http://informahealthcare.com/drd ISSN: 1071-7544 (print), 1521-0464 (electronic) Drug Deliv, Early Online: 1–8 ! 2014 Informa Healthcare USA, Inc. DOI: 10.3109/10717544.2014.910566
RESEARCH ARTICLE
Preparation of surface multiple-coated polylactide acid drug-loaded nanoparticles for intranasal delivery and evaluation on its brain-targeting efficiency Drug Delivery Downloaded from informahealthcare.com by Memorial University of Newfoundland on 06/04/14 For personal use only.
Junjie Bian1*, Zhixiang Yuan2*, Xiaoliang Chen1, Yuan Gao1, Chaoqun Xu2, and Jianyou Shi3 1
College of Pharmacy, Chengdu University of Traditional Chinese Medicine, Chengdu, Sichuan, China, 2Institute of Pharmacy, Sichuan Academy of Chinese Medicine Sciences, Chengdu, Sichuan, China, and 3Sichuan Academy of Medical Sciences, Sichuan Provincial People’s Hospital, Chengdu, Sichuan, China Abstract
Keywords
Purpose: To prepare a mixture of multiple-coated aniracetam nasal polylactic-acid nanoparticles (M-C-PLA-NP) and evaluate its stability preliminarily in vitro and its brain-targeting efficiency in vivo. Methods: The solvent diffusion–evaporation combined with magnetic stirring method has been chosen for the entrapment of aniracetam. The M-C-PLA-NP was characterized with respect to its morphology, particle size, size distribution and aniracetam entrapment efficiency. The in vivo distribution was studied in male SD rats after an intranasal administration. Results: In vitro release of M-C-PLA-NP showed two components with an initial rapid release due to the surface-associated drug and followed by a slower exponential release of aniracetam, which was dissolved in the core. The AUC0!30 min of M-C-PLA-NP in brain tissues resulted in a 5.19-fold increase compared with aniracetam solution. The ratios of AUC in brain to that in other tissues obtained after nasal application of M-C-PLA-NP were significantly higher than those of aniracetam solution. Conclusion: Therefore, it can be concluded that M-C-PLA-NP demonstrated its potential on increasing the brain-targeting efficiency of drugs and will be used as novel brain-targeting agent for nasal drug delivery.
Aniracetam, brain-targeting, intranasal delivery, surface multiple-coated nanoparticles
Introduction The brain-targeting technology can be divided into three categories. The first category is the invasive injury administration method, such as hypertonic shock, carotid injection of vasoactive substances and direct intraventricular injection. Although these methods are effective, they can cause the infection of the brain, the blood–brain barrier (BBB) damage and surgical injury. The second category method is to increase the ability that the drug through the BBB?including lipidization and chemical delivery systems. These methods have stringent requirements on physical and chemical properties of the drug. Therefore, they have lots of limitations. The third category is using the intranasal delivery system by-pass the BBB to achieve the brain-targeting. Intranasal administration with the characteristics of high bioavailability, rapid absorption, non-invasive and rapid-onset, etc., has become one of the hottest areas in pharmaceutical studies. *These authors contributed equally to this study. Address for correspondence: Chaoqun Xu, Institute of Pharmacy, Sichuan Academy of Chinese Medicine Sciences, Chengdu, Sichuan, China. Tel: +86-28-85213973. E-mail: [email protected]; Jianyou Shi, Sichuan Academy of Medical Sciences, Sichuan Provincial People’s Hospital, Chengdu, Sichuan, China. Tel: +86-028-8739-3234. E-mail: [email protected]
History Received 20 February 2014 Revised 28 March 2014 Accepted 28 March 2014
The human olfactory system is linked to a channel in the brain and the surrounding environment (Graff & Pollack, 2005). After nasal administration, the drug can directly through the olfactory region to reach the cerebrospinal fluid or even the brain. Modern studies have shown that the high degree of bioavailability of the certain drugs nasal delivery, and can even be comparable to the injection, but most of the drug after intranasal administration will be absorbed through the nasal mucosa into the systemic circulation, thus can be engulfed by the body’s reticuloendothelial system. In order to avoid the reticuloendothelial system engulfed, and improve the concentration of drug in the brain, a long cycle modification with PEG-20000 for M-C-PLA-NP is necessary, so the distribution of M-C-PLA-NP in other tissues such as liver and spleen which are distributed richly of the reticuloendothelial system is greatly reduced. Pharmaceutical experiments certificate that the brain–targeting efficiency greatly improved if the M-C-PLA-NP was coated with Tween-80. Studies have shown that Tween-80 have been adsorbed by M-C-PLA-NP surface, which can inhibit P-glycoprotein in brain capillary endothelial cell membrane’s drug efflux function (Alpesh et al., 2009). It has been reported that the PLA-NP under the action of the nasal enzymes and nasal mucosa will lead to aggregation, clearance and degradation (Tobı´o et al., 2000). Some studies also indicate that
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M-C-PLA-NP surface coated with chitosan hydrochloride, the positively charged nature of chitosan with powerful biological adhesion, can increase the stability of PLA-NP and extend its nasal residence time, enhance the absorption of M-C-PLA-NP through the membrane (Calvo et al., 1997). The borneol/ mentholum eutectic mixture reported in the literature shows strong penetration, and can improve the drug concentration in the brain. It has demonstrated that the speed and extent through the nose into the brain were greatly increased after M-C-PLA-NP coated by borneol/mentholum eutectic mixture (Chai et al., 2009). Based on the previous studies, we designed the aniracetam nasal M-C-PLA-NP for braintargeting studies. Aniracetam is a lactam drug, which can be dissolved in organic solvent such as acetone and not soluble in water. It can improve brain function and enhance brain metabolism. Aniracetam has been used for treatment of senile cognitive disorders and intellectual and memory impairment. Orally administered aniracetam has been used in clinical trials at daily doses of 1000–1500 mg for the treatment of cognitive impairment in patients with SDAT, and at doses of 600–1500 mg for the treatment of various symptoms including memory disorders in elderly patients with cerebrovascular disease (Lee & Benfield, 1994). Currently, the capsule, tablet and granule of aniracetam are the most common pharmaceutical formulations on the market. However, it is very rapidly eliminated from the body and plasma elimination half-time is 30 min. The absolute systemic bioavailability is only about 0.2% (Roncari, 1993). What is more, these kind of aniracetam formulations show non-specific distribution after oral administration. Therefore, the aniracetam pharmaceutical formulations were limited to an important role for the treatment of brain diseases. In this study, the aniracetam has been chosen as the model drug and the solvent diffusion–evaporation combined with magnetic stirring method has been chosen to prepare M-C-PLA-NP as a novel brain-targeting agent for nasal drug delivery, aim of increasing aniracetam brain-targeting efficiency for the treatment of brain diseases. The cumulative release characteristic, stability in vitro, tissue distribution of M-C-PLA-NP in rats are evaluated and the brain-targeting efficiency is preliminary evaluated by quantitative analytic mean.
Methods Materials Polylactic acid (PLA, MW ¼ 15 kDa) were purchased from Shandong Key Laboratory of Medical Polymer Materials (Shandong, China). Chitosan hydrochloride (degree of deacetylation, 80–90%, viscosity, 30 mPa s) and Lysozyme were obtained from Shanghai KABO Trading Company (Shanghai, China). Aniracetam (purity 99.5%), Betamethasone and Poloxamer 188(F-68, MW ¼ 8350 ± 1000) were supplied by Sigma Chemical Company. Polyethylene Glycol (PEG, MW ¼ 20 kDa), Oleum Menthae Dementhola Tum, Borneol and Tween-80 were from Chengdu Kelong Chemical Company (Chengdu, China). Other chemical reagents and solvents were of analytical or spectroscopic grade.
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Sprague–Dawley rats (7–8 weeks of age, body weight 210– 230 g) were obtained from the Experimental Animal Center of Sichuan Academy of Chinese Medicine Sciences. These animals were allowed to acclimatize in environmentally controlled quarters (24 ± 1 C and 12:12 h light–dark cycle) for at least 5 days before being used for experiments. All procedures of the animal studies were approved by animal ethical experimentation committee, according to the requirements of the National Act on the use of experimental animals (People’s Republic of China). Preparation of M-C-PLA-NP and PLA-NP M-C-PLA-NPs were prepared using the solvent diffusion– evaporation combined with magnetic stirring method. Different formulation variables such as the ratio of the drug to PLA (3:10, 1:5, 2:15 and 1:10 ), concentration of poloxamer 188 as stabilizer (0.5, 1, 2 and 3% w/v), the aqueous phase volume (10, 15, 20 and 25 ml), nanoparticle solution volume after rotary evaporated (7, 5, 4 and 3 ml), ethanol:acetone ratio (3:2, 1:1, 2:3 and 1:2) were varied, and the effects on particle size, zeta potential and entrapment efficiency were evaluated according to the previous study (Li et al., 2001). Only one parameter was changed at a time in each set of experiments. The optimized formulation was prepared by dissolving PLA (150 mg), aniracetam (20 mg, aniracetam was not added in the preparation of PLA-NP) and borneol/mentholum eutectic mixture in 20 ml of ethanol/ acetone (3:2; Chai et al., 2009). This organic phase was added at the rate of 1 ml/min to 20 ml of 1% w/v poloxamer 188 and 100 mg PEG20000 solution with continuous stirring on a magnetic stirrer at room temperature (Patil et al., 2009). After the organic phase was added completely, vacuum rotary evaporating was continued to allow complete evaporation of the organic solvent. Then, 100 mg Tween-80 was added to the nanoparticle solution, and continued rotary evaporation until the volume of the suspension was less than 3 ml. And, the nanoparticles were collected by centrifugation and washed thrice with ultrapure water. After that, the collected nanoparticles re-dispersed in 1% F-68 solution and 5 mg of chitosan hydrochloride was added into the suspension under magnetic stirring. Finally, nanoparticle suspension was concentrated at 30 C until the volume of the nanoparticle suspension is less than 3 ml (Li et al., 2008). In this way, M-C-PLA-NPs were obtained. PLA-NPs were prepared without coating process. Drug loading and entrapment efficiency Ultrafiltration method was used to determine drug loading and entrapment efficiency of M-C-PLA-NP (Govender et al., 1999). M-C-PLA-NP suspension was taken into the Millipore ultrafiltration tube (Mw ¼ 10000) and centrifuged at 3500 rpm for 35 min. Then the filtered liquid was collected and the amount of the free drug in the water phase was determined by HPLC. The total amount of the drug in the suspension was extracted ultrasonic and diluted with ethanol, which was also detected by HPLC system. The amount of drug inside M-C-PLA-NP was calculated by subtracting the free drug in the water phase of the suspension from the total
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DOI: 10.3109/10717544.2014.910566
amount of the drug in the suspension. The entrapment efficiency (EE) and drug loading (DL) of drug were calculated by the following equation: EEð%Þ ¼ ðW1 W2 Þ=W1 100%
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DLð%Þ ¼ ðW1 W2 Þ=W3 100% where W1 is the total amount of the drug in the suspension, W2 is the amount of the free drug in the water phase and W3 is the total amount of the drug plus the amount of PLA. HPLC condition was as follows: with a reversed-phase column, Welchrom-C18 reversed-phase column (150 4.6 mm, 5 mm), the separations were achieved using methanol and water at the ratio of 40:60 as the mobile phase with flow rate of 1 ml/min and column temperature of 30 C. The detection wavelength was at 284 nm. Particle size analysis and zeta potential The size analysis, polydispersity index and zeta potential distribution of the M-C-PLA-NP were determined using a Malvern Zetasizer Nano ZS90 (Malvern Instruments, UK; Seju et al., 2011). Each sample was suitably dispersed with distilled water and placed in a disposable sizing cuvette. The polydispersity index was studied to determine the narrowness of the size distribution. The size analysis of a sample consisted of three measurements, and the results were expressed as mean size ± SD. In order to determine the zeta potential distribution, each sample was suitably diluted five times with filtered distilled water and placed in a disposable zeta cell. The average of three measurements of each sample was used to derive the average zeta potential. Nanoparticle morphology Morphology of the M-C-PLA-NP was visualized with scanning electron microscopy (SEM). About 50 ml of 0.1% (w/v) particle suspension was air dried overnight on a metal stub. Particles were gold/palladium sputtered using a sputter coater device and analyzed with scanning electron microscope (Hitachi, S4800; Bram et al., 2010). Stability and cumulative release of M-C-PLA-NP To determine the stability of the M-C-PLA-NP, lysozyme (1 mg) was added into the prepared M-C-PLA-NP suspension (1 ml). The M-C-PLA-NP were incubated at 37 C for 2 h under continuous stirring, then the changes in particle size were compared before and after incubation (Zhang et al., 2006). In vitro release of M-C-PLA-NP Cumulative release characteristics were also observed. A certain amount volume of M-C-PLA-NP suspension transferred to dialysis bags (MWCO: 3500 Da) placed in 100 ml of PBS (pH 4.4 or pH 7.4) with stirring at 37 C. At appropriate time points (0, 5 min, 30 min, 1 h, 2 h, 4 h, 8 h, 12 h and 24 h), a certain amount of the environmental buffer solution (1 ml) was replaced with fresh buffer solution, and the concentration of the released aniracetam in the removed buffer solution was determined using HPLC.
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Evaluation of brain uptake following intranasal administration Animal experiment Forty-two SD rats were divided into two groups: control group and nanoparticle group. After weighing, the rats were anesthetized by intraperitoneal injected of 7% chloral in accordance with the 0.5 ml/100 g standard (Zhang et al., 2006). Then, the rat was fixed on the surgical board to a supine posture, cut neck, isolated trachea and esophagus. An incision was made by the surgical scissor in the rat tracheal, a size suitable hose was inserted for breathing and at the same time the esophagus was ligated to prevent drug into the stomach. The front part of a micro-injector with hose, which is used to unilateral intranasal administrated of M-C-PLA-NP. The preparations were administered at the openings of the nostrils using a polyethylene 10 (PE 10) tube attached to a microlitre syringe. The procedure was performed gently and slowly, which lasted for about 2 min, allowing the rats to inhale all of the preparations (Gao et al., 2006). Rats in nanoparticle group were administrated with M-C-PLA-NP (50 ml, containing 6.667 mg/ml aniracetam); rats in control group were received with 50 ml aniracetam solution (equal to the dose of M-C-PLA-NP; Jing, 1994). At each of the time points (1, 3, 5, 7.5, 10, 15 and 30 min) following administration, three animals were sacrificed by bloodletting from the cervical artery. And, then blood was collected and put into the tubes with heparin. Finally, the skulls were cut open and brain tissue and other tissue samples excised. All the samples were stored at 40 C until assay. Analytical procedures To prepare the samples for analysis, the tissue samples were homogenized with 1-fold volume of distilled water and the homogenate (1 ml) was taken into EP tube. Then 5 ml of betamethasone (0.1 mg/ml, internal standard) and 3 ml of ethyl acetate were added to 1 ml of the homogenate to extract the aniracetam and betamethasone from the samples (Xu et al., 2007). Intensely vortexed for 5 min, the mixture was subjected to centrifuge at 15 000 g for 10 min. The supernatant was collected and evaporated under a stream of nitrogen (Zhang et al., 2006). The residue was dissolved in 200 ml of methanol, and 20 ml was injected into the HPLC system. The whole blood sample was subjected to centrifuge at 3500 g for 15 min. The plasma was collected without the addition of water and treated in the same manner. The uptake of aniracetam associated to the nanoparticles by brain tissues and the circulation were analyzed by the HPLC system (Agilent 1200). With a reversed-phase column, Welchrom-C18 reversed-phase column (150 4.6 mm, 5 mm), the separations were achieved using methanol and water at the ratio of 40:60 (0 min)–40:60 (10 min)–60:40 (20 min) as the mobile phase with flow rate of 1 ml/min and column temperature of 30 C. The mixture was monitored with UV detection at 240 nm. Data analysis All data were dose-normalized and plotted as drug concentration–time curves in the plasma, brain and other tissues.
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The Cmax values were read directly from the concentrationtime profile and the area under the concentration–time curve (AUC0!t) was calculated by the DAS software (published by the Mathematical Pharmacology Professional Committee of China). The brain/plasma and other tissues AUC0!t ratios for aniracetam carried by M-C-PLA-NP and aniracetam suspension were calculated to evaluate the drug-targeting efficiency (DTE). The three parameters of targeting efficiency (Te), concentration ratio efficiency (Ce) and relative targeting efficiency (Re) were selected to determine the brain-targeting efficiency of the M-C-PLA-NP. Drug target tissues can be judged by the two parameters of Te and Re. The parameter of Ce was used to judge M-C-PLA-NP and aniracetam solution-targeting efficiency. The equations are as follows. Te ¼ ðAUCÞT =ðAUCÞNT Ce ¼ ðCmax Þp =ðCmax Þs Re ¼ ðAUCÞp =ðAUCÞs where T is the mean of target tissue, NT is the mean of nontarget tissue, p is the mean of targeting drug delivery system, S is the mean of non-targeting drug delivery system.
Results and discussion Determination of aniracetam Quantitative analysis of aniracetam was done by HPLC. In a different concentration of the sample, the regression equation was Y ¼ 14.639X + 15.764 (r ¼ 0.9989) in the linear range of 2.52–126 mgml1 after linear regression of its peak area. Figure 1. Scheme of preparing M-C-PLANPs coated with various kinds of materials.
Under the above conditions, the within-day precision and between-day precision is of less than 2%. Amount of aniracetam solution (including high, medium and low concentrations of aniracetam solution) were taken into blank M-C-PLA-NP suspension into the Millipore ultrafiltration tube (Mw ¼ 10 000). After that, the nanoparticles suspension was centrifuged at 3500 rpm for 35 min. Then, the aniracetam content in filtered fluid was determined by HPLC and the recovery result of aniracetam solution in blank M-C-PLA-NP was more than 95% (RSD52.0%). Physicochemical characterization and effect of formulation variables Nanoparticles and liposomes are wildly used in drugtargeting system, especially in brain drug delivery. Nanoparticles show many advantages in brain-targeting systems, such as good stability in cavity and smaller size. The chitosan coating nanoparticles has some important properties such as biocompatibility, non-toxicity and mucoadhesivity which render it an interesting biomaterial (Lehr et al., 1992). From a physicochemical point of view, chitosan has the special quality of gelling upon contact with anions (Calvo et al., 1997). There are several research papers showed nanoparticles and chitosan based nanoparticles developed for brain-targeting systems (Olivier, 2005; Wang et al., 2008; Al-Ghananeem et al., 2010). In our experiment, chitosan plays an important role in prolonging the residence time of nanoparticles in nasal cavity due to its positive charge and biological adhesiveness, with PEG20000 avoiding the reticuloendothelial system engulfed, Tween-80 enhancing the brain-targeting efficiency, borneol and mentholum eutectic opening the blood-brain barrier
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DOI: 10.3109/10717544.2014.910566
(Figure 1). Thus, it can add the absorption of drugs in nasal mucosa and increase the dose transferring to brain. After preliminary study, the ratio of ethanol to acetone (2:3) was selected as the organic solvent, as it gave maximum entrapment efficiency and minimum particle size. poloxamer 188 was selected as the stabilizer. Poloxamer 188 is a swellable, hydrophilic polymer and therefore the higher amount of residual poloxamer 188 at the surface of nanoparticles would account for their hydrophilicity. The prepared nanoparticles aggregation was observed without stabilizer. Poloxamer 188 was added ranging from 0.5% to 3% (w/v). The entrapment efficiency of nanoparticle increased from 79.54 ± 6.83% to 88.50 ± 5.70%. Especially, the entrapment efficiency reached peak value of 92.56 ± 8.15% with 1.0% poloxamer 188. When poloxamer 188 with higher concentration (2% w/v) was used, the entrapment efficiency decreased and particle size became larger. This phenomenon can be better understood in terms of an increase in solubility of the drug in aqueous medium. An optimum concentration of stabilizer led to a reduced size of M-C-PLA-NP, as the insufficient amount of stabilizer was unable to cover the dispersed M-C-PLA-NP completely, failing to stabilize and causing aggregation, leading to larger M-C-PLA-NP (Feng & Huang, 2001). Thus, 1.0% poloxamer 188 was considered optimum. To clarify the influence of amount ratio of drug to polymer on the nanoparticles, different ratios of drug to polymer were studied. With the increased ratio from 1:10 to 3:10, however, the drug entrapment of M-C-PLA-NP was decreased and the particle size got bigger, which was consistent with previous reports (Budhian et al., 2007; Shah et al., 2009). An increase in the polymer concentration led to an increase in the viscosity of the organic phase, which resulted in rapid diffusion of the organic phase into the aqueous phase. Coarse dispersions were obtained at higher polymer concentrations, possibly due to the insufficient amount of stabilizer presented in the aqueous phase for that particular polymer amount, which led to bigger particle size. The influence of volume of aqueous phase on the size and entrapment of the M-C-PLA-NP was also observed in the study. Using the volume of aqueous phase from 10 to 25 ml, the entrapment efficiency was improved from 64.97 ± 2.49% to 90.23 ± 2.01%. The particle size was also found to decrease from 342.6 ± 12.45 to 149.3 ± 7.8 nm. Further increase in volume of the aqueous phase induced the lower entrapment efficiency (575%) of M-C-PLA-NP. The reason may be that the polymer rapidly dispersed to large volume of aqueous phase to form smaller particles in comparison with low volume of aqueous phase. Hence, the optimal volume of aqueous phase was 25 ml. The entrapment efficiency was also found to decrease with increasing the volume of nanoparticles after rotary evaporation. This may be due to the lower volume of water, the drug which is rapidly mixes with the PLA, thus producing high entrapment efficiency after rotary evaporated (3 ml; Muthu et al., 2009). When the volume ratio of ethanol to acetone was increased from 1:1 and 2:3, the obtained entrapment efficiency was increased from 87.63 ± 2.33% to 95.31 ± 2.85%, without obvious change in particle size (145 nm). Further increasing the ratio of acetone produced
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the entrapment of585% and particle size4150 nm of the M-C-PLA-NP. Therefore, the volume ratio of ethanol to acetone (2:3) was adopted. Finally, using optimized parameters mentioned above, the prepared M-C-PLA-NP showed the entrapment efficiency of 96.40 ± 1.77% and drug loading of 11.57 ± 0.21%, with mean diameter of 145.6 ± 9.54 nm. Characterization, particle size and zeta potential of M-C-PLA-NP Nanoparticles were gold sputtered using a sputter coater device and analyzed with scanning electron microscope (Hitachi, S4800; Bram et al., 2010). Figure 2 shows
Figure 2. Scanning electron micrographs of M-C-PLA-NP (105 magnification, A); Size distribution chart of M-C-PLA-NP (B) and Zeta potential distribution chart of M-C-PLA-NP (C).
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Stability of M-C-PLA-NP The conformational stability of M-C-PLA-NP in the nasal cavity environment is of great importance from the pharmaceutical point of view. In this study, lysozyme was incubated with the prepared M-C-PLA-NP suspension for 2 h to determine the stability of the M-C-PLA-NP (Figure 3A). Size distribution chart of multiple M-C-PLA-NP after lysozyme incubated for 2 h (Figure 3B). The particle size of the M-C-PLA-NP after incubated was 149.1 ± 7.2 nm with polydispersity index of 0.082 ± 0.012, which had no obvious changes compared with the M-C-PLA-NP before the incubation. The results demonstrated that the M-C-PLA-NP was thus expected to be stable in the nasal cavity environment. In vitro release of M-C-PLA-NP The cumulative release of aniracetam was calculated as a function of time as shown in Figure 4. The drug release from M-C-PLA-NP followed the biphasic model, with an initial
burst attributed to the drug-associated near-particle surface and sustained release. Within 4 h, about 70% aniracetam released in buffer solution, and then, the release of M-C-PLANP reached the platform. After 24 h, the percentage of the accumulated release was 80–85%. Thus, it was clear that incorporation in M-C-PLA-NP could significantly sustain the release of aniracetam. Evaluation of brain uptake following intranasal administration To evaluate the drug brain-targeting efficiency of the M-C-PLA-NP after intranasal administration, the tissue distribution of aniracetam incorporated in M-C-PLA-NP and aniracetam solution at different time points were shown in Figure 5. The uptake of aniracetam incorporated in M-C-PLA-NP and aniracetam solution in the different tissues presented Cmax at the time point of 3 min. This could be related to the M-C-PLA-NP coated materials chitosan hydrochloride and borneol/mentholum eutectic mixture, which were cutaneous permeable agents that can promote the absorption of aniracetam in the nasal mucosa (Zhang et al., 2006). As time progressed, the concentration in the brain still remained at a constantly higher level from intranasal administrated of the M-C-PLA-NP, but a lower level in the case of aniracetam solution by the same route. For 15 and 30 min, the concentration of aniracetam incorporated in M-C-PLA-NP group showed 5.47 and 11.41 times higher in brain than those of control group with intranasal administration. The data suggested that though the systemic adsorption of aniracetam associated with both M-C-PLA-NP and aniracetam solution exhibited similar concentration–time profiles with the arrival of maximum concentration 3 min later (Figure 5), the AUC0–30 min of aniracetam incorporated in M-C-PLA-NP in the brain was about 1.12-fold at least compared with other tissues, while the AUC0–30 min of aniracetam solution in the brain was about less than 0.87 compared with other tissues except lung (Table 1). The date preliminary demonstrated that the targeting tissue of the M-C-PLA-NP was the brain, but aniracetam solution was not. As shown in Table 1, Cmax in the brain of aniracetam incorporated in M-C-PLA-NP was 3.21-fold than that of aniracetam solution in the brain, while Cmax of aniracetam incorporated in M-C-PLA-NP in the spleen and liver were about 0.55- and 0.42-fold, respectively, compared with that of aniracetam solution. The results further demonstrated
100 Cumulative realease(%)
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the scanning electron micrographs of M-C-PLA-NP (105 magnification). Obviously, M-C-PLA-NP was generally spherical and the surface appeared to be smooth and without pores, uniform size, and each M-C-PLA-NP showed a better dispersibility. The adhesion-like substances should be M-C-PLA-NP coated materials. The mean diameter of M-CPLA-NP was 145.6 ± 9.54 nm with a polydispersity index of 0.077 ± 0.01 and the zeta potential was + 20.4 mv (Figure 2B and C). The zeta potential of M-C-PLA-NP not only determines its colloidal stability, but also influences the effectiveness of the interaction with negatively charged cell membranes. The uptake efficiency of M-C-PLA-NP into brain cell is thus expectable.
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80 60 40
pH7.4
20
pH4.0
0 0
4
8
12
16
20
24
Time(h)
Figure 3. Scanning electron micrograph of M-C-PLA-NP after lysozyme incubated for 2 h (105 magnification, A) and Size distribution chart of M-C-PLA-NP after lysozyme incubated for 2 h (B).
Figure 4. The curve of M-C-PLA-NP cumulative release percentage in pH 7.4 or pH 4.0 PBS. Data represented the mean ± SD.
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3
Brain
3.5 3
Concentration(µg/ml)
Concentration(µg/ml)
Figure 5. Different tissues concentrationtime profiles of aniracetam following nasal administration of M-C-PLA-NP (g) and aniracetam solution (—) at a dose of M-C-PLA-NP of 50 ml (aniracetam content of 6.667 mg/ml). Data represented the mean ± SD.
2.5 2 1.5 1 0.5
Plasma
2.5 2 1.5 1 0.5
0
0 0
10
20
30
0
10
T(min) 3
Heart
2
30
1.5 1 0.5
20
30
20
30
Liver
2.5 2 1.5 1 0.5 0
0 0
10
20
30
0
10
T(min) 1.2
T(min) 1
Spleen Concentration(µg/ml)
Concentration(µg/ml)
20 T(min)
Concentration(µg/ml)
Concentration(µg/ml)
2.5
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1 0.8 0.6 0.4 0.2
0.6 0.4 0.2
0 0
10
20
Lung
0.8
0
30
0
10 T(min)
T(min)
Concentration( µg/m l
3.5 Kidney
3 2.5 2 1.5 1 0.5 0
0
10
20
30
T(min)
Table 1. The results of targeting efficiency evaluation (n ¼ 3). M-C-PLA-NP
Aniracetam solution
Tissues
AUC
Te
Cmax
AUC
Te
Cmax
Ce
Re
Brain Heart Spleen Plasma Lung Liver Kidney
34.36 ± 0.99* 19.27 ± 0.72 5.49 ± 0.46 30.6 ± 0.65 8.96 ± 0.19 7.40 ± 0.82 16.72 ± 0.81
– 1.783 ± 0.33 6.259 ± 0.18 1.123 ± 0.21 3.835 ± 0.27 4.643 ± 1.31 2.055 ± 0.34
3.37 ± 0.15* 2.15 ± 0.08 0.54 ± 0.03 2.32 ± 0.37 0.82 ± 0.06 0.91 ± 0.33 2.44 ± 0.59
6.62 ± 0.81 8.36 ± 0.48 7.67 ± 0.23 7.90 ± 0.56 5.41 ± 0.32 33.50 ± 0.30 13.31 ± 0.42
– 0.767 ± 0.11 0.863 ± 0.13 0.838 ± 0.05 1.223 ± 0.10 0.198 ± 0.02 0.497 ± 0.05
1.05 ± 0.10 1.42 ± 0.08 0.98 ± 0.02 1.20 ± 0.17 0.40 ± 0.08 2.16 ± 0.28 1.83 ± 0.25
3.21 ± 0.33 1.51 ± 0.05 0.55 ± 0.04 1.93 ± 0.30 2.05 ± 0.08 0.42 ± 0.19 1.33 ± 0.20
5.19 ± 0.58 2.31 ± 0.21 0.72 ± 0.07 3.87 ± 0.74 1.66 ± 0.12 0.22 ± 0.06 1.26 ± 0.12
Data represented the mean ± SD (n ¼ 3). AUC and Cmax in M-C-PLA-NP group exhibited significant increase compared with that in control group, *p50.05.
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that M-C-PLA-NP showed a fine brain-targeting efficiency and M-C-PLA-NP was rarely accumulated in other tissues. Compared with control group, the AUC0–30 min of M-C-PLANP in the brain increased 5.19-fold. Such obvious evidence strongly suggested that M-C-PLA-NP might increase the brain-targeting efficiency of aniracetam by intranasal administration. It is believed that the uptake of M-C-PLA-NP into the brain from the nasal mucosa can be achieved via two different pathways: a systemic pathway of absorption into the circulation then into the brain across the BBB and an olfactory pathway of a fraction of drug direct delivery to the brain tissues (Graff & Pollack, 2005). The AUC0–30 min of M-C-PLA-NP in the brain was still higher than that in the plasma, while a part of M-C-PLA-NP may enter into the circulation. This could be related to the M-C-PLA-NP coated materials Tween-80 (Kreuter, 2001), which can inhibit P-glycoprotein in brain capillary endothelial cell membrane for drug efflux function, therefore, increasing the concentration of aniracetam in the brain. As indicated in the Table 1, the AUC0–30 min of spleen and liver of M-C-PLA-NP group were about 0.72- and 0.22-fold, respectively, compared with that of control group. This phenomenon can be explained by the function of coating material PEG 20000, which can avoid the M-C-PLA-NP engulfed by reticuloendothelial system in the systemic circulation. Therefore, not only the M-C-PLA-NP might increase the brain-targeting efficiency of the aniracetam but also decrease the distribution of the M-C-PLA-NP in other tissues.
Conclusion M-C-PLA-NP as a novel brain-targeting agent for nasal drug delivery was established in this study. The prepared M-C-PLA-NP showed high entrapment, smaller particle size and proper zeta potential and exhibited sustained release. The particle size of M-C-PLA-NP incubated with lysozyme for 2 h did not change significantly, which showed that the M-C-PLA-NP in the nasal cavity environment was stable. From the results of in vivo studies, the results indicated that the M-C-PLA-NP not only provide a higher brain concentration of aniracetam after intranasal administrated but also reduced the distribution of the M-C-PLA-NP in other tissues compared with free aniracetam. Also, M-C-PLA-NP provided a longer stay of the drug in the brain compared with aniracetam solution. Thus, it can be concluded that M-C-PLA-NP are capable of providing brain transport after intranasal administrated of drugs, thereby enhancing drugs brain-targeting efficiency.
Declaration of interest The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this article. This study was supported by the National Natural Science Foundation of China (81202489).
References Al-Ghananeem AM, Saeed H, Florence R, et al. (2010). Intranasal drug delivery of didanosine-loaded chitosan nanoparticles for brain
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targeting: an attractive route against infections caused by aids viruses. J Drug Target 18:381–8. Alpesh M, Snjezana S, Lisbeth I. (2009). Nanoparticles for direct noseto-brain delivery of drugs. Int J Pharm 379:146–57. Bram S, Suzanne B, Chantal K, et al. (2010). Nasal vaccination with N-trimethyl chitosan and PLGA based nanoparticles: nanoparticle characteristics determine quality and strength of the antibody response in mice against the encapsulated antigen. Vaccine 28:6282–91. Budhian A, Siegel SJ, Winey KI. (2007). Haloperidol-loaded PLGA nanoparticles: systematic study of particle size and drug content. Int J Pharm 336:367–75. Calvo P, Remunan-Lopez C, Vila-Jato JL, et al. (1997). Novel hydrophilic chitosan-polyethylene oxide nanoparticles as protein carriers. J Appl Polym Sci 63:125–32. Chai GB, Fan YF, Li FZ. (2009). Effect of borneol/mentholum eutectic mixture on nasal-brain delivery of neurotoxin loaded nanoparticles. China J Chi Mater Med 34:698–701. Feng S, Huang G. (2001). Effects of emulsifiers on the controlled release of paclitaxel (Taxol) from nanospheres of biodegradable polymers. J Control Release 71:53–69. Gao XL, Tao WX, Lu W, et al. (2006). Lectin-conjugated PEG–PLA nanoparticles: preparation and brain delivery after intranasal administration. Biomaterials 27:3482–90. Govender T, Stolnik S, Garnett MC, et al. (1999). PLGA nanoparticles preparation by nanoprecipitation: drug loading and release studies of a water soluble drug. J Control Release 57:171–85. Graff CL, Pollack GM. (2005). Nasal drug administration: potential for targeted central nervous system delivery. J Pharm Sci 94: 1187–95. Jing YS. (1994). New nootropics aniracetam. World Pharm 15: 327–40. Kreuter J. (2001). Nanoparticulate system for brain delivery of drugs. Adv Drug Deliv Rev 47:65–81. Lee CR, Benfield P. (1994). Aniracetam. Drugs Aging 4:257–73. Lehr CM, Boustra JA, Schacht EH, et al. (1992). In vitro evaluation of mucoadhesive properties of chitosan and some other natural polymers. Int J Pharm 78:43–8. Li Y-P, Pei Y-Y, Zhang X-Y, et al. (2001). PEGylated PLGA nanoparticles as protein carriers: synthesis, preparation, and biodistribution in rats. J Control Release 71:203–11. Li XM, Xu YL, Chen GG, et al. (2008). PLGA Nanoparticles for the oral delivery of 5-fluorouracil using high pressure homogenization-emulsification as the preparation method and in vitro/in vivo studies. Drug Dev Ind Pharm 34:107–15. Muthu MS, Rawat MK, Mishra A. (2009). PLGA nanoparticle formulations of risperidone: preparation and neuropharmacological evaluation. Nanomedicine 5:323–33. Olivier J-C. (2005). Drug transport to brain with targeted nanoparticles. Blood-Brain Barrier Neurother 2:108–19. Patil YB, Toti US, Khdair A, et al. (2009). Single-step surface functionalization of polymeric nanoparticles for targeted drug delivery. Biomaterials 30:859–66. Roncari G. (1993). Human pharmacokinetics of aniracetam. Drug Invest 5:68–72. Seju U, Kumar A, Sawant KK. (2011). Development and evaluation of olanzapine-loaded PLGA nanoparticles for nose-to-brain delivery: In vitro and in vivo studies. Acta Biomater 7:4169–76. Shah N, Chaudhari K, Dantuluri P, et al. (2009). Paclitaxel-loaded PLGA nanoparticles surface modified with transferrin and Pluronic P85, an in vitro cell line and in vivo biodistribution studies on rat model. J Drug Target 17:533–42. Tobı´o M, Sa´nchez A, Vila A, et al. (2000). The role of PEG on the stability in digestive fluids and in vivo fate of PEG-PLA nanoparticles following oral administration. Colloid Surf B 18:315–23. Wang X, Chi N, Tang X. (2008). Preparation of estradiol chitosan nanoparticles for improving nasal absorption and brain targeting. Eur J Pharm Biopharm 70:735–40. Xu L, Sun Y, Xu P, et al. (2007). Assay of levodopa in brain by CE. Artif Cells Nanomed Biotechnol 35:415–20. Zhang QZ, Wang MN, Cao SL, et al. (2006). Preparation of chitosancoated PLA nanoparticles for intranasal delivery and evaluation on its brain targeting efficiency. Chin Pharm Assoc 1:25–49.
RESEARCH ARTICLE
Preparation of surface multiple-coated polylactide acid drug-loaded nanoparticles for intranasal delivery and evaluation on its brain-targeting efficiency Drug Delivery Downloaded from informahealthcare.com by Memorial University of Newfoundland on 06/04/14 For personal use only.
Junjie Bian1*, Zhixiang Yuan2*, Xiaoliang Chen1, Yuan Gao1, Chaoqun Xu2, and Jianyou Shi3 1
College of Pharmacy, Chengdu University of Traditional Chinese Medicine, Chengdu, Sichuan, China, 2Institute of Pharmacy, Sichuan Academy of Chinese Medicine Sciences, Chengdu, Sichuan, China, and 3Sichuan Academy of Medical Sciences, Sichuan Provincial People’s Hospital, Chengdu, Sichuan, China Abstract
Keywords
Purpose: To prepare a mixture of multiple-coated aniracetam nasal polylactic-acid nanoparticles (M-C-PLA-NP) and evaluate its stability preliminarily in vitro and its brain-targeting efficiency in vivo. Methods: The solvent diffusion–evaporation combined with magnetic stirring method has been chosen for the entrapment of aniracetam. The M-C-PLA-NP was characterized with respect to its morphology, particle size, size distribution and aniracetam entrapment efficiency. The in vivo distribution was studied in male SD rats after an intranasal administration. Results: In vitro release of M-C-PLA-NP showed two components with an initial rapid release due to the surface-associated drug and followed by a slower exponential release of aniracetam, which was dissolved in the core. The AUC0!30 min of M-C-PLA-NP in brain tissues resulted in a 5.19-fold increase compared with aniracetam solution. The ratios of AUC in brain to that in other tissues obtained after nasal application of M-C-PLA-NP were significantly higher than those of aniracetam solution. Conclusion: Therefore, it can be concluded that M-C-PLA-NP demonstrated its potential on increasing the brain-targeting efficiency of drugs and will be used as novel brain-targeting agent for nasal drug delivery.
Aniracetam, brain-targeting, intranasal delivery, surface multiple-coated nanoparticles
Introduction The brain-targeting technology can be divided into three categories. The first category is the invasive injury administration method, such as hypertonic shock, carotid injection of vasoactive substances and direct intraventricular injection. Although these methods are effective, they can cause the infection of the brain, the blood–brain barrier (BBB) damage and surgical injury. The second category method is to increase the ability that the drug through the BBB?including lipidization and chemical delivery systems. These methods have stringent requirements on physical and chemical properties of the drug. Therefore, they have lots of limitations. The third category is using the intranasal delivery system by-pass the BBB to achieve the brain-targeting. Intranasal administration with the characteristics of high bioavailability, rapid absorption, non-invasive and rapid-onset, etc., has become one of the hottest areas in pharmaceutical studies. *These authors contributed equally to this study. Address for correspondence: Chaoqun Xu, Institute of Pharmacy, Sichuan Academy of Chinese Medicine Sciences, Chengdu, Sichuan, China. Tel: +86-28-85213973. E-mail: [email protected]; Jianyou Shi, Sichuan Academy of Medical Sciences, Sichuan Provincial People’s Hospital, Chengdu, Sichuan, China. Tel: +86-028-8739-3234. E-mail: [email protected]
History Received 20 February 2014 Revised 28 March 2014 Accepted 28 March 2014
The human olfactory system is linked to a channel in the brain and the surrounding environment (Graff & Pollack, 2005). After nasal administration, the drug can directly through the olfactory region to reach the cerebrospinal fluid or even the brain. Modern studies have shown that the high degree of bioavailability of the certain drugs nasal delivery, and can even be comparable to the injection, but most of the drug after intranasal administration will be absorbed through the nasal mucosa into the systemic circulation, thus can be engulfed by the body’s reticuloendothelial system. In order to avoid the reticuloendothelial system engulfed, and improve the concentration of drug in the brain, a long cycle modification with PEG-20000 for M-C-PLA-NP is necessary, so the distribution of M-C-PLA-NP in other tissues such as liver and spleen which are distributed richly of the reticuloendothelial system is greatly reduced. Pharmaceutical experiments certificate that the brain–targeting efficiency greatly improved if the M-C-PLA-NP was coated with Tween-80. Studies have shown that Tween-80 have been adsorbed by M-C-PLA-NP surface, which can inhibit P-glycoprotein in brain capillary endothelial cell membrane’s drug efflux function (Alpesh et al., 2009). It has been reported that the PLA-NP under the action of the nasal enzymes and nasal mucosa will lead to aggregation, clearance and degradation (Tobı´o et al., 2000). Some studies also indicate that
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M-C-PLA-NP surface coated with chitosan hydrochloride, the positively charged nature of chitosan with powerful biological adhesion, can increase the stability of PLA-NP and extend its nasal residence time, enhance the absorption of M-C-PLA-NP through the membrane (Calvo et al., 1997). The borneol/ mentholum eutectic mixture reported in the literature shows strong penetration, and can improve the drug concentration in the brain. It has demonstrated that the speed and extent through the nose into the brain were greatly increased after M-C-PLA-NP coated by borneol/mentholum eutectic mixture (Chai et al., 2009). Based on the previous studies, we designed the aniracetam nasal M-C-PLA-NP for braintargeting studies. Aniracetam is a lactam drug, which can be dissolved in organic solvent such as acetone and not soluble in water. It can improve brain function and enhance brain metabolism. Aniracetam has been used for treatment of senile cognitive disorders and intellectual and memory impairment. Orally administered aniracetam has been used in clinical trials at daily doses of 1000–1500 mg for the treatment of cognitive impairment in patients with SDAT, and at doses of 600–1500 mg for the treatment of various symptoms including memory disorders in elderly patients with cerebrovascular disease (Lee & Benfield, 1994). Currently, the capsule, tablet and granule of aniracetam are the most common pharmaceutical formulations on the market. However, it is very rapidly eliminated from the body and plasma elimination half-time is 30 min. The absolute systemic bioavailability is only about 0.2% (Roncari, 1993). What is more, these kind of aniracetam formulations show non-specific distribution after oral administration. Therefore, the aniracetam pharmaceutical formulations were limited to an important role for the treatment of brain diseases. In this study, the aniracetam has been chosen as the model drug and the solvent diffusion–evaporation combined with magnetic stirring method has been chosen to prepare M-C-PLA-NP as a novel brain-targeting agent for nasal drug delivery, aim of increasing aniracetam brain-targeting efficiency for the treatment of brain diseases. The cumulative release characteristic, stability in vitro, tissue distribution of M-C-PLA-NP in rats are evaluated and the brain-targeting efficiency is preliminary evaluated by quantitative analytic mean.
Methods Materials Polylactic acid (PLA, MW ¼ 15 kDa) were purchased from Shandong Key Laboratory of Medical Polymer Materials (Shandong, China). Chitosan hydrochloride (degree of deacetylation, 80–90%, viscosity, 30 mPa s) and Lysozyme were obtained from Shanghai KABO Trading Company (Shanghai, China). Aniracetam (purity 99.5%), Betamethasone and Poloxamer 188(F-68, MW ¼ 8350 ± 1000) were supplied by Sigma Chemical Company. Polyethylene Glycol (PEG, MW ¼ 20 kDa), Oleum Menthae Dementhola Tum, Borneol and Tween-80 were from Chengdu Kelong Chemical Company (Chengdu, China). Other chemical reagents and solvents were of analytical or spectroscopic grade.
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Sprague–Dawley rats (7–8 weeks of age, body weight 210– 230 g) were obtained from the Experimental Animal Center of Sichuan Academy of Chinese Medicine Sciences. These animals were allowed to acclimatize in environmentally controlled quarters (24 ± 1 C and 12:12 h light–dark cycle) for at least 5 days before being used for experiments. All procedures of the animal studies were approved by animal ethical experimentation committee, according to the requirements of the National Act on the use of experimental animals (People’s Republic of China). Preparation of M-C-PLA-NP and PLA-NP M-C-PLA-NPs were prepared using the solvent diffusion– evaporation combined with magnetic stirring method. Different formulation variables such as the ratio of the drug to PLA (3:10, 1:5, 2:15 and 1:10 ), concentration of poloxamer 188 as stabilizer (0.5, 1, 2 and 3% w/v), the aqueous phase volume (10, 15, 20 and 25 ml), nanoparticle solution volume after rotary evaporated (7, 5, 4 and 3 ml), ethanol:acetone ratio (3:2, 1:1, 2:3 and 1:2) were varied, and the effects on particle size, zeta potential and entrapment efficiency were evaluated according to the previous study (Li et al., 2001). Only one parameter was changed at a time in each set of experiments. The optimized formulation was prepared by dissolving PLA (150 mg), aniracetam (20 mg, aniracetam was not added in the preparation of PLA-NP) and borneol/mentholum eutectic mixture in 20 ml of ethanol/ acetone (3:2; Chai et al., 2009). This organic phase was added at the rate of 1 ml/min to 20 ml of 1% w/v poloxamer 188 and 100 mg PEG20000 solution with continuous stirring on a magnetic stirrer at room temperature (Patil et al., 2009). After the organic phase was added completely, vacuum rotary evaporating was continued to allow complete evaporation of the organic solvent. Then, 100 mg Tween-80 was added to the nanoparticle solution, and continued rotary evaporation until the volume of the suspension was less than 3 ml. And, the nanoparticles were collected by centrifugation and washed thrice with ultrapure water. After that, the collected nanoparticles re-dispersed in 1% F-68 solution and 5 mg of chitosan hydrochloride was added into the suspension under magnetic stirring. Finally, nanoparticle suspension was concentrated at 30 C until the volume of the nanoparticle suspension is less than 3 ml (Li et al., 2008). In this way, M-C-PLA-NPs were obtained. PLA-NPs were prepared without coating process. Drug loading and entrapment efficiency Ultrafiltration method was used to determine drug loading and entrapment efficiency of M-C-PLA-NP (Govender et al., 1999). M-C-PLA-NP suspension was taken into the Millipore ultrafiltration tube (Mw ¼ 10000) and centrifuged at 3500 rpm for 35 min. Then the filtered liquid was collected and the amount of the free drug in the water phase was determined by HPLC. The total amount of the drug in the suspension was extracted ultrasonic and diluted with ethanol, which was also detected by HPLC system. The amount of drug inside M-C-PLA-NP was calculated by subtracting the free drug in the water phase of the suspension from the total
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DOI: 10.3109/10717544.2014.910566
amount of the drug in the suspension. The entrapment efficiency (EE) and drug loading (DL) of drug were calculated by the following equation: EEð%Þ ¼ ðW1 W2 Þ=W1 100%
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DLð%Þ ¼ ðW1 W2 Þ=W3 100% where W1 is the total amount of the drug in the suspension, W2 is the amount of the free drug in the water phase and W3 is the total amount of the drug plus the amount of PLA. HPLC condition was as follows: with a reversed-phase column, Welchrom-C18 reversed-phase column (150 4.6 mm, 5 mm), the separations were achieved using methanol and water at the ratio of 40:60 as the mobile phase with flow rate of 1 ml/min and column temperature of 30 C. The detection wavelength was at 284 nm. Particle size analysis and zeta potential The size analysis, polydispersity index and zeta potential distribution of the M-C-PLA-NP were determined using a Malvern Zetasizer Nano ZS90 (Malvern Instruments, UK; Seju et al., 2011). Each sample was suitably dispersed with distilled water and placed in a disposable sizing cuvette. The polydispersity index was studied to determine the narrowness of the size distribution. The size analysis of a sample consisted of three measurements, and the results were expressed as mean size ± SD. In order to determine the zeta potential distribution, each sample was suitably diluted five times with filtered distilled water and placed in a disposable zeta cell. The average of three measurements of each sample was used to derive the average zeta potential. Nanoparticle morphology Morphology of the M-C-PLA-NP was visualized with scanning electron microscopy (SEM). About 50 ml of 0.1% (w/v) particle suspension was air dried overnight on a metal stub. Particles were gold/palladium sputtered using a sputter coater device and analyzed with scanning electron microscope (Hitachi, S4800; Bram et al., 2010). Stability and cumulative release of M-C-PLA-NP To determine the stability of the M-C-PLA-NP, lysozyme (1 mg) was added into the prepared M-C-PLA-NP suspension (1 ml). The M-C-PLA-NP were incubated at 37 C for 2 h under continuous stirring, then the changes in particle size were compared before and after incubation (Zhang et al., 2006). In vitro release of M-C-PLA-NP Cumulative release characteristics were also observed. A certain amount volume of M-C-PLA-NP suspension transferred to dialysis bags (MWCO: 3500 Da) placed in 100 ml of PBS (pH 4.4 or pH 7.4) with stirring at 37 C. At appropriate time points (0, 5 min, 30 min, 1 h, 2 h, 4 h, 8 h, 12 h and 24 h), a certain amount of the environmental buffer solution (1 ml) was replaced with fresh buffer solution, and the concentration of the released aniracetam in the removed buffer solution was determined using HPLC.
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Evaluation of brain uptake following intranasal administration Animal experiment Forty-two SD rats were divided into two groups: control group and nanoparticle group. After weighing, the rats were anesthetized by intraperitoneal injected of 7% chloral in accordance with the 0.5 ml/100 g standard (Zhang et al., 2006). Then, the rat was fixed on the surgical board to a supine posture, cut neck, isolated trachea and esophagus. An incision was made by the surgical scissor in the rat tracheal, a size suitable hose was inserted for breathing and at the same time the esophagus was ligated to prevent drug into the stomach. The front part of a micro-injector with hose, which is used to unilateral intranasal administrated of M-C-PLA-NP. The preparations were administered at the openings of the nostrils using a polyethylene 10 (PE 10) tube attached to a microlitre syringe. The procedure was performed gently and slowly, which lasted for about 2 min, allowing the rats to inhale all of the preparations (Gao et al., 2006). Rats in nanoparticle group were administrated with M-C-PLA-NP (50 ml, containing 6.667 mg/ml aniracetam); rats in control group were received with 50 ml aniracetam solution (equal to the dose of M-C-PLA-NP; Jing, 1994). At each of the time points (1, 3, 5, 7.5, 10, 15 and 30 min) following administration, three animals were sacrificed by bloodletting from the cervical artery. And, then blood was collected and put into the tubes with heparin. Finally, the skulls were cut open and brain tissue and other tissue samples excised. All the samples were stored at 40 C until assay. Analytical procedures To prepare the samples for analysis, the tissue samples were homogenized with 1-fold volume of distilled water and the homogenate (1 ml) was taken into EP tube. Then 5 ml of betamethasone (0.1 mg/ml, internal standard) and 3 ml of ethyl acetate were added to 1 ml of the homogenate to extract the aniracetam and betamethasone from the samples (Xu et al., 2007). Intensely vortexed for 5 min, the mixture was subjected to centrifuge at 15 000 g for 10 min. The supernatant was collected and evaporated under a stream of nitrogen (Zhang et al., 2006). The residue was dissolved in 200 ml of methanol, and 20 ml was injected into the HPLC system. The whole blood sample was subjected to centrifuge at 3500 g for 15 min. The plasma was collected without the addition of water and treated in the same manner. The uptake of aniracetam associated to the nanoparticles by brain tissues and the circulation were analyzed by the HPLC system (Agilent 1200). With a reversed-phase column, Welchrom-C18 reversed-phase column (150 4.6 mm, 5 mm), the separations were achieved using methanol and water at the ratio of 40:60 (0 min)–40:60 (10 min)–60:40 (20 min) as the mobile phase with flow rate of 1 ml/min and column temperature of 30 C. The mixture was monitored with UV detection at 240 nm. Data analysis All data were dose-normalized and plotted as drug concentration–time curves in the plasma, brain and other tissues.
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The Cmax values were read directly from the concentrationtime profile and the area under the concentration–time curve (AUC0!t) was calculated by the DAS software (published by the Mathematical Pharmacology Professional Committee of China). The brain/plasma and other tissues AUC0!t ratios for aniracetam carried by M-C-PLA-NP and aniracetam suspension were calculated to evaluate the drug-targeting efficiency (DTE). The three parameters of targeting efficiency (Te), concentration ratio efficiency (Ce) and relative targeting efficiency (Re) were selected to determine the brain-targeting efficiency of the M-C-PLA-NP. Drug target tissues can be judged by the two parameters of Te and Re. The parameter of Ce was used to judge M-C-PLA-NP and aniracetam solution-targeting efficiency. The equations are as follows. Te ¼ ðAUCÞT =ðAUCÞNT Ce ¼ ðCmax Þp =ðCmax Þs Re ¼ ðAUCÞp =ðAUCÞs where T is the mean of target tissue, NT is the mean of nontarget tissue, p is the mean of targeting drug delivery system, S is the mean of non-targeting drug delivery system.
Results and discussion Determination of aniracetam Quantitative analysis of aniracetam was done by HPLC. In a different concentration of the sample, the regression equation was Y ¼ 14.639X + 15.764 (r ¼ 0.9989) in the linear range of 2.52–126 mgml1 after linear regression of its peak area. Figure 1. Scheme of preparing M-C-PLANPs coated with various kinds of materials.
Under the above conditions, the within-day precision and between-day precision is of less than 2%. Amount of aniracetam solution (including high, medium and low concentrations of aniracetam solution) were taken into blank M-C-PLA-NP suspension into the Millipore ultrafiltration tube (Mw ¼ 10 000). After that, the nanoparticles suspension was centrifuged at 3500 rpm for 35 min. Then, the aniracetam content in filtered fluid was determined by HPLC and the recovery result of aniracetam solution in blank M-C-PLA-NP was more than 95% (RSD52.0%). Physicochemical characterization and effect of formulation variables Nanoparticles and liposomes are wildly used in drugtargeting system, especially in brain drug delivery. Nanoparticles show many advantages in brain-targeting systems, such as good stability in cavity and smaller size. The chitosan coating nanoparticles has some important properties such as biocompatibility, non-toxicity and mucoadhesivity which render it an interesting biomaterial (Lehr et al., 1992). From a physicochemical point of view, chitosan has the special quality of gelling upon contact with anions (Calvo et al., 1997). There are several research papers showed nanoparticles and chitosan based nanoparticles developed for brain-targeting systems (Olivier, 2005; Wang et al., 2008; Al-Ghananeem et al., 2010). In our experiment, chitosan plays an important role in prolonging the residence time of nanoparticles in nasal cavity due to its positive charge and biological adhesiveness, with PEG20000 avoiding the reticuloendothelial system engulfed, Tween-80 enhancing the brain-targeting efficiency, borneol and mentholum eutectic opening the blood-brain barrier
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DOI: 10.3109/10717544.2014.910566
(Figure 1). Thus, it can add the absorption of drugs in nasal mucosa and increase the dose transferring to brain. After preliminary study, the ratio of ethanol to acetone (2:3) was selected as the organic solvent, as it gave maximum entrapment efficiency and minimum particle size. poloxamer 188 was selected as the stabilizer. Poloxamer 188 is a swellable, hydrophilic polymer and therefore the higher amount of residual poloxamer 188 at the surface of nanoparticles would account for their hydrophilicity. The prepared nanoparticles aggregation was observed without stabilizer. Poloxamer 188 was added ranging from 0.5% to 3% (w/v). The entrapment efficiency of nanoparticle increased from 79.54 ± 6.83% to 88.50 ± 5.70%. Especially, the entrapment efficiency reached peak value of 92.56 ± 8.15% with 1.0% poloxamer 188. When poloxamer 188 with higher concentration (2% w/v) was used, the entrapment efficiency decreased and particle size became larger. This phenomenon can be better understood in terms of an increase in solubility of the drug in aqueous medium. An optimum concentration of stabilizer led to a reduced size of M-C-PLA-NP, as the insufficient amount of stabilizer was unable to cover the dispersed M-C-PLA-NP completely, failing to stabilize and causing aggregation, leading to larger M-C-PLA-NP (Feng & Huang, 2001). Thus, 1.0% poloxamer 188 was considered optimum. To clarify the influence of amount ratio of drug to polymer on the nanoparticles, different ratios of drug to polymer were studied. With the increased ratio from 1:10 to 3:10, however, the drug entrapment of M-C-PLA-NP was decreased and the particle size got bigger, which was consistent with previous reports (Budhian et al., 2007; Shah et al., 2009). An increase in the polymer concentration led to an increase in the viscosity of the organic phase, which resulted in rapid diffusion of the organic phase into the aqueous phase. Coarse dispersions were obtained at higher polymer concentrations, possibly due to the insufficient amount of stabilizer presented in the aqueous phase for that particular polymer amount, which led to bigger particle size. The influence of volume of aqueous phase on the size and entrapment of the M-C-PLA-NP was also observed in the study. Using the volume of aqueous phase from 10 to 25 ml, the entrapment efficiency was improved from 64.97 ± 2.49% to 90.23 ± 2.01%. The particle size was also found to decrease from 342.6 ± 12.45 to 149.3 ± 7.8 nm. Further increase in volume of the aqueous phase induced the lower entrapment efficiency (575%) of M-C-PLA-NP. The reason may be that the polymer rapidly dispersed to large volume of aqueous phase to form smaller particles in comparison with low volume of aqueous phase. Hence, the optimal volume of aqueous phase was 25 ml. The entrapment efficiency was also found to decrease with increasing the volume of nanoparticles after rotary evaporation. This may be due to the lower volume of water, the drug which is rapidly mixes with the PLA, thus producing high entrapment efficiency after rotary evaporated (3 ml; Muthu et al., 2009). When the volume ratio of ethanol to acetone was increased from 1:1 and 2:3, the obtained entrapment efficiency was increased from 87.63 ± 2.33% to 95.31 ± 2.85%, without obvious change in particle size (145 nm). Further increasing the ratio of acetone produced
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the entrapment of585% and particle size4150 nm of the M-C-PLA-NP. Therefore, the volume ratio of ethanol to acetone (2:3) was adopted. Finally, using optimized parameters mentioned above, the prepared M-C-PLA-NP showed the entrapment efficiency of 96.40 ± 1.77% and drug loading of 11.57 ± 0.21%, with mean diameter of 145.6 ± 9.54 nm. Characterization, particle size and zeta potential of M-C-PLA-NP Nanoparticles were gold sputtered using a sputter coater device and analyzed with scanning electron microscope (Hitachi, S4800; Bram et al., 2010). Figure 2 shows
Figure 2. Scanning electron micrographs of M-C-PLA-NP (105 magnification, A); Size distribution chart of M-C-PLA-NP (B) and Zeta potential distribution chart of M-C-PLA-NP (C).
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Stability of M-C-PLA-NP The conformational stability of M-C-PLA-NP in the nasal cavity environment is of great importance from the pharmaceutical point of view. In this study, lysozyme was incubated with the prepared M-C-PLA-NP suspension for 2 h to determine the stability of the M-C-PLA-NP (Figure 3A). Size distribution chart of multiple M-C-PLA-NP after lysozyme incubated for 2 h (Figure 3B). The particle size of the M-C-PLA-NP after incubated was 149.1 ± 7.2 nm with polydispersity index of 0.082 ± 0.012, which had no obvious changes compared with the M-C-PLA-NP before the incubation. The results demonstrated that the M-C-PLA-NP was thus expected to be stable in the nasal cavity environment. In vitro release of M-C-PLA-NP The cumulative release of aniracetam was calculated as a function of time as shown in Figure 4. The drug release from M-C-PLA-NP followed the biphasic model, with an initial
burst attributed to the drug-associated near-particle surface and sustained release. Within 4 h, about 70% aniracetam released in buffer solution, and then, the release of M-C-PLANP reached the platform. After 24 h, the percentage of the accumulated release was 80–85%. Thus, it was clear that incorporation in M-C-PLA-NP could significantly sustain the release of aniracetam. Evaluation of brain uptake following intranasal administration To evaluate the drug brain-targeting efficiency of the M-C-PLA-NP after intranasal administration, the tissue distribution of aniracetam incorporated in M-C-PLA-NP and aniracetam solution at different time points were shown in Figure 5. The uptake of aniracetam incorporated in M-C-PLA-NP and aniracetam solution in the different tissues presented Cmax at the time point of 3 min. This could be related to the M-C-PLA-NP coated materials chitosan hydrochloride and borneol/mentholum eutectic mixture, which were cutaneous permeable agents that can promote the absorption of aniracetam in the nasal mucosa (Zhang et al., 2006). As time progressed, the concentration in the brain still remained at a constantly higher level from intranasal administrated of the M-C-PLA-NP, but a lower level in the case of aniracetam solution by the same route. For 15 and 30 min, the concentration of aniracetam incorporated in M-C-PLA-NP group showed 5.47 and 11.41 times higher in brain than those of control group with intranasal administration. The data suggested that though the systemic adsorption of aniracetam associated with both M-C-PLA-NP and aniracetam solution exhibited similar concentration–time profiles with the arrival of maximum concentration 3 min later (Figure 5), the AUC0–30 min of aniracetam incorporated in M-C-PLA-NP in the brain was about 1.12-fold at least compared with other tissues, while the AUC0–30 min of aniracetam solution in the brain was about less than 0.87 compared with other tissues except lung (Table 1). The date preliminary demonstrated that the targeting tissue of the M-C-PLA-NP was the brain, but aniracetam solution was not. As shown in Table 1, Cmax in the brain of aniracetam incorporated in M-C-PLA-NP was 3.21-fold than that of aniracetam solution in the brain, while Cmax of aniracetam incorporated in M-C-PLA-NP in the spleen and liver were about 0.55- and 0.42-fold, respectively, compared with that of aniracetam solution. The results further demonstrated
100 Cumulative realease(%)
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the scanning electron micrographs of M-C-PLA-NP (105 magnification). Obviously, M-C-PLA-NP was generally spherical and the surface appeared to be smooth and without pores, uniform size, and each M-C-PLA-NP showed a better dispersibility. The adhesion-like substances should be M-C-PLA-NP coated materials. The mean diameter of M-CPLA-NP was 145.6 ± 9.54 nm with a polydispersity index of 0.077 ± 0.01 and the zeta potential was + 20.4 mv (Figure 2B and C). The zeta potential of M-C-PLA-NP not only determines its colloidal stability, but also influences the effectiveness of the interaction with negatively charged cell membranes. The uptake efficiency of M-C-PLA-NP into brain cell is thus expectable.
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80 60 40
pH7.4
20
pH4.0
0 0
4
8
12
16
20
24
Time(h)
Figure 3. Scanning electron micrograph of M-C-PLA-NP after lysozyme incubated for 2 h (105 magnification, A) and Size distribution chart of M-C-PLA-NP after lysozyme incubated for 2 h (B).
Figure 4. The curve of M-C-PLA-NP cumulative release percentage in pH 7.4 or pH 4.0 PBS. Data represented the mean ± SD.
Preparation of surface M-C-PLA-NP
DOI: 10.3109/10717544.2014.910566
4
3
Brain
3.5 3
Concentration(µg/ml)
Concentration(µg/ml)
Figure 5. Different tissues concentrationtime profiles of aniracetam following nasal administration of M-C-PLA-NP (g) and aniracetam solution (—) at a dose of M-C-PLA-NP of 50 ml (aniracetam content of 6.667 mg/ml). Data represented the mean ± SD.
2.5 2 1.5 1 0.5
Plasma
2.5 2 1.5 1 0.5
0
0 0
10
20
30
0
10
T(min) 3
Heart
2
30
1.5 1 0.5
20
30
20
30
Liver
2.5 2 1.5 1 0.5 0
0 0
10
20
30
0
10
T(min) 1.2
T(min) 1
Spleen Concentration(µg/ml)
Concentration(µg/ml)
20 T(min)
Concentration(µg/ml)
Concentration(µg/ml)
2.5
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7
1 0.8 0.6 0.4 0.2
0.6 0.4 0.2
0 0
10
20
Lung
0.8
0
30
0
10 T(min)
T(min)
Concentration( µg/m l
3.5 Kidney
3 2.5 2 1.5 1 0.5 0
0
10
20
30
T(min)
Table 1. The results of targeting efficiency evaluation (n ¼ 3). M-C-PLA-NP
Aniracetam solution
Tissues
AUC
Te
Cmax
AUC
Te
Cmax
Ce
Re
Brain Heart Spleen Plasma Lung Liver Kidney
34.36 ± 0.99* 19.27 ± 0.72 5.49 ± 0.46 30.6 ± 0.65 8.96 ± 0.19 7.40 ± 0.82 16.72 ± 0.81
– 1.783 ± 0.33 6.259 ± 0.18 1.123 ± 0.21 3.835 ± 0.27 4.643 ± 1.31 2.055 ± 0.34
3.37 ± 0.15* 2.15 ± 0.08 0.54 ± 0.03 2.32 ± 0.37 0.82 ± 0.06 0.91 ± 0.33 2.44 ± 0.59
6.62 ± 0.81 8.36 ± 0.48 7.67 ± 0.23 7.90 ± 0.56 5.41 ± 0.32 33.50 ± 0.30 13.31 ± 0.42
– 0.767 ± 0.11 0.863 ± 0.13 0.838 ± 0.05 1.223 ± 0.10 0.198 ± 0.02 0.497 ± 0.05
1.05 ± 0.10 1.42 ± 0.08 0.98 ± 0.02 1.20 ± 0.17 0.40 ± 0.08 2.16 ± 0.28 1.83 ± 0.25
3.21 ± 0.33 1.51 ± 0.05 0.55 ± 0.04 1.93 ± 0.30 2.05 ± 0.08 0.42 ± 0.19 1.33 ± 0.20
5.19 ± 0.58 2.31 ± 0.21 0.72 ± 0.07 3.87 ± 0.74 1.66 ± 0.12 0.22 ± 0.06 1.26 ± 0.12
Data represented the mean ± SD (n ¼ 3). AUC and Cmax in M-C-PLA-NP group exhibited significant increase compared with that in control group, *p50.05.
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J. Bian et al.
that M-C-PLA-NP showed a fine brain-targeting efficiency and M-C-PLA-NP was rarely accumulated in other tissues. Compared with control group, the AUC0–30 min of M-C-PLANP in the brain increased 5.19-fold. Such obvious evidence strongly suggested that M-C-PLA-NP might increase the brain-targeting efficiency of aniracetam by intranasal administration. It is believed that the uptake of M-C-PLA-NP into the brain from the nasal mucosa can be achieved via two different pathways: a systemic pathway of absorption into the circulation then into the brain across the BBB and an olfactory pathway of a fraction of drug direct delivery to the brain tissues (Graff & Pollack, 2005). The AUC0–30 min of M-C-PLA-NP in the brain was still higher than that in the plasma, while a part of M-C-PLA-NP may enter into the circulation. This could be related to the M-C-PLA-NP coated materials Tween-80 (Kreuter, 2001), which can inhibit P-glycoprotein in brain capillary endothelial cell membrane for drug efflux function, therefore, increasing the concentration of aniracetam in the brain. As indicated in the Table 1, the AUC0–30 min of spleen and liver of M-C-PLA-NP group were about 0.72- and 0.22-fold, respectively, compared with that of control group. This phenomenon can be explained by the function of coating material PEG 20000, which can avoid the M-C-PLA-NP engulfed by reticuloendothelial system in the systemic circulation. Therefore, not only the M-C-PLA-NP might increase the brain-targeting efficiency of the aniracetam but also decrease the distribution of the M-C-PLA-NP in other tissues.
Conclusion M-C-PLA-NP as a novel brain-targeting agent for nasal drug delivery was established in this study. The prepared M-C-PLA-NP showed high entrapment, smaller particle size and proper zeta potential and exhibited sustained release. The particle size of M-C-PLA-NP incubated with lysozyme for 2 h did not change significantly, which showed that the M-C-PLA-NP in the nasal cavity environment was stable. From the results of in vivo studies, the results indicated that the M-C-PLA-NP not only provide a higher brain concentration of aniracetam after intranasal administrated but also reduced the distribution of the M-C-PLA-NP in other tissues compared with free aniracetam. Also, M-C-PLA-NP provided a longer stay of the drug in the brain compared with aniracetam solution. Thus, it can be concluded that M-C-PLA-NP are capable of providing brain transport after intranasal administrated of drugs, thereby enhancing drugs brain-targeting efficiency.
Declaration of interest The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this article. This study was supported by the National Natural Science Foundation of China (81202489).
References Al-Ghananeem AM, Saeed H, Florence R, et al. (2010). Intranasal drug delivery of didanosine-loaded chitosan nanoparticles for brain
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targeting: an attractive route against infections caused by aids viruses. J Drug Target 18:381–8. Alpesh M, Snjezana S, Lisbeth I. (2009). Nanoparticles for direct noseto-brain delivery of drugs. Int J Pharm 379:146–57. Bram S, Suzanne B, Chantal K, et al. (2010). Nasal vaccination with N-trimethyl chitosan and PLGA based nanoparticles: nanoparticle characteristics determine quality and strength of the antibody response in mice against the encapsulated antigen. Vaccine 28:6282–91. Budhian A, Siegel SJ, Winey KI. (2007). Haloperidol-loaded PLGA nanoparticles: systematic study of particle size and drug content. Int J Pharm 336:367–75. Calvo P, Remunan-Lopez C, Vila-Jato JL, et al. (1997). Novel hydrophilic chitosan-polyethylene oxide nanoparticles as protein carriers. J Appl Polym Sci 63:125–32. Chai GB, Fan YF, Li FZ. (2009). Effect of borneol/mentholum eutectic mixture on nasal-brain delivery of neurotoxin loaded nanoparticles. China J Chi Mater Med 34:698–701. Feng S, Huang G. (2001). Effects of emulsifiers on the controlled release of paclitaxel (Taxol) from nanospheres of biodegradable polymers. J Control Release 71:53–69. Gao XL, Tao WX, Lu W, et al. (2006). Lectin-conjugated PEG–PLA nanoparticles: preparation and brain delivery after intranasal administration. Biomaterials 27:3482–90. Govender T, Stolnik S, Garnett MC, et al. (1999). PLGA nanoparticles preparation by nanoprecipitation: drug loading and release studies of a water soluble drug. J Control Release 57:171–85. Graff CL, Pollack GM. (2005). Nasal drug administration: potential for targeted central nervous system delivery. J Pharm Sci 94: 1187–95. Jing YS. (1994). New nootropics aniracetam. World Pharm 15: 327–40. Kreuter J. (2001). Nanoparticulate system for brain delivery of drugs. Adv Drug Deliv Rev 47:65–81. Lee CR, Benfield P. (1994). Aniracetam. Drugs Aging 4:257–73. Lehr CM, Boustra JA, Schacht EH, et al. (1992). In vitro evaluation of mucoadhesive properties of chitosan and some other natural polymers. Int J Pharm 78:43–8. Li Y-P, Pei Y-Y, Zhang X-Y, et al. (2001). PEGylated PLGA nanoparticles as protein carriers: synthesis, preparation, and biodistribution in rats. J Control Release 71:203–11. Li XM, Xu YL, Chen GG, et al. (2008). PLGA Nanoparticles for the oral delivery of 5-fluorouracil using high pressure homogenization-emulsification as the preparation method and in vitro/in vivo studies. Drug Dev Ind Pharm 34:107–15. Muthu MS, Rawat MK, Mishra A. (2009). PLGA nanoparticle formulations of risperidone: preparation and neuropharmacological evaluation. Nanomedicine 5:323–33. Olivier J-C. (2005). Drug transport to brain with targeted nanoparticles. Blood-Brain Barrier Neurother 2:108–19. Patil YB, Toti US, Khdair A, et al. (2009). Single-step surface functionalization of polymeric nanoparticles for targeted drug delivery. Biomaterials 30:859–66. Roncari G. (1993). Human pharmacokinetics of aniracetam. Drug Invest 5:68–72. Seju U, Kumar A, Sawant KK. (2011). Development and evaluation of olanzapine-loaded PLGA nanoparticles for nose-to-brain delivery: In vitro and in vivo studies. Acta Biomater 7:4169–76. Shah N, Chaudhari K, Dantuluri P, et al. (2009). Paclitaxel-loaded PLGA nanoparticles surface modified with transferrin and Pluronic P85, an in vitro cell line and in vivo biodistribution studies on rat model. J Drug Target 17:533–42. Tobı´o M, Sa´nchez A, Vila A, et al. (2000). The role of PEG on the stability in digestive fluids and in vivo fate of PEG-PLA nanoparticles following oral administration. Colloid Surf B 18:315–23. Wang X, Chi N, Tang X. (2008). Preparation of estradiol chitosan nanoparticles for improving nasal absorption and brain targeting. Eur J Pharm Biopharm 70:735–40. Xu L, Sun Y, Xu P, et al. (2007). Assay of levodopa in brain by CE. Artif Cells Nanomed Biotechnol 35:415–20. Zhang QZ, Wang MN, Cao SL, et al. (2006). Preparation of chitosancoated PLA nanoparticles for intranasal delivery and evaluation on its brain targeting efficiency. Chin Pharm Assoc 1:25–49.
