Rutin-encapsulated chitosan nanoparticles targeted to the brain in the treatment of Cerebral Ischemia

https://doi.org/10.1016/j.ijbiomac.2016.06.001Get rights and content

Abstract

Objective

Rutin, a potent antioxidant, has been reported to reduce the risk of ischemic disease. Our study aims to prepare rutin-encapsulated-chitosan nanoparticles (RUT-CS-NPs) via ionic gelation method and determine its results, based on different parameters i.e. surface morphology characterization, in-vitro or ex-vivo release, dynamic light scattering and differential scanning calorimetry (DSC), for treating cerebral ischemia.

Methods

UPLC-ESI-Q-TOF-MS/MS was used to evaluate the optimized RT-CS-NPs1 for brain-drug uptake as well as to follow-up the pharmacokinetics, bio-distrbution, brain-targeting efficiency and potential after intranasal administration (i.n.).

Key findings

A particle size of <100 nm for the formulation, significantly affected by drug:CS ratio, and entrapment efficiency and loading capacity of 84.98% ± 4.18% and 39.48% ± 3.16%, respectively were observed for RUT. Pharmacokinetics, bio-distribution, brain-targeting efficiency (1443.48 ± 39.39%) and brain drug-targeting potential (93.00 ± 5.69%) showed enhanced bioavailability for RUT in brain as compared to intravenous administration. In addition; improved neurobehavioral activity, histopathology and reduced infarction volume effects were observed in middle cerebral artery occlusion (MCAO) induced cerebral ischemic rats model after i.n. administration of RUT-CS-NPs.

Conclusion

A significant role of mucoadhesive-RT-CS-NPs1 as observed after high targeting potential and efficiency of the formulation prove; RUT-CS-NPs are more effectively accessed and target easily the brain.

Introduction

The third largest neurological disease resulting long term disability worldwide and a cause of morbidity and mortality is considered to be Stroke [1], [2], [3], [38]. Cerebral ischemia, a reason for stroke, generally causes permanent deterioration of the central nervous system (CNS) [4]. It is believed that once cerebral ischemia is developed, it’s the determinant factor (released due to inflammatory process stimulation) which leads towards serious cerebral damage alongwith morbidity and mortality [3], [5], [38]. The literature cited here reports; alongwith aforementioned causes, oxidative stress also plays an important role in cerebral ischemia reperfusion injury. The strive for high amount of oxygen consumption after oxidative stress leads to production of free radicals and reactive oxygen species (ROS) acting as a source of injury [1], [2], [6]. Due to improper developed antioxidant defense mechanism in brain, oxidants or free radicals release from inflammatory cells intimidates tissue viability in the vicinity of ischemic core resulting a good source of pathogenesis for ischemic-reperfusion [1], [7]. Hence it indicates that pharmacological modification for treating oxidative damage may be an important part of management.

Neuroglobin (Ngb), newly discovered globin, have been reported with protection as offered against hypoxic/ischemic cell injury and thus plays a role in brain protection [57], [58], [59], [60], [61], [62], [63]. According to research hypoxic/ischemic injury results in accumulation of reactive oxygen (ROS) and nitrogen (RNS) species which were effectively encountered by Ngb hence improve neuronal cell survival [57]. Ubiquilin-1 (biomarker) as applied in RNS and ROS can oxidize different proteins which lead to misfolding as well as changes in three dimensional structures of protein after MCAO insult brain [64].

Numerous antioxidant drugs i.e. Thymoquinone, Curcumin, Ropinirole, Thioperamide with well-established mechanisms i.e. reducing ROS-mediated reactions and rescue the neurons from reperfusion-induced neuronal loss in animal models of cerebral ischemia have been reported [1], [8], [9], [10]. Recently, research studies have reported Rutin (found in Carpobrotus edulis and Ruta graveolens) a lipophilic drug to be a potent source of treatment in cerebral ischemia [11], [12], [13], [14], [15] but still different factors exists as hindrance while using Rutin i.e. low water solubility and hence less bioavailability, chemical and enzymatic degradation in gastrointestinal tract due to lipophilic nature thus posing permeability complications and extensive hepatic first pass metabolism [16], [17], [18], [19], [20]. Likewise, another important factor posing problems related to drug discovery and development of an effective formulation in order to treat and manage cerebral ischemia is blood brain barrier (BBB) i.e. a protective natural barrier surrounding the central nervous system (CNS) that hinders the delivery of drugs to the brain. Even the most powerful drug-delivery systems (DDS) i.e. Intravenous, buccal and transdermal route are unable to affect BBB [21], [22]. Hence different magnetic drug targeting and drug carrier systems approaches i.e. liposomes, antibodies, nanoemulsions, nanoparticles (NPs) or nanomicelles have been adopted to overcome the aforementioned problems. Amongst aforementioned approaches, Nanoparticles preparation may be best utilized to prepare controlled and targeted delivery system for drugs with different nature i.e. hydrophilic, hydrophobic natural and synthetic drugs, vaccines, proteins as well as biological macromolecules [23], [24]. Similarly, out of the available route of drug administrations for brain targeting, Intranasal route (i.n) showed numerous advantages i.e. significant amount of drug transportation into cerebrospinal fluid (CSF) and olfactory bulb via olfactory sensory neurons, noninvasiveness, effective, safe and painless route, less skilled required for drug delivery as well as property of localized therapeutic effect with less side effects observed [1], [2], [25].

In our study we utilized the same approach of i.n drug delivery system and brain targeting in order to achieve therapeutic goals via eliminating different factors which can lead to less amount of Rutin access in brain i.e. avoiding first pass metabolism and distribution to non-targeted sites and hence reducing the peripheral side effects. Thus, Rutin loaded Mucoadhesive polymeric nanoparticles (NPs) were prepared in order to enhance the nasal residence, slow release of drug supply to brain at a constant rate (problems encountered with convention i.n route of drug delivery) [2], [26], [27]. The other major objective for this study is; determination of Rutin loaded NPs pharmacokinetics in brain and plasma after i.n and i.v. administration in Wistar rats and hence evaluate direct nose-to-brain transport pathway.

Section snippets

Materials and methods

Chitosan (medium mol. wt. chemical with degree of acetylation of 85%), Sodium tripolyphosphate (TPP) and cellophane tube with specifications; cut off mol. wt., 12,000 Da, flat (25 mm), diameter (16 mm) and capacity (60 mL/ft) alongwith LC–MS grade solvents i.e. Acetonitrile, Methanol and Formic acid were obtained from Sigma-Aldrich (St Louis, MO). Acetic acid (glacial) was obtained from IOL Chemical Ltd. (Mumbai, India) whereas Methanol, Ethanol, sodium hydroxide (NaOH), Potassium dihydrogen

In-vivo study

The in-vivo pharmacokinetics, biodistribution and pharmacodynamics study was performed after proper approval (protocol approval No: 847) Animal Ethical Committee, Jamia Hamdard (New Delhi, India) which ensures to confirm according to National Guidelines on the Care and Use of Laboratory Animals. One week before experiments; Wistar rats (n:6, weight: 300–400 g and age: 8–10 weeks) were maintained in an environment with controlled room temperature (25 ± 2 °C) and humidity (60 ± 5%) for 12 h dark–light

Statistical analysis

All the results were expressed as mean ± standard error of mean (SEM) whereas students’ t-test was applied for statistical difference between unpaired observations via ANOVA with p-value <0.05.

Preparation and characterization of RUT-CS-NPs

Different compositions trials were tried to find the optimized formulation (based mainly on the clarity and system aggregation as mentioned in Table 1) which improved the lower particle size and PDI, high process yield, EE and LC (Table 2). The high EE may be due to the ionic interaction between negatively charged rutin with positively charged CS. Results in detail shows; the selected formulation C4 (90.86 nm, 0.309, and 69.64%) had a smaller minimum particle size, optimum PDI, and higher

Discussion

Ionotropic gelation technique was used for the development of RUT-CS-NPs and optimized with the help of different factors mentioned in Tables 1, 2 and 3. For the optimum particle size for intranasal to brain drug delivery to obtain i.e. below 100 nm with a PDI closer to 0.206 as mentioned [1], [47], different parameters were checked such as CS concentration, TTP concentration, stirring speed and pH to obtain small particle size, maximum loading with maximum encapsulation efficiency. The values

Conclusion

Optimized Rutin loaded CS-NPs formulated via ionotropic gelation method having optimum particle size, loading capacity, and entrapment efficiency showed a sustained release (over 24 h) in-vitro drug release profile. UPLC/ESI-Q-TOF-MS/MS based bioanalytical method for CS-NPs was developed, validated and successfully applied for the pharmacokinetic and biodistribution studies resulting in an improved brain bioavailability and thus effective treatment of cerebral ischemia. The Pharmacodynamic

Conflict of interests

No conflict of interest exists among authors.

Funding

No grants were received.

Acknowledgment

I (Dr. Niyaz Ahmad) am grateful to Prof. (Dr.) Farhan Jalees Ahmad for the collaboration research study in between University of Dammam, Dammam, Saudi Arabia and Jamia Hamdard (Hamdard University), New Delhi, India.

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      However, its poor aqueous solubility is the cause of its low bioavailability [4]. Many nanotechnology-based attempts have been devoted to enhancing Rutin's solubility and biological performance for different therapeutic purposes via different routes of administration [5–11]. Over the years, significant efforts have been devoted to innovate new functional materials to formulate successful drug delivery vehicles.

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