INTRODUCTION:
In certain cases, oral route fails to deliver therapeutic amount of drug to brain due the presence of certain interfaces like blood–brain barrier (BBB), blood-cerebrospinal fluid barrier and efflux transporters1. These barriers regulate the exchange between peripheral blood circulation and cerebrospinal fluid (CSF) circulatory system. Other factors like physicochemical properties of drug also create hindrance in central nervous system (CNS) delivery2.
Thus, various approaches like BBB disruption, drug manipulation and alternative route of drug administration like intra cerebroventricular, intrathecal, and olfactory pathways (intranasal route) are being used for targeting of drugs to the brain3. In the present scenario, the intranasal route to bypass the BBB is an upcoming field, as this route caters a novel, practical, simple and non-invasive approach to bypass the BBB and reduce the systemic exposure and thus systemic side-effects associated with drug4. Drug after intranasal administration reaches the olfactory epithelium region of the nasal mucosa that acts as a gateway for substances entering the CNS due to neural connection between the nasal mucosa and the brain5.
Many formulation strategies have been developed to overcome the BBB via intranasal route but all have their own limitations. On this note, the concept of lipid based nanoparticles like solid lipid nanoparticles (SLNs) matured.
SLNs were introduced at the beginning of 1991 as submicron colloidal carriers (50–1000 nm). They are used for delivery of both hydrophilic and lipophilic drug (s) being trapped in biocompatible material core made up of a lipid or combination of lipids and stabilized by surfactant which is present at the outer shell6. SLNs showed preferential benefits like targeted drug delivery, controlled drug release and increased bioavailability hence reduced dose and side effects of drug7. They have good tolerability and biodegradability, lack of acute and chronic toxicity, scalability to large scale production8. SLNs furnish an improvement in nose-to-brain drug delivery since they are able to protect the encapsulated drug from biological and/or chemical degradation and may also increase nasal retention time due to the occlusive effect, good application properties and adhesion to mucous membranes8.
Donepezil (DPL) (Fig. 1) is a piperidine-based, reversible and non-competitive inhibitor of the enzyme acetylcholinesterase (AChE)9. It is the second drug approved by the Food and Drug Administration (FDA) for the treatment of mild to moderate dementia of the Alzheimer’s type10. DPL is postulated to exert its therapeutic effect by enhancing cholinergic function. This is accomplished by increasing the concentration of acetylcholine (ACh) through reversible inhibition of its hydrolysis by AChE.
Currently, tablets or capsules (5 or 10 mg/day) of DPL are available in the market for oral delivery. But these dosage forms provide non targeted delivery resulting in numerous gastrointestinal side effects such as diarrhoea, nausea, anorexia, gastric bleeding, muscle convulsions etc11. Another limitation of oral delivery is restricted entry of DPL into brain due to its hydrophilicity (freely soluble in water) 12, thereby entailing frequent dosing resulting in severe cholinergic side effects. As per the previous findings, DPL exhibited hepatotoxicity (but less as related to its processor tacrine) and undergo first pass metabolism, again a restraint for oral delivery.
Fig. 1: Structure of donepezil
As a result of above mentioned conditions, it will be beneficial to formulate a non-oral delivery system of DPL to restrict the side effects associated with oral delivery, prevention of systemic exposure as well as distribution of drug to non-targeted sites. As the target site for DPL is brain, thereby developed system should also provide the therapeutic concentration of drug at the site of action. Consequently, intranasal route was selected for the delivery of DPL to target site (brain) in the form of solid lipid nanoparticles. Moreover, DPL possess acceptable log p value (4.14), molecular weight (379.492 g mol-1) and small dose (5-10 mg/day) necessary for brain targeting by means of intranasal administration.
In the present study, Glyceryl behenate based SLNs containing DPL were developed for brain targeting via nasal route. The applicability of Glyceryl behenate as a lipid for brain delivery is already reported by Jose and co-workers13. SLNs were evaluated for particle size, poly dispersity index (PDI), zeta potential, entrapment efficiency (%), drug loading (%) and in vitro release. In vivo studies were performed on albino wistar rat model. Stability studies were performed by storing the optimized formulation at 4±2 °C (refrigerator), 25±2 °C /60 ±5% RH and 40±2 °C /75±5% RH up to six months.
MATERIALS AND METHODS:
Materials:
DPL was received as a gift sample from Jubilant Life Sciences, Noida, India. Glyceryl behenate (Compritol ATO 888), and Glyceryl palmitostearate (Precirol ATO 5) were obtained as a gift sample from Gattefosse, Witten, Germany. Stearic acid, Palmitic acid, Acetonitrile (ACN), Triethylamine (TEA), o-Phosphoric acid (o-PA), Tween 80 and Poloxamer 188 along with all other chemicals were purchased from Sigma-Aldrich, New Delhi, India. Dialysis membrane (molecular weight cut off 12000–14000 D), Membrane filter and Syringe filter (0.22 µm) were purchased from HiMedia Laboratories Pvt. Ltd., Mumbai, India. ACN, TEA, o-PA were of HPLC grade while all other solvents and chemicals used were of analytical grade.
Methods:
Selection of Lipid:
Solubility of drug in melted lipid is one of the most important factors that determine the encapsulation efficiency of the drug in the lipid. However, equilibrium solubility studies cannot be carried out in this case. Hence, we used a modified method to identify the solid lipid having better solubilisation potential for drug14. Glyceryl behenate, glyceryl palmitostearate, stearic acid and palmitic acid were screened for their potential to solubilise DPL.
DPL (20 mg) was taken in screw capped vial. The solid lipids were separately heated above their melting point. These lipid melts were gradually added in portions to the vial containing DPL with continuous stirring using vortex mixer and maintaining the same temperature (above the melting point of lipid). The end point of the solubility was the formation of clear, pale yellow solution of molten lipid. The amount of molten lipid required to solubilise the DPL was noted visually. The experiment was perform in triplicate (n=3).
Preparation of DPL Loaded SLNs:
In a preliminary laboratory study, various factors like drug to lipid ratio (1:4, 50 mg: 200 mg), surfactant concentration (Tween 80: Poloxamer 188, 2 % w/v), chloroform: ethanol ratio (1:1, 5% v/v) as the solvent for drug and lipid, homogenization time (30 min), stirring time (2.5 h) and stirring speed (2500 rpm), sonication time (5 min) were fixed and their effect on particle size, entrapment efficiency were observed. Factors like drug to lipid ratio, surfactant concentration, and stirring speed were further optimized in this study. All experiments were performed in triplicate and averages were considered as the response. Table1 shows the composition of various batches.
DPL- loaded SLNs (DPL-SLNs) were prepared by solvent emulsification–diffusion technique8. Accurately weighed lipid (200 mg) was dissolved in mixture of ethanol and chloroform (1:1, 5ml) as the internal oil phase. Drug (50 mg) was dispersed in the above solution. This reaction mixture was heated above the melting point of lipid (700C). This organic phase was then poured drop by drop into a homogenizer tube containing 20 ml of hot aqueous surfactant solution of tween 80: poloxamer 188 (1:1, 2.25 %). The mixture was then homogenized (Remi Instruments Pvt. Ltd, India) for 30 min at 3,000 rpm to form primary emulsion (o/w). The above emulsion was poured into 80 ml of ice-cold water (2-30C) containing surfactant (mixture tween 80: poloxamer 188 in 1:1 ratio, 2.5 %) followed by stirring to extract the organic solvent into the continuous phase and for proper solidification of SLNs. The stirring was continued (2.75 h) at 3,000 rpm to get SLN dispersion. The dispersion was then centrifuged (Remi Instruments Pvt, Ltd, India) at 18,000 rpm (20 min) to separate the solid lipid material containing the drug. These SLNs were washed with deionized water and then re-dispersed in aqueous surfactant mixture (Tween 80: Poloxamer 188, 1:1) and sonicated for 5 min (1 cycle, 100 % amplitude, Bandelin sonoplus, Germany) to obtain the SLN dispersion of uniform size.
Table 1: Composition of various batches of DPL-SLNs
|
Formulation code |
Variables |
||||
|
Drug (mg) |
Lipid (mg) |
Surfactant % (w/v) |
Stirring time (h) |
Stirring Speed (rpm) |
|
|
DPL1 |
50 |
200 |
2 |
2.5 |
2500 |
|
DPL2 |
50 |
250 |
2 |
2.5 |
2500 |
|
DPL3 |
50 |
200 |
2.25 |
2.5 |
2500 |
|
DPL4 |
50 |
200 |
2 |
2.75 |
2500 |
|
DPL5 |
50 |
200 |
2.25 |
2.75 |
2500 |
|
DPL6 |
50 |
200 |
2.25 |
2.75 |
3000 |
Evaluation of Optimized Formulation:
Optimized DPL-SLN formulation (DPL6) was characterized for the following parameters:
Particle size, zeta potential and morphological study:
Average particle size, PDI and zeta potential were measured by photon correlation spectroscopy (PCS; Zetasizer, HAS 3000; Malvern Instruments, Malvern, UK). Measurements were carried out at an angle of 90 degrees at 250C. Surface morphology of optimized SLN formulation was done by using transmission electron microscope (TEM, Philips CM 10, Holland). To perform the TEM observation , SLNs dispersion ( approx 10 µl) were dropped on a 300 mesh copper grid coated with carbon film, allowing sitting for 10 min until air-dried. After complete drying, the sample was stained with 2% w/v phosphotungstic acid solution several times and dried at room temperature. Digital micrograph and soft imaging viewer software were used to perform the image capture and analysis.
Determination of drug loading (%) and entrapment efficiency (%):
A fixed quantity of DPL-SLNs dispersion (10 mL) was centrifuged (Remi Instruments, Pvt. Ltd, India) at 18,000 rpm for 20 min at 20 °C. The supernatant was analyzed spectrophotometrically at λmax 270.5 nm (Shimadzu 1800, Japan) for determination of unencapsulated drug15. The drug loading (%) and drug entrapment efficiency (%) were calculated by using equation (1) and (2).
(Wt – Ws)x 100
Drug loading (%) =----------------------- (1)
(Wt – Ws + WL)
Where Wt is the total weight of drug used, Ws is weight of drug in the supernatant, and WL is the weight of the lipid used in preparing the SLNs.
Differential Scanning Calorimetry (DSC) analysis:
The thermograms of drug, lipid and optimized DPL-SLNs were recorded with DSC (Pyris 6 DSC Perkin Elmer, CT, USA) under an inert atmosphere. Sufficient amount of samples (5 mg) were loaded into an aluminium pan and an empty aluminium pan was used as a reference. Samples were heated at a scanning rate of 10 °C/min over a temperature range of 40–300 °C and the thermograms were recorded16.
In vitro release and release kinetic studies:
In vitro release studies from DPL-SLNs were carried out to evaluate the release of drug from the optimized formulation and comparing it with the pure drug. It was performed by dialysis bag diffusion technique employing a dialysis membrane17. An accurate amount of DPL-SLNs and DPL solution (DPL-Sol.) each containing the drug equivalent to 5 mg was transferred to dialysis bag and sealed at both ends. The sealed bag was then suspended in a beaker containing 100 mL of phosphate buffer (pH 7.4, corresponding to cerebrospinal fluid pH) and stirred at a constant speed (50 rpm) at 37±0.5 °C. Aliquots (5 mL) were withdrawn at predetermined time intervals up to 24 h from receiver compartment (beaker) and replaced with an equal volume of fresh medium to maintain sink condition. The samples were analyzed spectrophotometrically at λmax of 270.5 nm.
In vitro release data of optimized formulation was fitted to zero order, first order and Higuchi release model17. To find out the mechanism of drug release, data was fitted in Korsmeyer–Peppas model (Mt/M∞=Ktn) and value of n (exponent) was determined.
Stability studies:
The stability studies were carried out to determine the effect of the presence of formulation additives on the stability of drug and also to determine the physical stability of the prepared formulation under conditions of storage temperature and relative humidity17.
The optimized DPL-SLNs were subjected to stability studies and the studies were performed in triplicate. The storage conditions used for stability testing were 4±2 0C (refrigerator), 25±2 0C /60 ±5 % RH, 40±2 0C /75 ±5 % RH in stability chamber (Hicon instruments, N. Delhi). The samples were withdrawn after a period of 0, 1, 3 and 6 months and effect on particles size, PDI, zeta potential, entrapment efficiency, loading capacity was determined.
In vivo studies:
In vivo studies of optimized DPL- SLNs and DPL-Sol. were performed on male albino wistar rats (Adult/weighing 200-250 g). A protocol for studies was approved by Institutional Animal Ethical Committee (IAEC). The animals were kept under standard laboratory conditions i.e. temperature of 22±3°C and relative humidity of 30%–70%. The animals were housed in polypropylene cages, 6 animals per cage with free access to standard laboratory diet and water ad libitum.
Pharmacokinetic studies:
To conduct pharmacokinetic studies, rats were divided in three different groups: Group A, positive control for intravenous (i.v.) drug administration (DPL-Sol.); Group B, positive control for intranasal (i.n.) drug administration (DPL-Sol.); and Group C for intranasal (i.n.) SLN formulation administration (DPL-SLNs). Each group was divided into 7 subgroups on time basis as given below and each subgroup contained 6 animals.
Subgroup 1: drug was administered at time 0 and sacrifice was done after 0.5 h
Subgroup 2: drug was administered at time 0 and sacrifice was done after 1 h
Subgroup 3: drug was administered at time 0 and sacrifice was done after 2 h
Subgroup 4: drug was administered at time 0 and sacrifice was done after 4 h
Subgroup 5: drug was administered at time 0 and sacrifice was done after 6 h
Subgroup 6: drug was administered at time 0 and sacrifice was done after 8 h
Subgroup 7: drug was administered at time 0 and sacrifice was done after 24 h
Procedure of drug administration:
Drug solution (positive control), containing 0.09 mg (for 200 g of rat) of DPL (equivalent to 0.45 mg/kg body weight), was injected through the tail vein (10 µL) in one group of wistar rats. Similarly, drug solution and drug formulation (DPL-SLNs) containing 0.09 mg of DPL were administered in each nostril in the other two groups with the help of micropipette (10–100 µL) with 0.1 mm internal diameter at the delivery site. The rats were anaesthetized prior to nasal administration by pentobarbital sodium (35–50 mg/kg, i.p.) and held firmly from the back in a slanted position during nasal administration.
Blood sampling, separation and processing of brain: The rats were killed humanely by overdose of pentobarbital sodium at different time intervals ( 0.5, 1, 2, 4, 6, 8 and 24 h) and the blood was collected by cardiac puncture in EDTA coated Eppendrof tubes. The blood was centrifuged at 5000 rpm for 15 min and aliquots of the supernatant separated and stored at -21 °C until drug analysis was carried out using High performance liquid chromatography (HPLC)18.
At the same interval of blood collection, the rats were sacrificed and brain separated. The brain was rinsed twice with normal saline to make free from adhering tissue/fluid and weighed. Cold normal saline solution was added (brain weight: normal saline, 1:5) to brain and homogenized on ice. The homogenate was centrifuged at 5000 rpm for 15 min at a temperature of 4 °C and aliquots of the supernatant were separated and stored at–21 °C until drug analysis was carried using HPLC.
Extraction of DPL from plasma and brain: Chromatographic separation was achieved with a Cosmosil C18 column (250 mm×4.6 mm, particle size 5 μm). Before quantification of drug by HPLC, the method was developed and validated. The mobile phase consisted of 0.02 M phosphate buffer (pH 7.4)–methanol–acetonitrile, 40:50:10 v/v/v and the pH was adjusted with o-phosphoric acid. 0.1 % triethylamine was added to reduce tailing. The mobile phase was sonicated for 15 min and filtered through 0.22 μm membrane filter before use. Flow rate of mobile phase was maintained at 1.2 mL/min and eluents were monitored at 280 nm. 20 μL of sample was injected using a HPLC injector. All determinations were performed at ambient temperature for a run time of 10 min. The method was linear in drug concentration range of 25-600 ng/ml with a correlation coefficient of 0.992. % RSD value of intraday and interday precision was found to be 0.73-1.27 and 0.85-1.89 respectively. The accuracy (recovery) of the developed method was 97.68%-102.39%.
For the determination of DPL, plasma sample and homogenized brain tissues (100 µL) were taken in test tube separately and mixed with 20 µL (1µg/mL) internal standard (Ondansetron) solution. The tubes were vortexed (S.M. scientific instruments Pvt. Ltd., Delhi, India) and 4 ml of 1% acetic acid was added. The content of each tube was vigorously shaken for 10 min and then centrifuged at 5000 rpm until a clear organic layer was separated (approximately 5 min). Organic layer from each tube was evaporated to dryness at room temperature. After complete removal of organic solvent, the dried samples were then reconstituted separately with 100 µL of mobile phase and evaluated by HPLC for the presence of DPL.
Analysis of pharmacokinetic parameters:
Plasma concentration–time profiles of DPL after i.n. and i.v. delivery were evaluated by pharmacokinetic software (PK Functions for Microsoft Excel, Pharsight Corporation, Mountain View, CA, USA). Various pharmacokinetic parameters as Cmax (Peak of maximum concentration), Tmax (Time of peak concentration), AUC0-∞ (Area under the curve from time 0 to time infinity), AUMC0-∞ (Area under the first movement curve from time 0 to time infinity), Ke (Elimination rate constant) and MRT (Mean residence time) were calculated. The data was statistically compared by one way ANOVA.
Brain targeting studies:
The extent of nose-to-brain delivery, bypassing the BBB, due to direct connection between the nose and the brain via the olfactory region of the nasal cavity could be evaluated by many parameters19 (i) The brain/blood ratio, at 0.5 h, following intranasal and intravenous administration (ii) The relative bioavailability (RB) percentage following the intranasal administration in the blood and brain (iii) The drug targeting index (DTI) (iv) The drug targeting efficiency (DTE) percentage and (v) The nose-to-brain direct transport percentage (DTP)20. All five parameters were determined to check the authenticity of developed formulation for brain targeting.
RESULTS AND DISCUSSION:
Selection of Lipid:
Excipients used for the formulation development should be pharmaceutically acceptable, non-irritant and non-sensitizing in nature. They should be generally regarded as safe (GRAS). For the SLNs development selection of suitable lipid and surfactant is important. Solubility of drug in the lipid is a determinant of encapsulation efficiency of the lipid nanoparticles. It is expected that high lipid solubility can result in high encapsulation efficiency17, 21.
Result of solubility study indicated that amongst the Glyceryl behenate, glyceryl palmitostearate, stearic acid and palmitic acid, the Glyceryl behenate effectively solubilised the DPL (Table 2). The applicability of Glyceryl behenate in brain drug delivery had been already reported by Jose and co-workers13.
Table 2: Solubility of DPL in various lipids
|
Lipid |
Melting point of lipid (°C) |
Amount(mg) of lipid required to solubilize 20 mg DPL # |
|
Glyceryl behenate |
70 |
52.83± 7.24* |
|
Precirol ATO 5 |
56 |
61.38± 4.26** |
|
Palmitic acid |
63 |
131.93± 3.17*** |
|
Stearic acid |
69 |
148.74± 7.93 *** |
#Values are mean± SD, n=3, SD= Standard deviation, *P < 0.05 versus Glyceryl behenate, **P< 0.01versus Glyceryl behenate,
***P< 0.001versus Glyceryl behenate, *P < 0.05 results are significant, **P< 0.01 results are moderately significant,
***P < 0.001 results are highly significant
Preparation of SLNs:
Various batches of SLNs were prepared by modified solvent emulsification diffusion technique according to table 1 and various observed responses are given in table 3.
Evaluation of Optimized Formulation:
Optimized DPL-SLN formulation (DPL6) was evaluated for different parameters with following results:
Particle size, zeta potential and morphological study:
Particle size distribution and zeta potential curve of optimized formulation (DPL6) are shown in Fig. 2a and 2b respectively. The mean particle size of different batches of SLNs ranges from 99.42±7.26nm to 217.19±23.49 nm (Table 3). The average particle size of optimized formulation was 99.42±7.26nm which was found to be suitable for brain targeting through nasal route. All batches had particles in the nano range which is well evident from the values of PDI (Polydispersity index). PDI value of optimized formulation was found be 0.173 indicating uniformity in particle size of developed SLNs22. The zeta potential indicates the degree of charge present on suspended particles in SLNs dispersion. A suitably high value of zeta potential (+ 30 mV to -30 mV) confers stability because particles resist aggregation. The value of zeta potential (-24.5 mV) of optimized SLN formulation indicated good stability23, 24. TEM image (Fig. 2c) indicated a dense roughly spherical pattern of optimized SLNs. The result of particle size was found to be in good agreement with the result established by TEM study.
(a) Particle size distribution curve
(b) Zeta potential curve and
(c) TEM image of optimized formulation
Fig. 2:
Drug loading (%) and entrapment efficiency (%):
The entrapment efficiency and drug loading of optimized formulation were found to be 67.95±1.58 % and 12.15±0.98 % respectively (Table 3).