Controlled release of a model protein drug ovalbumin from thiolated hyaluronic acid matrix

Journal of Drug Delivery Science and Technology 30 (2015) 74e81

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Research paper

Controlled release of a model protein drug ovalbumin from thiolated hyaluronic acid matrix Jinping Du, Fazhao Fu, Xinyue Shi, Zongning Yin* Key Laboratory of Drug Targeting and Drug Delivery Systems, West China School of Pharmacy, Sichuan University, Chengdu, Sichuan Province, PR China

a r t i c l e i n f o

a b s t r a c t

Article history: Received 9 August 2015 Received in revised form 14 September 2015 Accepted 21 September 2015 Available online 25 September 2015

The objectives of this work were to investigate the properties of the thiolated hyaluronic acid (HA) with different molecular weights (MWs) and to assess the effects of MW and dissolution medium on the release of ovalbumin from thiolated HA matrix tablets. Four HA-cysteine (HA-Cys) conjugates with increasing MWs (47, 101, 260 and 550 kDa) were synthesized. The resulting HA-Cys conjugates exhibited increased stability against hyaluronidase degradation, and such an effect became stronger with the increase of polymer MW. Swelling and erosion studies of HAs or HA-Cys conjugates based matrix tablets were performed in various media. We found that the water uptake capacity and erosion rate were strongly dependent on MW of HA, ionic strength and pH of the medium. Release studies showed that all investigated factors exerted effects on the drug release. Most of the release data based on HA matrix tablets followed super Case II transport but conformed to non-Fickian diffusion with HA-Cys conjugates. Taken together, this work managed to develop HA-Cys conjugates-based matrix tablets as controlled release systems for protein with tunable release rate through varying MWs of polymer. © 2015 Elsevier B.V. All rights reserved.

Keywords: HA-Cys Molecular weight Swelling Erosion Drug release Matrix tablets

1. Introduction Oral delivery of peptide and protein drugs is of great convenience and patient compliance. However, development of oral controlled delivery for peptide and protein drugs faces tremendous challenges [1], including low stability, low permeability of large molecules, and short half-time. Moreover, these drugs can be easily destroyed by enzymes in the gastrointestinal tract, leading to extremely low oral bioavailability and therapeutic efficacy of most peptides and proteins. Fortunately, various approaches have been explored to enhance the oral bioavailability of therapeutic proteins, such as the use of absorption enhancers [2], enzyme inhibitors [3], mucoadhesive systems [4], and some special drug delivery system [5] et al. During the last decade, thiolated polymers, the introduction of thiol functional groups to polymers, have displayed excellent mucoadhesive properties [6], permeation-enhancing effect and efflux pump inhibitory properties [7]. Additionally, thiomers showed effective resistance towards the activity of carboxypeptidase A or B, chymotrypsin and aminopeptidase, and therefore they

* Corresponding author. No. 17, Block 3, Southern Renmin Road, Chengdu 610041, PR China. Tel./fax: þ86 2885502917. E-mail address: [email protected] (Z. Yin). http://dx.doi.org/10.1016/j.jddst.2015.09.015 1773-2247/© 2015 Elsevier B.V. All rights reserved.

can protect the incorporated drugs, especially peptides and proteins, from the enzymatic degradation in the intestine [8e10]. It has been demonstrated that polycarbophilecysteine as carrier matrix of insulin can guarantee a controlled drug release over 10 h among thiolated polymers [11]. Release studies of insulin showed an almost zero-order release kinetic due to its in situ gelling properties [12]. Therefore, all above-mentioned advantages favor thiomers as a highly suitable hydrophilic matrix for the oral controlled delivery of proteins. However, it is known that drug release from hydrophilic matrix tablets is a complex process, including dissolution, diffusion and erosion mechanisms [13], and such a process depends on many factors (drug, polymer and dissolution medium) that affect the water diffuse through the matrix and erosion [14,15]. Hyaluronic acid (HA), a natural liner polysaccharide [16], is endowed with properties of strong hydration, high biocompatibility, viscoelasticity and low immunogenicity [17]. However, natural HA is sensitive to strong acid, alkali, heat, free radicals and hyaluronidase, and it is easy to be degraded, which limits its application in prolonged release formulations. Chemically modified HA [18] has been widely accepted as an alternative to efficiently solve this defect. For this purpose, thiol-modified HA has been synthesized and applied in wound healing [19], tissue engineering [20] and drug delivery [21]. This new system exhibited significant improvement in mucoadhesion, the inhibition effect of the

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conjugate towards peptidase and sustained release property [22]. Moreover, HA has a wide molecular weight (MW) range of 103e107 Da [23], and it has been known to play different roles in the body depending on its MW [24]. However, matrix tablets of thiolated HA with different MWs have not been elucidated yet, and the drug release mechanisms remain unclear. Swelling and erosion behavior information of polymer matrix may be useful to understand drug release kinetics. In the present study, we aimed to synthesize HA-cysteine (HACys) conjugates with increasing MWs (HA47k-Cys, HA101k-Cys, HA260k-Cys and HA550k-Cys), to investigate the stability of thiol groups, enzymatic degradation and the effect of the MW and dissolution medium on the swelling and erosion, to determine the predominant release mechanisms via drug release in vitro, and to discuss their applicability as controlled release systems for protein. 2. Materials and methods

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solutions were incubated at 37 ± 0.5  C under continuous shaking (100 rpm). At predetermined time points, aliquots were withdrawn, and the amount of remaining free thiol groups was determined using a spectrophotometer with Ellman's reagent as abovementioned. 2.3.4. Degradation by hyaluronidase The amount of hyaluronic acid was carried out by colorimetric reaction of HA with cetyltrimethyl ammonium bromide (CTAB) according to the method described by Ferrante [27]. In order to determine the enzymatic degradation, solutions of HAs or HA-Cys conjugates (0.5 mg/ml) were prepared with 0.2 M acetate buffer (pH 6.0) containing 0.15 M NaCl, followed by addition of 1 mg/ml hyaluronidase solution (1.0 mL), respectively. The mixtures were incubated at 37 ± 0.5  C under continuous shaking (100 rpm), and 1 mL aliquots of the mixture were withdrawn at different times. Percentage of enzymatic degradation was calculated using equation as follows Eq. (1):

2.1. Materials

Pð%Þ ¼ 100*ðM0  Mt Þ=M0 HA with MWs of 47, 101, 260 and 550 kDa were purchased was from Freda (Shandong Freda Co. Ltd., China). L-cysteine, 1-ethyl-3(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDAC) and N-hydroxy-succinamide (NHS) were obtained from Aladdin (Shanghai, China). 5,50 -Dithiobis (2-nitrobenzoic acid) (DTNB) was supplied from Sigma (St. Louis, MO, USA). Ovalbumin (OVA) was provided by Sinparm Chemical Reagent Co., Ltd. Hyaluronidase (type I, 300e500 U/mg) was purchased from bovine testes. All other reagents were of analytical grade unless otherwise stated. 2.2. Synthesis of HA-Cys conjugates HA-Cys conjugates (47, 101, 260 and 550 kDa) were synthesized by the covalent attachment of cysteine to HA according to a previously described method [22]. Briefly, 1 g of each HA (47, 101, 260 and 550 kDa) was separately hydrated in deionized water followed by the addition of EDAC/NHS at a final concentration of 125 mM and 100 mM, respectively. Subsequently, the pH was adjusted to 5.5 using 0.1 M HCl. After incubation at room temperature for 45 min under stirring, L-cysteine (nLCys:nCOOH ¼ 5:1) was added, and the pH was adjusted to 5.0. Reaction mixtures were incubated at room temperature for 4 h under stirring. The resulting conjugates were dialyzed in dialysis bags (MW cut off of 12 kDa). Controls were prepared and isolated as the polymer conjugates using the same way except that EDAC and NHS were omitted during coupling reaction. 2.3. Characterization 2.3.1. FT-IR spectroscopy Samples pellets prepared with HA or HA-Cys conjugates and KBr were subjected to FT-IR spectroscopy using a Fourier-transform infrared spectrophotometer (Nicolet FT-IR, 20SXB, USA) with a frequency range of 4000e500 cm1. 2.3.2. Determination of the thiol group and disulfide contents The number of thiol groups immobilized on the polymer conjugates was spectrophotometrically determined using Ellman's reagent as previously described [25]. The disulfide contents were determined using NaBH4 for reduction [26] and Ellman's method. 2.3.3. Oxidation of thiol groups HA-Cys (47, 101, 260 and 550 kDa) conjugates were respectively dissolved in phosphate buffer (pH 5.0) and phosphate buffer (pH 7.4) containing 0.9% NaCl at a final concentration of 5 mg/ml. The

(1)

where P is the percentage of enzymatic degradation, M0 is the original amount of HA, and Mt is the amount of remaining HA after the degradation. 2.4. Preparation of matrix tablets and drug-loaded matrix tablets Briefly, 30 mg freeze-drying HA-Cys (47, 101, 260 and 550 kDa) conjugates and the corresponding unmodified HAs were compressed into flat-faced tablets (5.0 mm diameter) by single-punch tablet press (Type TDP, Shang Hai). In order to prepare OVA-loaded HA-Cys or HA matrix tablets, OVA was previously added to a polymeric solution of HA-Cys or HA in water, in a 1:5weight ratio (OVA: polymer). The solution was stirred for 10 min and freeze-dried. After that, the freeze-drying mixture were compressed into flat-faced tablets under similar conditions. The compaction pressure was maintained at 8e9 kN during the preparation of all tablets. 2.5. Swelling and erosion studies Swelling studies were performed using a dissolution apparatus (ZRS-8G, Tianjin, China) with the basket method (2010 Chinese Pharmacopoeia) in the dissolution medium of distilled water, 0.1 M saline solution, 0.2 M saline solution, HCl (pH1.2), or phosphate buffer (pH6.8). First, the dry matrix tablets without drug were weighed, and then the matrix tablets were immersed in test medium at 37 ± 0.5  C and stirred at 50 rpm. After 5, 10, 20, 40, 60, 90 and 120 min, the hydrated matrix was weighted after the surface solution was blotted with a tissue. The swelling ratio could be calculated at each time point via Eq. (2) as follows, where W0 is the initial weight, and Wt is weight of the test tablets at the time point t [28].

Swelling ¼ ½ðWt  W0 Þ=W0 

(2)

During the swelling studies, the amount of matrix erosion was analyzed at predetermined time periods via spectrophotometrically determining (UV1000, Techcomp Ltd., China) the cumulative contents of HA dissolved in test media as described above. 2.6. In-vitro drug release studies OVA release experiments were conducted under “sink” conditions by immersing the above-mentioned drug-loaded matrix

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tablets in a glass bottle filled with the dissolution medium of 10 mL distilled water, 0.1 M saline solution, 0.2 M saline solution, HCl (pH1.2) or phosphate buffer (pH6.8) at 37 ± 0.5  C under continuous shaking (100 rpm). At the suitable time intervals, 200 ml sample was withdrawn and the concentration of OVA was spectrophotometrically qualified with Bradford method [29].

3.2. Oxidation of thiol groups

2.7. Modeling of drug release profiles The difference between obtained release profiles was compared using similarity factor (f2), which was calculated according to the Eq. (3) as follows [30], where n is the number of time points, Rt is the dissolution value of the reference at time point t, and Tt is the dissolution value of the test at time point t. The release profiles were significantly different if f2 < 50. Only one measurement should be considered after 85% dissolution of both two contrastive formulations [31].

(" f2 ¼ 50g

1þ

n 1X ðRt  TtÞ2 n t1

NHS did not result in polymers with detectable thiol groups. Fig. 1 illustrates the IR spectra of HA and HA-Cys. Compared with HA, the IR spectra of HA-Cys exhibited the characteristic peak of bNH group at 1561 cm1, indicating the linkage of L-Cys to HA through amide bond.

)

#0:5  100

(3)

To further characterize the underlying mechanism of drug release from matrix tablets, the results were analyzed according to the Ritger-Peppas model Eq. (4) [32]. Only 60% of release data were used for calculation. The goodness of fit was evaluated using the correlation coefficient of determination, where the closer the value is to 1, the better the data fit to the model [33].

Mt=M∞ ¼ ktn

(4)

where Mt/M∞ is the fraction of drug released at time point t; while k is a constant incorporating structural and geometric characteristics of matrix tablet, and n is the release exponent depending on the release mechanism and the shape of the tested matrix, indicative of the drug release pattern. For cylindrical hydrophilic matrices (tablet), the n ¼ 0.45 is indicative drug release through Fickian diffusion, the values between 0.45 and 0.89 are an indication of anomalous (non-Fickian) diffusion, in which diffusion and erosion control the drug release. Values above 0.89 indicate Super Case II (mainly erosion controlled) transport [34]. 3. Results and discussion 3.1. Characterization of HA-Cys conjugates

The oxidation of thiol groups is of great importance on mucoadhesive, cohesive and in situ gelling properties of thiomers. Fig. 2 shows the oxidation of thiol groups within HA-Cys conjugates (47, 101, 260 and 550 kDa) at different pH. At pH 7.4, the amount of free thiol groups within HA47k-Cys was significantly decreased within 8 h, because more than 90% of the thiol groups were oxidized. Similarly, 80%, 73% and 60% of the thiol groups were also oxidized on HA101k-Cys, HA260k-Cys and HA550k-Cys, respectively. In contrast, the thiol displayed a lower oxidation rate at pH 5.0, and at least 80% of the thiol groups on HA550k-Cys remained stable during 24 h. Moreover, our study confirmed that the thiol groups of HACys conjugates could be easily oxidized at a higher pH value (pH 7.4) due to higher amount of negative thiolate anions (S) when the pH value was increased [35]. Fig. 2 further states that increases in MW of HA led to a reduction in oxidized rate, which could be explained by the fact that only closely located thiol groups can form disulfide bonds faster compared with the remaining isolated thiol groups [36]. However, as the MW of the polymer was increased, the chain entanglements could also be higher, resulting in a decreased flexibility and mobility of polymers [37]. Therefore, HA47k-Cys with a higher chain flexibility exhibited a higher oxidized rate. 3.3. Enzyme inhibition studies In the present study, we also evaluated the degradation of HAs and HA-Cys with hyaluronidase. Fig. 3 shows that significant difference was observed from the in vitro enzymatic degradation between HAs and HA-Cys conjugates. The amount of HA was rapidly decreased as the reaction proceeded: HAs (47, 101 and 260 kDa) were all completely hydrolyzed, and 83% of HA550k was also decreased in 1 h. In contrast, HA-Cys conjugates (47, 101, 260 and 550 kDa) displayed a reduction of 67%, 56%, 39% and 31% within 60 min, respectively, demonstrating that HA-Cys conjugates resisted hyaluronidase compared with unmodified HAs. In addition, we observed that the higher the MW, the lower the degradation rate.

The HA-Cys (47, 101, 260 and 550 kDa) conjugates were synthesized following the preparation procedures of thiolated polyacrylates. The obtained HA-Cys conjugates displayed 238.34 ± 5.49, 244.65 ± 8.48, 266.32 ± 9.09 and 212.59 ± 5.78 mmol thiol groups per gram, and their disulfide bonds were 103.45 ± 4.97, 115.07 ± 4.94, 104.46 ± 3.12 and 93.81 ± 4.15 mmol per gram of polymer, respectively. Moreover, the relative standard deviation value (RSD) of less than 5% of synthesis performed in triplicate, the synthesis method had a great reproducibility, as described in Table 1. However, incubation of L-cysteine with HA without EDC/

Table 1 Reproducibility of the synthesis method (n ¼ 3). Free thiol groups (mmol/g)

47 k

101 k

260 k

550 k

1 2 3 Average RSD%

234.18 236.27 244.56 238.34 2.3

235.33 246.74 252.29 244.79 3.53

267.71 265.99 244.97 259.56 4.88

210.87 210.84 213.48 211.73 0.72

Fig. 1. IR spectrum of HA and HA-Cys.

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Fig. 2. Decrease in thiol group content in HA-Cys(47, 101, 260 and 550 kDa) 0.5% (w/v) solution in pH5.0 (A) and pH7.4 (B), respectively, at 37 ± 0.5  C.Values are mean ± S.D. of three experiments.

Fig. 3. Comparison of in vitro enzymatic degradation between HAs and HA-Cys conjugates with different molecular weights, values are mean ± S.D. of three experiments.

The degradation tests showed a mechanical stability of the matrix in a following order: HA47k < HA101k < HA260k < HA550k z HA47kCys < HA101k-Cys < HA260k-Cys < HA550k-Cys. M.K and co-workers reported that the shorter polymeric chain presents more stretch, whereas longer polymeric chain length with higher MW obviously appears thread structure [38], which together with the thiol moieties results in the stronger steric effect towards hyaluronidase. 3.4. Swelling and erosion of the matrix tablets The swelling and erosion behavior of polymers have a great impact on their cohesiveness, stability and release of embedded drugs [39]. Fig. 4 shows the swelling and erosion profiles of HAs or HA-Cys conjugates (47, 101, 260 and 550 kDa) based matrix tablets in the dissolution medium of pH1.2 or pH6.8. In an acidic environment, the weight of HA260k-Cys and HA550k-Cys matrix tablets was increased 5-fold and 6-fold within 3 h, respectively. The pKa of HA (by virtue of the carboxyl groups on the components of uronic acid residues) is 3.21 [40], in acidic medium (pH1.2) the primarily unionized carboxyl groups of HAs or HA-Cys were able to form hydrogen bonds [41], and the share of protonated carboxylic acid groups was increased, leading to a relatively more amount of hydrophobic polymer [42], which both induced lower polymer swelling and erosion rate of matrix tablets at pH1.2 compared with pH6.8. As the pH was increased to 6.8, the weight of HA260k-Cys and HA550k-Cys matrix tablets was increased for 18-fold and 24-fold, respectively. This was likely because enhanced ionization of HAs or

HA-Cys resulted in electrostatic repulsive, relaxed the polymer chains and increased the macromolecular pore size, promoting more water infiltration [43] and better hydration of the matrix [44], which induced a higher weight loss of matrix at higher pH at same time. After 3 h, 35% and 64% mass loss of HA260k-Cys were detected at pH1.2 and pH6.8, respectively. On the other hand, the higher reactivity of the thiol groups on HA-Cys conjugates could be oxidized to form intramolecular disulphide bond [45], leading to the formation of a polymeric network within the thiomers. This could strongly improve the cohesiveness of the polymer and favor the polymer swelling and water absorption in multiples of their own weights without erosion or dissolution when hydrated [46]. In fact, matrix tablets of unmodified HAs (47, 101, 260 and 550 kDa) displayed poor water uptake, and they were dissolved very quickly due to rapid swelling and weak cohesiveness (data not shown) as reported for thiolated PAA [47]. Moreover, Fig. 4 also reveals the MW-dependent swelling and erosion behavior of matrix. The tablets prepared from HA-Cys with high MW (550 kDa) showed the greatest swelling and slowest erosion in both HCl (pH1.2) and phosphate buffer (pH6.8), followed by HA260k-Cys, HA101k-Cys and HA47k-Cys. It is reasonable since water molecules are easier to access through low MW HA structure with high hydrophilicity, causing loose gel and high erosion rate. Longer polymeric chains of HA-Cys at higher MW possess lower solubility in the release medium [48] and can form viscous and thick gel layer, resulting in low erosion rate [49]. Considering the varying ionic strength conditions, which a matrix formulation may encounter in the gastrointestinal lumen, swelling and erosion studies were conducted with the various matrices [50]. Fig. 5(A) indicates that HA550k or HA550k-Cys conjugates exhibited the fastest swelling rate in distilled water, and the swelling rate of polymer was deceased with the increase of ionic strength. Additionally, a similar behavior was observed for 47, 101 and 260 kDa (data not shown). This effect could be explained by the fact that when the ionic strength of the medium is increased, the HA or HA-Cys molecular chains lose water of hydration due to ion competition for the available water [51]. Fig. 5(B) shows that the erosion of the matrix was decreased with the addition of ions in dissolution, which could be attributed to the “salting out” of the polymer by the ions present in the dissolution media [52].

3.5. Drug release profile from matrix tablets Drug release from polymer matrix tablets is largely associated with many factors, such as structure, composition and MW of

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Fig. 4. Swelling degree(A)and erosion behavior(B)of HA-Cys(47, 101, 260 and 550 kDa) based matrix tablets at different pH, values are mean ± S.D. of three experiments.

Fig. 5. Swelling degree(A)and erosion behavior(B)of HA550k or HA550k-Cys based matrix tablets at different ionic strength, values are mean ± S.D. of three experiments.

Fig. 6. Release profiles of OVA from tablets based HAs or HA-Cys conjugates with molecular weight of (A) 47 kDa, (B) 101 kDa, (C)260 kDa, (D)550 kDa at different pH, values are mean ± S.D. of three experiments.

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between different pH also verified this finding. HA260k-Cys matrix showed higher swelling and erosion behavior in neutral medium, which was advantageous for the diffusion of drug molecules out of matrix, favoring faster release rate. It is recognized that the ionic strength significantly varies in real physiological circumstances [55]. Fig. 7 indicates that the presence of ions in the medium caused a marked decrease in the release rate for all tablets (f2 < 50). This result was consistent with the swelling and erosion studies, showing that increase in ionic strength brought a decrease in swelling and matrix erosion rate. However, when the ionic strength was increased to 0.2 M NaCl, the drug release had no significant change compared with 0.1 M NaCl (f2 > 50).

Fig. 7. Release profiles of OVA from tablets based HA260k or HA260k-Cys conjugates at different ionic strength, values are mean ± S.D. of three experiments.

polymer matrix, and pH as well as the ionic strength of the medium may also affect the drug release [53]. Fig. 5 shows the effect of polymer composition on the release behavior of model drugs in dissolution medium with different pH. Unmodified HA (47, 101, 260 and 550 kDa) tablets exhibited a strong swelling, and the total drug release was very quick. For instance, in the buffer with a pH of 6.8, 87% of OVA were liberated from HA260k tablets at 3 h in comparison to 55% from HA260k-Cys tablets. Its hydrophilic nature and the nonexistence of a cross-linking process might be the reason for a rapid penetration and dissolution of the HA260k matrix tablet as well as consequent faster release of the drug. HA-Cys (47, 101, 260 and 550 kDa) showed retarded OVA release in both media. It was possibly due to the formation of inter- and intra-molecular disulfide bonds within the thiomer and thiol of OVA combination with thiol of HA-Cys, a more entangled system was developed and the stability of the polymeric matrix tablets could be strongly improved. According to this finding, the release kinetics of OVA was affected, and a sustained release could be obtained based on HACys matrix. In addition, Fig. 6 shows that the release profile of OVA was dependent on the average molecular mass of the polymers, and the tablets prepared from HA-Cys with 47, 101, 260 and 550 kDa released 86%, 64%, 55% and 43% of OVA in the buffer with a pH of 6.8 within 3 h, respectively, which might be explained by that the increased MW of polymers resulted in slower dissolution and erosion rate of the tablet [54]. Fig. 6 shows that 72% of OVA were liberated from HA260k-Cys within 12 h at pH6.8 in comparison to 51% at pH4.5 and 17% at the pH1.2. The release results suggested a marked influence of the pH on the drug release, and the calculated similarity factor f2 < 50

3.6. Mechanism of drug release It has been known that drug release from hydrophilic matrix tablets is a complex interaction between dissolution, diffusion and erosion mechanisms [13]. Table 2 shows that the value of the exponent n was calculated using the Ritger-Peppas model. HA47k and HA101k matrix tablets did not fit to Ritger-Peppas model, because these tablets exhibited strong erosion, and their total drug release was very quick. Similar to previous findings with pectin/ calcium matrix [56], exponent n values bigger than 1.0 for tablets with HA260k and HA550k in all media, was also ascribed to a Super Case II transport, in which drug release seemed to be controlled by polymer relaxation. The good correlation with high coefficient (r) of over 0.99 between the matrix erosion and drug release for HA260k and HA550k matrix tablets (see Fig. 8), further indicating erosion controlled release mechanisms [57]. Erosion leads to the formation of new surfaces, contributing to dissolution of the drug present at the new surfaces and shortening the drug diffusional path length [58]. The release exponent ranged from 0.45 to 0.89 for HA-Cys matrix tablets, exhibiting an anomalous or non-Fickian transport. This suggested that more than one mechanism were involved in matrix erosion and diffusion of the drug in the hydrated HA-Cys matrices. 4. Conclusions In the present study, HA-Cys with increasing MW (47, 101, 260 and 550 kDa) were synthesized. They showed inhibition towards enzyme degradation, and the degradation rate was decreased with the increase of polymer MW. Swelling and erosion rate of HAs or HA-Cys conjugates matrix tablets were strongly dependent on MW of HA, ionic strength, and pH of the medium. It was found that the drug release results were consistent with the swelling and erosion

Fig. 8. Linearity between erosion and cumulative release for HA260k (A) and HA550k (B) matrix tablets at pH6.8.

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Table 2 Mathematic modeling and drug release kinetics of OVA at different media (n ¼ 3).

HA47k

HA47k-Cys

HA101k

HA101k-Cys

HA260k

HA260k-Cys

HA550k

HA550k-Cys

Media

r

n

Order of release

Media

r

n

Order of release

pH1.2 pH4.5 pH6.8 pH1.2 pH4.5 pH6.8 pH1.2 pH4.5 pH6.8 pH1.2 pH4.5 pH6.8 pH1.2 pH4.5 pH6.8 pH1.2 pH4.5 pH6.8 pH1.2 pH4.5 pH6.8 pH1.2 pH4.5 pH6.8

/ / / 0.9798 0.9896 0.9894 / / / 0.9683 0.9781 0.9843 0.9990 0.9937 0.9974 0.9494 0.9634 0.9999 0.9821 0.9813 0.9818 0.9076 0.9653 0.9920

/ / / 0.5869 0.6439 0.6250 / / / 0.6247 0.6117 0.6153 1.1706 1.3395 1.7333 0.5415 0.4558 0.8758 1.5244 1.8285 1.8505 0.7656 0.5195 0.7133

/ / / Non-Fickian Non-Fickian Non-Fickian / / / Non-Fickian Non-Fickian Non-Fickian Super case II Super case II Super case II Non-Fickian Non-Fickian Non-Fickian Super case II Super case II Super case II Non-Fickian Non-Fickian Non-Fickian

Water 0.1MNaCl 0.2MNaCl Water 0.1MNaCl 0.2MNaCl Water 0.1MNaCl 0.2MNaCl water 0.1MNaCl 0.2MNaCl Water 0.1MNaCl 0.2MNaCl water 0.1MNaCl 0.2MNaCl water 0.1MNaCl 0.2MNaCl water 0.1MNaCl 0.2MNaCl

/ / / / 0.9836 0.9833 / / / / 0.9509 0.9731 / 0.9788 0.9924 0.9666 0.9311 0.9583 0.9851 0.9775 0.9993 0.9849 0.9770 0.9099

/ / / / 1.2105 0.9133 / / / / 1.7696 1.1062 / 2.1151 1.5151 1.2622 0.7951 0.8182 1.4036 1.4376 1.5850 0.8841 0.6074 0.8565

/ / / / Super case II Super case II / / / / Super case II Super case II / Super case II Super case II Super Case II Non-Fickian Non-Fickian Super case II Super case II Super case II Non-Fickian Non-Fickian Non-Fickian

“/” not available.

studies. Ionic strength and pH of the medium exerted a great influence on drug release (f2 < 50), such a behavior was desirable for the development of oral pH-controlled delivery of protein, which could protect protein by preventing its delivery until higher pH environment of the small intestine. HA matrix tablets exhibited a strong erosion, and the total drug release very quick with Super Case II transport model. In contrast, HA-Cys swelled greater and offered low erodible gels compared with HA, resulting in slower drug release with non-Fickian (anomalous) kinetic. Taking into account all the parameters, which influenced the drug release, HACys conjugates could be used as controlled release systems for proteins, and desired release profiles might be achieved by manipulating MW of polymer.

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Acknowledgments [15]

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