Patients using ophthalmic drops are facing with frequent dosing schedules and difficult drop instillation. Therefore, the aim of the present investigation was to investigate and prepare a long lasting pilocarpine loaded chitosan nanoparticles. Nanoparticles were prepared by ionic gelation technique. The obtained nanoparticles were evaluated for their size, entrapment efficiency, zeta potential, release rate and biological response like IOP test, miotic tests and compared with pilocarpine in solution with different concentration. The nanoparticles were about 231 to 557 nm in size and percentage yield between 52.5 to 68%. The encapsulation efficiency was found to be 83.1%. Zeta potential of naoparticles was found to be between +40.6 Â± 4.7 to +47.1 Â± 1.6 mV. The in vitro release was fond to be formulation FM-1 to FM-6, 62.8%, to 72.3%. Biological response of nanoparticles suspension was measured by reduction in IOP and miotic response in albino rabbit eyes.
Key words: Chitosan, Nanoparticles, ionic gelation method, Betamethasone model, Ocular drug delivery, Glaucoma.
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Eye disease can cause discomfort and anxiety in patients, with the ultimate fear of loss of vision or even facial disfigurement. Many regions of the eye are relatively inaccessible to systemically administered drug and, as a result topical drug delivery remains the preferred route in most cases. Drug may be delivered to treat the precorneal region for such infection as conjunctivitis and blepharitis, or to provide intraocular treatment (IOT) via the cornea for diseases such as glaucoma and uveitis. (Le bourlais C, et al, 1998)
Most ophthalmic drugs are administered topically in the form of eyedrops. Although convenient and inexpensive, this type of delivery system yields low therapeutic efficacy due to the dynamics of the lachrimal system (i.e. blinking, lachrymal secretion and nasolachrimal drainage). The low efficacy necessitates more frequent administration to achieve the desired therapeutic effect. This can increase the frequency and severity of both ocular and systemic side effects. Therefore, it is necessary to develop safer, efficacious and more acceptable ocular delivery systems. Delivery systems that are capable of releasing the drug in a prolonged manner are of interest because they can improve the ocular residence time. An increase in ocular residence time maximizes the duration for topical or local action and also minimizes the systemic side effects. Additionally, a controlled release preparation requires fewer instillations and therefore will lead to increased patient compliance. (Simamora P et al,1998)
Pilocarpine, a parasympathomimitic, remains a miotic of choice for open angle glaucoma because it increases the out flow of aqueous humour, the drug penetrates the eye well, with miosis beginning 15-30 min after topical application and lasting for 4-8 h (Zimmerman 1981). Pilocarpine ophthalmic drops are administered as 1 or 2 drops par dose, with six drops per day as maximum recommended dosage.
Ocular bioavailability of topically applied pilocarpine is only 0.1-3% (Lazare and Horlington, 1975) and the drug must be administered as eye-drops three to four times per day which impairs patient compliance (Kass et al., 1986). The poor bioavailability is attributed to the low lipophilicity of pilocarpineas well as to rapid loss of the drug from the precorneal area via drainage and conjunctival absorption. (Sznitowska M et al, 1999)
Chitosan (CS; (1,4)-[2-amino -2 deoxy -Î²- D- glucan] ) a mixture of glucosamine and N -acetyl-glucosamine, is a cationic polysaccharide obtained from the chitin of crustacean shells. Chitosan is biocompatible, biodegradable, not highly toxic and mucoadhesive. (Lin HR et al 2006). The small size and positive charges of chitosan nanoparticles may improve their interaction with negatively charged biological membranes. (De la Torre at al 2003; torrado et al, 2004).
In this study we report an approach for preparing pilocarpine loaded chitosan nanoparticles. We evaluated the physicochemical characterization of nanoparticles using particle size, zeta potential, entrapment efficiency in vitro release and in vivo study.
2. Materials and methods
Pilocarpine nitrate was purchased from Medicine traders Mumbai India. Chitosan was obtained as gift sample from Indian Sea Foods, Cochin India. Sodium Tripolyphosphate was purchased from Loba Chemicals, Mumbai, Acetic Acid from Ranbaxy Fine Chemical Ltd Mumbai, Acetone, Sodium hydroxide pellets, Potassium dihydrogen orthophosphate were purchased from S.D. Fine Chemicals Ltd., Mumbai, Ranbaxy Fine Chemical Limited, New Delhi, Himedia Lab, Mumbai respectively.
2.2.1 Preparation of pilocarpine loaded chitosan Nanoparticles
Pilocarpine loaded chitosan Nanoparticles were prepared by ionic gelation method. Chitosan solution was prepared in 1%v/v acetic acid aqueous solution then, TPP in distilled water at 1 or 2 mg / ml. Finally, 2 ml of TPP solution was added to 5 ml chitosan solution and the drug solution were added through the syringe and stirred using mechanical stirrer at room temperature and was further examined as nanoparticles. (Yongmei Xu et al 2003). Details of formulation shown in table 1 (Jorg Kreuter et al, 1995).
2.3 Evaluation of pilocarpine loaded chitosan Nanoparticles
2.3.1 Percentage yield
Always on Time
Marked to Standard
Percentage practical yield is calculated to know about percentage yield or efficiency of any method, thus it helps in selection of appropriate method of production. Practical yield was calculated as the weight of nanoparticles recovered from each batch in relation to the sum of starting material (Sunit KS et al.2005).
2.3.2 Zeta potential
Zeta potential was measured by using zeta potentiometer (Zeta 3.0+ meter, USA). Samples were diluted with KCl (0.1mM) and placed in electrophoretic cell where the electric field of 15.2 V/cm was applied. Each sample was analyzed in triplicate. (Bekerman, T et al, 2004).
2.3.3 Particle Size Analysis
The size distributions along the volume mean diameters of the suspending particles were measured by dynamic scattering particle size analyzer (Nanotrac Particle Analyzer 150, Microtrac Inc., PA, USA). The range of measurement of Nanotrac Particle Analyser 150 is 0.8 nm ~ 6.54 Âµm. (Lin Y, et al).
2.3.4 Scanning Electron Microscopy (SEM)
Surface morphology of the specimens will be determined by using a scanning electron microscope (SEM), Hitachi model SU 1500. The dried samples were mounted on brass specimen studies, using double sided adhesive tape. Gold-palladium alloy of 1200A knees was coated on the sample using sputter coating unit ( JEOL JFC-Model 1100E, Japan) in Argon at ambient of 8-10 Pascal with plasma voltage about 20MA. The sputtering was done for nearly 5 minutes. The SEM was operated at low accelerating voltage of about 15KV with load current of about 80MA. The condenser lens position was maintained between 4.4-5.1Â° and the working distance WD=39mm. (Ye, J et al, 2008).
2.3.5 Drug Entrapment Efficiency
The percentage of incorporated pilocarpine nitrate (entrapment efficiency) was determined spectrophotometrically at 215 nm. After centrifugation of the aqueous suspension, amount of free drug was detected in the supernatant and the amount of incorporated drug was determined as the result of the initial drug minus the free. (Lin HR et al 2006).
2.3.6 In Vitro Drug Release Studies
The in vitro release of pilocarpine nitrate from the formulation was studied through Dialysis membrane-110 (cut-off: 3500 Da) using modified apparatus. The dissolution medium used was freshly prepared simulated tear fluid (pH 7.4). Dialysis membrane-110, previously soaked overnight in the dissolution medium was tied to one end of a specifically designed glass cylinder (open at both ends). 5 ml of formulation was placed into this assembly. The cylinder was attached to a stand and suspended in 50 ml of dissolution medium maintained at 37Â±ï€ 1°C so that the membrane just touched the receptor medium surface. The dissolution medium was stirred at low speed using magnetic stirrer. Aliquots, of 3 ml were withdrawn at predetermined intervals and replaced with an equal volume of the medium. The aliquots were suitably diluted with the receptor medium and analyzed by UV-Vis spectrophotometry at 215 nm. (Srividya B et al, 2001).
2.3.7 In Vivo Studies:
In vivo studies were performed on groups of six male New Zealand albino rabbits weighing 1.8-2.2 kg, and with no signs of ocular inflammation or gross abnormalities. The animal's procedures are fallowed as per CPCSEA guidelines.
Measurement of intra ocular pressure (lOP) reduction
The experiments were carried out in New Zealand albino rabbits weighing 1.8-2.2 kg. Ocular hypertension was induced by subconjunctival injections of 0.45 ml betamethasone suspension into the left eye, repeated weekly over a period of 3 weeks. After the third week of glucocorticoid treatment, an increased ocular pressure, stable for 2 weeks, was obtained. Only rabbits responding to the treatment with IOP increases above 25 mmHg were chosen for the experiments. Baseline tonometric measurements were taken at various time points over a period of 7.5 h in order to ensure daily stability of the IOP. Ten Âµl of Ophtetic Â® solution was administered before each measurement in order to anaesthetize the corneal surface. After a free interval of 24 h, a new series of tonometries was performed to avoid interference from possible corneal damage. The preparations were then tested by instilling 50Âµl into the lower conjunctival sac and measuring the IOP after predetermined time intervals. All experiments were carried out at the same hours of the day in order to comply with the circadian rhythm (Jorg Kreuter et al, 1995).
The experiments were carried out in male New Zealand strain albino rabbits weighing 2.2 kg; all tests were performed in the same room with constant artificial lighting. After 45 min of acclimatization in restraining boxes.FM-5, 1% Piocarpine Nitrate solution and 2% Piocarpine was then tested at least in 6 animals by instilling a dose of 50Âµl on the everted lower lid of the left eye. After dosing, the lids were gently held together for a few seconds in order to avoid loss of dosage form. Measurements of pupil diameter were carried out by the same operator, with a micrometer held at a fixed distance from the rabbits' eyes. (Jorg Kreuter et al, 1995).
2.3.8 Short term stability study
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Information on the stability of drug substance is an integral part of the systemic approach to formulation evaluation. The Purpose of stability testing is to provide evidence on how the quality of a drug substance or drug product varies with time under influence of variety of factors such as temperature, humidity and light, and to establish a re-test period for drug substance or a shelf life for the drug product and recommended storage conditions. Nanoparticle sample was divided into 3 samples of sets and stored at: 4OC in refrigerator, 37 0C Â± 2 OC, /65OC % Â± 5 % RH in humidity control ovens and Room temperature for 1month.
3. Result and discussion
3.1 Percentage yield
Percent practical yield increased as the amount of polymer added to each formulation increased, although it may not be dependent upon drug concentration in the formulation. The percentage yield was found to be 52.5% to 68.0%.
3.2 Particle Size
In this study photon correlation spectroscopy was used for routine particle sizing. Table 2 summarizes the results obtained for pilocarpine loaded chitosan nanoparticles. The mean diameter of all nanoparticle preparations was within a size range of about252 to557 nm. Scanning electron photomicrographs of nanoparticles are shown in fig. 1a, 1b and 1c. Magnification of 7,500- 20,000 X was used while taking these photographs. This was performed to study the surface morphology of the particles. Nanoparticles have Smooth textured and dispersing nanoparticles & the particles were spherical in shape.
3.3 Zeta potential
Zeta potential is the difference in electrical potential between a tightly bound layer of ions on particle surfaces and bulk liquid in which the particles are suspended. It can be quantified by tracking the charged particles when they migrate in voltage field, as measured in zeta potential analyzer. The zeta potential of the nanoparticles was between the +40.6 Â± 4.7 to +47.1 Â± 1.6mV table 2. The positive surface charges of nanoparticles make it easier for them to interact with the biological membranes in the eye.
3.4 Drug entrapment efficiency
The amount of drug bound per 1 ml of nanoparticles was determined for all formulations and the values of total entrapment efficiency of drug are shown in Table 2. It was observed that as the polymer concentration increased in the internal phase, an increase in drug entrapment efficiency was seen. The drug encapsulation efficiency increased from 69.76 % to 83.10 %.
3.5 In vitro drug release
Pilocarpine loaded in eye drops was released very quickly, and more than 95% of the loaded pilocarpine was released and reached at a plateau within 4 h .pilocarpine loaded chitosan nanoparticles showed an initial burst release fallowed by a continuous and sustained releasefor 24 h. fig 2. The in vitro drug release of FM-1 to FM-6 was found to be 59.5% to 72.3 % table 3.
3.6 In vivo study:
Intra ocular pressure (IOP)
Since pilocarpine exhibits IOP lowering effects only in hypertensive eyes, hypotensive activity tests must be carried out on animals with artificially increased IOP. In this study, ocular hypertension was induced in rabbits by the method of (Bonomi et al.1978), consisting of repeated subconjunctival injections of betamethasone. Systems containing nanoparticles, were further tested in the ocularly hypertensive rabbits. IOP values were recorded 0.5, 1, 2, 4, 6, 8, 10, 12 and 24 hr after instillation of the selected preparations.
The effect of IOP decreasing was studied by using tonometer response of the preparations was studied and compared with pure drug and marketed preparation of 2% Pilocarpine solutions and in control group no reduction in IOP. IOP activity data is given in table 4. The maximum duration of response was found with FM-5 up to 24 hrs. 1% pure drug shows effect up to 4hrs and 2% Pilocarpine marketed preparation (eye drops) shows increases the magnitude of response but not the duration of response figure 3.
In vivo miotic study
Mioitc activity of nanoparticles, 1% pure pilocarpine nitrate and 2% pilocarpine nitrate marketed preparation (eye drop) was studied it shows reduction in pupil diameter the effect of nanoparticles lasts up to 24 hrs when compared to 1% pure pilocarpine nitrate and 2% marketed preparation of pilocarpine solution table 5, figure 4. The in vivo study of pilocarpine nitrate nanoparticles demonstrates the extended decrease in IOP and miosis effect.
3.7 Stability studies
Stability studies of the prepared nanoparticles were carried out, by storing at 4°C in refrigerator, 25Â±2Â°C/60%Â±5% RH and 37° C Â± 2°C, 65% Â± 5% RH in humidity control oven for thirty days. Two parameters namely residual percent drug content and in vitro release studies were carried out. The results of drug content after 30 days are shown in Table 6. These studies revealed that there is a reduction in drug content after storage for thirty days at 4° C, 25Â±2Â°C/60%Â±5% and 37° C Â± 2°C/65% Â± 5% RH. It was also revealed that the one stored at 4° C showed maximum residual drug followed by the one stored at ambient temperature and humidity and 37° C Â± 2°C/65% Â± 5% RH.
The present study has been a satisfactory attempt to formulate nanoparticles of pilocarpine nitrate with a view to improving bioavailability and giving a controlled/ sustained release of drug and to reduce dosing amounts, frequency of administration, and adverse effects while maintaining the drug efficiency. Nanoparticles were successfully prepared by ionic gelation method. Chitosan is a good biodegradable polymer and is a promising agent for ocular delivery. The concentration of TPP increase up to 2 Mg/ml increases the entrapment efficiency of the Pilocarpine Nitrate. The in vitro release studies showed biphasic release pattern for all formulations, with an initial burst effect, which may be attributed to the drug loaded on the surface of the particles. In vivo study shows the maximum duration of response over a period of 24 hrs by maintaining the IOP at normal average pressure comparatively 1%pure drug and 2% solution of marketed preparation and Stability studies revealed that 4Â°C is the optimal temperature for storage.
The authors are thankful to Principal, K.L.E.S's college of pharmacy Belgaum, Medicine traders for pilocarpine nitrate, Indian sea foods for polymer, and Shrddha analytical services for providing the facility of SEM.