Formation of Nano-carriers by the Depressurization of Expanded Solution into an Aqueous Media (DESAM)

Transcription

1 Formation of Nano-carriers by the Depressurization of Expanded Solution into an Aqueous Media (DESAM) Chau Chun Beh, Raffaella Mammucari, Neil Russell Foster* School of Chemical Engineering, University of New South Wales, NSW 2052, Australia *Author to whom correspondence should be addressed; Tel: ; Fax: s: n.foster@unsw.edu.au (N.R. Foster) Abstract A novel dense gas processing platform, known as DESAM, was developed to produce nanocarriers such as micelles and vesicles (liposomes and polymersomes) more effectively than conventional methods and with minimal residual solvent. The DESAM process is a fast, one-step process that can overcome the limitations of most of the conventional processing methods, which include time-consuming procedures and harsh conditions that can degrade the nano-carriers and be detrimental to encapsulation efficiency. Introduction Nano-carriers such as liposomes, polymersomes and micelles find many useful applications, particularly in recent years. Liposomes are self-assembled vesicles generated from natural and biocompatible phospholipids (as shown in Figure 1 (Bioteach, 2000)), which consist of a hydrophilic head and a hydrophobic tail with an aqueous volume enclosed within a lipid bilayer membrane (as shown in Figure 2 (Bioteach, 2000)). The formation of a bilayer membrane can help decrease unfavorable interactions between the bulk aqueous phase and the long hydrocarbon fatty acid chains from the phospholipids. The lipid bilayer membrane is composed of hydrophilic and hydrophobic domains. Hydrophilic compounds can be included in the aqueous domains enclosed in the structure of vesicles, while hydrophobic compounds can be encapsulated within the lipid bilayer membrane as shown in Figure 3 (Bioteach, 2000). Hence, they can be used as carriers for a wide range of molecules including active pharmaceutical ingredients (APIs) and imaging agents. However, compared to polymersomes, liposomes have limited applications as drug carriers because of shorter half-lives in vivo, and difficulties in achieving high drug encapsulation efficiency without involving covalent chemical modification (Discher and Ahmed, 2006). Selfassembled vesicles generated from synthetic block copolymers (polymersomes) can be prepared with enhanced stability compared with conventional liposomal formations. Polymersomes offer a wider range of chemical and physical properties, and are stable toward degradation over longer timescales (Ghoroghchian et al., 2006). The formation of micelles occurs when a system-specific critical concentration of amphiphilic moieties is reached. The geometry of the constituent monomers, intermolecular interactions, and the conditions of the bulk solution such as concentration, ionic strength, ph, and temperature all contribute to determine the size and shape of the micelles (Svenson, 2005). Micelles have singlelayer membranes, which enable the encapsulation of hydrophobic compounds as shown in Figure 4 (Bioteach, 2000).

2 Many different conventional nano-carrier production techniques have been used over the past decades. However, most of the conventional methods have drawbacks such as complex and time consuming procedures involving organic solvents, and harsh conditions that can result in denaturation of the nano-carrier raw materials and drugs, and poor drug encapsulation efficiency (Discher and Ahmed, 2006). Figures Descriptions Figure 1: Single amphiphilic/phospholipids molecule (Bioteach, 2000) Figure 2: Amphiphilic/phospholipids molecules forming bilayer membrane (Bioteach, 2000) Figure 3: A bilayer vesicle (liposomes/polymersomes) encapsulating both hydrophilic and hydrophobic compounds (Bioteach, 2000) Figure 4: A micelle encapsulating hydrophobic compounds (Bioteach, 2000) The use of organic solvents as required by most of the conventional nano-carrier production methods presents occupational and environmental issues and is not ideal for pharmaceutical applications. Therefore, removal of organic solvent would be ideal for pharmaceutical preparations (Kunastitchai et al., 2006). However, the processing costs are high for additional purification and waste disposal steps. The scale of processing cost is also dependent on the unit size of the production plant. Hence, removal of organic solvent as post-processing of nano- 2

3 carriers is a substantial issue. Poor control of morphology of nano-carriers in conventional methods also explains the emergence of dense gas technologies for producing nano-carriers with better control over morphologies and minimal residual solvent. Dense gas technology can provide environmentally acceptable and economic methods for producing nano-carriers. Dense gases are ideal for many different processes due to their properties such as liquid-like densities, and diffusivities and viscosities intermediate to liquids and gases. The solvent strength of a dense gas is proportional to its density. Density is extremely responsive to changes in temperature and pressure around the critical point. By inducing small fluctuations in temperature and pressure, it is possible to manipulate the solvation power of a fluid in the vicinity of the critical point. Dense gas techniques for the production of vesicles provide the potential to reduce, or even eliminate, the amount of organic solvent required by conventional methods; consequently they are well suited to pharmaceutical applications. Dense gas processing can also offer sterile operating conditions and the potential for one-step production processes, which is convenient in transferring the technology to larger scale operations. Carbon dioxide is often used as an environmentally friendly replacement for organic solvents. It is non-toxic, non-flammable, non-corrosive, environmentally acceptable, economical, and has easily accessible critical temperature and pressure. Carbon dioxide has a low critical temperature of approximately 31.1 C, and a critical pressure of 73.8bar. The Depressurization of an Expanded Solution into Aqueous Media (DESAM) is a dense gas technique designed to generate nano-carriers in a single-step process. In the DESAM process, a dense gas is used to expand a liquid solution and then the gas expanded liquid is released into an aqueous medium via a nozzle. Nano-carriers are produced as the gas expanded lipid solution is released into the aqueous medium. The experimental aspects and principles of DESAM are discussed in later sections. Aim of Study The aim of the study was to produce nano-carriers, which include micelles and vesicles such as liposomes and polymersomes by a novel process developed by the Supercritical Fluids Research Group at UNSW. Encapsulation efficiency of hydrophilic compounds within the vesicles was also investigated. The suspension product was analyzed by Transmission Electron Microscopy (TEM), Cryogenic Transmission Electron Microscopy (Cryo-TEM), Photon Correlation Spectroscopy (PCS) using dynamic light scattering, Ultraviolet-visible Spectroscopy (UV-Vis), and Gas Chromatography (GC). Depressurization of Expanded Solution into an Aqueous Media (DESAM) The DESAM process is used to produce nano-carriers, starting from expanding a solution of nano-carrier raw materials in liquid solvent by a dense gas, and then releasing and depressurizing the expanded solvent into an aqueous media via a nozzle. The raw materials of liposomes are phospholipids and cholesterol, whilst the raw material of polymersomes and polymer micelles is poly(butadiene)-block-poly(ethylene oxide) (PBd-PEO) with different number of block units of monomers, which will be discussed in later section. The 3

4 nano-carrier raw materials are first dissolved in a suitable organic solvent and expanded in a pressurization vessel well below the supersaturation point where the precipitation of solutes would occur. The expanded mixture of solutes, conventional solvent and dense gas is allowed to sit in the pressurization vessel for approximately 10 minutes to ensure the attainment of equilibrium. The mixture is then released by a pressure gradient into a precipitation vessel that is filled with aqueous media (deionised water) at a temperature between the glass transition temperature of nano-carriers raw materials and the boiling point of the aqueous medium. The dense gas is used to expand the nano-carriers raw materials solution and assist in homogeneous mixing during depressurization. The expanded solution is sprayed and dispersed into the aqueous media in the form of fine droplets, which provides a better interaction between the compounds. Small and homogeneous nano-carriers in suspension are then formed by self-assembly. The aqueous medium is usually deionised water or a water solution of a hydrophilic guest compound. Hence, when the vesicles form, the hydrophilic compounds can be entrapped within the aqueous space in the vesicles. The removal of residual solvent from the vesicle suspension generated in the precipitation vessel is achieved by a flow of dense gas through the suspension. Materials Industrial grade carbon dioxide (CO 2 ) gas with minimum purity of 99.5% was purchased from COREGAS in Australia. Liposome formation: Absolute Ethanol (gradient HPLC grade) was purchased from Scharlau and used as received. Cholesterol 99+% was purchased from Aldrich Chemical Company, Inc. and used as received. 1,2-distearoyl-sn-glycero-3-phosphatidylcholine (Phospholipids) was purchased from Avanti and used as received. Polymersome and micelle formation: Dichloromethane (DCM) was purchased from Ajax Chemicals and used as received. Poly(butadiene)-block-poly(ethylene oxide) was purchased from Polymer Source, Inc. and used as received. Block copolymers with different numbers of monomers per block were purchased as indicated below: Block copolymer 1: Monomers in butadiene block unit: 36 Monomers Ethylene Oxide Block Unit: 20. Block copolymer 2: Monomers in butadiene block unit: 406 Monomers Ethylene Oxide Block Unit:

5 Methods Figure 5: Schematic diagram of the DESAM rig A solution of nano-carrier raw materials is placed in a high pressure vessel (pressurization vessel). The solution is expanded by introducing compressed carbon dioxide from the bottom of the vessel. Carbon dioxide is sparged through a frit until the pressure reaches 35 bars. The expansion of organic solutions by carbon dioxide can trigger the precipitation of solutes; hence preliminary studies have been conducted to identify the threshold pressure to avoid the precipitation of solutes in the pressurization vessel. Materials precipitated in the pressurization vessel would not be delivered to the precipitation vessel and, therefore, they would not participate in nano-carrier production. The gas expanded solution is then delivered through a nozzle into the precipitation vessel by a pressure gradient. The precipitation vessel contains a water solution heated to a temperature that is above the transition temperature of the nano-carriers raw materials below the boiling point (between 50 C and 80 C). The aqueous phase is cooled by immersion of the precipitation vessel in water at around 4 C whilst CO 2 is bubbled through the nano-carrier suspension for an hour. The flux of CO 2 is controlled by a valve and kept at an average flowrate of 773.3L/min (measured at ambient conditions by the wet gas meter). A flux of carbon dioxide is maintained between the vessels to keep the aqueous phase agitated and to facilitate the elimination of the solvent left in the aqueous phase. The whole process is completed in approximately 1.5 hours. Hydrophilic Compound (Isoniazid) Encapsulation Isoniazid (INH) is an anti-tuberculosis drug. It was chosen as a model encapsulating hydrophilic compound in the liposomes and polymersomes vesicles. Aqueous solutions of INH were loaded in the precipitation vessel and heated up to the experimental temperature. 5

6 Analysis of Product Transmission Electron Microscopy (TEM) Transmission Electron Microscopy (JEOL 1400, 100kV accelerating voltage) was used with negative staining to investigate the morphology and size of the nano-carriers produced. The nano-carriers are transparent under the electron microscope since the components consist of low weight atoms, such as carbon, hydrogen and oxygen. Hence, negative staining was required to give contrast to electron-transparent samples. Providing a bright area with dark stains as the background under the microscope gives the name negative staining. The stain selected was uranyl acetate, due to its ability to stabilize the membranes of nano-carriers by cross-linking molecules and reducing any adverse effect when the sample is dried. Cryogenic-Transmission Electron Microscopy (Cryo-TEM) Cryogenic-Transmission Electron Microscopy (JEOL 2100) was able to provide information on morphology and size of nano-carriers. Cryo-TEM is conducted on samples quickly frozen; the embedded ice confers high contrast in the samples. Care must be taken in sample preparation to avoid ice formation on the sample that could result in imaging difficulty. Due to its ability to freeze the samples, therefore preserving their original state, Cryo-TEM can avoid the undesirable interactions that can occur during negative staining in TEM. Undesired stain-product interactions can result in unsatisfactory imaging. Therefore, Cryo-TEM is used to confirm the morphology of nano-carriers. Photon Correlation Spectroscopy (PCS) Photon Correlation Spectroscopy (Brookhaven ZetaPlus) is able to measure particle size range between 3nm and 3µm. Each sample was tested at room temperature. Ultraviolet-Visible Spectroscopy (UV-Vis) The model hydrophilic compound, isoniazid (INH) has an UV absorbance; hence, the encapsulation efficiency of INH within the vesicles can be determined by UV-Vis. The unencapsulated free INH in the vesicle suspension was separated by filtration through 10kDa membrane filters. Preliminary studies were conducted to ensure that encapsulated INH was not extracted. The centrifugation process was conducted at 12,000 g for 6 minutes. The total amount of encapsulated INH within the vesicles was calculated by subtracting the un-encapsulated INH in suspension (u) from the total amount of INH used in the experiment (W). Therefore, Gas Chromatography (GC) The residual solvent in the product suspension was investigated by gas chromatography using a Shimadzu GC-2010, equipped with a flame ionization detector (FID). The GC column used was polyethylene glycol (SGE, BP20, 25m length, 0.53mm i.d., 1µm film thickness). Solid phase extraction was used to remove the solutes before injecting the solution into GC for residual solvent analysis. Triton X-100 was added to the nano-carrier product suspension to break the membranes of vesicles or micelles. The solutions were then filtered through Maxi- 6

7 C.C. Beh, R. Mammucari, N.R. Foster Clean cartridges with C8 sorbent to remove the solutes. Before filtration was carried out, the cartridges were conditioned by methanol to activate the sorbent ligands, followed by deionised water to equilibrate or wash the sorbent bed. The first few ml of solution were purged through the filter before collection for GC analysis. Results The results were tabulated as follow: Sample TEM Image Liposomes produced by the DESAM at 35bar, 75 C Cryo-TEM Image Figure 7 Figure 6 Scale bar: 200nm Average hydrodynamic diameter measured by PCS: 180.2nm Average encapsulation efficiency of isoniazid: ± 0.58 % Residual Solvent volume fraction measured by GC: 2.21 ± 0.06 % v/v Ethanol Polymersomes produced by the DESAM at 35bar, 55 C Figure 9 Figure 8 Average hydrodynamic diameter measured by PCS: 291.9nm Average encapsulation efficiency of isoniazid: ± 1.23 % Residual Solvent volume fraction measured by GC: No residual solvent was detected. 7

8 Polymer micelles produced by the DESAM at 35bar, 55 C Figure 10 Figure 11 Average hydrodynamic diameter measured by PCS: 203.0nm Residual Solvent volume fraction measured by GC: No residual solvent was detected. Both TEM and Cryo-TEM were used to investigate the morphology of nano-carriers produced by the DESAM process. Cryo-TEM images confirmed the morphology of the nano-carriers. The vesicles (liposomes and polymersomes as shown in Figure 7 and Figure 9 respectively) have bilayer membranes, while polymer micelles have single layer membranes (Figure 11). The nanocarriers produced are spherical in shape. Liposomes samples presented some rod or coffee beanshaped formulations (Figure 6), which were very likely to be collapsed vesicles. The average hydrodynamic diameters for different nano-carriers were measured by PCS. A small difference can be observed between the results from PCS and TEM/Cryo-TEM. If small vesicles are present in the samples that are close to the lower detection limit of the instrument, PCS may show a deceptive size distribution (Lasch et al., 2003). Photon Correlation Spectroscopy therefore provides accurate results for small vesicles analysis only if they are present in large populations. For instance, the liposomes from TEM/Cryo-TEM were observed to fall within the size range of 50 to 250nm with a prevalence of 100nm, while PCS measured an average hydrodynamic diameter of 180.2nm. The residual solvent volume fractions measured by GC for each nano-carrier sample were considered to be low, or not detected, compared to other conventional processes. Both liposomes and polymersomes have been proven to successfully encapsulate the model hydrophilic compound (isoniazid) with relatively high encapsulation efficiencies, which are ± 0.58 % and ± 1.23 % respectively. In comparison, the conventional method of producing liposomes with INH encapsulation achieved a range of encapsulation efficiency between 8 % and 12 % (Pandey et al., 2004). Conclusion The DESAM technique is a non-complicated and fast method of producing nano-carriers, including vesicles such as liposomes and polymersomes, and polymer micelles in suspension with considerably low residual solvent volume fraction. 8

9 Reference BIOTEACH Use of lipid-encapsulated drugs for targeting cancer cells [Online]. Available: [Accessed 18 January 2010]. DISCHER, D. E. & AHMED, F Polymersomes. Annual Review of Biomedical Engineering, 8, GHOROGHCHIAN, P. P., LI, G., LEVINE, D. H., DAVIS, K. P., BATES, F. S., HAMMER, D. A. & THERIEN, M. J Bioresorbable vesicles formed through spontaneous selfassembly of amphiphilic poly(ethylene oxide)-block-polycaprolactone. Macromolecules, 39, KUNASTITCHAI, S., PICHERT, L., SARISUTA, N. & MÜLLER, B. W Application of aerosol solvent extraction system (ASES) process for preparation of liposomes in a dry and reconstitutable form. International Journal of Pharmaceutics, 316, LASCH, J., WEISSIG, V. & BRANDL, M Preparation of liposomes. In: TORCHILIN, V. W., V. (ed.) Liposomes: A practical approach. 2 ed.: Oxford University Press, PANDEY, R., SHARMA, S. & KHULLER, G. K Liposome-based antitubercular drug therapy in a guinea pig model of tuberculosis. International Journal of Antimicrobial Agents, 23, SVENSON, S Advances in particulate polymeric drug delivery. In: SVENSON, S. (ed.) Polymeric drug delivery I: particulate drug carriers. American Chemical Society, Washington, DC,