Development and evaluation of rivastigmine loaded chitosan nanoparticles for brain targeting

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Abstract

The rivastigmine (RHT) loaded chitosan nanoparticles (CS-RHT NPs) were prepared by ionic gelation method to improve the bioavailability and enhance the uptake of RHT to the brain via intranasal (i.n.) delivery. CS-RHT NPs were characterized for particles size, particle size distribution (PDI), encapsulation efficiency, zeta potential and in vitro release study. Nose-to-brain delivery of placebo nanoparticles (CS-NPs) was investigated by confocal laser scanning microscopy technique using rhodamine-123 as a marker. The brain/blood ratio of RHT for different formulations were 0.235, 0.790 and 1.712 of RHT (i.v.), RHT (i.n.), and CS-RHT NPs (i.n.) respectively at 30 min are indicative of direct nose to brain transport bypassing the BBB. The brain concentration achieved from i.n. administration of CS-NPs (966 ± 20.66 ng ml−1; tmax 60 min) was significantly higher than those achieved after i.v. administration of RHT sol (387 ± 29.51 ng ml−1; tmax 30 min), and i.n. administration of RHT solution (508.66 ± 22.50 ng ml−1; tmax 60 min). The higher drug transport efficiency (355 ± 13.52%) and direct transport percentage (71.80 ± 6.71%) were found with CS-RHT NPs as compared to other formulation. These results suggest that CS-RHT NPs have better brain targeting efficiency and are a promising approach for i.n. delivery of RHT for the treatment and prevention of Alzheimer’s disease (AD).

Graphical abstract

A pharmacokinetic study in rats indicates that RHT loaded CS-NPs administered intranasally (i.n.) have higher concentration in brain as compared to RHT solution (i.n.) and (i.v.).

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Introduction

Alzheimer’s disease (AD) is a progressive neurodegenerative disorder of the central nervous system (CNS) and is the most common cause of dementia in the elderly population (Ray and Lahiri, 2009) in which there is a progressive deterioration of intellectual and social functions, memory loss, personality changes and inability for self care (Kosasa et al., 1999). The exact pathogenesis of the neuronal degeneration and cognitive impairment in AD still remains to unclear. AD is multisystemic in nature and this presents numerous difficulties for the potential treatment of this disorder. Various factors hinder the discovery and development of effective drug delivery systems (DDS) for the delay, treatment, and prevention of AD, the inability to deliver drugs effectively to the brain due to the numerous protective barriers surrounding the CNS i.e., blood–brain barrier (BBB) being a major concern (Lockman et al., 2002). Many strategies which include development of chemical delivery systems, magnetic drug targeting, or drug carrier systems such as antibodies, liposomes, or nanoparticles (NPs) have been developed to overcome these problems. Among the various DDS developed polymeric NPs have attracted great attention as potential drug delivery systems in the CNS because they have the ability to deliver a wide range of drugs to targeting areas of the body for controlled drug release and site-specific drug targeting (Sahni et al., 2011). Many authors have delivered a variety of drugs such as hydrophilic drugs, hydrophobic drugs, proteins, vaccines, and biological macromolecules using NPs (NPs) as carriers (Hans and Lowman, 2002). NPs have a further advantage over larger bulk materials, because they have a higher surface-to-volume ratio and therefore the dose and frequency of administration is reduced hence increasing patient compliance, thereby making them better suited for intranasal (i.n.) delivery (Wilson et al., 2010).

CNS drug delivery strategy that has received relatively little attention is the administration of drug by i.n. route. Drugs delivered intranasally are transported along olfactory sensory neurons to yield significant concentrations in the CSF and olfactory bulb. Recent evidence of direct nose-to-brain transport and direct access to CSF of neuropeptides bypassing the bloodstream has been shown in human trials, despite the inherent difficulties in delivery (Misra et al., 2003). i.n. delivery is non-invasive, essentially painless, does not require sterile preparation, and can be easily and readily administered by the patients themselves or by a physician, e.g., in an emergency setting. Drug delivery systems are designed with rationale of promoting the therapeutic effect of a drug and minimizing its toxic side effects, which is achieved by optimizing the amount and duration of the drug in the vicinity of the target cells while reducing the drug exposure to non-target cells.

There are many approaches for the treatment of AD, but cholinergic hypothesis has special attention. Rivastigmine (RHT) was chosen as a drug candidate for AD as it is inhibitor of both acetylcholinestrase (AChE) and butrylcholinestrase (BuChE) enzyme and is 4–17 times more specific for inhibiting AChE in brain as compared to heart and blood. However limitation with its oral drug delivery is its restricted entry into brain due to its hydrophilicity, thereby necessitating frequent dosing resulting in severe cholinergic side effects. Other investigators have worked on RHT loaded poly(n-butyl cyanoacrylate) NPs for brain targeting through i.v. route. The results showed that surfactant coated poly(n-butylcyanoacrylate) NPs significantly transported the drug RHT in comparison with the free drug to the brain. The high concentrations of RHT achieved in the brain may be a significant improvement for treating AD. But the approach is also associated with several disadvantages like possibility of systemic side effects, patient non-compliance as it is a painful techniques beside possibility of distribution to non targeted site.

The present investigation was aimed to formulate nanoparticulate system of RHT that will be targeted to brain through nasal route to avoid first pass metabolism and avoid the distribution to non-targeted sites thus leading to decrease peripheral side effects. The mucoadhesive polymeric NPs of RHT so developed are also expected to offer many advantages over conventional nasal dosage forms, like increased nasal residence and possibility of drug release at slow and constant rate.

Section snippets

Materials

Chitosan with medium molecular weight (Mw = 750,000 Da) and degree of deacetylation about 85% and sodium tripolyphosphates (TPP) were purchased from Sigma–Aldrich (Bangalore, India). RHT having molecular weight (Mw = 400.43 g/mol) was received as a gift sample from Torrent Pharmaceuticals Ltd. (Himachal Pradesh, India) having molecular weight. Potassium dihydrogen phosphate, Methanol, sodium hydroxide (NaOH) and 1-Octanol were all purchased from S.D. Fine Chemicals, Ltd. (Mumbai, India). Glacial

Preparation of chitosan NPs

CS-NPs were prepared according to the ionic gelation process (Calvo et al., 1997, Vila et al., 2002, Aktas et al., 2005). CS-NPs were obtained upon the addition of a TPP aqueous solution (2 mg/ml) to a CS solution (1.75 mg/ml) stirred at room temperature. The formation of NPs was a result of the ionic interaction between the positively charged amino groups of chitosan and negative groups of TPP. The ratio of chitosan/TPP was established according to the preliminary studies. CS-RHT NPs were

Physiochemical characterization of chitosan NPs

The surface morphology of the prepared NPs was determined for by using transmission electron microscopy (TEM). The nanosuspension samples were prepared by dispersing a small amount of NPs into distilled water. A drop of nanosuspension was placed on a paraffin sheet and carbon coated grid was placed on sample and left for 1 min to allow CS-NPs to adhere on the carbon substrate. The remaining suspension was removed by adsorbing the drop with the corner of a piece of filter paper. Then the grid was

In vitro permeability studies

Fresh nasal tissues were carefully removed from the nasal cavity of porcine obtained from the local slaughter house. The tissue samples were fixed in Logan instrument (Logan Instrument Carporation, NJ, USA) cells displaying a permeation area of 0.785 cm2. Twenty milliliters of phosphate buffer saline (PBS) pH 6.4 was added to the receptor chamber. To ensure oxygenation and agitation, a mixture of 95% O2 and 5% CO2 was bubbled through the system. The temperature was maintained at 37 °C. After a

In vivo study

Wistar rats (aged, 4–5 months) of either sex weighing between 200 and 250 g were selected for the study of biodistribution and pharmacokinetic studies. The protocol for animal studies was approved by the Institutional Animal Ethical Committee of Jamia Hamdard, New Delhi, India, and study was carried out in accordance with principles of laboratory animal care and the approved protocol.

Statistical analysis

All data are reported as mean ± S.E.M. and the differences between the groups were tested using Student’s t-test at the level of P < 0.05. More than two groups were compared using ANOVA and the difference greater at P < 0.05 was considered significant.

Preparation and characterization of chitosan NPs

Different concentrations of CS and TPP were used to optimize the best CS/TPP ratio on the basis of particle size, polydispersity index (PDI), zeta potential and process yield. The mean particle size, PDI and process yield of different formulations of CS-NPs are shown in Table 1. The process yield of different formulation of CS-NPs ranged between 40.56 ± 2.30% and 88.42 ± 3.25%. The mean particle size and PDI varied from 143.1 ± 9.2 to 3300 ± 7.0 nm and 0.424 ± 0.012 to 0.985 ± 0.032 respectively depending

Preparation and characterization of CS-RHT NPs

The chitosan/TPP particles prepared with different concentrations of chitosan or TPP were studied. The results indicated that with a increase in the concentration of either CS or TPP particle size increased. Fan and coworker found that the formation of chitosan/TPP nanoparticles was only possible for some specific concentrations of CS and TPP (Fan et al., 2012). On the basis of experimental data analysis it was observed that the ratio between CS and TPP 2.19/1 to 2.5/1 was found optimum in

Conclusion

The present research work proposed a novel nanoparticulate formulation for the intranasal delivery of RHT. The effect of different variables on NPs preparation was investigated. The encapsulation efficiency, typically 85.3 ± 3.5%, and the reproducibility of the preparations were satisfactory. The in vitro release found to be 89.27 ± 2.672 over 24 h indicated a controlled and sustained release profile of CS-RHT NPs. An enhanced brain uptake of CS–RHT NPs was clearly observed following nose to brain

Acknowledgements

The authors are grateful to University Grant Commission (UGC), Government of India for providing fellowship to Mohammad Fazil as financial assistance and are also grateful to Department of Science and Technology (DST), New Delhi for providing DST INSPIRE Fellowship to Shadab Md as financial assistance.

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