Exploration of hydrophobic modification degree of chitosan-based nanocomplexes on the oral delivery of enoxaparin

European Journal of Pharmaceutical Sciences 50 (2013) 263–271

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European Journal of Pharmaceutical Sciences journal homepage: www.elsevier.com/locate/ejps

Exploration of hydrophobic modification degree of chitosan-based nanocomplexes on the oral delivery of enoxaparin Linlin Wang a, Liang Li a, Yujiao Sun a, Ye Tian a, Ying Li a, Conghao Li a, Varaporn B. Junyaprasert b, Shirui Mao a,⇑ a b

School of Pharmacy, Shenyang Pharmaceutical University, 103 Wenhua Road, Shenyang 110016, China Department of Pharmacy, Faculty of Pharmacy, Mahidol University, 447 Sri-Autthaya, Rajathavee, Bangkok 10400, Thailand

a r t i c l e

i n f o

Article history: Received 30 May 2013 Received in revised form 11 July 2013 Accepted 18 July 2013 Available online 26 July 2013 Keywords: Chitosan Glyceryl monostearate Oral Enoxaparin Polyelectrolyte nanocomplexes

a b s t r a c t The objective of this paper is to elucidate the influence of lipophilic modification degree of chitosan on the peroral absorption of enoxaparin. A series of novel chitosan grafted glyceryl monostearate (GM) copolymers with different GM substitution degree were synthesized and the successful synthesis was confirmed by 1H NMR, FTIR and X-ray diffraction. Enoxaparin loaded nanocomplexes with different carriers were prepared by self-assembly process. Influence of GM substitution degree and chitosan molecular weight in the copolymer on the properties of the nanocomplexes was investigated. Morphology of the nanocomplexes was observed by atomic force microscopy. Mucoadhesive properties of the nanocomplexes were characterized using mucin particle method. Initially, mucoadhesion of the nanocomplexes increased with the increase of GM substitution degree and it started to decrease when the substitution degree was up to 18.6%. A good linear relationship between GM substitution degree and in vivo absorption of enoxaparin in fasted rats was established in the substitution degree range of 0–11.1%. In agreement with mucoadhesion data, further increasing GM substitution degree to 18.6% caused a decrease in oral absorption. In conclusion, oral bioavailability of enoxaparin can be enhanced by structure modification of the carriers and the bioavailability is hydrophobic modification degree dependent. Ó 2013 Elsevier B.V. All rights reserved.

1. Introduction Heparin, a highly sulfated natural polysaccharide, has been successfully used in the prevention and therapy of deep venous thromboembolism (DVT), venous thrombosis and pulmonary embolism (PE) (Grabovac and Bernkop-Schnürch, 2006). Its potential anticancer effect was also reported (Mellema et al., 2011). In the past years, low molecular weight heparin (LMWH), such as enoxaparin, has replaced unfractionated heparin (UFH) owning to its several clinical advantages including less-frequent dosing and elimination of the need for monitoring (Merli, 2005). However, LMWH is still poorly absorbed in the gastrointestinal tract (GIT) owing to its high anionic charge, large molecular size, enzymatic degradation and first pass effect (Paliwal et al., 2012). Consequently, it has to be administered via the parenteral route. So far, various approaches have been proposed in order to achieve better oral absorption of enoxaparin, including coadministration with absorption enhancers, using nanocarriers, lipidization of the drug and protection the drug against acidic pH of the stomach (Motlekar ⇑ Corresponding author. Address: School of Pharmacy Shenyang Pharmaceutical University 103, Wenhua Road, 32#, 110016 Shenyang, China. Tel./fax: +86 24 23986358. E-mail address: [email protected] (S. Mao). 0928-0987/$ - see front matter Ó 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ejps.2013.07.009

and Youan, 2006). Amongst them, nanoparticles as peroral delivery carriers exhibited many advantages including (1) protecting bioactive macromolecules against in vivo acid and enzymatic degradation; (2) increasing the membrane permeability; (3) increasing the contact and absorption area in the GI tract (Jung et al., 2000). Recently, nanoparticles based on amphiphilic copolymers have been demonstrated to be quite effective in improving the peroral absorption of hydrophilic macromolecular drugs. For example, a blood glucose reduction of 34% was reported with lauryl succinyl chitosan based insulin particles compared to 17% with native chitosan particles (Rekha and Sharma, 2009). Likewise, oleoyl chitosan based insulin particles exhibited significantly improved uptake than that of chitosan (Sonia et al., 2011). Especially, hydrophobic interaction is known to play a key role in binding of amphiphilic compounds to biological and artificial lipid membranes, followed by enhanced endocytosis (Liu et al., 2010). Hoffart et al. reported that by using a hydrophobic modified oral polymeric nanoparticle delivery system, the oral bioavailability of LMWH was increased up to 51% (Hoffart et al., 2006). However, influence of hydrophobic modification degree on the absorption of LMWH is unclear and needs to be explored. Chitosan (CS), a natural cationic polysaccharide derived by partial deacetylation of chitin (Rekha and Sharma, 2009), has drawn significant attention in the pharmaceutical and biomedical fields

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owing to its excellent biocompatibility, biodegradability, strong mucoadhesion and reactive functional groups for easy modification (Sonia et al., 2011). Importantly, chitosan owns two advantages for peroral drug delivery including mucoadhesion and absorption enhancement. Our previous work demonstrated that chitosan and its derivatives based polyelectrolyte nanocomplexes (PECs) enhanced the oral absorption of enoxaparin significantly. However, the oral bioavailability achieved was only 7.24% (Sun et al., 2010). Hydrophobic modification of the carrier might be an effective way to further increase the absorption of enoxaparin. Here glyceryl monostearate (GM) was selected as the hydrophobic part since its fatty acid chains can interact with the phospholipids component of the membrane thereby leading to increased permeability (Sonia et al., 2011). It is assumed that increasing the lipophilicity of chitosan based nanocarrier by glyceryl monostearate modification may be an effective way to further enhance the peroral absorption of enoxaparin and the absorption extent might be modification degree dependent. Therefore, to test this hypothesis, a series of chitosan graft glyceryl monostearate (CS-GM) copolymers were synthesized in this study and copolymer based enoxaparin nanocomplexes were prepared by self-assembly process. It is anticipated that, by combining the distinctive advantages of nanoparticle delivery system with enhanced lipophilicity, the absorption enhancing function of chitosan and GM, the transport of enoxaparin across the intestinal epithelium can be further improved. For this purpose, properties of the nanocomplexes were characterized including particle size, morphology, zeta potential, entrapment efficiency, in vitro release, mucoadhesion and oral absorption in rats. Influence of hydrophobic modification degree of the carriers on the oral absorption of enoxaparin nanocomplexes was elucidated. To the best of our knowledge, this is the first time that chitosan graft glyceryl monostearate copolymers were synthesized and being used as the carrier of LMWH. 2. Materials and methods 2.1. Materials Chitosan (400 kDa) was purchased from Weifang Kehai Chitin Co., Ltd. (Shandong, China) with a degree of deacetylation (DD) of 86.5%. Enoxaparin (mean MW 4251 Da) was purchased from Hangzhou Jiuyuan Gene Engineering Co., Ltd. (Hangzhou, China). Glyceryl monostearate was purchased from TianJin Bodi Chemical Holding Co., Ltd. (Tianjin, China). 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) and N-Hydroxysuccinimide (NHS) were purchased from Shanghai Medpep Co., Ltd. (Shanghai, China). Type III mucin from porcine stomach was from Sigma. (Beijing, China). Activated partial thromboplastin time (APTT) assay kits were purchased from Shanghai Sunbio Co., Ltd. (Shanghai, China). All other chemicals were of analytical grade. 2.2. Synthesis of CS-GM Briefly, 1.0 g of succinic anhydride and 3.58 g of GM (feed ratio 1:1, mol/mol) were added into a 100 ml round bottomed flask and reacted at 170 °C for 10 h under vacuum and stirring. The temperature was decreased to 80 °C after the reaction to remove unreacted succinic anhydride. Finally, succinyl-GM was collected by filtration. Thereafter, succinyl-GM was reacted with EDC and NHS in 100 ml methanol to activate the carboxyl groups of succinyl-GM at 50 °C for 2 h. The feeding mole ratio of EDC and NHS to GM was 1.5:1 (Kim et al., 2010). Then, the CS solution was mixed with activated succinyl-GM solution in methanol and reacted for 24 h at

50 °C to form CS-GM. The feeding mole ratio of chitosan and succinyl GM was varied from 1:0.05 to 1:0.25. The reaction mixture was cooled down at 20 °C for 6 h after the reaction to precipitate the unreacted succinyl-GM. Dehydrated alcohol was added into the filtrate to precipitate the CS-GM. The copolymer was collected and dried in oven at 40°Cfor 24 h. 2.3. Characterization of CS-GM The successful synthesis of CS-GM copolymer was confirmed by using 1H NMR, FTIR and X-ray diffraction. Tetramethylsilane was used as an internal standard. 1H NMR spectra of the CS and CS-GM were measured by dissolving the polymers in mixed solvents of D2O and CF3COOD (D2O/CF3COOD = 1:1 v/v), whereas GM and succinyl-GM were measured in CDCl3 by using a ARX-300 MHz spectrometer (Bruker, Germany) at 25 °C. The amount of GM covalently bounded to chitosan (DS) was determined by the peak areas of –CH3 of GM (A(–CH3)) and –COCH3 (A(–COCH3)) of CS, and the deacetylation degree of CS (DA) (Du et al., 2011).

DS ð%Þ ¼

AðCH3 Þ  100% AðCOCH3 Þ=DA

Powder samples (CS and CS-GM) were compressed into KBr disks and conducted in the range of 4000 and 400 cm1 for the IFS-55 FTIR (Bruker, Switzerland) measurement. X-ray diffraction (XRD) spectrometry was performed by using a Bruker AXS D8 powder diffractometer. A Cu Ka radiation at 40 kV and 40 mA was used. Diffractograms were observed from the initial angle 2h = 5° to the final angle 2h = 55° with steps of 0.02° and at a scanning speed of 12°/min (2h). 2.4. Preparation of CS-GM/enoxaparin nanocomplexes First of all, the specific CS-GM copolymer was dissolved in 0.25% acetic acid solution under gentle stirring, then the sample solution was treated by probe-type ultrasonicator (activated every 6 s for a 3 s duration) at 200 W for 10 min under ice bath. Thereafter, the solution was passed through 0.8 lm Millipore membrane. For nanocomplexes preparation, enoxaparin dissolved in distilled water (1 mg/ml) was added into the CS-GM solution (2.5 mg/ml) under magnetic stirring. The system pH was adjusted to 6 and then incubated for further 30 min at room temperature. All experiments were performed in triplicate at ambient temperature. Freshly prepared solutions were used in each experiment. 2.5. Characterization of CS-GM/enoxaparin nanocomplexes The particle size of the prepared nanocomplexes were measured by photon correlation spectroscopy (PCS) with a Malvern Zetasizer 4 (Malvern Instruments, UK) at 25 °C with a scattering angle of 90°, and the zeta potential measurements were carried out using the Malvern Zetasizer 4 by electrophoretic laser doppler anemometry at 25 °C. The amount of enoxaparin entrapped in nanocomplexes was determined by calculating the total amount of enoxaparin added and the quantity of free enoxaparin left in the supernatant after PEC formation. Briefly, the nanocomplexes were centrifuged at 40,000 rpm/min (31000g) for 1 h at room temperature. The concentration of enoxaparin in the supernatant was determined with Azure II colorimetric method (Jiao et al., 2002), and enoxaparin encapsulation efficiency was calculated using the following equation (Chen et al., 2009). All samples were measured in triplicate.

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EE ð%Þ ¼ fractotal amount of heparin added -free heparin left in supernatanttotal amount of heparin added  100%

2.6. Visualization of heparin nanocomplexes The morphology and size of the nanocomplexes were observed by atomic force microscopy (AFM) (Agilent Technologies, Tempe, AZ, USA). The samples were diluted with ultra-pure water, and 5 ll of diluted sample was applied to a freshly cleaved mica surface and allowed to adhere to the surface for a few minutes. The samples were allowed to air-dry. The microscope was vibrationdamped. All measurements were performed in tapping mode in order to avoid damage of the sample surface. 2.7. In vitro release studies In vitro release of enoxaparin from the nanocomplexes was studied using dialysis method (MWCO: 8000–14000). Briefly, 5 ml of enoxaparin-loaded nanoparticles based on different carriers was transferred in dialysis bags and drug release was investigated in 30 ml of pH 1.2 SGF (in the first 2 h) or pH 6.8 SIF (in the followed 4 h) at 37 °C. At appropriate time intervals (0 h, 1 h, 2 h, 3 h, 4 h, 5 h and 6 h), samples were withdrawn followed by replacement with equal volume of fresh medium and the samples were passed through 0.45 lm Millipore membrane. Enoxaparin content in the filtrate was measured according to the Azure II colorimetric method (Jiao et al., 2002). All experiments were performed in triplicate. 2.8. Mucoadhesion measurement Mucoadhesive properties of various PECs were evaluated by using mucin particle method (Takeuchi et al., 2005). Briefly, the coarse mucin particles (1% w/v) was prepared by suspending and continuously stirring mucin type III powder in 10 mM Tris buffer, pH 6.8, for 10 h. The coarse mucin particle was then incubated at 37 °C overnight. The precisely controlled submicron-sized mucin (ss-mucin, ca. 300 ± 20 nm in diameter) suspension was prepared by ultrasonication (activated every 6 s for a 3 s duration) at 200 W for 20 min. It was then centrifuged at 4000 rpm for 20 min to extract submicron-sized mucin particles in the supernatant (Jintapattanakit et al.,2008). Then 0.6 ml of various nanocomplexes samples were added into 1 ml of 1% w/v extracted ss-mucin suspension and incubated at 37 °C for 2 h. Thereafter the extent of change in particle size was presented, which was calculated by dividing the size of the aggregates by that of the original ss-mucin. The particle size increase depended on the mucoadhesion of the particles (Takeuchi et al., 2005). All experiments were performed in triplicate. 2.9. Pharmacokinetic study in rats In vivo absorption of the selected enoxaparin nanocomplexes were studied in rats. All animal studies were approved by the University Ethics Committee and were carried out in accordance with the Principle of Laboratory Animal Care. The Sprague Dawley rats (male, 200 ± 20 g) were fasted for 12 h with free access to water before the administration of various formulations (n = 5). All kinds of aqueous suspension of CS-GM/enoxaparin nanocomplexes with different amino substitution degree of GM and chitosan molecular weight were administered through an oral gavage tube that was carefully passed down through the esophagus into the stomach

of rats at a single oral dose (1365 IU/kg). A saline solution of enoxaparin administered orally was used as control (1365 IU/kg). Blood samples (about 450 ll) were withdrawn from retro-orbital plexus at 0 h, 0.5 h, 1 h, 2 h, 3 h, 4 h, 6 h and 8 h after administration of each sample and centrifuged at 3000 rmp (1600g) for 15 min. The plasma enoxaparin concentration was estimated by measuring the activated partial thromboplastin time (APTT) value of the plasma sample with a standard commercial kit (Cushing et al., 2010). Freshly prepared plasma was used for APTT analyses. The absolute bioavailability (F) of orally administered formulations was calculated by comparing their AUC with that of intravenous injection of 1 mg/ml enoxaparin saline solution (2 mg/kg) corrected by the administered dose using DAS 2.1.1 software.

2.10. Statistical analysis Results are depicted as mean value ± standard deviation (SD) from at least three measurements. Significance of difference was evaluated using one-way ANOVA at the probability level of 0.05.

3. Results and discussion 3.1. Preparation and characterization of CS-GM copolymers To increase the lipophilicity of chitosan, CS-GM copolymers with different GM ratios were synthesized in this paper. First of all, chitosans with different molecular weights were prepared by depolymerization method (Mao et al., 2004). The synthesis scheme of CS-GM copolymers is presented in Fig. 1. For the chemical coupling of GM onto CS, the terminal hydroxyl group of GM was firstly converted to carboxyl group with the help of succinic anhydride (Fig. 1a), then the carboxyl group of succinyl GM was bonded to the amino group of CS to form CS-GM copolymer in the presence of EDC and NHS (Fig. 1b) (Wang et al., 2011). CS-GM copolymers with different hydrophobicity were obtained by controlling the feed ratio of CS and GM. Meanwhile, by keeping GM graft ratio at approximately 10%, copolymers with different chitosan molecular weight were also prepared. The following nomenclature was adopted for the copolymers: CS (a)-GM (b%), where a denotes the molecular weight of CS in kDa and b represents the degree of substitution (DS), defined as the number of GM groups per 100 amino groups of CS. Successful synthesis of succinyl GM and CS-GM was confirmed by 1H NMR spectrum, as shown in Fig. 2. Compared with GM (Fig. 2a), a multiplet at 2.65 ppm observed in the spectrum of succinyl GM (Fig. 2b) may be attributed to the two adjacent methylene groups of the succinyl moiety, illustrating that succinyl group was attached to GM successfully (Shaikh et al., 1998). Fig. 2d exemplifies the 1H NMR spectra of CS(50)-GM(11.1%). The proton assignment of CS-GM is as follows: d0.9 = CH3 (methyl group of GM); d1.2 = CH2 (methylene of GM); d2.1 = CH3 (acetyl group carbon a of chitosan); d3.2 = CH (carbon c of chitosan); d3.7 = CH (carbon e of chitosan); d3.9–4.0 = CH (carbon d of chitosan); d4.9 = CH (carbon b of chitosan). The peak at 0.9 ppm is characteristic methyl protons of GM grafted on the back bone of chitosan (Hu et al., 2006). The amount of GM covalently bounded to chitosan was determined by the peak areas of –CH3 of GM and –COCH3 of CS and the deacetylation degree of CS based on the 1H NMR spectra (Du et al., 2011).

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Fig. 1. The scheme of chitosan grafted glyceryl monostearate copolymers synthesis.

FTIR spectra provided further evidence for the successful synthesis of CS-GM. The FTIR spectra of CS(50) and CS(50)GM(11.1%) are shown in Fig. 3. In comparison with the FTIR spectrum of CS(50) (Fig. 3a), a new absorption peak at around 1739 cm1, corresponding to the carbonyl groups of GM grafts, was found in the spectra of CS(50)-GM(11.1%) (Fig. 3b). Moreover, a significant increase in the intensity at about 2920–2850 cm1 can

Fig. 2. 1H NMR spectra of glyceryl monostearate (a), succinyl glyceryl monostearate (b), CS(50) (c) and CS(50)-GM(11.1%) (d).

be attributed to –C–H stretching vibrations from the large number of methylene groups of GM moiety. Moreover, it was found that the relative absorption strength of the amide groups at 1651 and 1565 cm1 of the graft copolymers were enhanced in comparison with that of chitosan (Hao and Chang, 2006), implying that the amino groups of chitosan decreased by graft polymerization of GM. All the above results suggested that GM has been grafted onto the chitosan backbone successfully. In order to investigate the changes of nature and crystallinity of CS-GM, X-ray diffraction studies were performed. The X-ray diffraction spectra of succinic anhydride (a), GM (b), succinyl GM (c), CS(50) (d) and CS(50)-GM(11.1%) (e) are shown in Fig. 4. Succinic anhydride showed intense characteristic peaks at 2h of 14.9°, 17.9°, 20°, 22.1°, 26.8° and 29.9°, which disappeared in the diffractogram of succinyl GM, indicating the succinylation of GM disrupted the crystal structure of both succinic anhydride and GM successfully. For the native chitosan, three crystalline reflections were observed at 2h = 22°, 2h = 20.1° and 2h = 10.3°, which was assigned to the crystal form (Zhang et al., 2003). In contrast to native chitosan, the intensity of the peak at 2h = 20.1° decreased obviously and the peak at 2h = 10.3° and 22° disappeared in the

Fig. 3. FTIR spectra of CS(50) (a) and CS(50)-GM(11.1%) (b).

L. Wang et al. / European Journal of Pharmaceutical Sciences 50 (2013) 263–271

Fig. 4. X-RD spectra of succinic anhydride (a), glyceryl monostearate (b), succinyl glyceryl monostearate (c), CS(50) (d) and CS(50)-GM(11.1%) (e).

diffraction pattern of CS(50)-GM(11.1%), indicating that its ability of forming hydrogen bond might be decreased after chemical modification and the chitosan derivatives could be amorphous (Zhang et al., 2003). The similar changes in diffractogram have also been observed when deoxycholic acid was grafted onto O-carboxymethylated chitosan (Wang et al., 2011). These results further demonstrated indirectly that GM has been conjugated to the chitosan backbone successfully and the crystalline structure of native chitosan has been disrupted owning to the introduction of GM. 3.2. Preparation and characterization of CS-GM/enoxaparin nanocomplexes CS-GM/enoxaparin nanocomplexes were prepared by selfassembly method based on the electrostatic interaction between the positively charged copolymer and negatively charged enoxaparin as reported previously (Chen et al., 2009). The optimal concentration and mass ratio of chitosan or chitosan derivative with enoxaparin was selected based on the property of the obtained nanocomplexes in reference to previously published method (Sun et al., 2008). Finally, polymer/enoxaparin mass ratio 2.5 and enoxaparin 1 mg/ml was selected to prepare various nanocomplexes in this study. First of all, keeping chitosan molecular weight 50 kDa, influence of hydrophobic modification on the properties of the nanocomplexes was investigated. As presented in Table 1, at lower GM graft ratio up to 11.1%, no remarkable change in particle size was found, further increase of GM graft ratio to 18.6% caused significant increase in particle size (P < 0.05), from 285 nm for chitosan to 323 nm for this copolymer. This can probably be explained by the fact that conjugation of GM in chitosan backbone to a critical point decreased the positive charge density of the copolymer,

Table 1 Properties of CS-GM/enoxaparin nanocomplexes.

a

Polymer

Size(nm)

Zeta (mv)

EE(%)

CS(50) CS(50)-GM(3.7%) CS(50)-GM(11.1%) CS(50)-GM(18.6%) CS(20)-GM(9.8%) CS(100)-GM(11.4%)

285 ± 10.4 279.7 ± 5.8 291.8 ± 6.5 323.5 ± 8.9a 250.3 ± 15.3 400.6 ± 21.6

18.4 ± 0.9 18.35 ± 1.6 17.4 ± 0.4 16.4 ± 0.3 17 ± 0.9 17.7 ± 0.8

99.07 ± 0.17 99.23 ± 0.38 99.16 ± 0.11 99.21 ± 0.44 99.14 ± 0.11 98.92 ± 0.19

Indicate these data are statistically different compared to CS50. (P < 0.05).

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leading to weaker electrostatic interaction between the carrier and enoxaparin and therefore particle size increase. Similar results were reported by Bravo-Osuna et al (Bravo-Osuna et al., 2006). As to the change of surface charge, a slight decrease in zeta potential was observed with the increase of GM graft ratio, but no statistical change was found (P > 0.05) and all the complexes were positively charged, implying enoxaparin was encapsulated in the core of the nanocomplexes. This positive charge is advantageous for better interaction with mucus and therefore may facilitate nanoparticles uptake. As to drug loading efficiency, in the range studied, the copolymer structure had no influence on drug loading in the nanocomplexes. Thereafter, by keeping GM graft ratio at approximately 10%, influence of chitosan molecular weight on the properties of the nanocomplexes was further investigated. Increase in chitosan molecular weight from 20 kDa to 100 kDa caused particle size change of the nanocomplexes from 250 nm to 400 nm, but no influence on particle surface charge and drug loading was found. The increased particle size may be explained by the increased viscosity in combination with increased rigidity of chitosan chain along with the increase of molecular weight (Kim et al., 2001). 3.3. Visualization of enoxaparin nanocomplexes Influence of copolymer structure on the morphology of the nanocomplexes was observed by AFM. Images of various nanocomplexes based on CS50, CS(50)-GM(11.1%), CS(50)-GM(18.6%) and CS(100)-GM(11.4%) are presented in Fig. 5. All the nanocomplexes are spherical or sub spherical in shape and the particle size obtained from AFM is much smaller than that determined by DLS (Table 1), which can be explained by the different measurement mechanism and sample treatment. For the dynamic light scattering, nanoparticles were measured in the hydrated state in aqueous medium, so the molecular chains were well stretched. On the other hand, nanocomplexes characterized by AFM were firstly dried at room temperature, and shrinkage of nanocomplexes were anticipated (Sun et al., 2008). As shown in Fig. 5a, majority of chitosan based particles have an average diameter of 50–100 nm and were well separated from each other, suggesting that these particles were possibly stabilized against agglomeration. Similar phenomenon was observed for PECs based on CS(50)-GM(11.1%) and CS(50)GM(18.6%) as shown in Fig. 5b and 5c. Three dimensional images (Fig. 5d) further indicated that most of the nanocomplexes were spherical or sub spherical with smooth surface. 3.4. In vitro release studies To improve the stability of nanocomplexes in the GIT, it is anticipated that limited enoxaparin was released and most of the nanocomplexes can be uptaken by the cells. The in vitro release of enoxaparin from various PECs is shown in Fig. 6. It was noted that enoxaparin release in SGF was very limited probably due to the strong electrostatic interaction between the positively charged CS/CS-GM and negatively charge enoxaparin, which is of interest for peroral administration. In SIF, slight drug release was observed, contributed to the decreased charge density of chitosan or its copolymer. However, still, the total amount of drug release was less than 15% in 6 h, indicating good stability of the nanocomplexes in the GIT. In regard to the influence of hydrophobic modification on drug release, compared to the homopolymer chitosan based particles, statistical change in release was only observed with CS50-GM18.6% based nanocomplexes after 5 h (P < 0.05), which can be explained by its larger particle size (Table 1) and therefore better fluid penetration. As to the influence of chitosan molecular weight, a faster drug release from CS20-GM9.8% based particles was observed compared with that

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Fig. 5. Atomic force microscopy images of polymer/enoxaparin complexes: (a) CS(50), (b) CS(50)-GM(11.1%), (c) CS(50)-GM(18.6%), and (d) CS(100)-GM(11.4%).

Fig. 6. Release profiles of various enoxaparin nanocomplexes in simulated gastrointestinal tract (pH 1.2 for 2 h then change to pH 6.8 SIF) (n = 3).

from CS100-GM11.4% based particles, which can be attributed to the different viscosity of the gel layer around nanoparticles upon contact with the dissolution medium (Yang and Hon, 2009). However, no difference in release was observed between CS50GM11.1% and CS100-GM11.4% based particles after 5 h (P > 0.05). 3.5. Mucoadhesion of various nanocomplexes To predict the in vivo absorption of different nanocomplexes, mucoadhesive properties of the nanocomplexes was evaluated using mucin particle method based on the change of particle size

Fig. 7. Evolution of the particle size of ss-mucin particles upon mixed with various nanocomplexes. ⁄Indicates these data are statistically different from each other.

and zeta potential (Takeuchi et al., 2005). It has been reported that the viscoelastic and gel-forming properties of purified mucin are comparable to native mucus gels (Norris et al., 1998) and results from hydrated commercial mucin has no significant difference with that obtained with native mucus (Leitner et al., 2003). Porcine gastric mucin type III, a highly heterogeneous glycoprotein extracted from porcine stomach mucin, was used in this study. Mucin contains some hydrophobic composition which can interact with hydrophobic surface of nanocomplexes through the hydrophobic

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Fig. 8. Schematic diagram of nanocomplexes formation with varied CS-GM copolymers.

Fig. 9. Mean DAPTT over time after a single oral administration of enoxaparinloaded nanocomplexes (1365 IU/kg) based on chitosan and various CS-GMo copolymers in rats (n = 5).

Table 2 Main pharmacokinetic parameters after peroral administration of various enoxaparin-loaded nanocomplexes in rats at dose of 1365 IU/kg. Formulation

Tmax (h)

AUC0–8h (s h)

F (%)

iv. solution Oral solution CS(50) CS(50)-GM(3.7%) CS(50)-GM(11.1%) CS(50)-GM(18.6%) CS(20)-GM(9.8%) CS(100)-GM(11.4%)

– – 3 3 3 2 2 3

107.83 7.24 30.161 34.863 46.819 41.972 33.157 69.351

100 1.34 5.64 6.52 8.75 7.85 6.2 12.96

interaction (Sonia et al., 2011). The stronger the surface hydrophobicity, the better the hydrophobic interaction and therefore stronger mucoadhesion. It was expected that ss-mucin particles would aggregate when mixed with a system that had a strong affinity to the particles (Jintapattanakit et al., 2008). In this study, the interaction between varied nanocomplexes and ss-mucin particles was characterized at pH 6.8 in Tris buffer. When 1 ml of ss-mucin particle suspension was mixed with 0.6 ml of nanocomplexes solution, the mucin particles started to aggregate. Importantly, it has been confirmed that ss-mucin particles keep stable for at least 2 h, so the aggregate of particles did not own to the self-aggregation of ss-mucin particles, but the mucoadhesive property of the nanocomplexes. Fig. 7 shows the size increase of mucin particles after adding 0.6 ml of 1 mg/ml various nanocomplexes solution and incubated at 37 °C for 2 h. It was noted that hydrophobic modification of chitosan with GM increased the mucoadhesion of the nanoparticles significantly (P < 0.05) compared with that of chitosan, but no statistical

difference in mucoadhesion was found when GM graft ratio increased from 3.7% to 11.1% (P > 0.05). However, further increasing the DS of GM to 18.6% caused a decrease in mucoadhesion (P < 0.05). This can probably be explained by the structure change of the nanoparticles along with the increase of GM graft ratio. Chitosan is a highly hydrophilic polymer, at low degrees of GM modification (0% to about 11.1%), water-soluble CS-GM copolymers were obtained. Similar phenomenon was observed at low degrees of fatty acid/cholesterol modification to dextran-spermine (Azzam et al., 2004). Nanocomplexes formation based on watersoluble CS-GM copolymers is mainly controlled by the electrostatic interaction between positively charged CS-GM and the negatively charged enoxaparin and the hydrophobic GM moiety was mainly exposed at the out layer of the particles. However, further increasing GM ratio over the critical value, a good hydrophile–lipophile balance can be achieved in the copolymer, and self-assembled micelle like particles could be formed upon copolymer dispersion, with the hydrophobic GM forming the inner core and hydrophilic chitosan forming the shell, with limited GM chains remained on the particle surface (Azzam et al., 2004; Eliyahu et al., 2004; Thompson et al., 2009). Therefore, when enoxaparin was added into the self-aggregates, probably it was mainly encapsulated into the shell section owing to its electrostatic interaction with chitosan without affecting the built-in micelle structure (Thompson et al., 2009; Wittemann et al., 2007; Zhang et al., 2012). Thus, decreased GM density at CS(50)-GM(18.6%) based particle surface might explain its reduced mucoadhesion compared with CS(50)GM(11.1%) based particles. Fig. 8 schemically presented the hypothesis for the influence of GM graft ratio on the structure of the nanoparticles. The data in Table 1 showed that zeta potentials of various nanocomplexes have no statistical difference (P > 0.05), further indicating that the difference in mucoadhesion is not contributed to electric charge but difference in surface hydrophobicity of the particles. Fig. 8 schemically described the structure of the nanocomplexes prepared with copolymers with different GM modification degree. At low degrees of GM modification (0% to about 11.1%), the hydrophobic GM moiety was mainly exposed at the out layer of the particles, and increasing GM substitution could prohibit the chain relaxation of the copolymer, leading to limited drug release (Martin et al., 2002). Further increasing the DS of GM to 18.6% caused a structure change of the nanocomplexes, with GM as the core and chitosan as the shell, while enoxaparin was mainly distributed in the shell section, leading to faster drug release in SIF especially after 4 h. However, it should be noted that in addition to hydrophobic interaction mentioned above, flexibility of the copolymer chains could also affect the mucoadhesion. It has been reported that by the introduction of long alkyl chains, intermolecular and intramolecular hydrogen bonding between chitosan backbone chains was weakened, and in turn, flexibility of the molecular chains increased (Wang and Ye, 2012), which was in agreement with the fact that enoxaparin nanocomplexes based on CS-GM exhibited increased

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mucoadhesion compared with that based on native chitosan. It is anticipated that amphiphilic CS-GM containing octadecyl alkyl chains owned extended chain conformation at lower DS, whereas collapsed conformation was forced at higher DS by stronger hydrophobic interaction (Kang et al., 2001). The extended chain increased chain flexibility and chain mean area in lipid membranes of epithelial cell at lower DS (Binder and Gawrisch, 2001). However, octadecyl alkyl chains shows abnormal behavior due to the nature of the physical cross-linker of frozen octadecyl chains at higher DS, which limited the chain flexibility and decreased the endocytosis efficiency (Kang et al., 2001). Moreover, by keeping the DS of GM at approximately 10%, influence of chitosan molecular weight on the mucoadhesion of the nanocomplexes was also investigated. As shown in Fig 7, a significant increase in bioadhesion was observed when the molecular weight of chitosan was increased from 20 kDa to 100 kDa (p < 0.05), probably due to the fact that high molecular weight chitosan offered multiple sites for mucin attachment (Qaqish and Amiji, 1999). This is in good agreement with our previous study that the mucoadhesion of chitosan increased significantly with the increase of molecular weight (Sun et al., 2010). 3.6. In vivo absorption in rats The contribution of hydrophobic modification on the in vivo absorption of enoxaparin was further studied in fasted rats. After a single peroral administration of different polymer based enoxaparin nanocomplexes, enoxaparin concentration in plasma was estimated by APTT assay and shown in Fig. 9. The main pharmacokinetic parameters were calculated and listed in Table 2. Compared with oral enoxaparin saline solution, all the nanocomplexes investigated increased APTT value significantly (P < 0.05). It was noted that lipophilic modification of chitosan with GM enhanced the in vivo absorption of the nanocomplexes remarkably compared with chitosan homopolymer based nanocomplexes. And initially, the in vivo absorption of enoxaparin increased with the increase of GM substitution. In terms of absolute bioavailability (F), the nanocomplexes with different DS of GM 0%, 3.7% and 11.1%, showed different in vivo absorption, and a good correlation between DS% (x) and absolute bioavailability (y) could be established with the equation y = 0.2832x + 5.5729 (n = 3, r = 0.97). However, further increasing the substitution degree to 18.6% caused a decrease in AUC compared with CS(50)-GM(11.1%) based nanocomplexes (P < 0.05). This can probably be explained by the decreased mucoadhesion of the particles due to the internal structure change and the limited polymer chain flexibility at higher DS of GM. The decreased in vivo absorption at high DS of GM is also in good agreement with the mucoadhesion data. As reported, the oral bioavailability of nanoparticles in vivo is related to the particle size, zeta potential and surface hydrophobicity (Chen et al., 2011). As presented in Table 1, particle size and zeta potential of the nanoparticles based on modified chitosan 50KDa with different DS of GM are very similar, indicating that these two factors cannot affect the oral absorption significantly. The enhanced oral absorption of enoxaparin based on CS-GM copolymers might be related to the lipophilicity of GM and the excellent properties of chitosan. The free-rotation property of the saturated carbon atoms in stearic acid was expected to give the molecule more flexibility to move inward or project outward PEC (Alshamsan et al., 2008). These hydrophobic stearic acid chain can enhance bioadhesion with mucus layer and strengthen PECs-plasma membrane interactions, which may facilitate endocytosis process, and, in turn, increase the peroral bioavailability (Sonia et al., 2011). In contrast, decreased surface hydrophobicity at high DS and limited polymer chain flexibility explained the lower bioavailability of CS(50)-GM(18.6%)/ enoxaparin PECs. Similarly, it was reported that compared to

unmodified dextran-spermine, oleate-modified dextran-spermine enhanced the gene expression in serum rich media remarkably (Eliyahu et al., 2004). Moreover, gene expression increased with oleate content at a range of 0–10%, however, further increasing oleate content to 20%, caused gene expression decrease (Azzam et al., 2004). This is in good agreement with our results. It has been reported that molecular weight of chitosan influenced the stability of the biomolecule/chitosan complex, the efficiency of cell uptake once in contact with the cellular membrane and the dissociation of active molecule from the complex after subsequent endocytosis (Bravo-Osuna et al., 2007a), therefore, it is essential to evaluate the influence of chitosan molecule weight on the bioavailability of CS-GM based complexes. For this purpose, keeping the DS of GM at approximately 10%, influence of chitosan molecular weight, 20, 50, 100 kDa, on the in vivo absorption of enoxaparin was further investigated. As shown in Table 2, the AUC values increased with the increase of chitosan molecular weight and the highest bioavailability of 12.96% was achieved with CS(100)-GM(11.4%) based nanocomplexes, compared to 6.2% of CS(20)-GM(11.4%) based ones. This tendency is in consistent with mucoadhesion data of PECs based on various molecular weight CSGM, which can be explained by the fact that the longer chitosan chains owned increased tendency to interpenetrate and entangle with the mucus protein chains (Bravo-Osuna et al., 2007b). These studies further demonstrated that mucoadhesive properties are of special importance for the in vivo absorption of nanoparticles. In this paper, the enhanced absorption of CS-GM based enoxaparin nanocomplexes is regarded as a combination of the superior property of chitosan, the lipophilicity of GM and the distinctive advantages of nanoparticle delivery system to enhance the transport of enoxaparin across the intestinal epithelium. Chitosan, a mucoadhesive polymer, are thought to be able to interpenetrate into the mucous layer, thus reaching the physical barrier and facilitating transport of hydrophilic molecules across the mucous layer (Junginger and Verhoef, 1998). In addition, the cationic property of chitosan can interact with anionic mucosa, prolonging the interactive time and aid these nanocomplexes interpenetrating into the mucous layer. CS-GM was used as carriers to form nanocomplexes with enoxaparin to enhance the lipophilicity of enoxaparin and facilitate membrane partitioning (Alshamsan et al., 2008). Nanoparticles have been reported to be absorbed by endocytosis, paracellular transport, or by the gut associated lymphoid tissue (Norris et al., 1998). Based on our in vitro mucoadhesion data, the nanocomplexes based on CS-GM can probably be adsorbed on the cell surface due to the combination of ionic and hydrophobic interaction, leading to a higher level of internalization. 4. Conclusion In this study, novel amphiphilic copolymers, chitosan graft glyceryl monostearate (CS-GM) were synthesized and used as the nanocarriers for oral delivery of enoxaparin. The hydrophobic modification of chitosan could significantly influence the resultant nanocomplexes’ lipophilicity, the mucoadhesive properties and therefore the oral bioavailability of enoxaparin, but in a glyceryl monostearate substitution degree dependent manner. The maximal absolute bioavailability of 12.96% was achieved with PECs prepared using CS(100)-GM(11.4%) copolymers. In conclusion, the oral bioavailability of enoxaparin can be enhanced by structure modification of the carrier and the bioavailability is hydrophobic modification degree dependent. Acknowledgements This project is financially supported by the Natural Science Foundation of China (Grant No. 81273446); Sino-Thai joint

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