extracted hydrogels were again dried to a constant weight (G1) and gel fraction (GF) was determined by using eq 1.

remaining insoluble due to the physical or chemical crosslinking of individual polymer chains.12,13 Ideally, a hydrogel system for oral protein delivery should be biocompatible, pH responsive to protect proteins from the harsh stomach environment, mucoadhesive to prolong the hydrogel residence time in the intestines, and capable of decreasing permeation barriers in the intestine.14 Stimuli-responsive hydrogels undergo dramatic changes in swelling and network structure in response to environmental stimuli such as pH, temperature, ionic strength, enzymes, and light. Stimuli-responsive behavior, particularly pH responsiveness, has been shown to increase the hydrogel ability to protect proteins from the harsh stomach environment and eventual release in the intestine. In addition, incorporation of mucoadhesive polymers into the hydrogel structure has been shown to prolong the duration of hydrogel residence in the intestine, thereby enhancing the absorbance of protein by maintaining intimate contact with mucus.7,13 Recently, numerous studies investigating natural and synthetic hydrogels for oral protein delivery have emerged.15−17 We have recently fabricated stimuli-responsive hydrogels from bacterial cellulose (BC) and acrylic acid (AA) using electron beam (EB) irradiation. The individual components of these hydrogels (BC and AA) impart appealing characteristics for the oral delivery of proteins. BC synthesized by bacteria, such as Acetobacter xylinium spp., is superior to other cellulose sources in terms of purity, structure, mechanical strength, degree of polymerization, water absorption, and resistance to corrosive chemicals. In addition to various biomedical and pharmaceutical applications, BC has also recently been investigated for protein delivery.18−22 AA is widely used in hydrogel formulations to provide pH responsive behavior; additionally, AA and its analogues have been reported to augment mucoadhesion, enzyme inhibition, and paracellular transport.13 The use of EB irradiation to prepare the bacterial cellulose-g-poly(acrylic acid) (BC-g-P(AA) hydrogels was advantageous because strong hydrogels could be produced under mild reaction conditions without the use of (toxic) crosslinking agents.26 Moreover, the degree of cross-linking, swelling, pore size, mechanical strength, and drug release profile of the hydrogels could be fine-tuned by varying the EB radiation dose, as demonstrated in previous reports.23,29 In the present work, we investigated the oral protein delivery potential of BC-g-P(AA) hydrogels. Hydrogels were synthesized by EB irradiation and the controlled release of bovine serum albumin (BSA) protein was investigated in vitro in simulated gastric fluid (SGF) and simulated intestinal fluid (SIF). Subsequently, the structural stability and bioactivity of the released BSA was conformed. Moreover, mucoadhesion and ex vivo protein penetration was investigated to estimate the hydrogel potential to increase the bioavailability of proteins. Finally, the cytocompatibility and acute oral toxicity of the hydrogels was assessed to investigate the safety of hydrogels for in vivo applications.

%GF = (G1/Go) × 100% B

(1)



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Switzerland) at 595 nm. The entrapment efficiency (EE) of BSA in the hydrogels was calculated using eq 3

The hydrogel sheets were then cut into disks of 1 cm diameter and stored in desiccators. The detailed composition of hydrogels and their representative codes are shown in Table 1. 2.4. Hydrogel Characterization. Solid-state cross-polarization/magic angle spinning (CPMAS) 13C NMR spectra of the hydrogels were recorded using an NMR spectrometer (Avance 400; Bruker, Germany). Hydrogels were pulverized (Pulverisette 14, Frisch, Idar-Oberstein, Germany), and 100 mg of samples were packed into the 4 mm inner diameter cylindrical zirconium oxide MAS rotor with an O-ring seal and end-cap. Chemical shifts were recorded in ppm with reference 29.50 ± 0.10 ppm (CH) and 38.56 ± 0.10 ppm (CH2) using adamantine as the external standard. X-ray diffractrograms of the micronized hydrogels were recorded using an X-ray diffractometer (D8-Advance, Bruker AXS, Germany) with Cu Kα radiation at 40 kV and 50 mA in the differential angle range of 5−80° (2θ). Scanning electron microscope (SEM) images of hydrogels were acquired using an SEM (Quanta 200, FEI, The Netherlands). The swollen freeze-dried BC-g-P(AA) hydrogel disks were sputter-coated with gold in an argon atmosphere and then imaged at 20 kV. Rheological characterization of hydrogels was performed using a Bohlin Gemini II rheometer (Malvern Instruments, U.K.). The oscillatory frequency sweep measurements were recorded in a frequency range of 0.1−15 Hz at constant strain of 0.1%. The measurements were recorded using 20 mm parallel plate geometry with a gap of 2 mm at 30 °C. 2.5. Dynamic Swelling/Deswelling. The dynamic swelling studies of hydrogels were performed by alternatively immersing the hydrogel disks in SGF and SIF at 37 °C. Initially, the dried hydrogel disks were immersed in SGF (pH 1.2) and weighed after blotting at 10, 20, 40, and 60 min. Then, the same hydrogels were transferred from SGF to SIF (pH 6.8) and weighed at 70, 80, 100, and 120 min. The percent swelling ratio (% SR) of the hydrogels at different time points was calculated using eq 2. This pulsatile swelling/deswelling behavior of the hydrogels in SGF and SIF was observed for 8 h, and the responsive index (RI) of the hydrogels was calculated using eq 2a %SR = (Gs − Gd /Gd) × 100

EE = (Wo − Wf )/Wo × 100

where Wo is the total amount of BSA in the loading solution and Wf is the residual amount of BSA in the solution after loading. Measurement of the BSA released from loaded hydrogel disks was performed by immersing the loaded hydrogel in 25 mL of SGF for 2 h and then transferring it to SIF until maximum release. The release studies were performed at 37 °C with constant agitation at 50 rpm. After fixed intervals, 20 μL samples were drawn from the release medium and replaced with fresh medium. The concentration of BSA in the samples was determined according to the BSA standard calibration curve prepared using the Bradford reagent. 2.7. Conformational Stability Studies of BSA Released from Hydrogels. 2.7.1. SDS-PAGE Analysis. The integrity of BSA released from the hydrogels was analyzed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDSPAGE). The release studies of BSA were performed as mentioned in section 2.6, and aliquots (200 μL) were drawn from the release medium after 8 h. The BSA concentration was determined using the Bradford reagent and adjusted to 10 μg/ mL using SIF. These BSA samples were then mixed with Laemmli sample buffer (1:1) and incubated at 95 °C for 4−5 min. Subsequently, 10 μL aliquots were loaded on a 4% stacking and 10% resolving gel (Mini-PROTEAN Tetra Cell: Bio-Rad Laboratories, Germany) and run at a constant voltage of 200 V (Power Pac 1000; Bio-Rad) in running buffer for 1 h. The gel was then carefully removed, washed, and stained with Coomassie blue for 2 h, then destained in methanol/water/acid acetic 50:40:10 (v/v/v) for 12h. 2.7.2. Circular Dichroism Spectropolarimetry. The stability of the secondary and tertiary structures of BSA released from the hydrogels was confirmed by circular dichroism (CD) spectropolarimetry (Jasco J-810 spectropolarimeter). The concentration of BSA released from the hydrogels was adjusted to 0.01% and 0.2% (w/v) for secondary and tertiary structure analysis, respectively. The CD analysis was performed using a 0.1 cm path length quartz cell, and parameters were as follows: 1 nm bandwidth, 10 mdeg sensitivity, 0.2 nm resolution, 4 s response time, and 100 nm min−1 scanning. The samples were scanned over a far-UV range of 200−240 nm for secondary structure analysis and a near-UV range of 250−350 nm for tertiary structure analysis. The secondary structure of BSA was estimated using the K2D program from Dichroweb online server.24,25 2.7.3. BSA Bioactivity. The esterase activity of the native and released BSA samples was determined to confirm the retention of the bioactivity of the BSA released from the hydrogels. The assay was performed by adding 20 mM of released BSA (at different time intervals in SIF) in 50 mM of substrate (pnitrophenyl acetate) solution in 0.1 M PBS (pH 7.4) at 37 °C. The formation of product (p-nitrophenol) from the substrate was measured at 405 nm using a UV spectrophotometer. The esterase activity of the native BSA solution was considered to be 100%, and activity of released BSA was compared to that value. 2.8. Mucoadhesion Study. Mucoadhesion of the hydrogel disks was determined using a texture analyzer (CT3, Brookfield, U.S.A.) with accessories including a fixture base table (TA-BT KIT), dual extrusion cell probe (TA-DEC), and using tension

(2)

where Gs and Gd represent the swollen and dried weights of the hydrogels, respectively RI = %SR(6.8), t = 120 − %SR (pH1.2), t = 180

(3)

(2a)

where SR (pH 6.8), t = 120 and SR (pH 1.2), t = 180 are the swelling ratios at t = 120 min (pH 6.8) and t = 180 min (pH 1.2), respectively. 2.6. Protein Release from Hydrogels. BC-g-P(AA) hydrogels were loaded with BSA, used as the model protein drug, using a swelling diffusion method. Dry hydrogel disks were weighed and immersed in 25 mL of BSA solution (0.5% in PBS at pH 7.4) and incubated at 37 °C for 24 h. Hydrogels disks were then removed from the BSA solution and washed with 0.1 N HCl and dried to a constant weight at 25 °C. The residual BSA loading solution was diluted to 50 mL, and the concentration of BSA in the residual loading solution was quantified with Bradford reagent using a BSA standard calibration curve. The standard calibration curve was prepared using BSA solutions in PBS (0.25, 0.5, 0.75, 1.0, and 1.4 mg/ mL) and the Bradford reagent for detection. The absorbance was measured using a plate reader (Tecan infinite M200 Pro, C



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type test in “withhold time” mode. The test parameters were as follows: 10 kg load cell, 0.5 mm s−1 test speed, 60 s contact time, and 1 N applied force. The experiments were performed on the jejunum and colon sections of goat intestine. Briefly, the jejunum and colon sections of the goat intestine were obtained immediately after slaughter at a local slaughter house and washed with normal saline to remove food content; sections were used within 6 h. The intestine sections were fixed to the base stage and maintained at 37 °C during the test in 20 mL of SIF. Prehydrated hydrogel disks (in SIF for 30 min) were attached to the cylindrical probe (20 mm diameter) using double adhesive tape. During testing, the upper probe was lowered into the test medium and allowed to contact the intestinal tissue for 60 s. After 60 s, the probe was vertically raised, and adhesiveness was determined using Texture Pro CT V 1.3 Build 15 software (Brookfield, U.S.A.). 2.9. Cytocompatibility Studies. 2.9.1. Cell Culture. The Caco-2 cell line was used to assess the cytocompatibility of the BC-g-P(AA) hydrogels. Cells were cultured in DMEM supplemented with 10% FBS and 1% penicillin-streptomycin at 37 °C, in a humidified 5% CO2 air atmosphere. 2.9.2. Cell Viability Assay. The effect of the hydrogels on the viability of the Caco-2 cells was assessed using a direct contact method. Prior to analysis, hydrogel disks were autoclaved and swelled in a 24-well plate using culture medium. Cells were harvested at 80% confluence using 0.05% Trypsin/EDTA, seeded in 24-well plates at density of 1 × 105 cells per well, and incubated for 24 h. After 24 h, the swollen hydrogel disks were placed over the cells in direct contact and incubated. The cell viability was determined using the Alamar Blue assay at 24, 48, and 96 h. Cells were incubated with 10% (v/v) Alamar Blue for 4 h, after which 100 μL of the supernatant from the culture medium was transferred to a 96-well plate in triplicates, and the absorbance was measured using a plate reader (Tecan infinite M200 Pro, Switzerland) at 570 and 600 nm. The untreated cells were used as control. 2.9.3. Live/Dead Assay. The hydrogel cytotoxicity to the Caco-2 cells was qualitatively assessed using a LIVE/DEAD Viability/Cytotoxicity Kit (Life Technologies, U.S.A.). After treatment with the hydrogels for 24 h, the culture medium and hydrogels were removed and the cells were rinsed with PBS. Then, 200 μL of 2 μM calcein-AM and 4 μM of ethidium homodimer-1 solutions were added and incubated for 1 h. Subsequently, wells were washed three times with PBS and observed using an Evos FLoid microscope (Invitrogen, U.S.A.). 2.10. Ex Vivo BSA Penetration. An ex vivo BSA penetration study into the intestinal mucosa was performed using freshly excised goat intestine. The jejunum section of the goat intestine was obtained from a local slaughter house, gently washed with isotonic phosphate buffer, cut into small pieces of adequate size, spread over a 0.45 μm cellulose ester filter, and mounted on a Franz diffusion cell (0.95 cm2). To observe the transport of the BSA across the intestinal mucosa, hydrogels were loaded with FITC-BSA and placed over the mucosa in the donor compartment. The donor and recipient compartments were filled with PBS (pH 7.4) and maintained at 37 °C in a water bath under constant stirring. The FITC-BSA standard solution alone was used as the control and experiments were performed in triplicate. Tissue samples were removed from the Franz diffusion cell after 4 h, and 7 μm thick cross vertical slices were prepared using a cryostat (Leica CM1900, Wetzlar, Germany). The nuclei of the tissue samples were stained with (1:10 000) PI solution. The slides were mounted using

poly(vinyl alcohol) mounting medium with DABCO. The slides visualized using a fluorescence microscope (Carl Zeiss, Germany). 2.11. Acute Oral Toxicity in Rats. Acute oral toxicity studies were performed in healthy male Wistar rats. No lethal dose or median lethal dose (LD50) was detected in the preliminary experiments due to the great biocompatibility of the hydrogels. Therefore, the acute oral toxicity of the BC-gP(AA) hydrogels was estimated using the maximum tolerance dose (MTD). Twenty rats (250−300 g) were divided into 2 groups and fasted overnight with ad libitum water. Rats were administered the BC-g-P(AA) hydrogel powder dispersion (in deionized water) through oral gavages 2 times at a total dosage of 1000 mg/kg/day. The rats were given free access to food 4 h after administering the second dose. They were monitored for the next 2 weeks for mortality, behavior patterns, feces, hair, weight gain, injury, activity, and other clinical signs of illness. After 14 days, the rats were sacrificed by cervical dislocation, and organs (heart, liver spleen, kidney, stomach, and intestine) were collected for histopathological analysis, where tissue sections were stained with hematoxylin and eosin stain (H&E stain). 2.12. Statistical Analysis. Statistical data analyses were performed by using SPSS 19.0 software. The differences between the treated and control groups were analyzed by using Student’s t-test. One-way analysis of variance (ANOVA), followed by posthoc Tukey’s analysis, was used to compare multiple groups. P values 0.6), another approach needed to be considered. The general advection-diffusion equation for a growing domain with a constant diffusion equation, D (eq 5) can be used to define the concentration of BSA within hydrogel at time t39

Table 2. BSA Entrapment Efficiencies into Hydrogels and Both Diffusion Exponents and Diffusion Coefficients of BSA Release from Hydrogels hydrogels

entrapment efficiency (%)

diffusion exponents (n)

diffusion coefficients ( × 10−5 cm2s−1)

208035 307035 406035

54.5 ± 2.72 44.4 ± 3.12 39.7 ± 2.97

0.47 0.48 0.55

2.14 0.59 0.35

∂C = D∇2 C − ∇•(Cu) ∂t

where C is the concentration function and u is the function that represents the velocity of the growing domain. For the case C = C(r,t) and u = u(r, t) where r is a point on the radius of the hydrogel disk, the second term in eq 5 becomes

fluids (Figure 3b). BSA-loaded hydrogels were first immersed in SGF for 2 h to investigate the BSA release in simulated gastric environment.33,34 A duration of 2 h was selected for the investigation of BSA release in order to approximately mimic the gastric emptying (GE) time of human; however, GE may vary from person to person and according to an individual’s food intake.35 Furthermore, mucoadhesion of a drug delivery carrier on the surface of the stomach wall can also prolong its residence time. Initially, limited BSA release ( 0 ∂r

(7b)

by M̃ 0. Upon substituting the volume of the cylinders with Vd = πrd2h and Vc = πrc2h into the resulting equations, the formula was then simplified to

( ) r

⎧ M͠ ⎪ 0 , 0 < r ≤ rd C(r , 8h) = ⎨ Vd ⎪ rd < r < rc ⎩ 0,

2 d n=1 J α 1 rc n 2 2 rd2 M͠ t e−Dg αn t / rc = 1 − 2 − 4∑ 2 2 M͠ 0 rc J ( ) α α n 0 n ∞

M͠ 0rd2(Vc − Vd) rc2Vd

−

4 M͠ 0rd2Vc rc2Vd

n=1

∑ ∞

J12

(7c)

( α )e rd rc n

αn2J02 (αn)

−Dαn2t / rc2

(9)

The unknown parameter, D, was then determined by fitting 9 to the experimental release data using a weighted nonlinear least-squares method to minimize the fitting error:

where, C(r, t) is the concentration at radius r and time t, and Vdis the volume of the hydrogel disk at t = 8 h. M̃ 0 in eq 7c is the remainder mass in the hydrogel disk at 8 h. The above system was used to find the cumulative protein released, starting from 8 h onward. The solution for the above system, C(r,t) was then integrated from t = 8 h until time t to obtain the formula for the cumulative released mass due to diffusion M̃ t =

when t > 8 h

k=J

E(tc , D) =

∑ K

⎛ M̃ e t ⎞2 M̃ tk k ⎜⎜ (D)⎟⎟ wk − M̃ 0 ⎝ M̃ 0 ⎠

where ((M̃ etk)/(M̃ 0)) = ((Metk − Me8 hour)/(M0 − Me8 hour)) with Metkwas the experimental value at time tk, ((M̃ tk)/(M̃ 0)(D)) was the value from the formula at time tk, when the parameter D was used and {wk}K1 were positive weights. In this study, the infinite series of 9 was truncated for the first 62 terms to evaluate ((Mtk)/(M0)(D)). The weights chosen were proportional to the interval between two data points. These computation procedures were performed using MATLAB. A similar procedure was applied to data for the 208035 hydrogel, except the t = 8 h was replaced with 5 h

whent > 8 h

(8)

where Vc is the volume of the release medium in the container, J1is the first-order Bessel function, and αnis the root for J1(αn) = 0 for n = 0,1,2,.... The fractional releasewas found by dividing 8 H



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its analogues, are widely used mucoadhesive polymers in pharmaceutical formulations.44 Because BC-g-P(AA) hydrogels contain PAA that plays an important role in the mucoadhesion of hydrogels, we investigated the adhesiveness of hydrogels to goat intestinal tissue using a texture analyzer. The adhesiveness of the BC-g-P(AA) hydrogels to small intestine and colon tissue is shown in Figure 6. As expected, the hydrogels exhibited

because the fractional release achieved 0.6 at that time for that particular hydrogel. Table 2 lists the estimated effective diffusion coefficients for BSA release experiments from hydrogels 208035, 307035, and 406035 in SIF for release after ((Mt)/(M0)) > 0.6. It is shown that the 208035 hydrogel has the highest effective diffusion coefficient, followed by the 307035 and the 406035 hydrogels. Figure 4b shows the fractional release curves for the 208035, 307035, and 406035 hydrogels with the corresponding fitted curves after ((Mt)/ (M0)) > 0.6. 3.5. Conformational Stability Studies. 3.5.1. SDS-PAGE Analysis. The integrity of BSA released from the hydrogels after 8 h (section 2.4) was investigated by SDS-PAGE analysis (Figure 5a). No degradation product or formation of BSA aggregates was observed in SDS-PAGE analysis, suggesting that the structural integrity of BSA, after being loaded into hydrogels followed by exposure to harsh SGF and then release into SIF, was preserved. However, SDS-PAGE only provides information about the primary structure of BSA, therefore, the stability of secondary and tertiary structures needed to be confirmed. 3.5.2. Circular Dichroism Spectropolarimetry. The ability of the hydrogels to preserve the secondary and tertiary structures of BSA was evaluated using CD spectropolarimetry. Far-UV and near-UV spectra of native BSA and BSA released from the hydrogels in SIF are shown in Figure 5b and 5c, where each spectrum shown is an average of 5 scans, and all spectra were background corrected and smoothened. Figure 5b shows that the far-UV spectra of BSA released from hydrogels almost lines up with native BSA, suggesting that hydrogels preserved the secondary structure of BSA during the loading and release process. Furthermore, α-helix contents of the release and native BSA were calculated to be between 65−68%, which were consistent with the literature.41 Similarly, no significant difference was observed in the near-UV spectra (Figure 5c) of released and native BSA, indicating that the tertiary structure of BSA was preserved in hydrogels. 2.5.3. BSA Bioactivity. The preservation of protein bioactivity is essential for a protein delivery carrier. BSA possesses a remarkable functional activity to catalyze the hydrolysis of p-nitrophenyl acetate, known as “esterase like” activity. This activity is localized to the III A domain of the albumin structure.42,43 An esterase activity assay of BSA released from the hydrogels was performed to analyze the capability of the BC-g-P(AA) hydrogels to protect the protein bioactivity during loading and release processes. The results of the esterase activity assay for BSA released from the hydrogels are presented as the % bioactivity of native BSA in Figure 5d. These results demonstrate that over 90% esterase activity was retained by BSA released from the hydrogels. The BSA released from 406035 hydrogels retained the highest esterase activity, which was likely due to that hydrogel having the lowest swelling in SGF, resulting in lower exposure of entrapped BSA to SGF. It can be suggested from these results that hydrogels were capable of preserving the bioactivity of BSA during the loading and release processes, further indicating their potential as efficient oral drug delivery carriers for proteins. 3.6. Mucoadhesion Study. The mucoadhesion of oral drug delivery systems can play an important role in enhancing the bioavailability of protein drugs. Hydrophilic polymers with a negative charge have been reported to augment mucoadhesion and hydrogen-bond-forming groups can produce high mucoadhesion. Therefore, such anionic polymers, like AA and

Figure 6. Mucoadhesiveness of BC-g-P(AA) hydrogels to the freshly excised goat intestinal tissues (mean ± SD, n = 3). The asterisks (*) represent significant difference (*p < 0.05) from 208035 and 406035 and hash (#) represents significant difference (#p < 0.05) of colon from jejunum.

higher adhesiveness in the colon than in the jejunum, which is likely attributed to the higher mucus content in the colon. Furthermore, 307035 hydrogels exhibited the highest adhesiveness among the hydrogels (5.6 ± 0.79 N or 571 ± 80 g), likely due to better interaction between the carboxylic group of hydrogels and the mucus at this specific PAA concentration and cross-linking. Ivarsson and Wahlgren 2012, compared the mucoadhesiveness of six well-known mucoadhesive polymers: sodium carboxymethyl cellulose (CMC), hydroxyethyl cellulose (HEC), chitosan, polyvinylpyrrolidone (PVP) and two crosslinked polyacrylic acids, Noveon (hydrophobically modified) and Carbopol.45 They found that by using tensile strength method, (texture analyzer) HEC exhibited the highest adhesiveness (556 ± 141g). However, the adhesiveness of 307035 hydrogel in the present study is even higher than HEC, suggesting that this hydrogel hold high potential for the development of a mucoadhesive drug delivery carrier. 3.7. Hydrogel Cytocompatibility. Cytotoxicity, is defined as the “in vitro evaluation of toxicological risks of a material using cell culture”.46 Cytotoxicity assay is used to investigate in vitro biocompatibility of materials intended to be used for drug delivery application. This is a useful tool to scrutinize a material for in vivo application. Furthermore, determining the cytotoxicity of hydrogel systems is important because the presence of unreacted monomers, initiators, and cross-linkers may produce cytotoxicity.26 Therefore, an in vitro cytotoxicity test of hydrogels was performed using Caco-2 cells to assess the cytotoxicity of the hydrogels. As shown in Figure 7, the cell viability of all the hydrogel-treated cell lines, at the tested times, was well above 90%. The higher cyto-compatibility of the I



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3.8. Ex Vivo BSA Penetration. Ex vivo BSA penetration into intestinal mucosa was qualitatively observed by placing FITC-BSA-loaded hydrogels over goat intestinal mucosa tissue in a Franz diffusion cell. The fluorescence images from tissue sections of the intestine are shown in Figure 9. Hydrogels loaded with FITC-BSA conjugate are represented with green fluorescence, whereas the nuclei of the cells were stained red by propidium iodide. FITC-BSA conjugate solution was used as the control. We observed that the FITC-BSA solution (control) did not penetrate a greater extent into the tissue; however, the FITC-BSA release penetrated into the intestinal mucosa, as represented by the stronger green fluorescence visible in Figure 8. Furthermore, lower BSA penetration was observed from the 208035 hydrogel to the 406035 hydrogel, consistent with findings from the release study that indicated that the 406035 hydrogel exhibited the lowest release. The increased of BSA penetration into the intestinal tissue as compared to the control might be due to the presence of PAA in the hydrogel formulation. AA, and its analogues have been reported to enhance the paracellular transport of protein drugs by augmenting the opening of epithelial tight junction in intestinal epithelia.10,13,49 3.9. Acute Oral Toxicity in Rats. Although cytotoxicity provides initial toxicity information about the safety of a drug delivery system, its findings cannot replace in vivo evaluation of toxicity because many physiological and metabolic processes occur simultaneously in actual bodily systems that cannot be simulated in vitro. Therefore, acute oral toxicity studies of BC/ AA hydrogels were performed in Wistar rats using an MTD method. Rats were orally administered 1 g/KG/day of BC/AA hydrogel dispersion. The dose was selected as the maximum possible dose that can be administered orally due to the high viscosity of the swelled hydrogels at the maximum volume of oral gavage. The rats were monitored for 2 weeks for general conditions (weight, activity, mortality, hair, teeth, behavior, feces, and signs of illness). No toxic effects were observed in any of the animals at the given dose during the observation period; no mortality was observed during the study, and the behavior pattern, energy level, hair, and teeth of the BC/AAtreated group were all similar to the control group. The treated rats also gained weight similar to the control group, their eating

Figure 7. Cell viability of BC-g-P(AA) hydrogels in Caco-2 cell determined by direct contact method using Alamar Blue assay (mean ± SD, n = 6). The asterisks represent significant difference (* p < 0.05) from control.

hydrogels might be suggested due to the presence of BC that has been reported to enhance cell proliferation and cell attachment.47,48 In addition, no initiator or cross-linker was used to fabricate these BC-g-PAA hydrogels, thus eliminating the chances of cytotoxic effects due to an unreacted initiator or cross-linker.26 The presence of unreacted AA monomer, however, could have produced a cytotoxic effect. To ensure the removal unreacted AA monomers, we washed the hydrogels in DI water for 7 days (as described in sections 2.2 and 3.1). The treated and controlled cells were stained with Live/Dead assay kit (Life Technologies, U.S.A.) to visualize the cytotoxicity of the cells. The kit contains calcein-AM that produces green fluorescence in live cells, and ethidium homodimer-1 that produces red fluorescence in dead cells. As shown in Figure 8, there were not a significant number of red cells observed in the treated or control cells after 24 and 48 h of treatment, confirming the results of the cell viability assay. Therefore, these findings suggest that the BC-g-P(AA) hydrogels did not affect the metabolic activity of the Caco-2 cells and that none of the hydrogel formulations were cytotoxic to Caco-2 cells.

Figure 8. Live/dead fluorescent images of control and hydrogel treated cells, where calcein-AM (green) represents live cells, and ethidiumhomodimer-1 (red) represents dead cells. J



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Figure 9. Penetration of FITC-BSA (control) and FITC-BSA released from BC-g-P(AA) into intestinal tissues (cross vertical sections). Green fluorescence refers to FITC-BSA, and red fluorescence refers to propidium iodide.

Figure 10. Acute oral toxicity of BC-g-P(AA) hydrogels in Wistar Rats: light microscope images of hematoxylin−eosin stained tissue section dissected after 14 days of oral administration of hydrogels.

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behavior was normal, and the fecal matter was free of pus or blood. The histopathological images of the H&E-stained rat organs are shown in Figure 10. No significant histopathological changes were observed in the control or hydrogel-treated rat groups. All organs exhibited normal tissue histology without signs of necrosis, inflammation, edema, or any other pathological changes. As these hydrogels were intended for oral delivery, we also observed the histology of the stomach and small intestine to assess any localized toxic effects from the hydrogels. No ulceration or hemorrhage was observed in histograms of the stomach and intestine, suggesting that the hydrogels were safe for oral administration and could be used as an oral drug delivery system.

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel: + 6 03-92897690. Fax: +6 03 2698 3271. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors gratefully thank the Ministry of Higher Education, Malaysia (UKM-Farmasi-02-FRGS0192-2010) and Ministry of Agriculture, Malaysia (MoA: 05-01-02- SF1023) for funding this research.

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4. CONCLUSION Here, we prepared stimuli-responsive BC-g-P(AA) hydrogels by EB irradiation, and demonstrated their potential for site-specific delivery of protein-based drugs in the intestine. These hydrogels efficiently protected the structural integrity and bioactivity of proteins. Moreover, the hydrogels exhibited excellent mucoadhesive potential, which can play an important role in increasing residence time and enhance drug absorption, as demonstrated by enhanced protein penetration in the penetration studies. Furthermore, cytotoxicity and acute oral toxicity studies indicated the safety of these hydrogels for in vivo applications. On the basis of these findings, it can be concluded that BC-g-P(AA) hydrogels possess promising potential for further development as oral protein delivery carrier, however, further pharmacokinetic and pharmacodynamic studies within in vivo models are recommended.

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