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Development and Evaluation of Perfluorocarbon Nanobubbles for Apomorphine Delivery
Development and Evaluation of Perfluorocarbon Nanobubbles for Apomorphine Delivery TSONG-LONG HWANG,1 YIN-KU LIN,2,3 CHEN-HSIEN CHI,4 TSE-HUNG HUANG,3,5 JIA-YOU FANG4 1
Cell Pharmacology Laboratory, Graduate Institute of Natural Products, Chang Gung University, Kweishan, Taoyuan, Taiwan 2
Graduate Institute of Clinical Medical Sciences, Chang Gung University, Kweishan, Taoyuan, Taiwan
3
Department of Traditional Chinese Medicine, Chang Gung Memorial Hospital, Keelung, Taiwan
4
Pharmaceutics Laboratory, Graduate Institute of Natural Products, Chang Gung University, Kweishan, Taoyuan, Taiwan
5
Graduate Institute of Traditional Chinese Medicine, Chang Gung University, Kweishan, Taoyuan, Taiwan
Received 14 September 2008; revised 16 November 2008; accepted 16 December 2008 Published online 20 January 2009 in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/jps.21687
ABSTRACT: Apomorphine is a dopamine receptor agonist for treating Parkinson’s disease. However, its clinical application is limited by its instability and the need for frequent injections. The aim of the present work was to develop acoustically active perfluorocarbon nanobubbles (PNs) for encapsulation of both apomorphine HCl and base forms to circumvent these delivery problems. The PNs were prepared using coconut oil and perfluoropentane as the inner phase, which was emulsified by phospholipids and cholesterol. The morphology, size, zeta potential, and drug release of the PNs were characterized. The particle size ranged from 150 to 380 nm, with differences in the oil or perfluorocarbon ratio in the formulations. Atomic force microscopy confirmed oval- or raisin-shaped particles and a narrow size distribution of these systems (polydispersity index ¼ 0.25–0.28). The stability experimental results indicated that PNs could protect apomorphine from degradation. Evaporation of the PNs at 378C was also limited. Apomorphine HCl and base in PNs showed retarded and sustained release profiles. Ultrasound imaging confirmed the echogenic activity of PNs developed in this study. The apomorphine HCl release by insonation at 1 MHz showed enhancements of two- to fourfold compared to the non-ultrasound group, illustrating a possible drug-targeting effect. On the contrary, apomorphine base showed a decreased release profile with ultrasound application. Apomorphine-loaded PNs showed promising stability and safety. They were successful in sustaining apomorphine delivery. ß 2009 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 98:3735–3747, 2009
Keywords: apomorphine; perfluorocarbon nanobubbles; drug delivery system; drug targeting; ultrasound
INTRODUCTION
Correspondence to: Jia-You Fang (Telephone: 886-32118800 ext. 5521; Fax: 886-3-2118236; E-mail: [email protected]) Journal of Pharmaceutical Sciences, Vol. 98, 3735–3747 (2009) ß 2009 Wiley-Liss, Inc. and the American Pharmacists Association
Parkinson’s disease affects 1% of people over 65 years old and 3% of those over 85 years old; it is a chronic neurodegenerative disease caused by the loss of pigmented mesostriatal dopaminergic neurons linking the substantia nigra to the neostriatum.1,2 Apomorphine as a mixed D1 and D2 dopamine receptor agonist has
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been shown to be a very potent drug for treating patients with idiopathic Parkinson’s disease.3 However, its inherent instability, negligible oral bioavailability, and short half-life (41 min) have complicated its application in clinical practice.3,4 A high demand on the apomorphine delivery system is necessary to resolve these problems. Drug encapsulation systems such as liposomes and emulsions have been introduced as parenteral drug carriers offering sustained release and long residence times.5,6 Microbubbles represent a new class of encapsulation systems with both diagnostic and therapeutic applications. Microbubbles are spherical voids or cavities filled by a gas encapsulated by a coating material such as phospholipids, surfactants, albumin, or polymers.7 Drug-loaded microbubbles combined with ultrasound application can potentially target drugs to specific sites. Microbubbles are generally unstable and their mean diameter of 1–6 mm is too large for intravascular applications.8 Liquid perfluorocarbons and oils can be used to formulate microbubbles, which are strictly defined as perfluorocarbon nanobubbles (PNs). PNs can be much smaller compared to microbubbles, for example, with mean diameter of 200 nm.7,9 The blood–brain barrier (BBB) is a major limitation to delivering drugs to the central nervous system (CNS) for treating brain cancer, stroke, ischemia, and degenerative disorders such as Parkinson’s and Alzheimer’s diseases.10 The use of nanosystems such as lipid-based liposomes and solid lipid nanoparticles can resolve the problem of the inability of some drugs to cross the BBB.11 Ultrasound pulses combined with microbubbles or PNs can temporarily disrupt the BBB with negligible associated effects on the brain.7,12,13 This phenomenon can be exploited as a non-invasive means of targeting drug delivery to the CNS. Although many studies have investigated the in vivo or clinical pharmacological actions of apomorphine, few investigations of formulation design have been conducted. The aim of this present work was to develop novel PN systems for apomorphine to circumvent problems of instability and a short half-life, thus effectively targeting the drug to specific sites. The feasibility of using apomorphine-loaded PNs as a parenteral formulation was demonstrated through extensive characterization of the size, charge, appearance, drug release, and hemolysis. Both the HCl and base forms of apomorphine were utilized in this study to compare their delivery from PNs.
MATERIALS AND METHODS Materials Apomorphine HCl, coconut oil, and cholesterol were purchased from Sigma–Aldrich Chemical (St. Louis, MO). Perfluoropentane (96%) was obtained from Strem Chemicals (Newburyport, MA). Hydrogenated soybean phosphatidylcholine (SPC, Phospholipon1 80H) was supplied by American Lecithin Company (Oxford, CT). Cellulose membranes (Cellu-Sep1 T2, with a molecular weight cutoff of 6000–8000) were purchased from Membrane Filtration Products (Seguin, TX).
Preparation of Apomorphine Base Apomorphine base was obtained using a method of precipitation. After a saturated solution of Na2CO3 (1.4 g/mL) was added drop by drop to an apomorphine HCl solution in deionized water, apomorphine base was precipitated. The precipitate was then filtered and washed several times with deionized water to remove the Na2CO3. After drying, the residual apomorphine base was obtained and verified by infrared (IR) and nuclear magnetic resonance (NMR) analyses.
Preparation of Perfluorocarbon Nanobubbles (PNs) SPC, cholesterol, and the drug (3.6 mM of the final product) were dissolved in a 5-mL volume of chloroform: methanol (2:1) solution. The organic solvent was evaporated in a rotary evaporator at 508C, and solvent traces were removed by maintaining the lipid film under a vacuum overnight. The film was hydrated with deionized water using a probe-type sonicator (VCX600, Sonics and Materials, Newtown, CT) for 10 min at 608C. Then coconut oil was added to the system, followed by high-shear homogenization (Pro250, Pro Scientific, Monroe, CT) for 4 h. The resulting dispersion was cooled to 208C, then perfluoropentane was incorporated into the system and sonicated for 10 min. The PNs developed in the study are listed in Table 1.
Preparation of Liposomes Liposomes containing SPC (3.5%, w/v) and cholesterol (0.5%, w/v) were prepared by a rotary evaporation method. SPC and cholesterol were dissolved in a minimum amount of chloroform:
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Table 1. The Compositions and Their Percentages (%, w/v) of Perfluorocarbon Nanoparticles Code Coconut Oil C5F12 SPC Cholesterol F1 F2 F3 F4
4.0 10 4.0 4.0
15 15 25 15
3.5 3.5 3.5 3.5
1.0 1.0 1.0
Water Add Add Add Add
to to to to
100% 100% 100% 100%
SPC, soybean phosphatidylcholine.
methanol solution (2:1). The organic solvent was evaporated in a rotary evaporator at 408C, and solvent traces were removed by maintaining the lipid film under a vacuum overnight. The films were hydrated with deionized water containing 3.6 mM of the drug using a probe-type sonicator (VCX600, Sonics and Materials) for 30 min. The liposomal suspension was centrifuged at 48000g and 48C for 30 min in a Beckman Optima MAX1 ultracentrifuge (Beckman Coulter, Fullerton, CA) in order to separate the incorporated drug from the free form. The supernatant was analyzed by high-performance liquid chromatography (HPLC) to determine the encapsulation percentage.
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nitrile tips (SSS-SEIH-20, Nanoworld, Neuchaˆ tel, Switzerland). Tapping mode was applied to drive the cantilever at near its resonance frequency by a small piezoelectric element mounted in the tip holder. The samples were monitored in triplicate.
Ultrasound Imaging PNs were placed in a latex tube and positioned in a water bath. The probe (6 MHz) of an ultrasound imaging system (Aplio1 XG/SSA-790A, Toshiba Medical Systems, Tokyo, Japan) was positioned under the water bath, and the PNs were imaged in a motile form.
Evaporation of PNs Two milliliters of PNs was pipetted into a cylindrical vial with an opening diameter of 2.5 cm. The sample vial was positioned in an incubator at 378C. At determined periods, the vial was weighed, and the weight of the PNs remaining in the vial (%) was measured. The total duration of the experiment was 12 h. Water and neat perfluoropentane were also examined as the controls.
Determination of the Size and Zeta Potential The mean particle size (z-average) and zeta potential of PNs were measured by a laser scattering method (Nano ZS1 90, Malvern, Worcestershire, UK). The PNs were diluted 100fold with double-distilled water to achieve the measurement of both size and surface charge. The determination was repeated three times per sample for three independent batches.
Determination of Surface Tension Surface tension of the PNs was measured by the Wilhemy plate method using a thin platinum plate attached to a transducer amplifier (CBVPA3, Kyowa, Saitama, Japan).
Atomic Force Microscopic (AFM) Examination Experiments were performed using a SPA 300 HV AFM (Seiko Instruments, Tokyo, Japan). Freshly prepared PNs were immobilized onto poly-L-lysine slides (Dako, Glostrup, Denmark). After several minutes, normal saline was used to wash the sample. Imaging was carried out using silicon DOI 10.1002/jps
In Vitro Apomorphine Release Apomorphine release from the PNs was measured using a Franz diffusion cell. A cellulose membrane was mounted between the donor and receptor compartments. The donor medium consisted of 0.5 mL of vehicle containing camptothecin. The receptor medium consisted of 5.5 mL of pH 7.4 buffer. The available diffusion area between cells was 1.13 cm2. The stirring rate and temperature were kept at 600 rpm and 378C, respectively. At appropriate intervals, 300-mL aliquots of the receptor medium were withdrawn and immediately replaced with an equal volume of fresh buffer. The amount of drug released was determined by HPLC. In the study examining the influence of ultrasound on apomorphine release, the donor phase was exposed to ultrasound using a 1-MHz probe (Rich-Mar Sonitron1 2000, Inola, OK) with a 2.0W/cm2 intensity and a 50% duty cycle. A 0.5-mL sample of plasma was added to the donor compartment. The head of the transducer was immersed in the PN mixture with plasma in the donor compartment. The distance between the
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probe and cellulose membrane was 1 cm. Ultrasound was applied for 1 h starting at the beginning of the in vitro release experiment.
HPLC Analysis of Apomorphine The HPLC system for apomorphine included a Hitachi L-7100 pump (Tokyo, Japan), a Hitachi L-7200 sample processor, and a Hitachi L-7400 UV detector. A 25-cm long, 4-mm inner diameter stainless steel RP-18 column (Merck, Darmstadt, Germany) was used. The mobile phase was an acetonitrile: pH 3 aqueous solution adjusted with phosphoric acid (20:80) at a flow rate of 1.0 mL/ min. The UV wavelength was set to 212 nm.
In Vitro Apomorphine Stability in Plasma The stability of apomorphine in human plasma was tested in vitro. Human plasma was obtained as described previously.14 Briefly, blood was taken from healthy human donors (20–32 years old) by venipuncture, using a protocol approved by the Institutional Review Board at Chang Gung Memorial Hospital. A stock solution was prepared by dissolving a weighed amount of apomorphine HCl or base in water or PNs. A volume of 0.1 mL of the vehicle was added to 3.9 mL of prewarmed (378C) plasma. The resulting dispersion was incubated at 378C for 1 or 6 h. The reaction mixture was withdrawn, and acetonitrile was rapidly added to stop the process; then the mixture was stored in a refrigerator (208C). After thawing and filtration, the concentration of apomorphine was measured by HPLC.
Erythrocyte Hemolysis Blood samples were obtained from a healthy donor by venipuncture and collected into test tubes containing 124 mM sodium citrate (one volume of sodium citrate solution þ nine volumes of blood). This experiment was approved by the Institutional Review Board at Chang Gung Memorial Hospital. Erythrocytes were immediately separated by centrifugation at 2000g for 5 min and washed three times with four volumes of a normal saline solution. Erythrocytes collected from 1 mL of blood were resuspended in 10 mL of normal saline. Immediately thereafter, 2.5 mL of 2% (w/v) dispersions of the formulations and mixtures thereof in saline were incubated with 0.1 mL of
the erythrocyte suspension. Incubations were carried out at 378C while the test tubes were gently tumbled. After 1 h of incubation, the samples were centrifuged for 5 min at 2000g. The absorbance of the supernatant was measured at 415 nm to determine the percentage of cells undergoing hemolysis. Hemolysis induced by double-distilled water was taken as 100%.
Statistical Analysis The statistical analysis of differences among various treatments was performed using unpaired Student’s t-test. A 0.05 level of probability was taken as the level of significance. An analysis of variance (ANOVA) test was also used if necessary.
RESULTS Physicochemical Characteristics of PNs PNs were prepared with perfluoropentane (with a boiling point of 298C) as the inner core. Coconut oil and SPC were respectively used in the present work as the oil phase and interfacial membrane of the PNs. Based on the additives and preparation procedures, the PNs were formulated to load the interior with perfluorocarbon, which was then surrounded by coconut oil. SPC and cholesterol as the emulsifiers were located in the oil/water interface.15 On visual inspection, the PNs were white and homogeneous. In apomorphine-loaded dispersions, we observed no distinct, undissolved crystals. Even though the exact solubility of apomorphine in the inner phase could not be measured, it appeared that most of input drug had been solubilized. Table 2 summarizes the physicochemical characteristics of the PNs. There was a significant difference in particle size among these formulations. The standard PNs (F1) had a relatively small size of 155 nm. The increment of oil from 4% to 10% (F2) in the PNs resulted in a particle size increase to 290 nm ( p < 0.05). Also, the increment of perfluoropentane from 15% to 25% (F3) led to an enlargement of particle size (311 nm, p < 0.05). The size of PNs without cholesterol (F4) was 2.4-fold greater ( p < 0.05) than that with cholesterol (F1), indicating the important role of cholesterol in PN emulsification. The absolute zeta potential of the standard system (F1) had a value of -83 mV as given in Table 1. The pH of the vehicles at the status of measurement was 4.4–5.6. A higher oil loading
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Table 2. The Characterization of Perfluorocarbon Nanoparticles by Mean Diameter, Zeta Potential, and Surface Tension Code F1 F2 F3 F4
Mean Diameter (nm)
Zeta Potential (mV)
Surface Tension (dyne/cm)
155.2 2.9 289.8 3.5 311.4 4.0 378.7 23.1
83.4 3.0 66.5 1.8 53.5 7.0 80.8 1.2
24.6 0.1 24.6 0.4 27.3 0.5 35.1 0.9
Each value represents the mean SD (n ¼ 3).
(F2) produced lower surface charges ( p < 0.05) compared to the standard system (F1). A further reduction in surface charges was observed by increasing the perfluoropentane ratio (F3). No remarkable difference ( p > 0.05) was detected between the zeta potential of formulations with or those without cholesterol (F1 vs. F4). The surface tension of water is about 72 dyne/cm. Introducing perfluorocarbon, oil, and emulsifiers to form PNs reduced the surface tension. As shown in Table 2, PNs with cholesterol (F1–F3) generally showed values of 25–27 dyne/cm. Changes in the oil and perfluoropentane contents did not significantly ( p > 0.05) alter the surface tension. The surface tension increased to 35 dyne/ cm following the exclusion of cholesterol from the PNs (F4, p < 0.05).
Imaging of PNs by AFM and Ultrasound To characterize the microscopic structure of the produced PNs, an AFM technique was applied to observe diverse samples (F3). Figure 1A–D represents the field of vision from wide to narrow. As shown in Figure 1A,B, the dispersion investigated using AFM contained oval- or raisin-shaped particles. A narrow size distribution of PNs was observed, indicating a quite-homogeneous population of particles. The polydispersity index (PDI) determined from the laser scattering method also showed a low value of 0.25–0.28. No significant difference ( p > 0.05) of PDI was observed for all formulations tested. Figure 1C,D shows the structure of one PN particle. This particle had a mean diameter of 330 nm, which was in good agreement with the light scattering results. Figure 1E shows the distribution of particles from F3 in a 3D image. In order to examine the acoustically active feature of PNs developed in this work, ultrasound imaging was used to monitor the dispersion of PNs (F3). PNs in the latex tube, shown in DOI 10.1002/jps
Figure 2A within the frame, were observed to oscillate in response to acoustic pulses. There was no signal when monitoring water was placed in the latex tube (data not shown). The color-coded spots reflected echogenic movement signals of PNs. This observation confirmed the acoustical activity of PNs. As shown in Figure 2B, the echo signals with color-coded spots of particles almost faded out after a 15-min ultrasound application.
Evaporation of PNs Gas loss represents the primary destructive mechanism of microbubbles. The evaporation of PNs was evaluated by incubating them in a 378C oven. As shown in Figure 3, formulations with higher oil and perfluorocarbon contents (F2 and F3) demonstrated more-rapid evaporation than the standard one (F1). However, no significant difference ( p > 0.05) in the weight remaining was detected between water and these formulations (F1–F3) at the end of the experiment (12 h). The exclusion of cholesterol (F4) significantly accelerated ( p < 0.05) the evaporation rate of PNs compared to those with water and the standard system.
In Vitro Apomorphine Stability in Plasma Apomorphine is known to be chemically and biologically unstable. The inclusion of apomorphine in PNs may improve its stability, thus prolonging its half-life. Figure 4 shows the amounts of apomorphine HCl and base remaining after incubation in plasma for 1 and 6 h. Marked degradation of both apomorphine HCl and base in the free form occurred under the reaction conditions. There was no significant difference ( p > 0.05) in the amount of apomorphine remaining after the 1- and 6-h incubations (Fig. 4A). Inclusion in PNs generally produced significant protection ( p < 0.05) for apomorphine HCl except
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Figure 1. Images of atomic force microscopy of perfluorocarbon nanobubbles (F3) showing the particle appearance. (A–D) Represent the field of vision from wide to narrow; and (E) shows the distribution of particles from F3 in a 3D image.
with the F1 formulation at 1 h. The protective abilities of three delivery systems were similar ( p > 0.05). The standard formulation (F1) and the formulation with a high perfluorocarbon loading (F3) protected apomorphine base (Fig. 4B). However, no effect was found for PNs with high oil loading (F2, p > 0.05) compared to the free form.
In Vitro Apomorphine Release For development of PNs loaded with apomorphine, it is important to optimize the ability to release the drug from the particles. The ability of PNs to deliver apomorphine was examined by determining the drug release as shown in
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Figure 2. Ultrasonography of perfluorocarbon nanobubbles (F3). (A) Representative image taken immediately after beginning the ultrasound application (6 MHz); and (B) representative image taken after ultrasound application (6 MHz) for 15 min.
Figure 5. Both the HCl and base forms of apomorphine were incorporated into PNs for this test. The release of apomorphine from double-distilled water was used as the control.
Figure 3. Percentage weight remaining of perfluorocarbon nanobubbles as a function of time in the evaporation experiment at 378C. Each value represents the mean and SD (n ¼ 4). DOI 10.1002/jps
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Figure 4. Percentage of apomorphine remaining in the stability experiment by incubating perfluorocarbon nanobubbles with plasma at 378C for 1 and 6 h. (A) Represents the profiles of apomorphine HCl; and (B) represents the profiles of apomorphine base. Each value is presented as the mean and SD (n ¼ 6).
F4 was excluded from this experiment because of its low stability. As shown in Figure 5A, apomorphine HCl in an aqueous solution demonstrated the highest release. Release from the aqueous solution showed an initial burst, then leveled off after 2 h of administration. All PNs retarded the release of apomorphine HCl, with the formulation containing a high oil content (F2) showing the slowest drug delivery rate. Only 20% of the apomorphine base dose in the aqueous solution was released from the donor as depicted in Figure 5B. Similar to apomorphine HCl, the PNs released apomorphine base in a sustained manner. Discrepancies among the release rates of these three nanoparticle systems were not large for the base form. Ultrasound application of PNs has the potential to deliver apomorphine to target specific areas in the CNS. To explore this novel opportunity, we examined the amount of apomorphine released under the influence of ultrasound application. Plasma was added to the donor phase and mixed
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Figure 5. In vitro release-time profiles of apomorphine across a cellulose membrane from an aqueous solution (control) and perfluorocarbon nanobubbles. (A) Represents the release of apomorphine HCl; and (B) represents the release of apomorphine base. Each value is presented as the mean and SD (n ¼ 4).
with PNs to simulate in vivo conditions, The amounts of apomorphine HCl released in the absence and presence of ultrasound are summarized in Figure 6. There was no or negligible apomorphine detected in the receptor when using the aqueous solution as the vehicle, possibly due to the instability of free apomorphine in plasma. The effects of ultrasound on F1, F2, and F3 were evaluated as shown in Figure 6A–C. Apomorphine HCl release significantly increased ( p < 0.05) after applying ultrasound in all systems. The drug release percentage at the end of the experiment (8 h) by insonation showed enhancements of 1.9-, 3.6-, and 3.9-fold compared to the non-ultrasound group for F1, F2, and F3, respectively. A contrary result was observed for apomorphine base. The application of ultrasound decreased the release of apomorphine base from all PNs (Fig. 7).
Figure 6. Effect of ultrasound at 1 MHz at an intensity of 2.0 W/cm2 for 1 h (with a duty cycle of 50%) on the in vitro release of apomorphine HCl across a cellulose membrane from perfluorocarbon nanobubbles. (A) Represents the standard formulation (F1); (B) represents the formulation with a high oil ratio (F2); and (C) represents the formulation with a high perfluoropentane ratio (F3). A 0.5-mL aliquot of plasma was added to the donor compartment of a Franz diffusion cell. Each value is presented as the mean and SD (n ¼ 4).
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Erythrocyte Hemolysis To use PNs for parenteral administration, rigorous demands on the non-toxicity of the formulations must be imposed. To evaluate the safety of PNs themselves, the hemolytic activity was determined. Percentages of human erythrocytes undergoing hemolysis induced by the PNs are shown in Table 3. The standard PNs (F1) showed a hemolysis percentage of 11%. Adding oil and perfluoropentane in PNs (F2 and F3) led to reductions in hemolysis ( p < 0.05), especially for the system with a high oil ratio.
DISCUSSION
Figure 7. Effect of ultrasound at 1 MHz at an intensity of 2.0 W/cm2 for 1 h (with a duty cycle of 50%) on the in vitro release of apomorphine base across a cellulose membrane from perfluorocarbon nanobubbles. (A) Represents the standard formulation (F1); (B) represents the formulation with a high oil ratio (F2); and (C) represents the formulation with a high perfluoropentane ratio (F3). A 0.5-mL plasma aliquot was added to the donor compartment of a Franz diffusion cell. Each value is presented as the mean and SD (n ¼ 4).
We are attempting to develop an approach, which will permit utilization of apomorphine, which has some disadvantages in clinical practice due to the lack of a suitable drug carrier system. The use of PNs is possibly permit effective formulations and utilization of this potent agent in Parkinson’s disease since ultrasound application may target the drug-loaded PNs to the brain. Improvements in clinical outcomes can be achieved by modifying the physicochemical properties of the nanoparticles, especially for drugs delivered through the BBB.16 The small size of PNs, with substantial populations of 150–380 nm, is an important factor in their efficacy of parenteral administration. A higher oil ratio (F2) and fluorocarbon ratio (F3) led to increases in the size of the particles. The monolayer shell composed of SPC and cholesterol imparts stability to the microbubbles.17 Since the contents of emulsifiers in these systems were the same, the increase in diameter may have resulted from impoverishment of the emulsifiers at the interface with an increasing volume of the dispersed oil and perfluorocarbon phases. Our data also indicate the necessity of cholesterol for PN stability, since larger particle sizes were observed in the absence of cholesterol. Cholesterol was proven to provide a stable and rigid structure Table 3. Hemolysis Percentage (%) After 1 h of Incubation at 378C With Perfluorocarbon Nanoparticles Code F1 F2 F3
Hemolysis (%) 11.18 0.40 2.08 0.29 6.46 1.55
Each value represents the mean SD (n ¼ 4). DOI 10.1002/jps
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when incorporated into phospholipid membranes.18,19 Another observation was that the surface tension significantly increased when cholesterol was excluded from PNs, suggesting that cholesterol enhances the emulsification of PNs which stabilizes the entire system. That is, SPC alone cannot provide sufficient emulsification power for PNs. The SPC used in this study contained 80% PC, which is neutral. Other phospholipids components (20%), such as phosphatidylserine, phosphatidic acid, phosphatidylglycerol, and phosphatidylinositol, were responsible for the negative zeta potential of the PNs. Increments of the oil and perfluorocarbon ratios in these systems contributed to reductions in surface charges. It was found that PNs (F1–F3) showed an inverse relationship between size and zeta potential. The larger total surface area of the smaller particle systems may explain this phenomenon. However, this trend was not observed for nanobubbles without cholesterol (F4). The absence of cholesterol did not significantly reduce the zeta potential, although the particle size greatly increased. This may have been due to this formulation not being a stable and typical system compared to other PNs. Another indication is that the surface charge was not a predominant factor govering the instability of F4 since cholesterol played a negligible role on zeta potential. Regarding the long-term stability, the negative zeta potential of a stable colloidal system should not fall below 30 mV.20,21 As a result, the PNs developed in this study would likely show acceptable stability in normal use. AFM and ultrasound imaging, respectively confirmed the microscopic appearance/distribution and echogenic features of the PNs. Insonation with ultrasound energy can be used to stimulate the transition of the inner perfluoropentane core from a liquid to a gas. As the particles become gas bubbles, the acoustic properties change, and the activity as a cavitation nucleus is increased.7,22 After 15 min of insonation, the echo signals of PNs decreased to a much lower level. This was possibly due to ultrasound disrupting PNs by inducing cavitations after a determined period.8,23 Ideal microbubbles or PNs should be stable enough in both storage conditions and the vascular system with respect to the particles and the incorporated drug molecules. In the present study, the evaporation test and apomorphine stability in plasma were respectively used to examine the stability of the dispersions and loaded drug. The weight loss of PNs in the
evaporation experiment showed that there were no significant differences among the systems with cholesterol and water, suggesting that perfluorocarbon loss by PNs with cholesterol was limited. Perfluoropentane completely evaporated in a 50-min period. A phospholipids/ cholesterol monolayer and oil shell may have adequately protected the inner core from evaporation. PNs quickly evaporated after cholesterol was removed. This again confirms that cholesterol can produce a rigid membrane with SPC to maintain stability. One of the difficulties with apomorphine administration is its inherent instability.3,24 Autooxidation of apomorphine to quinone species is a potentially important factor in apomorphine metabolism.25 Moreover, internal conversion from the R to the S form at physiological pH values is another concern.4 The S form is the therapeutically inactive form. Apomorphine HCl and base in the free form showed comparable stabilities. The results suggest that apomorphine was quickly oxidized in plasma, since no great difference between 1- and 6-h incubation periods was observed. Apomorphine was rather stable when PNs were used as the delivery systems. Hence PNs can effectively prevent the degradation of apomorphine, thus prolonging the residence time in the circulation. The pore size of the permeated membrane used was below 10 nm,26 and free molecules were able to permeate across the membrane. The amounts of apomorphine HCl and base released from the aqueous solution were limited, with 40% and 20% of the drug being released over 8 h, respectively. This may have been due to the use of the in vitro Franz cell. Since a drug is released to the definitive space of the receptor (5.5 mL) and diffusion area (1.13 cm2), drug loading in the receptor compartment is limited. There might no longer be a concentration gradient between the donor and receptor compartments. Nevertheless, this setup is still useful for differentiating the relative release capabilities of various formulations.27,28 The release of free apomorphine HCl was higher than that of the free base form. The lipophilic base form at a dose of 3.6 mM did not completely dissolve in water. Since the compound should be released in a dissolved form, its low solubility retarded its release from the aqueous solution. Incorporation of apomorphine in PNs had slower release profiles compared to the free form, indicating that apomorphine did not diffuse freely
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when entrapped in the particles. The sustained release of an incorporated drug in a delivery system is an important feature quite often correlated with improved pharmacokinetics and efficacy.9,27 The clinical relevance of this is that the frequency of apomorphine administration can be reduced. This would be very beneficial for patients requiring up to 10–15 injections per day.29 It was found that the systems with a high oil or perfluoropentane percentage (F2 and F3) showed lower drug release profiles compared to that with a low percentage (F1) for both the HCl and base forms. This may have been due to the larger particle diameters of the formulations with higher oil and perfluoropentane contents. Drug release is generally slower from systems with larger particles since they have a decreased total surface area where drug diffusion can occur.30,31 Plasma was mixed with PNs in the release experiment with ultrasound application to mimic an actual in vivo status. Plasma is a viscous fluid consisting of about 91% water and 9% other substances such as proteins, ions, nutrients, and waste products. In the presence of plasma, drug release was reduced because of the higher viscosity and longer path lengths for drug diffusion. The decreased drug concentration gradient between the donor and receptor is another mechanism. It is apparent that insonation accelerated the release rate of apomorphine HCl from PNs. As ultrasound pressure waves interact with the particles, they begin to oscillate or resonate. This effect results in particle rupture,32 and abrupt drug release. PNs with a higher oil or perfluorocarbon load produced more-significant apomorphine HCl release enhancement after insonation. The size of the PNs may be important. As microbubbles become larger, they become more echogenic.33,34 It was surprising that apomorphine base exhibited a contrary result compared to the HCl form. Since particles loaded with the HCl and base forms did not show great differences in their physicochemical properties, the different locations of apomorphine HCl and base in the PNs may explain this noticeable discrepancy. Apomorphine HCl is more hydrophilic than apomorphine base. Hydrophilic molecules are difficult to encapsulate into the inner phase of particles composed of oil and perfluorocarbon. However, the slower release of apomorphine HCl from PNs compared to the aqueous solution may indicate that some interactions occurred between the particles and the HCl form. Drugs can be DOI 10.1002/jps
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incorporated into microbubbles in a number of different ways, including binding the drug to the emulsifier monolayer.35 Apomorphine HCl has positively ionized format pH values of <7.2.3 PNs prepared in this study had pH values of 5.6–6.2. Interfacial films with high negative charges may offer affinity to apomorphine HCl. On the other hand, lipophilic drugs are always incorporated into the oil layer of microbubbles.15,36 Non-ionic apomorphine base possibly is such a case. In order to elucidate the location of apomorphine in PNs, liposomes composed of SPC and cholesterol were prepared to examine their mean diameter and encapsulation of apomorphine HCl and base. As shown in Table 4, apomorphine HCl had an encapsulation percentage of 47% in liposomes. Comparable encapsulation percentages of apomorphine HCl and base forms were detected ( p > 0.05). However, entrapment of the base form in liposomes should be analyzed with caution since it did not completely dissolve in this system. The 54% rate of encapsulation of apomorphine base was overestimated, because undissolved molecules were included in the encapsulated drug due to the ultracentrifuge method used in this study. Hence apomorphine HCl may actually show greater entrapment compared to apomorphine base. This is reasonable since the positive apomorphine HCl can interact with the negative phospholipid bilayers due to electrostatic forces. The vesicle size of liposomes loaded with apomorphine HCl was 9-fold greater than that of blank liposomes. It is expected that the HCl form will largely interact with the film, enlarging the volume of the bilayers. Another observation is a great deviation of the size data (931.9 209.8 nm). This may be due to the poor stability of this drug-loaded liposomes and the large variation of the drug-phospholipids interaction among batches. Previous studies 15,34,37,38 suggested that phospholipids-based microbubbles are destroyed by Table 4. The Characterization of SPC Liposomes Loaded With Apomorphine HCl or Apomorphine Base by Mean Diameter and Encapsulation Ratio
Drug None Apomorphine HCl Apomorphine base
Encapsulation (%)
Mean Diameter (nm)
— 46.6 6.5 53.6 10.6
104.2 1.8 931.9 209.8 94.1 1.9
Each value represents the mean SD (n ¼ 3).
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ultrasound, due to rupture and loosening of the membranes. Encapsulated apomorphine HCl in membranes may be subsequently released by insonation. Insonation seemed to be less applicable to the oil phase. Fluorocarbon-filled microbubbles provide enhanced echogenic effects compared to air-filled microbubbles.37 With ultrasonic oscillation, air can diffuse through the shell and quickly dissolve in the surrounding medium because of its low boiling point and high Ostwald coefficient (the velocity with which a gas leaves a bubble). On the contrary, fluorocarbon and oil have much higher boiling points and lower Ostwald coefficients than air. After oscillation, apomorphine base might still be retained in the oil phase for a determined period. As the membrane has already been ruptured by insonation, the release of apomorphine base becomes more difficult since the direct partitioning from the oil to the aqueous phase is much lower in the absence of a phospholipid interface for lipophilic drugs. This results in reduced apomorphine base release from PNs under ultrasound influence. The mechanisms of this effect were not exactly determined in the present study and need to be further explored. Phospholipids are known to cause erythrocyte hemolysis.39 Our results show that the standard PNs (F1) produced a hemolysis percentage of 11%. Hemolysis occurs because of penetration of free phospholipids in the aqueous phase of the emulsions.38 The percentage of hemolysis decreased with an increased oil or perfluorocarbon concentration since most phospholipids resided in the interface and emulsified the inner phase with a larger volume. According to the hemolysis result, PNs developed in this study are generally biologically safe (hemolysis percentage <10%) and do not pose a toxicological risk.
CONCLUSIONS PNs are feasible to be carriers for apomorphine delivery according to the experimental results in the present work. Ultrasound pulses induced marked enhancement of apomorphine HCl release, thus allowing the bioactive drug to be targeted to specific sites such as the brain. However, that was not the case for apomorphine base. This suggests that selecting optimized drugs for PNs is important to achieve the aim of drug targeting. Particles with different ratios of the inner phase, including oil and perfluorocarbon,
were found to greatly influence the physicochemical characteristics, safety, and sensitivity to ultrasound. We also elucidated the importance of cholesterol to the interfacial films to stabilize these systems. The advantages of the prepared PNs warrant further investigations of this approach. In vivo studies are planned in order to evaluate the potential use of apomorphine loaded in PNs as parenteral drug delivery systems.
REFERENCES 1. Stacy M, Silver D. 2008. Apomorphine for the acute treatment of ‘‘off’’ episodes in Parkinson’s disease. Parkinsonism Relat Disord 14:85–92. 2. Chin YW, Balunas MJ, Chai HB, Kinghorn AD. 2006. Drug discovery from natural sources. AAPS J 8:239–253. 3. Li GL, Danhof M, Bouwstra JA. 2001. Iontophoretic delivery of apomorphine in vitro: Physicochemic considerations. Pharm Res 18:1509–1513. 4. Subramony JA. 2006. Apomorphine in dopaminergic therapy. Mol Pharm 3:380–385. 5. Kawakami S, Yamashita F, Hashida M. 2000. Deposition characteristics of emulsions and incorporated drugs after systemic or local injection. Adv Drug Deliv Rev 45:77–88. 6. Torchilin VP. 2005. Recent advances with liposomes as pharmaceutical carriers. Nat Rev Drug Discov 4:145–160. 7. Unger EC, Porter T, Culp W, Labell R, Matsunaga T, Zutshi R. 2004. Therapeutic applications of lipidcoated microbubbles. Adv Drug Deliv Rev 56:1291– 1314. 8. Suzuki R, Takizawa T, Negishi Y, Hagisawa K, Tanaka K, Sawamura K, Utoguchi N, Nishioka T, Maruyama K. 2007. Gene delivery by combination of novel liposomal bubbles with perfluoropropane and ultrasound. J Control Release 117:130–136. 9. Fang JY, Hung CF, Liao MH, Chien CC. 2007. A study of the formulation design of acoustically active lipospheres as carriers for drug delivery. Eur J Pharm Biopharm 67:67–75. 10. Tosi G, Costantino L, Ruozi B, Forni F, Vandelli MA. 2008. Polymeric nanoparticles for the drug delivery to the central nervous system. Expert Opin Drug Deliv 5:155–174. 11. Kreuter J. 2007. Nanoparticles—A historical perspective. Int J Pharm 331:1–10. 12. Treat LH, McDannold N, Zhang Y, Vykhodtseva N, Hynynen K. 2007. Targeted delivery of doxorubicin to the rat brain at therapeutic levels using MRIguided focused ultrasound. Int J Cancer 121:901– 907.
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13. McDannold N, Vykhodtseva N, Hynynen K. 2008. Blood-brain barrier disruption induced by focused ultrasound and circulating preformed microbubbles appears to be characterized by the mechanical index. Ultrasound Med Biol 34:834–840. 14. Wang JJ, Sung KC, Yeh CH, Fang JY. 2008. The delivery and antinociceptive effects of morphine and its ester prodrugs from lipid emulsions. Int J Pharm 353:95–104. 15. Liu Y, Miyoshi H, Nakamura M. 2006. Encapsulated ultrasound microbubbles: Therapeutic application in drug/gene delivery. J Control Release 114: 89–99. 16. Yu YB. 2006. Fluorocarbon nanoparticles as multifunctional drug delivery vehicles. J Drug Target 14:663–669. 17. Borden MA, Pu G, Runner GJ, Longo ML. 2004. Surface phase behavior and microstructure of lipid/ PEG-emulsifier monolayer-coated microbubbles. Colloid Surf B: Biointerface 35:209–223. 18. Nagayasu A, Uchiyama K, Kiwada H. 1999. The size of liposomes: A factor which affects their targeting efficiency to tumors and therapeutic activity of liposomal antitumor drugs. Adv Drug Deliv Rev 40:75–87. 19. Fang JY, Hwang TL, Huang YL, Fang CL. 2006. Enhancement of the transdermal delivery of catechins by liposomes incorporating anionic surfactants and ethanol. Int J Pharm 310:131–138. 20. Buszello K, Harnisch S, Mu¨ ller RH, Mu¨ ller BW. 2000. The influence of alkali fatty acids on the properties and the stability of parenteral O/W emulsions modified with Solutol HS151. Eur J Pharm Biopharm 49:143–149. 21. Chen H, Chang X, Du D, Liu W, Liu J, Weng T, Yang Y, Xu H, Yang X. 2006. Podophyllotoxinloaded solid lipid nanoparticles for epidermal targeting. J Control Release 110:296–306. 22. Kleirolomoom A, Dayton PA, Lum AFH, Little E, Paoli EE, Zheng H, Ferrara KW. 2007. Acousticallyactive microbubbles conjugated to liposomes: Characterization of a proposed drug delivery vehicle. J Control Release 118:275–284. 23. Pisani E, Tsapis N, Paris J, Nicolas V, Cattel L, Fattal E. 2006. Polymeric nano/microcapsules of liquid perfluorocarbons for ultrasonic imaging: Physical characterization. Langmuir 22:4397– 4402. 24. Ng Kin Ying NMK, Lal S, Thavundayil JX. 2001. Stability of apomorphine hydrochloride in aqueous sodium bisulphate solutions. Prog Neuropsychopharmacol Biol Psychiatry 25:1461–1468. 25. Ingram WM, Priston MJ, Sewell GT. 2006. Improved assay for R(-)-apomorphine with application to clinical pharmacokinetic studies in Parkinson’s disease. J Chromatogr B 831:1–7. 26. Fang JY, Lee WR, Shen SC, Huang YL. 2006. Effect of liposome encapsulation of tea catechins on their
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Cell Pharmacology Laboratory, Graduate Institute of Natural Products, Chang Gung University, Kweishan, Taoyuan, Taiwan 2
Graduate Institute of Clinical Medical Sciences, Chang Gung University, Kweishan, Taoyuan, Taiwan
3
Department of Traditional Chinese Medicine, Chang Gung Memorial Hospital, Keelung, Taiwan
4
Pharmaceutics Laboratory, Graduate Institute of Natural Products, Chang Gung University, Kweishan, Taoyuan, Taiwan
5
Graduate Institute of Traditional Chinese Medicine, Chang Gung University, Kweishan, Taoyuan, Taiwan
Received 14 September 2008; revised 16 November 2008; accepted 16 December 2008 Published online 20 January 2009 in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/jps.21687
ABSTRACT: Apomorphine is a dopamine receptor agonist for treating Parkinson’s disease. However, its clinical application is limited by its instability and the need for frequent injections. The aim of the present work was to develop acoustically active perfluorocarbon nanobubbles (PNs) for encapsulation of both apomorphine HCl and base forms to circumvent these delivery problems. The PNs were prepared using coconut oil and perfluoropentane as the inner phase, which was emulsified by phospholipids and cholesterol. The morphology, size, zeta potential, and drug release of the PNs were characterized. The particle size ranged from 150 to 380 nm, with differences in the oil or perfluorocarbon ratio in the formulations. Atomic force microscopy confirmed oval- or raisin-shaped particles and a narrow size distribution of these systems (polydispersity index ¼ 0.25–0.28). The stability experimental results indicated that PNs could protect apomorphine from degradation. Evaporation of the PNs at 378C was also limited. Apomorphine HCl and base in PNs showed retarded and sustained release profiles. Ultrasound imaging confirmed the echogenic activity of PNs developed in this study. The apomorphine HCl release by insonation at 1 MHz showed enhancements of two- to fourfold compared to the non-ultrasound group, illustrating a possible drug-targeting effect. On the contrary, apomorphine base showed a decreased release profile with ultrasound application. Apomorphine-loaded PNs showed promising stability and safety. They were successful in sustaining apomorphine delivery. ß 2009 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 98:3735–3747, 2009
Keywords: apomorphine; perfluorocarbon nanobubbles; drug delivery system; drug targeting; ultrasound
INTRODUCTION
Correspondence to: Jia-You Fang (Telephone: 886-32118800 ext. 5521; Fax: 886-3-2118236; E-mail: [email protected]) Journal of Pharmaceutical Sciences, Vol. 98, 3735–3747 (2009) ß 2009 Wiley-Liss, Inc. and the American Pharmacists Association
Parkinson’s disease affects 1% of people over 65 years old and 3% of those over 85 years old; it is a chronic neurodegenerative disease caused by the loss of pigmented mesostriatal dopaminergic neurons linking the substantia nigra to the neostriatum.1,2 Apomorphine as a mixed D1 and D2 dopamine receptor agonist has
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been shown to be a very potent drug for treating patients with idiopathic Parkinson’s disease.3 However, its inherent instability, negligible oral bioavailability, and short half-life (41 min) have complicated its application in clinical practice.3,4 A high demand on the apomorphine delivery system is necessary to resolve these problems. Drug encapsulation systems such as liposomes and emulsions have been introduced as parenteral drug carriers offering sustained release and long residence times.5,6 Microbubbles represent a new class of encapsulation systems with both diagnostic and therapeutic applications. Microbubbles are spherical voids or cavities filled by a gas encapsulated by a coating material such as phospholipids, surfactants, albumin, or polymers.7 Drug-loaded microbubbles combined with ultrasound application can potentially target drugs to specific sites. Microbubbles are generally unstable and their mean diameter of 1–6 mm is too large for intravascular applications.8 Liquid perfluorocarbons and oils can be used to formulate microbubbles, which are strictly defined as perfluorocarbon nanobubbles (PNs). PNs can be much smaller compared to microbubbles, for example, with mean diameter of 200 nm.7,9 The blood–brain barrier (BBB) is a major limitation to delivering drugs to the central nervous system (CNS) for treating brain cancer, stroke, ischemia, and degenerative disorders such as Parkinson’s and Alzheimer’s diseases.10 The use of nanosystems such as lipid-based liposomes and solid lipid nanoparticles can resolve the problem of the inability of some drugs to cross the BBB.11 Ultrasound pulses combined with microbubbles or PNs can temporarily disrupt the BBB with negligible associated effects on the brain.7,12,13 This phenomenon can be exploited as a non-invasive means of targeting drug delivery to the CNS. Although many studies have investigated the in vivo or clinical pharmacological actions of apomorphine, few investigations of formulation design have been conducted. The aim of this present work was to develop novel PN systems for apomorphine to circumvent problems of instability and a short half-life, thus effectively targeting the drug to specific sites. The feasibility of using apomorphine-loaded PNs as a parenteral formulation was demonstrated through extensive characterization of the size, charge, appearance, drug release, and hemolysis. Both the HCl and base forms of apomorphine were utilized in this study to compare their delivery from PNs.
MATERIALS AND METHODS Materials Apomorphine HCl, coconut oil, and cholesterol were purchased from Sigma–Aldrich Chemical (St. Louis, MO). Perfluoropentane (96%) was obtained from Strem Chemicals (Newburyport, MA). Hydrogenated soybean phosphatidylcholine (SPC, Phospholipon1 80H) was supplied by American Lecithin Company (Oxford, CT). Cellulose membranes (Cellu-Sep1 T2, with a molecular weight cutoff of 6000–8000) were purchased from Membrane Filtration Products (Seguin, TX).
Preparation of Apomorphine Base Apomorphine base was obtained using a method of precipitation. After a saturated solution of Na2CO3 (1.4 g/mL) was added drop by drop to an apomorphine HCl solution in deionized water, apomorphine base was precipitated. The precipitate was then filtered and washed several times with deionized water to remove the Na2CO3. After drying, the residual apomorphine base was obtained and verified by infrared (IR) and nuclear magnetic resonance (NMR) analyses.
Preparation of Perfluorocarbon Nanobubbles (PNs) SPC, cholesterol, and the drug (3.6 mM of the final product) were dissolved in a 5-mL volume of chloroform: methanol (2:1) solution. The organic solvent was evaporated in a rotary evaporator at 508C, and solvent traces were removed by maintaining the lipid film under a vacuum overnight. The film was hydrated with deionized water using a probe-type sonicator (VCX600, Sonics and Materials, Newtown, CT) for 10 min at 608C. Then coconut oil was added to the system, followed by high-shear homogenization (Pro250, Pro Scientific, Monroe, CT) for 4 h. The resulting dispersion was cooled to 208C, then perfluoropentane was incorporated into the system and sonicated for 10 min. The PNs developed in the study are listed in Table 1.
Preparation of Liposomes Liposomes containing SPC (3.5%, w/v) and cholesterol (0.5%, w/v) were prepared by a rotary evaporation method. SPC and cholesterol were dissolved in a minimum amount of chloroform:
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Table 1. The Compositions and Their Percentages (%, w/v) of Perfluorocarbon Nanoparticles Code Coconut Oil C5F12 SPC Cholesterol F1 F2 F3 F4
4.0 10 4.0 4.0
15 15 25 15
3.5 3.5 3.5 3.5
1.0 1.0 1.0
Water Add Add Add Add
to to to to
100% 100% 100% 100%
SPC, soybean phosphatidylcholine.
methanol solution (2:1). The organic solvent was evaporated in a rotary evaporator at 408C, and solvent traces were removed by maintaining the lipid film under a vacuum overnight. The films were hydrated with deionized water containing 3.6 mM of the drug using a probe-type sonicator (VCX600, Sonics and Materials) for 30 min. The liposomal suspension was centrifuged at 48000g and 48C for 30 min in a Beckman Optima MAX1 ultracentrifuge (Beckman Coulter, Fullerton, CA) in order to separate the incorporated drug from the free form. The supernatant was analyzed by high-performance liquid chromatography (HPLC) to determine the encapsulation percentage.
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nitrile tips (SSS-SEIH-20, Nanoworld, Neuchaˆ tel, Switzerland). Tapping mode was applied to drive the cantilever at near its resonance frequency by a small piezoelectric element mounted in the tip holder. The samples were monitored in triplicate.
Ultrasound Imaging PNs were placed in a latex tube and positioned in a water bath. The probe (6 MHz) of an ultrasound imaging system (Aplio1 XG/SSA-790A, Toshiba Medical Systems, Tokyo, Japan) was positioned under the water bath, and the PNs were imaged in a motile form.
Evaporation of PNs Two milliliters of PNs was pipetted into a cylindrical vial with an opening diameter of 2.5 cm. The sample vial was positioned in an incubator at 378C. At determined periods, the vial was weighed, and the weight of the PNs remaining in the vial (%) was measured. The total duration of the experiment was 12 h. Water and neat perfluoropentane were also examined as the controls.
Determination of the Size and Zeta Potential The mean particle size (z-average) and zeta potential of PNs were measured by a laser scattering method (Nano ZS1 90, Malvern, Worcestershire, UK). The PNs were diluted 100fold with double-distilled water to achieve the measurement of both size and surface charge. The determination was repeated three times per sample for three independent batches.
Determination of Surface Tension Surface tension of the PNs was measured by the Wilhemy plate method using a thin platinum plate attached to a transducer amplifier (CBVPA3, Kyowa, Saitama, Japan).
Atomic Force Microscopic (AFM) Examination Experiments were performed using a SPA 300 HV AFM (Seiko Instruments, Tokyo, Japan). Freshly prepared PNs were immobilized onto poly-L-lysine slides (Dako, Glostrup, Denmark). After several minutes, normal saline was used to wash the sample. Imaging was carried out using silicon DOI 10.1002/jps
In Vitro Apomorphine Release Apomorphine release from the PNs was measured using a Franz diffusion cell. A cellulose membrane was mounted between the donor and receptor compartments. The donor medium consisted of 0.5 mL of vehicle containing camptothecin. The receptor medium consisted of 5.5 mL of pH 7.4 buffer. The available diffusion area between cells was 1.13 cm2. The stirring rate and temperature were kept at 600 rpm and 378C, respectively. At appropriate intervals, 300-mL aliquots of the receptor medium were withdrawn and immediately replaced with an equal volume of fresh buffer. The amount of drug released was determined by HPLC. In the study examining the influence of ultrasound on apomorphine release, the donor phase was exposed to ultrasound using a 1-MHz probe (Rich-Mar Sonitron1 2000, Inola, OK) with a 2.0W/cm2 intensity and a 50% duty cycle. A 0.5-mL sample of plasma was added to the donor compartment. The head of the transducer was immersed in the PN mixture with plasma in the donor compartment. The distance between the
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probe and cellulose membrane was 1 cm. Ultrasound was applied for 1 h starting at the beginning of the in vitro release experiment.
HPLC Analysis of Apomorphine The HPLC system for apomorphine included a Hitachi L-7100 pump (Tokyo, Japan), a Hitachi L-7200 sample processor, and a Hitachi L-7400 UV detector. A 25-cm long, 4-mm inner diameter stainless steel RP-18 column (Merck, Darmstadt, Germany) was used. The mobile phase was an acetonitrile: pH 3 aqueous solution adjusted with phosphoric acid (20:80) at a flow rate of 1.0 mL/ min. The UV wavelength was set to 212 nm.
In Vitro Apomorphine Stability in Plasma The stability of apomorphine in human plasma was tested in vitro. Human plasma was obtained as described previously.14 Briefly, blood was taken from healthy human donors (20–32 years old) by venipuncture, using a protocol approved by the Institutional Review Board at Chang Gung Memorial Hospital. A stock solution was prepared by dissolving a weighed amount of apomorphine HCl or base in water or PNs. A volume of 0.1 mL of the vehicle was added to 3.9 mL of prewarmed (378C) plasma. The resulting dispersion was incubated at 378C for 1 or 6 h. The reaction mixture was withdrawn, and acetonitrile was rapidly added to stop the process; then the mixture was stored in a refrigerator (208C). After thawing and filtration, the concentration of apomorphine was measured by HPLC.
Erythrocyte Hemolysis Blood samples were obtained from a healthy donor by venipuncture and collected into test tubes containing 124 mM sodium citrate (one volume of sodium citrate solution þ nine volumes of blood). This experiment was approved by the Institutional Review Board at Chang Gung Memorial Hospital. Erythrocytes were immediately separated by centrifugation at 2000g for 5 min and washed three times with four volumes of a normal saline solution. Erythrocytes collected from 1 mL of blood were resuspended in 10 mL of normal saline. Immediately thereafter, 2.5 mL of 2% (w/v) dispersions of the formulations and mixtures thereof in saline were incubated with 0.1 mL of
the erythrocyte suspension. Incubations were carried out at 378C while the test tubes were gently tumbled. After 1 h of incubation, the samples were centrifuged for 5 min at 2000g. The absorbance of the supernatant was measured at 415 nm to determine the percentage of cells undergoing hemolysis. Hemolysis induced by double-distilled water was taken as 100%.
Statistical Analysis The statistical analysis of differences among various treatments was performed using unpaired Student’s t-test. A 0.05 level of probability was taken as the level of significance. An analysis of variance (ANOVA) test was also used if necessary.
RESULTS Physicochemical Characteristics of PNs PNs were prepared with perfluoropentane (with a boiling point of 298C) as the inner core. Coconut oil and SPC were respectively used in the present work as the oil phase and interfacial membrane of the PNs. Based on the additives and preparation procedures, the PNs were formulated to load the interior with perfluorocarbon, which was then surrounded by coconut oil. SPC and cholesterol as the emulsifiers were located in the oil/water interface.15 On visual inspection, the PNs were white and homogeneous. In apomorphine-loaded dispersions, we observed no distinct, undissolved crystals. Even though the exact solubility of apomorphine in the inner phase could not be measured, it appeared that most of input drug had been solubilized. Table 2 summarizes the physicochemical characteristics of the PNs. There was a significant difference in particle size among these formulations. The standard PNs (F1) had a relatively small size of 155 nm. The increment of oil from 4% to 10% (F2) in the PNs resulted in a particle size increase to 290 nm ( p < 0.05). Also, the increment of perfluoropentane from 15% to 25% (F3) led to an enlargement of particle size (311 nm, p < 0.05). The size of PNs without cholesterol (F4) was 2.4-fold greater ( p < 0.05) than that with cholesterol (F1), indicating the important role of cholesterol in PN emulsification. The absolute zeta potential of the standard system (F1) had a value of -83 mV as given in Table 1. The pH of the vehicles at the status of measurement was 4.4–5.6. A higher oil loading
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Table 2. The Characterization of Perfluorocarbon Nanoparticles by Mean Diameter, Zeta Potential, and Surface Tension Code F1 F2 F3 F4
Mean Diameter (nm)
Zeta Potential (mV)
Surface Tension (dyne/cm)
155.2 2.9 289.8 3.5 311.4 4.0 378.7 23.1
83.4 3.0 66.5 1.8 53.5 7.0 80.8 1.2
24.6 0.1 24.6 0.4 27.3 0.5 35.1 0.9
Each value represents the mean SD (n ¼ 3).
(F2) produced lower surface charges ( p < 0.05) compared to the standard system (F1). A further reduction in surface charges was observed by increasing the perfluoropentane ratio (F3). No remarkable difference ( p > 0.05) was detected between the zeta potential of formulations with or those without cholesterol (F1 vs. F4). The surface tension of water is about 72 dyne/cm. Introducing perfluorocarbon, oil, and emulsifiers to form PNs reduced the surface tension. As shown in Table 2, PNs with cholesterol (F1–F3) generally showed values of 25–27 dyne/cm. Changes in the oil and perfluoropentane contents did not significantly ( p > 0.05) alter the surface tension. The surface tension increased to 35 dyne/ cm following the exclusion of cholesterol from the PNs (F4, p < 0.05).
Imaging of PNs by AFM and Ultrasound To characterize the microscopic structure of the produced PNs, an AFM technique was applied to observe diverse samples (F3). Figure 1A–D represents the field of vision from wide to narrow. As shown in Figure 1A,B, the dispersion investigated using AFM contained oval- or raisin-shaped particles. A narrow size distribution of PNs was observed, indicating a quite-homogeneous population of particles. The polydispersity index (PDI) determined from the laser scattering method also showed a low value of 0.25–0.28. No significant difference ( p > 0.05) of PDI was observed for all formulations tested. Figure 1C,D shows the structure of one PN particle. This particle had a mean diameter of 330 nm, which was in good agreement with the light scattering results. Figure 1E shows the distribution of particles from F3 in a 3D image. In order to examine the acoustically active feature of PNs developed in this work, ultrasound imaging was used to monitor the dispersion of PNs (F3). PNs in the latex tube, shown in DOI 10.1002/jps
Figure 2A within the frame, were observed to oscillate in response to acoustic pulses. There was no signal when monitoring water was placed in the latex tube (data not shown). The color-coded spots reflected echogenic movement signals of PNs. This observation confirmed the acoustical activity of PNs. As shown in Figure 2B, the echo signals with color-coded spots of particles almost faded out after a 15-min ultrasound application.
Evaporation of PNs Gas loss represents the primary destructive mechanism of microbubbles. The evaporation of PNs was evaluated by incubating them in a 378C oven. As shown in Figure 3, formulations with higher oil and perfluorocarbon contents (F2 and F3) demonstrated more-rapid evaporation than the standard one (F1). However, no significant difference ( p > 0.05) in the weight remaining was detected between water and these formulations (F1–F3) at the end of the experiment (12 h). The exclusion of cholesterol (F4) significantly accelerated ( p < 0.05) the evaporation rate of PNs compared to those with water and the standard system.
In Vitro Apomorphine Stability in Plasma Apomorphine is known to be chemically and biologically unstable. The inclusion of apomorphine in PNs may improve its stability, thus prolonging its half-life. Figure 4 shows the amounts of apomorphine HCl and base remaining after incubation in plasma for 1 and 6 h. Marked degradation of both apomorphine HCl and base in the free form occurred under the reaction conditions. There was no significant difference ( p > 0.05) in the amount of apomorphine remaining after the 1- and 6-h incubations (Fig. 4A). Inclusion in PNs generally produced significant protection ( p < 0.05) for apomorphine HCl except
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Figure 1. Images of atomic force microscopy of perfluorocarbon nanobubbles (F3) showing the particle appearance. (A–D) Represent the field of vision from wide to narrow; and (E) shows the distribution of particles from F3 in a 3D image.
with the F1 formulation at 1 h. The protective abilities of three delivery systems were similar ( p > 0.05). The standard formulation (F1) and the formulation with a high perfluorocarbon loading (F3) protected apomorphine base (Fig. 4B). However, no effect was found for PNs with high oil loading (F2, p > 0.05) compared to the free form.
In Vitro Apomorphine Release For development of PNs loaded with apomorphine, it is important to optimize the ability to release the drug from the particles. The ability of PNs to deliver apomorphine was examined by determining the drug release as shown in
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Figure 2. Ultrasonography of perfluorocarbon nanobubbles (F3). (A) Representative image taken immediately after beginning the ultrasound application (6 MHz); and (B) representative image taken after ultrasound application (6 MHz) for 15 min.
Figure 5. Both the HCl and base forms of apomorphine were incorporated into PNs for this test. The release of apomorphine from double-distilled water was used as the control.
Figure 3. Percentage weight remaining of perfluorocarbon nanobubbles as a function of time in the evaporation experiment at 378C. Each value represents the mean and SD (n ¼ 4). DOI 10.1002/jps
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Figure 4. Percentage of apomorphine remaining in the stability experiment by incubating perfluorocarbon nanobubbles with plasma at 378C for 1 and 6 h. (A) Represents the profiles of apomorphine HCl; and (B) represents the profiles of apomorphine base. Each value is presented as the mean and SD (n ¼ 6).
F4 was excluded from this experiment because of its low stability. As shown in Figure 5A, apomorphine HCl in an aqueous solution demonstrated the highest release. Release from the aqueous solution showed an initial burst, then leveled off after 2 h of administration. All PNs retarded the release of apomorphine HCl, with the formulation containing a high oil content (F2) showing the slowest drug delivery rate. Only 20% of the apomorphine base dose in the aqueous solution was released from the donor as depicted in Figure 5B. Similar to apomorphine HCl, the PNs released apomorphine base in a sustained manner. Discrepancies among the release rates of these three nanoparticle systems were not large for the base form. Ultrasound application of PNs has the potential to deliver apomorphine to target specific areas in the CNS. To explore this novel opportunity, we examined the amount of apomorphine released under the influence of ultrasound application. Plasma was added to the donor phase and mixed
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Figure 5. In vitro release-time profiles of apomorphine across a cellulose membrane from an aqueous solution (control) and perfluorocarbon nanobubbles. (A) Represents the release of apomorphine HCl; and (B) represents the release of apomorphine base. Each value is presented as the mean and SD (n ¼ 4).
with PNs to simulate in vivo conditions, The amounts of apomorphine HCl released in the absence and presence of ultrasound are summarized in Figure 6. There was no or negligible apomorphine detected in the receptor when using the aqueous solution as the vehicle, possibly due to the instability of free apomorphine in plasma. The effects of ultrasound on F1, F2, and F3 were evaluated as shown in Figure 6A–C. Apomorphine HCl release significantly increased ( p < 0.05) after applying ultrasound in all systems. The drug release percentage at the end of the experiment (8 h) by insonation showed enhancements of 1.9-, 3.6-, and 3.9-fold compared to the non-ultrasound group for F1, F2, and F3, respectively. A contrary result was observed for apomorphine base. The application of ultrasound decreased the release of apomorphine base from all PNs (Fig. 7).
Figure 6. Effect of ultrasound at 1 MHz at an intensity of 2.0 W/cm2 for 1 h (with a duty cycle of 50%) on the in vitro release of apomorphine HCl across a cellulose membrane from perfluorocarbon nanobubbles. (A) Represents the standard formulation (F1); (B) represents the formulation with a high oil ratio (F2); and (C) represents the formulation with a high perfluoropentane ratio (F3). A 0.5-mL aliquot of plasma was added to the donor compartment of a Franz diffusion cell. Each value is presented as the mean and SD (n ¼ 4).
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Erythrocyte Hemolysis To use PNs for parenteral administration, rigorous demands on the non-toxicity of the formulations must be imposed. To evaluate the safety of PNs themselves, the hemolytic activity was determined. Percentages of human erythrocytes undergoing hemolysis induced by the PNs are shown in Table 3. The standard PNs (F1) showed a hemolysis percentage of 11%. Adding oil and perfluoropentane in PNs (F2 and F3) led to reductions in hemolysis ( p < 0.05), especially for the system with a high oil ratio.
DISCUSSION
Figure 7. Effect of ultrasound at 1 MHz at an intensity of 2.0 W/cm2 for 1 h (with a duty cycle of 50%) on the in vitro release of apomorphine base across a cellulose membrane from perfluorocarbon nanobubbles. (A) Represents the standard formulation (F1); (B) represents the formulation with a high oil ratio (F2); and (C) represents the formulation with a high perfluoropentane ratio (F3). A 0.5-mL plasma aliquot was added to the donor compartment of a Franz diffusion cell. Each value is presented as the mean and SD (n ¼ 4).
We are attempting to develop an approach, which will permit utilization of apomorphine, which has some disadvantages in clinical practice due to the lack of a suitable drug carrier system. The use of PNs is possibly permit effective formulations and utilization of this potent agent in Parkinson’s disease since ultrasound application may target the drug-loaded PNs to the brain. Improvements in clinical outcomes can be achieved by modifying the physicochemical properties of the nanoparticles, especially for drugs delivered through the BBB.16 The small size of PNs, with substantial populations of 150–380 nm, is an important factor in their efficacy of parenteral administration. A higher oil ratio (F2) and fluorocarbon ratio (F3) led to increases in the size of the particles. The monolayer shell composed of SPC and cholesterol imparts stability to the microbubbles.17 Since the contents of emulsifiers in these systems were the same, the increase in diameter may have resulted from impoverishment of the emulsifiers at the interface with an increasing volume of the dispersed oil and perfluorocarbon phases. Our data also indicate the necessity of cholesterol for PN stability, since larger particle sizes were observed in the absence of cholesterol. Cholesterol was proven to provide a stable and rigid structure Table 3. Hemolysis Percentage (%) After 1 h of Incubation at 378C With Perfluorocarbon Nanoparticles Code F1 F2 F3
Hemolysis (%) 11.18 0.40 2.08 0.29 6.46 1.55
Each value represents the mean SD (n ¼ 4). DOI 10.1002/jps
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when incorporated into phospholipid membranes.18,19 Another observation was that the surface tension significantly increased when cholesterol was excluded from PNs, suggesting that cholesterol enhances the emulsification of PNs which stabilizes the entire system. That is, SPC alone cannot provide sufficient emulsification power for PNs. The SPC used in this study contained 80% PC, which is neutral. Other phospholipids components (20%), such as phosphatidylserine, phosphatidic acid, phosphatidylglycerol, and phosphatidylinositol, were responsible for the negative zeta potential of the PNs. Increments of the oil and perfluorocarbon ratios in these systems contributed to reductions in surface charges. It was found that PNs (F1–F3) showed an inverse relationship between size and zeta potential. The larger total surface area of the smaller particle systems may explain this phenomenon. However, this trend was not observed for nanobubbles without cholesterol (F4). The absence of cholesterol did not significantly reduce the zeta potential, although the particle size greatly increased. This may have been due to this formulation not being a stable and typical system compared to other PNs. Another indication is that the surface charge was not a predominant factor govering the instability of F4 since cholesterol played a negligible role on zeta potential. Regarding the long-term stability, the negative zeta potential of a stable colloidal system should not fall below 30 mV.20,21 As a result, the PNs developed in this study would likely show acceptable stability in normal use. AFM and ultrasound imaging, respectively confirmed the microscopic appearance/distribution and echogenic features of the PNs. Insonation with ultrasound energy can be used to stimulate the transition of the inner perfluoropentane core from a liquid to a gas. As the particles become gas bubbles, the acoustic properties change, and the activity as a cavitation nucleus is increased.7,22 After 15 min of insonation, the echo signals of PNs decreased to a much lower level. This was possibly due to ultrasound disrupting PNs by inducing cavitations after a determined period.8,23 Ideal microbubbles or PNs should be stable enough in both storage conditions and the vascular system with respect to the particles and the incorporated drug molecules. In the present study, the evaporation test and apomorphine stability in plasma were respectively used to examine the stability of the dispersions and loaded drug. The weight loss of PNs in the
evaporation experiment showed that there were no significant differences among the systems with cholesterol and water, suggesting that perfluorocarbon loss by PNs with cholesterol was limited. Perfluoropentane completely evaporated in a 50-min period. A phospholipids/ cholesterol monolayer and oil shell may have adequately protected the inner core from evaporation. PNs quickly evaporated after cholesterol was removed. This again confirms that cholesterol can produce a rigid membrane with SPC to maintain stability. One of the difficulties with apomorphine administration is its inherent instability.3,24 Autooxidation of apomorphine to quinone species is a potentially important factor in apomorphine metabolism.25 Moreover, internal conversion from the R to the S form at physiological pH values is another concern.4 The S form is the therapeutically inactive form. Apomorphine HCl and base in the free form showed comparable stabilities. The results suggest that apomorphine was quickly oxidized in plasma, since no great difference between 1- and 6-h incubation periods was observed. Apomorphine was rather stable when PNs were used as the delivery systems. Hence PNs can effectively prevent the degradation of apomorphine, thus prolonging the residence time in the circulation. The pore size of the permeated membrane used was below 10 nm,26 and free molecules were able to permeate across the membrane. The amounts of apomorphine HCl and base released from the aqueous solution were limited, with 40% and 20% of the drug being released over 8 h, respectively. This may have been due to the use of the in vitro Franz cell. Since a drug is released to the definitive space of the receptor (5.5 mL) and diffusion area (1.13 cm2), drug loading in the receptor compartment is limited. There might no longer be a concentration gradient between the donor and receptor compartments. Nevertheless, this setup is still useful for differentiating the relative release capabilities of various formulations.27,28 The release of free apomorphine HCl was higher than that of the free base form. The lipophilic base form at a dose of 3.6 mM did not completely dissolve in water. Since the compound should be released in a dissolved form, its low solubility retarded its release from the aqueous solution. Incorporation of apomorphine in PNs had slower release profiles compared to the free form, indicating that apomorphine did not diffuse freely
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when entrapped in the particles. The sustained release of an incorporated drug in a delivery system is an important feature quite often correlated with improved pharmacokinetics and efficacy.9,27 The clinical relevance of this is that the frequency of apomorphine administration can be reduced. This would be very beneficial for patients requiring up to 10–15 injections per day.29 It was found that the systems with a high oil or perfluoropentane percentage (F2 and F3) showed lower drug release profiles compared to that with a low percentage (F1) for both the HCl and base forms. This may have been due to the larger particle diameters of the formulations with higher oil and perfluoropentane contents. Drug release is generally slower from systems with larger particles since they have a decreased total surface area where drug diffusion can occur.30,31 Plasma was mixed with PNs in the release experiment with ultrasound application to mimic an actual in vivo status. Plasma is a viscous fluid consisting of about 91% water and 9% other substances such as proteins, ions, nutrients, and waste products. In the presence of plasma, drug release was reduced because of the higher viscosity and longer path lengths for drug diffusion. The decreased drug concentration gradient between the donor and receptor is another mechanism. It is apparent that insonation accelerated the release rate of apomorphine HCl from PNs. As ultrasound pressure waves interact with the particles, they begin to oscillate or resonate. This effect results in particle rupture,32 and abrupt drug release. PNs with a higher oil or perfluorocarbon load produced more-significant apomorphine HCl release enhancement after insonation. The size of the PNs may be important. As microbubbles become larger, they become more echogenic.33,34 It was surprising that apomorphine base exhibited a contrary result compared to the HCl form. Since particles loaded with the HCl and base forms did not show great differences in their physicochemical properties, the different locations of apomorphine HCl and base in the PNs may explain this noticeable discrepancy. Apomorphine HCl is more hydrophilic than apomorphine base. Hydrophilic molecules are difficult to encapsulate into the inner phase of particles composed of oil and perfluorocarbon. However, the slower release of apomorphine HCl from PNs compared to the aqueous solution may indicate that some interactions occurred between the particles and the HCl form. Drugs can be DOI 10.1002/jps
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incorporated into microbubbles in a number of different ways, including binding the drug to the emulsifier monolayer.35 Apomorphine HCl has positively ionized format pH values of <7.2.3 PNs prepared in this study had pH values of 5.6–6.2. Interfacial films with high negative charges may offer affinity to apomorphine HCl. On the other hand, lipophilic drugs are always incorporated into the oil layer of microbubbles.15,36 Non-ionic apomorphine base possibly is such a case. In order to elucidate the location of apomorphine in PNs, liposomes composed of SPC and cholesterol were prepared to examine their mean diameter and encapsulation of apomorphine HCl and base. As shown in Table 4, apomorphine HCl had an encapsulation percentage of 47% in liposomes. Comparable encapsulation percentages of apomorphine HCl and base forms were detected ( p > 0.05). However, entrapment of the base form in liposomes should be analyzed with caution since it did not completely dissolve in this system. The 54% rate of encapsulation of apomorphine base was overestimated, because undissolved molecules were included in the encapsulated drug due to the ultracentrifuge method used in this study. Hence apomorphine HCl may actually show greater entrapment compared to apomorphine base. This is reasonable since the positive apomorphine HCl can interact with the negative phospholipid bilayers due to electrostatic forces. The vesicle size of liposomes loaded with apomorphine HCl was 9-fold greater than that of blank liposomes. It is expected that the HCl form will largely interact with the film, enlarging the volume of the bilayers. Another observation is a great deviation of the size data (931.9 209.8 nm). This may be due to the poor stability of this drug-loaded liposomes and the large variation of the drug-phospholipids interaction among batches. Previous studies 15,34,37,38 suggested that phospholipids-based microbubbles are destroyed by Table 4. The Characterization of SPC Liposomes Loaded With Apomorphine HCl or Apomorphine Base by Mean Diameter and Encapsulation Ratio
Drug None Apomorphine HCl Apomorphine base
Encapsulation (%)
Mean Diameter (nm)
— 46.6 6.5 53.6 10.6
104.2 1.8 931.9 209.8 94.1 1.9
Each value represents the mean SD (n ¼ 3).
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ultrasound, due to rupture and loosening of the membranes. Encapsulated apomorphine HCl in membranes may be subsequently released by insonation. Insonation seemed to be less applicable to the oil phase. Fluorocarbon-filled microbubbles provide enhanced echogenic effects compared to air-filled microbubbles.37 With ultrasonic oscillation, air can diffuse through the shell and quickly dissolve in the surrounding medium because of its low boiling point and high Ostwald coefficient (the velocity with which a gas leaves a bubble). On the contrary, fluorocarbon and oil have much higher boiling points and lower Ostwald coefficients than air. After oscillation, apomorphine base might still be retained in the oil phase for a determined period. As the membrane has already been ruptured by insonation, the release of apomorphine base becomes more difficult since the direct partitioning from the oil to the aqueous phase is much lower in the absence of a phospholipid interface for lipophilic drugs. This results in reduced apomorphine base release from PNs under ultrasound influence. The mechanisms of this effect were not exactly determined in the present study and need to be further explored. Phospholipids are known to cause erythrocyte hemolysis.39 Our results show that the standard PNs (F1) produced a hemolysis percentage of 11%. Hemolysis occurs because of penetration of free phospholipids in the aqueous phase of the emulsions.38 The percentage of hemolysis decreased with an increased oil or perfluorocarbon concentration since most phospholipids resided in the interface and emulsified the inner phase with a larger volume. According to the hemolysis result, PNs developed in this study are generally biologically safe (hemolysis percentage <10%) and do not pose a toxicological risk.
CONCLUSIONS PNs are feasible to be carriers for apomorphine delivery according to the experimental results in the present work. Ultrasound pulses induced marked enhancement of apomorphine HCl release, thus allowing the bioactive drug to be targeted to specific sites such as the brain. However, that was not the case for apomorphine base. This suggests that selecting optimized drugs for PNs is important to achieve the aim of drug targeting. Particles with different ratios of the inner phase, including oil and perfluorocarbon,
were found to greatly influence the physicochemical characteristics, safety, and sensitivity to ultrasound. We also elucidated the importance of cholesterol to the interfacial films to stabilize these systems. The advantages of the prepared PNs warrant further investigations of this approach. In vivo studies are planned in order to evaluate the potential use of apomorphine loaded in PNs as parenteral drug delivery systems.
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