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Surface-modified PLGA nanoparticles with chitosan for oral delivery of tolbutamide
Colloids and Surfaces B: Biointerfaces 161 (2018) 67–72
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Surface-modified PLGA nanoparticles with chitosan for oral delivery of tolbutamide Yongli Shi a,∗ , Jintao xue a , Liyun Jia b , Qian Du a , Jie Niu a , Dongyang Zhang a a b
College of Pharmacy, Xinxiang Medical University, 453003, Xinxiang, PR China Hebei Junsheng Inspection Technology co., 05000, LTD, Shijiazhuang, PR China
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Article history: Received 3 July 2017 Received in revised form 25 September 2017 Accepted 12 October 2017 Available online 12 October 2017 Keywords: PLGA Chitosan Tolbutamide Diabetic Hypoglycemic effect
a b s t r a c t The main purpose of present study was to develop novel chitosan-modified polylactic-co-glycolicacid nanoparticles (CS@PLGA NPs) for improving the bio-availability of tolbutamide (TOL). The TOL-loaded CS@PLGA NPs (TOL-CS@PLGA NPs) were fabricated with the solvent evaporation method. The cargo-free CS@PLGA NPs showed a diameter of 228.3 ± 2.5 nm monitored with a laser light particlesizer, and the transmission electron microscope (TEM) photographs revealed their “core-shell” structures. The Zeta potential of the original PLGA NPs and the cargo-free CS@PLGA NPs was measured to be −20.2 ± 3.21 mV and 24.2 ± 1.1 mV, respectively. The changes in Zeta potential indicated the CS chains were coated on the surfaces of the original PLGA NPs. The thermal gravity analysis (TGA) curves suggested that the CS chains improved the thermostability of the original PLGA NPs. The results of cells viability indicated the cargo-free CS@PLGA NPs were nontoxicity. The in vitro release profiles suggested that TOL-CS@PLGA NPs could release TOL in pH 7.4 phosphate buffer solution (PBS) at a sustained manner. Streptozotocin (STZ) was employed to build the diabetic rat models. The physiological changes in the islet  cells confirmed the obtaining of diabetic rats. After treatment by gavage, the TOL-CS@PLGA NPs showed an excellent hypoglycemic effect. Therefore, the TOL-CS@PLGA NPs had a potential application in oral delivery of TOL. © 2017 Published by Elsevier B.V.
1. Introduction Polylactic-co-glycolic acid (PLGA) is a random copolymer of polylactic and polyglycolic acid [1]. Due to its excellent biocompatibility and biodegradability, PLGA is widely used as the matrices of drug delivery systems [2]. Significantly, the PLGA has been approved by FDA for human therapy [3]. As a amphipathic copolymer, PLGA can be developed into NPs easily [4]. Due to their nano-scaled diameters, PLGA NPs can smoothly across the system barriers [5]. However, their negative potential will inhibit PLGA NPs from adsorbing by the intestinal mucosas. It is well known that the mucin of the intestinal surface contains sialic acid, which is a negatively charged sugar, forming a negatively charged layer [6]. Therefore, the negative potential of PLGA NPs may destroy their mucoadhesive properties and decrease their bio-availability. Hence, rising the surface potential of PLGA NPs will improve their mucoadhesive. For example: Pawar et al. prepare chitosan and glycol chitosan (GC) coated PLGA NPs [7]. Studies find that the zeta
∗ Corresponding author. E-mail addresses: [email protected], [email protected] (Y. Shi). https://doi.org/10.1016/j.colsurfb.2017.10.037 0927-7765/© 2017 Published by Elsevier B.V.
potentials of the PLGA NPs, CS-PLGA NPs, and GC-PLGA NPs are −15.79 ± 1.6 mV, 14.44 ± 1.7 mV, and 17.12 ± 1.3 mV, respectively. It is obvious that the CS and GC chains improve the zeta potentials of PLGA NPs. The stronger immune response of CS-PLGA NPs may be due to their better mucoadhesive ability. In addition, in vivo immunogenicity studies suggest that the CS-PLGA NPs can induce significantly high systemic and mucosal immunere sponse. Akl et al. coat CS chains onto the surfaces of the PLGA NPs to enhance their mucoadhesion and cellular uptake [8]. The CS-modified PLGA NPs switch their zeta potential from negative (-36.8 mV) to positive (31.2 mV). Moreover, the modification of CS chains improve the mucoadhesion and interaction of CS-PLGA NPs with porcine gastric mucin with 3.4-fold compared to the original PLGA NPs. CS is the N-deacetylated derivative of chitin which is the most abundant natural amino polysaccharide [9]. CS is an important natural polymer for its combination of properties like biocompatibility, biodegradability, and bio-activity [10]. Hence, CS is widely used in various areas, such as: bio-medical products [11], food processing [12], cosmetics [13], and drug delivery system [14]. The abundant amino groups make CS molecules can be used to surface modify NPs containing carboxyl groups. For example: Li et al. conjugate paclitaxel (PTX) to hyaluronic acid (HA), and fabricate the HA-PTX NPs [15]. To improve the oral bio-availability of PTX,
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the HA-PTX NPs are surface modified with CS chains. The CS/HAPTX NPs show an increased extent of cellular uptake for HepG2 cells than free PTX. In vitro cytotoxicity tests suggest that the PTX can be released from CS/HA-PTX NPs without losing their cytotoxicity to HepG2 cells. Significantly, the ex vivo bio-distribution experiments demonstrate that PTX effectively accumulate in the tumor sites after oral administration of CS/HA-PTX CNPs. Furthermore, many insulin-loaded NPs are surface coated with CS chains to improve the effects of oral insulin [16]. The CS modification promote NPs to be adhered and infiltrated into muscus members. Then, the intestinal epithelial cells transiently open a tight junction. The released insulin will penetrate through the paracellular pathway and enter bloodstream. Therefore, the CS-modified NPs improve the bio-availability of insulin, and avoid their degradation by pepsase. Tolbutamide (TOL) as the 1st generation hypoglycemic agent, has excellent hypoglycemic effect. After oral administration (0.5 g/d, two times), TOL will be found in the blood after 30 min of administration. Its plasma concentration will reach maximum after treatment of 3 − 4 h, showing a biological half-life of 4.5 −6.5 h. The TOL is mainly metabolized in liver, and over 85% of metabolic products are excreted by kidney. However, its high dosage will result in serious gastrointestinal reactions, which limits its further application [17–19]. In this study, TOL was loaded into PLGA NPs, and their surfaces were modified with CS. It was expected the TOL-CS@PLGA NPs could improve the bio-availability of TOL and reduce its dosage frequency. The FTIR, TGA, and TEM studies were applied to confirm the fabrication of cargo-free CS@PLGA NPs. The cytotoxicity of cargo-free CS@PLGA NPs were reflected by cell viability. The TOL release profiles and the hypoglycemic effect were also performed.
(1.0 mg/mL) were injected into a cuvette, and the measurements were performed at 25 ◦ C in triplicate. The surface morphology of the original PLGA NPs and the cargofree CS@PLGA NPs were observed via a JEM-1011 TEM (JEOL, Japan). Before observation, a droplet of NPs suspension (1.0 mg/mL) was covered onto a copper net. Then, the net was dried with an infrared lamp, and their morphology was observed with a JEM-1011 TEM (JEOL, Japan). To investigate the thermostability of CS, PLGA NPs, and the cargo-free CS@PLGA NPs, the TGA studies were carried out with a Mettler-Toledo TGA/DSC-2 (Switzerland). 3.0 −5.0 mg of samples were loaded into ceramic pans (50 L), and their TGA curves were recorded under a dynamic atmosphere of high purity nitrogen (flow rate of 20 mL/min) heating from 25 to 500 ◦ C at the rate of 10 ◦ C/min. 2.4. Cell viability The cytotoxicity of cargo-free CS@PLGA NPs was reflected with the cell viability, and the HePG2 cells were used in the studies. HePG2 cells were seeded in 96-well plates at the density of 8, 000 cells/mL. To remove bacteria, the lyophilized powder of nanoparticles (100 mg) was re-dissolved into 100 mL sterile water. Then, 100 L of NPs suspensions were added into the cell culture medium, and the cells were incubated for 24 h. Before harvest, the medium was replaced by 100 L fresh medium containing 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, 5.0 mg/mL). 4 h later, the absorbance was measured at 570 nm using a microplate reader (Bio-Rad Model 680, UK). 2.5. DSC patterns
2. Experimental 2.1. Materials PLGA, TOL, and streptozotocin (STZ) were purchased from Aladdin Industrial Corporation (Shanghai, China). CS (AR) was obtained from Tianjin Yongda chemical reagent Co., Ltd (Tianjin, China). Other organic reagents were bought from Tianjin Yongda chemical reagent Co., Ltd (Tianjin, China), and used as received without any further purification. 2.2. Preparation of CS@PLGA NPs The original PLGA NPs were fabricated with a solvent evaporation method [20]. 100 mg PLGA was dissolved in a binary solvent of 4.0 mL acetone and 2.0 mL anhydrous alcohol by vortexing and sonication. Then, the transparent solution was added dropwise into 100 mL ultrapure water under magnetic stirring. The suspension was dispersed with an emulsifying machine for 10 min at the rate of 25, 000 rpm. Then, the dispersed suspension was stirred for another 12 h under ambient temperature to completely remove the organic solvents. To obtain the CS@PLGA NPs suspension, 5.0 mL of the PLGA NPs suspension (1.0 mg/mL) was injected into 1.0 mL of the CS solution (pH 5.0, 1.0 mg/mL). After lyophilization, the CS@PLGA NPs powder was recovered and used for further studies. To prepare the TOL-CS@PLGA NPs, 20 mg TOL and 100 mg PLGA dissolved into the organic solvents, and other process was following the same methods described above. 2.3. Characterization on NPs A Nano-ZS90 Malvern Mastersizer (Malvern Instruments Ltd, Malvern, UK) was employed to monitor the particle size distributions and zeta potentials of original PLGA NPs and the cargo-free CS@PLGA NPs. The cargo-free CS@PLGA NPs suspensions
DSC studies were carried out to investigate the distribution state of TOL in NPs matrices. 10 mg of samples were loaded into the ceramic pans, and their thermal behaviors were recorded by a DZ3335 differential scanning calorimetry (Jiangsu, China). The scanning range was covering from 25 ◦ C to 250 ◦ C at a scanning rate of 7 ◦ C/min. 2.6. In vitro release profiles The in vitro release profiles of TOL were studied with pH 1.2 and 7.4 phosphate buffer solutions (PBS) as the medium. 0.1 g TOLCS@PLGA NPs powders were sealed into the dialysis bags (MD44, molecular cut off = 3, 500, USA), and then they were immersed into 35 mL PBS. To retard microbial growth, the buffers were added 0.02% w/v of sodium azide. These samples were vibrated in a ZHWY-100H water-bathing constant temperature vibrator (Shanghai, China) at 37 ± 1 ◦ C. At proper intervals, 5.0 mL PBS were taken off and replaced with equivalent fresh buffers. The TOL concentrations were measured with high performance liquid chro® matography (HPLC) [21]. HPLC were carried out in a Luna C18(2) (150 mm × 4.6 mm I.D.; 5 m) column, and the column oven was set at 40 ◦ C. The mobile phase consisted of 10 mM monobasic potassium phosphate (pH 4.5) and acetonitrile (65:35, v/v) and was filtered through a 0.45 m filter (Supelco, Bellefonte, PA, USA) before use. Absorbance of the eluent was monitored at 245 nm. 2.7. Animal experiments To investigate the hypoglycemic effects of TOL-CS@PLGA NPs, the adult Sprague-Dawley (SD) rats were used to build the diabetes models. The SD rats (300 g − 350 g, 8 weeks) were bought from Laboratory Animal Center, Xinxiang medical University (Xinxiang, China). Before modeling, the SD rats were fasted overnight but allowed to drink water adlibitum. Then, the rats in the normal
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group (n = 12) were fed a standard diet and water, while the rats in modeling group (n = 12) were fed with high fat diet (containing 9 wt.% glucose and 12 wt.% coconut oil) for 4 weeks. After 12 h of fasting, the rats were treated with 1.0% STZ in citric acid − sodium citrate buffer (pH 4.2-4.5) via intraperitoneal injection at a dose of 40 mg·kg−1 [22]. A droplet of blood was collected from the caudal veins, and their blood glucose levels were monitored with a home use glucometer. If the blood glucose level was over 16.7 mmol/L, the diabetic rats were obtained. After fasting overnight, the diabetic rats (n = 6) were treated with TOL-CS@PLGA NPs (200 mg/kg of TOL) by gavage. By contrast, the diabetic rats (n = 6) treated with metformin tablets (10 mg/kg of metformin) by gavage were applied as the positive control. The negative controls were fed with normal saline with the same volumes. The blood glucose levels of the diabetic rats were monitored at the 2 h, 4 h, 8 h, and 12 h, after treatment. All rats were fed in the controlled temperature (20 ± 2 ◦ C) and lighting (08:00 − 20:00) conditions with food and water available ad libitum. Animal experiments were carried out in accordance with the People’s Republic of China National Standard (GB/T 16886.6-1997). 3. Results and discussions 3.1. FTIR spectra and TGA curves FTIR spectra (Fig. 1a) were used to analyze the surface chemical composition between PLGA NPs and CS chains. The FTIR spectrum of CS, the peaks assigning to −OH stretching was observed at 3400 cm−1 . The bonds according to −CH-, −CH2 , and −CH3 stretching were found at the range of 2897–2986 cm−1 [23]. Importantly, the amide I and II vibration were observed at 1667 cm−1 and 1600 cm−1 , respectively. The bonds between 916 cm−1 and 1187 cm−1 was assigned to N H stretching vibration. In the FTIR spectrum of PLGA NPs, the bonds attributing to the carbonyl ( C O) and C O C stretching vibration were observed at 1713 cm−1 and 1094 cm−1 , respectively [24]. By contrast, the spectrum of cargo-free CS@PLGA NPs gave all the characteristic bands of the CS and PLGA molecules: −OH stretching (3400 cm−1 ), −CH, −CH2 , and −CH3 stretching (2773–2995 cm−1 ), carbonyl C O stretching vibration (C, 1708 cm−1 ), amide II vibration (1531 cm−1 ), and C O C stretching (1094 cm−1 ). These data indicated the coexistence of CS and PLGA. Fig. 1b described the TGA curves of the CS, PLGA NPs, and the cargo-free CS@PLGA NPs, respectively. In the TGA curve of CS chains, their dehydration, acetylation and deploymerization were observed at the temperature range of 250.2 ◦ C − 399.4 ◦ C [25]. However, the main decomposition event of PLGA NPs was observed at 209.6 ◦ C − 295.1 ◦ C with a mass loss of 71.7%. On the contrary, the thermostability of the cargo-free CS@PLGA NPs was between those of CS chains and the original PLGA NPs. These changes indicated that the CS shells improved the thermodynamic stability of PLGA NPs. In other words, these data further indicated the CS chains coated on the surfaces of the PLGA NPs.
Fig. 1. (a) FTIR spectra of CS, PLGA, and cargo-free CS@PLGA NPs. The bands attributed to the stretching of carbonyl groups showed a blue shift, which indicated the CS chains coated on the surfaces of PLGA NPs; (b) TGA curves of CS, PLGA NPs and the cargo-free CS@PLGA NPs. The thermostability of cargo-free CS@PLGA NPs was improved by the modification of CS chains. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
free CS@PLGA NPs was increased to 24.7 ± 4.5 mV, comparing to that of −20.1 ± 0.66 mV (original PLGA NPs). Importantly, the positive potential could prevent the cargo-free CS@PLGA NPs from aggregation, and increased their stability. 3.3. Cell viability The cytotoxicity of the cargo-free CS@PLGA NPs was reflected by the cell viability and assessed with the MTT method. In the teats, the un-treated HepG2 cells were used as the control, and their viability was recorded as 100%. It could be seen from Fig. 3 that, after 24 h of incubation, the viability of HepG2 cells were 121.4 ± 5.8% (0.05 mg/mL), 117.4 ± 12.0% (0.1 mg/mL), 116.5 ± 10.5% (0.25 mg/mL), 114.4 ± 16.1% (0.5 mg/mL), and 105.4 ± 15.0% (1.0 mg/mL), respectively. The high cell viability indicated the nontoxicity of the cargo-freeCS@PLGA NPs.
3.2. TEM photographs and zeta-potentials 3.4. TOL release profiles The surface morphology of the PLGA and the cargo-free CS@PLGA NPs were observed with TEM, and photographs were shown in Fig. 2. It could be seen that the original PLGA NPs had regular spherical shapes with a diameter around 100 nm. By contrast, the core-shell structures of the cargo-free CS@PLGA NPs were clearly observed (Fig. 2b). The differences between both kinds of NPs illustrated that the CS chains coated onto the surface of PLGA NPs. Due to the modification, the surface zeta potential of the cargo-
The TOL release profiles were carried out in 1.2 and 7.4 PBS. It found that the TOL possibly distributed in the TOL-CS@PLGA NPs with an amorphous or molecular state (as shown in Fig. S1), which was beneficial to TOL release form these NPs. The release profiles were depicted in Fig. 4. After 24 h of releasing, 74.1 ± 2.2% of encapsulated TOL was released into pH 7.4 PBS. However, only 52.9 ± 2.2% TOL was diffused into pH 1.2 PBS. The difference
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Fig. 2. TEM photographs of original PLGA NPs (a), amplified PLGA NPs (insert), and cargo-free CS@PLGA NPs (b). The PLGA NPs showed spherical shapes, and the cargo-free CS@PLGA NPs had “core-shell” structures. These photographs further confirm the modification of CS chains on the PLGA NPs.
Scheme 1. Schematic presentation for the fabrication and application of the TOLCS@PLGA NPs. Fig. 3. Cell viability of the cargo-free CS@PLGA NPs suspensions. The high cell viability indicated the nontoxicity of the cargo-free CS@PLGA NPs even in a high concentration (1.0 mg/mL).
Fig. 5. Kaplan-Meier survival curves of four groups of diabetic rats. These rats were administrated by gavage of normal saline (negative control), metformin tables (positive control), and the TOL-CS@PLGA NPs, respectively (n = 6). Fig. 4. In vitro release profiles of TOL in 1.2 and 7.4 buffers. The TOL-CS@PLGA NPs could continuously release TOL in 7.4 buffer for 24 h. A significant difference was observed between the both TOL release profiles (P < 0.01, **).
between both release profiles might be due to the special structures of the CS@PLGA NPs (as depicted in Scheme 1). The CS coated onto the PLGA surfaces mainly by the adsorption capacity between the COOH (PLGA NPs) and NH3 + groups (CS chains). In pH 1.2 buffer, the adsorption between CS and PLGA kept their core-shell structures, which prevented TOL from diffusion. However, in pH 7.4 buffer, the NH3 + groups lost a proton to generate −NH2 segments. Therefore, the adsorption was decreased and part of CS chains fell
off from PLGA NPs’ surfaces. Subsequently, more TOL molecules were released. The similar results were reported in previous lecture [15]. 3.5. Survival curves The survival rates of four groups of rats were depicted in Fig. 5. After 18 days of treatment, two rats died in the negative control. Another three rats died in the 20th and 23rd day, and their survival rate was only 25%. By contrast, two rats in the positive control, and their survival rate was 80%. One death of TOL-CS@PLGA NPs
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Fig. 6. (a) and (b) Representative histopathological changes noted at the islet cells of normal rats and the diabetic rats. These histopathological changes in islet cells illustrated the successful modeling of the diabetic rats; (c) Hypoglycemic effects of TOL-CS@PLGA NPs on blood glucose levels in STZ-introduced diabetic rats. Each diabetic rat was treated with the TOL-CA-PLGA NPs (60 mg TOL/kg, n = 6).
treated rat was happened at the 23rd day, which reflected a survival rate of 90%. After t-test analysis, there were no significant differences between four groups (P > 0.05). These data indicated that TOL-CS@PLGA NPs could improve the diabetes complications, and improve the survival rates of diabetic rats.
8 h, which was longer than that of metformin tablets (*, P < 0.05). Therefore, the TOL-CS@PLGA NPs had long-acting hypoglycemic effects, which could improve the bio-availability of TOL and reduce its dosing frequency.
4. Conclusions 3.6. Hypoglycemic effect In the modeling of diabetic rats, the pancreatic ˇ cells of SD rats were partly destroyed by STZ. Therefore, the treated rats showed the hallmarks of type II diabetes. The pancreatic islands of the normal and diabetic rat were taken out, and their pathological slices were shown in Fig. 6. It could be seen that the normal islet ˇ cells were in the oval or round shapes (Fig. 6a). However, the lesion or abnormality of ˇ cells was distributed in diabetes rats (Fig. 6b). These pathological structures of islet ˇ cells further confirmed the obtaining of the diabetic rats. The average blood glucose levels of diabetic rats at different intervals after treatment of TOL-CS@PLGA NPs were shown in Fig. 6c. For comparison, the rats of the positive controls were treated with 10 mg/kg body weight of metformin tablet, while the negative controls were administrated normal saline. No difference in blood glucose levels was observed between the positive controls and TOL-CS@PLGA NPs at the first 2 h after gavage, which illustrated that both preparations had the similar hypoglycemic effect. 4 h later, the blood glucose level of rats in the positive groups was decreased to 9.2 ± 3.8 mmol/L, while that of the rats treated with TOL-CS@PLGA NPs was dropped to 5.9 ± 0.78 mmol/L. 8 h later, the hypoglycemic effect of the positive groups could not be observed from Fig. 6c. On the contrary, the TOL-CS@PLGA NPS showed a longacting hypoglycemic effect. Their hypoglycemic effects lasted over
The PLGA NPs were fabricated with a solvent evaporation method. The CS@PLGA NPs were obtained by injecting PLGA NPs suspension (1.0 mg/mL) into CS solutions (1.0 mg/mL). The FTIR spectra revealed the new interactions between the PLGA NPs and CS chains. TEM photographs revealed the “core-shell” structures of the cargo-free CS@PLGA NPs. The TGA curves indicated that the CS chains improved the thermostability of the original PLGA NPs. The high cell viability indicated the nontoxicity of the cargofree CS@PLGA NPs. The TOL release profiles showed TOL had a high release amounts in 7.4 buffer. Animal experiments suggested that the TOL-CS@PLGA NPs had an excellent hypoglycemic effect. These data suggested that the TOL-CS@PLGA NPs had a promising prospect in oral TOL delivery and improving its bio-availability.
Acknowledgments This work was supported by the National Science Foundation for Young Scientists of China (Grant No. 81703458), Scientific Research Found of Xinxiang Medical University (2014QN147), and the Startup Foundation for Doctors of Xinxiang medical university (505038, 505095). This work was also financed through the university key research projects of Henan province(17A350001, 17A360026), the Scientific & Technological Projects of Henan
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province (172102310616), and the National Undergraduate Training Program for Innovation and Entrepreneurship (201510472045). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at https://doi.org/10.1016/j.colsurfb.2017.10. 037. References [1] F. Danhier, E. Ansorena, J.M. Silva, R. Coco, A.L. Breton, V. Préat, PLGA-based nanoparticles: an overview of biomedical applications, J. Controlled Release 161 (2012) 505–522. [2] A.N. Ford Versypt, D.W. Pack, R.D. Braatz, Mathematical modeling of drug delivery from autocatalytically degradable PLGA microspheres–a review, J. Controlled Release 165 (2012) 29–37. [3] C.E. Astete, C.M. Sabliov, Synthesis and characterization of PLGA nanoparticles, J. Biomater. Sci. Polym. Ed. 17 (2006) 247–289. [4] I. Bala, S. Hariharan, M.N. Kumar, PLGA nanoparticles in drug delivery: the state of the art, Crit. Rev. Ther. Drug Carrier Syst. 21 (2004) 387–422. [5] M. Kroubi, Characterization of endocytosis of transferrin-coated PLGA nanoparticles by the blood-brain barrier, Int. J. Pharm. 379 (2009) 285. [6] B.S. Kang, J.S. Choi, S.E. Lee, J.K. Lee, T.H. Kim, W.S. Jang, A. Tunsirikongkon, J.K. Kim, J.S. Park, Enhancing the in vitro anticancer activity of albendazole incorporated into chitosan-coated PLGA nanoparticles, Carbohydr. Polym. 159 (2017) 39–47. [7] D. Pawar, S. Mangal, R. Goswami, K.S. Jaganathan, Development and characterization of surface modified PLGA nanoparticles for nasal vaccine delivery: effect of mucoadhesive coating on antigen uptake and immune adjuvant activity, Euro. J. Pharm. Biopharm. 85 (2013) 550. [8] M.A. Akl, A. Kartal-Hodzic, T. Oksanen, H.R. Ismael, M.I. Afouna, M. Yliperttula, T. Viitala, A.M. Samy, Enhanced Mucoadhesion and cellular uptake of Curcumin delivered in Chitosan modified PLGA nanosphere, J. Med. Life 4 (2016) 39–54. [9] M.N.V.R. Kumar, A review of chitin and chitosan applications, React. Funct. Polym. 46 (2000) 1–27. [10] A.J. Varma, S.V. Deshpande, J.F. Kennedy, Metal complexation by chitosan and its derivatives: a review, Carbohydr. Polym. 55 (2004) 77–93. [11] R.M. Abdel-Rahman, A.M. Abdel-Mohsen, R. Hrdina, L. Burgert, Z. Fohlerova, ˇ D. Pavlinák, O.N. Sayed, J. Jancar, Wound dressing based on chitosan/hyaluronan/nonwoven fabrics: preparation, characterization and medical applications, Int. J. Biol. Macromol. 89 (2016) 725.
[12] I. Khan, S. Ullah, D.H. Oh, Chitosan grafted monomethyl fumaric acid as a potential food preservative, Carbohydr. Polym. 152 (2016) 87. [13] A. Jimtaisong, N. Saewan, Utilization of carboxymethyl chitosan in cosmetics, Int. J. Cosmet. Sci. 36 (2013) 12–21. [14] M.A. Elgadir, M.S. Uddin, S. Ferdosh, A. Adam, A.J.K. Chowdhury, M.Z.I. Sarker, Impact of chitosan composites and chitosan nanoparticle composites on various drug delivery systems: a review, J. Food Drug Anal. 23 (2014) 619–629. [15] J. Li, P. Huang, L. Chang, X. Long, A. Dong, J. Liu, L. Chu, F. Hu, J. Liu, L. Deng, Tumor targeting and pH-responsive polyelectrolyte complex nanoparticles based on hyaluronic acid-paclitaxel conjugates and Chitosan for oral delivery of paclitaxel, Macromol. Res. 21 (2013) 1331–1337. [16] P. Mukhopadhyay, R. Mishra, D. Rana, P.P. Kundu, Strategies for effective oral insulin delivery with modified chitosan nanoparticles: a review, Prog. Polym. Sci. 37 (2012) 1457–1475. [17] M. Yamasaki, T. Konishi, Y. Maeda, S. Saionji, Y. Takeda, S. Fukuhara, S. Arihara, T. Ueda, S. Tsukiai, A. Maruhashi, Condition for use of antidiabetic drugs and side effects, Jpn. J. Pharm. Health Care Sci. 27 (2001) 291–297. [18] K. Nakashima, S. Yasuda, A. Nishihara, Y. Yamashita, Adsorption and excimer formation of 4-(1-pyrenyl)-butyltrimethylammonium bromide on non-porous silica nanoparticles in aqueous dispersions, Colloids Surf. A Physicochem. Eng. Aspects 139 (1998) 251–257. [19] H. Takeuchi, T. Handa, Y. Kawashima, Spherical solid dispersion containing amorphous tolbutamide embedded in enteric coating polymers or colloidal silica prepared by spray-drying technique, Chem. Pharm. Bull. 35 (1987) 3800–3806. [20] X. Niu, W. Zou, C. Liu, N. Zhang, C. Fu, Modified nanoprecipitation method to fabricate DNA-loaded PLGA nanoparticles, Drug Dev. Ind. Pharm. 35 (2009) 1375–1383. [21] J. Jurica, L.Z. Konecny, J. Zahradnikova, J. Tomandl, Simultaneous HPLC determination of tolbutamide, phenacetin and their metabolites as markers of cytochromes 1A2 and 2C6/11 in rat liver perfusate, J. Pharm. Biomed. Anal. 52 (2010) 557–564. [22] X. Jintao, Y. Liming, L. Yufei, L. Chunyan, C. Han, Noninvasive and fast measurement of blood glucose in vivo by near infrared (NIR) spectroscopy, Spectrochim. Acta Part A Mol. Biomol. Spectrosc. 179 (2017) 250–254. [23] V. Kvande, P. Kiekens, Structure analysis and degree of substitution of chitin, chitosan and dibutyrylchitin by FT-IR spectroscopy and solid state 13C NMR, Carbohydr. Polym. 58 (2004) 409–416. [24] P.N. Thanki, D. Edith, J.L. Six, Surface characteristics of PLA and PLGA films, Appl. Surf. Sci. 253 (2006) 2758–2764. [25] K. Yodkhum, T. Phaechamud, Thermal analysis of chitosan-Lactate and chitosan-Aluminum monostearate composite system, Adv. Mater. Res. 506 (2012) 278–281.
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Full length article
Surface-modified PLGA nanoparticles with chitosan for oral delivery of tolbutamide Yongli Shi a,∗ , Jintao xue a , Liyun Jia b , Qian Du a , Jie Niu a , Dongyang Zhang a a b
College of Pharmacy, Xinxiang Medical University, 453003, Xinxiang, PR China Hebei Junsheng Inspection Technology co., 05000, LTD, Shijiazhuang, PR China
a r t i c l e
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Article history: Received 3 July 2017 Received in revised form 25 September 2017 Accepted 12 October 2017 Available online 12 October 2017 Keywords: PLGA Chitosan Tolbutamide Diabetic Hypoglycemic effect
a b s t r a c t The main purpose of present study was to develop novel chitosan-modified polylactic-co-glycolicacid nanoparticles (CS@PLGA NPs) for improving the bio-availability of tolbutamide (TOL). The TOL-loaded CS@PLGA NPs (TOL-CS@PLGA NPs) were fabricated with the solvent evaporation method. The cargo-free CS@PLGA NPs showed a diameter of 228.3 ± 2.5 nm monitored with a laser light particlesizer, and the transmission electron microscope (TEM) photographs revealed their “core-shell” structures. The Zeta potential of the original PLGA NPs and the cargo-free CS@PLGA NPs was measured to be −20.2 ± 3.21 mV and 24.2 ± 1.1 mV, respectively. The changes in Zeta potential indicated the CS chains were coated on the surfaces of the original PLGA NPs. The thermal gravity analysis (TGA) curves suggested that the CS chains improved the thermostability of the original PLGA NPs. The results of cells viability indicated the cargo-free CS@PLGA NPs were nontoxicity. The in vitro release profiles suggested that TOL-CS@PLGA NPs could release TOL in pH 7.4 phosphate buffer solution (PBS) at a sustained manner. Streptozotocin (STZ) was employed to build the diabetic rat models. The physiological changes in the islet  cells confirmed the obtaining of diabetic rats. After treatment by gavage, the TOL-CS@PLGA NPs showed an excellent hypoglycemic effect. Therefore, the TOL-CS@PLGA NPs had a potential application in oral delivery of TOL. © 2017 Published by Elsevier B.V.
1. Introduction Polylactic-co-glycolic acid (PLGA) is a random copolymer of polylactic and polyglycolic acid [1]. Due to its excellent biocompatibility and biodegradability, PLGA is widely used as the matrices of drug delivery systems [2]. Significantly, the PLGA has been approved by FDA for human therapy [3]. As a amphipathic copolymer, PLGA can be developed into NPs easily [4]. Due to their nano-scaled diameters, PLGA NPs can smoothly across the system barriers [5]. However, their negative potential will inhibit PLGA NPs from adsorbing by the intestinal mucosas. It is well known that the mucin of the intestinal surface contains sialic acid, which is a negatively charged sugar, forming a negatively charged layer [6]. Therefore, the negative potential of PLGA NPs may destroy their mucoadhesive properties and decrease their bio-availability. Hence, rising the surface potential of PLGA NPs will improve their mucoadhesive. For example: Pawar et al. prepare chitosan and glycol chitosan (GC) coated PLGA NPs [7]. Studies find that the zeta
∗ Corresponding author. E-mail addresses: [email protected], [email protected] (Y. Shi). https://doi.org/10.1016/j.colsurfb.2017.10.037 0927-7765/© 2017 Published by Elsevier B.V.
potentials of the PLGA NPs, CS-PLGA NPs, and GC-PLGA NPs are −15.79 ± 1.6 mV, 14.44 ± 1.7 mV, and 17.12 ± 1.3 mV, respectively. It is obvious that the CS and GC chains improve the zeta potentials of PLGA NPs. The stronger immune response of CS-PLGA NPs may be due to their better mucoadhesive ability. In addition, in vivo immunogenicity studies suggest that the CS-PLGA NPs can induce significantly high systemic and mucosal immunere sponse. Akl et al. coat CS chains onto the surfaces of the PLGA NPs to enhance their mucoadhesion and cellular uptake [8]. The CS-modified PLGA NPs switch their zeta potential from negative (-36.8 mV) to positive (31.2 mV). Moreover, the modification of CS chains improve the mucoadhesion and interaction of CS-PLGA NPs with porcine gastric mucin with 3.4-fold compared to the original PLGA NPs. CS is the N-deacetylated derivative of chitin which is the most abundant natural amino polysaccharide [9]. CS is an important natural polymer for its combination of properties like biocompatibility, biodegradability, and bio-activity [10]. Hence, CS is widely used in various areas, such as: bio-medical products [11], food processing [12], cosmetics [13], and drug delivery system [14]. The abundant amino groups make CS molecules can be used to surface modify NPs containing carboxyl groups. For example: Li et al. conjugate paclitaxel (PTX) to hyaluronic acid (HA), and fabricate the HA-PTX NPs [15]. To improve the oral bio-availability of PTX,
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the HA-PTX NPs are surface modified with CS chains. The CS/HAPTX NPs show an increased extent of cellular uptake for HepG2 cells than free PTX. In vitro cytotoxicity tests suggest that the PTX can be released from CS/HA-PTX NPs without losing their cytotoxicity to HepG2 cells. Significantly, the ex vivo bio-distribution experiments demonstrate that PTX effectively accumulate in the tumor sites after oral administration of CS/HA-PTX CNPs. Furthermore, many insulin-loaded NPs are surface coated with CS chains to improve the effects of oral insulin [16]. The CS modification promote NPs to be adhered and infiltrated into muscus members. Then, the intestinal epithelial cells transiently open a tight junction. The released insulin will penetrate through the paracellular pathway and enter bloodstream. Therefore, the CS-modified NPs improve the bio-availability of insulin, and avoid their degradation by pepsase. Tolbutamide (TOL) as the 1st generation hypoglycemic agent, has excellent hypoglycemic effect. After oral administration (0.5 g/d, two times), TOL will be found in the blood after 30 min of administration. Its plasma concentration will reach maximum after treatment of 3 − 4 h, showing a biological half-life of 4.5 −6.5 h. The TOL is mainly metabolized in liver, and over 85% of metabolic products are excreted by kidney. However, its high dosage will result in serious gastrointestinal reactions, which limits its further application [17–19]. In this study, TOL was loaded into PLGA NPs, and their surfaces were modified with CS. It was expected the TOL-CS@PLGA NPs could improve the bio-availability of TOL and reduce its dosage frequency. The FTIR, TGA, and TEM studies were applied to confirm the fabrication of cargo-free CS@PLGA NPs. The cytotoxicity of cargo-free CS@PLGA NPs were reflected by cell viability. The TOL release profiles and the hypoglycemic effect were also performed.
(1.0 mg/mL) were injected into a cuvette, and the measurements were performed at 25 ◦ C in triplicate. The surface morphology of the original PLGA NPs and the cargofree CS@PLGA NPs were observed via a JEM-1011 TEM (JEOL, Japan). Before observation, a droplet of NPs suspension (1.0 mg/mL) was covered onto a copper net. Then, the net was dried with an infrared lamp, and their morphology was observed with a JEM-1011 TEM (JEOL, Japan). To investigate the thermostability of CS, PLGA NPs, and the cargo-free CS@PLGA NPs, the TGA studies were carried out with a Mettler-Toledo TGA/DSC-2 (Switzerland). 3.0 −5.0 mg of samples were loaded into ceramic pans (50 L), and their TGA curves were recorded under a dynamic atmosphere of high purity nitrogen (flow rate of 20 mL/min) heating from 25 to 500 ◦ C at the rate of 10 ◦ C/min. 2.4. Cell viability The cytotoxicity of cargo-free CS@PLGA NPs was reflected with the cell viability, and the HePG2 cells were used in the studies. HePG2 cells were seeded in 96-well plates at the density of 8, 000 cells/mL. To remove bacteria, the lyophilized powder of nanoparticles (100 mg) was re-dissolved into 100 mL sterile water. Then, 100 L of NPs suspensions were added into the cell culture medium, and the cells were incubated for 24 h. Before harvest, the medium was replaced by 100 L fresh medium containing 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, 5.0 mg/mL). 4 h later, the absorbance was measured at 570 nm using a microplate reader (Bio-Rad Model 680, UK). 2.5. DSC patterns
2. Experimental 2.1. Materials PLGA, TOL, and streptozotocin (STZ) were purchased from Aladdin Industrial Corporation (Shanghai, China). CS (AR) was obtained from Tianjin Yongda chemical reagent Co., Ltd (Tianjin, China). Other organic reagents were bought from Tianjin Yongda chemical reagent Co., Ltd (Tianjin, China), and used as received without any further purification. 2.2. Preparation of CS@PLGA NPs The original PLGA NPs were fabricated with a solvent evaporation method [20]. 100 mg PLGA was dissolved in a binary solvent of 4.0 mL acetone and 2.0 mL anhydrous alcohol by vortexing and sonication. Then, the transparent solution was added dropwise into 100 mL ultrapure water under magnetic stirring. The suspension was dispersed with an emulsifying machine for 10 min at the rate of 25, 000 rpm. Then, the dispersed suspension was stirred for another 12 h under ambient temperature to completely remove the organic solvents. To obtain the CS@PLGA NPs suspension, 5.0 mL of the PLGA NPs suspension (1.0 mg/mL) was injected into 1.0 mL of the CS solution (pH 5.0, 1.0 mg/mL). After lyophilization, the CS@PLGA NPs powder was recovered and used for further studies. To prepare the TOL-CS@PLGA NPs, 20 mg TOL and 100 mg PLGA dissolved into the organic solvents, and other process was following the same methods described above. 2.3. Characterization on NPs A Nano-ZS90 Malvern Mastersizer (Malvern Instruments Ltd, Malvern, UK) was employed to monitor the particle size distributions and zeta potentials of original PLGA NPs and the cargo-free CS@PLGA NPs. The cargo-free CS@PLGA NPs suspensions
DSC studies were carried out to investigate the distribution state of TOL in NPs matrices. 10 mg of samples were loaded into the ceramic pans, and their thermal behaviors were recorded by a DZ3335 differential scanning calorimetry (Jiangsu, China). The scanning range was covering from 25 ◦ C to 250 ◦ C at a scanning rate of 7 ◦ C/min. 2.6. In vitro release profiles The in vitro release profiles of TOL were studied with pH 1.2 and 7.4 phosphate buffer solutions (PBS) as the medium. 0.1 g TOLCS@PLGA NPs powders were sealed into the dialysis bags (MD44, molecular cut off = 3, 500, USA), and then they were immersed into 35 mL PBS. To retard microbial growth, the buffers were added 0.02% w/v of sodium azide. These samples were vibrated in a ZHWY-100H water-bathing constant temperature vibrator (Shanghai, China) at 37 ± 1 ◦ C. At proper intervals, 5.0 mL PBS were taken off and replaced with equivalent fresh buffers. The TOL concentrations were measured with high performance liquid chro® matography (HPLC) [21]. HPLC were carried out in a Luna C18(2) (150 mm × 4.6 mm I.D.; 5 m) column, and the column oven was set at 40 ◦ C. The mobile phase consisted of 10 mM monobasic potassium phosphate (pH 4.5) and acetonitrile (65:35, v/v) and was filtered through a 0.45 m filter (Supelco, Bellefonte, PA, USA) before use. Absorbance of the eluent was monitored at 245 nm. 2.7. Animal experiments To investigate the hypoglycemic effects of TOL-CS@PLGA NPs, the adult Sprague-Dawley (SD) rats were used to build the diabetes models. The SD rats (300 g − 350 g, 8 weeks) were bought from Laboratory Animal Center, Xinxiang medical University (Xinxiang, China). Before modeling, the SD rats were fasted overnight but allowed to drink water adlibitum. Then, the rats in the normal
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group (n = 12) were fed a standard diet and water, while the rats in modeling group (n = 12) were fed with high fat diet (containing 9 wt.% glucose and 12 wt.% coconut oil) for 4 weeks. After 12 h of fasting, the rats were treated with 1.0% STZ in citric acid − sodium citrate buffer (pH 4.2-4.5) via intraperitoneal injection at a dose of 40 mg·kg−1 [22]. A droplet of blood was collected from the caudal veins, and their blood glucose levels were monitored with a home use glucometer. If the blood glucose level was over 16.7 mmol/L, the diabetic rats were obtained. After fasting overnight, the diabetic rats (n = 6) were treated with TOL-CS@PLGA NPs (200 mg/kg of TOL) by gavage. By contrast, the diabetic rats (n = 6) treated with metformin tablets (10 mg/kg of metformin) by gavage were applied as the positive control. The negative controls were fed with normal saline with the same volumes. The blood glucose levels of the diabetic rats were monitored at the 2 h, 4 h, 8 h, and 12 h, after treatment. All rats were fed in the controlled temperature (20 ± 2 ◦ C) and lighting (08:00 − 20:00) conditions with food and water available ad libitum. Animal experiments were carried out in accordance with the People’s Republic of China National Standard (GB/T 16886.6-1997). 3. Results and discussions 3.1. FTIR spectra and TGA curves FTIR spectra (Fig. 1a) were used to analyze the surface chemical composition between PLGA NPs and CS chains. The FTIR spectrum of CS, the peaks assigning to −OH stretching was observed at 3400 cm−1 . The bonds according to −CH-, −CH2 , and −CH3 stretching were found at the range of 2897–2986 cm−1 [23]. Importantly, the amide I and II vibration were observed at 1667 cm−1 and 1600 cm−1 , respectively. The bonds between 916 cm−1 and 1187 cm−1 was assigned to N H stretching vibration. In the FTIR spectrum of PLGA NPs, the bonds attributing to the carbonyl ( C O) and C O C stretching vibration were observed at 1713 cm−1 and 1094 cm−1 , respectively [24]. By contrast, the spectrum of cargo-free CS@PLGA NPs gave all the characteristic bands of the CS and PLGA molecules: −OH stretching (3400 cm−1 ), −CH, −CH2 , and −CH3 stretching (2773–2995 cm−1 ), carbonyl C O stretching vibration (C, 1708 cm−1 ), amide II vibration (1531 cm−1 ), and C O C stretching (1094 cm−1 ). These data indicated the coexistence of CS and PLGA. Fig. 1b described the TGA curves of the CS, PLGA NPs, and the cargo-free CS@PLGA NPs, respectively. In the TGA curve of CS chains, their dehydration, acetylation and deploymerization were observed at the temperature range of 250.2 ◦ C − 399.4 ◦ C [25]. However, the main decomposition event of PLGA NPs was observed at 209.6 ◦ C − 295.1 ◦ C with a mass loss of 71.7%. On the contrary, the thermostability of the cargo-free CS@PLGA NPs was between those of CS chains and the original PLGA NPs. These changes indicated that the CS shells improved the thermodynamic stability of PLGA NPs. In other words, these data further indicated the CS chains coated on the surfaces of the PLGA NPs.
Fig. 1. (a) FTIR spectra of CS, PLGA, and cargo-free CS@PLGA NPs. The bands attributed to the stretching of carbonyl groups showed a blue shift, which indicated the CS chains coated on the surfaces of PLGA NPs; (b) TGA curves of CS, PLGA NPs and the cargo-free CS@PLGA NPs. The thermostability of cargo-free CS@PLGA NPs was improved by the modification of CS chains. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
free CS@PLGA NPs was increased to 24.7 ± 4.5 mV, comparing to that of −20.1 ± 0.66 mV (original PLGA NPs). Importantly, the positive potential could prevent the cargo-free CS@PLGA NPs from aggregation, and increased their stability. 3.3. Cell viability The cytotoxicity of the cargo-free CS@PLGA NPs was reflected by the cell viability and assessed with the MTT method. In the teats, the un-treated HepG2 cells were used as the control, and their viability was recorded as 100%. It could be seen from Fig. 3 that, after 24 h of incubation, the viability of HepG2 cells were 121.4 ± 5.8% (0.05 mg/mL), 117.4 ± 12.0% (0.1 mg/mL), 116.5 ± 10.5% (0.25 mg/mL), 114.4 ± 16.1% (0.5 mg/mL), and 105.4 ± 15.0% (1.0 mg/mL), respectively. The high cell viability indicated the nontoxicity of the cargo-freeCS@PLGA NPs.
3.2. TEM photographs and zeta-potentials 3.4. TOL release profiles The surface morphology of the PLGA and the cargo-free CS@PLGA NPs were observed with TEM, and photographs were shown in Fig. 2. It could be seen that the original PLGA NPs had regular spherical shapes with a diameter around 100 nm. By contrast, the core-shell structures of the cargo-free CS@PLGA NPs were clearly observed (Fig. 2b). The differences between both kinds of NPs illustrated that the CS chains coated onto the surface of PLGA NPs. Due to the modification, the surface zeta potential of the cargo-
The TOL release profiles were carried out in 1.2 and 7.4 PBS. It found that the TOL possibly distributed in the TOL-CS@PLGA NPs with an amorphous or molecular state (as shown in Fig. S1), which was beneficial to TOL release form these NPs. The release profiles were depicted in Fig. 4. After 24 h of releasing, 74.1 ± 2.2% of encapsulated TOL was released into pH 7.4 PBS. However, only 52.9 ± 2.2% TOL was diffused into pH 1.2 PBS. The difference
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Fig. 2. TEM photographs of original PLGA NPs (a), amplified PLGA NPs (insert), and cargo-free CS@PLGA NPs (b). The PLGA NPs showed spherical shapes, and the cargo-free CS@PLGA NPs had “core-shell” structures. These photographs further confirm the modification of CS chains on the PLGA NPs.
Scheme 1. Schematic presentation for the fabrication and application of the TOLCS@PLGA NPs. Fig. 3. Cell viability of the cargo-free CS@PLGA NPs suspensions. The high cell viability indicated the nontoxicity of the cargo-free CS@PLGA NPs even in a high concentration (1.0 mg/mL).
Fig. 5. Kaplan-Meier survival curves of four groups of diabetic rats. These rats were administrated by gavage of normal saline (negative control), metformin tables (positive control), and the TOL-CS@PLGA NPs, respectively (n = 6). Fig. 4. In vitro release profiles of TOL in 1.2 and 7.4 buffers. The TOL-CS@PLGA NPs could continuously release TOL in 7.4 buffer for 24 h. A significant difference was observed between the both TOL release profiles (P < 0.01, **).
between both release profiles might be due to the special structures of the CS@PLGA NPs (as depicted in Scheme 1). The CS coated onto the PLGA surfaces mainly by the adsorption capacity between the COOH (PLGA NPs) and NH3 + groups (CS chains). In pH 1.2 buffer, the adsorption between CS and PLGA kept their core-shell structures, which prevented TOL from diffusion. However, in pH 7.4 buffer, the NH3 + groups lost a proton to generate −NH2 segments. Therefore, the adsorption was decreased and part of CS chains fell
off from PLGA NPs’ surfaces. Subsequently, more TOL molecules were released. The similar results were reported in previous lecture [15]. 3.5. Survival curves The survival rates of four groups of rats were depicted in Fig. 5. After 18 days of treatment, two rats died in the negative control. Another three rats died in the 20th and 23rd day, and their survival rate was only 25%. By contrast, two rats in the positive control, and their survival rate was 80%. One death of TOL-CS@PLGA NPs
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Fig. 6. (a) and (b) Representative histopathological changes noted at the islet cells of normal rats and the diabetic rats. These histopathological changes in islet cells illustrated the successful modeling of the diabetic rats; (c) Hypoglycemic effects of TOL-CS@PLGA NPs on blood glucose levels in STZ-introduced diabetic rats. Each diabetic rat was treated with the TOL-CA-PLGA NPs (60 mg TOL/kg, n = 6).
treated rat was happened at the 23rd day, which reflected a survival rate of 90%. After t-test analysis, there were no significant differences between four groups (P > 0.05). These data indicated that TOL-CS@PLGA NPs could improve the diabetes complications, and improve the survival rates of diabetic rats.
8 h, which was longer than that of metformin tablets (*, P < 0.05). Therefore, the TOL-CS@PLGA NPs had long-acting hypoglycemic effects, which could improve the bio-availability of TOL and reduce its dosing frequency.
4. Conclusions 3.6. Hypoglycemic effect In the modeling of diabetic rats, the pancreatic ˇ cells of SD rats were partly destroyed by STZ. Therefore, the treated rats showed the hallmarks of type II diabetes. The pancreatic islands of the normal and diabetic rat were taken out, and their pathological slices were shown in Fig. 6. It could be seen that the normal islet ˇ cells were in the oval or round shapes (Fig. 6a). However, the lesion or abnormality of ˇ cells was distributed in diabetes rats (Fig. 6b). These pathological structures of islet ˇ cells further confirmed the obtaining of the diabetic rats. The average blood glucose levels of diabetic rats at different intervals after treatment of TOL-CS@PLGA NPs were shown in Fig. 6c. For comparison, the rats of the positive controls were treated with 10 mg/kg body weight of metformin tablet, while the negative controls were administrated normal saline. No difference in blood glucose levels was observed between the positive controls and TOL-CS@PLGA NPs at the first 2 h after gavage, which illustrated that both preparations had the similar hypoglycemic effect. 4 h later, the blood glucose level of rats in the positive groups was decreased to 9.2 ± 3.8 mmol/L, while that of the rats treated with TOL-CS@PLGA NPs was dropped to 5.9 ± 0.78 mmol/L. 8 h later, the hypoglycemic effect of the positive groups could not be observed from Fig. 6c. On the contrary, the TOL-CS@PLGA NPS showed a longacting hypoglycemic effect. Their hypoglycemic effects lasted over
The PLGA NPs were fabricated with a solvent evaporation method. The CS@PLGA NPs were obtained by injecting PLGA NPs suspension (1.0 mg/mL) into CS solutions (1.0 mg/mL). The FTIR spectra revealed the new interactions between the PLGA NPs and CS chains. TEM photographs revealed the “core-shell” structures of the cargo-free CS@PLGA NPs. The TGA curves indicated that the CS chains improved the thermostability of the original PLGA NPs. The high cell viability indicated the nontoxicity of the cargofree CS@PLGA NPs. The TOL release profiles showed TOL had a high release amounts in 7.4 buffer. Animal experiments suggested that the TOL-CS@PLGA NPs had an excellent hypoglycemic effect. These data suggested that the TOL-CS@PLGA NPs had a promising prospect in oral TOL delivery and improving its bio-availability.
Acknowledgments This work was supported by the National Science Foundation for Young Scientists of China (Grant No. 81703458), Scientific Research Found of Xinxiang Medical University (2014QN147), and the Startup Foundation for Doctors of Xinxiang medical university (505038, 505095). This work was also financed through the university key research projects of Henan province(17A350001, 17A360026), the Scientific & Technological Projects of Henan
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province (172102310616), and the National Undergraduate Training Program for Innovation and Entrepreneurship (201510472045). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at https://doi.org/10.1016/j.colsurfb.2017.10. 037. References [1] F. Danhier, E. Ansorena, J.M. Silva, R. Coco, A.L. Breton, V. Préat, PLGA-based nanoparticles: an overview of biomedical applications, J. Controlled Release 161 (2012) 505–522. [2] A.N. Ford Versypt, D.W. Pack, R.D. Braatz, Mathematical modeling of drug delivery from autocatalytically degradable PLGA microspheres–a review, J. Controlled Release 165 (2012) 29–37. [3] C.E. Astete, C.M. Sabliov, Synthesis and characterization of PLGA nanoparticles, J. Biomater. Sci. Polym. Ed. 17 (2006) 247–289. [4] I. Bala, S. Hariharan, M.N. Kumar, PLGA nanoparticles in drug delivery: the state of the art, Crit. Rev. Ther. Drug Carrier Syst. 21 (2004) 387–422. [5] M. Kroubi, Characterization of endocytosis of transferrin-coated PLGA nanoparticles by the blood-brain barrier, Int. J. Pharm. 379 (2009) 285. [6] B.S. Kang, J.S. Choi, S.E. Lee, J.K. Lee, T.H. Kim, W.S. Jang, A. Tunsirikongkon, J.K. Kim, J.S. Park, Enhancing the in vitro anticancer activity of albendazole incorporated into chitosan-coated PLGA nanoparticles, Carbohydr. Polym. 159 (2017) 39–47. [7] D. Pawar, S. Mangal, R. Goswami, K.S. Jaganathan, Development and characterization of surface modified PLGA nanoparticles for nasal vaccine delivery: effect of mucoadhesive coating on antigen uptake and immune adjuvant activity, Euro. J. Pharm. Biopharm. 85 (2013) 550. [8] M.A. Akl, A. Kartal-Hodzic, T. Oksanen, H.R. Ismael, M.I. Afouna, M. Yliperttula, T. Viitala, A.M. Samy, Enhanced Mucoadhesion and cellular uptake of Curcumin delivered in Chitosan modified PLGA nanosphere, J. Med. Life 4 (2016) 39–54. [9] M.N.V.R. Kumar, A review of chitin and chitosan applications, React. Funct. Polym. 46 (2000) 1–27. [10] A.J. Varma, S.V. Deshpande, J.F. Kennedy, Metal complexation by chitosan and its derivatives: a review, Carbohydr. Polym. 55 (2004) 77–93. [11] R.M. Abdel-Rahman, A.M. Abdel-Mohsen, R. Hrdina, L. Burgert, Z. Fohlerova, ˇ D. Pavlinák, O.N. Sayed, J. Jancar, Wound dressing based on chitosan/hyaluronan/nonwoven fabrics: preparation, characterization and medical applications, Int. J. Biol. Macromol. 89 (2016) 725.
[12] I. Khan, S. Ullah, D.H. Oh, Chitosan grafted monomethyl fumaric acid as a potential food preservative, Carbohydr. Polym. 152 (2016) 87. [13] A. Jimtaisong, N. Saewan, Utilization of carboxymethyl chitosan in cosmetics, Int. J. Cosmet. Sci. 36 (2013) 12–21. [14] M.A. Elgadir, M.S. Uddin, S. Ferdosh, A. Adam, A.J.K. Chowdhury, M.Z.I. Sarker, Impact of chitosan composites and chitosan nanoparticle composites on various drug delivery systems: a review, J. Food Drug Anal. 23 (2014) 619–629. [15] J. Li, P. Huang, L. Chang, X. Long, A. Dong, J. Liu, L. Chu, F. Hu, J. Liu, L. Deng, Tumor targeting and pH-responsive polyelectrolyte complex nanoparticles based on hyaluronic acid-paclitaxel conjugates and Chitosan for oral delivery of paclitaxel, Macromol. Res. 21 (2013) 1331–1337. [16] P. Mukhopadhyay, R. Mishra, D. Rana, P.P. Kundu, Strategies for effective oral insulin delivery with modified chitosan nanoparticles: a review, Prog. Polym. Sci. 37 (2012) 1457–1475. [17] M. Yamasaki, T. Konishi, Y. Maeda, S. Saionji, Y. Takeda, S. Fukuhara, S. Arihara, T. Ueda, S. Tsukiai, A. Maruhashi, Condition for use of antidiabetic drugs and side effects, Jpn. J. Pharm. Health Care Sci. 27 (2001) 291–297. [18] K. Nakashima, S. Yasuda, A. Nishihara, Y. Yamashita, Adsorption and excimer formation of 4-(1-pyrenyl)-butyltrimethylammonium bromide on non-porous silica nanoparticles in aqueous dispersions, Colloids Surf. A Physicochem. Eng. Aspects 139 (1998) 251–257. [19] H. Takeuchi, T. Handa, Y. Kawashima, Spherical solid dispersion containing amorphous tolbutamide embedded in enteric coating polymers or colloidal silica prepared by spray-drying technique, Chem. Pharm. Bull. 35 (1987) 3800–3806. [20] X. Niu, W. Zou, C. Liu, N. Zhang, C. Fu, Modified nanoprecipitation method to fabricate DNA-loaded PLGA nanoparticles, Drug Dev. Ind. Pharm. 35 (2009) 1375–1383. [21] J. Jurica, L.Z. Konecny, J. Zahradnikova, J. Tomandl, Simultaneous HPLC determination of tolbutamide, phenacetin and their metabolites as markers of cytochromes 1A2 and 2C6/11 in rat liver perfusate, J. Pharm. Biomed. Anal. 52 (2010) 557–564. [22] X. Jintao, Y. Liming, L. Yufei, L. Chunyan, C. Han, Noninvasive and fast measurement of blood glucose in vivo by near infrared (NIR) spectroscopy, Spectrochim. Acta Part A Mol. Biomol. Spectrosc. 179 (2017) 250–254. [23] V. Kvande, P. Kiekens, Structure analysis and degree of substitution of chitin, chitosan and dibutyrylchitin by FT-IR spectroscopy and solid state 13C NMR, Carbohydr. Polym. 58 (2004) 409–416. [24] P.N. Thanki, D. Edith, J.L. Six, Surface characteristics of PLA and PLGA films, Appl. Surf. Sci. 253 (2006) 2758–2764. [25] K. Yodkhum, T. Phaechamud, Thermal analysis of chitosan-Lactate and chitosan-Aluminum monostearate composite system, Adv. Mater. Res. 506 (2012) 278–281.