http://informahealthcare.com/ddi ISSN: 0363-9045 (print), 1520-5762 (electronic) Drug Dev Ind Pharm, Early Online: 1–8 ! 2014 Informa Healthcare USA, Inc. DOI: 10.3109/03639045.2014.991400
RESEARCH ARTICLE
Design, characterization, and evaluation of intranasal delivery of ropinirole-loaded mucoadhesive nanoparticles for brain targeting
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Omidreza Jafarieh1, Shadab Md1,2, Mushir Ali1, Sanjula Baboota1, J. K. Sahni1,3, Bhavna Kumari1,4, Aseem Bhatnagar5, and Javed Ali1 1
Department of Pharmaceutics, Faculty of Pharmacy, Jamia Hamdard, Hamdard Nagar, New Delhi, India, 2Department of Pharmaceutical Technology, School of Pharmacy, International Medical University (IMU), Bukit Jalil, Kuala Lumpur, Malaysia, 3Department of Pharmaceutics, Khalsa College of Pharmacy, Amritsar, India, 4Department of Pharmaceutics, Dehradun Institute of Technology (DIT), Dehradun, Uttaranchal, India, and 5Department of Radiopharmaceuticals, Institute of Nuclear Medicine and Allied Sciences (INMAS), New Delhi, India Abstract
Keywords
Context: Parkinson disease (PD) is a common, progressive neurodegenerative disorder, characterized by marked depletion of striatal dopamine and degeneration of dopaminergic neurons in the substantia nigra. Objective: The purpose of the present study was to investigate the possibility of targeting an anti-Parkinson’s drug ropinirole (RH) to the brain using polymeric nanoparticles. Materials and methods: Ropinirole hydrochloride (RH)-loaded chitosan nanoparticles (CSNPs) were prepared by an ionic gelation method. The RH-CSNPs were characterized for particle size, polydispersity index (PDI), zeta potential, loading capacity, entrapment efficiency in vitro release study, and in vivo distribution after intranasal administration. Results and discussion: The RH-CSNPs showed sustained release profiles for up to 18 h. The RH concentrations (% Radioactivity/g) in the brain following intranasal administration (i.n.) of RH-CSNPs were found to be significantly higher at all the time points compared with RH solution. The concentration of RH was highest in the liver (7.210 ± 0.52), followed by kidneys (6.862 ± 0.62), intestine (4.862 ± 0.45), and lungs (4.640 ± 0.92) in rats following i.n. administration of RH-CSNPs. Gamma scintigraphy imaging in rats was performed to ascertain the localization of drug in the brain following intranasal administration of formulations. The brain/ blood ratios obtained (0.251 ± 0.09 and 0.386 ± 0.57 of RH (i.n.) and RH-CSNPs (i.n.), respectively) at 0.5 h are indicative of direct nose to brain transport, bypassing the blood–brain barrier (BBB). Conclusion: The novel formulation showed the superiority of nose to brain delivery of RH using mucoadhesive nanoparticles compared with other delivery routes reported earlier.
Brain targeting, biodistribution, gamma scintigraphy, nanoparticles, Parkinson’s disease
Introduction Parkinson disease (PD) is a common, progressive neurodegenerative disorder characterized by marked depletion of striatal dopamine (DA) and degeneration of dopaminergic (DAergic) neurons in the substantia nigra1,2. Clinically, the disease is manifested by both motor and non-motor symptoms. The cardinal motor symptoms include bradykinesia, resting tremor, rigidity, and disturbance of posture and gait; non-motor symptoms include a diminished sense of smell, depression, and sleep disturbance. The prevalence of PD is set to rise significantly in the coming years. In the near future, the patents of some important PD drugs
Address for correspondence: Dr Javed Ali, Department of Pharmaceutics, Faculty of Pharmacy, Jamia Hamdard, Hamdard Nagar, New Delhi 110062, India. Tel: +91 9811312247. Fax: +011 26059633. E-mail: [email protected]
History Received 3 July 2014 Revised 7 November 2014 Accepted 11 November 2014 Published online 11 December 2014
will expire. While new products may be developed, which of these will be successful will remain a challenge3. Ropinirole hydrochloride (RH) is a non-ergot D2/D3 DA agonist with the greatest affinity at the D3 receptors and is used in patients with moderate or advanced PD. It is absorbed rapidly with peak plasma concentrations occurring in 1–2 h. Oral ropinirole therapy is associated with nausea, vomiting, and gastrointestinal disturbances and ropinirole possess low oral bioavailability (approx. 50%) because of the hepatic first pass effect; its adverse effects and frequent dosing schedule contribute to non-compliance among patients4. In 2008, the FDA approved a once daily, prolonged release formulation of RH. This is the first once daily oral DA agonist approved for both early and advanced PD in the US. Once-daily administration resulted in improved compliance and adherence. However, since the target site of ropinirole is the brain, a strategy is desired that not only improves the bioavailability by preventing first pass metabolism but also provides targeting to the receptor site and bypasses the
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blood–brain barrier (BBB). Such a strategy would help to achieve the desired drug concentration at the site of action, as well as reducing availability at non-targeting sites, thus reducing adverse effects. In recent years, the intranasal (i.n.) route has received a great deal of attention as a convenient and reliable method for the systemic administration of drugs. It offers the advantages of simple, cost effective, and convenient administration. Additionally, i.n. delivery has been explored as a route to target drugs directly to the brain via the olfactory neurons, providing more opportunities for drugs to enter the central nervous system (CNS). However, various formulation factors should be considered while designing an intranasal drug delivery system5. The formulation should provide a rapid transport of drug across the nasal mucosa and a longer residence time in the nasal cavity to overcome nasal mucociliary clearance6. Nasal mucociliary clearance is one of the most important limiting factors for nasal drug delivery. It severely limits the time allowed for drug absorption to occur and effectively rules out sustained nasal drug administration. However, mucoadhesive polymers can be used to increase the nasal residence time, thus allowing longer absorption times, and achieve a more intimate contact with the nasal mucosa, which in turn can result in a higher concentration gradient and subsequent increased absorption of drugs7. Another important limiting factor in nasal application is the low permeability of the nasal mucosa for the drugs. An absorption enhancement mechanism is required for co-administration of drugs with either mucoadhesive polymers or penetration enhancers or combinations of the two. An increase in bioavailability of up to 82% was obtained by developing a mucoadhesive temperaturemediated in situ gel of ropinirole using CS and hydroxyl propyl methyl cellulose as a gelling agent to enhance intranasal delivery to the brain8. Mustafa and associates developed a nanoemulsion (o/w) of ropinirole with Sefsol 218, tween 80, Transcutol and water as matrix, surfactant, cosurfactant, and aqueous phase. Ex vivo nasal permeation of drug from the nanoemulsion showed highly significant permeation compared with a ropinirole suspension9. In the present study, it was planned to incorporate ropinirole into nanoparticles, which have been shown to protect drugs from the degrading milieu in the nasal cavity, as well as to facilitate their transport across the mucosal barriers10,11. The residence time of nanoparticles in nasal cavity is limited by mucociliary clearance, thus limiting the complete release and absorption of drugs from i.n. administered nanoparticles compared with gels and nanoemulsions. All these limitations have made mucoadhesive nanoparticles with enhanced permeability a better alternative. Fazil and associates, Haque and associates, and Md. and associates reported improved nose-to-brain delivery of various agents using nanoparticles that resulted in increased brain uptake after nasal administration, enhancing bioavailability to achieve a high benefit to risk ratio12–14. The main purpose of this study was to prepare and characterize ropinirole-loaded chitosan nanoparticles (CSNPs) and evaluate their performance in vitro and in vivo in rats using biodistribution studies.
Materials and methods
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molybdenum-99 (99 m) by solvent extraction method, was provided by Regional Center for Radiopharmaceutical Division (Northern Region), Board of Radiation and Isotope Technology, New Delhi, India. All other reagents and chemicals used were of analytical reagent grade without further purification. Preparation of CSNPs and drug-loaded nanoparticles The CSNPs were prepared by ionic gelation of chitosan with TPP as reported by Calvo and coworkers15. For this purpose, chitosan was dissolved in 2% (V/V) acetic acid at various concentrations of 0.5, 1, 1.5, and 2 mg/ml. CSNPs were prepared by the drop wise addition of different volumes of TPP (1, 2, 3, 4, and 5 ml) solution at concentrations of 0.5, 1, 1.5, and 2.0 mg/ml to 10 ml chitosan solution at room temperature under mechanical agitation for 30 min. By visual observation, the systems were classified as clear solutions, opalescent suspension, and aggregates. A clear solution was observed when both the chitosan and the TPP concentration were too small, whereas aggregates were formed spontaneously when they were too large. A zone of opalescent suspension of colloidal particles was found when the chitosan and TPP concentrations were appropriate. Placebo CSNPs were formed upon incorporation of 4 ml of TPP solution (0.571 mg/ml) into 10 ml of chitosan solution (1.428 mg/ml). For the preparation of RH-loaded CSNPs (RH-CSNPs), drug was added in different amounts (1, 2, 3, 4, and 5 mg) in CS solution with constant stirring prior to the addition of TPP solution. The resultant NPs were concentrated by ultra-centrifugation at 10 000 g at 4 C for 40 min. The supernatant was used to determine encapsulation and drug-loading (DL) efficiency and the pellets were washed with distilled water and then freeze-dried for further characterization study16. Physicochemical characterization of CSNPs Particle size analysis and zeta potential Photon correlation spectroscopy using a Zetasizer Nano ZS (Malvern Instruments, Worcestershire, UK) was used to measure the average nanoparticle size and particles size distribution (polydispersity index) of CSNPs and RH-CSNPs. PCS technique is a powerful and versatile tool for estimating the particle size distribution of fine-particle materials ranging from a few nanometers to several micrometers. The zeta potential is a very useful way of evaluating the stability of any colloidal system. It was determined based on an electrophoretic light scattering (ELS) technique with the Zetasizer Nano ZS. The particle size and the shape were determined for the prepared NPs by using transmission electron microscopy (TEM) (Morgagni 2680 TEM, Boston, MA). A drop of nanosuspension was placed on a paraffin sheet and carbon-coated grid was put on the sample and left for 1 min to allow NPs to adhere on the carbon substrate. The remaining drug suspension was removed by adsorbing the drop using the corner of a piece of filter paper. Then the grid was placed on the drop of phosphotungstate (1%) for 10 s. The remaining solution was removed by absorbing the liquid with a piece of filter paper and the sample was air dried. The sample was examined by TEM.
Materials
Encapsulation and drug-loading efficiency of RH CSNPs
RH was obtained as a gift from IND-SWIFT Laboratories, Chandigarh, India. Chitosan (85% deacetylated) and tripolyphosphate (TPP) were purchased from Sigma-Aldrich, St. Louis, MO. Stannous chloride dihydrate (SnCl22H2O) was purchased from Sigma-Aldrich, St. Louis, MO. Instant thin layer chromatography silica gel strips were purchased from Gelman Science Inc, Anne Arbor, MI. Sodium pertechnetate, separated from
The encapsulation efficiency (EE) and drug-loading (DL) efficiency of NPs were determined by separation of NPs from the aqueous medium containing non-associated RH by ultracentrifugation at 10 000 g at 4 C for 40 min. The amount of free RH in the supernatant was measured by HPLC (Shimadzu LC-4A, Kyoto, Japan) equipped with a UV detector and reversed phase column (250 mm 4.6 mm, a particle size of 5 mm). The mobile
Intranasal delivery of ropinirole-loaded nanoparticles
DOI: 10.3109/03639045.2014.991400
phase used was a mixture of methanol and ammonium acetate pH 7. The flow rate was 1.0 ml/min at 25 C, and the wavelength was set at 250 nm. The EE and DL of drug-loaded CSNPs were calculated as per equations given below: Encapsulation efficiancy ðEEÞ ¼
Drug loading ðDLÞ ¼
Total drug Free drug 100 Total drug
Total drug Free drug 100 Nanaoparticles weight
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In vitro release studies The in vitro release studies of drug (RH) from CSNPs were performed using cellulose membrane dialysis tubing (mol. wt. cut-off: 12 000 Da, flat with 25 mm, a diameter of 16 mm, a capacity of 60 ml ft) (Sigma Aldrich Chemicals, Pvt. Ltd, St. Louis, MO). The RH-CSNPs samples (2 ml) were enclosed in dialysis bags and incubated in 100 ml phosphate buffer (pH 7.4) with shaking in water bath at 37 C. At predetermined time intervals, 5 ml sample was withdrawn from the incubation medium and analyzed for drug (RH) content by UV spectrophotometry (Shimadzu, Kyoto, Japan). The samples (5 ml) withdrawn were always replaced with an equivalent amount of release media. The amount of drug released was calculated using a calibration curve of RH in phosphate buffer at pH 7.417. Release kinetics Data obtained from the in vitro release studies of RH-CSNPs were fitted to various kinetic models namely zero order (fraction drug release versus time), first order (log % drug remaining versus time), Higuchi model (fraction drug release versus square root of time), and Peppas model (%R ¼ Ktn, where %R is the percentage drug release, K is the kinetic constant and n is the release exponent and is a measure of release mechanism) to interpret the mechanism and kinetics of drug release18. R2 values were calculated for the linear curves obtained by regression analysis of the above plots. The n-values were obtained from the slope of above equation. If the value of n is 0.43 or less, the release of drugs from the formulation follows a Fickian diffusion mechanism, while the higher values of n (0.435n50.85) indicate a nonFickian mechanism of drug release (anomalous transport). The non-Fickian mechanism corresponds to coupled diffusion/polymer relaxation. If the n value is 0.85, the drug release follows zero order and case II transport. The case II transport generally refers to the dissolution of the polymeric matrix due to the relaxation of the polymer chain. However, the mechanism of drug release is regarded as super case-II transport if n values are higher than 0.85. This mechanism could result from increased plasticization at the relaxing boundary (gel layer)19. Ex vivo nasal permeation studies The permeation study was performed using porcine nasal mucosa obtained from the local slaughter house by the diffusion cell system (Electrolab, Mumbai, India) having 11 ml capacity and 2.404 cm2 permeation area. The mucosal layer faced the drug compartment of the cell, whereas the non-mucosal side was in contact with the receptor compartment. The permeation study was performed using phosphate buffer (pH 7.4) as a dissolution medium with continuous stirring with a magnetic beads at a speed of 100 rpm in a hot air oven maintained at 37 ± 0.5 C. At predetermined time points, 1 ml samples were withdrawn from the receptor chamber. The withdrawn samples were replaced with an equivalent amount of phosphate buffer solution pH 7.4 after each
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sampling, for a period of 24 h. The samples withdrawn were filtered and used for analysis. The amount of drug permeated was determined using the UV–VIS spectrophotometer. All the experiment was performed in triplicate and averaged. Pharmacoscintigraphy study in rats Gamma scintigraphy studies were carried out to determine the location of nanoparticles following i.n. administration and the extent of transit through the nose to the brain. For this study, the nanoparticles were labeled with 99mTc-pertechnetate. Permission for the use of experimental animals was obtained from institutional animal ethical committees (INMAS, Delhi, India). The protocol number of ethical committee permission was INM/TS/IEC/007(07). The experimental animals used were Swiss albino rats (male, aged 4–5 months) weighing between 200 and 250 g. Preparation of radiolabeled-drug formulation of RH and RH-CSNPs RH solutions and ropinirole-loaded CSNP (RH-CSNP) suspension were radiolabeled using 99mTc by a direct labeling method. One milliliter of either RH (5 mg/ml) or RH-CSNPs (5 mg/ml) was taken and stannous chloride dihydrate solution (1 mg/ml in 1N HCl) was added. The pH was adjusted to 6–7 using 1 N sodium hydroxide solution. About 1 ml of sterile 99mTcpertechnetate (200 mCi/100 ml) was added gradually over a period of 60 s to the resultant mixture, with continuous mixing. The mixture was incubated at room temperature for 30 min with continuous nitrogen purging. The final volume was made up to 2.50 ml using normal saline. The radiolabeling efficiency was assessed using ascending instant thin layer chromatography. Instant thin layer chromatography-silica gel (ITLC-SG) strips were used as the stationary phase and acetone 100% as the mobile phase. Free technetium moves with the solvent front whereas any reduced or hydrolyzed technetium stays at the base along with the conjugated one thereby effecting partial separation with regard to the purification. The radioactivity counts of the radiolabeled formulation on the strip were determined using the gamma counter and its percent radiolabeling was calculated by using following equation20:
radioactivity ðcountsÞ retained in the lower half of the strip 100 % radiolabeling ¼ initial radioactivity associated ðtotal count presentÞ with the strip The radiochemical purity of 99mTc-RH (Technetium-99 m labeled RH) and 99mTc-RH-CSNPs (Technetium-99 m labeled RH-CSNPs) was also assessed using ascending instant thin layer chromatography. Silica gel-coated fiber glass sheets (Gelman Sciences, Inc., Ann Arbor, MI) and dual solvent systems consisting of acetone and pyridine:acetic acid:water (3:5:1.5) were used as mobile phases. The reduced/hydrolyzed technetium remained at the point of application, whereas free pertechnetate and labeled complex moved with the solvent front. The counts on the strip were determined using the gamma counter and the percent radiochemical purity was calculated by using following equation: %
R ðcounts present in the lower part of the strip 100Þ ¼ H ðtotal count present in the stripÞ
The effect of incubation time, pH, and stannous chloride concentration on radiolabeling efficiency was studied to achieve optimum reaction conditions. The optimized radiolabeled
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formulations were assessed for in vitro stability in normal saline and in rat serum. Consequently, the optimized stable radiolabeled formulations were used to study bio-distribution in rats.
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Biodistribution studies Three rats for each formulation per time point were used in the study. The radiolabeled drug formulation of 99mTc-RH (200 mCi/ 100 ml) containing 0.017–0.03 mg RH (equivalent to 0.09 mg/kg body weight) was administered (50 ml) in each nostril. Formulations of RH solution and mucoadhesive nanoparticles (RH-CSNPs) suspension were instilled into the nostrils with the help of canula (size 18/20) at the delivery site. The rats were held from the back, in upright position during nasal administration. The rats were anesthetized with 10% chloral hydrate solution and sacrificed humanely by the cervical dislocation method at different time intervals and the blood was collected using cardiac puncture. Subsequently, brain and other tissues (heart, liver, lungs, spleen, intestine, and kidney) were dissected, washed twice using normal saline, made free from adhering tissue/fluid, and weighed. The radioactivity present in each tissue/organ was measured using a shielded well-type gamma scintillation counter. Radiopharmaceutical uptake per gram in each tissue/organ was calculated as a fraction of administered dose using the equation given below21: %
Radioactivity Count in sample of tissue ¼ 100 g Wt of sample Total count injected
Gamma scintigraphic studies Radiolabeled formulation of 99mTc-RH (200 mCi/100 ml) containing 0.017–0.03 mg RH (equivalent to 0.09 mg/kg body weight) was administered (50 ml) in each nostril. Formulations of control RH and nanoparticulate RH were instilled into the nostrils with the help of a cannula (size 18/20), having 0.1 mm internal diameter at the delivery site. The rats were held from the back, in an upright position during nasal administration. The rats were then placed on a board and images were captured using single positron emission computerized tomography (SPECT, eNTEGRA version, Entegra, Lincoln, MA) gamma camera22,23. The scintigraphy images of RH solution and mucoadhesive nanoparticulate RH-CSNPs were observed at different time intervals.
Statistical analysis All data are reported as mean ± SD and the differences between the groups were tested using Student’s t-test at the level of p50.05. More than two groups were compared using ANOVA and the difference greater at p50.05 was considered significant.
Result and discussions Particle size analysis and zeta potential In the present work, CSNPs of RH were prepared by the ionic gelation method. Placebo CSNP formulas were first established with different polymer concentrations and cross-linker concentrations, so as to obtain a small particle size and a mono-modal particle size distribution. These NPs were optimized on the basis of visual observation (clear solution, opalescent suspension, and aggregates). A zone of opalescent suspension corresponding to a suspension of small particles as a function of CS and TPP concentration in the final suspension was observed. For this purpose, polymer solution at various concentrations of 0.5, 1, 1.5, and 2 mg/ml and different volumes of TPP (1, 2, 3, 4, and 5 ml) solution at concentrations of 0.5, 1, 1.5, and 2.0, mg/ml were taken for preparation of NPs. After visual observation of all formulations, three formulations with an opalescent color and without any precipitation were selected (Table 1). These three preparations (F1, F2, and F3) were composed of 1 mg/ml TPP, 1 mg/ml CS (3.3:1), 1.5 mg/ml TPP, 1.5 mg/ml CS (2.5:1), and 2 mg/ml TPP, 2 mg/ml CS (2.5:1). The selected formulations were further characterized by PCS to observe particle size, particle size distribution, and finally polydispersity index (PDI) to confer unimodal particle size distributions (Table 2). The results of PCS revealed that the particle size of placebo containing F1, F2, and F3 formulations were 446.3 ± 3.34, 380.9 ± 2.73, and 156 ± 2.48 nm particle sizes. The value of PDI can range from 0.00 (for mono-dispersed systems) to 1.00 (for highly dispersed systems), where PDI value greater than 0.50 indicates a relatively broader size distribution24. The PDI of the three formulations F1, F2, and F3 was 0.582 ± 0.12, 0.536 ± 0.65, and 0.41 ± 0.23, respectively (Table 2). The comparatively lower value of PDI (0.41) shows unimodal particle size distribution. From the result of PCS, it was concluded that the optimized concentrations of chitosan and TPP for the formation of nanoparticles were 1.428 mg/ml and 0.571 mg/ml, respectively, and the ratio of CS/ TPP was 2.5:1. For formulation of drug-loaded NPs, different concentrations (1, 2, 3, 4, and 5 mg/ml) of RH were added to the
Table 1. Results of effect of different concentrations of CS and TPP on the basis of visual observation. Formulation code F1 F2 F3
CS/TPP ratio
Volume of CS added (ml)
Volume of TPP added (ml)
Final concentration of CS (mg/ml)
Final concentration of TPP (mg/ml)
Visual observation
3.3:1 2.5:1 2.5:1
10 10 10
3 4 4
0.769 1.071 1.428
0.230 0.428 0.571
Opalescent without ppt Opalescent without ppt Opalescent without ppt
Table 2. Particle size and particle size distribution of placebo formulation on the basis of PCS. Formulation code F1 F2 F3
CS/TPP ratio
Volume of CS added (ml)
Volume of TPP added (ml)
Final concentration of CS (mg/ml)
Final concentration of TPP (mg/ml)
Particle* size (nm) (DLS)
Polydispersity index*
3.3:1 2.5:1 2.5:1
10 10 10
3 4 4
0.769 1.071 1.428
0.230 0.428 0.571
446.3 ± 3.34 380.9 ± 2.73 156.5 ± 2.48
0.582 ± 0.12 0.536 ± 0.65 0.41 ± 0.23
*Each experiment was performed in triplicate (n ¼ 3).
Intranasal delivery of ropinirole-loaded nanoparticles
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DOI: 10.3109/03639045.2014.991400
optimized placebo formulation (F3) to produce formulations F4, F5, F6, F7, and F8, respectively. These selected formulations were also characterized by PCS to observe particle size, particle size distribution, and finally PDI to confer unimodal particle size distributions (Table 3). Of these formulations, F8 was observed to have better particle size and PDI (173.7 ± 2.32 nm and 0.39 ± 0.03, respectively) (Figure 1). The particle size of optimized CSNPs containing 1.428 mg/ml CS and 0.571 mg/ml TPP was less compared with the particle size of optimized RH-CSNPs (p50.05). The probable reason could be that the size of RH-CSNPs increased when RH was encapsulated, because some RH also could be absorbed on the surface of NPs14. The particle size and the shape of optimized drug-loaded CSNPs were further characterized by TEM. The particles size was found in the range of 82–129 nm (Figure 2). This apparent discrepancy between PCS and TEM can be explained by the dehydration of CSNPs during sample preparation for TEM imaging. The PCS measures the apparent size of particles, including hydrodynamic layers that form around the hydrophilic particles leading to an overestimation of NPs size. The TEM images indicated that RH-CSNPs formed using the ionic gelation method were spherical in shape, having rough surface25. The zeta potential is a measure of the charge of the particles; as such the larger the absolute value of the zeta potential, the larger the amount of charge of the surface. Thus, the zeta potential represents an index for particle stability26. A physically stable nanosuspension solely stabilized by electrostatic repulsion will have a minimum zeta potential of ± 30 mV27. The mean zeta potential of drug-loaded CSNPs (formulation F8) was +32.7 ± 1.5 mV which revealed that prepared formulation was stable. CSNPs are all positively charged which is a typical characteristic of chitosan/TPP particles. This can be explained by the particle formation mechanism. The positively charged amine groups are neutralized by their interaction with the negative charge in TPP molecules. The residual amino groups would be
responsible for the positive potential. The higher zeta potential in a certain range implied that RH-CSNPs are more stable28. Encapsulation efficiency (EE) and drug loading (DL) of ropinirole-loaded nanoparticles The DL and EE of drug-loaded NPs are very important to evaluate the process efficiency of different methods of preparation of NPs in order to achieve higher drug loads (where the polymeric NPs work as the carriers or reservoirs for sustained drug delivery and to reduce the drug loss). The EE and DL of drug-loaded formulations increased from 52.2 to 69.6 % and from 7.6 to 13.8 % when the concentration of RH was increased from 1 to 5 mg/ml (Table 3). An exception to this was formulation F6, in which an increased drug concentration (3 mg/ml) resulted in a reduced DL29. It was concluded that by increasing the initial concentration of drug the DL of NPs increased but the EE of NPs decreased. Based on the results of particle size, PDI, zeta potential, DL and EE of formulation, F8 was taken as the optimized formulation. The composition and the characterization of the optimized formula for drug-loaded CSNPs are given in Table 4. In vitro release studies For brain targeted RH-loaded nanoparticles, which were meant for nasal administration, PBS, pH 7.4 was used as the release medium. The release profile of RH from CSNPs is shown in Figure 3. The percentage drug release profile showed a sustained release of the drug from the formulation. It was apparent that RH released from the formulation in vitro showed a rapid initial release for 1 h (35.10% ± 1.56) followed by slow drug release for 18 h (89.18 ± 2.43)30. The initial rapid dissolution process and burst release of the drug suggested that the release medium penetrated into the particles due to the hydrophilic nature of chitosan and dissolved the entrapped drug. In addition, the NPs could adsorb RH due to their huge specific surface area. These adsorptions of RH also contribute to burst release of the drug31.
Table 3. Effect of ropinirole hydrochloride concentration on drug-loading and encapsulation efficiencies. Formulation code F4 F5 F6 F7 F8
CS/TPP ratio
Amount of drug added (mg)
Particle* size (mean ± S.D) (nm)
DL* (wt%) (mean ± SD)
EE* (wt%) (mean ± SD)
2.5:1 2.5:1 2.5:1 2.5:1 2.5:1
1 2 3 4 5
142.65 ± 1.63 158.12 ± 1.71 162.37 ± 2.69 166.94 ± 2.55 173.7 ± 2.32
7.6 ± 0.9 12.3 ± 1.1 9.4 ± 0.9 13.1 ± 1.2 13.8 ± 1.7
52.2 ± 3.3 57.3 ± 4.1 59.1 ± 3.2 68.5 ± 3.1 69.6 ± 3.3
*Each experiment was performed in triplicate (n ¼ 3).
Figure 1. Particle size and particle size distribution of optimized ropinirole-loaded chitosan nanoparticles (F8, p50.05).
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Table 4. The optimized formula for ropinirole-loaded chitosan nanoparticles (RH-CSNPs).
Formulation code F8
CS/TPP ratio
Final concentration of CS (mg/ml)
Final concentration of TPP (mg/ml)
Amount of drug added (mg)
Particle size* (mean ± SD) (nm)
2.5:1
1.428
0.571
5
173.7 ± 2.32
PDI*
DL* (wt%) (mean ± SD)
EE* (wt%) (mean ± SD)
Mean Zeta potential (mV)
0.39 ± 0.03
13.8 ± 1.7
69.6 ± 3.3
+32.7 ± 1.5
*Each experiment was performed in triplicate (n ¼ 3).
Table 5. Percentage labeling efficiency of RH and RH-CSNPs in saline and serum in 100 ml of SnCl2 (1 mg/ml).
100 90 % drug release
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Figure 2. TEM photographs of optimized ropinirole loaded chitosan nanoparticles (F8).
80 70
% LE of RH
60 50 40 30 20 10 0 0
5
10
15
20
Time (hr)
Figure 3. In vitro release studies of optimized ropinirole-loaded chitosan nanoparticles.
Release kinetics The release kinetics was characterized by fitting the data obtained from in vitro release studies of NPs into standard release equations (zero order, first-order, Higuchi model, and Korsmeyer–Peppas model). The model that best fits the release data was selected based on the correlation coefficient value of various models. The results indicated that release of drug from NPs was diffusion controlled, as indicated by higher r2 values (0.95) in the Higuchi model. When the release data were analyzed using the Korsmeyer–Peppas equation, the value of release exponent n was less than 0.43 and indicated that the mechanism of drug release from the RH-CSNPs was by Fickian diffusion18. Ex vivo nasal permeation The cumulative amount of drug that permeated (CADP) through nasal mucosa from RH-loaded NPs was 76.1 ± 2.32% whereas only 35.6 ± 1.73% was found to permeate from pure drug solution. The steady-state flux and permeability coefficient of drug solution through the nasal mucosa were 4.10 mgcm2 h1 and
% LE of RH-CSNPs
Time (h)
Serum
Saline
Serum
Saline
0.5 1 2 4 6 20 24
97.58 97.79 98.63 99.23 99.12 97.73 96.38
98.36 98.54 99.23 99.75 97.54 94.86 94.78
96.05 99.59 99.46 99.65 92.47 99.20 96.54
97.02 98.86 99.6 99.7 96.28 93.28 91.45
0.41 102 cm2 h1, respectively, whereas the steady-state flux and permeability coefficient of drug-loaded CSNPs were 10.62 mgcm2 h1 and 1.06 102 cm2 h1, respectively. The overall improvement ratio was 2.59. Biodistribution studies RH was effectively labeled using 99mTc and the radiolabeled RH, and RH-CSNPs formulation was optimized for maximum labeling efficiency and stability. The radiochemical purity achieved for RH and RH-CSNPs was found to be 97.62% and 95.5%, respectively, when evaluated for reduced/hydrolyzed 99mTc and free 99mTc. The pH range of 6.0–7.0 and 100 ml of stannous chloride with an incubation time of 60 min for RH-CSNPs and 30 min for RH were selected as conditions for the optimum radiolabeling. The radiolabeled complexes of RH formulations were found to be stable in saline and in rat serum up to 4 h (Table 5). Thus, it was concluded that the drug can be investigated for biodistribution studies using a radiolabeling technique in a nanoparticulate formulation. Biodistribution studies of 99mTc-RH formulations following intranasal administration (RH and RH-CSNPs) were performed
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DOI: 10.3109/03639045.2014.991400
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Table 6. Biodistribution data of 99mTc-RH (i.n.) and 99mTc-RH-CSNPs (i.n.) at different time intervals in normal Swiss albino rats. Formulation RH (i.n.) RH-CSNPs (i.n.) RH (i.n.) RH-CSNPs (i.n.)
Organs
0.5 h
1h
2h
4h
6h
8h
Brain Blood Brain Blood Brain/blood Brain/blood
0.808 ± 0.03 3.221 ± 0.32 0.202 ± 0.04 0.522 ± 0.07 0.251 ± 0.09 0.386 ± 0.57
0.952 ± 0.07 4.379 ± 0.12 0.278 ± 0.09 0.803 ± 0.39 0.217 ± 0.58 0.346 ± 0.23
0.947 ± 0.02 4.809 ± 0.22 0.264 ± 0.02 1.219 ± 0.13 0.196 ± 0.09 0.216 ± 0.15
0.422 ± 0.02 2.498 ± 0.09 0.245 ± 0.05 1.209 ± 0.39 0.168 ± 0.22 0.202 ± 0.12
0.217 ± 0.03 1.427 ± 0.34 0.218 ± 0.08 1.186 ± 0.34 0.152 ± 0.08 0.183 ± 0.23
0.107 ± 0.05 0.826 ± 0.54 0.176 ± 0.04 0.980 ± 0.36 0.129 ± 0.09 0.179 ± 0.11
Each experiment was performed in triplicate (n ¼ 3).
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Figure 4. Gamma scintigraphic images for RH solution administered intranasally in Swiss albino rat.
Figure 5. Gamma scintigraphic images for ropinirole-loaded chitosan nanoparticles (RH-CSNPs) formulation administered intranasally in Swiss albino rat.
using Swiss albino rats and the radioactivity was estimated at different intervals up to 8 h. The major accumulation of the activity in terms of % administered dose per whole organ was in the liver (7.210 ± 0.52), followed by the kidneys (6.862 ± 0.62), intestine (4.862 ± 0.45), and the lungs (4.640 ± 0.92). The brain– blood ratio of the drug at all sampling time points for different formulations was also calculated (Table 6). The RH concentrations in the brain following the i.n. of RH-CSNPs were found to be significantly higher at all the time points compared with RH solution. The brain–blood ratios of 0.251 ± 0.09 and 0.386 ± 0.57 of RH (i.n.) and RH-CSNPs (i.n.), respectively, at 0.5 h are indicative of direct nose to brain transport, bypassing the BBB, hence demonstrating the superiority of nose to brain delivery of RH by mucoadhesive nanoparticles32,33. Gamma scintigraphic studies In order to visualize brain uptake via the i.n. route, gamma scintigraphic studies were performed in rats, with the 99mTc-RH
solution and RH-CSNPs being instilled in each nostril. Gamma scintigraphic images were taken at 30 min, 1 h, and 2 h for both formulations (Figures 4 and 5). The scintigrams clearly demonstrated the accumulation of the formulations in the brain following intranasal administration. In the case of the RH solution, the activity was higher in the brain initially and the presence of some radioactivity in the esophagus following i.n. administration suggested that a part of the formulation could have been absorbed from the gastrointestinal tract. The radioactivity in the brain decreased at 1 h and 2 h (Figure 4). However, in the case of RH-CSNPs, the formulation showed a sustained effect for 2 h in the brain; this may be attributed to the mucoadhesiveness of the prepared formulation (Figure 5). The scintigraphy images were consistent with the results shown in Table 4 and a high uptake of RH-CSNPs into the brain was observed. The gamma scintigraphic images revealed mucoadhesion of the drug was greater when administered as a polymeric nanoparticulate formulation, compared with the administration as a drug solution; this could lead to maximum drug retention at the site of action, with a slow release
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from the nasal pathway34. Thus, it can be concluded from the in vivo studies that i.n. administration of nanoparticulate RH will deliver more drug to the target site compared with an RH solution, with a consequent increase in bioavailability.
Conclusion
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A polymeric nanoparticulate carrier system developed and characterized for brain targeting of RH by intranasal administration showed sustained release profiles for up to 18 h and were optimized for various performance parameters, including particle size, % encapsulation efficiency, and % drug loading. It can be concluded from the animal studies that i.n. administration of RHCSNPs will deliver higher drug to the target site compared with that delivered by an RH solution, with a consequent increase in bioavailability.
Acknowledgements The authors thank Professor Brian L. Furman (University of Strathclyde, Glasgow, UK) for critical reading of the manuscript and English editing.
Declaration of interest The authors report no conflict of interest. The authors are responsible for the content and writing of the article. The authors are thankful to All India Council of Technical Education (AICTE), New Delhi, India for providing financial assistance to project.
References 1. Paulus W, Jellinger K. The neuropathologic basis of different clinical subgroups of Parkinson’s disease. J Neuropathol Exp Neurol 1991;50:743–55. 2. Hely MA, Morris J, Reid WGJ. Sydney multicenter study of Parkinson’s disease: non-motor problems dominate at 15 years. Mov Disord 2005;20:190–9. 3. Md S, Haque S, Sahni JK, et al. New non-oral drug delivery systems for Parkinson’s disease treatment. Expert Opin Drug Deliv 2011;8: 359–74. 4. Kaye CM, Nicholls B. Clinical pharmacokinetics of ropinirole. Clin Pharmacokinet 2000;39:243–54. 5. Md S, Kumar M, Baboota S, et al. Preparation, characterization and evaluation of bromocriptine loaded chitosan nanoparticles for intranasal delivery. Sci Adv Mater 2012;4:961–5. 6. Ugwoke MI, Verbeke N, Kinget R. The biopharmaceutical aspects of nasal mucoadhesive drug delivery. J Pharm Pharmacol 2001;53: 3–21. 7. Critchley H, Davis SS, Farraj NF. Nasal absorption of desmopressin in rats and sheep. Effect of a bioadhesive microsphere delivery system. J Pharm Pharmacol 1994;46:651–6. 8. Khan S, Patil K, Bobade N, et al. Formulation of intranasal mucoadhesive temperature-mediated in situ gel containing ropinirole and evaluation of brain targeting efficiency in rats. J Drug Target 2010;18:223–34. 9. Mustafa G, Baboota S, Ali J, Ahuja A. Formulation development of chitosan coated intra nasal ropinirole nanoemulsion for better management option of Parkinson: an in vitro–ex vivo evaluation. Curr Nanosci 2012;29:293–300. 10. Dondeti P, Zia H, Needham TE. Bioadhesive and formulation parameters affecting nasal absorption. Int J Pharm 1996;127: 115–33. 11. Illum L, Farraj N, Critchley H. Nasal administration of gentamicin using a novel microsphere delivery system. Int J Pharm 1988;46: 261–5. 12. Fazil M, Md S, Haque S, et al. Development and evaluation of rivastigmine loaded chitosan nanoparticles for brain targeting. Eur J Pharm Sci 2012;47:6–15.
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13. Haque S, Md S, Fazil M, et al. Venlafaxine loaded chitosan NPs for brain targeting: pharmacokinetic and pharmacodynamic evaluation. Carbohydr Polym 2012;89:72–9. 14. Md S, Khan RA, Mustafa G, et al. Bromocriptine loaded chitosan nanoparticles intended for direct nose to brain delivery: pharmacodynamic, pharmacokinetic and scintigraphy study in mice model. Eur J Pharm Sci 2013;48:393–405. 15. Calvo P, Remunan-Lopez C, Vila-Jata JL, Alonso MJ. Chitosan and chitosan: ethylene oxide–propylene oxide block copolymer nanoparticles as novel carriers for proteins and vaccines. Pharm Res 1997;14:1431–6. 16. Papadimitriou S, Bikiaris D, Avgoustakis K, et al. Chitosan nanoparticles loaded with dorzolamide and pramipexole. Carbohydr Polym 2008;73:44–54. 17. Marchal-Heussler L, Maincent P, Hoffman M, et al. Antiglaucomatous activity of betaxolol chlorhydrate sorbed onto different isobutyl cyanoacrylate nanoparticles preparations. Int J Pharm 1990;58:115–22. 18. Korsemeyer RW, Gurny R, Docler E, et al. Mechanism of solute release from porous hydrophilic polymer. Int J Pharm 1983;15: 25–35. 19. Ritger PL, Peppas NA. A simple equation for description of solute release II. Fickian and anomalous release from swellable devices. J Control Release 1987;5:37–42. 20. Samad A, Sultana Y, Khar RK, et al. Radiolabeling and evaluation of alginate blend-isoniazid microspheres by 99mTc for the treatment of tuberculosis in rabbit model. J Drug Target 2008;16: 509–15. 21. Saha GB, ed. Methods of radiolabeling. In: Physics and radiobiology of nuclear medicine. New York: Springer-verlag; 1993:100–6. 22. Koziara JM, Lockman PR, Allen DD, Mumper RJ. In situ blood– brain barrier transport of nanoparticles. Pharm Res 2003;20:1772–8. 23. Pietrowky R, Thieman A, Kern W, et al. A nose–brain pathway for psychotropic peptides, evidence from a brain evolved potential study with cholecystokinin. Psychoneuroendocrinology 1996;21:559–72. 24. Avadi MR, Sadeghi AMM, Mohammadpour N, et al. Preparation and characterization of insulin nanoparticles using chitosan and Arabic gum with ionic gelation method. Nanomed: Nanotech Biol Med 2010;6:58–63. 25. Motwani SK, Chopra S, Talegaonkar S, et al. Chitosan-sodium alginate nanoparticles as submicroscopic reservoirs for ocular delivery: formulation, optimization and in vitro characterization. Eur J Pharm Biopharm 2008;68:513–25. 26. Hans ML, Lowman AM. Biodegradable nanoparticles for drug delivery and targeting. Curr Opin Solid State Mater Sci 2002;6: 319–27. 27. Muller RH, Jacobs C, Kayser O. Nanosuspensions as particulate drug formulations in therapy, rationale for development and what we can expect for the future. Adv Drug Deliv Rev 2001;47: 3–19. 28. Wang X, Chi N, Tang X. Preparation of estradiol chitosan nanoparticles for improving nasal absorption and brain targeting. Eur J Pharm Biopharm 2008;70:735–40. 29. Wan A, Gao Li SY, Huili L. Preparation of aspirin and probucol in combination loaded chitosan nanoparticles and in vitro release study. Carbohydr Polym 2009;75:566–74. 30. Zhou SB, Deng XM, Li XH. Investigation on a novel core-coated microspheres protein delivery system. J Control Release 2001;75: 27–36. 31. Sadeghi A, Dorkoosh FA, Avadi MR, et al. Preparation, characterization and antibacterial activities of chitosan, TMC and DEMC nanoparticles loaded with insulin using both the ionotropic gelation and polyelectrolyte complexation methods. Int J Pharm 2008;355: 299–306. 32. Qizhi Z, Jiang X, Xiang W, et al. Preparation of nimodipine loaded microemulsion for intranasal delivery and evaluation of the targeting efficiency to brain. Int J Pharm 2004;275:85–96. 33. Vyas TK, Shahiwala A, Marathe S, Misra A. Intranasal drug delivery for brain targeting. Curr Drug Deliv 2005;2:165–75. 34. Vyas TK, Babber AK, Sharma RK, et al. Preliminary brain targeting studies on intranasal mucoadhesive microemulsions of sumatriptan. AAPS PharmSciTech 2006;7:1–9.
RESEARCH ARTICLE
Design, characterization, and evaluation of intranasal delivery of ropinirole-loaded mucoadhesive nanoparticles for brain targeting
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Omidreza Jafarieh1, Shadab Md1,2, Mushir Ali1, Sanjula Baboota1, J. K. Sahni1,3, Bhavna Kumari1,4, Aseem Bhatnagar5, and Javed Ali1 1
Department of Pharmaceutics, Faculty of Pharmacy, Jamia Hamdard, Hamdard Nagar, New Delhi, India, 2Department of Pharmaceutical Technology, School of Pharmacy, International Medical University (IMU), Bukit Jalil, Kuala Lumpur, Malaysia, 3Department of Pharmaceutics, Khalsa College of Pharmacy, Amritsar, India, 4Department of Pharmaceutics, Dehradun Institute of Technology (DIT), Dehradun, Uttaranchal, India, and 5Department of Radiopharmaceuticals, Institute of Nuclear Medicine and Allied Sciences (INMAS), New Delhi, India Abstract
Keywords
Context: Parkinson disease (PD) is a common, progressive neurodegenerative disorder, characterized by marked depletion of striatal dopamine and degeneration of dopaminergic neurons in the substantia nigra. Objective: The purpose of the present study was to investigate the possibility of targeting an anti-Parkinson’s drug ropinirole (RH) to the brain using polymeric nanoparticles. Materials and methods: Ropinirole hydrochloride (RH)-loaded chitosan nanoparticles (CSNPs) were prepared by an ionic gelation method. The RH-CSNPs were characterized for particle size, polydispersity index (PDI), zeta potential, loading capacity, entrapment efficiency in vitro release study, and in vivo distribution after intranasal administration. Results and discussion: The RH-CSNPs showed sustained release profiles for up to 18 h. The RH concentrations (% Radioactivity/g) in the brain following intranasal administration (i.n.) of RH-CSNPs were found to be significantly higher at all the time points compared with RH solution. The concentration of RH was highest in the liver (7.210 ± 0.52), followed by kidneys (6.862 ± 0.62), intestine (4.862 ± 0.45), and lungs (4.640 ± 0.92) in rats following i.n. administration of RH-CSNPs. Gamma scintigraphy imaging in rats was performed to ascertain the localization of drug in the brain following intranasal administration of formulations. The brain/ blood ratios obtained (0.251 ± 0.09 and 0.386 ± 0.57 of RH (i.n.) and RH-CSNPs (i.n.), respectively) at 0.5 h are indicative of direct nose to brain transport, bypassing the blood–brain barrier (BBB). Conclusion: The novel formulation showed the superiority of nose to brain delivery of RH using mucoadhesive nanoparticles compared with other delivery routes reported earlier.
Brain targeting, biodistribution, gamma scintigraphy, nanoparticles, Parkinson’s disease
Introduction Parkinson disease (PD) is a common, progressive neurodegenerative disorder characterized by marked depletion of striatal dopamine (DA) and degeneration of dopaminergic (DAergic) neurons in the substantia nigra1,2. Clinically, the disease is manifested by both motor and non-motor symptoms. The cardinal motor symptoms include bradykinesia, resting tremor, rigidity, and disturbance of posture and gait; non-motor symptoms include a diminished sense of smell, depression, and sleep disturbance. The prevalence of PD is set to rise significantly in the coming years. In the near future, the patents of some important PD drugs
Address for correspondence: Dr Javed Ali, Department of Pharmaceutics, Faculty of Pharmacy, Jamia Hamdard, Hamdard Nagar, New Delhi 110062, India. Tel: +91 9811312247. Fax: +011 26059633. E-mail: [email protected]
History Received 3 July 2014 Revised 7 November 2014 Accepted 11 November 2014 Published online 11 December 2014
will expire. While new products may be developed, which of these will be successful will remain a challenge3. Ropinirole hydrochloride (RH) is a non-ergot D2/D3 DA agonist with the greatest affinity at the D3 receptors and is used in patients with moderate or advanced PD. It is absorbed rapidly with peak plasma concentrations occurring in 1–2 h. Oral ropinirole therapy is associated with nausea, vomiting, and gastrointestinal disturbances and ropinirole possess low oral bioavailability (approx. 50%) because of the hepatic first pass effect; its adverse effects and frequent dosing schedule contribute to non-compliance among patients4. In 2008, the FDA approved a once daily, prolonged release formulation of RH. This is the first once daily oral DA agonist approved for both early and advanced PD in the US. Once-daily administration resulted in improved compliance and adherence. However, since the target site of ropinirole is the brain, a strategy is desired that not only improves the bioavailability by preventing first pass metabolism but also provides targeting to the receptor site and bypasses the
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blood–brain barrier (BBB). Such a strategy would help to achieve the desired drug concentration at the site of action, as well as reducing availability at non-targeting sites, thus reducing adverse effects. In recent years, the intranasal (i.n.) route has received a great deal of attention as a convenient and reliable method for the systemic administration of drugs. It offers the advantages of simple, cost effective, and convenient administration. Additionally, i.n. delivery has been explored as a route to target drugs directly to the brain via the olfactory neurons, providing more opportunities for drugs to enter the central nervous system (CNS). However, various formulation factors should be considered while designing an intranasal drug delivery system5. The formulation should provide a rapid transport of drug across the nasal mucosa and a longer residence time in the nasal cavity to overcome nasal mucociliary clearance6. Nasal mucociliary clearance is one of the most important limiting factors for nasal drug delivery. It severely limits the time allowed for drug absorption to occur and effectively rules out sustained nasal drug administration. However, mucoadhesive polymers can be used to increase the nasal residence time, thus allowing longer absorption times, and achieve a more intimate contact with the nasal mucosa, which in turn can result in a higher concentration gradient and subsequent increased absorption of drugs7. Another important limiting factor in nasal application is the low permeability of the nasal mucosa for the drugs. An absorption enhancement mechanism is required for co-administration of drugs with either mucoadhesive polymers or penetration enhancers or combinations of the two. An increase in bioavailability of up to 82% was obtained by developing a mucoadhesive temperaturemediated in situ gel of ropinirole using CS and hydroxyl propyl methyl cellulose as a gelling agent to enhance intranasal delivery to the brain8. Mustafa and associates developed a nanoemulsion (o/w) of ropinirole with Sefsol 218, tween 80, Transcutol and water as matrix, surfactant, cosurfactant, and aqueous phase. Ex vivo nasal permeation of drug from the nanoemulsion showed highly significant permeation compared with a ropinirole suspension9. In the present study, it was planned to incorporate ropinirole into nanoparticles, which have been shown to protect drugs from the degrading milieu in the nasal cavity, as well as to facilitate their transport across the mucosal barriers10,11. The residence time of nanoparticles in nasal cavity is limited by mucociliary clearance, thus limiting the complete release and absorption of drugs from i.n. administered nanoparticles compared with gels and nanoemulsions. All these limitations have made mucoadhesive nanoparticles with enhanced permeability a better alternative. Fazil and associates, Haque and associates, and Md. and associates reported improved nose-to-brain delivery of various agents using nanoparticles that resulted in increased brain uptake after nasal administration, enhancing bioavailability to achieve a high benefit to risk ratio12–14. The main purpose of this study was to prepare and characterize ropinirole-loaded chitosan nanoparticles (CSNPs) and evaluate their performance in vitro and in vivo in rats using biodistribution studies.
Materials and methods
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molybdenum-99 (99 m) by solvent extraction method, was provided by Regional Center for Radiopharmaceutical Division (Northern Region), Board of Radiation and Isotope Technology, New Delhi, India. All other reagents and chemicals used were of analytical reagent grade without further purification. Preparation of CSNPs and drug-loaded nanoparticles The CSNPs were prepared by ionic gelation of chitosan with TPP as reported by Calvo and coworkers15. For this purpose, chitosan was dissolved in 2% (V/V) acetic acid at various concentrations of 0.5, 1, 1.5, and 2 mg/ml. CSNPs were prepared by the drop wise addition of different volumes of TPP (1, 2, 3, 4, and 5 ml) solution at concentrations of 0.5, 1, 1.5, and 2.0 mg/ml to 10 ml chitosan solution at room temperature under mechanical agitation for 30 min. By visual observation, the systems were classified as clear solutions, opalescent suspension, and aggregates. A clear solution was observed when both the chitosan and the TPP concentration were too small, whereas aggregates were formed spontaneously when they were too large. A zone of opalescent suspension of colloidal particles was found when the chitosan and TPP concentrations were appropriate. Placebo CSNPs were formed upon incorporation of 4 ml of TPP solution (0.571 mg/ml) into 10 ml of chitosan solution (1.428 mg/ml). For the preparation of RH-loaded CSNPs (RH-CSNPs), drug was added in different amounts (1, 2, 3, 4, and 5 mg) in CS solution with constant stirring prior to the addition of TPP solution. The resultant NPs were concentrated by ultra-centrifugation at 10 000 g at 4 C for 40 min. The supernatant was used to determine encapsulation and drug-loading (DL) efficiency and the pellets were washed with distilled water and then freeze-dried for further characterization study16. Physicochemical characterization of CSNPs Particle size analysis and zeta potential Photon correlation spectroscopy using a Zetasizer Nano ZS (Malvern Instruments, Worcestershire, UK) was used to measure the average nanoparticle size and particles size distribution (polydispersity index) of CSNPs and RH-CSNPs. PCS technique is a powerful and versatile tool for estimating the particle size distribution of fine-particle materials ranging from a few nanometers to several micrometers. The zeta potential is a very useful way of evaluating the stability of any colloidal system. It was determined based on an electrophoretic light scattering (ELS) technique with the Zetasizer Nano ZS. The particle size and the shape were determined for the prepared NPs by using transmission electron microscopy (TEM) (Morgagni 2680 TEM, Boston, MA). A drop of nanosuspension was placed on a paraffin sheet and carbon-coated grid was put on the sample and left for 1 min to allow NPs to adhere on the carbon substrate. The remaining drug suspension was removed by adsorbing the drop using the corner of a piece of filter paper. Then the grid was placed on the drop of phosphotungstate (1%) for 10 s. The remaining solution was removed by absorbing the liquid with a piece of filter paper and the sample was air dried. The sample was examined by TEM.
Materials
Encapsulation and drug-loading efficiency of RH CSNPs
RH was obtained as a gift from IND-SWIFT Laboratories, Chandigarh, India. Chitosan (85% deacetylated) and tripolyphosphate (TPP) were purchased from Sigma-Aldrich, St. Louis, MO. Stannous chloride dihydrate (SnCl22H2O) was purchased from Sigma-Aldrich, St. Louis, MO. Instant thin layer chromatography silica gel strips were purchased from Gelman Science Inc, Anne Arbor, MI. Sodium pertechnetate, separated from
The encapsulation efficiency (EE) and drug-loading (DL) efficiency of NPs were determined by separation of NPs from the aqueous medium containing non-associated RH by ultracentrifugation at 10 000 g at 4 C for 40 min. The amount of free RH in the supernatant was measured by HPLC (Shimadzu LC-4A, Kyoto, Japan) equipped with a UV detector and reversed phase column (250 mm 4.6 mm, a particle size of 5 mm). The mobile
Intranasal delivery of ropinirole-loaded nanoparticles
DOI: 10.3109/03639045.2014.991400
phase used was a mixture of methanol and ammonium acetate pH 7. The flow rate was 1.0 ml/min at 25 C, and the wavelength was set at 250 nm. The EE and DL of drug-loaded CSNPs were calculated as per equations given below: Encapsulation efficiancy ðEEÞ ¼
Drug loading ðDLÞ ¼
Total drug Free drug 100 Total drug
Total drug Free drug 100 Nanaoparticles weight
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In vitro release studies The in vitro release studies of drug (RH) from CSNPs were performed using cellulose membrane dialysis tubing (mol. wt. cut-off: 12 000 Da, flat with 25 mm, a diameter of 16 mm, a capacity of 60 ml ft) (Sigma Aldrich Chemicals, Pvt. Ltd, St. Louis, MO). The RH-CSNPs samples (2 ml) were enclosed in dialysis bags and incubated in 100 ml phosphate buffer (pH 7.4) with shaking in water bath at 37 C. At predetermined time intervals, 5 ml sample was withdrawn from the incubation medium and analyzed for drug (RH) content by UV spectrophotometry (Shimadzu, Kyoto, Japan). The samples (5 ml) withdrawn were always replaced with an equivalent amount of release media. The amount of drug released was calculated using a calibration curve of RH in phosphate buffer at pH 7.417. Release kinetics Data obtained from the in vitro release studies of RH-CSNPs were fitted to various kinetic models namely zero order (fraction drug release versus time), first order (log % drug remaining versus time), Higuchi model (fraction drug release versus square root of time), and Peppas model (%R ¼ Ktn, where %R is the percentage drug release, K is the kinetic constant and n is the release exponent and is a measure of release mechanism) to interpret the mechanism and kinetics of drug release18. R2 values were calculated for the linear curves obtained by regression analysis of the above plots. The n-values were obtained from the slope of above equation. If the value of n is 0.43 or less, the release of drugs from the formulation follows a Fickian diffusion mechanism, while the higher values of n (0.435n50.85) indicate a nonFickian mechanism of drug release (anomalous transport). The non-Fickian mechanism corresponds to coupled diffusion/polymer relaxation. If the n value is 0.85, the drug release follows zero order and case II transport. The case II transport generally refers to the dissolution of the polymeric matrix due to the relaxation of the polymer chain. However, the mechanism of drug release is regarded as super case-II transport if n values are higher than 0.85. This mechanism could result from increased plasticization at the relaxing boundary (gel layer)19. Ex vivo nasal permeation studies The permeation study was performed using porcine nasal mucosa obtained from the local slaughter house by the diffusion cell system (Electrolab, Mumbai, India) having 11 ml capacity and 2.404 cm2 permeation area. The mucosal layer faced the drug compartment of the cell, whereas the non-mucosal side was in contact with the receptor compartment. The permeation study was performed using phosphate buffer (pH 7.4) as a dissolution medium with continuous stirring with a magnetic beads at a speed of 100 rpm in a hot air oven maintained at 37 ± 0.5 C. At predetermined time points, 1 ml samples were withdrawn from the receptor chamber. The withdrawn samples were replaced with an equivalent amount of phosphate buffer solution pH 7.4 after each
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sampling, for a period of 24 h. The samples withdrawn were filtered and used for analysis. The amount of drug permeated was determined using the UV–VIS spectrophotometer. All the experiment was performed in triplicate and averaged. Pharmacoscintigraphy study in rats Gamma scintigraphy studies were carried out to determine the location of nanoparticles following i.n. administration and the extent of transit through the nose to the brain. For this study, the nanoparticles were labeled with 99mTc-pertechnetate. Permission for the use of experimental animals was obtained from institutional animal ethical committees (INMAS, Delhi, India). The protocol number of ethical committee permission was INM/TS/IEC/007(07). The experimental animals used were Swiss albino rats (male, aged 4–5 months) weighing between 200 and 250 g. Preparation of radiolabeled-drug formulation of RH and RH-CSNPs RH solutions and ropinirole-loaded CSNP (RH-CSNP) suspension were radiolabeled using 99mTc by a direct labeling method. One milliliter of either RH (5 mg/ml) or RH-CSNPs (5 mg/ml) was taken and stannous chloride dihydrate solution (1 mg/ml in 1N HCl) was added. The pH was adjusted to 6–7 using 1 N sodium hydroxide solution. About 1 ml of sterile 99mTcpertechnetate (200 mCi/100 ml) was added gradually over a period of 60 s to the resultant mixture, with continuous mixing. The mixture was incubated at room temperature for 30 min with continuous nitrogen purging. The final volume was made up to 2.50 ml using normal saline. The radiolabeling efficiency was assessed using ascending instant thin layer chromatography. Instant thin layer chromatography-silica gel (ITLC-SG) strips were used as the stationary phase and acetone 100% as the mobile phase. Free technetium moves with the solvent front whereas any reduced or hydrolyzed technetium stays at the base along with the conjugated one thereby effecting partial separation with regard to the purification. The radioactivity counts of the radiolabeled formulation on the strip were determined using the gamma counter and its percent radiolabeling was calculated by using following equation20:
radioactivity ðcountsÞ retained in the lower half of the strip 100 % radiolabeling ¼ initial radioactivity associated ðtotal count presentÞ with the strip The radiochemical purity of 99mTc-RH (Technetium-99 m labeled RH) and 99mTc-RH-CSNPs (Technetium-99 m labeled RH-CSNPs) was also assessed using ascending instant thin layer chromatography. Silica gel-coated fiber glass sheets (Gelman Sciences, Inc., Ann Arbor, MI) and dual solvent systems consisting of acetone and pyridine:acetic acid:water (3:5:1.5) were used as mobile phases. The reduced/hydrolyzed technetium remained at the point of application, whereas free pertechnetate and labeled complex moved with the solvent front. The counts on the strip were determined using the gamma counter and the percent radiochemical purity was calculated by using following equation: %
R ðcounts present in the lower part of the strip 100Þ ¼ H ðtotal count present in the stripÞ
The effect of incubation time, pH, and stannous chloride concentration on radiolabeling efficiency was studied to achieve optimum reaction conditions. The optimized radiolabeled
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formulations were assessed for in vitro stability in normal saline and in rat serum. Consequently, the optimized stable radiolabeled formulations were used to study bio-distribution in rats.
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Biodistribution studies Three rats for each formulation per time point were used in the study. The radiolabeled drug formulation of 99mTc-RH (200 mCi/ 100 ml) containing 0.017–0.03 mg RH (equivalent to 0.09 mg/kg body weight) was administered (50 ml) in each nostril. Formulations of RH solution and mucoadhesive nanoparticles (RH-CSNPs) suspension were instilled into the nostrils with the help of canula (size 18/20) at the delivery site. The rats were held from the back, in upright position during nasal administration. The rats were anesthetized with 10% chloral hydrate solution and sacrificed humanely by the cervical dislocation method at different time intervals and the blood was collected using cardiac puncture. Subsequently, brain and other tissues (heart, liver, lungs, spleen, intestine, and kidney) were dissected, washed twice using normal saline, made free from adhering tissue/fluid, and weighed. The radioactivity present in each tissue/organ was measured using a shielded well-type gamma scintillation counter. Radiopharmaceutical uptake per gram in each tissue/organ was calculated as a fraction of administered dose using the equation given below21: %
Radioactivity Count in sample of tissue ¼ 100 g Wt of sample Total count injected
Gamma scintigraphic studies Radiolabeled formulation of 99mTc-RH (200 mCi/100 ml) containing 0.017–0.03 mg RH (equivalent to 0.09 mg/kg body weight) was administered (50 ml) in each nostril. Formulations of control RH and nanoparticulate RH were instilled into the nostrils with the help of a cannula (size 18/20), having 0.1 mm internal diameter at the delivery site. The rats were held from the back, in an upright position during nasal administration. The rats were then placed on a board and images were captured using single positron emission computerized tomography (SPECT, eNTEGRA version, Entegra, Lincoln, MA) gamma camera22,23. The scintigraphy images of RH solution and mucoadhesive nanoparticulate RH-CSNPs were observed at different time intervals.
Statistical analysis All data are reported as mean ± SD and the differences between the groups were tested using Student’s t-test at the level of p50.05. More than two groups were compared using ANOVA and the difference greater at p50.05 was considered significant.
Result and discussions Particle size analysis and zeta potential In the present work, CSNPs of RH were prepared by the ionic gelation method. Placebo CSNP formulas were first established with different polymer concentrations and cross-linker concentrations, so as to obtain a small particle size and a mono-modal particle size distribution. These NPs were optimized on the basis of visual observation (clear solution, opalescent suspension, and aggregates). A zone of opalescent suspension corresponding to a suspension of small particles as a function of CS and TPP concentration in the final suspension was observed. For this purpose, polymer solution at various concentrations of 0.5, 1, 1.5, and 2 mg/ml and different volumes of TPP (1, 2, 3, 4, and 5 ml) solution at concentrations of 0.5, 1, 1.5, and 2.0, mg/ml were taken for preparation of NPs. After visual observation of all formulations, three formulations with an opalescent color and without any precipitation were selected (Table 1). These three preparations (F1, F2, and F3) were composed of 1 mg/ml TPP, 1 mg/ml CS (3.3:1), 1.5 mg/ml TPP, 1.5 mg/ml CS (2.5:1), and 2 mg/ml TPP, 2 mg/ml CS (2.5:1). The selected formulations were further characterized by PCS to observe particle size, particle size distribution, and finally polydispersity index (PDI) to confer unimodal particle size distributions (Table 2). The results of PCS revealed that the particle size of placebo containing F1, F2, and F3 formulations were 446.3 ± 3.34, 380.9 ± 2.73, and 156 ± 2.48 nm particle sizes. The value of PDI can range from 0.00 (for mono-dispersed systems) to 1.00 (for highly dispersed systems), where PDI value greater than 0.50 indicates a relatively broader size distribution24. The PDI of the three formulations F1, F2, and F3 was 0.582 ± 0.12, 0.536 ± 0.65, and 0.41 ± 0.23, respectively (Table 2). The comparatively lower value of PDI (0.41) shows unimodal particle size distribution. From the result of PCS, it was concluded that the optimized concentrations of chitosan and TPP for the formation of nanoparticles were 1.428 mg/ml and 0.571 mg/ml, respectively, and the ratio of CS/ TPP was 2.5:1. For formulation of drug-loaded NPs, different concentrations (1, 2, 3, 4, and 5 mg/ml) of RH were added to the
Table 1. Results of effect of different concentrations of CS and TPP on the basis of visual observation. Formulation code F1 F2 F3
CS/TPP ratio
Volume of CS added (ml)
Volume of TPP added (ml)
Final concentration of CS (mg/ml)
Final concentration of TPP (mg/ml)
Visual observation
3.3:1 2.5:1 2.5:1
10 10 10
3 4 4
0.769 1.071 1.428
0.230 0.428 0.571
Opalescent without ppt Opalescent without ppt Opalescent without ppt
Table 2. Particle size and particle size distribution of placebo formulation on the basis of PCS. Formulation code F1 F2 F3
CS/TPP ratio
Volume of CS added (ml)
Volume of TPP added (ml)
Final concentration of CS (mg/ml)
Final concentration of TPP (mg/ml)
Particle* size (nm) (DLS)
Polydispersity index*
3.3:1 2.5:1 2.5:1
10 10 10
3 4 4
0.769 1.071 1.428
0.230 0.428 0.571
446.3 ± 3.34 380.9 ± 2.73 156.5 ± 2.48
0.582 ± 0.12 0.536 ± 0.65 0.41 ± 0.23
*Each experiment was performed in triplicate (n ¼ 3).
Intranasal delivery of ropinirole-loaded nanoparticles
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DOI: 10.3109/03639045.2014.991400
optimized placebo formulation (F3) to produce formulations F4, F5, F6, F7, and F8, respectively. These selected formulations were also characterized by PCS to observe particle size, particle size distribution, and finally PDI to confer unimodal particle size distributions (Table 3). Of these formulations, F8 was observed to have better particle size and PDI (173.7 ± 2.32 nm and 0.39 ± 0.03, respectively) (Figure 1). The particle size of optimized CSNPs containing 1.428 mg/ml CS and 0.571 mg/ml TPP was less compared with the particle size of optimized RH-CSNPs (p50.05). The probable reason could be that the size of RH-CSNPs increased when RH was encapsulated, because some RH also could be absorbed on the surface of NPs14. The particle size and the shape of optimized drug-loaded CSNPs were further characterized by TEM. The particles size was found in the range of 82–129 nm (Figure 2). This apparent discrepancy between PCS and TEM can be explained by the dehydration of CSNPs during sample preparation for TEM imaging. The PCS measures the apparent size of particles, including hydrodynamic layers that form around the hydrophilic particles leading to an overestimation of NPs size. The TEM images indicated that RH-CSNPs formed using the ionic gelation method were spherical in shape, having rough surface25. The zeta potential is a measure of the charge of the particles; as such the larger the absolute value of the zeta potential, the larger the amount of charge of the surface. Thus, the zeta potential represents an index for particle stability26. A physically stable nanosuspension solely stabilized by electrostatic repulsion will have a minimum zeta potential of ± 30 mV27. The mean zeta potential of drug-loaded CSNPs (formulation F8) was +32.7 ± 1.5 mV which revealed that prepared formulation was stable. CSNPs are all positively charged which is a typical characteristic of chitosan/TPP particles. This can be explained by the particle formation mechanism. The positively charged amine groups are neutralized by their interaction with the negative charge in TPP molecules. The residual amino groups would be
responsible for the positive potential. The higher zeta potential in a certain range implied that RH-CSNPs are more stable28. Encapsulation efficiency (EE) and drug loading (DL) of ropinirole-loaded nanoparticles The DL and EE of drug-loaded NPs are very important to evaluate the process efficiency of different methods of preparation of NPs in order to achieve higher drug loads (where the polymeric NPs work as the carriers or reservoirs for sustained drug delivery and to reduce the drug loss). The EE and DL of drug-loaded formulations increased from 52.2 to 69.6 % and from 7.6 to 13.8 % when the concentration of RH was increased from 1 to 5 mg/ml (Table 3). An exception to this was formulation F6, in which an increased drug concentration (3 mg/ml) resulted in a reduced DL29. It was concluded that by increasing the initial concentration of drug the DL of NPs increased but the EE of NPs decreased. Based on the results of particle size, PDI, zeta potential, DL and EE of formulation, F8 was taken as the optimized formulation. The composition and the characterization of the optimized formula for drug-loaded CSNPs are given in Table 4. In vitro release studies For brain targeted RH-loaded nanoparticles, which were meant for nasal administration, PBS, pH 7.4 was used as the release medium. The release profile of RH from CSNPs is shown in Figure 3. The percentage drug release profile showed a sustained release of the drug from the formulation. It was apparent that RH released from the formulation in vitro showed a rapid initial release for 1 h (35.10% ± 1.56) followed by slow drug release for 18 h (89.18 ± 2.43)30. The initial rapid dissolution process and burst release of the drug suggested that the release medium penetrated into the particles due to the hydrophilic nature of chitosan and dissolved the entrapped drug. In addition, the NPs could adsorb RH due to their huge specific surface area. These adsorptions of RH also contribute to burst release of the drug31.
Table 3. Effect of ropinirole hydrochloride concentration on drug-loading and encapsulation efficiencies. Formulation code F4 F5 F6 F7 F8
CS/TPP ratio
Amount of drug added (mg)
Particle* size (mean ± S.D) (nm)
DL* (wt%) (mean ± SD)
EE* (wt%) (mean ± SD)
2.5:1 2.5:1 2.5:1 2.5:1 2.5:1
1 2 3 4 5
142.65 ± 1.63 158.12 ± 1.71 162.37 ± 2.69 166.94 ± 2.55 173.7 ± 2.32
7.6 ± 0.9 12.3 ± 1.1 9.4 ± 0.9 13.1 ± 1.2 13.8 ± 1.7
52.2 ± 3.3 57.3 ± 4.1 59.1 ± 3.2 68.5 ± 3.1 69.6 ± 3.3
*Each experiment was performed in triplicate (n ¼ 3).
Figure 1. Particle size and particle size distribution of optimized ropinirole-loaded chitosan nanoparticles (F8, p50.05).
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Table 4. The optimized formula for ropinirole-loaded chitosan nanoparticles (RH-CSNPs).
Formulation code F8
CS/TPP ratio
Final concentration of CS (mg/ml)
Final concentration of TPP (mg/ml)
Amount of drug added (mg)
Particle size* (mean ± SD) (nm)
2.5:1
1.428
0.571
5
173.7 ± 2.32
PDI*
DL* (wt%) (mean ± SD)
EE* (wt%) (mean ± SD)
Mean Zeta potential (mV)
0.39 ± 0.03
13.8 ± 1.7
69.6 ± 3.3
+32.7 ± 1.5
*Each experiment was performed in triplicate (n ¼ 3).
Table 5. Percentage labeling efficiency of RH and RH-CSNPs in saline and serum in 100 ml of SnCl2 (1 mg/ml).
100 90 % drug release
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Figure 2. TEM photographs of optimized ropinirole loaded chitosan nanoparticles (F8).
80 70
% LE of RH
60 50 40 30 20 10 0 0
5
10
15
20
Time (hr)
Figure 3. In vitro release studies of optimized ropinirole-loaded chitosan nanoparticles.
Release kinetics The release kinetics was characterized by fitting the data obtained from in vitro release studies of NPs into standard release equations (zero order, first-order, Higuchi model, and Korsmeyer–Peppas model). The model that best fits the release data was selected based on the correlation coefficient value of various models. The results indicated that release of drug from NPs was diffusion controlled, as indicated by higher r2 values (0.95) in the Higuchi model. When the release data were analyzed using the Korsmeyer–Peppas equation, the value of release exponent n was less than 0.43 and indicated that the mechanism of drug release from the RH-CSNPs was by Fickian diffusion18. Ex vivo nasal permeation The cumulative amount of drug that permeated (CADP) through nasal mucosa from RH-loaded NPs was 76.1 ± 2.32% whereas only 35.6 ± 1.73% was found to permeate from pure drug solution. The steady-state flux and permeability coefficient of drug solution through the nasal mucosa were 4.10 mgcm2 h1 and
% LE of RH-CSNPs
Time (h)
Serum
Saline
Serum
Saline
0.5 1 2 4 6 20 24
97.58 97.79 98.63 99.23 99.12 97.73 96.38
98.36 98.54 99.23 99.75 97.54 94.86 94.78
96.05 99.59 99.46 99.65 92.47 99.20 96.54
97.02 98.86 99.6 99.7 96.28 93.28 91.45
0.41 102 cm2 h1, respectively, whereas the steady-state flux and permeability coefficient of drug-loaded CSNPs were 10.62 mgcm2 h1 and 1.06 102 cm2 h1, respectively. The overall improvement ratio was 2.59. Biodistribution studies RH was effectively labeled using 99mTc and the radiolabeled RH, and RH-CSNPs formulation was optimized for maximum labeling efficiency and stability. The radiochemical purity achieved for RH and RH-CSNPs was found to be 97.62% and 95.5%, respectively, when evaluated for reduced/hydrolyzed 99mTc and free 99mTc. The pH range of 6.0–7.0 and 100 ml of stannous chloride with an incubation time of 60 min for RH-CSNPs and 30 min for RH were selected as conditions for the optimum radiolabeling. The radiolabeled complexes of RH formulations were found to be stable in saline and in rat serum up to 4 h (Table 5). Thus, it was concluded that the drug can be investigated for biodistribution studies using a radiolabeling technique in a nanoparticulate formulation. Biodistribution studies of 99mTc-RH formulations following intranasal administration (RH and RH-CSNPs) were performed
Intranasal delivery of ropinirole-loaded nanoparticles
DOI: 10.3109/03639045.2014.991400
7
Table 6. Biodistribution data of 99mTc-RH (i.n.) and 99mTc-RH-CSNPs (i.n.) at different time intervals in normal Swiss albino rats. Formulation RH (i.n.) RH-CSNPs (i.n.) RH (i.n.) RH-CSNPs (i.n.)
Organs
0.5 h
1h
2h
4h
6h
8h
Brain Blood Brain Blood Brain/blood Brain/blood
0.808 ± 0.03 3.221 ± 0.32 0.202 ± 0.04 0.522 ± 0.07 0.251 ± 0.09 0.386 ± 0.57
0.952 ± 0.07 4.379 ± 0.12 0.278 ± 0.09 0.803 ± 0.39 0.217 ± 0.58 0.346 ± 0.23
0.947 ± 0.02 4.809 ± 0.22 0.264 ± 0.02 1.219 ± 0.13 0.196 ± 0.09 0.216 ± 0.15
0.422 ± 0.02 2.498 ± 0.09 0.245 ± 0.05 1.209 ± 0.39 0.168 ± 0.22 0.202 ± 0.12
0.217 ± 0.03 1.427 ± 0.34 0.218 ± 0.08 1.186 ± 0.34 0.152 ± 0.08 0.183 ± 0.23
0.107 ± 0.05 0.826 ± 0.54 0.176 ± 0.04 0.980 ± 0.36 0.129 ± 0.09 0.179 ± 0.11
Each experiment was performed in triplicate (n ¼ 3).
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Figure 4. Gamma scintigraphic images for RH solution administered intranasally in Swiss albino rat.
Figure 5. Gamma scintigraphic images for ropinirole-loaded chitosan nanoparticles (RH-CSNPs) formulation administered intranasally in Swiss albino rat.
using Swiss albino rats and the radioactivity was estimated at different intervals up to 8 h. The major accumulation of the activity in terms of % administered dose per whole organ was in the liver (7.210 ± 0.52), followed by the kidneys (6.862 ± 0.62), intestine (4.862 ± 0.45), and the lungs (4.640 ± 0.92). The brain– blood ratio of the drug at all sampling time points for different formulations was also calculated (Table 6). The RH concentrations in the brain following the i.n. of RH-CSNPs were found to be significantly higher at all the time points compared with RH solution. The brain–blood ratios of 0.251 ± 0.09 and 0.386 ± 0.57 of RH (i.n.) and RH-CSNPs (i.n.), respectively, at 0.5 h are indicative of direct nose to brain transport, bypassing the BBB, hence demonstrating the superiority of nose to brain delivery of RH by mucoadhesive nanoparticles32,33. Gamma scintigraphic studies In order to visualize brain uptake via the i.n. route, gamma scintigraphic studies were performed in rats, with the 99mTc-RH
solution and RH-CSNPs being instilled in each nostril. Gamma scintigraphic images were taken at 30 min, 1 h, and 2 h for both formulations (Figures 4 and 5). The scintigrams clearly demonstrated the accumulation of the formulations in the brain following intranasal administration. In the case of the RH solution, the activity was higher in the brain initially and the presence of some radioactivity in the esophagus following i.n. administration suggested that a part of the formulation could have been absorbed from the gastrointestinal tract. The radioactivity in the brain decreased at 1 h and 2 h (Figure 4). However, in the case of RH-CSNPs, the formulation showed a sustained effect for 2 h in the brain; this may be attributed to the mucoadhesiveness of the prepared formulation (Figure 5). The scintigraphy images were consistent with the results shown in Table 4 and a high uptake of RH-CSNPs into the brain was observed. The gamma scintigraphic images revealed mucoadhesion of the drug was greater when administered as a polymeric nanoparticulate formulation, compared with the administration as a drug solution; this could lead to maximum drug retention at the site of action, with a slow release
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from the nasal pathway34. Thus, it can be concluded from the in vivo studies that i.n. administration of nanoparticulate RH will deliver more drug to the target site compared with an RH solution, with a consequent increase in bioavailability.
Conclusion
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A polymeric nanoparticulate carrier system developed and characterized for brain targeting of RH by intranasal administration showed sustained release profiles for up to 18 h and were optimized for various performance parameters, including particle size, % encapsulation efficiency, and % drug loading. It can be concluded from the animal studies that i.n. administration of RHCSNPs will deliver higher drug to the target site compared with that delivered by an RH solution, with a consequent increase in bioavailability.
Acknowledgements The authors thank Professor Brian L. Furman (University of Strathclyde, Glasgow, UK) for critical reading of the manuscript and English editing.
Declaration of interest The authors report no conflict of interest. The authors are responsible for the content and writing of the article. The authors are thankful to All India Council of Technical Education (AICTE), New Delhi, India for providing financial assistance to project.
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