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intravenous administration [6]. Risperidone, an atypical antipsychotic agent, has been used in the treatment of psychotic dis- orders. It has been approved by the ...

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Preparation and characterization of nanoparticles containing an atypical antipsychotic agent Sanjay Singh1† & Madaswamy S Muthu1,2 †Author

for correspondence of Pharmaceutics, Institute of Technology, Banaras Hindu University, Varanasi – 221005, India Tel.: + 91 542 231 5871; Fax: + 91 542 236 8428; E-mail: [email protected] rediffmail.com [email protected] 1Department

Keywords: in vitro release studies, nanoprecipitation method, poly(ε-caprolactone) nanoparticles, reverse dialysis technique, risperidone part of

Aim: The aim of this work was to prepare poly(ε-caprolactone) nanoparticles of risperidone and to characterize them. Methods: Risperidone-loaded poly(ε-caprolactone) nanoparticles were prepared by the nanoprecipitation method using poly(ethylene oxide)–poly(propylene oxide)–poly(ethylene oxide) triblock polymeric stabilizer (Pluronic® F-68). The particles were characterized for particle size by photon correlation spectroscopy and transmission electron microscopy. The free dissolved drug in the nanosuspension was determined by the bulk equilibrium reverse dialysis bag technique. In vitro release studies were carried out using the dialysis bag diffusion technique. Results: The particle size of the prepared nanoparticles ranged from 90 to 300 nm. Nanoparticles of risperidone were obtained with high encapsulation efficiency (70–80%). The drug release from the risperidone nanoparticles was sustained in some batches for more than 24 h with 80% drug release, whereas release from risperidone in polyethylene glycol 400 solution showed release within 2 h. Conclusion: These studies suggest the feasibility of formulating risperidone-loaded poly(ε-caprolactone) nanoparticles for the treatment of psychotic disorders.

Technological advancements have brought us many new innovative drug delivery systems. Among these, polymeric nanoparticulate systems from biodegradable and biocompatible polymers are interesting options for controlled drug delivery and drug targeting [1–3]. These nanoparticles have been investigated especially in drug delivery systems for drug targeting owing to their particle size (10–1000 nm) and long circulation in the blood [4]. Thus, if the carrier size is under 1 µm, an intravenous injection (the diameter of the smallest blood capillaries is 4 µm) is enabled, minimizing any possible irritant reactions [5]. Poly(ε-caprolactone) (PCL), a biodegradable and biocompatible polymer, has been used extensively for developing an array of drug delivery systems [6–8]. Among US FDA-approved polyesters, PCL possess unique properties, such as higher hydrophobicity and neutral biodegradation end products, which do not disturb the pH balance of the degradation medium [9,10]. The rapid clearance of circulating nanoparticles from the bloodstream coupled with their high uptake by liver and spleen can be overcome by making the particle surface hydrophilic with poloxamers and poloxamines [11]. PCL nanoparticles loaded with tamoxifen also showed high and selective biodistribution after intravenous administration [6]. Risperidone, an atypical antipsychotic agent, has been used in the treatment of psychotic disorders. It has been approved by the FDA as an

10.2217/17435889.2.2.233 © 2007 Future Medicine Ltd ISSN 1743-5889

atypical antipsychotic agent because it causes less extrapyramidal effects than do conventional antipsychotics. It is practically insoluble in water and undergoes significant ‘first-pass’ metabolism; oral bioavailability is 70% (coefficients of variation [CV] = 25%). The active metabolite of risperidone is 9-hydroxy risperidone. The halflife of risperidone and its metabolite 9-hydroxy risperidone is 3 and 21 h, respectively [12–14]. Animal studies suggest that the blood–brain barrier may be penetrable preferentially to risperidone over 9-hydroxy risperidone, with blood:brain ratios of 0.22 and 0.04, respectively. This difference in penetrability may provide clinical importance to risperidone [15]. Therefore, maintenance of risperidone (owing to its short half-life) in plasma using long circulating nanoparticles via the intravenous route may enhance the bioavailability and biodistribution of risperidone and improve the pharmacotherapy of psychotic disorders. An injectable risperidone formulation was approved recently by the FDA as the first atypical long-acting antipsychotic medication. Longacting risperidone has been available in high doses of 25 and 50 mg for gluteal injection every 2 weeks. It has the drawback of a lack of initial drug release ( 0.05). The data in Table 2 suggest that an increase in polymer concentration increases the size of the nanoparticles. However, the polydispersity of the nanoparticles decreased with an increase in PCL concentration and particle size of the nanoparticles (Table 2). External morphological study

The external morphological study revealed that all nanoparticles (electron-dense spheres) were spherical in shape. Matrices of fibrous matter were detected nearer to nanoparticles (Figure 1A). Placebo nanoparticles (without drug) showed an absence of any matrix of fibrous matter nearer to nanoparticles (Figure 1B). The nanoparticle size, as observed by TEM, correlated well with the size measured by PCS. Determination of total drug content, free dissolved drug content of nanosuspension & encapsulation efficiency of nanoparticles

The total drug content in the nanosuspensions varied from 5.94 ± 0.09 to 6.68 ± 0.04 mg (RNS1–3) and 7.92 ± 0.02 to 8.25 ± 0.05 mg (RNS4–6). The total drug content in the nanosuspension increased with an increase in the concentration of PCL (Table 3). The amount of free dissolved drug in the nanosuspensions ranged from 0.68 ± 0.03 to 0.65 ± 0.08 mg (RNS1–3) and 0.84 ± 0.07 to 0.76 ± 0.06 mg (RNS4–6). This was due to the

Table 2. Particle size and polydispersity of risperidone nanoparticles. Batches

Particle size (nm) (mean ± SD*)

Polydispersity (mean ± SD*)

92.4 ± 15.6

0.6299 ± 0.049

RNS0 RNS1

96.7 ± 12.4

0.6112 ± 0.039

RNS2

202.7 ± 15.1

0.4211 ± 0.032

RNS3

298.5 ± 19.3

0.3421 ± 0.013

RNS4

98.3 ± 11.4

0.6213 ± 0.059

RNS5

213.1 ± 14.2

0.4227 ± 0.037

RNS6

300.8 ± 17.4

0.3538 ± 0.022

*n

= 3. SD: Standard deviation for three determinations.

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Nanoparticles containing an atypical antipsychotic agent – RESEARCH ARTICLE

Figure 1. TEM micrographs of PCL nanoparticles. A

100 nm

B

100 nm

(A) Risperidone-loaded PCL nanoparticles magnified 37,000 × and (B) placebo nanoparticles magnified 37,000 ×. PCL: Poly(ε-caprolactone); TEM: Transmission electron microscopy.

limited solubility of risperidone in the aqueous phase. Free dissolved drug in the nanosuspension decreased with an increase in the PCL concentration. As expected, the free drug content increased as the drug loading was increased from 7.5 to 10 mg (Table 3). The increase in risperidone loading decreased the encapsulation efficiency. Encapsulation efficiency was also inversely related to free dissolved drug; however, it was directly related to the amount of PCL used. Although encapsulation efficiency increased with an increase in the PCL amount, it was not significant (p > 0.05) (Table 3). future science group

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In vitro drug-release studies

show the percentage release of risperidone from different batches (RNS1–6) in PBS (pH 7.4). The release profile of risperidone control solutions (risperidone in PEG 400) indicates very rapid diffusion of risperidone with nearly 50% release in 30 min and 80% in 2 h. The nanosuspensions (batches RNS1–6) initially showed burst release followed by sustained release up to 24 h. After 24 h of dialysis in PBS (pH 7.4), the percentages of risperidone released were 95, 91 and 84% for batches RNS1, RNS2 and RNS3,, respectively (Figure 2), and, for batches RNS4, RNS 5 and RNS6, it was 100, 92 and 86%, respectively (Figure 3). The effect of drug loading on risperidone release was also studied. 7.5 and 10 mg of risperidone-loaded nanoparticles showed a difference in drug-release kinetics. The t80% for batches RNS1 and RNS4 was 5 and 4 h, respectively, in PBS (pH 7.4) (Figures 2 & 3). Similar drug-release profiles were observed with other batches. The data indicate that the rate of release of risperidone from the nanoparticles can be faster with an increase in drug loading. The PCL content also affected the drug-release kinetics. The increase in PCL content in the nanosuspensions resulted in slower kinetics. The t50% in PBS (pH 7.4) was approximately 0.5, 1 and 1.5 h for batches RNS1, RNS2 and RNS3 containing 120, 200 and 280 mg of PCL, respectively (Figure 2). Similar results were obtained for batches RNS4–6 (Figure 3). Figures 2 & 3

Discussion In the present work, risperidone-loaded PCL nanoparticles and placebo nanoparticles were prepared by the nanoprecipitation method. The nanoprecipitation protocols utilize an organic phase composed of volatile solvents, whose elimination is readily achieved by evaporation. Risperidone was dissolved in acetone along with the different quantities of PCL and introduced into aqueous medium containing surfactant (Pluronic F-68), which resulted in the formation of nanoparticles (polymeric matrix with drug). Drug was adsorbed on the nanoparticles and/or dispersed into the matrix of nanoparticles because it gets precipitated when put into aqueous phase owing to its limited solubility in the water [17]. 237

RESEARCH ARTICLE – Singh & Muthu

Table 3. Total drug content, free dissolved drug in the nanosuspensions and encapsulation efficiency of the nanoparticles. Batches

Total drug content (mg) (mean ± SD*)

Free dissolved drug (mg) (mean ± SD*)

Encapsulation efficiency (%) (mean ± SD*)

RNS1

5.94 ± 0.09

0.68 ± 0.03

70.14 ± 0.88

RNS2

6.58 ± 0.05

0.67 ± 0.05

78.84 ± 0.60

RNS3

6.68 ± 0.04

0.65 ± 0.08

80.44 ± 0.67

RNS4

7.92 ± 0.02

0.84 ± 0.07

70.77 ± 0.85

RNS5

8.14 ± 0.03

0.82 ± 0.09

73.33 ± 1.02

RNS6

8.25 ± 0.05

0.76 ± 0.06

74.86 ± 0.90

*n

= 3. SD: Standard deviation for three determinations.

Matrices of fibrous matter were detected nearer to nanoparticles during TEM analysis of risperidone nanoparticles (Figure 1A). TEM analysis of placebo nanoparticles (without drug) showed an absence of any matrix of fibrous matter nearer to nanoparticles (Figure 1B). This indicates that the matrices of fibrous matter nearer to nanoparticles are the free drug crystals (Figures 1A & 1B). The presence of some portion of free drug outside the nanoparticles is also indicated by rapid initial in vitro drug release (Figures 2 & 3). The size of the nanoparticles (electron dense spheres) as observed by TEM correlated with PCS data. The matrices of fibrous matter (free drug crystals) present are larger than nanoparticles (>200 nm) because Figure 2. In vitro drug release from risperidone nanosuspensions and control risperidone solution (0.75 mg/ml) in phosphate-buffered saline (pH 7.4).

100

Drug released (%)

80

60

Risperidone in PEG 400 RNS1 RNS2 RNS3

40

20

0

0

5

10

15

20

Time (h)

PEG: Polyethylene glycol; RNS: Risperidone nanosuspension.

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Nanomedicine (2007) 2(2)

these free drug crystals are obtained from free dissolved drug during the drying of nanosuspension for the TEM analysis. The main requirements for a particulate formulation intended for parenteral use include biocompatibility of its ingredients and suitable drug carrier size. Pluronic F-68 had been used for stabilization of PCL nanoparticles [6]. Pluronic is regarded as nontoxic for parenteral use, therefore no special purification step is required for its elimination from the final formulation [18]. Particle size must be strictly controlled in nanoparticulate formulation intended for intravascular delivery. Since the estimated diameter of the smallest blood capillaries in the human body is 4–7 µm [23], the particle size should optimally be kept in the submicronial range to prevent capillary occlusion. The results of this study show that the nanoparticle size is influenced by several formulative variables, such as concentration of the polymer and drug. The effect of the polymer concentration on the nanoparticle size may be due to the higher resultant organic phase viscosity, which leads to larger nanodroplet formation [24]. The total drug content in the nanosuspension was less than the drug loaded (Table 3). Drug losses from the final nanosuspensions were due to the separation of risperidoneloaded polymeric aggregates by centrifugation during the preparation of the nanosuspension. Free dissolved drug present in the nanosuspension increased with an increase in the risperidone loading. As the drug loading increases, the concentration of both non-entrapped free dissolved drug and free insoluble drug in the nanosuspensions increases, although the latter is removed during the preparation of nanosuspensions (Table 3).

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Nanoparticles containing an atypical antipsychotic agent – RESEARCH ARTICLE

Figure 3. In vitro drug release from risperidone nanosuspensions and control risperidone solution (1 mg/ml) in phosphate-buffered saline (pH 7.4).

100

Drug released (%)

80

60

40

Risperidone in PEG 400 RNS4 RNS5 RNS6

20

0 0

5

10

15

20

Time (h)

PEG: Polyethylene glycol; RNS: Risperidone nanosuspension.

The release of drug from polymeric nanoparticles was studied by the dialysis bag diffusion technique. This is a very popular method to study the release of drugs from colloidal suspensions [21]. Since the aim of the study was to administer the nanoparticles by the intravenous route, the release studies were carried out with PBS (pH 7.4) to mimic the in vivo conditions. The release rate of the risperidone from the nanoparticles and its appearance in the dissolution medium was governed by the partition coefficient of the drug between the polymeric phase and the aqueous environment in the dialysis bag and also by the diffusion of the drug across the membrane. The dialysis bag retained nanoparticles and allowed the diffusion of the drug immediately into the receiver compartment [21]. Executive summary • Risperidone-loaded, poly(ε-caprolactone) (PCL) nanoparticles were prepared by the nanoprecipitation method. • Nanoparticles were characterized for particle size analysis, total drug content, free dissolved drug of nanosuspensions, encapsulation efficiency of nanoparticles and in vitro drug-release studies. • Pluronic® F-68 was used for the stabilization of PCL nanoparticles. • Drug-release profiles of the prepared polymeric nanoparticles indicate that drug release can be controlled for more than 24 h, thereby improving the dosage regimen.

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The nanoparticle size was also associated with changes in drug-release kinetics. The smaller-sized nanoparticles prepared with lower amounts of PCL exhibited higher drug-release rates. This release behavior of the small-sized nanoparticles may be explained by a corresponding increase in the total nanoparticle surface, resulting in a larger drug fraction exposed to the leaching medium. Smaller nanoparticle size also leads to a shorter average diffusion path of the matrix-entrapped drug molecules [25]. The initial burst effect on the release of risperidone from different nanoparticle batches (RNS1–6) (Figures 2 & 3) may be due to the free dissolved drug observed with nanosuspensions and the free drug adsorbed on the nanoparticles. The release of drug adsorbed on the nanoparticle surface was considerably faster compared with the encapsulated drug [17]. The increase of PCL content in the risperidone nanosuspensions increases the size of the polymeric nanoparticles (Table 2), improves the encapsulation of the drug (Table 3) and results in slower drug release (Figures 2 & 3). On the basis of the results of this study, it can be predicted that the risperidone-loaded, biodegradable polymeric nanoparticles (PCL based) may prolong the antipsychotic effect of the drug after intravenous administration. Conclusion This study confirms that the nanoprecipitation technique is suitable for the preparation of risperidone nanoparticles with high encapsulation efficiency. This formulation approach can be used to improve the therapeutic efficacy of poorly soluble drugs. The changes in nanoparticle size and drugrelease kinetics were affected by changes in polymer concentration. The controlled drug release from the risperidone nanoparticles suggests that the frequency of administration, dose and adverse effects of this molecule could be reduced, if administered via the intravenous route. We can conclude that there is large scope for improving the use of risperidone for psychotic treatments through parenteral administration. Future perspective This nanoprecipitation method will offer reproducible carrier size in the nanometer range and use of ingredients with low toxic potential, especially important for intravascular delivery of poorly soluble drugs. Risperidone-loaded PCL nanoparticles were prepared at the concentrations of 0.75 and 1 mg/ml. Therefore, a volume 239

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selection for intravenous administration will be easy. A general low oral effective dose of risperidone is 0.5–2.0 mg/day. A dose of 0.5–1.0 ml/day of risperidone nanosuspension for intravenous administration will be effective for the management of manifestations of psychotic disorders. In the future, in vivo pharmacodynamic studies will be carried out to assess the efficacy of risperidone-loaded PCL nanoparticles. The inhibition of apomorphine-induced

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