Abstract
The aim of this study is (i) to fabricate a nanoparticle formulation of ketoprofen (Keto) using a relatively new family of macrocycles as the carrier for drug delivery: cucurbit[6]uril (CB[6]), (ii) to evaluate its in vitro dissolution and (iii) to investigate its in vivo pharmaceutical property. The CB[6]–Keto nanoparticles were prepared by emulsion solvent evaporation method. Morphology and size of the successfully prepared nanoparticles were then confirmed using a transmission electron microscope and dynamic light scattering. It was shown that they are spherical with hydrodynamic diameter of 200–300 nm. The in vitro dissolution studies of CB[6]–Keto nanoparticles were conducted at pH 1.2 and 7.4. The results indicated that there is a significant increase in Keto concentration at pH 7.4 compared to pH 1.2. For the in vivo assessment, CB[6]–Keto nanoparticles and referential profenid were administered by oral gavages to rabbits. The results implied that CB[6]–Keto nanoparticles remarkably increased area under the curve compared to profenid.
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1. Introduction
Ketoprofen (Keto) is one type of analgesic drug, classified into non-steroidal anti-inflammatory group. It is commonly used to treat rheumatism and arthritis. However, the conventional capsule formulation of Keto has several disadvantages such as short half-life, low bioavailability and side effects [1]. Moreover, poor permeability is also one of the major factors that limit oral bioavailability of several drugs. Owing to poor permeability, some drugs have to be administered at significantly higher doses [2].
In order to overcome these drawbacks, during the last few decades several new approaches using polymers for preparing Keto nanoparticles have been studied. Besides the sustained release ability [3–5], polymeric nanoparticle formulations present the drug in very fine nanodroplets offering very high surface area for absorption, leading to quick absorption of the drug, thus improved oral bioavailability. In addition, the improvement in bioavailability can be translated into reduction in the drug dose and dose-related side effects of many hydrophobic drugs. In previous studies the polymers often chosen are poly(lactic-co-glycolic acid), methacrylic polymers, chitosan, albumin [6] etc. In this study we used a new material: cucurbit[6]uril (CB[6]), a pharmaceutically acceptable material for this approach. This agent is methylene-linked six glycoluril monomers. Reported since 2000 and used as a drug delivery vehicle since 2004, the application of CB in this field has grown rapidly. CB has hydrophobic core that is able to entrap hydrophobic molecules to protect them from the surrounding environment. There have been several investigations on the formation of drug-CB host guest complexes [7].
In our study Keto was entrapped into CB[6], and CB[6]–Keto nanoparticles were prepared using emulsion solvent evaporation method. The nanoparticle sizes were controlled by changing CB[6] concentration. The CB[6]–Keto nanoparticles' morphologies and sizes were confirmed using TEM and DLS. The Keto entrapment ability of CB[6] was evaluated using UV–Vis spectroscopy. Moreover, the in vitro drug release behaviors at pH 1.2 and 7.4 and the in vivo assessment of pharmaceutical properties of CB[6]–Keto nanoparticles were also conducted.
2. Experimental
2.1. Materials
The ketoprofen (3-benzoyl-α-methylbenzeneacetic acid), a widely used non-steroidal anti-inflammatory phamaceutical drug (NSAID), was purchased from Rohm (Rohm Pharma, Darmstadt, Germany) and used without further purification.
The cucurbit[6]uril chosen in this study is a pharmaceutically acceptable material and has been used for oral formulation. This agent is methylene-linked six glycoluril monomers [ = C4H2N4O2 = ], poorly soluble in water. We obtained this material from Merck (Germany) and used it as-received.Phosphate buffer saline (PBS) was purchased from Sigma Aldrich. All other chemicals used were procured from Merck (Germany).
2.2. Preparation of nanoparticles
Nanoparticles containing Keto and CB[6] were prepared by emulsion solvent evaporation method. Briefly, acetone (20 ml) containing the drug (2.5 g) and the polymer CB[6] (3.3 g) dissolved in it was taken as the organic phase. The aqueous phase (30 ml DI water) was added to the organic phase following the sonication using an ultra-sonicator for 30 min, power 40% to form a w/o emulsion. The residual solvent was completely evaporated for 1.5 h. Suspension obtained was added by lactose and glucose (with a ratio of 1:1) prior to the freeze-drying stage.
2.3. In vitro dissolution studies
An amount of 100 mg dry powder was weighed and filled into a gelatin capsule. Round-bottomed cylindrical glass vessels having a total volume of 1000 ml were used as released chambers. The solutions were kept in a water bath at 37 ± 0.5 °C and stirred at a speed of 75 rpm. For the test in acid medium, 700 ml of HCl 0.1 N was used to obtain pH 1.2 of the release medium. For the test in base medium, 700 ml of PBS (pH 7.4) was used as release medium. Aliquot (10 ml) was withdrawn at appropriate times and immediately replaced with fresh medium equilibrated at 37 °C. The amount of released Keto was determined by measuring UV absorption at wavelength of 258 nm. The percentage of released Keto was determined from the following equation:
The measurements were performed three times and the values reported are mean values. The repeatability of the method was evaluated by analyzing three parallel samples. N22 and N25, which have drug contents of 22 and 25%, respectively, were samples in this study. The release behaviors of N22 and N25 were also compared to that of referential profenid.
2.4. In vivo assessment of oral administration
2.4.1. In vivo absorption study.
CB[6]–Keto formulations were administered by oral gavages to rabbits. All rabbits were kept fasting 18 h prior to the dose administration and remained fasting until 4 h after dose administration. The 100 mg of CB[6]–Keto containing 25% of Keto was filled in a gelatin capsule and then administered to rabbits by oral gavages (two capsules for each). Blood samples of 2.5 ml were collected into vacutainer tubes containing ethylendiamin tetracetic acid (EDTA) prior to and 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 6.0, 8.0, 12.0, 24.0 and 36.0 h after administration. After this collection, the blood samples were centrifuged at approximately 3500 rpm at 2–8 °C for about 15 min. Each plasma specimen was collected and stored at −20 °C until analysis. For the comparison, profenid containing 32.4% of Keto was also administered to rabbits by oral gavages following similar procedures. The amount of Keto for oral administration was 50 mg for both CB[6]–Keto and profenid.
2.4.2. Preparation of plasma samples for determination of Keto in rabbit plasma by HPLC.
Keto in the plasma samples was determined by high-performance liquid chromatography (HPLC). Piroxicam was used as internal standard. A solution of internal standard was prepared with methanol at 50 μg ml−1.
Samples were prepared as follows: 50 μl of plasma was extracted with 1 ml of internal standard solution in polypropylene tubes containing 100 μl of H3PO4 and 3 ml of tertbutyl methyl ether. Samples were then sonicated for 5 min, sequence vortexed for 5 min and then centrifuged at 4500 rpm for 5 min. Supernatants were transferred into glass test tubes. A blank (50 μl of blank plasma extracted with internal standard) and double blank (50 μl of blank plasma extracted with blank methanol) were also prepared. Samples were dried under nitrogen at 40 °C, reconstituted with 500 μl of methanol, and transferred into a glass insert in an autosampler vial for HPLC assay.
2.5. Characterization
Particle sizes and morphological examinations of the nanoparticles were investigated through Dynamic Light Scattering (LB502, Japan), Transmission electron microscope (JEM 1400, Japan) UV–Vis (U-3010, Japan) and HPLC (Shimadazu PDA LC-M20A, Japan).
3. Results and discussion
3.1. Preparation and physicochemical characterization of CB[6]–Keto
Keto is an anti-inflammatory drug and a great candidate for transdermal delivery. However, it has limited use because of its adverse side effects, poor solubility and short half-life after oral administration. It was previously reported that drug-loaded nanoparticles may provide a number of advantages compared to the free drug, such as increasing bioavailability and drug skin penetration, ensuring controlled drug delivery, and delayed and prolonged drug action in the application site. Our study is to prepare Keto-loaded CB[6] nanoparticles with the aim to enhance the solubility of Keto and assess the absorption in vivo of rabbits. CB[6] has been known to entrap hydrophobic molecules into their hydrophobic core. Hence, in this paper CB[6] was used for entrapping Keto and used as drug carrier. The CB[6]–Keto nanoparticles were prepared using emulsion solvent evaporation method. The morphology of the obtained CB[6]–Keto was confirmed to be spherical, smooth and nano-size using TEM. Figure 1 shows a TEM image of Keto-loaded CB[6]. The result indicates that CB[6]–Keto nanoparticles have a size of 100–200 nm.
Figure 1. Exemplary TEM image of Ketoprofen-loaded CB[6] particles.
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Standard imageThe hydrodynamic diameter of nanoparticles was determined using DLS and the result is illustrated in figure 2. The mean particle size of CB[6]–Keto nanoparticles measured by DLS is 250 nm, which is slightly bigger than that determined by TEM. This result is quite reasonable since particle size determined by DLS represents its hydrodynamic diameter, whereas that obtained by TEM is related to the collapsed nanoparticles after water evaporation.
Figure 2. Hydrodynamic diameter of CB[6]–Keto nanoparticles measured by DLS.
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Standard image3.2. Effect of CB[6] concentration on particle size
In the previous section, the nano-size of CB[6]–Keto particles was already confirmed by TEM and DLS. In this section, the effect of CB[6] concentrations on particle size was performed. The experiments were conducted with three CB[6] concentrations of 7.5, 10.0 and 20.0 mg mL−1 while keeping other processing parameters at standard conditions and the results are shown in table 1 and figure 3.
Figure 3. Influence of polymer concentration on the particle size of submicron particles prepared by the emulsion evaporation method.
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Standard imageTable 1. Effect of CB[6] concentration on particle size of CB[6]–Keto nanoparticles prepared by the emulsion evaporation method.
| Run | Polymer concentrationa (mg ml−1) | Particle sizeb (nm) | Size distributionb (nm) |
|---|---|---|---|
| 1 | 7.5 | 233.5 | 76.2–668.7 |
| 2 | 10.0 | 260.2 | 87.3–668.7 |
| 3 | 20.0 | 306.6 | 197.1–668.7 |
The increase in the polymer concentration leads to a gradual increase in nanoparticle diameters. This phenomenon can be explained based on the viscosity of dispersed phase. The increasing polymer concentration is usually accompanied by increasing viscosity of dispersed phase. As a result, the droplet is formed bigger leading to the bigger nanoparticle diameters. The control of nanoparticle sizes by changing polymer concentrations has previously been reported [8, 9]. It was explained that the increase in polymer concentration leads to an increase in the viscous forces resisting droplet breakdown by sonication. The viscous forces oppose the shear stresses in the organic phase and the final size and size distribution of particles depends on the net shear stress available for droplet breakdown [10]. Figure 3 illustrates this effect.
3.3. In vitro dissolution studies
3.3.1. In the acidic medium (pH 1.2).
The results of in vitro release study in acidic medium of CB[6]–Keto nanoparticles and profenid were shown in table 2 and plotted in figure 4. The percentages of Keto released from profenid were almost negligible (less than 5%) after 2 h. On the other hand, for the first 30 min, the percentages of Keto released from N22 and N25 were less than 10% and then gradually increased with the increasing time. However, the percentages of Keto released were nonlinear function of time. It implies that the Keto released from CB[6]–Keto nanoparticles is due to the diffusion of Keto from the outer shells of nanoparticles or/and due to the partial dissolution of CB[6] in acidic medium. The diffusion of drugs from the outer shells of nanoparticles was confirmed in the previous publications [11, 12]. Keto in the outer shells was poorly entrapped in the CB[6] matrix leading to easy diffusion of Keto. Wheate and co-workers [13] proclaimed that although CB[6] is poorly soluble in water, it still has solubility of 1–4 mM at pH 1–3 of gastric fluid resulting in the release of Keto from CB[6] matrix. However, because the solubility of CB[6] in acidic medium is low, the percentage of released Keto is correlatively low. The above-mentioned can explain why Keto was released from nanoparticles but its released percentage was less than 27% after 2 h in acidic medium.
Figure 4. In vitro release of CB[6]–Keto nanoparticles and profenid in the acidic medium.
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Standard imageTable 2. In vitro release of CB[6]–Keto nanoparticles and profenid in the acidic medium (pH 1.2)a.
| Time (h) | N22b | N25c | Profenidd |
|---|---|---|---|
| 0 | 0 | 0 | 0 |
| 0.5 | 8.4 | 7.8 | 1.7 |
| 1.0 | 14.1 | 15.2 | 2.1 |
| 1.5 | 19.7 | 22.1 | 2.9 |
| 2.0 | 24.6 | 26.9 | 3.4 |
aRelease (%), determined by equation 1, bKeto content 22%. cKeto content 25%. dKeto content 32.4%.
3.3.2. In the basic medium (pH 7.4).
The results of in vitro release study in basic medium of CB[6]–Keto nanoparticles and profenid are shown in table 3 and plotted in figure 5. The profenid released almost 100% of Keto after 1 h while N22 and N25 released 69.6 and 61.9% of Keto, respectively. This implies that the profenid released Keto faster than the CB[6]–Keto nanoparticles when it was dissolved in basic medium.
Figure 5. In vitro release of CB[6]–Keto nanoparticles and profenid in the basic medium.
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Standard imageTable 3. In vitro release of CB[6]–Keto nanoparticles and profenid in the basic medium (pH 7.4a).
| Time (h) | N22b | N25c | Profenidd |
|---|---|---|---|
| 0 | 0 | 0 | 0 |
| 0.5 | 51.2 | 38.3 | 60.5 |
| 1.0 | 69.6 | 61.9 | 104.3 |
| 1.5 | 76.3 | 87.6 | 106.2 |
| 2.0 | 81.4 | 90.8 | 106.1 |
| 2.5 | 85.3 | 94.4 | 105.3 |
| 3.0 | 90.4 | 96.6 | 104.3 |
| 3.5 | 93.3 | 95.6 | 104.5 |
| 4.0 | 101.7 | 100.7 | 105.3 |
aRelease (%), determined by equation 1. bKeto content 22%. cKeto content 25%. dKeto content 32.4%.
The percentages of Keto released from CB[6]–Keto nanoparticles in basic medium were significantly higher than those in acidic medium with the correlative times. For example, N22 released 38.3% of Keto at basic pH but only 8.4% of Keto at acidic pH after 30 min. This can be explained based on the solubility of CB[6] in basic medium. Wheate and co-workers [14] also proclaimed that the solubility of CB[6] can be increased up to 45 mM in basic medium [14]. The solubility of CB[6] is increased in the presence of cations [15, 16]. It forms the complexes with cations resulting in the charged complex which is hydrated more easily than the neutral molecule. The more CB[6] is dissolved, the more Keto is released. As a result, the percentage of released Keto in basic medium was higher than that in acidic medium due to the higher solubility of CB[6] in the basic environment. Moreover, the higher percentage of released Keto in basic medium can also be explained based on the solubility of Keto in the basic environment. The carboxylic acid group in Keto is ionized in basic medium leading to increasing solubility of Keto. As a result, Keto is released readily from nanoparticles leading to increasing percentage of released Keto.
3.4. In vivo assessment of oral administration of CB[6]–Keto nanoparticles
The in vivo assessment of oral administration of CB[6]–Keto nanoparticles was tested in rabbits with the aim to investigate the absorption ability of CB[6]–Keto nanoparticles, the maximum Keto concentration in plasma (Cmax), time of maximum concentration (Tmax) and the enhancement ratio of CB[6]–Keto noparticles. CB[6]–Keto nanoparticles and profenid were administered by oral gavages to rabbits with the amount of Keto of 50 mg for each rabbit. The oral administration procedures and the preparation of plasma are stated in section 2.4. The plasma concentration-time profiles of Keto after oral administration in rabbits are shown in figure 6. A comparison of Tmax between N25 and profenid indicates that the concentration of Keto in plasma increased rapidly in both cases and peaks were observed after 2 h. This results imply that Keto released in the intestine from CB[6]–Keto nanoparticles and profenid penetrated through the intestine into the bloodstream of rabbits and the Tmax of CB[6]–Keto nanoparticles and profenid were almost the same.
Figure 6. Plasma concentration-time profiles of Keto after oral administration in rabbits of N25 and profenid.
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Standard imagePharmacokinetic parameters following oral administration of the CB[6]–Keto nanoparticles and profenid are presented in table 4. Interestingly, the CB[6]–Keto nanoparticles remarkably increased AUC and the maximum drug concentration (Cmax) values of Keto, 1.3-fold (enhancement ratio) compared to the respective value of profenid. The in vivo absorptions of Keto were significantly improved by CB[6]–Keto nanoparticles compared to those of profenid.
Table 4. Pharmacokinetic parameters of Keto following oral administration of CB[6]–Keto nanoparticles and profenida.
| Drug formulation | Cmaxb (μg ml−1) | Tmaxc (h) | AUCd (μg h ml−1) | Enhancement ratioe |
|---|---|---|---|---|
| Profenid | 59.3 | 2 | 546.89 | 1.0 |
| N25 | 85.9 | 2 | 692.311 | 1.3 |
a50 mg of dose of Keto for each rabbit. bCmax denotes maximum drug concentration. cTmax denotes time of maximum concentration. dAUC area under the plasma concentration-time curve. eThis is the ratio AUC of Keto/AUC of profenid.
4. Conclusion
The Keto loaded CB[6] nanoparticles were successfully prepared by emulsion solvent evaporation method. The obtained CB[6]–Keto particles were confirmed to be spherical, smooth and nano-size using TEM and DLS. The particle sizes of CB[6]–Keto were controlled by changing CB[6] concentration. The result showed that the particle sizes increased with increasing CB[6] concentration. The in vitro dissolution studies of CB[6]–Keto nanoparticles were conducted at pH 1.2 and 7.4. The results indicated that there is a significant increase in released Keto concentration at pH 7.4 compared to pH 1.2 due to the higher solubilities of CB[6] and Keto in the basic environment compared to those in acidic medium. For the in vivo assessment, CB[6]–Keto nanoparticles and referential profenid were administered by oral gavages to rabbits. The results implied that CB[6]–Keto nanoparticles remarkably increased AUC compared to profenid. These initial results demonstrate that the proposed carrier CB[6] is a very promising vehicle for drug delivery.