http://informahealthcare.com/drd ISSN: 1071-7544 (print), 1521-0464 (electronic) Drug Deliv, Early Online: 1–7 ! 2014 Informa Healthcare USA, Inc. DOI: 10.3109/10717544.2014.916765
RESEARCH ARTICLE
Preparation and in vitro evaluation of thienorphine-loaded PLGA nanoparticles Yang Yang1, Xiang Yang Xie1,2, and Xing Guo Mei1
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Beijing Institute of Pharmacology and Toxicology, Beijing, PR China and 2Wuhan General Hospital of Guangzhou Military Command, Wuhan, PR China Abstract
Keywords
Poly (D,L-lactic-co-glycolide) nanoparticles (PLGA-NPs) have attracted considerable interest as new delivery vehicles for small molecules, with the potential to overcome issue such as poor drug solubility and cell permeability. However, their negative surface charge decreases bioavailability under oral administration. Recently, cationically modified PLGA-NPs has been introduced as novel carriers for oral delivery. In this study, our aim was to introduce and evaluate the physiochemical characteristics and bioadhesion of positively charged chitosancoated PLGA-NPs (CS-PLGA-NPs), using thienorphine as a model drug. These results indicated that both CS-PLGA-NPs and PLGA-NPs had a narrow size distribution, averaging less than 130 nm. CS-PLGA-NPs was positively charged (+42.1 ± 0.4 mV), exhibiting the cationic nature of chitosan, whereas PLGA-NPs showed a negative surface charge (2.01 ± 0.3 mV). CS-PLGA-NPs exhibited stronger bioadhesive potency than PLGA-NPs. Furthermore, the transport of thienorphine-CS-PLGA-NPs by Caco-2 cells was higher than thienorphine-PLGA-NPs or thienorphine solution. CS-PLGA-NPs were also found to significantly enhance cellular uptake compared with PLGA-NPs on Caco-2 cells. An evaluation of cytotoxicity showed no increase in toxicity in either kind of nanoparticles during the formulation process. The study proves that CS-PLGA-NPs can be used as a vector in oral drug delivery systems for thienorphine due to its positive surface charge and bioadhesive properties.
Chitosan, nanoparticles, oral administration, poly (D,L-lactic-co-glycolide), thienorphine
Introduction Opioid abuse and dependence remains a serious worldwide health problem. The drugs currently in clinical use for treating opioid dependence are either full-opioid agonist methadone or antagonist naltrexone (Johnson et al., 2003). However, the agonist merely substitutes one addiction for another, and the antagonist is unable to retain patients in treatment due to a lack of desired positive subjective effects (Fudala et al., 1998). Therefore, there is an urgent requirement for developing new practical opioid agonists with long duration of action and safety. In the search for such compounds, thienorphine [N-cyclopropylmethyl-7(-[(R)-1-hydroxy-1-methyl-3-(thien-2yl)-propyl]-6, 14-endo-ethano-tetrahydronororipavine], a novel analog of buprenorphine, was synthesized by the chemists in our institute (Figure 1). The pharmacology studies showed that thienorphine is a potent, long-acting partial opioid agonist and may have a possible application in treating addiction (Liu et al., 2005). As peroral administration is the
Address for correspondence: Professor XingGuo Mei, Beijing Institute of Pharmacology and Toxicology, 27 Taiping Road, Beijing 100850, PR China. Tel & Fax: 86-010-6693 2654 (o). E-mail: [email protected]
History Received 23 February 2014 Revised 12 April 2014 Accepted 16 April 2014
most convenient route, developing a viable means of oral administration is of great value. However, thienorphine has poor oral bioavailability, which might contribute to the low compliance. Our main objective was to develop an oral delivery system of thienorphine that can overcome the problem and increase patient compliance. Recently, polymeric nanoparticles (NPs) have attracted considerable interest as new delivery vehicles for small molecules, with the potential to overcome issue such as poor drug solubility and cell permeability. Among polymers, polyesters based on polylactic acid (PLA), polyglycolic acid (PGA), and their copolymers, poly (lactic-co-glycolic) acid (PLGA), are some of the best defined biomaterials as far as design and performance are concerned (Hulse et al., 2005; De et al., 2008). PLGA is FDA-approved biodegradable polymer, which has an excellent biocompatibiltiy. PLGA has been studied extensively as a polymeric carrier for NPs (Makhlof et al., 2011; Shi et al., 2009). However, the slight negative surface charge of PLGA nanoparticles (PLGA-NPs) tend to limit their ability to interact with negatively charged plasmids and intracellular uptake, which leads to lower bioavailability (He et al., 2009). Physical and chemical modifications of PLGA-NPs were studied for practical requirements. One of the most promising approaches is to use chitosan, a polycationic polymer retrieved from biological
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Methods Preparation and characteristics of PLGA-NPs and CS-PLGA-NPs Preparation
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Figure 1. The chemical structures of buprenorphine and thienorphine.
sources. Chitosan is also a biodegradable and biocompatible polymer, and due to its cationic nature, it has good mucoadhesive and membrane-permeability-enhancing properties. Mucoadhesive properties play an important role in oral drug delivery systems by prolonging the duration of residence of drug carriers and increasing the closeness of contact between drugs and mucous membranes at absorption sites, thus enhancing permeability, reducing degradation, and eventually improving oral bioavailability (Thirawong et al., 2007; He et al., 2013). Hence, chitosan has been extensively studied for its potential as an absorption enhancer across intestinal epithelium (Merwe et al., 2004; Guo et al., 2014). The advantages of chitosan-modified PLGA-NPs (CSPLGA-NPs) include decreased burst effect of the encapsulated drug, better cellular adhesion and longer site-retention of NPs (Zhang et al., 2012). Many researchers have evaluated common PLGA-NPs as drug carriers, but only a few have focused on CS-PLGA-NPs. The purpose of this paper is to discuss two kinds of thienorphine-encapsulated NPs formulations, negatively charged PLGA-NPs and positively charged CS-PLGANPs, and to evaluate their physicochemical properties, such as particle size, zeta-potential, drug entrapment efficiency, release in vitro, mucosa adhesion, permeability and cellular uptake. Cytotoxicity was also evaluated because the toxicity of NPs is extremely relevant to their development and use. These investigations will further understanding of biological activity in vivo of thienorphine-CS-PLGA-NPs or thienorphinePLGA-NPs for oral delivery of thienorphine.
Experimental materials PLGA (50: 50, Av. MW 15 000, with one acid per end group) was purchased from the Shandong Institute of Medical Instruments (Shandong, China). Thienorphine (99% purity) was supplied by Beijing Institute of Pharmacology and Toxicology (Beijing, China). Chitosan (deacetylation degree 91.10%, MW4250 kDa) was a gift from Qingdao Scitech Co., Ltd. (Qingdao, China) Dichloromethane (DCM) and Hank’s balanced salt solution (HBSS) were obtained from Beijing Chemical Reagents Company (Beijing, China). Dulbecco’s Modified Eagle’s Medium (DMEM) and fetal bovine serum (FBS) were obtained from GIBCO, Invitrogen Corp. (Carlsbad, CA). Trypsin was obtained from Amresco, Inc., (Solon, OH) and 6-coumarin (COU, purity499%) and 3(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) were purchased from Sigma-Aldrich (Saint Louis, MO). All reagents used for high-performance liquid chromatography (HPLC) were of chromatographic purity. Other chemicals used were of analytical grade.
PLGA-NPs were prepared by a single emulsion (o/w) method commonly known as solvent evaporation technique (Perez et al., 2001). Briefly, 100 mg of PLGA and 10 mg of thienorphine (or 1 mg of 6-cumarin) were dissolved in DCM to obtain the organic phase. This organic solution was then added dropwise into 1% (w/v) PVA aqueous solution precooled at 4 C under magnetic stirring. The coarse emulsion was sonicated by ultrasonic probe (SCIENTZ-IID, Scientz Biotechnology Co., Ningbo, China) and then subjected to rotary evaporation to remove the organic solvent. Drugs that were un-encapsulated in the samples were removed by centrifugation (5000 rpm 5 min). The NPs in the supernatant were precipitated by centrifugation (15 000 rpm 40 min) and the precipitant was washed 5 times with distilled water by centrifugation following overnight freeze-drying. The NP suspensions was incubated (1:1, v/v) with chitosan solution (1 mg/mL) prepared by 1% (v/v) acetic acid for 10 min under agitation to prepare CS-PLGA-NPs. Morphology observations The morphology of thienorphine-PLGA-NPs or thienorphineCS-PLGA-NPs was observed using transmission electron microscopy (TEM, HITACHI, H-7650, Tokyo, Japan) and atomic force microscopy (AFM, NanoWizarc, JPK Ltd., Berlin, Germany). To prepare the samples for TEM, the NPs were suspended in water and stained with 1% uranyl acetate. To prepare the samples for AFM, the NPs were diluted with distilled water, followed by dropping the samples onto a silicon surface. The samples were air-dried at room temperature. Particles size and zeta potential of NPs The particle size (Z-average mean) and zeta potential of the NPs were measured by photon correlation spectroscopy and laser doppler anemometry, respectively, using a Zetasizer Nano Analyzer (ZS90, Malvern Instruments, London, UK). For size measurements, the PLGA-NPs or CS-PLGA-NPs suspensions was diluted in water and measured for a minimum of 180 s. Raw data were subsequently correlated to mean hydrodynamic size by cumulant analysis. For the measurement of zeta potential, samples were diluted in 0.1 mM KCl and measured in automatic mode. All measurements were performed in triplicate. Determination of encapsulation efficiency and loading efficiency After weighed amounts of dried thienorphine-PLGA-NPs or thienorphine-CS-PLGA-NPs were dissolved in DCM, a clear solution was obtained by vigorous vortex. The total amount of thienorphine in PLGA-NPs or CS-PLGA-NPs was estimated by HPLC analysis (1211, Agilent Technologies Inc., Avondale, PA). The chromatographic conditions were as follows: C18 column (250 mm 4.6 mm, 5 mm)
Thienorphine-loaded PLGA-NPs
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(Agilent Technologies Inc., Avondale, PA), a mixture of acetonitrile-methanol-0.02 mol l1 phosphate (40:15:45) buffer containing 0.2% triethylamine (pH ¼ 3) as eluant, detection at 220 nm. The polymers did not interfere with absorbance of the drug at the specified wavelength. Encapsulation efficiency (EE) was expressed as the ratio of thienorphine in PLGA-NPs or CS-PLGA-NPs to the initial amount of thienorphine used in the formulation. Loading efficiency (LE) corresponds to the ratio of amount of thienorphine entrapped in PLGA-NPs or CSPLGA-NPs recovered to the total amount of polymer and thienorphine. Each measurement was performed in triplicate. Release in vitro To determine the amount of drug released from the NPs, a dynamic dialysis technique was used. The thienorphine concentration in the aliquots was analyzed using the HPLC as described above. Two kinds of NPs, the thienorphineloaded common NPs and adhesive NPs, in which thienorphine was calculated as 0.5 mg, were placed in the dialysis bag (MWCO 10,000; Spectrum, Los Angeles, LA) containing 5 ml of phosphate buffer solution (PBS, 0.1M, pH 7.4). The dialysis bag was filled with the nanosuspensions and immersed in receiver solution (50 ml) identical to the inner solution and then incubated in a water-bath shaker at 37 C with constant orbital mixing (100 rpm). At specified time points (0.5, 1, 2, 3, 5, 7, 10 and 12 h) an aliquot (200 ml) of the receiver solution was removed and replaced with fresh medium. The thienorphine concentrations of the aliquots were determined by using HPLC, and the cumulative amount of thienorphine released from the NPs was calculated. The cumulative percentage of thienorphine released was plotted versus time. Each data point is calculated from three measurements. Residual DCM content Gas chromatography (HP 5890, USA) was used to determine the residual DCM in the thienorphine-PLGA-NPs or thienorphine-CS-PLGA-NPs. 1 ml N,N-dimethyl formamide of 0.05 ml of ethyl acetate was used as internal standard. Approximately 25 mg of NPs were dissolved in 1 ml of internal standard. GC conditions were as follows: DB-624 capillary column (30 m 0.25 mm 0.25 mm, HP, Palo Alto, CA); high purity nitrogen (99.99%) as carrier gas with flow rate of 30 mlmin1; injection temperature at 270 C, with FLD detector. Bioadhesion
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Bioadhesive studies Rats were fed 2 ml aqueous dispersions of the test formulations containing 0.5 mg thienorphine. The rats in group 1 were given free thienorphine, those in group 2 were given thienorphine-PLGA-NPs, and those in group 3 were given thienorphine-CS-PLGA-NPs. The animals were killed by cervical dislocation at 0.5, 1, 2, 3, 5 and 7 h after administration, three rats per time points. The abdominal cavity of each rat was opened and the small intestinal tract (including duodenum, jejunum, and ileum, to a fixed length of 20 cm) was removed. Then, the gut was rinsed with 40 ml of physiological saline in order to recover the non-adhered fraction. The rinse liquids were centrifuged at 25 000 g for 30 min, and thienorphine from the supernatants was quantified and termed ‘‘free thienorphine’’. The pellets were digested in 1 ml 3 mol/l NaOH for 24 h and centrifuged at 25 000 g for 30 min. The amounts of thienorphine in the supernatants were assayed by the HPLC and termed ‘‘unreleased thienorphine’’. The free and unreleased thienorphine levels were added together to form the total amount of thienorphine in the lumen in order to estimate the bioadhesive characteristics of the different formulations. In vitro cell assays Cell culture A Caco-2 cell line was obtained from the American Type Culture Collection (Rockville, MD). Cells were grown routinely on 75 cm2 plastic culture flasks (Corning, NY) in DMEM containing 25 mM D-glucose, 25 mM N-2-hydroxyethyl piperazine-N/-2-ethane sulfonate buffer (HEPES), 44 mM NaHCO3, supplemented with 10% (v/v) fetal bovine serum (FBS), 1% (v/v) non-essential amino acid solution, 1% (v/v) L-glutamine, 1% (v/v) penicillin-streptomycin at 37 C in an atmosphere of 5% CO2 and 90% relative humidity. The medium was replaced every 2 days after incubation. Cells were passaged, 1:5, approximately every 5 days (at 80–85% confluence) using 0.25% trypsin and 0.02% ethylenediaminetetraacetic acid (EDTA). Caco-2 cells of passage numbers 32–38 were used in these experiments. Cellular uptake To visualize the cellular uptake of PLGA-NPs or CS-PLGANPs labeled by 6-cumarin, 3 105 Caco-2 cells were grown on cover slips placed in 6-well plates. Cells were treated with the PLGA-NPs or CS-PLGA-NPs for different time intervals (0.5, 1 and 2 h), fixed in 4% paraformaldehyde (PFA) and finally examined under a confocal laser scanning microscopy (UltraVIEW Vox, PerkinElmer, Waltham, MA).
Animals Fifty-four Wistar rats (adult male, 220 ± 20 g, Beijing Institute of Pharmacology and Toxicology) were used for the bioadhesive study and they were randomly divided into three groups. All of them were fasted overnight, but had free access to water. All animal experiments were justified in detail and were approved by the Institutional Animal Care and Use Committee of Beijing Institute of Pharmacology and Toxicology.
Transport experiments For transport experiments, Caco-2 cells were cultivated on bicameral inserts TranswellÕ , Corning, Inc., New York following standards protocols (Garinot et al., 2007). On the day of the transport experiments, the cells were washed with HBSS for the apical and basolateral sides, respectively. The cell monolayers were subjected to transepithelial electrical resistance (TEER) determination and only cell monolayer
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with TEER higher than 450 /cm2 were used for the transport experiments. Solutions from the apical side of the monolayers were replaced by the different thienorphine formulations (1 mg/ml), and samples were collected from the basolateral side. Samples of 500 ml were taken from the acceptor chamber at predesigned time and replaced by 500 ml of fresh experimental culture medium. The thienorphine in samples was quantified by an HPLC. The apparent permeability coefficient (Papp) was calculated from the slope of the accumulated concentration versus time curve using the following relation: Papp ¼ dQ/dt/(AC0), where dQ is the amount of thienorphine in the basolateral side at time t, A is the diffusion area, and C0 is the initial concentration of thienorphine in the apical side. MTT assay NPs cytotoxicity in vitro was evaluated using MTT assay with Caco-2 cells, as described previously (Viholal et al., 2005). Briefly, Caco-2 cells were seeded on 96-well plates at a density of 10 000 cells per well and were cultured for 5 days. Then the culture medium was replaced with PLGA material, blank PLGA-NPs, or blank CS-PLGA-NPs, and all were diluted with culture medium to a concentration of 1.0, 5.0, and 25.0 mg/ml, respectively. After 2 h of incubation at 37 C, the PLGA polymer, blank PLGA-NPs and blank CS-PLGANPs were replaced with 100 ml of MTT (5 mg/ml in HBSS) solutions, and the cells were incubated for a further 4 h at 37 C. The test solution was decanted and the formazan crystals were dissolved in dimethyl sulfoxide (200 ml/well). Absorption was measured using a microplate reader (Tecan Deutschland GmbH, Crailsheim, Germany) at 590 nm. Cell viability was expressed as percentage of absorbance relative to control. Control cells were not exposed to the PLGA materials. Experiments were performed in triplicate, with eight replicate wells for each sample and control per assay. Statistical analysis Data are presented as the mean ± standard deviation (SD). The difference between any two groups was determined using ANOVA. p50.05 was considered statistically significant.
Results and discussion Characterization of NPs The particle size, zeta potential, EE and LE of the two kinds of NPs are listed in Table 1. The results show that the EE of CS-PLGA-NPs was 54.16 ± 3.18%, which was higher than PLGA-NPs, 43.65 ± 2.73%. The particle size of CS-PLGANPs was 121.1 ± 10 nm, slightly greater than that of PLGANPs (118.5 ± 13 nm), and there was no significant difference between them (p40.05). However, the zeta potentials of the two kinds of NPs were opposite to each other. PLGA-NPs has Table 1. Characteristics of PLGA-NPs and CS-PLGA-NPs (mean ± SD, n ¼ 3).
Type of NPs
Particle size (nm)
PLGA-NPs 118.5 ± 13 CS-PLGA-NPs 121.1 ± 10
Zeta potential (mV) 2.01 ± 0.3 +42.1 ± 0.4
EE (%)
LE (%)
43.65 ± 2.73 1.24 ± 0.46 54.16 ± 3.18 1.59 ± 0.39
a negative charge (2.01 mV) due to the nature characteristics of PLGA polymer and CS-PLGA-NPs has a positive charge (+42.1 mV) due to chitosan coated on the surface. Positively charged particles have a higher potency for interaction with biological membranes than negatively charged particles. This is because the gastrointestinal tract is made up of smooth muscle cells and its surface charge is about 50 mV (He et al., 2013). Interactions between positively charged NPs and negatively charged cells will lead to alterations in the permeability and ease of transport through electrostatic attraction. As shown in Figure 6, the difference in zeta potential from the negatively charged PLGA-NPs to positively charged CS-PLGA-NPs did cause a significant increase in cell uptake. This showed that particle surface charge is a vital factor in manipulating the process of particle entry into the body. This is in agreement with the data presented in existing literature (Chung et al., 2010). The TEM image of PLGA-NPs or CS-PLGA-NPs is illustrated in Figure 2(A) and (B). The overall morphological appearance for the two types of NPs was observed to be similar. The particle size range and the observed spheroidal morphology did not change significantly upon chitosan coating, suggesting the formation of a polymer layer on PLGA-NPs that was too thin to be detected in this analysis. However, the AFM images of the two kinds of NPs are depicted in Figure 2(C) and (D). Using the AFM technique, we clearly visualized the three-dimensional group of NPs. The particle surface of PLGA-NPs was relatively smooth, while that of the CS-PLGA-NPs had small globular protuberances. Release in vitro Figure 3 shows the release profiles of thienorphine from CS-PLGA-NPs and PLGA-NPs at PBS (pH 7.4). It is here apparent that thienorphine release in vitro underwent a very rapid initial burst (23.84 ± 1.43%), suggesting that some thienorphine was localized on the surface of PLGA-NPs. During the coating process in the preparation of CS-PLGANPs, in which negative PLGA-NPs is coated with positively charged chitosan, thienorphine absorbed on the surface of PLGA-NPs can be loaded into CS-PLGA-NPs. As a result, the burst effect is less pronounced (14.29 ± 1.24%) for CS-PLGANPs than for pure PLGA-NPs (23.84 ± 1.43%). The size of the decrease in burst release suggests that the bulk of the thienorphine is encapsulated inside the matrix. In this way, the EE of CS-PLGA-NPs can be said to be higher than that of PLGA-NPs. As shown in Figure 3, the effect of chitosan on the release behavior in vitro is most significant before 5 h, showing a significant difference between CS-PLGA-NPs and PLGA-NPs (p50.05), but the effect become less dramatic over time. There was no remarkable difference between the two kinds of NPs at 12 h (p40.05). Residual DCM contents The residual DCM contents in all thienorphine loaded CS-PLGA-NP and PLGA-NP were below 600 ppm, which was in accord with the requirements of the ICH standard (International Conference on Harmonization, 2005).
Thienorphine-loaded PLGA-NPs
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DOI: 10.3109/10717544.2014.916765
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Figure 2. Morphological appearance of NPs observed using TEM (A: PLGA-NPs; B: CS-PLGA-NPs) and AFM (C: PLGA-NPs; D: CS-PLGA-NPs).
Figure 3. Drug release from PLGA-NPs and CS-PLGA-NPs in vitro (mean ± SD, n ¼ 3).
Figure 4. amount of thienorphine in the lumen content of the gastrointestinal tract after oral administration of 2 ml solution or NPs dispersion containing 0.5 mg thienorphine. Each experiment was performed in triplicate.
Bioadhesiveness Besides particle size and zeta potential, mucosal adhesion is the most important characteristic of NP drug delivery systems. Prolonging the duration of drug retention on the mucosa may result in a higher bioavailability. Chitosan is a linear polycationic macromolecule linked by b(1,4)-glycoside bonds. Its mucoadhesive properties are thought to be mediated by electrostatic interactions between the positively charged D-glucosamine residue and the negatively charged sialic acid residue of mucin. In this study, Figure 4 shows changes in total thienorphine in the lumen content over time. For free thienorphine PBS solution, it was found that the amounts of thienorphine decreased rapidly to about 20% of the total dose in about 7 h. It may be possible that the encapsulation into NPs slowed the elimination of
thienorphine from the gastrointestinal tract, thus causing a statistically significant difference between free thienorphine solution and the two kinds of NP formulations (p50.05). Adhesive CS-PLGA-NPs slowed this process further. The amounts of remaining thienorphine in CS-PLGA-NPs and PLGA-NPs were 43.8% and 58.1%, respectively, at 7 h. Cellular uptake In order to visualize the cellular uptake of NPs, the high fluorescent 6-coumarin has proved to be useful in confocal microscopy studies because of its low loading requirement. The time course of 6-coumarin-PLGA-NPs and 6-coumarinCS-PLGA-NPs internalization is shown in Figure 5. At the same time point, the uptake of CS-PLGA-NPs was much
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Figure 5. Fluorescent images of intracellular uptake of 6-coumarin-PLGA-NPs and 6-coumarin-CS-PLGA-NPs.
Figure 6. Effect of NPs on the permeability of thienorphine (mean ± SD, n ¼ 3). Figure 7. MTT assay evaluation of cytotoxicity after 2 h of incubation with PLGA polymer, blank PLGA-NPs, and blank CS-PLGA-NPs.
higher than that of PLGA-NPs. This difference could be a direct consequence of surface modifications, because chitosan coating changes the negative surface charge to a positive one. It has been reported that the internalization of cationic chitosan particles appears to occur predominantly by adsorptive endocytosis, suggesting that the positively charged particle surface can facilitate adherence to the negatively charged cell membrane. Transport experiments Figure 6 shows that cumulative amount of thienorphine transported from the apical side to the basolateral side of the Caco-2 monolavers incubated with thienorphine, thienorphine-PLGA-NPs and thienorphine-CS-PLGA-NPs. The Papp value of thienorphine-PLGA-NPs was 2.16 ± 0.26 cm/ s 107, which was significantly higher by 2.2 folds as compared to that of free thienorphine (0.97 ± 0.08 cm/ s 107). On the other hand, loading of thienorphine into CS-PLGA-NPs resulted in a Papp value of
4.38 ± 0.31 cm/s 107 with 4.5 folds improvement in thienorphine permeation. It could be related to the higher positive charge which is a critical factor for penetration enhancement towards cells. MTT assay The toxicity of NP drug delivery systems has been a prominent concern over the past 10 years (Nahar et al., 2008; Jain et al., 2011). Related studies have shown that NPs enhance therapeutic effects but can also increase toxicity. This study mainly concerns the cytotoxicity caused by the residual solvent and uses MTT to evaluate the cell survival rate. The cytotoxicities of CS-PLGA-NPs, PLGA-NPs and PLGA polymer were evaluated after incubation with the aforementioned cells for 2 h. As shown in Figure 7, cell viability did not decrease even after 2 h of incubation at 37 C while polymer/NP concentration was increased from
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DOI: 10.3109/10717544.2014.916765
1 to 25 mg/ml. Compared with the PLGA polymer group, the viability values of the Caco-2 cells exposed to the two NP formulations, blank PLGA and blank PLGA-NP, showed no significant differences (p40.05). The results indicated that the residue of organic solvent induced in the process of preparing NPs was thin, so the formulations showed no particular toxicity and did not influence the normal growth of Caco-2 cells within this dose range (25 mg/ml). And compared with negatively charged PLGA-NPs, the positive properties of CS-PLGA-NPs did not increase the cytotoxicity to Caco-2 cells after 2 h of incubation at 37 C. The main reason for this is that chitosan is biocompatible, biodegradable, and has low cell toxicity. This is why it has been widely applied in tissue engineering, gene therapy, drug delivery and other fields (Jayakumar et al., 2010).
Conclusions In this study, bioadhesive NPs containing thienorphine have been prepared. In vitro studies show that the NPs have attractive properties, such as a positive charge, mucosal adhesion and absorption promotion, which can prolong the duration of residence of thienorphine for oral delivery. CS-PLGA-NPs were found to significantly enhance cellular uptake compared with PLGA-NPs on Caco-2 cells. MTT test reveals that CS-PLGA-NPs did not increase cytotoxicity; both CS-PLGA-NPs and PLGA-NPs were well tolerated at concentrations below 25.0 mg/ml in Caco-2 cell cultures. In addition, the cellular transport of thienorphine showed higher permeation enhancing profiles of CS-PLGA-NPs, as compared with thienorphine soluton or PLGA-NPs. Compared with negatively charged PLGA-NPs, the positively charged CS-PLGA-NPs delivery system may bring us closer to a safe, general system for oral administration of thienorphine. Also, we are currently evaluating the bioavailability of thienorphine-CS-PLGA-NPs or thienorphine-PLGA-NPs in beagle dogs.
Declaration of interest We are grateful to the financial support from the National Natural Science Foundation of China (Grant No. 81202466) and the Important National Science & Technology Specific Projects (Grant No. 2012ZX09301003-001-009) of China.
References Johnson RE, Strain EC, Amass L. (2003). Buprenorphine: how to use it right. Drug Alcohol Depend 70:S59–77. Fudala PJ, Yu E, Macfadden W, et al. (1998). Effects of buprenorphine and naloxone in morphine-stabilized opioid addicts. Drug Alcohol Depend 50:1–8.
Thienorphine-loaded PLGA-NPs
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Liu CH, Liu H, Han XY, et al. (2005). Synthesis and characterization of thienorphine and its glucuronide conjugate. Synth Commun 35: 701–10. De Jong WH, Borm PJ. (2008). Drug delivery and nanoparicles: applications and hazards. Int J Nanomed 3:133–49. Hulse GK, Stalenberg V, McCallum D, et al. (2005). Histological changes over time around the site of sustained release naltrexonepoly(DL-lactide) implants in humans. J Control Release 108:43–55. 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. Shi K, Cui F, Yamamoto H, Kawashima Y. (2009). Optimized formulation of high-payload PLGA nanoparicles containing insulinlauryl sulfate complex. Drug Dev Ind Pharm 35:177–84. He B, Lin P, Jia ZR, et al. (2013). The transport mechanisms of polymer nanoparticles in Caco-2 epithelial cells. Biomaterials 34:6082–98. Thirawong N, Nunthanid J, Puttipipatkhachorn Satit, et al. (2007). Mucoadhesive properties of various pectins on gastrointestinal mucosa: an in vitro evaluation using texture analyzer. Eur J Pharm Biopharm 67:132–40. He B, Jia ZR, Du WW, et al. (2013). The transport pathways of polymer nanoparticles in MDCK epithelial cells. Biomaterials 34:4309–26. Merwe SM, Verhoef JC, Kotze´ AF, et al. (2004). N-Trimethyl chitosan chloride as absorption ehancer in oral peptide drug delivery development and characterization of minitablet and granule formulations. Eur J Pharm Biopharm 57:85–91. Guo XH, Zhang XX, Ye L, et al. (2014). Inhalable microspheres embedding chitosan-coated PLGA nanoparticles for 2-methoxyestradiol. J Drug Target published online:1–7. Zhang XY, Sun MZ, Zheng AP, et al. (2012). Preparation and characterization of insulin-loaded bioadhesive PLGA nanoparticles for oral administration. Eur J Pharm Sci 45:632–8. Perez C, Sanchez A, Putnam D, et al. (2001). Poly (lactic acid)-poly (ethylene glycol) nanoparticles as new carriers for the delivery of plasmid DNA. J Control Release 75:211–24. Garinot M, Fievez V, Pourcelle V, et al. (2007). PEGylated PLGA-based nanoparticles targeting M cells for oral vaccination. J Control Release 120:195–204. Viholal H, Laukkanen A, Valtola L, et al. (2005). Cytotoxicity of thermosensitive polymers poly (N-isopropylacrylamide), poly(N-vinylcaprolactam) and amphiphilically modified poly(Nvinylcaprolactam). Biomaterials 26:3055–64. Chung YL, Kim JC, Kim YH, et al. (2010). The effect of surface functionalization of PLGA nanoparticles by heparin- or chitosanconjugated pluronic on tumor targeting. J Control Release 143: 374–82. International Conference on Harmonization (ICH) harmonized tripartite guideline. (2005). Impurities: guideline for residual solvents, Q3C (R3). In International Conference on Harmonisation harmonised tripartite guideline Current Step 4 version. Available at:http:// www.ich.org/LOB/media/MEDIA423.pdf. Nahar M, Mishra D, Dubey V, et al. (2008). Development, characterization, and toxicity evaluation of amphotericin B-loaded gelatin nanoparticles. Nanomed Nanotech Bio Med 4:252–62. Jain AK, Swarnakar NK, Godugu C, et al. (2011). The effect of the oral administration of polymeric nanoparticles on the efficacy and toxicity of tamoxifen. Biomaterials 32:503–15. Jayakumar R, Chennazhi KP, Muzzarelli RAA, et al. (2010). Chitosan conjugated DNA nanoparticles in gene therapy. Carbohyd Polym 79: 1–8.
RESEARCH ARTICLE
Preparation and in vitro evaluation of thienorphine-loaded PLGA nanoparticles Yang Yang1, Xiang Yang Xie1,2, and Xing Guo Mei1
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Beijing Institute of Pharmacology and Toxicology, Beijing, PR China and 2Wuhan General Hospital of Guangzhou Military Command, Wuhan, PR China Abstract
Keywords
Poly (D,L-lactic-co-glycolide) nanoparticles (PLGA-NPs) have attracted considerable interest as new delivery vehicles for small molecules, with the potential to overcome issue such as poor drug solubility and cell permeability. However, their negative surface charge decreases bioavailability under oral administration. Recently, cationically modified PLGA-NPs has been introduced as novel carriers for oral delivery. In this study, our aim was to introduce and evaluate the physiochemical characteristics and bioadhesion of positively charged chitosancoated PLGA-NPs (CS-PLGA-NPs), using thienorphine as a model drug. These results indicated that both CS-PLGA-NPs and PLGA-NPs had a narrow size distribution, averaging less than 130 nm. CS-PLGA-NPs was positively charged (+42.1 ± 0.4 mV), exhibiting the cationic nature of chitosan, whereas PLGA-NPs showed a negative surface charge (2.01 ± 0.3 mV). CS-PLGA-NPs exhibited stronger bioadhesive potency than PLGA-NPs. Furthermore, the transport of thienorphine-CS-PLGA-NPs by Caco-2 cells was higher than thienorphine-PLGA-NPs or thienorphine solution. CS-PLGA-NPs were also found to significantly enhance cellular uptake compared with PLGA-NPs on Caco-2 cells. An evaluation of cytotoxicity showed no increase in toxicity in either kind of nanoparticles during the formulation process. The study proves that CS-PLGA-NPs can be used as a vector in oral drug delivery systems for thienorphine due to its positive surface charge and bioadhesive properties.
Chitosan, nanoparticles, oral administration, poly (D,L-lactic-co-glycolide), thienorphine
Introduction Opioid abuse and dependence remains a serious worldwide health problem. The drugs currently in clinical use for treating opioid dependence are either full-opioid agonist methadone or antagonist naltrexone (Johnson et al., 2003). However, the agonist merely substitutes one addiction for another, and the antagonist is unable to retain patients in treatment due to a lack of desired positive subjective effects (Fudala et al., 1998). Therefore, there is an urgent requirement for developing new practical opioid agonists with long duration of action and safety. In the search for such compounds, thienorphine [N-cyclopropylmethyl-7(-[(R)-1-hydroxy-1-methyl-3-(thien-2yl)-propyl]-6, 14-endo-ethano-tetrahydronororipavine], a novel analog of buprenorphine, was synthesized by the chemists in our institute (Figure 1). The pharmacology studies showed that thienorphine is a potent, long-acting partial opioid agonist and may have a possible application in treating addiction (Liu et al., 2005). As peroral administration is the
Address for correspondence: Professor XingGuo Mei, Beijing Institute of Pharmacology and Toxicology, 27 Taiping Road, Beijing 100850, PR China. Tel & Fax: 86-010-6693 2654 (o). E-mail: [email protected]
History Received 23 February 2014 Revised 12 April 2014 Accepted 16 April 2014
most convenient route, developing a viable means of oral administration is of great value. However, thienorphine has poor oral bioavailability, which might contribute to the low compliance. Our main objective was to develop an oral delivery system of thienorphine that can overcome the problem and increase patient compliance. Recently, polymeric nanoparticles (NPs) have attracted considerable interest as new delivery vehicles for small molecules, with the potential to overcome issue such as poor drug solubility and cell permeability. Among polymers, polyesters based on polylactic acid (PLA), polyglycolic acid (PGA), and their copolymers, poly (lactic-co-glycolic) acid (PLGA), are some of the best defined biomaterials as far as design and performance are concerned (Hulse et al., 2005; De et al., 2008). PLGA is FDA-approved biodegradable polymer, which has an excellent biocompatibiltiy. PLGA has been studied extensively as a polymeric carrier for NPs (Makhlof et al., 2011; Shi et al., 2009). However, the slight negative surface charge of PLGA nanoparticles (PLGA-NPs) tend to limit their ability to interact with negatively charged plasmids and intracellular uptake, which leads to lower bioavailability (He et al., 2009). Physical and chemical modifications of PLGA-NPs were studied for practical requirements. One of the most promising approaches is to use chitosan, a polycationic polymer retrieved from biological
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Methods Preparation and characteristics of PLGA-NPs and CS-PLGA-NPs Preparation
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Figure 1. The chemical structures of buprenorphine and thienorphine.
sources. Chitosan is also a biodegradable and biocompatible polymer, and due to its cationic nature, it has good mucoadhesive and membrane-permeability-enhancing properties. Mucoadhesive properties play an important role in oral drug delivery systems by prolonging the duration of residence of drug carriers and increasing the closeness of contact between drugs and mucous membranes at absorption sites, thus enhancing permeability, reducing degradation, and eventually improving oral bioavailability (Thirawong et al., 2007; He et al., 2013). Hence, chitosan has been extensively studied for its potential as an absorption enhancer across intestinal epithelium (Merwe et al., 2004; Guo et al., 2014). The advantages of chitosan-modified PLGA-NPs (CSPLGA-NPs) include decreased burst effect of the encapsulated drug, better cellular adhesion and longer site-retention of NPs (Zhang et al., 2012). Many researchers have evaluated common PLGA-NPs as drug carriers, but only a few have focused on CS-PLGA-NPs. The purpose of this paper is to discuss two kinds of thienorphine-encapsulated NPs formulations, negatively charged PLGA-NPs and positively charged CS-PLGANPs, and to evaluate their physicochemical properties, such as particle size, zeta-potential, drug entrapment efficiency, release in vitro, mucosa adhesion, permeability and cellular uptake. Cytotoxicity was also evaluated because the toxicity of NPs is extremely relevant to their development and use. These investigations will further understanding of biological activity in vivo of thienorphine-CS-PLGA-NPs or thienorphinePLGA-NPs for oral delivery of thienorphine.
Experimental materials PLGA (50: 50, Av. MW 15 000, with one acid per end group) was purchased from the Shandong Institute of Medical Instruments (Shandong, China). Thienorphine (99% purity) was supplied by Beijing Institute of Pharmacology and Toxicology (Beijing, China). Chitosan (deacetylation degree 91.10%, MW4250 kDa) was a gift from Qingdao Scitech Co., Ltd. (Qingdao, China) Dichloromethane (DCM) and Hank’s balanced salt solution (HBSS) were obtained from Beijing Chemical Reagents Company (Beijing, China). Dulbecco’s Modified Eagle’s Medium (DMEM) and fetal bovine serum (FBS) were obtained from GIBCO, Invitrogen Corp. (Carlsbad, CA). Trypsin was obtained from Amresco, Inc., (Solon, OH) and 6-coumarin (COU, purity499%) and 3(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) were purchased from Sigma-Aldrich (Saint Louis, MO). All reagents used for high-performance liquid chromatography (HPLC) were of chromatographic purity. Other chemicals used were of analytical grade.
PLGA-NPs were prepared by a single emulsion (o/w) method commonly known as solvent evaporation technique (Perez et al., 2001). Briefly, 100 mg of PLGA and 10 mg of thienorphine (or 1 mg of 6-cumarin) were dissolved in DCM to obtain the organic phase. This organic solution was then added dropwise into 1% (w/v) PVA aqueous solution precooled at 4 C under magnetic stirring. The coarse emulsion was sonicated by ultrasonic probe (SCIENTZ-IID, Scientz Biotechnology Co., Ningbo, China) and then subjected to rotary evaporation to remove the organic solvent. Drugs that were un-encapsulated in the samples were removed by centrifugation (5000 rpm 5 min). The NPs in the supernatant were precipitated by centrifugation (15 000 rpm 40 min) and the precipitant was washed 5 times with distilled water by centrifugation following overnight freeze-drying. The NP suspensions was incubated (1:1, v/v) with chitosan solution (1 mg/mL) prepared by 1% (v/v) acetic acid for 10 min under agitation to prepare CS-PLGA-NPs. Morphology observations The morphology of thienorphine-PLGA-NPs or thienorphineCS-PLGA-NPs was observed using transmission electron microscopy (TEM, HITACHI, H-7650, Tokyo, Japan) and atomic force microscopy (AFM, NanoWizarc, JPK Ltd., Berlin, Germany). To prepare the samples for TEM, the NPs were suspended in water and stained with 1% uranyl acetate. To prepare the samples for AFM, the NPs were diluted with distilled water, followed by dropping the samples onto a silicon surface. The samples were air-dried at room temperature. Particles size and zeta potential of NPs The particle size (Z-average mean) and zeta potential of the NPs were measured by photon correlation spectroscopy and laser doppler anemometry, respectively, using a Zetasizer Nano Analyzer (ZS90, Malvern Instruments, London, UK). For size measurements, the PLGA-NPs or CS-PLGA-NPs suspensions was diluted in water and measured for a minimum of 180 s. Raw data were subsequently correlated to mean hydrodynamic size by cumulant analysis. For the measurement of zeta potential, samples were diluted in 0.1 mM KCl and measured in automatic mode. All measurements were performed in triplicate. Determination of encapsulation efficiency and loading efficiency After weighed amounts of dried thienorphine-PLGA-NPs or thienorphine-CS-PLGA-NPs were dissolved in DCM, a clear solution was obtained by vigorous vortex. The total amount of thienorphine in PLGA-NPs or CS-PLGA-NPs was estimated by HPLC analysis (1211, Agilent Technologies Inc., Avondale, PA). The chromatographic conditions were as follows: C18 column (250 mm 4.6 mm, 5 mm)
Thienorphine-loaded PLGA-NPs
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(Agilent Technologies Inc., Avondale, PA), a mixture of acetonitrile-methanol-0.02 mol l1 phosphate (40:15:45) buffer containing 0.2% triethylamine (pH ¼ 3) as eluant, detection at 220 nm. The polymers did not interfere with absorbance of the drug at the specified wavelength. Encapsulation efficiency (EE) was expressed as the ratio of thienorphine in PLGA-NPs or CS-PLGA-NPs to the initial amount of thienorphine used in the formulation. Loading efficiency (LE) corresponds to the ratio of amount of thienorphine entrapped in PLGA-NPs or CSPLGA-NPs recovered to the total amount of polymer and thienorphine. Each measurement was performed in triplicate. Release in vitro To determine the amount of drug released from the NPs, a dynamic dialysis technique was used. The thienorphine concentration in the aliquots was analyzed using the HPLC as described above. Two kinds of NPs, the thienorphineloaded common NPs and adhesive NPs, in which thienorphine was calculated as 0.5 mg, were placed in the dialysis bag (MWCO 10,000; Spectrum, Los Angeles, LA) containing 5 ml of phosphate buffer solution (PBS, 0.1M, pH 7.4). The dialysis bag was filled with the nanosuspensions and immersed in receiver solution (50 ml) identical to the inner solution and then incubated in a water-bath shaker at 37 C with constant orbital mixing (100 rpm). At specified time points (0.5, 1, 2, 3, 5, 7, 10 and 12 h) an aliquot (200 ml) of the receiver solution was removed and replaced with fresh medium. The thienorphine concentrations of the aliquots were determined by using HPLC, and the cumulative amount of thienorphine released from the NPs was calculated. The cumulative percentage of thienorphine released was plotted versus time. Each data point is calculated from three measurements. Residual DCM content Gas chromatography (HP 5890, USA) was used to determine the residual DCM in the thienorphine-PLGA-NPs or thienorphine-CS-PLGA-NPs. 1 ml N,N-dimethyl formamide of 0.05 ml of ethyl acetate was used as internal standard. Approximately 25 mg of NPs were dissolved in 1 ml of internal standard. GC conditions were as follows: DB-624 capillary column (30 m 0.25 mm 0.25 mm, HP, Palo Alto, CA); high purity nitrogen (99.99%) as carrier gas with flow rate of 30 mlmin1; injection temperature at 270 C, with FLD detector. Bioadhesion
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Bioadhesive studies Rats were fed 2 ml aqueous dispersions of the test formulations containing 0.5 mg thienorphine. The rats in group 1 were given free thienorphine, those in group 2 were given thienorphine-PLGA-NPs, and those in group 3 were given thienorphine-CS-PLGA-NPs. The animals were killed by cervical dislocation at 0.5, 1, 2, 3, 5 and 7 h after administration, three rats per time points. The abdominal cavity of each rat was opened and the small intestinal tract (including duodenum, jejunum, and ileum, to a fixed length of 20 cm) was removed. Then, the gut was rinsed with 40 ml of physiological saline in order to recover the non-adhered fraction. The rinse liquids were centrifuged at 25 000 g for 30 min, and thienorphine from the supernatants was quantified and termed ‘‘free thienorphine’’. The pellets were digested in 1 ml 3 mol/l NaOH for 24 h and centrifuged at 25 000 g for 30 min. The amounts of thienorphine in the supernatants were assayed by the HPLC and termed ‘‘unreleased thienorphine’’. The free and unreleased thienorphine levels were added together to form the total amount of thienorphine in the lumen in order to estimate the bioadhesive characteristics of the different formulations. In vitro cell assays Cell culture A Caco-2 cell line was obtained from the American Type Culture Collection (Rockville, MD). Cells were grown routinely on 75 cm2 plastic culture flasks (Corning, NY) in DMEM containing 25 mM D-glucose, 25 mM N-2-hydroxyethyl piperazine-N/-2-ethane sulfonate buffer (HEPES), 44 mM NaHCO3, supplemented with 10% (v/v) fetal bovine serum (FBS), 1% (v/v) non-essential amino acid solution, 1% (v/v) L-glutamine, 1% (v/v) penicillin-streptomycin at 37 C in an atmosphere of 5% CO2 and 90% relative humidity. The medium was replaced every 2 days after incubation. Cells were passaged, 1:5, approximately every 5 days (at 80–85% confluence) using 0.25% trypsin and 0.02% ethylenediaminetetraacetic acid (EDTA). Caco-2 cells of passage numbers 32–38 were used in these experiments. Cellular uptake To visualize the cellular uptake of PLGA-NPs or CS-PLGANPs labeled by 6-cumarin, 3 105 Caco-2 cells were grown on cover slips placed in 6-well plates. Cells were treated with the PLGA-NPs or CS-PLGA-NPs for different time intervals (0.5, 1 and 2 h), fixed in 4% paraformaldehyde (PFA) and finally examined under a confocal laser scanning microscopy (UltraVIEW Vox, PerkinElmer, Waltham, MA).
Animals Fifty-four Wistar rats (adult male, 220 ± 20 g, Beijing Institute of Pharmacology and Toxicology) were used for the bioadhesive study and they were randomly divided into three groups. All of them were fasted overnight, but had free access to water. All animal experiments were justified in detail and were approved by the Institutional Animal Care and Use Committee of Beijing Institute of Pharmacology and Toxicology.
Transport experiments For transport experiments, Caco-2 cells were cultivated on bicameral inserts TranswellÕ , Corning, Inc., New York following standards protocols (Garinot et al., 2007). On the day of the transport experiments, the cells were washed with HBSS for the apical and basolateral sides, respectively. The cell monolayers were subjected to transepithelial electrical resistance (TEER) determination and only cell monolayer
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with TEER higher than 450 /cm2 were used for the transport experiments. Solutions from the apical side of the monolayers were replaced by the different thienorphine formulations (1 mg/ml), and samples were collected from the basolateral side. Samples of 500 ml were taken from the acceptor chamber at predesigned time and replaced by 500 ml of fresh experimental culture medium. The thienorphine in samples was quantified by an HPLC. The apparent permeability coefficient (Papp) was calculated from the slope of the accumulated concentration versus time curve using the following relation: Papp ¼ dQ/dt/(AC0), where dQ is the amount of thienorphine in the basolateral side at time t, A is the diffusion area, and C0 is the initial concentration of thienorphine in the apical side. MTT assay NPs cytotoxicity in vitro was evaluated using MTT assay with Caco-2 cells, as described previously (Viholal et al., 2005). Briefly, Caco-2 cells were seeded on 96-well plates at a density of 10 000 cells per well and were cultured for 5 days. Then the culture medium was replaced with PLGA material, blank PLGA-NPs, or blank CS-PLGA-NPs, and all were diluted with culture medium to a concentration of 1.0, 5.0, and 25.0 mg/ml, respectively. After 2 h of incubation at 37 C, the PLGA polymer, blank PLGA-NPs and blank CS-PLGANPs were replaced with 100 ml of MTT (5 mg/ml in HBSS) solutions, and the cells were incubated for a further 4 h at 37 C. The test solution was decanted and the formazan crystals were dissolved in dimethyl sulfoxide (200 ml/well). Absorption was measured using a microplate reader (Tecan Deutschland GmbH, Crailsheim, Germany) at 590 nm. Cell viability was expressed as percentage of absorbance relative to control. Control cells were not exposed to the PLGA materials. Experiments were performed in triplicate, with eight replicate wells for each sample and control per assay. Statistical analysis Data are presented as the mean ± standard deviation (SD). The difference between any two groups was determined using ANOVA. p50.05 was considered statistically significant.
Results and discussion Characterization of NPs The particle size, zeta potential, EE and LE of the two kinds of NPs are listed in Table 1. The results show that the EE of CS-PLGA-NPs was 54.16 ± 3.18%, which was higher than PLGA-NPs, 43.65 ± 2.73%. The particle size of CS-PLGANPs was 121.1 ± 10 nm, slightly greater than that of PLGANPs (118.5 ± 13 nm), and there was no significant difference between them (p40.05). However, the zeta potentials of the two kinds of NPs were opposite to each other. PLGA-NPs has Table 1. Characteristics of PLGA-NPs and CS-PLGA-NPs (mean ± SD, n ¼ 3).
Type of NPs
Particle size (nm)
PLGA-NPs 118.5 ± 13 CS-PLGA-NPs 121.1 ± 10
Zeta potential (mV) 2.01 ± 0.3 +42.1 ± 0.4
EE (%)
LE (%)
43.65 ± 2.73 1.24 ± 0.46 54.16 ± 3.18 1.59 ± 0.39
a negative charge (2.01 mV) due to the nature characteristics of PLGA polymer and CS-PLGA-NPs has a positive charge (+42.1 mV) due to chitosan coated on the surface. Positively charged particles have a higher potency for interaction with biological membranes than negatively charged particles. This is because the gastrointestinal tract is made up of smooth muscle cells and its surface charge is about 50 mV (He et al., 2013). Interactions between positively charged NPs and negatively charged cells will lead to alterations in the permeability and ease of transport through electrostatic attraction. As shown in Figure 6, the difference in zeta potential from the negatively charged PLGA-NPs to positively charged CS-PLGA-NPs did cause a significant increase in cell uptake. This showed that particle surface charge is a vital factor in manipulating the process of particle entry into the body. This is in agreement with the data presented in existing literature (Chung et al., 2010). The TEM image of PLGA-NPs or CS-PLGA-NPs is illustrated in Figure 2(A) and (B). The overall morphological appearance for the two types of NPs was observed to be similar. The particle size range and the observed spheroidal morphology did not change significantly upon chitosan coating, suggesting the formation of a polymer layer on PLGA-NPs that was too thin to be detected in this analysis. However, the AFM images of the two kinds of NPs are depicted in Figure 2(C) and (D). Using the AFM technique, we clearly visualized the three-dimensional group of NPs. The particle surface of PLGA-NPs was relatively smooth, while that of the CS-PLGA-NPs had small globular protuberances. Release in vitro Figure 3 shows the release profiles of thienorphine from CS-PLGA-NPs and PLGA-NPs at PBS (pH 7.4). It is here apparent that thienorphine release in vitro underwent a very rapid initial burst (23.84 ± 1.43%), suggesting that some thienorphine was localized on the surface of PLGA-NPs. During the coating process in the preparation of CS-PLGANPs, in which negative PLGA-NPs is coated with positively charged chitosan, thienorphine absorbed on the surface of PLGA-NPs can be loaded into CS-PLGA-NPs. As a result, the burst effect is less pronounced (14.29 ± 1.24%) for CS-PLGANPs than for pure PLGA-NPs (23.84 ± 1.43%). The size of the decrease in burst release suggests that the bulk of the thienorphine is encapsulated inside the matrix. In this way, the EE of CS-PLGA-NPs can be said to be higher than that of PLGA-NPs. As shown in Figure 3, the effect of chitosan on the release behavior in vitro is most significant before 5 h, showing a significant difference between CS-PLGA-NPs and PLGA-NPs (p50.05), but the effect become less dramatic over time. There was no remarkable difference between the two kinds of NPs at 12 h (p40.05). Residual DCM contents The residual DCM contents in all thienorphine loaded CS-PLGA-NP and PLGA-NP were below 600 ppm, which was in accord with the requirements of the ICH standard (International Conference on Harmonization, 2005).
Thienorphine-loaded PLGA-NPs
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Figure 2. Morphological appearance of NPs observed using TEM (A: PLGA-NPs; B: CS-PLGA-NPs) and AFM (C: PLGA-NPs; D: CS-PLGA-NPs).
Figure 3. Drug release from PLGA-NPs and CS-PLGA-NPs in vitro (mean ± SD, n ¼ 3).
Figure 4. amount of thienorphine in the lumen content of the gastrointestinal tract after oral administration of 2 ml solution or NPs dispersion containing 0.5 mg thienorphine. Each experiment was performed in triplicate.
Bioadhesiveness Besides particle size and zeta potential, mucosal adhesion is the most important characteristic of NP drug delivery systems. Prolonging the duration of drug retention on the mucosa may result in a higher bioavailability. Chitosan is a linear polycationic macromolecule linked by b(1,4)-glycoside bonds. Its mucoadhesive properties are thought to be mediated by electrostatic interactions between the positively charged D-glucosamine residue and the negatively charged sialic acid residue of mucin. In this study, Figure 4 shows changes in total thienorphine in the lumen content over time. For free thienorphine PBS solution, it was found that the amounts of thienorphine decreased rapidly to about 20% of the total dose in about 7 h. It may be possible that the encapsulation into NPs slowed the elimination of
thienorphine from the gastrointestinal tract, thus causing a statistically significant difference between free thienorphine solution and the two kinds of NP formulations (p50.05). Adhesive CS-PLGA-NPs slowed this process further. The amounts of remaining thienorphine in CS-PLGA-NPs and PLGA-NPs were 43.8% and 58.1%, respectively, at 7 h. Cellular uptake In order to visualize the cellular uptake of NPs, the high fluorescent 6-coumarin has proved to be useful in confocal microscopy studies because of its low loading requirement. The time course of 6-coumarin-PLGA-NPs and 6-coumarinCS-PLGA-NPs internalization is shown in Figure 5. At the same time point, the uptake of CS-PLGA-NPs was much
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Figure 5. Fluorescent images of intracellular uptake of 6-coumarin-PLGA-NPs and 6-coumarin-CS-PLGA-NPs.
Figure 6. Effect of NPs on the permeability of thienorphine (mean ± SD, n ¼ 3). Figure 7. MTT assay evaluation of cytotoxicity after 2 h of incubation with PLGA polymer, blank PLGA-NPs, and blank CS-PLGA-NPs.
higher than that of PLGA-NPs. This difference could be a direct consequence of surface modifications, because chitosan coating changes the negative surface charge to a positive one. It has been reported that the internalization of cationic chitosan particles appears to occur predominantly by adsorptive endocytosis, suggesting that the positively charged particle surface can facilitate adherence to the negatively charged cell membrane. Transport experiments Figure 6 shows that cumulative amount of thienorphine transported from the apical side to the basolateral side of the Caco-2 monolavers incubated with thienorphine, thienorphine-PLGA-NPs and thienorphine-CS-PLGA-NPs. The Papp value of thienorphine-PLGA-NPs was 2.16 ± 0.26 cm/ s 107, which was significantly higher by 2.2 folds as compared to that of free thienorphine (0.97 ± 0.08 cm/ s 107). On the other hand, loading of thienorphine into CS-PLGA-NPs resulted in a Papp value of
4.38 ± 0.31 cm/s 107 with 4.5 folds improvement in thienorphine permeation. It could be related to the higher positive charge which is a critical factor for penetration enhancement towards cells. MTT assay The toxicity of NP drug delivery systems has been a prominent concern over the past 10 years (Nahar et al., 2008; Jain et al., 2011). Related studies have shown that NPs enhance therapeutic effects but can also increase toxicity. This study mainly concerns the cytotoxicity caused by the residual solvent and uses MTT to evaluate the cell survival rate. The cytotoxicities of CS-PLGA-NPs, PLGA-NPs and PLGA polymer were evaluated after incubation with the aforementioned cells for 2 h. As shown in Figure 7, cell viability did not decrease even after 2 h of incubation at 37 C while polymer/NP concentration was increased from
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1 to 25 mg/ml. Compared with the PLGA polymer group, the viability values of the Caco-2 cells exposed to the two NP formulations, blank PLGA and blank PLGA-NP, showed no significant differences (p40.05). The results indicated that the residue of organic solvent induced in the process of preparing NPs was thin, so the formulations showed no particular toxicity and did not influence the normal growth of Caco-2 cells within this dose range (25 mg/ml). And compared with negatively charged PLGA-NPs, the positive properties of CS-PLGA-NPs did not increase the cytotoxicity to Caco-2 cells after 2 h of incubation at 37 C. The main reason for this is that chitosan is biocompatible, biodegradable, and has low cell toxicity. This is why it has been widely applied in tissue engineering, gene therapy, drug delivery and other fields (Jayakumar et al., 2010).
Conclusions In this study, bioadhesive NPs containing thienorphine have been prepared. In vitro studies show that the NPs have attractive properties, such as a positive charge, mucosal adhesion and absorption promotion, which can prolong the duration of residence of thienorphine for oral delivery. CS-PLGA-NPs were found to significantly enhance cellular uptake compared with PLGA-NPs on Caco-2 cells. MTT test reveals that CS-PLGA-NPs did not increase cytotoxicity; both CS-PLGA-NPs and PLGA-NPs were well tolerated at concentrations below 25.0 mg/ml in Caco-2 cell cultures. In addition, the cellular transport of thienorphine showed higher permeation enhancing profiles of CS-PLGA-NPs, as compared with thienorphine soluton or PLGA-NPs. Compared with negatively charged PLGA-NPs, the positively charged CS-PLGA-NPs delivery system may bring us closer to a safe, general system for oral administration of thienorphine. Also, we are currently evaluating the bioavailability of thienorphine-CS-PLGA-NPs or thienorphine-PLGA-NPs in beagle dogs.
Declaration of interest We are grateful to the financial support from the National Natural Science Foundation of China (Grant No. 81202466) and the Important National Science & Technology Specific Projects (Grant No. 2012ZX09301003-001-009) of China.
References Johnson RE, Strain EC, Amass L. (2003). Buprenorphine: how to use it right. Drug Alcohol Depend 70:S59–77. Fudala PJ, Yu E, Macfadden W, et al. (1998). Effects of buprenorphine and naloxone in morphine-stabilized opioid addicts. Drug Alcohol Depend 50:1–8.
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Liu CH, Liu H, Han XY, et al. (2005). Synthesis and characterization of thienorphine and its glucuronide conjugate. Synth Commun 35: 701–10. De Jong WH, Borm PJ. (2008). Drug delivery and nanoparicles: applications and hazards. Int J Nanomed 3:133–49. Hulse GK, Stalenberg V, McCallum D, et al. (2005). Histological changes over time around the site of sustained release naltrexonepoly(DL-lactide) implants in humans. J Control Release 108:43–55. 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. Shi K, Cui F, Yamamoto H, Kawashima Y. (2009). Optimized formulation of high-payload PLGA nanoparicles containing insulinlauryl sulfate complex. Drug Dev Ind Pharm 35:177–84. He B, Lin P, Jia ZR, et al. (2013). The transport mechanisms of polymer nanoparticles in Caco-2 epithelial cells. Biomaterials 34:6082–98. Thirawong N, Nunthanid J, Puttipipatkhachorn Satit, et al. (2007). Mucoadhesive properties of various pectins on gastrointestinal mucosa: an in vitro evaluation using texture analyzer. Eur J Pharm Biopharm 67:132–40. He B, Jia ZR, Du WW, et al. (2013). The transport pathways of polymer nanoparticles in MDCK epithelial cells. Biomaterials 34:4309–26. Merwe SM, Verhoef JC, Kotze´ AF, et al. (2004). N-Trimethyl chitosan chloride as absorption ehancer in oral peptide drug delivery development and characterization of minitablet and granule formulations. Eur J Pharm Biopharm 57:85–91. Guo XH, Zhang XX, Ye L, et al. (2014). Inhalable microspheres embedding chitosan-coated PLGA nanoparticles for 2-methoxyestradiol. J Drug Target published online:1–7. Zhang XY, Sun MZ, Zheng AP, et al. (2012). Preparation and characterization of insulin-loaded bioadhesive PLGA nanoparticles for oral administration. Eur J Pharm Sci 45:632–8. Perez C, Sanchez A, Putnam D, et al. (2001). Poly (lactic acid)-poly (ethylene glycol) nanoparticles as new carriers for the delivery of plasmid DNA. J Control Release 75:211–24. Garinot M, Fievez V, Pourcelle V, et al. (2007). PEGylated PLGA-based nanoparticles targeting M cells for oral vaccination. J Control Release 120:195–204. Viholal H, Laukkanen A, Valtola L, et al. (2005). Cytotoxicity of thermosensitive polymers poly (N-isopropylacrylamide), poly(N-vinylcaprolactam) and amphiphilically modified poly(Nvinylcaprolactam). Biomaterials 26:3055–64. Chung YL, Kim JC, Kim YH, et al. (2010). The effect of surface functionalization of PLGA nanoparticles by heparin- or chitosanconjugated pluronic on tumor targeting. J Control Release 143: 374–82. International Conference on Harmonization (ICH) harmonized tripartite guideline. (2005). Impurities: guideline for residual solvents, Q3C (R3). In International Conference on Harmonisation harmonised tripartite guideline Current Step 4 version. Available at:http:// www.ich.org/LOB/media/MEDIA423.pdf. Nahar M, Mishra D, Dubey V, et al. (2008). Development, characterization, and toxicity evaluation of amphotericin B-loaded gelatin nanoparticles. Nanomed Nanotech Bio Med 4:252–62. Jain AK, Swarnakar NK, Godugu C, et al. (2011). The effect of the oral administration of polymeric nanoparticles on the efficacy and toxicity of tamoxifen. Biomaterials 32:503–15. Jayakumar R, Chennazhi KP, Muzzarelli RAA, et al. (2010). Chitosan conjugated DNA nanoparticles in gene therapy. Carbohyd Polym 79: 1–8.
