Formation of Nanocarrier Systems by Dense Gas Processing

Aug 27, 2014 - In this work, a novel dense gas technique known as depressurization of an expanded solution into aqueous media (DESAM) was used to prod...
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Formation of Nanocarrier Systems by Dense Gas Processing Chau Chun Beh, Raffaella Mammucari,* and Neil R. Foster* School of Chemical Engineering, University of New South Wales (UNSW), Sydney, New South Wales 2052, Australia

ABSTRACT: Nanocarrier systems, such as liposomes, polymersomes, and micelles, find applications in the delivery of a wide range of compounds, including targeted delivery of pharmaceuticals. Nanocarrier systems have the ability to increase the bioavailability, reduce toxicity, and avoid undesirable interactions of active pharmaceutical ingredients. In this work, a novel dense gas technique known as depressurization of an expanded solution into aqueous media (DESAM) was used to produce different types of nanocarrier systems. The effects of using different types of dense gases and different operating temperatures were investigated. Encapsulation of hydrophilic compounds in the vesicles (liposomes and polymersomes) was also studied. The highest encapsulation efficiencies in liposomes and polymersomes achieved were 10.2 and 9.7%, respectively. The DESAM process was also able to reduce the residual solvent content in the product to 2.2% (v/v), which is significantly lower than the solvent residual levels reported for conventional processing.

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INTRODUCTION Significant developments in the controlled release of therapeutics can be observed over the last few decades: from the most basic delayed release dosages in the 1960s to welldesigned self-regulated delivery systems in the 1990s.7 Controlled release drug delivery systems provide a more controllable rate of uptake of active pharmaceutical ingredients (APIs) by the body and the possibility of prolonging therapeutic action without increasing dosage.8 In conventional pharmaceutical systems, the APIs are released in the body without control. The amount of APIs delivered to the site of action depends upon the capacity of the organ to absorb the APIs. Conventional delivery systems tend to have an initial burst of APIs in the body, followed by the decay of the concentration over time. In contrast, controlled release delivery systems can maintain a constant drug concentration in blood or tissues for an extended period of time. Generally, controlled release delivery systems gradually release the API to maintain optimum concentration levels.9 Vesicles (liposomes and polymersomes) and micelles are nanocarrier systems commonly used for the controlled release of APIs. The nature of each type of nanocarrier system has been described elsewhere.10 They find useful application as carriers for many types of therapeutic compounds, including enzymes, proteins, peptides, and DNA and RNA fragments.11,12 The © 2014 American Chemical Society

nanocarrier systems are designed to maintain API concentrations at the required level and at the targeted location. Hence, they are expected to offer a more controllable rate of uptake of APIs by the body. In addition, over-dosage of APIs can be prevented while providing extended therapeutic action. Liposomes are self-assembled vesicles generated from biocompatible phospholipids with an aqueous volume enclosed within a lipid bilayer membrane. The lipid bilayer membrane is composed of hydrophilic and hydrophobic domains that allow for the encapsulation of both hydrophilic and hydrophobic compounds. Hydrophilic compounds can be included in aqueous domains enclosed in the structure of the vesicles, while hydrophobic compounds can be encapsulated within the lipid bilayer membranes. Vesicles are commonly used as carriers, particularly for APIs and imaging agents. Liposomes are widely used in pharmaceutical applications, especially targeted delivery. The bilayer membrane of liposomes is composed of natural phospholipids, which makes the membrane behave similarly to the endogenous cell membrane and can avoid uptake by the reticuloendothelial system (RES). The RES system is an important component of the immune Received: July 1, 2014 Revised: August 15, 2014 Published: August 27, 2014 11046

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dense gas technique, originally developed to generate liposomes in a single-step process at moderate temperatures and pressures. The process was developed in response to the limitations of the conventional methods for the production of liposomes, with the advantages of products with minimal residual solvent content. In the DESAM process, a dense gas is used to expand an organic solution containing the raw materials. The gas-expanded solution is then released into an aqueous medium via a nozzle. Liposomes are produced as the gas-expanded lipid solution is released into the aqueous medium. In the present study, the scope of DESAM was extended to the production of polymersomes, micelles, and drug-loaded vesicles (liposomes and polymersomes). In addition, the original DESAM process was modified to improve the removal of organic solvent. The present study has extended the application of the DESAM process and investigated the significance of pre-expansion of the organic solution. The effect of pre-expansion on product characteristics has been studied using nitrogen as an alternative to CO2. Nitrogen does not have the ability to expand organic solutions. The encapsulation efficiency of hydrophilic compounds within the vesicles was also investigated. The products were analyzed by transmission electron microscopy (TEM), cryogenic transmission electron microscopy (cryo-TEM), photon correlation spectroscopy (PCS) using dynamic light scattering, and gas chromatography (GC).

system that includes phagocytic cells. Phagocytic cells have the ability to engulf materials that are foreign to the body, such as bacteria and viruses. Therefore, liposomes have long circulation times in the body that can allow API delivery to the targeted sites. The flexibility in manipulating the use of liposomes in targeted delivery has been recognized. The liposomal products that are currently available on the market are described in the published literature.10 Literature also shows that liposomes are still under extensive research because their demand as carrier systems is evidently increased substantially in recent years.10,13 Liposomes are labile aggregates, which limits their applications as drug carriers.14 Polymersomes are similar to liposomes in that they have an enclosed aqueous media in the inner core that allows for encapsulation of hydrophilic compounds, while their hydrophobic membranes can encapsulate hydrophobic compounds but possess better mechanical properties than liposomes. Polymersomes are under extensive research for drug delivery applications because they offer a wider range of chemical and physical properties and are more stable toward degradation than liposomes.12,15 Micelles are different from liposomes and polymersomes because they only have hydrophobic inner cores to encapsulate hydrophobic compounds. Micelles can be composed from either natural compounds or amphiphilic block copolymers. Micelles are formed when a system-specific critical concentration of amphiphilic moieties is reached. Micelles possess single-layer membranes that enable the encapsulation of hydrophobic compounds. A number of different conventional nanocarrier system production techniques have been developed. Most of which have drawbacks, such as complex and time-consuming procedures involving organic solvents, and harsh process conditions that can result in denaturation of the nanocarrier systems forming materials and APIs and poor drug encapsulation efficiency.14 A critical issue is the use of organic solvents required by most of the conventional nanocarrier system production methods because they present occupational and environmental risks. Moreover, removal of organic solvent has to be ensured for pharmaceutical preparations with associated high processing cost for additional purification and waste disposal steps.16 Hence, removal of organic solvent as post-processing of nanocarrier system production is a substantial issue. Poor control of morphology of nanocarrier systems in conventional production methods also provides impetus for the emergence of dense gas technologies as a technological platform for the production of nanocarrier systems with improved morphologies and minimal residual solvent. Dense gas technology has become an alternative production technique that is more environmentally acceptable and economical. Dense gases are ideal processing media because of the properties, such as liquid-like densities, and diffusivities and viscosities intermediate to liquids and gases.10,17−21 Dense gases are fluids in the proximity of their critical point. The solvent strength of a dense gas is proportional to its density. Density is extremely responsive to changes in the temperature and pressure around the critical point. By induction of small fluctuations in the temperature and pressure, it is possible to manipulate the solvation power of a fluid in the vicinity of the critical point.17 A dense gas process, known as the depressurization of an expanded solution into aqueous media or the DESAM process, was developed by Meure et al.1,17 The DESAM process is a

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MATERIALS AND METHODS

Liposome Production. Absolute ethanol [gradient high-performance liquid chromatography (HPLC) grade] was purchased from Scharlau; cholesterol with 99% minimum purity was purchased from Sigma-Aldrich; and 1,2-distearoyl-sn-glycero-3-phosphatidylcholine (DSPC) with 99% minimum purity was purchased from Avanti. All compounds were used as received. Polymersome and Micelle Production. Dichloromethane (DCM) (99% minimum purity) was purchased from Ajax Chemicals. Poly(butadiene)-block-poly(ethylene oxide) (PBD−PEO) with a different number of unit block copolymers was purchased from Polymer Source, Inc. (Canada): (1) PBD36−PEO20 (block unit of PBD/PEO is 36:20) and (2) PBD406−PEO286 (block unit of PBD/ PEO is 406:286). All compounds were used as received. Encapsulation Study. Isoniazid [isonicotinic hydroxide (INH)] with 99% minimum purity was purchased from Sigma-Aldrich and used as received. Phase Transition Study of Raw Materials. A phase behavior study was conducted to investigate suitable operating conditions for the DESAM process. The DESAM process used carbon dioxide (CO2) to expand an organic solution by rapid diffusion of CO2 into the organic solution. The dense gas was used to expand the lipid solution and act as an aerosolization aid upon depressurization in a later step. The expanded organic solution subsequently became supersaturated and triggered the precipitation of solutes. Therefore, it was necessary to identify the threshold pressure point at which precipitation occurred. An experimental rig as illustrated in Figure 1 was commissioned to conduct the phase behavior study. Phase behavior experiments were conducted on organic solutions of the raw materials in a temperature-controlled water bath. A solution of raw materials in a suitable organic solvent was introduced into a highpressure vessel with a viewing window cell (Jerguson sight gauge). After the desired temperature was obtained, the system was pressurized with CO2 by passing it through a coil to allow for temperature equilibration before CO2 entered the Jerguson sight gauge from the bottom. The organic solution expanded when CO2 was fed into the Jerguson sight gauge. The pressure in the system was increased gradually as CO2 was fed to the vessel. When the saturation point of the organic solution was attained, the solution became cloudy 11047

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The gas-expanded solution was then delivered by a nozzle into a precipitation vessel through a pressure gradient. The precipitation vessel contained a water solution at atmospheric pressure and a temperature that was above the glass transition temperature of the nanocarrier system raw materials, preferably above the boiling point of the organic solvent, and below the boiling point of the aqueous phase. Temperatures higher than the transition temperature of the nanocarrier system raw materials were required for better formation of nanocarrier systems. Hence, the operating temperature of the water solution in the precipitation vessel ranged from 50 to 80 °C. The expanded solution was sprayed and dispersed into the aqueous media in the form of fine droplets, which provided a good contact between the compounds. Small and homogeneous nanocarrier systems in suspension were then formed by self-assembly. To encapsulate hydrophilic drug into the forming nanocarrier systems, the hydrophilic compound can be dissolved in the aqueous phase in advance, so that drug-encapsulating nanocarrier systems can be formed by selfassembly. After the formation of the nanocarrier systems, the vessel containing the aqueous phase was then immersed in water at 4 °C, while CO2 was bubbled though the nanocarrier system suspension for 1 h. The flux of CO2 was controlled by a valve and was maintained at an average flow rate of 0.77 L/min, measured at ambient conditions by a wet gas meter. The flux of CO2 was maintained to keep the aqueous phase agitated and to facilitate the elimination of the residual solvent left in the aqueous phase. The whole process was completed in approximately 1.5 h. Encapsulation of Isoniazid. Isoniazid or INH is an antituberculosis drug and was chosen as the model hydrophilic compound in the formation of API-loaded liposomes and polymersomes. Isoniazid is one of the first-line antituberculosis drugs extensively used in treatment and prophylactic programs for tuberculosis.22 A number of publications have reported the encapsulation of INH in liposomes and polymersomes. Liposomes and polymersomes have been successfully used for the delivery of antituberculosis drugs and have demonstrated good chemotherapeutic efficacy in vivo.23−28 Isoniazid was dissolved in deionized water, and then the resulting solution was loaded in the precipitation vessel and heated to the experimental temperature before conducting the DESAM process. The organic solution of vesicle raw materials was subsequently introduced into the aqueous medium via a nozzle. In this way, vesicles

Figure 1. Schematic diagram of the apparatus for the phase behavior study DESAM.

because of the precipitation of the solutes. The saturation pressure where the organic solution starts to become cloudy and precipitate is the threshold pressure. The value of the threshold pressure determined in the phase transition study was used to determine the operating pressure for the DESAM process. Operating the DESAM process at pressures less than the threshold level was to avoid precipitation of solutes when the organic solution was expanded by carbon dioxide, which could then result in materials precipitated in the pressurization vessel and not being delivered to the precipitation vessel. Therefore, nanocarrier system production would not succeed. A schematic diagram of the DESAM process is illustrated in Figure 2. The nanocarrier system raw materials were first dissolved in a suitable organic solvent and placed in a high-pressure vessel, which was referred to as the pressurization vessel. The pressurization vessel was at room temperature. The solution was expanded by introducing compressed carbon dioxide from the bottom of the vessel. Carbon dioxide was sparged through a frit until the pressure reaches 35 bar, which was below the threshold pressure for all of the starting solutions. The system was then allowed to sit for approximately 10 min for equilibration.

Figure 2. Schematic diagram of the DESAM system. 11048

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Table 1. Summary of Experimental Operating Conditions and Resultsa type of nanocarrier material type of dense gas used temperature of water media (°C) operating pressure (bar) type of organic solvent used concentration of DSPCb in ethanol (mg/mL) concentration of cholesterol in ethanol (mg/mL) concentration of PBD−PEO in DCM (mg/mL) morphology observed from TEM imaging average hydrodynamic diameter (nm) residual solvent (%, v/v) figure

liposomes DSPC,b cholesterol CO2 CO2 60 75 35 ethanol 4.7 4.8 1.44

1.32

N2 75

polymersomes PBD36−PEO20c CO2 CO2 40 55 35 dichloromethane

N2 55

polymeric micelles PBD406−PEO286d CO2 CO2 60 55 35 dichloromethane

N2 55

6.22

6.18

6.06

13.32

6.34

13.28

mostly spherical 340 not detected

spherical

mostly spherical 400

mostly spherical 190 not detected

spherical

mostly spherical 230

4.32 1.28

spherical

spherical

spherical

200 6.6

180 2.2 3a, 3b

160 2.3

330 3c, 3d

210 3e, 3f

a

In all experiments, the volumes of organic solution and water solution were 5 and 50 mL, respectively. bDSPC = 1,2-distearoyl-sn-glycero-3phosphatidylcholine (phospholipids). cPBD36−PEO20 = poly(butadiene)36−poly(ethylene oxide)20. dPBD406−PEO286 = poly(butadiene)406− poly(ethylene oxide)286. encapsulating INH within the aqueous phase were produced by selfassembly. Product Characterization. In this study, samples were characterized using two types of electron microscopy to determine their morphology and size: TEM and cryo-TEM. PCS was used to investigate the nanocarrier system size distribution. Encapsulation efficiency was determined gravimetrically with the aid of an ultracentrifugation filtering device. Lastly, GC was used to determine the residual solvent in the product. Characterization of Nanocarrier Morphology. TEM. The nanocarrier systems are transparent under electron microscopy; hence, negative staining was required to give contrast to electrontransparent samples.29 The negative stain material chosen was uranyl acetate, because of its ability to stabilize the membranes of nanocarrier systems by cross-linking molecules and reducing any adverse effects when the sample is dried.1,17,29 TEM (JEOL 1400, 100 kV accelerating voltage) was used with negative staining to investigate the morphology and size of the nanocarrier systems produced. During the sample preparation, a droplet of nanocarrier system suspension was placed on a copper grid, which was previously coated with a thin film of Formvar and allowed to sit for 60 s. The droplet was then dried with filter paper. A droplet of staining agent was subsequently placed on top of the grid and allowed to sit for 30 s and was then removed with filter paper. Before loading the copper grid into the sample holder, the JEOL 1400 TEM instrument was cooled with liquid nitrogen to avoid any evaporation of staining agent that may contaminate the TEM system. Cryo-TEM. The main difference between cryo-TEM and conventional TEM is that cryo-TEM can avoid the staining and drying artifacts that are commonly involved in conventional TEM. Cryo-TEM is able to retain the native state of a sample because of rapid freezing of the sample in liquid ethane and liquid nitrogen that aids in preserving the sample in a frozen hydrated state.30 Thus, cryo-TEM can avoid any undesirable interaction that can occur between the negative staining agent (uranyl acetate) used in conventional TEM and the sample during sample preparation. Any interactions between the sample and the staining agent could potentially lead to unsatisfactory TEM images. However, the freezing procedure used in sample preparation for cryoTEM has to be carried out cautiously to avoid ice formation. Ice formation on a sample can occur easily, leading to imaging difficulty. Cryo-TEM (JEOL 2100) was able to provide information on morphology and size of nanocarrier systems. Before conducting sample preparation, a copper grid was first hydrophilized by glowdischarge treatment. Sample preparation was then conducted in a controlled environment vitrification system by Vitrobot Mark IV supplied by the FEI Company, which was a custom-built chamber. A

controlled environment vitrification system was used to prevent water evaporation from samples by maintaining the humidity of the environment nearly saturated with water. The temperature of the isolated chamber where the sample preparation took place was set at 22.0 °C. A volume of 5 μL of sample was placed on the copper grid and then dried by a filter paper, which resulted in the formation of a thin film on the grid. The copper grid was subsequently rapidly vitrified with liquid ethane at its melting temperature, which is approximately at −183 °C. The remaining liquid ethane on the grid was dried with filter paper while remaining close to the liquid nitrogen environment to avoid any formation of ice. The grid was then stored in liquid nitrogen until it was loaded into the JEOL 2100 instrument.30,31 Nanocarrier Particle Size Distribution. PCS was used to examine the particle size distribution of the population of nanocarrier systems. Varying laser light intensity from scattering can be measured as a result of Brownian motion of particles in suspension. Larger particles have lower intensity fluctuation because larger particles move slower than smaller particles. The PCS instrument is able to measure the particle size range between 3 nm and 3 μm. The PCS measures the hydrodynamic diameter size of particles based on spherical particle correlation. Nanocarrier system sample suspensions were placed in a disposable polypropylene cuvette. Each sample was measured over 5 runs (where each run was completed in 30 s) at room temperature (25 °C) with a dust cutoff of 30%. Encapsulation Efficiency Measurements. The encapsulation efficiency of the model hydrophilic compound, INH, within the vesicles was determined gravimetrically. The unencapsulated free INH was separated from the vesicle suspension by centrifugation through a membrane filter with a 10 kDa cutoff. The centrifugal tube device was used as supplied by the Millipore Company.32 Studies were conducted on the period of time and centrifugation speed required for the separation. The study was essential to prevent INH encapsulated within the vesicles from leaking because of the breakage of vesicles. After centrifugation, the filtrate left in the inner section of the filter tube consisted of vesicles encapsulated with INH, while the fluid collected in the outer section of the filter tube was the remaining bulk solution with the unencapsulated INH. The centrifugation process was operated at 12000g for 30 min. The encapsulation efficiency of INH in vesicles can be calculated using the weight of filtrate divided by the total weight of filtrate and concentrate in a centrifugal tube. The assumptions underlying this calculation were as follows: (1) The concentration of INH in the volume entrapped by vesicles was the same as the concentration of INH in the bulk of the suspension, because the vesicles self-assemble in the INH solution at one concentration level. (2) The volume of vesicles was approximately the volume of the aqueous domain entrapped in them. Observation 11049

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can be made from the TEM images that the membranes of vesicles were extremely thin; thus, their contribution to the overall volume of the vesicles was negligible. (3) All fractions have the same density. Considering the small amount of polymer or phospholipids used in each experiment and that any difference in density would result from the presence of such compounds, the assumption was very realistic.

encapsulation efficiency of INH within vesicles encapsulated INH in suspension = × 100% total INH in suspension Characterization of Product Residual Solvent. The residual solvent in products was determined by GC using a Shimadzu GC-2010 with a flame ionization detector (FID). The GC column used was polyethylene glycol (SGE, BP20, 25 m length, 0.53 mm inner diameter, and 1 μm film thickness). Helium was used as the carrier gas, while hydrogen and air were supplied to the detector. A solid-phase extraction method was used in removing the solutes before injecting the solution into GC for residual solvent analysis. Triton X-100 was added to the nanocarrier system product suspensions to break the nanocarrier systems. The suspension was then filtered through Maxi-Clean cartridges with C8 sorbent (supplied by the Millipore Company) to remove the solutes, which were phospholipids and cholesterol for liposomes or block copolymers for polymersomes and micelles. Before filtration was carried out, the cartridges were conditioned by methanol to activate the sorbent ligands and deionized water to equilibrate or wash the sorbent bed. The first few milliliters of solution were passed through the filter before collection for GC analysis.

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RESULTS A summary of the experimental operating conditions and results is listed in Table 1. The effect of using different processing dense gases was also investigated. The effect of working at different operating temperatures was examined. The encapsulation study using INH as a model compound is discussed in the last section of this paper. Formation of Nanocarrier Systems. The morphology of nanocarrier systems produced by DESAM with CO2 was analyzed by TEM and cryo-TEM. The results are illustrated in Figure 3. The liposomes and polymersomes from PBD36− PEO20 (shown in panels b and d of Figure 3, respectively) have bilayer membranes, while polymeric micelles from PBD406− PEO286 have single-layer membranes (Figure 3f), as illustrated by the cryo-TEM images. The results are consistent with published data.13,31,33−36 The nanocarrier systems produced were mostly spherical in shape. Liposomes also presented a small number of rod and coffee bean shapes under the TEM imaging (Figure 3a). The images obtained were very similar to the liposomes produced by Meure et al.17 that described rod-shaped or coffee-beanshaped liposomes as collapsed vesicles. The polymersomes produced from PBD36−PEO20 and the polymeric micelles from PBD406−PEO286 have spherical bilayer and spherical singlelayer structures, as illustrated in panels d and f of Figure 3, respectively. Results were consistent with the findings by Jain and Bates.13 Liposomes and polymersomes in cryo-TEM images appear to be larger than in TEM images (Figure 3). This could be due to differences in sample preparation for the two microscopy techniques. TEM was conducted applying the conventional airdry negative technique. Cryo-TEM, instead, requires frozenhydrated/vitrified samples. Hence, variations in sample appearance and size can be related to the different moisture contents of the specimens.

Figure 3. (a) TEM and (b) cryo-TEM images of liposomes produced at 35 bar and 75 °C, (c) TEM and (d) cryo-TEM images of polymersomes produced at 35 bar and 55 °C, and (e) TEM and (f) cryo-TEM images of micelles produced at 35 bar and 55 °C.

The average hydrodynamic diameters for the different nanocarrier systems were measured by PCS. A small difference can be observed between the results from PCS and TEM/cryoTEM. If vesicles with sizes close to the lower detection limit of the instrument are present in the samples, PCS may show a deceptive size distribution.2 The data obtained from PCS therefore provides accurate results for small-particle analysis only if they are present in large populations with homogeneous dispersion. Analysis from TEM/cryo-TEM indicated that liposomes were within a size range of 50−250 nm with a prevalence of 100 nm, while PCS measured an average hydrodynamic diameter of 180 nm. PCS measures the average hydrodynamic diameters of nanoparticles in suspension based on their Brownian motion (hydrodynamic diameter). Thin layers of solvent can adhere to the surface of suspended nanoparticles, affecting their Brownian motion and the estimate of the hydrodynamic diameter. TEM and cryo-TEM generate images of solid samples and allow for the estimation of the projected area diameter. In this work, hydrodynamic diameter values measured by PCS are higher than diameters estimated by TEM. The occurrence can be ascribed to a combination of two factors: the solvation of the hydrophilic segments of amphiphilic molecules in the solution37−41 and aggregation of nanocarriers in suspension. The residual ethanol solvent measured by GC in liposome samples was 2.2% (v/v), while the polymersome and micelle samples had undetectable levels of residual dichloromethane. Hence, the DESAM process can produce nanocarrier systems 11050

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nanocarrier systems.17 The aim of having smaller nanocarrier systems was to increase their circulation time in the body when delivering drugs by avoiding uptake by the RES in the body. Effect of the Operating Temperature. The effect of the temperature of the aqueous media on the formation of nanocarrier systems by the DESAM process was investigated using CO2 as the processing aid. The formation of liposomes was conducted at 60 and 75 °C; the formation of polymersomes was conducted at 40 and 55 °C; and the formation of polymeric micelles was conducted at 55 and 60 °C. The range of the temperature was selected on the basis of the phase transition temperatures of the raw materials. The average hydrodynamic diameter of each nanocarrier produced at different operating temperatures is presented in Figure 5.

with relatively low residual solvent. In contrast, nanocarrier systems that were prepared by other manufacturing methods, such as the supercritical liposome method by Frederiksen et al., contained residual ethanol levels between 14 and 17% (v/v).42 Effect of the Type of Dense Gases Used. The effect of using different types of dense gases was investigated using carbon dioxide (CO2) and nitrogen (N2). Carbon dioxide has the ability to expand the solution of raw materials of nanocarrier systems (i.e., phospholipids and block copolymers) before mixing with the aqueous phase. Nitrogen, on the other hand, does not have the characteristic of expanding organic solutions. Rather, N2 is used as a process aid to deliver the nanocarrier raw materials into the aqueous media through a pressure gradient and to ensure mixing. Comparison of results between the use of CO2 and N2 helps elucidate the significance of pre-expanding the organic solutions in the DESAM process. The average hydrodynamic diameter of each type of nanocarrier system is shown in Figure 4.

Figure 5. Average hydrodynamic diameter of nanocarrier systems by DESAM with CO2 at different operating temperatures.

The effect of increments in the operating temperature is consistent for all of the samples: at a higher operating temperature, the nanocarrier systems produced had slightly lower average hydrodynamic diameter, which is consistent with the literature.38,43−45 The trend could be due to many factors, such as softening of the hydrogen bonding of water molecules to the amphiphilic molecule hydrophilic segment and selfassembly of the hydrophobic segment that results in a lower hydrodynamic diameter.43,44 Overall, the operating temperatures did not affect the shapes of nanocarrier systems produced and had moderate effects on their sizes. Residual solvent was below the detection limit for polymersomes and polymer micelles for all experimental conditions. However, 2.2% (v/v) of ethanol was detected in liposomes produced at 75 °C, while 6.6% (v/v) of ethanol was detected in liposomes produced at 60 °C. Encapsulation of the Hydrophilic Compound (Isoniazid). The encapsulation of isoniazid in the aqueous medium of liposomes and polymersomes was investigated using the DESAM process with CO2 as the process fluid. The vesicles encapsulating INH were produced at 35 bar. The operating temperature was 60 °C for the formation of liposomes and 55 °C for the formation of polymersomes. Each experiment was repeated 3 times. The presence of the model compound did not affect the morphology of the product. The results are tabulated in Tables 2 and 3. The encapsulation efficiency of isoniazid in liposomes at 60 °C was 10%. The polymersomes encapsulating isoniazid produced at an operating temperature of 55 °C achieved an

Figure 4. Average hydrodynamic diameter of nanocarrier systems by DESAM using CO2 and N2.

Polymersomes (PBD36−PEO20) produced using N2 had higher average hydrodynamic diameter measured by PCS than samples produced by CO2, with the difference in average hydrodynamic diameter of particles being approximately 18%. However, processing with CO2 or N2 yielded similar results in PCS measurement of the liposomes and micelles produced, as illustrated in Figure 4. The difference in average hydrodynamic diameter of liposomes and micelles produced using CO2 and N2 was approximately 10%. There was no dichloromethane residual solvent detected in polymersomes and polymeric micelles produced using both CO2 and N2. However, there was 2.2% (v/v) of ethanol detected in liposomes produced using CO2, while 2.3% (v/v) of ethanol was detected in liposomes produced with N2. In contrast to N2, CO2 is able to expand the lipid organic solutions and may act as an aerosolization aid during depressurization, where a spray of fine droplets or particles is formed, which can assist with the dispersion of lipid organic solutions in the aqueous media, hence, a better interaction between the components. This, in turn, can lead to a formation of small and homogeneous liposomes in suspension.17 Polymersomes and polymeric micelles produced with CO2 have a smaller particle size from TEM images and PCS measurements. Smaller nanocarrier systems have lower probability to absorb opsonins because of their high curvature radius in comparison to larger 11051

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Table 2. Summary of Liposomes Encapsulating Isoniazid Samples, with Each Concentration of Raw Material Involveda

a

liposome sample

cholesterol concentration in ethanol (mg/mL)

phospholipid concentration in ethanol (mg/mL)

isoniazid concentration in water (mg/mL)

encapsulation efficiency (%)

1 2 3

1.32 1.20 1.32

4.18 4.12 4.16

1.03 1.06 1.01

10.2 10.1 10.2

The operating temperature was 60 °C.

Table 3. Encapsulation Efficiency of Isoniazid in Polymersomes Indicating the Concentration of Raw Material Involveda

a

polymersome sample

PBD−PEO concentration in DCM, (mg/mL)

isoniazid concentration in water (mg/mL)

polymer/isoniazid mass ratio

encapsulation efficiency (%)

1 2 3

6.62 6.56 6.74

1.03 1.04 1.07

0.645 0.630 0.632

9.7 9.2 8.9

The operating temperature was 55 °C.

systems can perform more effectively when the diameter is less than 200 nm and have hydrophilic surfaces, which are surfaces with neutral or weak negative overall charge, to avoid the uptake by the RES.12 Therefore, the DESAM process produced nanocarrier systems that are suitable as drug carriers in pharmaceutical applications. The DESAM technique is a simple and fast method of producing different types of nanocarrier systems, such as vesicles (liposomes and polymersomes) and polymeric micelles in suspension with low residual solvent levels. The results obtained showed that the DESAM process can achieve similar encapsulation efficiencies for both liposomes and polymersomes to those achieved with conventional methods. Furthermore, the DESAM process has a major advantage compared to the conventional methods because it manages to reduce the residual solvent content in the product to 2.2% (v/ v), which is significantly lower than those reported for conventional processing.2−6,17

average encapsulation efficiency of about 9%. The results obtained in the present study show that both liposomes and polymersomes encapsulating a hydrophilic compound produced by the DESAM process have similar encapsulation efficiencies as obtained by the conventional manufacturing method used by Pandey and co-workers. The encapsulation efficiencies achieved by Pandey and co-workers were between 8 and 12%.46 The average hydrodynamic diameters of empty vesicles (liposomes and polymersomes) and the vesicles encapsulating isoniazid are compared in a bar chart (Figure 6). The incorporation of the hydrophilic guest compound had no significant effect on the hydrodynamic diameter of vesicles.

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AUTHOR INFORMATION

Corresponding Authors

*Telephone: +61-2-9385-5575. Fax: +61-2-93855966. E-mail: [email protected]. *Telephone: +61-2-9385-4341. Fax: +61-2-93855966. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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REFERENCES

(1) Meure, L. A.; Knott, R.; Foster, N. R.; Dehghani, F. The depressurization of an expanded solution into aqueous media for the bulk production of liposomes. Langmuir 2009, 25 (1), 326−337. (2) Lasch, J.; Weissig, V.; Brandl, M., Preparation of liposomes. In Liposomes: A Practical Approach, 2nd ed.; Torchilin, V. P., Weissig, V., Eds.; Oxford University Press: Oxford, U.K., 2003; pp 3−29. (3) New, R. C. C. Preparation of liposomes. In Liposomes: A Practical Approach; New, R. C. C., Ed.; IRL Press: Oxford, U.K., 1990; pp 33− 104. (4) Deamer, D. W. Preparation and properties of ether injection liposomes. Ann. N. Y. Acad. Sci. 1978, 308, 250−258. (5) Deamer, D.; Bangham, A. D. Large volume liposomes by an ether vaporization method. Biochim. Biophys. Acta, Biomembr. 1976, 443 (3), 629−634. (6) Szoka, F., Jr.; Papahadjopoulos, D. Procedure for preparation of liposomes with large internal aqueous space and high capture by reverse-phase evaporation. Proc. Natl. Acad. Sci. U. S. A. 1978, 75 (9), 4194−4198.

Figure 6. Comparison of average hydrodynamic diameters of both empty vesicles and isoniazid-encapsulated vesicles produced by DESAM with CO2 (liposomes were produced at 60 °C, while polymersomes were produced at 55 °C).

It has been widely reported47,48,49 that the encapsulation efficiency can be improved by decreasing the polymer/drug mass ratio. The ratio may be varied by either decreasing the amount of block copolymer or increasing the amount of isoniazid. Further investigation on this issue is warranted.

■

CONCLUSION The DESAM process successfully produced nanocarrier systems with diameters smaller than 200 nm and with hydrophilic surfaces. According to Svenson, nanocarrier 11052

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