http://informahealthcare.com/drd ISSN: 1071-7544 (print), 1521-0464 (electronic) Drug Deliv, Early Online: 1–9 ! 2014 Informa Healthcare USA, Inc. DOI: 10.3109/10717544.2014.880860
RESEARCH ARTICLE
Development, characterization and nasal delivery of rosmarinic acid-loaded solid lipid nanoparticles for the effective management of Huntington’s disease Rahul Bhatt1, Devendra Singh1, Atish Prakash2, and Neeraj Mishra1 Department of Pharmaceutics and 2Department of Pharmacology, I.S.F. College of Pharmacy, Moga, Punjab, India
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Abstract
Keywords
Objective: The objective of the present study was to investigate the potential use of solid lipid nanoparticles (SLNs) as a drug delivery system to enhance the brain-targeting efficiency of rosmarinic acid (RA) following intranasal (i.n.) administration. Materials and methods: The RA-loaded SLNs was prepared by the hot homogenization technique, in which glycerol monostearate (GMS) as lipid, tween 80 and soya lecithin were used as surfactant along with hydrogenated soya phosphatidyl choline (HSPC) as a stabilizer, and were characterized for particle size, zeta potential (ZP), in vitro study. Nasal delivery of the developed formulation followed by the study of behavioral (locomotor, narrow beam, body weight) and biochemical parameters (glutathione, lipid peroxidation, catalase and nitrite) in wistar rat was carried out. Results: Optimized RA-loaded SLNs using tween 80 (SLNPRT) have the mean size of (149.2 ± 3.2 nm), ZP (38.27 mV) entrapment efficiency (61.9 ± 2.2%). 3-NP-treated rat significantly increased behavioral alterations, oxidative damage as compared with the control group. SLNPRT treatment significantly improved behavioral abnormalities and attenuated the oxidative stress in 3NP-treated rats. However, the nasal delivery of SLNPRT produced significant therapeutic action as compared to intravenous application. In the organ distribution study, brain drug concentration was found to be 5.69 mg, in pharmacokinetic study Cmax, tmax, t1/2, AUC values were found to be 0.284 mg/ml, 1.5 h, 3.17 h, and 1.505 mg/ml/h, respectively. Conclusion: The encouraging results confirmed the developed optimized RA-loaded SLNs formulation following the non-invasive nose-to-brain drug delivery that is a promising therapeutic approach for the effective management in Huntington disease.
3-NP, HSPC, huntington, intra nasal, RA, SLNs
Introduction Huntington’s disease (HD) is an autosomal dominant, fully penetrant, progressive, and fatal neurodegenerative disease (Gudesblatt & Daniel, 2012) characterized by progressively worsening chorea, cognitive and psychiatric disturbance (Mehler & Gokhan, 2000; Bonelli & Hofmann, 2004). Oxidative injury, transcriptional dysregulation, glutamate exitotoxicity (Li et al., 2003), apoptotic huntingtin is expressed more intensely in neurons than in glial cells. The protein huntingtin (htt) is widely expressed within the central nervous system. Accumulation of proteolytic htt fragments and their aggregation trigger a cascade that leads to increasing neuronal dysfunction through signals, mitochondrial dysfunction and energy depletion (Beal & Ferrante, 2004). These changes are accompanied by neurochemical alterations,
Address for correspondence: Dr. Neeraj Mishra, Associate Professor, Department of Pharmaceutics, ISF College of Pharmacy, FerozepurRoad, GhalKalan, Moga, Punjab 142001, India. Tel: 01636324200. Fax: 01636-239515. E-mail: [email protected]
History Received 9 November 2013 Revised 3 January 2014 Accepted 4 January 2014
which involve not only glutamate receptors but also other receptors, such as the dopamine (DA) and adenosine receptors involved in motor functions (Teunissen et al., 2001; Pavese et al., 2003). It has been reported that Rosmarinic acid (RA) protects neurons from oxidative stress-induced disease condition (Kelsey et al., 2010a); RA significantly attenuated H2O2-induced reactive oxygen species (ROS) generation and apoptotic cell death (Lee et al., 2008); RA could contribute at least in part to neuroprotective effects because it exerts neuroprotective and anti-oxidative effects against neurotoxin insult in dopaminergic cells (Ren et al., 2009; Park et al., 2010); RA significantly protected neurons, these effects are mediated by the prevention of oxidative stress, intracellular Ca2+ overload and c-fos expression (Fallarini et al., 2009). The active constituents (RA and carnosic acid) of rosemary are generally considered to be antioxidants by directly scavenging free radicals or indirectly increasing endogenous cellular antioxidant defenses, for example, via activation of the nuclear factor erythroid-derived 2-related factor 2 (Nrf2) transcription factor pathway. Alternative mechanisms of action have also been suggested for the neuroprotective
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effects of these compounds, such as modulation of signal transduction cascades or effects on gene expression (Kelsey et al., 2010b). Solid lipid nanoparticles (SLNs) physicochemical characteristics are also particularly regarded in order to address the critical issues related to the development of suitable brain-targeting formulations. The uptake of the SLN by the brain might be explained by adsorption of a blood protein mediating the adherence to the endothelial cells of the blood–brain barrier (BBB) (Alyautdin et al., 1997). SLN were proposed for brain drug-targeting application independently by two research groups (i.e. Scho¨ler et al., 2001; Schubert et al., 2006). Delivering drugs to the CNS is impaired by the presence of the BBB that represents the main obstacle for CNS drug development. SLNs have been investigated as possible carriers for modified intravenous drug delivery and then to the central nervous system by various workers (Yang et al., 1999; Cavalli et al., 2000). The potential advantages of the use of SLNs accounted on the bases of a lower cytotoxicity, higher drug-loading capacity, and best production scalability. The use of NPs has been regarded as having great potential for the delivery of drugs into the CNS by several different approaches previously mentioned. The pharmacokinetics and biodistribution studies in mice after delivering risperidone (RSP)-loaded SLNs (RSLNs) showed that the brain/blood ratio 1 h post-administration of RSLNs (i.n.) was found to be 1.36 ± 0.06 (nearly 10- and 5-fold higher) as compared with 0.17 ± 0.05 for RS (i.v.) and 0.78 ± 0.07 for RSLNs (i.v.), respectively (Patel et al., 2011). Onset of HD is typically in middle age approximately 40 years; however, the age span includes those both young and old. The degenerative process primarily involves medium spiny striatal neurons and, to a lesser extent, cortical neurons. g-Amino butyric acid (GABA)ergic and enkephalin neurons of the basal ganglia are the most vulnerable in HD duration, typically from diagnosis to death, approximately 17 years. The neuro-pathological feature of HD is early and selective neuronal loss can be observed in the caudate and putamen striatum, the aspiny interneurons are relatively resistant to degeneration in HD. Loss of projection neurons in the cerebral cortex and hippocampus is also a feature of HD. As the disease progress, degenerative changes become more generalized and include other brain regions such as the globus pallidus, subthalamic nucleus, substantia nigra (SN), cerebellum and thalamus (Smith et al., 2004). Intranasal (i.n.) delivery of RA is a potential strategy to overcome the obstacles created by the BBB and is a promising option because of its non-invasiveness. Currently, nasal drug delivery has been recognized as a very promising route for brain delivery of therapeutic compounds including biopharmaceuticals. The concentration–time profiles achieved after nasal administration are often similar to those after intravenous administration, resulting in a rapid onset of pharmacological activity. In the case of SLN, rapid drug absorption via highly vascularized mucosa may improve the brain drug concentration, which may lead to site-specific delivery to brain ultimately minimizing the side effects. SLNs prepared using tween 80 (as a surfactant in hot homogenization technique of SLNs preparation) may be advantageous as tween 80 may develop the space between the tight paracellular space by interacting with cell membrane (Hongxia et al.,
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2007) and increase cellular uptake at the site of absorption at the nasal cavity which may result in absorption through transcellular (dues to lipophilic nature) as well as paracellular route. The main aim of our study is to develop and evaluate SLNs consisting of lipid cores as Glycerol Monosterate (GMS) using surfactant tween 80; investigate the potential of SLNs via intra nasal delivery; first time deliver the RA-loaded SLNs in HD in order to reduce the oxidative stress in brain. Furthermore, characterization of SLNs was carried out using several parameters such as shape and size, encapsulation efficiency, in vitro release study, organ distribution, pharmacokinetic study and brain homogenate study, i.e. biochemical parameter, 3-nitropropionic acid (3-Np) induce animal model for brain targeting to reduce the oxidative stress using Wistar rat.
Materials and Methods Chemicals Hydrogenated soya phosphatidyl choline (HSPC Obtained as a gift sample from Lipoid Germany), 3-nitropropionic acid (3-NP), Tween 80 and RA were purchased from SigmaAldrich (Sigma-Aldrich Corp. St. Louis, MO), and GMS was purchased from Central Drug House. All other chemicals were procured form Sigma and CDH. Method of preparation of SLNs RA-loaded SLNs were prepared by the hot homogenization method reported by Vivek et al. (2007), with slight modification. The lipid was melted at 69 C (10 C above the melting point of the GMS used as lipid), and RA (12 mg) was dissolved in lipid to obtain a drug–lipid mixture. In another beaker HSPC was dissolved in chloroform and added to the lipid–drug mixture. The mixture was then warmed at 70 C to completely evaporate the chloroform. The clear lipid melt containing HSPC was added to the hot aqueous surfactant solution (Soya Lecithin, Tween 80) preheated to 69 C above the lipid’s melting point under high-shear homogenization at 5000 rpm for 6 min to yield a crude emulsion. The crude emulsion was subsequently homogenized in a probe sonicator (Mesonix, NY) for 7 min. The hot nanoemulsion obtained was then cooled in ice cool water to recrystallize the lipid back to the solid state in the form of an aqueous SLN dispersion. Characterizations Particle size, polydispersity index and zeta potential measurement Particle size and polydispersity index (PI) of SLNs were measured by Zetasizer Desla Nano C (Malvern Instruments, Ltd., Malvern, UK), and Zeta potential (ZP) was also measured by the same instrument. In vitro drug release study In vitro drug release was evaluated using a dialysis bag diffusion technique (Yang & Zhu, 2002). A total of 5 ml of the sample was taken into a dialysis bag (10 kDa molecular cut-off) and placed in a beaker containing in 200 ml
HRA-loaded SLNs formulation
DOI: 10.3109/10717544.2014.880860
phosphate-buffered saline (pH 6.8) at 37 ± 1 C by using shaking incubator (Labtech, New Delh, India). An aliquot of 0.5 ml dissolution medium was removed at a series of various points. Samples were withdrawn and measuring the absorbance at 324 nm against a reagent blank, using the UV spectrophotometer (Parkin Elmer, UV/VIS spectrometer Lambda 25). Sink conditions were maintained throughout the experiment. The aliquots were diluted and analysed by UV. All the experiments were performed in triplicate.
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Encapsulation efficiency measurement Entrapment efficiency (EE) was determined by measuring the concentration of free drug (unentrapped) in an aqueous medium, as reported by Venkateswarlu & Manjunath (2004). SLNs were taken in the centrifugation tube and centrifuged at 20 000 rpm for 20 min. Then the supernatant was measured for the unentrapped drug. Experimental animals Male Wistar rats, weighing 180–250 g (3–5 month old), were obtained from Central Animal House facility of I.S.F. College of Pharmacy, Moga, Punjab, India. Animals were housed in group of three, in polypropylene cages with husk bedding under standard conditions of light and dark cycle with food and water ad libitum. Animals were acclimatized to laboratory conditions before the test. All the behavioral assessments were carried between 9:00 and 17:00 h. The experimental protocol was approved by the Institutional Animal Ethics Committee (IAEC) and was carried out in accordance with the guidelines of the Indian National Science Academy (INSA) for the use and care of the experimental animals.
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Behavioral assessment Rotarod activity Rotarod apparatus (INCO Medicraft, Ambala, India) is used for determining balance, motor co-ordination and muscle tone in rats. Motor function and grip strength of the rats were determined using this apparatus. Rats were placed individually on rotating rod and the cut-off time was 300 s and each rat had undergone three separate trials with a gap of 5 min. Briefly, the rats were exposed to prior training session on the rotarod (constant speed of 25 rpm; rod diameter 7 cm) for 1st, 5th, 10th, 15th day after 3-NP administration. The average fall of time was recorded and expressed as percentage fall off time (Kalonia et al., 2009). Measurement of body weight Animal body weight was recorded on the first and last day of the experimentation. Percent change in body weight was calculated as ½Body weight ð1st day 15th dayÞ 100= ½1st day body weight Locomotor activity The locomotor activity was monitored using an actophotometer (Medicraft, INCO, Ambala, Haryana) which operates on photoelectric cells which are connected in circuit with a counter. Before subjecting the animals to a cognitive task, all animals were individually placed in the activity meter and the total activity count was measured for 10 min. The locomotor activity was expressed in terms of total photobeam counts per 10 min per animal (Kalonia et al., 2009).
Drug treatment schedule 3-NP (Sigma chemicals, St. Louis, MO) was diluted with saline (adjust pH 7.4) and administered intraperitoneally (i.p.) to animals. SLNPRT was administered through two different routes of administration (Table 1), e.g. i.v. and i.n. through tail vein and nostrils (10 ml per nostril) by means of polyethylene tube connected to the Hamilton syringe, respectively. All drugs or vehicle were administered daily for 14 d. Animals were randomly divided into different groups, consisting of six animals in each. Study was conducted in multiple phases. On 1st, 5th, 10th, and 15th day different behavioral parameters were assessed and biochemical parameters were also measured on 15th d. Group I: Control (vehicle treated) Group II: 3-NP (10 mg/kg) Group III: 3-NP + RA (12 mg) i.n. Group IV: 3-NP + SLNPRT (i.v. 12 mg, SLN) Group V: 3-NP + SLNPRT (i.n. 12 mg, SLN)
Narrow beam The apparatus consists of 50 cm wooden strips supported by two pedestals at each end, with a height of 100 cm above the ground. The rats have to traverse a narrow beam which is suspended between a start platform and their home cage. It is important to make sure that the entire apparatus be placed at a height of at least 100 cm above the ground, so that the animal fears the height and really attempts to reach the goal box. All rats must be trained to walk over a beam for 5 d before testing. A ceiling of 120 s is employed at the end after which the rat is removed and placed in the cage by hand and receives, if it is the case, a score of 120 s. The test procedure is identical for all rats tested and performed in the same environment, preferably in the morning. Time taken to traverse rat from start platform to their home cage was measured along with their number of slipping errors (Kalonia et al., 2009). Dissection and homogenization
Table 1. Formulation code. S.no. Formulation code 1. 2. 3.
SLNP 3-NP SLNPRT
Description Solid lipid nanoparticle blank 3- Nitropropionic acid giving intra peritoneal RA-loaded SLNs using tween 80
On day 15, all the animals were sacrificed by decapitation immediately after behavioral assessments. The brains were removed, forebrain was dissected out and cerebellum was discarded. Brains were put on the ice, and the cortex and striatum regions were separated and weighed. A 10% (w/v) tissue homogenate was prepared in 0.1 mol/1 phosphate buffer
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(pH 7.4). Homogenate was centrifuged for 20 min at 20 000 rpm and the supernatant was stored in 80 C for assessing the biochemical parameters. Biochemical assessment Biochemical tests were conducted 24 h after last behavioral test. The animals were sacrificed by decapitation. Brains were removed and rinsed with ice-cold isotonic saline. Brains were then homogenized with ice-cold 0.1 mmol/L phosphate buffer (pH 7.4). The homogenate (10%w/v) was then centrifuged at 10 000 g for 15 min and the supernatant so formed was used for the biochemical estimations.
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Estimation of lipid peroxidation assay The extent of lipid peroxidation in the brain was determined quantitatively by performing the method as described by Wills (1966). The amount of malondialdehyde (MDA) was measured by reaction with thiobarbituric acid at 532 nm using Perkin Elmer Lambda 20 spectrophotometer. The values were calculated using the molar extinction co-efficient of chromophore (1.56 105 (mol/l)1 cm1; Wills, 1966). Estimation of nitrite The accumulation of nitrite in the supernatant, an indicator of the production of nitric oxide, was determined by a colorimetric assay with Greiss reagent (0.1% N-(1-napththyl) ethylene diamine dihydrochloride, 1% sulphanilamide and 5% phosphoric acid). Equal volumes of the supernatant and the Greiss reagent were mixed and the mixture was incubated for 10 min at room temperature in the dark. The absorbance was measured at 540 nm using Perkin Elmer Lambda 20 spectrophotometer. The concentration of nitrite in the supernatant was determined from the sodium nitrite standard curve (Green et al., 1982). Estimation of reduced glutathione Reduced glutathione was estimated according to the method described by Ellman. First, 1 ml of supernatant was precipitated with 1 ml of 4% sulphosalicylic acid and cold digested for 1 h at 4 C. The samples were then centrifuged at 1200 g for 15 min at 4 C. To 1 ml of the supernatant obtained, 2.7 ml of phosphate buffer (0.1 mmol/l, pH 8) and 0.2 ml of 5,50 -dithio-bis (2-nitrobenzoic acid) (DTNB) was added. The yellow color developed was measured at 412 nm using Perkin Elmer Lambda 20 spectrophotometer. Results were calculated using molar extinction co-efficient of the chromophore (1.36 104 (mol/l)1 cm1) (Ellman, 1959). Estimation of catalase activity Catalase activity was assessed by the method of Luck, wherein the breakdown of H2O2 is measured. Briefly, assay mixture consists of 3 ml of H2O2 phosphate buffer and 0.05 ml of the supernatant of the tissue homogenate. The change in absorbance was recorded for 2 min at 30 s interval at 240 nm using Perkin Elmer Lambda 20 spectrophotometer. The results were expressed as micromoles of hydrogen peroxide decomposed per min per mg of protein (Luck, 1971).
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Estimation of protein The protein content was estimated by the Biuret method (Gornall et al., 1949) using bovine serum albumin as a standard. Organ distribution study Five male Wistar rats were given four different doses of intra nasal administration of SLNPRT threw Cather tube. Tissues (brain) were promptly removed after 1 h post administration of SLNPRT (i.n.) and washed with saline. Each tissue sample was diluted with 0.9% saline solution as an internal standard and homogenized, and the homogenate was collected and stored at 20 C until analysis. Organ distribution study was carried out in order to determine the drug concentration reaches the brain with the help of HPLC. RA-loaded SLNs using Tween 80 formulation dose was 12 mg, which is delivered into the animal by the intra nasal rout. Pharmacokinetic study Three male Wistar rat were given 0.1 ml dose via i.n. route at 15, 30, 45 min, 1, 1.5, 4, 6, 24 h time interval. Sample was taken from both eye of retro orbital of rat. Pharmacokinetic parameters were determined, i.e. Cmax, Tmax, Thalf, AUC. Statistical analysis All the results are expressed as mean ± standard deviation. The treated groups were compared with control by applying the analysis of variance (ANOVA). The statistical analysis was carried out at Graph Pad Software Corp. (San Diego, CA). The p value50.05 was considered significant.
Result and discussion Particle size, polydispersity index Particle size measurement was required to confirm the production of particle in nano-range. The results indicate that particles were significantly influenced by most of the formulation and process variables. Among two different surfactants (soya lecithin and tween 80) used in the preparation of SLNs, the particle size was found to be 157.2 and 149.2 nm for soya lecithin and tween 80, respectively (Table 3). Hydrophilic and lipophilic balance value (HLB) of 12–16 is considered to be ideal for the production of stable o/w emulsion. The HLB value of tween 80 is 15 and soya lecithin is 4–9, difference in HLB values may be the reason of difference in the particle size using two different surfactants. Smaller particles are better able to reach a target tissue via the circulation. Small particle size is the requirement for the nasal delivery of SLNs to minimize the barrier-associated problems at the site of action. However, attention has been paid to particle size distribution in order to minimize the risk of embolism and other side effects due to the presence of large size SLNs. RA-loaded SLNs were successfully prepared and optimized on the basis of particle size and PDI of the nanoparticles. The process parameter includes the drug EE and for the lipid, soya lecithin, HSPC concentration was optimized.
HRA-loaded SLNs formulation
DOI: 10.3109/10717544.2014.880860
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Optimization of HSPC and lipid concentration
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and HSPC and on increasing concentration of tween 80 from 0.5, 1, 1.5 and 2 particles size reduce from 146.6 ± 11.2 to 123.1 ± 16.5. Relatively high emulsifier concentrations are required to stabilize the surface of small particles, and also to prevent problems associated with particle size stability. However, high concentration may led to toxic side effects.
Dispersion of the liquid melt into fine droplets in a hot, surfactant-containing aqueous phase-led droplet generation process is completely analogous to that during the preparation of submicron lipid emulsions; it means that all the technologies established for emulsion preparation can be used. In the preparation of SLNs, the prepared emulsion first forms the coarse particles which can be converted into nanoemulsion on applying energy and form SLNs on solidification. In the optimization process, the lipid concentration was optimized from F7 to F12 and HSPC was optimized from F1 to F6. The concentration of HSPC greatly affects the particle size and PDI, which was observed on changing the concentration during preparation of SLNs. At the initial phase on taking 1 mg of HSPC, the particle size and PDI values was found to be 793.5 ± 42.1 and 0.267 ± 0.023 (Table 2), respectively, while on increasing the HSPC concentration decrease in particle size with PDI was observed till 4 mg of HSPC, beyond that increment in particle size and PDI values was observed.
Optimization of entrapment of efficiency with soya lecithin and tween 80 The drug EE in SLNs was optimized by using soya lecithin and tween-80 as a surfactant having formulation code F21 and F22, respectively. The highest EE (61.9 ± 2.2%) was observed in case of tween 80 (Table 3) while in case of soya lecithin as surfactant the EE was being observed to 49.86 ± 3.1%. Surfactant as a tween 80 on 1% exhibits the highest EE while soya lecithin at 0.5% shows the highest EE. On the basis of higher EE and smaller size, formulation F23 was used for further in vitro characterization as code SLNPRT. Drug release study Sustain and control release of poorly water-soluble drugs may be important aspect of development of SLNs; however, it is already reported that SLNs provide the sustain/prolong release pattern. In the hypothesized formulation, the optimized formula is able to control the release the drug for longer period of time, in starting to 7 h. 75.02 ± 5.4% drug was release and at 14 h. 97.5 ± 2.9 drug was released (Figure 1). It may be the evidence that the drug is homogenously dispersed in solid lipid core of SLNs. Form the above statement it can also be concluded that the proposed formulation may able to maintain the drug concentration up to 14 h which may ultimately reduce the dosing frequency and may enhance the effectiveness of therapy. Crystallization behavior of the lipid carrier and high mobility of the drug lead to fast drug release in proposed formulation crystallization
Optimization of soya lecithin and tween 80 concentration The choice of emulsifier and its ratio to the matrix lipid are very important in the development of high-quality SLNs. The effect of soya lecithin and tween 80 concentration on nanoparticle size was evaluated by varying the concentration of both tween 80 and soya lecithin. The soya lecithin and Tween-80 were optimized from F13 to F16 and F17 to F20, respectively (Table 2). In the case of soya lecithin, the concentration varying from the 0.1, 0.2, 0.5, and 1, while maintaining the concentration of HSPC 4 mg lipid 3% each as expected results, revealed that when increasing the concentration of soya lecithin decrease in particle size decrease from 141.3 ± 13.2 to 126.1 ± 5.3 while on the same amount of lipid Table 2. Optimization of lipid, soya lecithin, tween-80, HSPC (surfactant). Formulation code (F) F1 F2 F3 F4 F5 F6 F7 F8 F9 F10 F11 F12 F13 F14 F15 F16 F17 F18 F19 F20
Lipid (%)
Soya lecithin (%)
Tween 80 (%)
HSPC (mg)
Homogenization time
Sonication time (min)
Size (nm)
PDI
1 1 1 1 1 1 1 2 3 4 5 6 3 3 3 3 3 3 3 3
0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.2 0.5 1 0.5 0.5 0.5 0.5
0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 .5 0.5 1.5 2
1 2 3 4 5 6 4 4 4 4 4 4 4 4 4 4 4 4 4 4
30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30
7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7
793.5 ± 42.1 697.4 ± 33.1 623.2 ± 23.4 544.1 ± 34.2 688.9 ± 18.3 756.9 ± 34.3 161.7 ± 24.3 133.2 ± 31.2 118.1 ± 26.2 181.1 ± 18.4 201.1 ± 22.3 231 ± 31.4 141.3 ± 13.2 139.2 ± 8.4 135.5 ± 11.9 126.1 ± 5.3 146.6 ± 11.2 139.2 ± 17.1 126.5 ± 13.0 123.1 ± 16.5
0.267 ± 0.023 0.285 ± 0.015 0.255 ± 0.023 0.250 ± 0.021 0.364 ± 0.012 0.384 ± 0.017 0.250 ± 0.02 0.391 ± 0.023 0.201 ± 0.019 0.380 ± 0.015 0.344 ± 0.013 0.312 ± 0.018 0.331 ± 0.013 0.301 ± 0.016 0.296 ± 0.025 0.325 ± 0.022 0.251 ± 15.3 0.282 ± 13.2 0.221 ± 11.8 0.347 ± 17.0
Optimization of HSPC, lipid, soya lecithin, tween-80. Formulation codes F1 to F6 were optimized for HSPC, F7 to F12 were optimized for lipid, F13 to F16 were optimized for soya lecithin, and F17 to F20 were optimized for tween-80. Bold values signifies optimum size of SLNs by using different concentrations of lipid, soya lecithin, tween 80 and HPSC.
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Table 3 Optimization of, drug loading using two different surfactant (soy lecithin and tween 80). Formulation code F21 F22
Lipid (%)
Soya lecithin (%)
Tween 80 (%)
HSPC (mg)
Drug (mg)
Size (nm)
PDI
EE (%)
3 3
0.5 0
0 1
4 4
12 12
167.4 ± 30.02 149.2 ± 18.2
0.289 ± 0.031 0.290 ± 0.021
40.46 ± 2.8 61.9 ± 2.2
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Final formulation selection and optimization of EE using two different surfactant (soy lecithin and tween 80). Formulation codes F21 and F22 were optimized for soya lecithin and Tween-80, respectively.
Figure 1. %Cumulative percentage release of drug from, i.e. SLNPRT.
Figure 3. Effect of SLNPRT on rotarod activity in 3-NP-treated rats. Values are expressed as mean ± SEM ap 0.05 as compared to control group. bp 0.05 as compared to 3NP treated group. cp 0.05 as compared to RA per se. dp 0.05 as compared to RA from i.v.
drug incorporated, structural properties of lipid and drug), production methods (Melike & Gulgun, 2007). It is proved by changing the surfactant soya lecithin with tween changes in drug entrapment, and also the effect of the drug loading, PDI and particle size on changing the surfactant was observed. Behavioral assessment Effect of RA (SLN) on body weight, rotarod activity, locomotor, and narrow beam in 3-NP-treated rats
Figure 2. Effect of SLNPRT on body weight in 3-NP-treated rats. Values are expressed as mean ± SEM ap 0.05 as compared to control group. b p 0.05 as compared to 3NP-treated group. cp 0.05 as compared to RA per se. dp 0.05 as compared to RA from i.v.
behavior and drug mobility may be limited because of controlled/sustain release pattern of SLNs. The particle size that affects drug release rate directly depends on various parameters such as composition of SLN formulation (such as surfactant/surfactant mixture, amount of
Systemic treatment with 3-NP significantly reduced body weight (Figure 2), motor co-ordination (rotarod) (Figure 3), impaired locomotor (Figure 4) and increased the latency to reach goal platform (narrow beam test) (Figure 5) (p50.05) on 5th, 10th and 15th d as compared with the vehicle-treated group, suggesting that the effects of 3-NP most probably mimic either the juvenile onset or late stages of HD-like behavior. Deficiency of GABAergic stimulation has been implicated in the pathophysiology of HD (Lee & Chang, 2004; Saulle et al., 2004). Study suggested the sequential neurodegenerative process in the striatal GABAergic efferent projections during increased neuropathological grades of HD (Allen et al., 2009). In the present study, i.n. administration of SLNPRT formulation significantly improved the behavioral abnormalities in rotarod and locomotor activities (p50.05) and decreased the latency to reach the goal platform in narrow beam walk (p50.05), indicating the neuroprotective effect. The hunting-like symptoms was reversed at variable extent on giving treatment using
HRA-loaded SLNs formulation
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DOI: 10.3109/10717544.2014.880860
Figure 4. Effect of SLNPRT on locomotor activity in 3-NP-treated rats. Values are expressed as mean ± SEM ap 0.05 as compared to control group. bp 0.05 as compared to 3NP-treated group. cp 0.05 as compared to RA per se. dp 0.05 as compared to RA from i.v.
Figure 6. Effect of SLNPRT on malondialdehyde (MDA) level striatum and cortex in 3-NP-treated rats. Values are expressed mean ± SEM ap 0.05 as compared to control group. bp 0.05 compared to 3NP-treated group. cp 0.05 as compared to RA per d p 0.05 as compared to RA from i.v.
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in as as se.
Figure 5. Effect of SLNPRT on balance beam walking performance in 3-NP-treated rats. Values are expressed as mean ± SEM ap 0.05 as compared to control group. bp 0.05 as compared to 3NP-treated group. cp 0.05 as compared to RA per se. dp 0.05 as compared to RA from i.v.
SLNs as a carrier of two different compositions. One is the SLNPRT and RA (without carrier) administered through the nasal and i.v, and also comparing the importance of selection of i.n. route of administration. As the figures of the behavioral activities show that the hunting-like symptoms disappeared on successful treatment with the RA in forms SLNs, on comparing the SLNPRT and RA (without carrier) the SLNPRT was found to be effective by the nasal route. Also, comparison of the same formulation given through the i.v. route shows the evidence of the importance of selection of nasal route; i.v. route in itself has the limitations like invasive, painful and also requires the assistance of the third person. The importance of selection of i.n. route is also observed in the assessment of biochemical parameter. The 3-NP experiment model mimics both the hyperkinetic and hypokinetic
Figure 7. Effect of RA (43) on nitrite level in striatum and cortex in 3-NP-treated rats. Values are expressed as mean ± SEM ap 0.05 as compared to control group. bp 0.05 as compared to 3NP-treated group. cp 0.05 as compared to RA per se. dp 0.05 as compared to RA from i.v.
symptoms of HD, depending upon the duration of administration (Borlongan et al., 1997). Biochemical assessment Effects of RA on lipid peroxidation, nitrite, catalase and GSH after 3-NP treatment Systemic administration of 3-NP significantly increased lipid peroxidation, nitrite concentration and depleted catalase and glutathione in striatum and cortex, and control vehicle treated found to be stable normal (Figures 6–9). SLNPRT
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Figure 8. Effect of SLNPRT on catalase in striatum and in 3-NP-treated rats. Values are expressed as mean ± SEM ap 0.05 as compared to control group. bp 0.05 as compared to 3NP-treated group. cp 0.05 as compared to RA per se. dp 0.05 as compared to RA from i.v.
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substantial evidence that oxidative damage significantly contributes to the pathogenesis of several neurodegenerative diseases including HD (Kumar et al., 2007). Disruption of the mitochondrial enzyme complex activity is associated with ROS. According to the immune histochemical data, biochemical assays in HD patients show significant increases in MDA and 4-hydroxynonenal brain levels, which are almost 8-fold greater than the control subjects (Stoy et al., 2005). Both nitrite and nitric oxide species were shown to be oxidizing DA into its quinone forms bound covalently to critical proteins (LaVoie & Hastings, 1999). From the above hypothesis, it can be correlated that 3-NP treatment increased the oxidative stress that is responsible for the damage of dopaminergic neuron (act as motor control), which is ultimately responsible for impairment in motor coordination, behavioral alterations. In organ distribution study, brain drug concentration of SLNPRT formulation was found to be 5.69 mg, in pharmacokinetic study Cmax, tmax, t1/2, AUC values were found to be 0.284 mg/ml, 1.5 h, 3.17 h, and 1.505 mg/ml/h, respectively.
Conclusion
Figure 9. Effect of SLNPRT on reduced GSH level in striatum & cortex in 3-NP-treated rats. Values are expressed as mean ± SEM ap 0.05 as compared to control group. bp 0.05 as compared to 3NP-treated group. cp 0.05 as compared to RA per se. dp 0.05 as compared to RA from i.v.
formulation significantly attenuated lipid peroxidation, nitrite concentration, and restored endogenous antioxidants enzyme (catalase and GSH) activities on 3-NP-treated animals. RA protects neuron from oxidative stress (Kelsey et al., 2010a,b). The increment on brain LPO and nitrite level is directly associated with neurodegenerative disease. It has been reported that RA significantly attenuate H2O2-induced ROS generation and apoptotic cell death (Lee et al., 2008). RA significantly protected neurons, these effects are mediated by the prevention of oxidative stress, intracellular Ca2+ overload and c-fos expression (Fallarini et al., 2009). Systemic administration of 3-NP in rats and non-human primates produces homogeneous blockade of complex II within the brain (Kumar et al., 2007), leading to preferential excitotoxic striatal degeneration associated with behavioral abnormalities that are highly reminiscent of HD (Brouillet et al., 1993, Brouillet et al., 1999; Bizat et al., 2003). There is
In this study, SLNs containing RA was successfully prepared by the hot homogenization method. The study suggests the importance of controlling the critical formulation parameters during formulation, which greatly influence the final product, such as particles size, PI and drug encapsulation efficiency. Sustain release was observed from the SLNs. RA-loaded SLNs treatment is able to significantly attenuate 3NP-induced deficits in body weight, beam walk, locomotor and motor coordination, and are also able to significantly attenuate 3NPinduced striatal oxidative stress. The optimum brain drug concentration signifies the importance of using nasal route compared to i.v. route. Nasal delivery also prevents the unwanted distribution and metabolism in other parts of body. From the present study, it can be concluded that nasal administration of RA-loaded SLNs may be a promising approach for the effective management of HD.
Declaration of interest The authors report no conflicts of interest. The author alone is responsible for the content and writing of the paper.
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Borlongan CV, Koutouzis TK, Sanberg PR. (1997). 3-Nitropropionic acid animal model and Huntington’s disease. Neurosci Biobehav Rev 21:289–293. Brouillet E, Conde F, Beal MF, Hantraye P. (1999). Replicating Huntington’s disease phenotype in experimental animals. Prog Neurobiol 59:427–468. Brouillet E, Jenkins BG, Hyman BT, et al. (1993). Age-dependent vulnerability of the striatum to the mitochondrial toxin 3-nitropropionic acid. J Neurochem 60:356–359. Cavalli R, Caputo O, Gasco MR. (2000). Preparation and characterization of solid lipid nanospheres containing paclitaxel. Eur J Pharmaceut Sci 10:305–9. Ellman GL. (1959). Tissue sulfhydryl groups. Arch Biochem Biophys 82:70–7. Fallarini S, Miglio G, Paoletti T, et al. (2009). Clovamide and rosmarinic acid induce neuroprotective effects in in vitro models of neuronal death. Br J Pharmacol 157:1072–84. Gornall AG, Bardawill CT, David MM. (1949). Determination of serum proteins by means of Biuret reaction. J Biol Chem 177: 751–66. Green LC, Wagner DA, Glagowski J. (1982). Analysis of nitrate, nitrite and (15 N) nitrate in biological fluids. J Biol Chem 193:265–75. Gudesblatt M, Daniel T. (2012). Huntington’s disease: a clinical review. Neurol Rev, S1–8. Hongxia L, Matthias G, Shengjie B, et al. (2007). Enhancing effect of surfactants on fexofenadine. HCl transport across the human nasal epithelial cell monolayer 330:23–31. Kalonia H, Kumar P, Kumar A, Nehru B. (2009). Effect of caffeic acid and rofecoxib and their combination against intrastriatal quinolinic acid induced oxidative damage, mitochondrial and histological alterations in rats. Inflammopharmacology 17:211–19. Kelsey NA, Wilkins HM, Linseman DA. (2010a). Nutraceutical antioxidants as novel neuroprotective agents. Molecules 15:7792–814. Kelsey NA, Wilkins HM, Linseman DA. (2010b). Nutraceutical antioxidants as novel neuroprotective agents. Molecules 15:7792–814. Kumar P, Padi SS, Naidu PS, Kumar A. (2007). Cyclooxygenase inhibition attenuates 3-nitropropionic acid-induced neurotoxicity in rats: possible antioxidant mechanisms. Fundam Clin Pharmacol 21:297–306. LaVoie MJ, Hastings TG. (1999). Peroxynitrite- and nitrite-induced oxidation of dopamine: implications for nitric oxide in dopaminergic cell loss. J Neurochem 73:2546–54. Lee WT, Chang C. (2004). Magnetic resonance imaging and spectroscopy in assessing 3-nitropropionic acid-induced brain lesions: an animal model of Huntington’s dis- ease. Prog. Neurobiol 72:87–110. Lee HJ, Cho H-S, Park E, et al. (2008). Rosmarinic acid protects human dopaminergic neuronal cells against hydrogen peroxide-induced apoptosis. Toxicology 250:109–15. Li J-Y, Plomann M, Brundin P. (2003). Huntington’s disease: a synaptopathy? Trends Mol Med 9:414–20. Luck H. (1971). Catalase. In Bergmeyer HJ, ed. Methods of enzymatic analysis. 3rd ed., Vol. III. New York (NY): Academic Press, 885–93.
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Mehler MF, Gokhan S. (2000). Mechanisms underlying neural cell death in neurodegenerative diseases: alterations of a developmentallymediated cellular rheostat. Trends Neurosci 23:599–605. Melike U, Gulgun Y. (2007). Importance of solid lipid nanoparticles (SLN) in various administration routes and future perspectives. Int J Nanomedicine 2007;2:289–300. Park DH, Park SJ, Kim JM, et al. (2010). Subchronic administration of rosmarinic acid, a natural prolyl oligopeptidase inhibitor, enhances cognitive performances. Fitoterapia 81:644–8. Patel S, Chavhan S, Soni H, et al. (2011). Brain targeting of risperidoneloaded solid lipid nanoparticles by intranasal route. J Drug Target 19: 468–74. Pavese N, Andrews TC, Brooks DJ, et al. (2003). Progressive striatal and cortical dopamine receptor dysfunction in Huntington’s disease: a PET study. Brain 126:1127–35. Ren P, Jiang H, Li R, et al. (2009). Rosmarinic acid inhibits 6-OHDAinduced neurotoxicity by anti-oxidation in MES23. 5 cells. J Mol Neurosci 39:220–5. Saulle E, Gubellini P, Picconi B, et al. (2004). Neuronal vulnerability following inhibition of mitochondrial complex II: a possible ionic mechanism for Huntington’s disease. Mol. Cell. Neurosci 25:9–20. Scho¨ler N, Olbrich C, Tabatt K, et al. (2001). Surfactant, but not the size of solid lipid nanoparticles (SLN) influences viability and cytokine production of macrophages. Int J Pharmaceut 221:57–67. Schubert MA, Harms M, Mu¨ller-Goymann CC. (2006). Structural investigations on lipid nanoparticles containing high amounts of lecithin. Eur J Pharmaceut Sci 27:226–36. Smith Y, Raju DV, Pare J-F, Sidibe M. (2004). The thalamostriatal system: a highly specific network of the basal ganglia circuitry. Trends Neurosci 27:520–7. Stoy N, Mackay GM, Forrest CM, et al. (2005). Tryptophan metabolism and oxidative stress in patients with Huntington’s disease. J Neurochem 93:611–23. Teunissen C, Steinbusch H, Angevaren M, et al. (2001). Behavioural correlates of striatal glial fibrillary acidic protein in the 3-nitropropionic acid rat model: disturbed walking pattern and spatial orientation. Neuroscience 105:153–67. Venkateswarlu V, Manjunath K. (2004). Preparation, characterization and in vitro release kinetics of clozapine solid lipid nanoparticles. J Control Rel 95:627–38. Vivek K, Reddy H, Murthy RS. (2007). Investigations of the effect of the lipid matrix on drug entrapment, in vitro release, and physical stability of olanzapine-loaded solid lipid nanoparticles. AAPS PharmSciTech 8:16–24. Wills ED. (1966). Mechanism of lipid peroxide formation in animal tissues. Biochemical J 99:667–76. Yang SC, Lu LF, Cai Y, et al. (1999). Body distribution in mice of intravenously injected camptothecin solid lipid nanoparticles and targeting effect on brain. J Control Rel 59:299–307. Yang SC, Zhu JB. (2002). Preparation and characterization of camptothecin solid lipid nanoparticles. Drug Dev Ind Pharm 28: 265–74.
RESEARCH ARTICLE
Development, characterization and nasal delivery of rosmarinic acid-loaded solid lipid nanoparticles for the effective management of Huntington’s disease Rahul Bhatt1, Devendra Singh1, Atish Prakash2, and Neeraj Mishra1 Department of Pharmaceutics and 2Department of Pharmacology, I.S.F. College of Pharmacy, Moga, Punjab, India
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Abstract
Keywords
Objective: The objective of the present study was to investigate the potential use of solid lipid nanoparticles (SLNs) as a drug delivery system to enhance the brain-targeting efficiency of rosmarinic acid (RA) following intranasal (i.n.) administration. Materials and methods: The RA-loaded SLNs was prepared by the hot homogenization technique, in which glycerol monostearate (GMS) as lipid, tween 80 and soya lecithin were used as surfactant along with hydrogenated soya phosphatidyl choline (HSPC) as a stabilizer, and were characterized for particle size, zeta potential (ZP), in vitro study. Nasal delivery of the developed formulation followed by the study of behavioral (locomotor, narrow beam, body weight) and biochemical parameters (glutathione, lipid peroxidation, catalase and nitrite) in wistar rat was carried out. Results: Optimized RA-loaded SLNs using tween 80 (SLNPRT) have the mean size of (149.2 ± 3.2 nm), ZP (38.27 mV) entrapment efficiency (61.9 ± 2.2%). 3-NP-treated rat significantly increased behavioral alterations, oxidative damage as compared with the control group. SLNPRT treatment significantly improved behavioral abnormalities and attenuated the oxidative stress in 3NP-treated rats. However, the nasal delivery of SLNPRT produced significant therapeutic action as compared to intravenous application. In the organ distribution study, brain drug concentration was found to be 5.69 mg, in pharmacokinetic study Cmax, tmax, t1/2, AUC values were found to be 0.284 mg/ml, 1.5 h, 3.17 h, and 1.505 mg/ml/h, respectively. Conclusion: The encouraging results confirmed the developed optimized RA-loaded SLNs formulation following the non-invasive nose-to-brain drug delivery that is a promising therapeutic approach for the effective management in Huntington disease.
3-NP, HSPC, huntington, intra nasal, RA, SLNs
Introduction Huntington’s disease (HD) is an autosomal dominant, fully penetrant, progressive, and fatal neurodegenerative disease (Gudesblatt & Daniel, 2012) characterized by progressively worsening chorea, cognitive and psychiatric disturbance (Mehler & Gokhan, 2000; Bonelli & Hofmann, 2004). Oxidative injury, transcriptional dysregulation, glutamate exitotoxicity (Li et al., 2003), apoptotic huntingtin is expressed more intensely in neurons than in glial cells. The protein huntingtin (htt) is widely expressed within the central nervous system. Accumulation of proteolytic htt fragments and their aggregation trigger a cascade that leads to increasing neuronal dysfunction through signals, mitochondrial dysfunction and energy depletion (Beal & Ferrante, 2004). These changes are accompanied by neurochemical alterations,
Address for correspondence: Dr. Neeraj Mishra, Associate Professor, Department of Pharmaceutics, ISF College of Pharmacy, FerozepurRoad, GhalKalan, Moga, Punjab 142001, India. Tel: 01636324200. Fax: 01636-239515. E-mail: [email protected]
History Received 9 November 2013 Revised 3 January 2014 Accepted 4 January 2014
which involve not only glutamate receptors but also other receptors, such as the dopamine (DA) and adenosine receptors involved in motor functions (Teunissen et al., 2001; Pavese et al., 2003). It has been reported that Rosmarinic acid (RA) protects neurons from oxidative stress-induced disease condition (Kelsey et al., 2010a); RA significantly attenuated H2O2-induced reactive oxygen species (ROS) generation and apoptotic cell death (Lee et al., 2008); RA could contribute at least in part to neuroprotective effects because it exerts neuroprotective and anti-oxidative effects against neurotoxin insult in dopaminergic cells (Ren et al., 2009; Park et al., 2010); RA significantly protected neurons, these effects are mediated by the prevention of oxidative stress, intracellular Ca2+ overload and c-fos expression (Fallarini et al., 2009). The active constituents (RA and carnosic acid) of rosemary are generally considered to be antioxidants by directly scavenging free radicals or indirectly increasing endogenous cellular antioxidant defenses, for example, via activation of the nuclear factor erythroid-derived 2-related factor 2 (Nrf2) transcription factor pathway. Alternative mechanisms of action have also been suggested for the neuroprotective
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effects of these compounds, such as modulation of signal transduction cascades or effects on gene expression (Kelsey et al., 2010b). Solid lipid nanoparticles (SLNs) physicochemical characteristics are also particularly regarded in order to address the critical issues related to the development of suitable brain-targeting formulations. The uptake of the SLN by the brain might be explained by adsorption of a blood protein mediating the adherence to the endothelial cells of the blood–brain barrier (BBB) (Alyautdin et al., 1997). SLN were proposed for brain drug-targeting application independently by two research groups (i.e. Scho¨ler et al., 2001; Schubert et al., 2006). Delivering drugs to the CNS is impaired by the presence of the BBB that represents the main obstacle for CNS drug development. SLNs have been investigated as possible carriers for modified intravenous drug delivery and then to the central nervous system by various workers (Yang et al., 1999; Cavalli et al., 2000). The potential advantages of the use of SLNs accounted on the bases of a lower cytotoxicity, higher drug-loading capacity, and best production scalability. The use of NPs has been regarded as having great potential for the delivery of drugs into the CNS by several different approaches previously mentioned. The pharmacokinetics and biodistribution studies in mice after delivering risperidone (RSP)-loaded SLNs (RSLNs) showed that the brain/blood ratio 1 h post-administration of RSLNs (i.n.) was found to be 1.36 ± 0.06 (nearly 10- and 5-fold higher) as compared with 0.17 ± 0.05 for RS (i.v.) and 0.78 ± 0.07 for RSLNs (i.v.), respectively (Patel et al., 2011). Onset of HD is typically in middle age approximately 40 years; however, the age span includes those both young and old. The degenerative process primarily involves medium spiny striatal neurons and, to a lesser extent, cortical neurons. g-Amino butyric acid (GABA)ergic and enkephalin neurons of the basal ganglia are the most vulnerable in HD duration, typically from diagnosis to death, approximately 17 years. The neuro-pathological feature of HD is early and selective neuronal loss can be observed in the caudate and putamen striatum, the aspiny interneurons are relatively resistant to degeneration in HD. Loss of projection neurons in the cerebral cortex and hippocampus is also a feature of HD. As the disease progress, degenerative changes become more generalized and include other brain regions such as the globus pallidus, subthalamic nucleus, substantia nigra (SN), cerebellum and thalamus (Smith et al., 2004). Intranasal (i.n.) delivery of RA is a potential strategy to overcome the obstacles created by the BBB and is a promising option because of its non-invasiveness. Currently, nasal drug delivery has been recognized as a very promising route for brain delivery of therapeutic compounds including biopharmaceuticals. The concentration–time profiles achieved after nasal administration are often similar to those after intravenous administration, resulting in a rapid onset of pharmacological activity. In the case of SLN, rapid drug absorption via highly vascularized mucosa may improve the brain drug concentration, which may lead to site-specific delivery to brain ultimately minimizing the side effects. SLNs prepared using tween 80 (as a surfactant in hot homogenization technique of SLNs preparation) may be advantageous as tween 80 may develop the space between the tight paracellular space by interacting with cell membrane (Hongxia et al.,
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2007) and increase cellular uptake at the site of absorption at the nasal cavity which may result in absorption through transcellular (dues to lipophilic nature) as well as paracellular route. The main aim of our study is to develop and evaluate SLNs consisting of lipid cores as Glycerol Monosterate (GMS) using surfactant tween 80; investigate the potential of SLNs via intra nasal delivery; first time deliver the RA-loaded SLNs in HD in order to reduce the oxidative stress in brain. Furthermore, characterization of SLNs was carried out using several parameters such as shape and size, encapsulation efficiency, in vitro release study, organ distribution, pharmacokinetic study and brain homogenate study, i.e. biochemical parameter, 3-nitropropionic acid (3-Np) induce animal model for brain targeting to reduce the oxidative stress using Wistar rat.
Materials and Methods Chemicals Hydrogenated soya phosphatidyl choline (HSPC Obtained as a gift sample from Lipoid Germany), 3-nitropropionic acid (3-NP), Tween 80 and RA were purchased from SigmaAldrich (Sigma-Aldrich Corp. St. Louis, MO), and GMS was purchased from Central Drug House. All other chemicals were procured form Sigma and CDH. Method of preparation of SLNs RA-loaded SLNs were prepared by the hot homogenization method reported by Vivek et al. (2007), with slight modification. The lipid was melted at 69 C (10 C above the melting point of the GMS used as lipid), and RA (12 mg) was dissolved in lipid to obtain a drug–lipid mixture. In another beaker HSPC was dissolved in chloroform and added to the lipid–drug mixture. The mixture was then warmed at 70 C to completely evaporate the chloroform. The clear lipid melt containing HSPC was added to the hot aqueous surfactant solution (Soya Lecithin, Tween 80) preheated to 69 C above the lipid’s melting point under high-shear homogenization at 5000 rpm for 6 min to yield a crude emulsion. The crude emulsion was subsequently homogenized in a probe sonicator (Mesonix, NY) for 7 min. The hot nanoemulsion obtained was then cooled in ice cool water to recrystallize the lipid back to the solid state in the form of an aqueous SLN dispersion. Characterizations Particle size, polydispersity index and zeta potential measurement Particle size and polydispersity index (PI) of SLNs were measured by Zetasizer Desla Nano C (Malvern Instruments, Ltd., Malvern, UK), and Zeta potential (ZP) was also measured by the same instrument. In vitro drug release study In vitro drug release was evaluated using a dialysis bag diffusion technique (Yang & Zhu, 2002). A total of 5 ml of the sample was taken into a dialysis bag (10 kDa molecular cut-off) and placed in a beaker containing in 200 ml
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phosphate-buffered saline (pH 6.8) at 37 ± 1 C by using shaking incubator (Labtech, New Delh, India). An aliquot of 0.5 ml dissolution medium was removed at a series of various points. Samples were withdrawn and measuring the absorbance at 324 nm against a reagent blank, using the UV spectrophotometer (Parkin Elmer, UV/VIS spectrometer Lambda 25). Sink conditions were maintained throughout the experiment. The aliquots were diluted and analysed by UV. All the experiments were performed in triplicate.
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Encapsulation efficiency measurement Entrapment efficiency (EE) was determined by measuring the concentration of free drug (unentrapped) in an aqueous medium, as reported by Venkateswarlu & Manjunath (2004). SLNs were taken in the centrifugation tube and centrifuged at 20 000 rpm for 20 min. Then the supernatant was measured for the unentrapped drug. Experimental animals Male Wistar rats, weighing 180–250 g (3–5 month old), were obtained from Central Animal House facility of I.S.F. College of Pharmacy, Moga, Punjab, India. Animals were housed in group of three, in polypropylene cages with husk bedding under standard conditions of light and dark cycle with food and water ad libitum. Animals were acclimatized to laboratory conditions before the test. All the behavioral assessments were carried between 9:00 and 17:00 h. The experimental protocol was approved by the Institutional Animal Ethics Committee (IAEC) and was carried out in accordance with the guidelines of the Indian National Science Academy (INSA) for the use and care of the experimental animals.
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Behavioral assessment Rotarod activity Rotarod apparatus (INCO Medicraft, Ambala, India) is used for determining balance, motor co-ordination and muscle tone in rats. Motor function and grip strength of the rats were determined using this apparatus. Rats were placed individually on rotating rod and the cut-off time was 300 s and each rat had undergone three separate trials with a gap of 5 min. Briefly, the rats were exposed to prior training session on the rotarod (constant speed of 25 rpm; rod diameter 7 cm) for 1st, 5th, 10th, 15th day after 3-NP administration. The average fall of time was recorded and expressed as percentage fall off time (Kalonia et al., 2009). Measurement of body weight Animal body weight was recorded on the first and last day of the experimentation. Percent change in body weight was calculated as ½Body weight ð1st day 15th dayÞ 100= ½1st day body weight Locomotor activity The locomotor activity was monitored using an actophotometer (Medicraft, INCO, Ambala, Haryana) which operates on photoelectric cells which are connected in circuit with a counter. Before subjecting the animals to a cognitive task, all animals were individually placed in the activity meter and the total activity count was measured for 10 min. The locomotor activity was expressed in terms of total photobeam counts per 10 min per animal (Kalonia et al., 2009).
Drug treatment schedule 3-NP (Sigma chemicals, St. Louis, MO) was diluted with saline (adjust pH 7.4) and administered intraperitoneally (i.p.) to animals. SLNPRT was administered through two different routes of administration (Table 1), e.g. i.v. and i.n. through tail vein and nostrils (10 ml per nostril) by means of polyethylene tube connected to the Hamilton syringe, respectively. All drugs or vehicle were administered daily for 14 d. Animals were randomly divided into different groups, consisting of six animals in each. Study was conducted in multiple phases. On 1st, 5th, 10th, and 15th day different behavioral parameters were assessed and biochemical parameters were also measured on 15th d. Group I: Control (vehicle treated) Group II: 3-NP (10 mg/kg) Group III: 3-NP + RA (12 mg) i.n. Group IV: 3-NP + SLNPRT (i.v. 12 mg, SLN) Group V: 3-NP + SLNPRT (i.n. 12 mg, SLN)
Narrow beam The apparatus consists of 50 cm wooden strips supported by two pedestals at each end, with a height of 100 cm above the ground. The rats have to traverse a narrow beam which is suspended between a start platform and their home cage. It is important to make sure that the entire apparatus be placed at a height of at least 100 cm above the ground, so that the animal fears the height and really attempts to reach the goal box. All rats must be trained to walk over a beam for 5 d before testing. A ceiling of 120 s is employed at the end after which the rat is removed and placed in the cage by hand and receives, if it is the case, a score of 120 s. The test procedure is identical for all rats tested and performed in the same environment, preferably in the morning. Time taken to traverse rat from start platform to their home cage was measured along with their number of slipping errors (Kalonia et al., 2009). Dissection and homogenization
Table 1. Formulation code. S.no. Formulation code 1. 2. 3.
SLNP 3-NP SLNPRT
Description Solid lipid nanoparticle blank 3- Nitropropionic acid giving intra peritoneal RA-loaded SLNs using tween 80
On day 15, all the animals were sacrificed by decapitation immediately after behavioral assessments. The brains were removed, forebrain was dissected out and cerebellum was discarded. Brains were put on the ice, and the cortex and striatum regions were separated and weighed. A 10% (w/v) tissue homogenate was prepared in 0.1 mol/1 phosphate buffer
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(pH 7.4). Homogenate was centrifuged for 20 min at 20 000 rpm and the supernatant was stored in 80 C for assessing the biochemical parameters. Biochemical assessment Biochemical tests were conducted 24 h after last behavioral test. The animals were sacrificed by decapitation. Brains were removed and rinsed with ice-cold isotonic saline. Brains were then homogenized with ice-cold 0.1 mmol/L phosphate buffer (pH 7.4). The homogenate (10%w/v) was then centrifuged at 10 000 g for 15 min and the supernatant so formed was used for the biochemical estimations.
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Estimation of lipid peroxidation assay The extent of lipid peroxidation in the brain was determined quantitatively by performing the method as described by Wills (1966). The amount of malondialdehyde (MDA) was measured by reaction with thiobarbituric acid at 532 nm using Perkin Elmer Lambda 20 spectrophotometer. The values were calculated using the molar extinction co-efficient of chromophore (1.56 105 (mol/l)1 cm1; Wills, 1966). Estimation of nitrite The accumulation of nitrite in the supernatant, an indicator of the production of nitric oxide, was determined by a colorimetric assay with Greiss reagent (0.1% N-(1-napththyl) ethylene diamine dihydrochloride, 1% sulphanilamide and 5% phosphoric acid). Equal volumes of the supernatant and the Greiss reagent were mixed and the mixture was incubated for 10 min at room temperature in the dark. The absorbance was measured at 540 nm using Perkin Elmer Lambda 20 spectrophotometer. The concentration of nitrite in the supernatant was determined from the sodium nitrite standard curve (Green et al., 1982). Estimation of reduced glutathione Reduced glutathione was estimated according to the method described by Ellman. First, 1 ml of supernatant was precipitated with 1 ml of 4% sulphosalicylic acid and cold digested for 1 h at 4 C. The samples were then centrifuged at 1200 g for 15 min at 4 C. To 1 ml of the supernatant obtained, 2.7 ml of phosphate buffer (0.1 mmol/l, pH 8) and 0.2 ml of 5,50 -dithio-bis (2-nitrobenzoic acid) (DTNB) was added. The yellow color developed was measured at 412 nm using Perkin Elmer Lambda 20 spectrophotometer. Results were calculated using molar extinction co-efficient of the chromophore (1.36 104 (mol/l)1 cm1) (Ellman, 1959). Estimation of catalase activity Catalase activity was assessed by the method of Luck, wherein the breakdown of H2O2 is measured. Briefly, assay mixture consists of 3 ml of H2O2 phosphate buffer and 0.05 ml of the supernatant of the tissue homogenate. The change in absorbance was recorded for 2 min at 30 s interval at 240 nm using Perkin Elmer Lambda 20 spectrophotometer. The results were expressed as micromoles of hydrogen peroxide decomposed per min per mg of protein (Luck, 1971).
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Estimation of protein The protein content was estimated by the Biuret method (Gornall et al., 1949) using bovine serum albumin as a standard. Organ distribution study Five male Wistar rats were given four different doses of intra nasal administration of SLNPRT threw Cather tube. Tissues (brain) were promptly removed after 1 h post administration of SLNPRT (i.n.) and washed with saline. Each tissue sample was diluted with 0.9% saline solution as an internal standard and homogenized, and the homogenate was collected and stored at 20 C until analysis. Organ distribution study was carried out in order to determine the drug concentration reaches the brain with the help of HPLC. RA-loaded SLNs using Tween 80 formulation dose was 12 mg, which is delivered into the animal by the intra nasal rout. Pharmacokinetic study Three male Wistar rat were given 0.1 ml dose via i.n. route at 15, 30, 45 min, 1, 1.5, 4, 6, 24 h time interval. Sample was taken from both eye of retro orbital of rat. Pharmacokinetic parameters were determined, i.e. Cmax, Tmax, Thalf, AUC. Statistical analysis All the results are expressed as mean ± standard deviation. The treated groups were compared with control by applying the analysis of variance (ANOVA). The statistical analysis was carried out at Graph Pad Software Corp. (San Diego, CA). The p value50.05 was considered significant.
Result and discussion Particle size, polydispersity index Particle size measurement was required to confirm the production of particle in nano-range. The results indicate that particles were significantly influenced by most of the formulation and process variables. Among two different surfactants (soya lecithin and tween 80) used in the preparation of SLNs, the particle size was found to be 157.2 and 149.2 nm for soya lecithin and tween 80, respectively (Table 3). Hydrophilic and lipophilic balance value (HLB) of 12–16 is considered to be ideal for the production of stable o/w emulsion. The HLB value of tween 80 is 15 and soya lecithin is 4–9, difference in HLB values may be the reason of difference in the particle size using two different surfactants. Smaller particles are better able to reach a target tissue via the circulation. Small particle size is the requirement for the nasal delivery of SLNs to minimize the barrier-associated problems at the site of action. However, attention has been paid to particle size distribution in order to minimize the risk of embolism and other side effects due to the presence of large size SLNs. RA-loaded SLNs were successfully prepared and optimized on the basis of particle size and PDI of the nanoparticles. The process parameter includes the drug EE and for the lipid, soya lecithin, HSPC concentration was optimized.
HRA-loaded SLNs formulation
DOI: 10.3109/10717544.2014.880860
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Optimization of HSPC and lipid concentration
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and HSPC and on increasing concentration of tween 80 from 0.5, 1, 1.5 and 2 particles size reduce from 146.6 ± 11.2 to 123.1 ± 16.5. Relatively high emulsifier concentrations are required to stabilize the surface of small particles, and also to prevent problems associated with particle size stability. However, high concentration may led to toxic side effects.
Dispersion of the liquid melt into fine droplets in a hot, surfactant-containing aqueous phase-led droplet generation process is completely analogous to that during the preparation of submicron lipid emulsions; it means that all the technologies established for emulsion preparation can be used. In the preparation of SLNs, the prepared emulsion first forms the coarse particles which can be converted into nanoemulsion on applying energy and form SLNs on solidification. In the optimization process, the lipid concentration was optimized from F7 to F12 and HSPC was optimized from F1 to F6. The concentration of HSPC greatly affects the particle size and PDI, which was observed on changing the concentration during preparation of SLNs. At the initial phase on taking 1 mg of HSPC, the particle size and PDI values was found to be 793.5 ± 42.1 and 0.267 ± 0.023 (Table 2), respectively, while on increasing the HSPC concentration decrease in particle size with PDI was observed till 4 mg of HSPC, beyond that increment in particle size and PDI values was observed.
Optimization of entrapment of efficiency with soya lecithin and tween 80 The drug EE in SLNs was optimized by using soya lecithin and tween-80 as a surfactant having formulation code F21 and F22, respectively. The highest EE (61.9 ± 2.2%) was observed in case of tween 80 (Table 3) while in case of soya lecithin as surfactant the EE was being observed to 49.86 ± 3.1%. Surfactant as a tween 80 on 1% exhibits the highest EE while soya lecithin at 0.5% shows the highest EE. On the basis of higher EE and smaller size, formulation F23 was used for further in vitro characterization as code SLNPRT. Drug release study Sustain and control release of poorly water-soluble drugs may be important aspect of development of SLNs; however, it is already reported that SLNs provide the sustain/prolong release pattern. In the hypothesized formulation, the optimized formula is able to control the release the drug for longer period of time, in starting to 7 h. 75.02 ± 5.4% drug was release and at 14 h. 97.5 ± 2.9 drug was released (Figure 1). It may be the evidence that the drug is homogenously dispersed in solid lipid core of SLNs. Form the above statement it can also be concluded that the proposed formulation may able to maintain the drug concentration up to 14 h which may ultimately reduce the dosing frequency and may enhance the effectiveness of therapy. Crystallization behavior of the lipid carrier and high mobility of the drug lead to fast drug release in proposed formulation crystallization
Optimization of soya lecithin and tween 80 concentration The choice of emulsifier and its ratio to the matrix lipid are very important in the development of high-quality SLNs. The effect of soya lecithin and tween 80 concentration on nanoparticle size was evaluated by varying the concentration of both tween 80 and soya lecithin. The soya lecithin and Tween-80 were optimized from F13 to F16 and F17 to F20, respectively (Table 2). In the case of soya lecithin, the concentration varying from the 0.1, 0.2, 0.5, and 1, while maintaining the concentration of HSPC 4 mg lipid 3% each as expected results, revealed that when increasing the concentration of soya lecithin decrease in particle size decrease from 141.3 ± 13.2 to 126.1 ± 5.3 while on the same amount of lipid Table 2. Optimization of lipid, soya lecithin, tween-80, HSPC (surfactant). Formulation code (F) F1 F2 F3 F4 F5 F6 F7 F8 F9 F10 F11 F12 F13 F14 F15 F16 F17 F18 F19 F20
Lipid (%)
Soya lecithin (%)
Tween 80 (%)
HSPC (mg)
Homogenization time
Sonication time (min)
Size (nm)
PDI
1 1 1 1 1 1 1 2 3 4 5 6 3 3 3 3 3 3 3 3
0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.2 0.5 1 0.5 0.5 0.5 0.5
0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 .5 0.5 1.5 2
1 2 3 4 5 6 4 4 4 4 4 4 4 4 4 4 4 4 4 4
30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30
7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7
793.5 ± 42.1 697.4 ± 33.1 623.2 ± 23.4 544.1 ± 34.2 688.9 ± 18.3 756.9 ± 34.3 161.7 ± 24.3 133.2 ± 31.2 118.1 ± 26.2 181.1 ± 18.4 201.1 ± 22.3 231 ± 31.4 141.3 ± 13.2 139.2 ± 8.4 135.5 ± 11.9 126.1 ± 5.3 146.6 ± 11.2 139.2 ± 17.1 126.5 ± 13.0 123.1 ± 16.5
0.267 ± 0.023 0.285 ± 0.015 0.255 ± 0.023 0.250 ± 0.021 0.364 ± 0.012 0.384 ± 0.017 0.250 ± 0.02 0.391 ± 0.023 0.201 ± 0.019 0.380 ± 0.015 0.344 ± 0.013 0.312 ± 0.018 0.331 ± 0.013 0.301 ± 0.016 0.296 ± 0.025 0.325 ± 0.022 0.251 ± 15.3 0.282 ± 13.2 0.221 ± 11.8 0.347 ± 17.0
Optimization of HSPC, lipid, soya lecithin, tween-80. Formulation codes F1 to F6 were optimized for HSPC, F7 to F12 were optimized for lipid, F13 to F16 were optimized for soya lecithin, and F17 to F20 were optimized for tween-80. Bold values signifies optimum size of SLNs by using different concentrations of lipid, soya lecithin, tween 80 and HPSC.
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Table 3 Optimization of, drug loading using two different surfactant (soy lecithin and tween 80). Formulation code F21 F22
Lipid (%)
Soya lecithin (%)
Tween 80 (%)
HSPC (mg)
Drug (mg)
Size (nm)
PDI
EE (%)
3 3
0.5 0
0 1
4 4
12 12
167.4 ± 30.02 149.2 ± 18.2
0.289 ± 0.031 0.290 ± 0.021
40.46 ± 2.8 61.9 ± 2.2
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Final formulation selection and optimization of EE using two different surfactant (soy lecithin and tween 80). Formulation codes F21 and F22 were optimized for soya lecithin and Tween-80, respectively.
Figure 1. %Cumulative percentage release of drug from, i.e. SLNPRT.
Figure 3. Effect of SLNPRT on rotarod activity in 3-NP-treated rats. Values are expressed as mean ± SEM ap 0.05 as compared to control group. bp 0.05 as compared to 3NP treated group. cp 0.05 as compared to RA per se. dp 0.05 as compared to RA from i.v.
drug incorporated, structural properties of lipid and drug), production methods (Melike & Gulgun, 2007). It is proved by changing the surfactant soya lecithin with tween changes in drug entrapment, and also the effect of the drug loading, PDI and particle size on changing the surfactant was observed. Behavioral assessment Effect of RA (SLN) on body weight, rotarod activity, locomotor, and narrow beam in 3-NP-treated rats
Figure 2. Effect of SLNPRT on body weight in 3-NP-treated rats. Values are expressed as mean ± SEM ap 0.05 as compared to control group. b p 0.05 as compared to 3NP-treated group. cp 0.05 as compared to RA per se. dp 0.05 as compared to RA from i.v.
behavior and drug mobility may be limited because of controlled/sustain release pattern of SLNs. The particle size that affects drug release rate directly depends on various parameters such as composition of SLN formulation (such as surfactant/surfactant mixture, amount of
Systemic treatment with 3-NP significantly reduced body weight (Figure 2), motor co-ordination (rotarod) (Figure 3), impaired locomotor (Figure 4) and increased the latency to reach goal platform (narrow beam test) (Figure 5) (p50.05) on 5th, 10th and 15th d as compared with the vehicle-treated group, suggesting that the effects of 3-NP most probably mimic either the juvenile onset or late stages of HD-like behavior. Deficiency of GABAergic stimulation has been implicated in the pathophysiology of HD (Lee & Chang, 2004; Saulle et al., 2004). Study suggested the sequential neurodegenerative process in the striatal GABAergic efferent projections during increased neuropathological grades of HD (Allen et al., 2009). In the present study, i.n. administration of SLNPRT formulation significantly improved the behavioral abnormalities in rotarod and locomotor activities (p50.05) and decreased the latency to reach the goal platform in narrow beam walk (p50.05), indicating the neuroprotective effect. The hunting-like symptoms was reversed at variable extent on giving treatment using
HRA-loaded SLNs formulation
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DOI: 10.3109/10717544.2014.880860
Figure 4. Effect of SLNPRT on locomotor activity in 3-NP-treated rats. Values are expressed as mean ± SEM ap 0.05 as compared to control group. bp 0.05 as compared to 3NP-treated group. cp 0.05 as compared to RA per se. dp 0.05 as compared to RA from i.v.
Figure 6. Effect of SLNPRT on malondialdehyde (MDA) level striatum and cortex in 3-NP-treated rats. Values are expressed mean ± SEM ap 0.05 as compared to control group. bp 0.05 compared to 3NP-treated group. cp 0.05 as compared to RA per d p 0.05 as compared to RA from i.v.
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in as as se.
Figure 5. Effect of SLNPRT on balance beam walking performance in 3-NP-treated rats. Values are expressed as mean ± SEM ap 0.05 as compared to control group. bp 0.05 as compared to 3NP-treated group. cp 0.05 as compared to RA per se. dp 0.05 as compared to RA from i.v.
SLNs as a carrier of two different compositions. One is the SLNPRT and RA (without carrier) administered through the nasal and i.v, and also comparing the importance of selection of i.n. route of administration. As the figures of the behavioral activities show that the hunting-like symptoms disappeared on successful treatment with the RA in forms SLNs, on comparing the SLNPRT and RA (without carrier) the SLNPRT was found to be effective by the nasal route. Also, comparison of the same formulation given through the i.v. route shows the evidence of the importance of selection of nasal route; i.v. route in itself has the limitations like invasive, painful and also requires the assistance of the third person. The importance of selection of i.n. route is also observed in the assessment of biochemical parameter. The 3-NP experiment model mimics both the hyperkinetic and hypokinetic
Figure 7. Effect of RA (43) on nitrite level in striatum and cortex in 3-NP-treated rats. Values are expressed as mean ± SEM ap 0.05 as compared to control group. bp 0.05 as compared to 3NP-treated group. cp 0.05 as compared to RA per se. dp 0.05 as compared to RA from i.v.
symptoms of HD, depending upon the duration of administration (Borlongan et al., 1997). Biochemical assessment Effects of RA on lipid peroxidation, nitrite, catalase and GSH after 3-NP treatment Systemic administration of 3-NP significantly increased lipid peroxidation, nitrite concentration and depleted catalase and glutathione in striatum and cortex, and control vehicle treated found to be stable normal (Figures 6–9). SLNPRT
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Figure 8. Effect of SLNPRT on catalase in striatum and in 3-NP-treated rats. Values are expressed as mean ± SEM ap 0.05 as compared to control group. bp 0.05 as compared to 3NP-treated group. cp 0.05 as compared to RA per se. dp 0.05 as compared to RA from i.v.
Drug Deliv, Early Online: 1–9
substantial evidence that oxidative damage significantly contributes to the pathogenesis of several neurodegenerative diseases including HD (Kumar et al., 2007). Disruption of the mitochondrial enzyme complex activity is associated with ROS. According to the immune histochemical data, biochemical assays in HD patients show significant increases in MDA and 4-hydroxynonenal brain levels, which are almost 8-fold greater than the control subjects (Stoy et al., 2005). Both nitrite and nitric oxide species were shown to be oxidizing DA into its quinone forms bound covalently to critical proteins (LaVoie & Hastings, 1999). From the above hypothesis, it can be correlated that 3-NP treatment increased the oxidative stress that is responsible for the damage of dopaminergic neuron (act as motor control), which is ultimately responsible for impairment in motor coordination, behavioral alterations. In organ distribution study, brain drug concentration of SLNPRT formulation was found to be 5.69 mg, in pharmacokinetic study Cmax, tmax, t1/2, AUC values were found to be 0.284 mg/ml, 1.5 h, 3.17 h, and 1.505 mg/ml/h, respectively.
Conclusion
Figure 9. Effect of SLNPRT on reduced GSH level in striatum & cortex in 3-NP-treated rats. Values are expressed as mean ± SEM ap 0.05 as compared to control group. bp 0.05 as compared to 3NP-treated group. cp 0.05 as compared to RA per se. dp 0.05 as compared to RA from i.v.
formulation significantly attenuated lipid peroxidation, nitrite concentration, and restored endogenous antioxidants enzyme (catalase and GSH) activities on 3-NP-treated animals. RA protects neuron from oxidative stress (Kelsey et al., 2010a,b). The increment on brain LPO and nitrite level is directly associated with neurodegenerative disease. It has been reported that RA significantly attenuate H2O2-induced ROS generation and apoptotic cell death (Lee et al., 2008). RA significantly protected neurons, these effects are mediated by the prevention of oxidative stress, intracellular Ca2+ overload and c-fos expression (Fallarini et al., 2009). Systemic administration of 3-NP in rats and non-human primates produces homogeneous blockade of complex II within the brain (Kumar et al., 2007), leading to preferential excitotoxic striatal degeneration associated with behavioral abnormalities that are highly reminiscent of HD (Brouillet et al., 1993, Brouillet et al., 1999; Bizat et al., 2003). There is
In this study, SLNs containing RA was successfully prepared by the hot homogenization method. The study suggests the importance of controlling the critical formulation parameters during formulation, which greatly influence the final product, such as particles size, PI and drug encapsulation efficiency. Sustain release was observed from the SLNs. RA-loaded SLNs treatment is able to significantly attenuate 3NP-induced deficits in body weight, beam walk, locomotor and motor coordination, and are also able to significantly attenuate 3NPinduced striatal oxidative stress. The optimum brain drug concentration signifies the importance of using nasal route compared to i.v. route. Nasal delivery also prevents the unwanted distribution and metabolism in other parts of body. From the present study, it can be concluded that nasal administration of RA-loaded SLNs may be a promising approach for the effective management of HD.
Declaration of interest The authors report no conflicts of interest. The author alone is responsible for the content and writing of the paper.
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