http://informahealthcare.com/drd ISSN: 1071-7544 (print), 1521-0464 (electronic) Drug Delivery, Early Online: 1–10 ! 2014 Informa Healthcare USA, Inc. DOI: 10.3109/10717544.2014.909908
RESEARCH ARTICLE
pH-responsive thiolated chitosan nanoparticles for oral low-molecular weight heparin delivery: in vitro and in vivo evaluation Bo Fan*, Yang Xing*, Ying Zheng, Chuan Sun, and Guixian Liang
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School of Pharmaceutical Science, Shanxi Medical University, Taiyuan, Shanxi, People’s Republic of China
Abstract
Keywords
The aim of present study was to investigate a pH-responsive and mucoadhesive nanoparticle system for oral bioavailability enhancement of low-molecular weight heparin (LMWH). The thioglycolic acid (TGA) was first covalently attached to chitosan (CS) with 396.97 ± 54.54 mmol thiol groups per gram of polymer and then the nanoparticles were prepared with thiolated chitosan (TCS) and pH-sensitive polymer hydroxypropyl methylcellulose phthalate (HPMCP) by ionic cross-linking method. The obtained nanoparticles were characterized for the shape, particle size, zeta potential, drug entrapment efficiency and loading capacity. In vitro results revealed the acid stability of pH-responsive nanoparticles, which had a significant control over LMWH release and could effectively protect entrapped drugs in simulated gastric conditions. By the attachment of the thiol ligand, an improvement of permeation-enhancing effect on freshly excised carp intestine (1.86-fold improvement) could be found. The mucoadhesive properties were evaluated using fluorescently labeled TCS or CS nanoparticles. As compared with the controls, a significant improvement of mucoadhesion on rat intestinal mucosa was observed in TCS/HPMCP nanoparticles via confocal laser scanning microscopy. The activated partial thromboplastin time (APTT) was significantly prolonged and an increase in the oral bioavailability of LMWH was turned out to be pronounced after oral delivered LMWH-loaded TCS/HPMCP nanoparticles in rats, which suggested enhanced anticoagulant effects and improved absorption of LMWH. In conclusion, pH-responsive TCS/HPMCP nanoparticles hold promise for oral delivery of LMWH.
Low-molecular weight heparin, mucoadhesive, oral drug delivery, pH-responsive, thiolated chitosan nanoparticles
Introduction Low-molecular weight heparin (LMWH) is clinically used in parenteral routes mainly as an anticoagulant for the prevention and treatment of deep vein thrombosis (DVT), pulmonary embolism (PE), and coronary syndromes (Arbit et al., 2006; Paliwal et al., 2011). Although oral drug delivery is the safest and most convenient means of drug administration, the main obstacles hindering oral route for LMWH are its low stability at acidic pH of the stomach and poor absorption in gastrointestinal tract due to its high anionic charge density, large molecular size, high water solubility and enzymatic degradation (Motlekar & Youan, 2006). Accordingly, various approaches have been investigated for oral delivery of heparin including penetration enhancers (Hayes et al., 2006), microparticles (Javot et al., 2009; Oliveira et al., 2011), polymeric nanoparticles (Chen et al., 2009), dendrimeric nanocarriers (Bai & Ahsan, 2009), chemical conjugates (Lee et al., 2006), etc. Among them, chitosan (CS)-based nanoparticles are particularly attractive (Paliwal et al., 2012; Bagre et al., 2013). *Bo Fan and Yang Xing contributed equally to this work. Address for correspondence: Guixian Liang, School of Pharmaceutical Science, Shanxi Medical University, No. 56, Xinjian Nan Road, Taiyuan 030001, Shanxi, People’s Republic of China. Tel/Fax: +86 351 4690137. E-mail: [email protected]
History Received 19 February 2014 Revised 25 March 2014 Accepted 26 March 2014
CS, a cationic polysaccharide, is widely regarded as a safe and efficient intestinal absorption enhancer of therapeutic macromolecules, owing to its inherent mucoadhesive feature and ability to modulate the integrity of epithelial tight junctions reversibly (Amidi et al., 2010). These mucoadhesive and permeation-enhancing properties of chitosan can be strongly improved by the immobilization of thiol groups on the polymer. The significantly improved mucoadhesive properties may be attributed to the formation of disulfide bonds between their thiol groups and the cysteine-rich subdomains of glycoproteins in the mucus layer, which is stronger than the non-covalent interaction between cationic CS and anionic mucosal substances (Kast & BernkopSchnu¨rch, 2001). However, the introduction of the thiol moieties does not modify the main limitation of CS nanoparticles for oral peptide and protein delivery that the polymer matrix can be easily dissolved in gastric juice by protonation of the amino groups at low pH values. To overcome the obstacle, pH-responsive enteric polymer could be introduced in the preparation of nanoparticles. Most commercially available enteric coating materials are soluble in the pH range from 5.0 to 7.0 (Kokubo et al., 1997). But as for drugs with poor and limited absorbability in the gastro-intestinal tract, it is desirable to ensure that the enteric polymer is dissolved as
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early as possible by reducing the dissolution pH, in order to maximize the drug absorption (Lappas & McKeehan, 1967). Hydroxypropyl methylcellulose phthalate (HPMCP, HP-55) is an enteric coating material which is soluble above pH 5.0 medium and the pKa of the free carboxylic groups of HPMCP is approximately 5.2 (Makhlof et al., 2011; Wang & Zhang, 2012). The free carboxylic acid becomes negatively charged at higher pH values and could interact with positively charged thiolated CS by the electrostatic driving force. Furthermore, the pH-sensitive solubility of HPMCP (HP-55) could enable nanoparticles to withstand the degradation of acidic gastric fluids, but readily dissolve in the mildly acidic to neutral environment of the small intestine, making for the LMWH release and absorption. Therefore, we employed HPMCP as the ionic cross-linking agent with the aim of protecting the loaded LMWH against acidic denaturation and enzymatic degradation. The TCS-HPMCP nanoparticles can be expected to exhibit both pH-responsive and mucoadhesive features. In this study, TCS nanoparticles were prepared by ionic cross-linking with HPMCP (HP-55). The obtained nanoparticles were characterized for the shape, particle size, zeta potential, drug entrapment efficiency, loading capacity and in vitro drug release in the simulated gastrointestinal conditions. The ability of nanoparticles to protect LMWH against gastric pepsin degradation was evaluated in vitro. The permeation enhancing effect was performed in Ussing-type chambers on freshly excised carp small intestinal mucosa. The intestinal mucoadhesion in vivo was studied by confocal laser scanning microscopy using fluorescence-labeled nanoparticles. The anticoagulant effects after oral administration of LMWHloaded TCS/HP-55 nanoparticles (TCS/HP-55 NPs) was estimated using activated partial thromboplastin time (APTT) assay in rats. The main pharmacokinetic parameters were calculated using trapezoidal method.
Materials and methods Materials The LMWH used was enoxaparin (average molecular weight 4.5 kDa and anti-FXa activity 102 IU/mg) provided by Hebei Changshan Biochemical Pharmaceutical Ltd. (Shijiazhuang, China). CS (degree of deacetylation490%) was purchased from Hongfeng Chemical Co., Ltd. (Qingdao, China). Ellman’s reagent (DTNB, 5,50 -Dithiobis-(2-nitrobenzoic acid)) and 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDAC) were purchased from Sigma Chemicals Co. (St. Louis, Mo, USA). TGA was obtained from Chinese medicine (group) company Shanghai chemical reagent (Shanghai, China). HP-55 was a kind gift from ShinEtsu Chemical Co., Japan. Sodium tripolyphosphate (TPP) was obtained from Tianjin Kermel Chemical Reagents Development Centre (Tianjin, China). Azure A was purchased from Nanjing Duly Biotech Co., Ltd (Nanjing, China). Pepsin from porcine stomach (3000 U/mg protein) was obtained from Shanghai Lanji Sci-Technology Development Co., Ltd. (Shanghai, China). Fluorescein-5-isothiocyanate (FITC) and dialysis membrane were purchased from Shanghai Yuanye Bio-Technology Co., Ltd. (Shanghai, China). APTT assay kits were purchased from Nanjing jiancheng Bioengineering
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Institute. (Nanjing, China). All other chemicals and reagents were analytical grade. The buffer solution was prepared according to Pharmacopoeia of the People’s Republic of China (the 2010 version). Synthesis of TCS TCS was synthesized using CS according to the reported literatures (Saboktakin et al., 2011). Briefly, 50 mg CS was dissolved in 50 mL 1% acetic acid. Then 200 mg EDAC was added in order to activate the carboxylic acid moieties of TGA. Thereafter, 30 mL TGA was added into the solution and the pH was adjusted to 5.0 with 3 mol/L NaOH. The reaction mixture was incubated for 5 h in dark at room temperature under constant stirring. To eliminate the unbonded TGA and isolate the polymer conjugates, the reaction mixture was dialyzed against 5 mmol/L HCl one time (MWCO 8–14 kDa) over a period of 3 d in the dark, then two times against 5 mmol/L HCl containing 1.0% NaCl to reduce ionic interactions between the cationic polymer and the anionic sulfhydryl compound, finally against 1.0 mmol/L HCl. After dialysis, the polymer was lyophilized and stored at 4 C for further use. Characterization of TCS Quantification of thiol group content by Ellman’s method The amount of thiol groups and disulfide bonds on TCS was determined using Ellman’s reagent (Anitha et al., 2011). Briefly, 50 mg TCS was dissolved in 2 mL 1% acetic acid. Then 1 mL solution above was added to 6.5 mL of Ellman’s reagent (250 mg of DTNB in 500 mL phosphate buffer pH 8.0). The reaction was allowed to proceed for 2 h in dark at room temperature. The absorbance was measured at a wavelength of 410 nm (TU-1901, Beijing Persee General Instrument Co., Ltd., Beijing, China). Control samples were elaborated with distilled water. The amount of thiol moieties was calculated from the corresponding standard curve elaborated between 0.68 and 2.72 mg/mL of TGA solution in water. Fourier transformed infrared spectroscopy (FTIR) FTIR spectra of TCS and CS were recorded in range 400 4000 cm1 on a Fourier transforms infrared spectrophotometer (Tensor27, Bruker Co., Germany) using KBr method. The new amide bond formation and thiol group substitution in TCS can be confirmed by FTIR, based on the presence of the characteristic peaks of newly formed amide bond and thiol groups. Preparation of nanoparticles The pH-sensitive TCS NPs were prepared using ionic crosslinking method as reported earlier (Makhlof et al., 2011). Briefly, TCS was dissolved in 0.1 mol/L acetic acid. Then HP55 solution in 0.1 mol/L NaOH (0.2%, w/v) containing LMWH was added drop-by-drop to TCS solution under magnetic stirring at room temperature. The pH of the final nanoparticle dispersion was adjusted to 4.0. Then the nanoparticles were separated from the solution by ultracentrifugation (TGL-16G, Anke Scientific Instrument Factory, Shanghai, China) at 13 000 rpm for 30 min.
DOI: 10.3109/10717544.2014.909908
The pH-nonsensitive TCS NPs were preparation with TPP as cross-linking agent. TPP (0.2%, w/v) was dissolved in 0.4 mol/L NaOH solutions. The rest operations were the same as above. In vitro characterization of nanoparticles Transmission electron microscopy (TEM)
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The nanoparticles were examined for their morphological examination by TEM (Nogueira et al., 2013). Briefly, 1 mL of the nanoparticles dispersed in 100 mL distilled water was placed on a carbon-coated copper grid, and the 2% (w/v) solution of phosphotungstic acid was used as a negative stain for 10 min. The images were obtained with a Jeol JEM-1011 electron microscope (Jeol Ltd., Tokyo, Japan) operating at an acceleration voltage of 80 kV.
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(ZRS-8G, Tiandatianfa Science and Technology Co., Ltd., Tianjin, China). At predetermined time points, samples of 5 mL were withdrawn from outside of the dialysis membrane (attached with 0.45-mm membrane filter) and replaced with equal volumes of the release medium. The sample was analyzed for LMWH content according to a previously described colorimetric method in section ‘‘Drug entrapment efficiency and loading capacity’’. LMWH stability against gastric degradation
The size and zeta potential of the nanoparticles were measured with a Zatesizer Nano ZEN3690 (Malvern Instruments Ltd., Malvern, UK) by dynamic light scattering technique. The particle-size distribution of the nanoparticles was reported as a polydispersity index (PDI). All measurements were performed in triplicate.
LMWH stability against pepsin degradation was evaluated in vitro to assess the protective effect of the nanoparticle formulations against gastric degradation (Zhang et al., 2010). Typically, pepsin solution was prepared in 0.05 mol/L HCl. Pepsin solution was added to the LMWH solution, TCS/TPP NPs or TCS/HP-55 NPs. The mixtures were incubated at 37 C in a shaking (100 rpm) water bath (SHZ-82, Changzhou Guohua Electric Appliance Co., Ltd., Jiangsu, China), and aliquots (100 mL) were withdrawn after specified time intervals. The enzymatic reaction was stopped immediately by the addition of 100 mL of NaOH (0.5%, w/v). After standing for 1 h and centrifugation at 4000 rpm for 10 min, 2 mL supernatant was assayed for LMWH content according to a previously described colorimetric method.
Drug entrapment efficiency and loading capacity
Penetration study in vitro
The entrapment efficiency of LMWH in nanoparticles was determined by measuring the amount of unentrapped drug in aqueous solution (supernatant) followed by centrifugation of dispersion using the Azure A colorimetric method reported earlier (Patel et al., 2012). Briefly, 20 mL suspension was ultracentrifugated at 13 000 rpm for 30 min to separate the free LMWH in the supernatant from that loaded in the nanoparticles. Then supernatant samples were reacted with Azure A solution (0.04% w/v) and were estimated spectrophotometrically with a UV spectrophotometer at 653 nm using a standard curve. All measurements were performed in triplicate. The entrapment efficiency and loading capacity of the nanoparticles were calculated as follows:
In vitro studies that would support the postulated mechanism for the permeation enhancing effect of thiomers were performed on freshly excised intestinal mucosa mounted in Ussing-type chambers (Watanabe et al., 2004; Liu et al., 2012). The studies were carried out in Franz diffusion cells with a 4 cm2 orifice area (RYJ-6A, Shanghai Huanghai testing instrument Co., Ltd., Shanghai, China). Fresh small intestine of killed carp (1000 1500 g) was excised, rinsed with NaCl 0.9 % (w/v), and cut into segments of 4 cm in length. Each segment was placed between the donor and acceptor chambers of the cell. The receptor chamber of the Franz cell was filled with 25 ± 0.5 C phosphate buffer (pH 6.8). After 30 min of pre-incubation, 2 mL TCS/HP-55 NPs in phosphate buffer at a LMWH concentration of 0.17 mg/mL were placed on the excised carp intestine in the donor chambers and then incubated at 25 ± 0.5 C under 200 rpm. At fixed time intervals, 2 mL samples were withdrawn from the acceptor and replaced with fresh buffer (attached with 0.45-mm membrane filter). The sample was assayed for LMWH content according to a previously described colorimetric method. As control the permeation test of LMWH solution and CS/HP-55 NPs in phosphate buffer at the same concentration was performed, respectively. CS/HP-55 NPs were prepared as the descriptions in section ‘‘Preparation of nanoparticles’’. The apparent permeability coefficients (Papp) for LMWH were calculated in accordance with the following equation:
Size and zeta potential
EEð%Þ ¼
ðtotal amount of LMWH-free LMWHÞ 100% total amount of LMWH
LCð%Þ ¼
ðtotal amount of LMWH-free LMWHÞ 100%: total dry weight of NPs
In vitro release study In vitro release assessments of LMWH from the prepared nanoparticles were evaluated in 0.1 mol/L HCl solution at pH 1.0 and phosphate buffered saline (PBS) at pH 6.8 using the dialysis method. A total of 10 mL TCS/HP-55 NPs suspension, 10 mL TCS/TPP NPs suspension and 5 mg LMWH were placed into ready-to-use dialysis membrane (MWCO 20kDa), respectively. The dialysis membrane was placed in beaker containing 50 mL of the release medium and incubated in an intelligent dissolution tester at 37 ± 0.5 C shaking water-bath under gentle magnetic stirring at 100 rpm
Papp ¼ Q = ðA c tÞ Where Papp is the apparent permeability coefficient (cm/s), Q is the total amount of LMWH permeated within the incubation time (mg), A is the diffusion area of the Ussing chamber
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(cm2), c is the initial concentration of LMWH in the donor compartment (mg/cm3), and t is the total time of the experiment (s). Transport enhancement ratios (R) were calculated from Papp values according to the following equation: R ¼Papp ðsampleÞ=Papp ðcontrolÞ In vivo mucosal adhension of the nanoparticles
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Preparation of FITC-labeled nanoparticles FITC-labeled TCS or CS was synthesized by the reaction between the isothiocyanate group of FITC and the primary amino group of TCS or CS (Fan et al., 2012). The TCS or CS solution was prepared by dissolving 100 mg of the polymer in 5 ml of 2% acetic acid, and then 1 mol/L NaOH was added till a pH value of 7.5. After that, 1 mL of FITC solution (1 mg/mL in methanol) was slowly added to TCS or CS solution under continuous stirring. The reaction was allowed to proceed in the dark at room temperature for 6 h and then precipitated by adding methanol/ammonia mixture (70:30, v/v) with continuous stirring for 5 min, centrifuged at 8000 rpm for 10 min, and extensively washed with methanol until there was no fluorescence in the washing solution wavelengths (EX:490nm, EM:520nm). Thereafter the labeled TCS or CS was redissolved in 20 mL 0.1 mol/L acetic acid and dialyzed in the dark against 3L ultrapure water for 3 d. Finally, the labeled TCS or CS was freeze-dried for further use. FITC-labeled TCS/HP-55 NPs, TCS/TPP NPs and CS/HP55 NPs were prepared as described in section ‘‘Preparation of nanoparticles’’. In vivo mucosal adhesion of the nanoparticles All animal experiments in the present study were ethically approved by Laboratory Animal Ethics Committee in Shanxi medical university. All experimental procedures were performed in conformity with institutional guidelines and protocols for the care and use of laboratory animals. Male Sprague Dawley(SD)rats (180 200g) were obtained from Shanxi medical university, China. The samples used for the in vivo mucoadhesion studies were prepared with FITC-labeled TCS or CS nanoparticles as previous descriptions. Mucoadhesion was evaluated by confocal laser scanning microscopy (CLSM, FV1000, Olympus, Japan) (Paliwal et al., 2012). The rats were fasted for 12 h with free access to water before the administration of various formulations. TCS/HP-55 NPs suspension, TCS/TPP NPs suspension, CS/HP-55 NPs suspension and TCS solution were administered, respectively, through an oral gavage tube that was carefully passed down through the esophagus into the stomach of rats. Then the rats were sacrificed either 2 h after oral administration. The small intestinal segments close to the stomach were resected, opened longitudinally, and slowly rinsed with saline. For CLSM visualization, the freshly excised tissues were put into petri dishes and the light was adjusted in the green fluorescence mode, which yielded an excitation wavelength at 490 nm and emission wavelengths at 520 nm. (Tomography scanning, in 80 mm magnification 10, observing the fluorescence intensity).
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In vivo anticoagulant activity The APTT was chosen as a widely accepted clinically relevant measure to assess the anticoagulant effect of heparin (Cushing et al., 2010). In this work, APTT activity was determined using a commercial kit according to the instructions of the manufacturer. Twelve male Sprague-Dawley (SD) rats (180 200g) were randomized into four groups and fasted for 12 h with free access to water before the administration of various formulations. TCS/HP-55 NPs suspension, CS/HP-55 NPs suspension, TCS/TPP NPs suspension and LMWH solution were administered, respectively, through an oral gavage tube that was carefully passed down through the esophagus into the stomach of rats at a single oral dose (50 mg/kg). Here, the LMWH solution, TCS/TPP NPs and CS/HP-55 NPs were used as control. At appropriate time intervals, blood (approximately 0.45 mL) was collected via the jugular vein catheter, and immediately transferred into EP tube containing 0.05 mL 0.109 mol/L sodium citrate. Plasma samples were harvested after centrifugation at 3000 rpm for 15 min at 4 C. According to the instructions, freshly prepared plasma (50 mL) and 50 mL APTT reagent were pre-incubated at 37 C for 5 min. Then coagulation was started by the addition of 50 mL of 25 mmol/L CaCl2 solution, and the clotting time was measured mechanically. The absolute bioavailability (F) was calculated by comparing the AUC of oral administration with that of intravenous injection corrected by the administered dose. Statistical data analysis Quantitative data were expressed as the mean ± standard deviation (SD). Statistical analysis was performed with SPSS v 13.0 software. Statistical data analyses were performed using the t test with p50.05 as the minimal level of significance.
Results and discussion Synthesis and characterization of TCS TCS was synthesized using TGA as the thiol reagent and the coupling reaction was catalyzed by EDAC. The obtained polymers appeared as white, odorless powder of fibrous structure. The amount of the thiol groups attached to the polymer by the Ellman’s test was 396.97 ± 54.54 mmol per gram polymer. Figure 1 represents the combined FTIR spectra of CS and TCS. According to spectrum of the TCS (Figure 1B), -OH stretching peak at 3475 cm1 indicated the presence of hydroxyl groups of chitosan; -SH stretching peak at 2346 cm1 was related to -SH groups of TGA. The amide band peak at 1513 cm1 confirmed the formation of amide bond in the chitosan-TGA conjugates. Preparation and characterization of nanoparticles Nanoparticles were produced by the ionic gelation of positively charged TCS with negatively charged HP-55 or TPP. The ratio of polymers to cross-linkers and the pH value of the final nanoparticles dispersion had a great impact on the formation of nanoparticles and the formulation design was
pH-responsive and mucoadhesive nanoparticle system
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Figure 1. FTIR spectra of CS (A) and TCS (B). Table 1. Particle sizes and zeta potentials of nanoparticles prepared with different TCS/polyanion weight ratios. TCS: polyanion (w/w)
Size (nm)
PDI
Zeta potential (mV)
TCS/HP-55 NPs
1:0.8 1:1 1:1.5 1.3.6
323.1 329.6 508.1 1360.67
0.169 0.110 0.276 0.305
35.5 34.8 23.6 13.3
97.75 12.16 97.81 12.22 95.12 9.55 89.57 4.80
TCS/TPP NPs
1:0.4 1:0.5 1:0.6
172.6 190.0 297.7
0.157 0.134 0.271
15.9 12.7 11.6
98.01 14.15 97.91 15.14 95.32 13.75
Formulation
EE (%)
LA (%)
optimized on the basis of particle size, PDI, Zeta potential, encapsulation efficiency and loading capacity. With the addition of polyanion solutions, TCS solution spontaneously changed from clear to opalescent solution and finally to turbid suspension, indicating the formation of nanoparticles then microparticles and eventually aggregates. As shown in Table 1, an increased volume of HP-55 or TPP solution resulted in increased particle size and less positive values of the zeta potential. Also, with the increasing weight ratio of TCS: HP-55 above 1:1, both loading capacity and encapsulation efficiency decreased. Positive charges of particles could give rise to a strong electrostatic interaction with negatively charged mucus layer (Bernkop-Schnurch et al., 2004), hence particles with a more positive zeta potential should display comparatively more pronounced mucoadhesive properties. In addition, we observed an enhanced yield of the nanoparticles by increasing the weight ratio of polyanion, but further increase in polyanion ratio resulted in aggregation of the particles. Based on the above considerations,
the optimal weight ratio between TCS and polyanions was found to be 1:1 for TCS/ HP-55 NPs, compared to 2:1 for TCS/TPP NPs. To study the effects of changing pH values on nanoparticle size and zeta potential, TCS nanoparticles were formulated at fixed weight ratio of TCS to polyanion. Figure 2 presents the characteristics of nanoparticles with the changing pH values. It was observed that further increase in the preparation pH resulted in an increase in the particle size of obtained dispersions and a corresponding decrease in their positive surface charge. In addition, due to the pH-sensitve feature of HP-55, both measured particle size and zeta potential of TCS/ HP-55 NPs were more sensitive to the changing pH values as compared with TCS/TPP NPs. As we know the nanoparticles are formed spontaneously upon mixing of polyanion and TCS solutions due to inter- and intra-molecular cross-linkages between positively charged amino groups of TCS and negatively charged polyanions. Because of the presence of amine groups TCS could carry a lot of positive charges at lower pH, making for ionic interaction with the polyanion. But with regard to the TCS/HP-55 NPs, if the pH of nanoparticles dispersion was too low, some of the -COOgroups on HP-55 became protonated (-COOH) and the electrostatic interaction between TCS and HP-55 was therefore relatively weaker, leading to a relatively larger nanoparticle. According to the outcome of numerous experiments, pH 4.0 was investigated as most suitable for the TCS/HP-55 and TCS/TPP NPs dispersion. Additionally, we investigated the influence of different rpm and time of ultracentrifugation on the characterizations of nanoparticles. The results showed that when the speed was less than 10 000 rpm or the time was less than 20 min, nanoparticles could not be separated completely and
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Figure 2. The effects of pH values of the final NPs dispersion on nanoparticle size (A) and zeta potential (B).
Figure 3. TEM of LMWH-loaded TCS/HP-55 nanoparticles.
efficiently. However, further increasing the centrifugal speed or time above 13 000 rpm or 30 min led to the decreases of both encapsulation efficiency and loading capacity, meaning that the structure of the nanoparticles had been destroyed. Therefore, 13 000 rpm and 20 min were chosen to separate the prepared nanopaticles from the solution. The morphological characterizations of the TCS/HP-55 NPs were evaluated by TEM (Figure 3). The nanoparticles had an almost spherical shape, similar nanometric dimension and were well dispersed without any aggregation. LMWH was efficiently loaded into TCS/HP-55 NPs with an encapsulation efficiency of 97.75 ± 0.02% and loading capacity of 12.22 ± 0.03%. In vitro release study In this study, the release of LMWH from the prepared nanoparticles was evaluated in simulated gastrointestinal fluid without enzymes and compared with the membrane diffusion of free LMWH. Figure 4(A) and (B) represents the cumulative release of LMWH at different time point in vitro from TCS/ HP-55 NPs, TCS/TPP NPs and LMWH solution, respectively. Sink conditions inside and outside the dialysis bags were guaranteed by using diluted dispersions of the nanoparticles. As shown in Figure 4(A) and (B), the diffusion of free LMWH through the dialysis bag was very fast and nearly complete release was observed within the fixed time.
Concerning the NPs formulation, the cases were different. In gastric fluid without enzymes (0.1 mol/L HCl solution) (Figure 4A), TCS/TPP NPs showed 26.8% and 56.7% release of LMWH in 2 h and 12 h, respectively. Whereas, TCS/HP-55 NPs showed 14.6% and 31.7% release of LMWH in 2 h and 12 h, respectively. There were significant differences at 2 h and 12 h (p50.05). However, in the case of release in PBS (pH 6.8) (Figure 4B), no noticeable differences could be observed in the drug release behavior between TCS/TPP NPs and TCS/HP-55 NPs (p40.05). The results could account for the different dissolution feature between TCS/HP-55 matrix and TCS/TPP matrix in acidic environment. Considering that HP-55 is insoluble in acidic conditions, the easy solubility of TCS in lower pH values is prevented by physical entanglement with the undissolved HP-55 polymeric structure. Therefore, the TCS/HP-55 NPs showed an improved physical stability at lower pH values without noticeable change in their size. A slow release behavior of LMWH from nanoparticles at pH 6.8 might be related to the insolubility of TCS at neutral pH. It has been reported that as the pKa value of chitosan is approximately 6.5, chitosan could be soluble at acidic pH due to the protonation of the amine group, yet aggregates at neutral pH (Chen et al., 2013). During the in vitro release experiments of TCS nanoparticles in PBS (pH 6.8), we observed white precipitation of aggregates in the bottom of dialysis bags. This finding suggested that because of the reduced degree of protonation at surface of particles at pH 6.8, decreasing electrostatic repulsion between the particles thereby led to the aggregation of nanoparticles, which resulted in the reduced release of LMWH from nanoparticles in PBS (pH 6.8). It is worth mentioning that the followed in vivo studies in the part of ‘‘Mucoadhesion study of the nanoparticles’’ and ‘‘In vivo anticoagulant effect’’ confirmed the effectivity of nanoparticles for the oral delivery of LMWH. Similarly, previously reported heparin-loaded nanoparticles also displayed a slow release in vitro but generated very positive absorption results in vivo (Jiao et al., 2002). Protection of LMWH study The potential ability of nanoparticles to protect the entrapped LMWH from the digestive gastric enzymes was investigated in the presence of pepsin. Figure 5 depicts the remaining
pH-responsive and mucoadhesive nanoparticle system
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Figure 4. In vitro release profile of LMWH-loaded nanoparticle systems in different simulated pH environments (A) 0.1 mol/L HCl (pH 1.0); (B) phosphate buffered saline (PBS, pH 6.8). Data represent mean ± SD (n ¼ 3). Table 2. In vitro apparent permeability coefficients (Papp) and absorption enhancement of NPs through excised carp intestine.
Formulation TCS/HP-55 NPs CS/HP-55 NPs a
Apparent permeability coefficient, Papp (106 cm/s) (means ± SD, n ¼ 3)
Enhancement ratio, R
4.92 ± 0.014a 3.83 ± 0.015a
1.86 1.45
The Papp enhancement ratio was 1.28-fold higher.
Penetration of the nanoparticles in vitro
Figure 5. Percentage of LMWH remaining after incubation of different samples with pepsin solutions in simulated gastric fluid. Data represent mean ± SD (n ¼ 3).
percentage of LMWH after incubation of LMWH solution, LMWH-loaded TCS/HP-55 NPs and TCS/TPP NPs with pepsin solution in 0.05 mol/L HCl. LMWH solution was rapidly degraded within 5 min. After an incubation period of 30 min with pepsin solution, the TCS/TPP NPs was degraded more quickly than TCS/HP-55 NPs. In this study, the remaining LMWH percentage after incubation of TCS/HP55 NPs, TCS/TPP NPs and LMWH solution at 120 min were 80.44 %, 70.28 %, 43.27 %, respectively. With regard to TCS/ HP-55 NPs, the protective effect was significantly higher than that of TCS/TPP NPs (p50.05). These results could be explained based on the ability of HPMCP to overcome the damages from gastric conditions. In this study, TCS/TPP NPs were rapidly dissociated with fast release of the entrapped drug, while TCS/HP-55 NPs were physically stable and retarded LMWH release in acid medium by their pH-sensitive property. Furthermore, HPMCP has relatively higher molecular weight chains (45 kDa) that can entangle with TCS molecules more efficiently. Due to the steric hindrance effect, the TCS/HP-55 NPs system could moderately protect the wrapped LMWH from stomach acid and digestive enzyme degradation.
The influence of thiolated nanoparticles on the transport of LMWH was investigated by using the Ussing chamber technique, which is particularly well adapted to study the effect of drug-loaded delivery system formulations on drug fluxes across the intestinal barriers. As control, CS/HP-55 NPs and LMWH solution were also used. Results of permeation studies on carp intestine mucosa are represented in Table 2 and Papp values with enhancement ratios are summarized. Within this study it could be demonstrated that nanoparticles could increase the transport of LMWH. The Papp enhancement ratio was 1.86-fold higher for the TCS/HP55 NPs and 1.45-fold higher for the CS/HP-55 NPs as compared with the control, which demonstrated the view that CS-based nanoparticles could mediate the opening of tight junctions between epithelial cells reversibly, thus facilitating the paracellular transport of hydrophilic macromolecules (Chen et al., 2013). Moreover, as compared with the CS formulation group, improved transport of LMWH in TCS formulation group was achieved on account of thiolation. (The Papp enhancement ratio was 1.28-fold higher). As shown by Kast et al. due to the formation of disulfide bonds between their thiol groups and the cysteine-rich subdomains of glycoproteins in the mucus layer, TCS could prolong the contact time of the polymer on mucosal tissue (Kast & Bernkop-Schnu¨rch, 2001), which considerably increased the drug concentration gradient at the absorption site and promoted the permeability. The results in Table 2 could typically support that the permeation enhancing effect of
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Figure 6. Fluorescence microscopic images of microtome section of intestinal epithelium after 2 h of oral administration of different samples (A) TCS solution; (B) CS/HP-55 NPs; (C)TCS/TPP NPs; (D)TCS/HP-55 NPs.
chitosan per se can be strongly improved by the immobilization of thiol groups on the CS backbone. Mucoadhesion study of the nanoparticles To further investigate a possible effect of mucoadhesion on intestinal permeation, we checked the influence of the mucoadhesive capacity of nanoparticles on the LMWH absorption. The mucosal adhesion and uptake properties of fluorescently labeled TCS or CS NPs were evaluated after oral administration to rats. This method did neither affect the polymer ability to conjugate with polyanions nor the properties of the obtained nanoparticles. Following oral administration of the fluorescent nanoparticles, the amount of FITC-labeled TCS or CS associated to the small intestine of rats was evaluated by CLSM. Figure 6 shows the intensity of green fluorescence in the small intestine after oral administration of FITC-labeled TCS solution, CS/HP-55 NPs, TCS/TPP NPs and TCS/HP-55 NPs. As illustrated, the highest intensity of green fluorescence could be detected in the small intestine of TCS/HP-55, while the TCS solution showed the lowest intensity. This result indicated that the TCS/HP-55 NPs possessed the most
effectively adhesive capability on the mucosal surface. It could be explained from two aspects. On the one hand, it might be attributed to the ability of HPMCP to protect TCS/HP-55 NPs from dissociation in the stomach by virtue of its pH-sensitive solubility. Therefore, higher percentage of intact particles could reach the small intestine and interact with the intestinal membrane as compared with the TCS/TPP NPs and TCS solution. On the other hand, the considerable improvement in mucoadhesion of TCS nanoparticles was based on the immobilisation of thiol groups on chitosan. Compared with the CS/HP-55 NPs, the enhancing attachment of TCS/HP-55 NPs to the mucosal side of the intestinal wall can prolong the residence time of the delivery system and achieve higher drug concentration in direct contact with the epithelial membrane. Moreover, this direct contact and/or adhesion of the nanoparticles to the mucosal surface are a prerequisite step before translocation of the intact particles can take place. In vivo anticoagulant effect After a single oral administration of different samples in rats, the coagulating effect on plasma by APTT assay is shown in
pH-responsive and mucoadhesive nanoparticle system
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increased permeation of LMWH via parcellular mechanism of drug absorption.
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Conclusions
Figure 7. The APTT profiles in male adult Sprague-Dawley rats following oral administration of free LMWH and LMWH-loaded nanoparticles (50 mg/kg) Data represent mean ± SD (n ¼ 3).
In the present work, a pH-responsive nanoparticle system composed of TCS and HP-55 was successfully prepared using a simple ionic gelation method. Our results indicated that these nanoparticles were stable at the acid conditions and could protect the entrapped LMWH against gastric degradation. Furthermore, the intestinal penetrative and mucoadhesive property of TCS/HP-55 nanoparticles was significantly improved in vitro and in vivo. The enhanced anticoagulant effects after oral administration of LMWH-loaded TCS/HP55 nanoparticles were also observed in rats. As expected before it could be demonstrated that TCS/HP-55 NPs have promising pH-responsive and mucoadhesive properties. Therefore, the current strategy using TCS/HP-55 nanoparticles as drug carriers seems to have potential applications for the oral delivery of LMWH.
Table 3. Main pharmacokinetic parameters of LMWH in rats following a single oral administration of different samples versus the LMWH solution administered intravenously.
Declaration of interest
Formulation
APTTmax(s)
Tmax(h)
AUC0–24 h(sh)
F(%)
iv. solution Oral solution CS/HP55 TCS/TPP TCS/HP55
– 30.1 45.9 44.9 75.3
– 1 8 5 8
77.67 28.15 298.7 226.0 559.3
100 1.09 11.5 8.73 21.6
This work was supported by the Shanxi Provincial Innovative Foundation for College Students (Grant No. 2012130). The authors report no conflict of interests.
Figure 7. The main pharmacokinetic parameters is calculated and listed in Table 3. As illustrated in Figure 7, the maximal APTT of 30.1s, 44.90s and 45.94s were occurred at 1, 5, and 8 h after dosing with LMWH solution, TCS/TPP NPs and CS/HP-55 NPs, respectively. And a significantly prolonged maximal APTT of 75.34 s was observed at 8 h after TCS/HP-55 NPs administration. Compared with the controls, the maximal APTT value of TCS/HP-55 NPs increased approximately 2-fold (p50.05), reflecting stronger anticoagulant activity of LMWH-loaded TCS/HP-55 NPs. It means that higher concentrations of LMWH in plasma were achieved via TCS/ HP-55 NPs delivery system. In addition, the TCS/HP-55 NPs reached the maximal APTT later than LMWH solution and TCS/TPP NPs, but the same as CS/HP-55 NPs. This typical behavior shows the efficiency of our nano-drug delivery system in controlling the release of LMWH in blood circulation. In terms of absolute bioavailability (Table 3), the TCS/HP55 NPs also showed the highest absorption and the duration of the anticoagulant effect was significantly longer. This result suggests that as compared with the LMWH solution, TCS/ TPP NPs and CS/HP-55 NPs, the TCS/HP-55 NPs could effectively promote drug absorptions probably attributing to the synergistic effects between the pH-sensitive protection of HPMCP and an inherent permeation enhancing feature of thiolated chitosans (TCS). Therefore, a higher drug concentration gradient was offered at the absorption sites and better intimate contacts of TCS/HP-55 NPs with intestinal mucosa
References Amidi M, Mastrobattista E, Jiskoot W, Hennink WE. (2010). Chitosanbased delivery systems for protein therapeutics and antigens. Adv Drug Deliv Rev 62:59–82. Anitha A, Deepa N, Chennazhi KP, et al. (2011). Development of mucoadhesive thiolated chitosan nanoparticles for biomedical applications. Carbohydr Polym 83:66–73. Arbit E, Goldberg M, Gomez-Orellana I, Majuru S. (2006). Oral heparin: status review. Thromb J 4:6. doi:10.1186/1477-9560-4-6. Bagre AP, Jain K, Jain NK. (2013). Alginate coated chitosan core shell nanoparticles for oral delivery of enoxaparin: in vitro and in vivo assessment. Int J Pharm 456:31–40. Bai S, Ahsan F. (2009). Synthesis and evaluation of pegylated dendrimeric nanocarrier for pulmonary delivery of low molecular weight heparin. Pharm Res 26:539–48. Bernkop-Schnurch A, Hornof M, Guggi D. (2004). Thiolated chitosans. Eur J Pharm Biopharm 57:9–17. Chen MC, Mi FL, Liao ZX, et al. (2013). Recent advances in chitosanbased nanoparticles for oral delivery of macromolecules. Adv Drug Deliv Rev 65:865–79. Chen MC, Wong HS, Lin KJ, et al. (2009). The characteristics, biodistribution and bioavailability of a chitosan-based nanoparticulate system for the oral delivery of heparin. Biomaterials 30:6629–37. Cushing DJ, Cooper WD, Cohen ML, et al. (2010). Reversal of heparininduced increases in aPTT in the rat by PM102, a novel heparin antagonist. Eur J Pharmacol 635:165–70. Fan W, Yan W, Xu Z, Ni H. (2012). Erythrocytes load of low molecular weight chitosan nanoparticles as a potential vascular drug delivery system. Colloids Surf B: Biointerf 95:258–65. Hayes PY, Ross BP, Thomas BG, Toth I. (2006). Polycationic lipophiliccore dendrons as penetration enhancers for the oral administration of low molecular weight heparin. Bioorg Med Chem 14:143–52. Javot L, Lecompte T, Rabiskova M, Maincent P. (2009). Encapsulation of low molecular weight heparins: influence on the anti-Xa/anti-IIa ratio. J Contr Rel 139:8–14. Jiao Y, Ubrich N, Marchand-Arvier M, et al. (2002). In vitro and in vivo evaluation of oral heparin-loaded polymeric nanoparticles in rabbits. Circulation 105:230–5. Kast CE, Bernkop-Schnu¨rch A. (2001). Thiolated polymers — thiomers: development and in vitro evaluation of chitosan–thioglycolic acid conjugates. Biomaterials 22:2345–52.
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Kokubo H, Obara S, Minemura K, Tanaka T. (1997). Development of cellulose derivatives as novel enteric coating agents soluble at pH 3.54.5 and higher. Chem Pharm Bull (Tokyo) 45:1350–3. Lappas LC, McKeehan W. (1967). Polymeric pharmaceutical coating materials. II. In vivo evaluation as enteric coatings. J Pharm Sci 56: 1257–61. Lee YK, Kim SK, Lee DY, et al. (2006). Efficacy of orally active chemical conjugate of low molecular weight heparin and deoxycholic acid in rats, mice and monkeys. J Control Release 111:290–8. Liu Y, Di Zang H, Kong M, et al. (2012). In vitro evaluation of mucoadhesion and permeation enhancement of polymeric amphiphilic nanoparticles. Carbohydrate Polymers 89:453–60. Makhlof A, Tozuka Y, Takeuchi H. (2011). Design and evaluation of novel pH-sensitive chitosan nanoparticles for oral insulin delivery. Eur J Pharm Sci 42:445–51. Motlekar NA, Youan BB. (2006). The quest for non-invasive delivery of bioactive macromolecules: a focus on heparins. J Control Release 113: 91–101. Nogueira DR, Tavano L, Mitjans M, et al. (2013). In vitro antitumor activity of methotrexate via pH-sensitive chitosan nanoparticles. Biomaterials 34:2758–72. Oliveira SSM, Oliveira FS, Gaitani CM, Marchetti JM. (2011). Microparticles as a strategy for low-molecular-weight heparin delivery. J Pharmaceut Sci 100:1783–92.
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Paliwal R, Paliwal SR, Agrawal GP, Vyas SP. (2011). Biomimetic solid lipid nanoparticles for oral bioavailability enhancement of low molecular weight heparin and its lipid conjugates: in vitro and in vivo evaluation. Mol Pharm 8:1314–21. Paliwal R, Paliwal SR, Agrawal GP, Vyas SP. (2012). Chitosan nanoconstructs for improved oral delivery of low molecular weight heparin: in vitro and in vivo evaluation. Int J Pharm 422:179–84. Patel B, Gupta V, Ahsan F. (2012). PEG-PLGA based large porous particles for pulmonary delivery of a highly soluble drug, low molecular weight heparin. J Control Release 162:310–20. Saboktakin MR, Tabatabaie RM, Maharramov A, Ramazanov MA. (2011). Development and in vitro evaluation of thiolated chitosan— poly(methacrylic acid) nanoparticles as a local mucoadhesive delivery system. Int J Biol Macromol 48:403–7. Wang X-Q, Zhang Q. (2012). pH-sensitive polymeric nanoparticles to improve oral bioavailability of peptide/protein drugs and poorly watersoluble drugs. Eur J Pharmaceut Biopharmaceut 82:219–29. Watanabe E, Takahashi M, Hayashi M. (2004). A possibility to predict the absorbability of poorly water-soluble drugs in humans based on rat intestinal permeability assessed by an in vitro chamber method. Eur J Pharm Biopharm 58:659–65. Zhang N, Li J, Jiang W, et al. (2010). Effective protection and controlled release of insulin by cationic beta-cyclodextrin polymers from alginate/chitosan nanoparticles. Int J Pharm 393:212–18.
RESEARCH ARTICLE
pH-responsive thiolated chitosan nanoparticles for oral low-molecular weight heparin delivery: in vitro and in vivo evaluation Bo Fan*, Yang Xing*, Ying Zheng, Chuan Sun, and Guixian Liang
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School of Pharmaceutical Science, Shanxi Medical University, Taiyuan, Shanxi, People’s Republic of China
Abstract
Keywords
The aim of present study was to investigate a pH-responsive and mucoadhesive nanoparticle system for oral bioavailability enhancement of low-molecular weight heparin (LMWH). The thioglycolic acid (TGA) was first covalently attached to chitosan (CS) with 396.97 ± 54.54 mmol thiol groups per gram of polymer and then the nanoparticles were prepared with thiolated chitosan (TCS) and pH-sensitive polymer hydroxypropyl methylcellulose phthalate (HPMCP) by ionic cross-linking method. The obtained nanoparticles were characterized for the shape, particle size, zeta potential, drug entrapment efficiency and loading capacity. In vitro results revealed the acid stability of pH-responsive nanoparticles, which had a significant control over LMWH release and could effectively protect entrapped drugs in simulated gastric conditions. By the attachment of the thiol ligand, an improvement of permeation-enhancing effect on freshly excised carp intestine (1.86-fold improvement) could be found. The mucoadhesive properties were evaluated using fluorescently labeled TCS or CS nanoparticles. As compared with the controls, a significant improvement of mucoadhesion on rat intestinal mucosa was observed in TCS/HPMCP nanoparticles via confocal laser scanning microscopy. The activated partial thromboplastin time (APTT) was significantly prolonged and an increase in the oral bioavailability of LMWH was turned out to be pronounced after oral delivered LMWH-loaded TCS/HPMCP nanoparticles in rats, which suggested enhanced anticoagulant effects and improved absorption of LMWH. In conclusion, pH-responsive TCS/HPMCP nanoparticles hold promise for oral delivery of LMWH.
Low-molecular weight heparin, mucoadhesive, oral drug delivery, pH-responsive, thiolated chitosan nanoparticles
Introduction Low-molecular weight heparin (LMWH) is clinically used in parenteral routes mainly as an anticoagulant for the prevention and treatment of deep vein thrombosis (DVT), pulmonary embolism (PE), and coronary syndromes (Arbit et al., 2006; Paliwal et al., 2011). Although oral drug delivery is the safest and most convenient means of drug administration, the main obstacles hindering oral route for LMWH are its low stability at acidic pH of the stomach and poor absorption in gastrointestinal tract due to its high anionic charge density, large molecular size, high water solubility and enzymatic degradation (Motlekar & Youan, 2006). Accordingly, various approaches have been investigated for oral delivery of heparin including penetration enhancers (Hayes et al., 2006), microparticles (Javot et al., 2009; Oliveira et al., 2011), polymeric nanoparticles (Chen et al., 2009), dendrimeric nanocarriers (Bai & Ahsan, 2009), chemical conjugates (Lee et al., 2006), etc. Among them, chitosan (CS)-based nanoparticles are particularly attractive (Paliwal et al., 2012; Bagre et al., 2013). *Bo Fan and Yang Xing contributed equally to this work. Address for correspondence: Guixian Liang, School of Pharmaceutical Science, Shanxi Medical University, No. 56, Xinjian Nan Road, Taiyuan 030001, Shanxi, People’s Republic of China. Tel/Fax: +86 351 4690137. E-mail: [email protected]
History Received 19 February 2014 Revised 25 March 2014 Accepted 26 March 2014
CS, a cationic polysaccharide, is widely regarded as a safe and efficient intestinal absorption enhancer of therapeutic macromolecules, owing to its inherent mucoadhesive feature and ability to modulate the integrity of epithelial tight junctions reversibly (Amidi et al., 2010). These mucoadhesive and permeation-enhancing properties of chitosan can be strongly improved by the immobilization of thiol groups on the polymer. The significantly improved mucoadhesive properties may be attributed to the formation of disulfide bonds between their thiol groups and the cysteine-rich subdomains of glycoproteins in the mucus layer, which is stronger than the non-covalent interaction between cationic CS and anionic mucosal substances (Kast & BernkopSchnu¨rch, 2001). However, the introduction of the thiol moieties does not modify the main limitation of CS nanoparticles for oral peptide and protein delivery that the polymer matrix can be easily dissolved in gastric juice by protonation of the amino groups at low pH values. To overcome the obstacle, pH-responsive enteric polymer could be introduced in the preparation of nanoparticles. Most commercially available enteric coating materials are soluble in the pH range from 5.0 to 7.0 (Kokubo et al., 1997). But as for drugs with poor and limited absorbability in the gastro-intestinal tract, it is desirable to ensure that the enteric polymer is dissolved as
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early as possible by reducing the dissolution pH, in order to maximize the drug absorption (Lappas & McKeehan, 1967). Hydroxypropyl methylcellulose phthalate (HPMCP, HP-55) is an enteric coating material which is soluble above pH 5.0 medium and the pKa of the free carboxylic groups of HPMCP is approximately 5.2 (Makhlof et al., 2011; Wang & Zhang, 2012). The free carboxylic acid becomes negatively charged at higher pH values and could interact with positively charged thiolated CS by the electrostatic driving force. Furthermore, the pH-sensitive solubility of HPMCP (HP-55) could enable nanoparticles to withstand the degradation of acidic gastric fluids, but readily dissolve in the mildly acidic to neutral environment of the small intestine, making for the LMWH release and absorption. Therefore, we employed HPMCP as the ionic cross-linking agent with the aim of protecting the loaded LMWH against acidic denaturation and enzymatic degradation. The TCS-HPMCP nanoparticles can be expected to exhibit both pH-responsive and mucoadhesive features. In this study, TCS nanoparticles were prepared by ionic cross-linking with HPMCP (HP-55). The obtained nanoparticles were characterized for the shape, particle size, zeta potential, drug entrapment efficiency, loading capacity and in vitro drug release in the simulated gastrointestinal conditions. The ability of nanoparticles to protect LMWH against gastric pepsin degradation was evaluated in vitro. The permeation enhancing effect was performed in Ussing-type chambers on freshly excised carp small intestinal mucosa. The intestinal mucoadhesion in vivo was studied by confocal laser scanning microscopy using fluorescence-labeled nanoparticles. The anticoagulant effects after oral administration of LMWHloaded TCS/HP-55 nanoparticles (TCS/HP-55 NPs) was estimated using activated partial thromboplastin time (APTT) assay in rats. The main pharmacokinetic parameters were calculated using trapezoidal method.
Materials and methods Materials The LMWH used was enoxaparin (average molecular weight 4.5 kDa and anti-FXa activity 102 IU/mg) provided by Hebei Changshan Biochemical Pharmaceutical Ltd. (Shijiazhuang, China). CS (degree of deacetylation490%) was purchased from Hongfeng Chemical Co., Ltd. (Qingdao, China). Ellman’s reagent (DTNB, 5,50 -Dithiobis-(2-nitrobenzoic acid)) and 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDAC) were purchased from Sigma Chemicals Co. (St. Louis, Mo, USA). TGA was obtained from Chinese medicine (group) company Shanghai chemical reagent (Shanghai, China). HP-55 was a kind gift from ShinEtsu Chemical Co., Japan. Sodium tripolyphosphate (TPP) was obtained from Tianjin Kermel Chemical Reagents Development Centre (Tianjin, China). Azure A was purchased from Nanjing Duly Biotech Co., Ltd (Nanjing, China). Pepsin from porcine stomach (3000 U/mg protein) was obtained from Shanghai Lanji Sci-Technology Development Co., Ltd. (Shanghai, China). Fluorescein-5-isothiocyanate (FITC) and dialysis membrane were purchased from Shanghai Yuanye Bio-Technology Co., Ltd. (Shanghai, China). APTT assay kits were purchased from Nanjing jiancheng Bioengineering
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Institute. (Nanjing, China). All other chemicals and reagents were analytical grade. The buffer solution was prepared according to Pharmacopoeia of the People’s Republic of China (the 2010 version). Synthesis of TCS TCS was synthesized using CS according to the reported literatures (Saboktakin et al., 2011). Briefly, 50 mg CS was dissolved in 50 mL 1% acetic acid. Then 200 mg EDAC was added in order to activate the carboxylic acid moieties of TGA. Thereafter, 30 mL TGA was added into the solution and the pH was adjusted to 5.0 with 3 mol/L NaOH. The reaction mixture was incubated for 5 h in dark at room temperature under constant stirring. To eliminate the unbonded TGA and isolate the polymer conjugates, the reaction mixture was dialyzed against 5 mmol/L HCl one time (MWCO 8–14 kDa) over a period of 3 d in the dark, then two times against 5 mmol/L HCl containing 1.0% NaCl to reduce ionic interactions between the cationic polymer and the anionic sulfhydryl compound, finally against 1.0 mmol/L HCl. After dialysis, the polymer was lyophilized and stored at 4 C for further use. Characterization of TCS Quantification of thiol group content by Ellman’s method The amount of thiol groups and disulfide bonds on TCS was determined using Ellman’s reagent (Anitha et al., 2011). Briefly, 50 mg TCS was dissolved in 2 mL 1% acetic acid. Then 1 mL solution above was added to 6.5 mL of Ellman’s reagent (250 mg of DTNB in 500 mL phosphate buffer pH 8.0). The reaction was allowed to proceed for 2 h in dark at room temperature. The absorbance was measured at a wavelength of 410 nm (TU-1901, Beijing Persee General Instrument Co., Ltd., Beijing, China). Control samples were elaborated with distilled water. The amount of thiol moieties was calculated from the corresponding standard curve elaborated between 0.68 and 2.72 mg/mL of TGA solution in water. Fourier transformed infrared spectroscopy (FTIR) FTIR spectra of TCS and CS were recorded in range 400 4000 cm1 on a Fourier transforms infrared spectrophotometer (Tensor27, Bruker Co., Germany) using KBr method. The new amide bond formation and thiol group substitution in TCS can be confirmed by FTIR, based on the presence of the characteristic peaks of newly formed amide bond and thiol groups. Preparation of nanoparticles The pH-sensitive TCS NPs were prepared using ionic crosslinking method as reported earlier (Makhlof et al., 2011). Briefly, TCS was dissolved in 0.1 mol/L acetic acid. Then HP55 solution in 0.1 mol/L NaOH (0.2%, w/v) containing LMWH was added drop-by-drop to TCS solution under magnetic stirring at room temperature. The pH of the final nanoparticle dispersion was adjusted to 4.0. Then the nanoparticles were separated from the solution by ultracentrifugation (TGL-16G, Anke Scientific Instrument Factory, Shanghai, China) at 13 000 rpm for 30 min.
DOI: 10.3109/10717544.2014.909908
The pH-nonsensitive TCS NPs were preparation with TPP as cross-linking agent. TPP (0.2%, w/v) was dissolved in 0.4 mol/L NaOH solutions. The rest operations were the same as above. In vitro characterization of nanoparticles Transmission electron microscopy (TEM)
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The nanoparticles were examined for their morphological examination by TEM (Nogueira et al., 2013). Briefly, 1 mL of the nanoparticles dispersed in 100 mL distilled water was placed on a carbon-coated copper grid, and the 2% (w/v) solution of phosphotungstic acid was used as a negative stain for 10 min. The images were obtained with a Jeol JEM-1011 electron microscope (Jeol Ltd., Tokyo, Japan) operating at an acceleration voltage of 80 kV.
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(ZRS-8G, Tiandatianfa Science and Technology Co., Ltd., Tianjin, China). At predetermined time points, samples of 5 mL were withdrawn from outside of the dialysis membrane (attached with 0.45-mm membrane filter) and replaced with equal volumes of the release medium. The sample was analyzed for LMWH content according to a previously described colorimetric method in section ‘‘Drug entrapment efficiency and loading capacity’’. LMWH stability against gastric degradation
The size and zeta potential of the nanoparticles were measured with a Zatesizer Nano ZEN3690 (Malvern Instruments Ltd., Malvern, UK) by dynamic light scattering technique. The particle-size distribution of the nanoparticles was reported as a polydispersity index (PDI). All measurements were performed in triplicate.
LMWH stability against pepsin degradation was evaluated in vitro to assess the protective effect of the nanoparticle formulations against gastric degradation (Zhang et al., 2010). Typically, pepsin solution was prepared in 0.05 mol/L HCl. Pepsin solution was added to the LMWH solution, TCS/TPP NPs or TCS/HP-55 NPs. The mixtures were incubated at 37 C in a shaking (100 rpm) water bath (SHZ-82, Changzhou Guohua Electric Appliance Co., Ltd., Jiangsu, China), and aliquots (100 mL) were withdrawn after specified time intervals. The enzymatic reaction was stopped immediately by the addition of 100 mL of NaOH (0.5%, w/v). After standing for 1 h and centrifugation at 4000 rpm for 10 min, 2 mL supernatant was assayed for LMWH content according to a previously described colorimetric method.
Drug entrapment efficiency and loading capacity
Penetration study in vitro
The entrapment efficiency of LMWH in nanoparticles was determined by measuring the amount of unentrapped drug in aqueous solution (supernatant) followed by centrifugation of dispersion using the Azure A colorimetric method reported earlier (Patel et al., 2012). Briefly, 20 mL suspension was ultracentrifugated at 13 000 rpm for 30 min to separate the free LMWH in the supernatant from that loaded in the nanoparticles. Then supernatant samples were reacted with Azure A solution (0.04% w/v) and were estimated spectrophotometrically with a UV spectrophotometer at 653 nm using a standard curve. All measurements were performed in triplicate. The entrapment efficiency and loading capacity of the nanoparticles were calculated as follows:
In vitro studies that would support the postulated mechanism for the permeation enhancing effect of thiomers were performed on freshly excised intestinal mucosa mounted in Ussing-type chambers (Watanabe et al., 2004; Liu et al., 2012). The studies were carried out in Franz diffusion cells with a 4 cm2 orifice area (RYJ-6A, Shanghai Huanghai testing instrument Co., Ltd., Shanghai, China). Fresh small intestine of killed carp (1000 1500 g) was excised, rinsed with NaCl 0.9 % (w/v), and cut into segments of 4 cm in length. Each segment was placed between the donor and acceptor chambers of the cell. The receptor chamber of the Franz cell was filled with 25 ± 0.5 C phosphate buffer (pH 6.8). After 30 min of pre-incubation, 2 mL TCS/HP-55 NPs in phosphate buffer at a LMWH concentration of 0.17 mg/mL were placed on the excised carp intestine in the donor chambers and then incubated at 25 ± 0.5 C under 200 rpm. At fixed time intervals, 2 mL samples were withdrawn from the acceptor and replaced with fresh buffer (attached with 0.45-mm membrane filter). The sample was assayed for LMWH content according to a previously described colorimetric method. As control the permeation test of LMWH solution and CS/HP-55 NPs in phosphate buffer at the same concentration was performed, respectively. CS/HP-55 NPs were prepared as the descriptions in section ‘‘Preparation of nanoparticles’’. The apparent permeability coefficients (Papp) for LMWH were calculated in accordance with the following equation:
Size and zeta potential
EEð%Þ ¼
ðtotal amount of LMWH-free LMWHÞ 100% total amount of LMWH
LCð%Þ ¼
ðtotal amount of LMWH-free LMWHÞ 100%: total dry weight of NPs
In vitro release study In vitro release assessments of LMWH from the prepared nanoparticles were evaluated in 0.1 mol/L HCl solution at pH 1.0 and phosphate buffered saline (PBS) at pH 6.8 using the dialysis method. A total of 10 mL TCS/HP-55 NPs suspension, 10 mL TCS/TPP NPs suspension and 5 mg LMWH were placed into ready-to-use dialysis membrane (MWCO 20kDa), respectively. The dialysis membrane was placed in beaker containing 50 mL of the release medium and incubated in an intelligent dissolution tester at 37 ± 0.5 C shaking water-bath under gentle magnetic stirring at 100 rpm
Papp ¼ Q = ðA c tÞ Where Papp is the apparent permeability coefficient (cm/s), Q is the total amount of LMWH permeated within the incubation time (mg), A is the diffusion area of the Ussing chamber
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(cm2), c is the initial concentration of LMWH in the donor compartment (mg/cm3), and t is the total time of the experiment (s). Transport enhancement ratios (R) were calculated from Papp values according to the following equation: R ¼Papp ðsampleÞ=Papp ðcontrolÞ In vivo mucosal adhension of the nanoparticles
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Preparation of FITC-labeled nanoparticles FITC-labeled TCS or CS was synthesized by the reaction between the isothiocyanate group of FITC and the primary amino group of TCS or CS (Fan et al., 2012). The TCS or CS solution was prepared by dissolving 100 mg of the polymer in 5 ml of 2% acetic acid, and then 1 mol/L NaOH was added till a pH value of 7.5. After that, 1 mL of FITC solution (1 mg/mL in methanol) was slowly added to TCS or CS solution under continuous stirring. The reaction was allowed to proceed in the dark at room temperature for 6 h and then precipitated by adding methanol/ammonia mixture (70:30, v/v) with continuous stirring for 5 min, centrifuged at 8000 rpm for 10 min, and extensively washed with methanol until there was no fluorescence in the washing solution wavelengths (EX:490nm, EM:520nm). Thereafter the labeled TCS or CS was redissolved in 20 mL 0.1 mol/L acetic acid and dialyzed in the dark against 3L ultrapure water for 3 d. Finally, the labeled TCS or CS was freeze-dried for further use. FITC-labeled TCS/HP-55 NPs, TCS/TPP NPs and CS/HP55 NPs were prepared as described in section ‘‘Preparation of nanoparticles’’. In vivo mucosal adhesion of the nanoparticles All animal experiments in the present study were ethically approved by Laboratory Animal Ethics Committee in Shanxi medical university. All experimental procedures were performed in conformity with institutional guidelines and protocols for the care and use of laboratory animals. Male Sprague Dawley(SD)rats (180 200g) were obtained from Shanxi medical university, China. The samples used for the in vivo mucoadhesion studies were prepared with FITC-labeled TCS or CS nanoparticles as previous descriptions. Mucoadhesion was evaluated by confocal laser scanning microscopy (CLSM, FV1000, Olympus, Japan) (Paliwal et al., 2012). The rats were fasted for 12 h with free access to water before the administration of various formulations. TCS/HP-55 NPs suspension, TCS/TPP NPs suspension, CS/HP-55 NPs suspension and TCS solution were administered, respectively, through an oral gavage tube that was carefully passed down through the esophagus into the stomach of rats. Then the rats were sacrificed either 2 h after oral administration. The small intestinal segments close to the stomach were resected, opened longitudinally, and slowly rinsed with saline. For CLSM visualization, the freshly excised tissues were put into petri dishes and the light was adjusted in the green fluorescence mode, which yielded an excitation wavelength at 490 nm and emission wavelengths at 520 nm. (Tomography scanning, in 80 mm magnification 10, observing the fluorescence intensity).
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In vivo anticoagulant activity The APTT was chosen as a widely accepted clinically relevant measure to assess the anticoagulant effect of heparin (Cushing et al., 2010). In this work, APTT activity was determined using a commercial kit according to the instructions of the manufacturer. Twelve male Sprague-Dawley (SD) rats (180 200g) were randomized into four groups and fasted for 12 h with free access to water before the administration of various formulations. TCS/HP-55 NPs suspension, CS/HP-55 NPs suspension, TCS/TPP NPs suspension and LMWH solution were administered, respectively, through an oral gavage tube that was carefully passed down through the esophagus into the stomach of rats at a single oral dose (50 mg/kg). Here, the LMWH solution, TCS/TPP NPs and CS/HP-55 NPs were used as control. At appropriate time intervals, blood (approximately 0.45 mL) was collected via the jugular vein catheter, and immediately transferred into EP tube containing 0.05 mL 0.109 mol/L sodium citrate. Plasma samples were harvested after centrifugation at 3000 rpm for 15 min at 4 C. According to the instructions, freshly prepared plasma (50 mL) and 50 mL APTT reagent were pre-incubated at 37 C for 5 min. Then coagulation was started by the addition of 50 mL of 25 mmol/L CaCl2 solution, and the clotting time was measured mechanically. The absolute bioavailability (F) was calculated by comparing the AUC of oral administration with that of intravenous injection corrected by the administered dose. Statistical data analysis Quantitative data were expressed as the mean ± standard deviation (SD). Statistical analysis was performed with SPSS v 13.0 software. Statistical data analyses were performed using the t test with p50.05 as the minimal level of significance.
Results and discussion Synthesis and characterization of TCS TCS was synthesized using TGA as the thiol reagent and the coupling reaction was catalyzed by EDAC. The obtained polymers appeared as white, odorless powder of fibrous structure. The amount of the thiol groups attached to the polymer by the Ellman’s test was 396.97 ± 54.54 mmol per gram polymer. Figure 1 represents the combined FTIR spectra of CS and TCS. According to spectrum of the TCS (Figure 1B), -OH stretching peak at 3475 cm1 indicated the presence of hydroxyl groups of chitosan; -SH stretching peak at 2346 cm1 was related to -SH groups of TGA. The amide band peak at 1513 cm1 confirmed the formation of amide bond in the chitosan-TGA conjugates. Preparation and characterization of nanoparticles Nanoparticles were produced by the ionic gelation of positively charged TCS with negatively charged HP-55 or TPP. The ratio of polymers to cross-linkers and the pH value of the final nanoparticles dispersion had a great impact on the formation of nanoparticles and the formulation design was
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Figure 1. FTIR spectra of CS (A) and TCS (B). Table 1. Particle sizes and zeta potentials of nanoparticles prepared with different TCS/polyanion weight ratios. TCS: polyanion (w/w)
Size (nm)
PDI
Zeta potential (mV)
TCS/HP-55 NPs
1:0.8 1:1 1:1.5 1.3.6
323.1 329.6 508.1 1360.67
0.169 0.110 0.276 0.305
35.5 34.8 23.6 13.3
97.75 12.16 97.81 12.22 95.12 9.55 89.57 4.80
TCS/TPP NPs
1:0.4 1:0.5 1:0.6
172.6 190.0 297.7
0.157 0.134 0.271
15.9 12.7 11.6
98.01 14.15 97.91 15.14 95.32 13.75
Formulation
EE (%)
LA (%)
optimized on the basis of particle size, PDI, Zeta potential, encapsulation efficiency and loading capacity. With the addition of polyanion solutions, TCS solution spontaneously changed from clear to opalescent solution and finally to turbid suspension, indicating the formation of nanoparticles then microparticles and eventually aggregates. As shown in Table 1, an increased volume of HP-55 or TPP solution resulted in increased particle size and less positive values of the zeta potential. Also, with the increasing weight ratio of TCS: HP-55 above 1:1, both loading capacity and encapsulation efficiency decreased. Positive charges of particles could give rise to a strong electrostatic interaction with negatively charged mucus layer (Bernkop-Schnurch et al., 2004), hence particles with a more positive zeta potential should display comparatively more pronounced mucoadhesive properties. In addition, we observed an enhanced yield of the nanoparticles by increasing the weight ratio of polyanion, but further increase in polyanion ratio resulted in aggregation of the particles. Based on the above considerations,
the optimal weight ratio between TCS and polyanions was found to be 1:1 for TCS/ HP-55 NPs, compared to 2:1 for TCS/TPP NPs. To study the effects of changing pH values on nanoparticle size and zeta potential, TCS nanoparticles were formulated at fixed weight ratio of TCS to polyanion. Figure 2 presents the characteristics of nanoparticles with the changing pH values. It was observed that further increase in the preparation pH resulted in an increase in the particle size of obtained dispersions and a corresponding decrease in their positive surface charge. In addition, due to the pH-sensitve feature of HP-55, both measured particle size and zeta potential of TCS/ HP-55 NPs were more sensitive to the changing pH values as compared with TCS/TPP NPs. As we know the nanoparticles are formed spontaneously upon mixing of polyanion and TCS solutions due to inter- and intra-molecular cross-linkages between positively charged amino groups of TCS and negatively charged polyanions. Because of the presence of amine groups TCS could carry a lot of positive charges at lower pH, making for ionic interaction with the polyanion. But with regard to the TCS/HP-55 NPs, if the pH of nanoparticles dispersion was too low, some of the -COOgroups on HP-55 became protonated (-COOH) and the electrostatic interaction between TCS and HP-55 was therefore relatively weaker, leading to a relatively larger nanoparticle. According to the outcome of numerous experiments, pH 4.0 was investigated as most suitable for the TCS/HP-55 and TCS/TPP NPs dispersion. Additionally, we investigated the influence of different rpm and time of ultracentrifugation on the characterizations of nanoparticles. The results showed that when the speed was less than 10 000 rpm or the time was less than 20 min, nanoparticles could not be separated completely and
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Figure 2. The effects of pH values of the final NPs dispersion on nanoparticle size (A) and zeta potential (B).
Figure 3. TEM of LMWH-loaded TCS/HP-55 nanoparticles.
efficiently. However, further increasing the centrifugal speed or time above 13 000 rpm or 30 min led to the decreases of both encapsulation efficiency and loading capacity, meaning that the structure of the nanoparticles had been destroyed. Therefore, 13 000 rpm and 20 min were chosen to separate the prepared nanopaticles from the solution. The morphological characterizations of the TCS/HP-55 NPs were evaluated by TEM (Figure 3). The nanoparticles had an almost spherical shape, similar nanometric dimension and were well dispersed without any aggregation. LMWH was efficiently loaded into TCS/HP-55 NPs with an encapsulation efficiency of 97.75 ± 0.02% and loading capacity of 12.22 ± 0.03%. In vitro release study In this study, the release of LMWH from the prepared nanoparticles was evaluated in simulated gastrointestinal fluid without enzymes and compared with the membrane diffusion of free LMWH. Figure 4(A) and (B) represents the cumulative release of LMWH at different time point in vitro from TCS/ HP-55 NPs, TCS/TPP NPs and LMWH solution, respectively. Sink conditions inside and outside the dialysis bags were guaranteed by using diluted dispersions of the nanoparticles. As shown in Figure 4(A) and (B), the diffusion of free LMWH through the dialysis bag was very fast and nearly complete release was observed within the fixed time.
Concerning the NPs formulation, the cases were different. In gastric fluid without enzymes (0.1 mol/L HCl solution) (Figure 4A), TCS/TPP NPs showed 26.8% and 56.7% release of LMWH in 2 h and 12 h, respectively. Whereas, TCS/HP-55 NPs showed 14.6% and 31.7% release of LMWH in 2 h and 12 h, respectively. There were significant differences at 2 h and 12 h (p50.05). However, in the case of release in PBS (pH 6.8) (Figure 4B), no noticeable differences could be observed in the drug release behavior between TCS/TPP NPs and TCS/HP-55 NPs (p40.05). The results could account for the different dissolution feature between TCS/HP-55 matrix and TCS/TPP matrix in acidic environment. Considering that HP-55 is insoluble in acidic conditions, the easy solubility of TCS in lower pH values is prevented by physical entanglement with the undissolved HP-55 polymeric structure. Therefore, the TCS/HP-55 NPs showed an improved physical stability at lower pH values without noticeable change in their size. A slow release behavior of LMWH from nanoparticles at pH 6.8 might be related to the insolubility of TCS at neutral pH. It has been reported that as the pKa value of chitosan is approximately 6.5, chitosan could be soluble at acidic pH due to the protonation of the amine group, yet aggregates at neutral pH (Chen et al., 2013). During the in vitro release experiments of TCS nanoparticles in PBS (pH 6.8), we observed white precipitation of aggregates in the bottom of dialysis bags. This finding suggested that because of the reduced degree of protonation at surface of particles at pH 6.8, decreasing electrostatic repulsion between the particles thereby led to the aggregation of nanoparticles, which resulted in the reduced release of LMWH from nanoparticles in PBS (pH 6.8). It is worth mentioning that the followed in vivo studies in the part of ‘‘Mucoadhesion study of the nanoparticles’’ and ‘‘In vivo anticoagulant effect’’ confirmed the effectivity of nanoparticles for the oral delivery of LMWH. Similarly, previously reported heparin-loaded nanoparticles also displayed a slow release in vitro but generated very positive absorption results in vivo (Jiao et al., 2002). Protection of LMWH study The potential ability of nanoparticles to protect the entrapped LMWH from the digestive gastric enzymes was investigated in the presence of pepsin. Figure 5 depicts the remaining
pH-responsive and mucoadhesive nanoparticle system
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DOI: 10.3109/10717544.2014.909908
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Figure 4. In vitro release profile of LMWH-loaded nanoparticle systems in different simulated pH environments (A) 0.1 mol/L HCl (pH 1.0); (B) phosphate buffered saline (PBS, pH 6.8). Data represent mean ± SD (n ¼ 3). Table 2. In vitro apparent permeability coefficients (Papp) and absorption enhancement of NPs through excised carp intestine.
Formulation TCS/HP-55 NPs CS/HP-55 NPs a
Apparent permeability coefficient, Papp (106 cm/s) (means ± SD, n ¼ 3)
Enhancement ratio, R
4.92 ± 0.014a 3.83 ± 0.015a
1.86 1.45
The Papp enhancement ratio was 1.28-fold higher.
Penetration of the nanoparticles in vitro
Figure 5. Percentage of LMWH remaining after incubation of different samples with pepsin solutions in simulated gastric fluid. Data represent mean ± SD (n ¼ 3).
percentage of LMWH after incubation of LMWH solution, LMWH-loaded TCS/HP-55 NPs and TCS/TPP NPs with pepsin solution in 0.05 mol/L HCl. LMWH solution was rapidly degraded within 5 min. After an incubation period of 30 min with pepsin solution, the TCS/TPP NPs was degraded more quickly than TCS/HP-55 NPs. In this study, the remaining LMWH percentage after incubation of TCS/HP55 NPs, TCS/TPP NPs and LMWH solution at 120 min were 80.44 %, 70.28 %, 43.27 %, respectively. With regard to TCS/ HP-55 NPs, the protective effect was significantly higher than that of TCS/TPP NPs (p50.05). These results could be explained based on the ability of HPMCP to overcome the damages from gastric conditions. In this study, TCS/TPP NPs were rapidly dissociated with fast release of the entrapped drug, while TCS/HP-55 NPs were physically stable and retarded LMWH release in acid medium by their pH-sensitive property. Furthermore, HPMCP has relatively higher molecular weight chains (45 kDa) that can entangle with TCS molecules more efficiently. Due to the steric hindrance effect, the TCS/HP-55 NPs system could moderately protect the wrapped LMWH from stomach acid and digestive enzyme degradation.
The influence of thiolated nanoparticles on the transport of LMWH was investigated by using the Ussing chamber technique, which is particularly well adapted to study the effect of drug-loaded delivery system formulations on drug fluxes across the intestinal barriers. As control, CS/HP-55 NPs and LMWH solution were also used. Results of permeation studies on carp intestine mucosa are represented in Table 2 and Papp values with enhancement ratios are summarized. Within this study it could be demonstrated that nanoparticles could increase the transport of LMWH. The Papp enhancement ratio was 1.86-fold higher for the TCS/HP55 NPs and 1.45-fold higher for the CS/HP-55 NPs as compared with the control, which demonstrated the view that CS-based nanoparticles could mediate the opening of tight junctions between epithelial cells reversibly, thus facilitating the paracellular transport of hydrophilic macromolecules (Chen et al., 2013). Moreover, as compared with the CS formulation group, improved transport of LMWH in TCS formulation group was achieved on account of thiolation. (The Papp enhancement ratio was 1.28-fold higher). As shown by Kast et al. due to the formation of disulfide bonds between their thiol groups and the cysteine-rich subdomains of glycoproteins in the mucus layer, TCS could prolong the contact time of the polymer on mucosal tissue (Kast & Bernkop-Schnu¨rch, 2001), which considerably increased the drug concentration gradient at the absorption site and promoted the permeability. The results in Table 2 could typically support that the permeation enhancing effect of
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Figure 6. Fluorescence microscopic images of microtome section of intestinal epithelium after 2 h of oral administration of different samples (A) TCS solution; (B) CS/HP-55 NPs; (C)TCS/TPP NPs; (D)TCS/HP-55 NPs.
chitosan per se can be strongly improved by the immobilization of thiol groups on the CS backbone. Mucoadhesion study of the nanoparticles To further investigate a possible effect of mucoadhesion on intestinal permeation, we checked the influence of the mucoadhesive capacity of nanoparticles on the LMWH absorption. The mucosal adhesion and uptake properties of fluorescently labeled TCS or CS NPs were evaluated after oral administration to rats. This method did neither affect the polymer ability to conjugate with polyanions nor the properties of the obtained nanoparticles. Following oral administration of the fluorescent nanoparticles, the amount of FITC-labeled TCS or CS associated to the small intestine of rats was evaluated by CLSM. Figure 6 shows the intensity of green fluorescence in the small intestine after oral administration of FITC-labeled TCS solution, CS/HP-55 NPs, TCS/TPP NPs and TCS/HP-55 NPs. As illustrated, the highest intensity of green fluorescence could be detected in the small intestine of TCS/HP-55, while the TCS solution showed the lowest intensity. This result indicated that the TCS/HP-55 NPs possessed the most
effectively adhesive capability on the mucosal surface. It could be explained from two aspects. On the one hand, it might be attributed to the ability of HPMCP to protect TCS/HP-55 NPs from dissociation in the stomach by virtue of its pH-sensitive solubility. Therefore, higher percentage of intact particles could reach the small intestine and interact with the intestinal membrane as compared with the TCS/TPP NPs and TCS solution. On the other hand, the considerable improvement in mucoadhesion of TCS nanoparticles was based on the immobilisation of thiol groups on chitosan. Compared with the CS/HP-55 NPs, the enhancing attachment of TCS/HP-55 NPs to the mucosal side of the intestinal wall can prolong the residence time of the delivery system and achieve higher drug concentration in direct contact with the epithelial membrane. Moreover, this direct contact and/or adhesion of the nanoparticles to the mucosal surface are a prerequisite step before translocation of the intact particles can take place. In vivo anticoagulant effect After a single oral administration of different samples in rats, the coagulating effect on plasma by APTT assay is shown in
pH-responsive and mucoadhesive nanoparticle system
DOI: 10.3109/10717544.2014.909908
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increased permeation of LMWH via parcellular mechanism of drug absorption.
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Conclusions
Figure 7. The APTT profiles in male adult Sprague-Dawley rats following oral administration of free LMWH and LMWH-loaded nanoparticles (50 mg/kg) Data represent mean ± SD (n ¼ 3).
In the present work, a pH-responsive nanoparticle system composed of TCS and HP-55 was successfully prepared using a simple ionic gelation method. Our results indicated that these nanoparticles were stable at the acid conditions and could protect the entrapped LMWH against gastric degradation. Furthermore, the intestinal penetrative and mucoadhesive property of TCS/HP-55 nanoparticles was significantly improved in vitro and in vivo. The enhanced anticoagulant effects after oral administration of LMWH-loaded TCS/HP55 nanoparticles were also observed in rats. As expected before it could be demonstrated that TCS/HP-55 NPs have promising pH-responsive and mucoadhesive properties. Therefore, the current strategy using TCS/HP-55 nanoparticles as drug carriers seems to have potential applications for the oral delivery of LMWH.
Table 3. Main pharmacokinetic parameters of LMWH in rats following a single oral administration of different samples versus the LMWH solution administered intravenously.
Declaration of interest
Formulation
APTTmax(s)
Tmax(h)
AUC0–24 h(sh)
F(%)
iv. solution Oral solution CS/HP55 TCS/TPP TCS/HP55
– 30.1 45.9 44.9 75.3
– 1 8 5 8
77.67 28.15 298.7 226.0 559.3
100 1.09 11.5 8.73 21.6
This work was supported by the Shanxi Provincial Innovative Foundation for College Students (Grant No. 2012130). The authors report no conflict of interests.
Figure 7. The main pharmacokinetic parameters is calculated and listed in Table 3. As illustrated in Figure 7, the maximal APTT of 30.1s, 44.90s and 45.94s were occurred at 1, 5, and 8 h after dosing with LMWH solution, TCS/TPP NPs and CS/HP-55 NPs, respectively. And a significantly prolonged maximal APTT of 75.34 s was observed at 8 h after TCS/HP-55 NPs administration. Compared with the controls, the maximal APTT value of TCS/HP-55 NPs increased approximately 2-fold (p50.05), reflecting stronger anticoagulant activity of LMWH-loaded TCS/HP-55 NPs. It means that higher concentrations of LMWH in plasma were achieved via TCS/ HP-55 NPs delivery system. In addition, the TCS/HP-55 NPs reached the maximal APTT later than LMWH solution and TCS/TPP NPs, but the same as CS/HP-55 NPs. This typical behavior shows the efficiency of our nano-drug delivery system in controlling the release of LMWH in blood circulation. In terms of absolute bioavailability (Table 3), the TCS/HP55 NPs also showed the highest absorption and the duration of the anticoagulant effect was significantly longer. This result suggests that as compared with the LMWH solution, TCS/ TPP NPs and CS/HP-55 NPs, the TCS/HP-55 NPs could effectively promote drug absorptions probably attributing to the synergistic effects between the pH-sensitive protection of HPMCP and an inherent permeation enhancing feature of thiolated chitosans (TCS). Therefore, a higher drug concentration gradient was offered at the absorption sites and better intimate contacts of TCS/HP-55 NPs with intestinal mucosa
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