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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.
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).
2.3.8 Short term stability study
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% to68.0%.
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.