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Process intensification: Nano-carrier formation by a continuous dense gas process
Accepted Manuscript Process Intensification: Nano-Carrier Formation by a Continuous Dense Gas Process Chau Chun Beh, Raffaella Mammucari, Neil R. Foster PII: DOI: Reference:
S1385-8947(14)01695-7 http://dx.doi.org/10.1016/j.cej.2014.12.072 CEJ 13073
To appear in:
Chemical Engineering Journal
Received Date: Revised Date: Accepted Date:
18 August 2014 8 December 2014 18 December 2014
Please cite this article as: C.C. Beh, R. Mammucari, N.R. Foster, Process Intensification: Nano-Carrier Formation by a Continuous Dense Gas Process, Chemical Engineering Journal (2015), doi: http://dx.doi.org/10.1016/j.cej. 2014.12.072
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Process Intensification: Nano-Carrier Formation by a Continuous Dense Gas Process Chau Chun Beh, Raffaella Mammucari*, Neil R. Foster* School of Chemical Engineering, UNSW Australia *Authors to whom correspondence should be addressed; Tel: +61 2 9385 4341; Fax: +61 2 93855966; Email: [email protected]; Tel: +61 2 9385 5575; Email: [email protected]
Abstract Formation of nano-carriers such as vesicles and micelles using dense gas processing has been under extensive research for decades. Several dense gas processes have been developed to produce nano-carriers, most of them being batch processes. In the present study, a novel continuous dense gas, known as Nano-carrier by a Continuous Dense Gas (NADEG) process was developed as an evolution of a dense gas batch process known as the Depressurization of an Expanded Solution into Aqueous Media (DESAM) process. Transforming a batch process into a continuous process is a main aspect of process intensification. The NADEG process developed in this work enhances the production output of the batch process while producing nano-carriers free of harmful residual organic solvent. The NADEG process is a one-step process for the production of nano-carriers with lower size and higher encapsulation efficiency than the nanocarriers produced by other batch processes. Encapsulation efficiencies as high as 15% were achieved using liposomes to encapsulate a model hydrophilic compound (isoniazid) while encapsulation efficiencies of 10% were achieved in polymersomes for the same model compound. Keywords: Liposomes; Polymersomes; Micelles; Dense Gas Technology; Supercritical Fluid Technology; Continuous process 1
1.
Introduction
Nano-carriers such as vesicles and micelles are widely used as delivery systems, especially for pharmaceutical applications. Production of nano-carriers using different techniques has been under extensive research for decades. Several conventional production methods including the Bangham method (also known as the hand-shaken or thin film hydration method), the detergent depletion process, the ethanol and ether injection technique, the reverse-phase evaporation vesicles (REV) method and the emulsion method have been discussed in the literature [1-6]. The large amount of organic solvents required by conventional techniques has always been a limitation in pharmaceutical applications. Post-removal of organic solvents after the processes is essential. The additional purification and waste disposal steps greatly contribute to production costs, especially on a large scale production plant. Dense gas technologies are among the techniques that have been under extensive research recently to improve the processing of nano-carriers, especially control of morphologies and sizes of nano-carriers and minimisation of residue organic solvent [7]. Dense gas techniques are capable of reducing the use of organic solvents in the production of nano-carriers. In addition, dense gas processes are conducted under sterile operating conditions and involved simple production steps, both advantages in scale up and critical aspects to commercial application. Several dense gas processes have been investigated for the production of nano-carriers in the last decade, such as: 1. Rapid Expansion of Supercritical Solutions (RESS) [8-14] 2. Supercritical Anti-Solvent (SAS) [8, 14-20] 3. Gas Antisolvent Precipitation (GAS) [10, 21] 4. Particles from Gas-Saturated Solutions (PGSS) [22, 23] 5. Supercritical Reverse Phase Evaporation Method (scRPE) [24-27] 6. Depressurization of an Expanded Solution into Aqueous Media (DESAM) [1, 28] 7. Depressurization of an Expanded Liquid Organic Solution (DELOS) [29, 30] 8. SuperFluids (SFS-CFN) [31] 9. Supercritical Liposome Method [32] 2
10. Supercritical Fluid Extraction of Emulsions (SFEE) [33] The dense gas processes listed above are mostly batch processes that generally involve intensive labor, multiple steps, limited production output and limited control of the formation of nano-carriers. The major advantage of continuous processing over batch operation is the increase in production rate while lowering labour demand. The feasibility of scaling up a process is ultimately based on economic evaluations such as the cost of materials, equipment associated with the process and its control. Generally, scaling up reduces production costs and thus brings economic advantage. In addition, process scale up increases the production load, which leads to faster entry into the market place or better product distribution or faster response to the market demands and correspondingly larger market-share [34]. There are two continuous dense gas processes for liposomes production by Lesoin et al. [35] and Santo et al. [36]. However, neither publication included residual solvent analysis. Given the substantial amount of organic solvent used, the assessment of residual solvent in the product can be crucial especially for pharmaceutical applications. Among the above mentioned batch dense gas processes, the DESAM process was developed in the Supercritical Fluid Research Group at UNSW Australia [1, 28]. The DESAM process has been successfully used to manufacture nano-carriers with minimal residual solvent at milder operating conditions than the other dense gas processes listed above [1, 7, 28, 37]. Polymersomes and polymeric micelles produced using the DESAM process had undetectable residual organic solvent while liposomes produced by the DESAM process had about 2% v/v residual ethanol, which is equivalent to 1.6x104 ppm in the products [1, 7, 28]. Despite the successful production of nano-carriers by the DESAM process, the process has relatively poor control over the formation of nano-carriers. In this study, the NADEG process was developed as an evolution of the batch DESAM process to overcome the limitations of the DESAM process in the production of nano-carrier systems (liposomes, polymersomes and polymeric micelles), with a better control over the formation of the nano-carriers and reduction of residual solvent levels in products. The NADEG process is an intermediate step to develop a continuous process for production of nano-carriers based on the 3
same process mechanism. The results obtained from the NADEG process were compared to the DESAM process. In the present work, the design of the NADEG process for the manufacturing and purification of nano-carriers suspensions is presented. Supercritical fluid (SCF) is used as an extraction tool in the NADEG process as discussed in detail in the following section. The implications of further developing the NADEG process are also discussed.
1.1. Supercritical Fluids as Extraction Media Supercritical fluids have the ability to remove organic residual solvents, which is essential in the manufacturing of any product for pharmaceutical application because of the toxicity of most organic solvents. Supercritical fluids (SCFs) are well-known for being able to extract organic compounds with which they are miscible or partially soluble [38, 39]. There are three main advantages of using supercritical fluids in pharmaceutical applications: 1. Removal of organic solvent contaminants from biodegradable polymers, labile compounds and from aqueous solutions [38, 39]. 2. Operation in inert environments at moderate temperatures, which is particularly advantageous for labile pharmaceutical ingredients [40]. 3. Total or partial replacement of toxic solvents with environmentally benign non-toxic dense gas solvents for processing [41]. The range of SCF solvents that can be used in extraction processes includes carbon dioxide (CO2), ethylene, ethane, nitrous oxide, propane, butane, pentane, and hexane. Among all the SCF solvents, CO2 is the most commonly used because it is non-toxic, non-flammable, economical, non-corrosive and environmentally acceptable. Removal of organic solvents from pharmaceutical product by dense gas CO2 was demonstrated recently and the removal of organic solvents could be performed without damaging the characteristics of drug carrier systems [40].
4
Several studies have demonstrated that ethanol can be extracted from aqueous solutions using scCO2 [38, 42-48]. The DESAM process utilised ethanol to dissolve the raw material (phospholipids) in the production of liposomes. Liposomes were formed by self-assembly in water. Supercritical CO2 (scCO2) was selected as the extraction tool in the NADEG process to remove the residual ethanol.
1.2. Multistage Extraction Multistage counter-current contacting is generally the most effective operation typology in an extraction process [49], hence multistage gas extraction in counter-current columns has also received attention in the pharmaceutical industry [7, 10, 16, 40, 42, 50]. Supercritical fluid extraction can be conducted in a multistage counter-current column operated similarly to liquid-liquid extraction methods. Multistage counter-current separation by SCF has been widely applied. Examples of application in the food industry are deacidification and deodorization of vegetable oils, separation or enhancement of flavors and fat-soluble vitamins, and organic components of aqueous solutions [39]. A counter-current contact extraction design maximizes mass transfer between phases compared to co-current flow exchange. Extraction occurs when there is a difference in the solubilities of the organic components between the two phases. When the feed and scCO2 come in contact, mass transfer of the organic compound takes place from the feed solution to the scCO2 phase and the organic compounds are extracted from the feed solution. The use of a packed column can help to increase the contacting surface between the feed solution and the scCO2. A sufficient residence time must be ensured to allow efficient extraction of the organic compounds. In this study, a 316L stainless steel sampling cylinder with double openings with a volume of 150 cm3 from Swagelok (Part number 316L-50DF4-150) was filled with ceramic packing and was used as the extraction column. A preliminary investigation was conducted to determine the experimental operating conditions for an efficient removal of residual organic solvent.
5
1.3. The NADEG Process The DESAM process firstly pre-expands a solution of raw materials (phospholipids and block copolymers) in a pressurization vessel then the expanded solution is delivered via a nozzle into a vessel containing aqueous media. The delivery of the expanded solution is guided by a pressure gradient. The formation of nano-carriers occurs by self-assembly when the raw materials come in contact with the aqueous media. The mixing of the raw materials is facilitated by a continuous flow of dense gas passing through the suspension. In the NADEG process, a Low Volume Mixing Tee (LVMT), as illustrated in Figure 1, was used to achieve a better mixing during the formation of nano-carriers when the raw materials solution came in contact with an aqueous solution, in comparison to the DESAM process [7, 37]. The production of nano-carriers using the NADEG process occurred in a LVMT, in a temperature controlled water bath. An organic solution containing the raw materials and a deionized water solution were fed simultaneously into the LVMT in a pressurized system. Nano-carriers formed by self-assembly when feed inlets came into contact. The resulting suspension of nano-carriers was then delivered into a packed vessel that was filled with raschig rings. The suspension was delivered to the packed bed from the top by a nozzle. A flow of CO2 was introduced into the system from the bottom of the vessel to remove the organic solvent. Figure 1 shows the LVMT with a 25 µL column mixer. The purpose of using a LVMT is to generate consistent mixing conditions when the nanocarriers self-assemble, which overcame the limitation of the DESAM process. A nozzle was used to deliver the mixture suspension into the vessel where the extraction took place, preventing the suspension from escaping through the exit vent that was placed at the top of the packed vessel. A counter-current flow of CO2 was introduced from the bottom of the vessel to extract the organic solvent as the newly formed suspension of nano-carriers dripped from the top of the vessel. Ceramic raschig rings were used as packings in the vessel to maximize the contact between the CO2 and the suspension thus increasing the efficiency of organic solvent extraction. A filter stone was connected to the CO2 inlet as a sparger to help homogeneity of CO2 distribution in the vessel.
6
The LVMT was a micro volume housing device purchased from Grace Davison Discovery Sciences (Part Number 30168) with 25µL mixer cartridge (Part Number 30166). The device is depicted in Figure 1.
Figure 1: The Low Volume Mixing Tee (housing) with a 25 µL column mixer
2.
Experimental Section
2.1. Materials 2.1.1. For liposome production: Absolute ethanol (gradient HPLC grade) was purchased from Scharlau, cholesterol with 99% minimum purity was purchased from Sigma Aldrich, and 1,2-distearoyl-sn-glycero-3phosphatidylcholine (DSPC) with 99% minimum purity was purchased from Avanti. All compounds were used as received.
2.1.2. For polymersome and micelle production: Dichloromethane (DCM) (99% minimum purity) was purchased from Ajax Chemicals and used as received. Poly(butadiene)-block-poly(ethylene oxide) (PBD-PEO) with 2 different numbers of block unit (PBD36-PEO20 and PBD406-PEO286) were purchased from Polymer Source, Inc. (Canada) and used as received.
7
2.1.3. For encapsulation study: Isoniazid (isonicotinic hydroxide, INH) with 99% minimum purity was purchased from Sigma Aldrich and used as received.
2.2. Experimental Operating Conditions The NADEG process was developed based on the operating conditions used for the batch DESAM process. The formation of polymersomes and micelles using the DESAM process has been successful with undetectable residual solvent [7, 37]. Hence, the operating conditions to produce polymersomes and micelles using the NADEG process were the same as the DESAM process, specifically 3.5MPa and 328K. The formation of liposomes using the DESAM process at 3.5MPa and 348K generated the product with 2% residual solvent (which is equivalent to 1.6x104 ppm (g/mL) [7, 37]. Ethanol is classified as low risk to human health. Ethanol residual level of 5x103 ppm ppm (0.5%) or below is considered acceptable without justification [51]. The operating condition for producing the liposomes using the NADEG process has been selected within the supercritical range for CO2 to improve solvent removal. The operating temperature was chosen between the phase transition temperature of the raw materials and the boiling point of the aqueous phase in the process. Operating above the phase transition temperature of the raw materials ensured better formation of nano-carriers because the materials were in the liquid-crystal state, thus molecules were mobile enough to ensure re-arrangement in the aqueous phase instead of solid state. The liposomes production by the NADEG process was set at 333K. Carbon dioxide was selected as the processing supercritical fluid. A preliminary study was conducted to determine the operating pressure for production of liposomes to ensure efficient removal of ethanol. The study was conducted using the NADEG process. Removal of ethanol was carried out at a constant temperature of 333K, and at pressures of 12MPa and 14MPa. Figure 2 summarizes the average residual ethanol and the volume ratio of CO2 to ethanol at 333K, and pressures of 12MPa and 14MPa. From Figure 2, it can be observed that working at 14MPa could allow for a more effective extraction of ethanol. Hence, the production of liposomes was conducted using the NADEG 8
process at 333K, and at pressure of 14MPa with approximately 121 of CO2:ethanol ratio To achieve complete ethanol removal a purification step was designed that consistent passing of CO2 at the working condition through the suspension for an additional 30 minutes before product collection. The production of polymersomes and polymeric micelles required the use of dichloromethane to solubilize the raw materials before feeding into the system. As no residual solvent was detected by GC analysis in the final products, the purification step was not required.
Ethanol Extraction by NADEG at 333K 25.00
Residual Ethanol %
20.00 15.00 12Mpa
10.00
14Mpa 5.00 0.00 0.00
50.00
100.00
150.00
200.00
250.00
300.00
350.00
CO2 used : Ethanol feed
Figure 2: Graph of residual ethanol against the volume ratio of CO2 used to ethanol feed at 333K (STD 0.1-0.5%).
2.3 Experimental Method Firstly, the water bath was set at the working temperature. The system was then pressurized with CO2 to the desired working pressure, via a sparger, from the bottom of the high pressure vessel that was pre-filled with raschig rings packings. Before being introduced into the vessel, CO2 was passed through a heating coil, so that it was at the same temperature as the water 9
bath. The system was allowed to run at constant pressure and temperature and with a constant CO2 flowrate. Carbon dioxide flowrate was regulated by valve V3 on the vent line. Raw materials for the nano-carrier were dissolved in a suitable organic solvent and introduced into the system to one side of the LVMT. Deionized water, or an aqueous solution of a hydrophilic guest compound was introduced into the system to the other side of the inlets of LVMT simultaneously to the organic solution. The water solution was passed through a heating coil before entering the LVMT so that the operating temperature could be reached. When the inlet feeds came in contact, nano-carrier was formed by self-assembly. The hydrophilic guest compound was encapsulated in the vesicles in the process. A continuous flow of CO2 was allowed to pass through the vessel counter-currently from the bottom of the vessel as droplets of nano-carrier suspension were dripping from the top of the vessel via a nozzle, as illustrated in Figure 3. After 30 minutes, both feed inlets of the solution of raw materials and deionized water were stopped. Carbon dioxide was allowed to flow through the system continuously from the bottom of the vessel for additional 30 minutes, to ensure complete removal of residual solvent. Then the CO2 flux was stopped and V3 was closed, and the product was collected by opening valve V4 (Figure 3). Product collection did not require the depressurization of the system and the process was resumed by simply reactivating the inputs and regulating the opening of V3 (Figure 3). The procedure was repeated for the collection of additional product fractions. In the present study, a total of 4 to 5 collections of product were made in each experiment. The operating pressure, temperature, CO2 flowrate and time required for extraction were determined by the preliminary study, as described in the previous section. The experimental conditions and results are tabulated in Table 1 (Section 4).
10
Figure 3: Schematic diagram of the NADEG process
11
3.
Product Characterization
The morphology and size of the nano-carriers were analysed by Transmission Electron Microscopy (TEM). The particle size distribution of the nano-carriers in suspension was investigated by Photon Correlation Spectroscopy (PCS). The residual solvent was quantified by Gas Chromatography (GC). Lastly, encapsulation of isoniazid in vesicles (liposomes and polymersomes) was determined gravimetrically aided by ultra-centrifugation filtering device.
3.1 Characterization of Nano-Carriers Morphology Nano-carriers are transparent under TEM, hence, a negative staining agent was required to give contrast to electron-transparent samples [18]. The negative staining agent used in this study was uranyl acetate. Transmission Electron Microscopy (JEOL 1400, 100 kV accelerating voltage) was used with negative staining to investigate the morphology and size of the nano-carriers. The details of the sample preparation has been reported elsewhere [37].
3.2 Particle Size Distribution Analysis Photon Correlation Spectroscopy (PCS) was used to analyse the particle size distribution of nano-carriers population. By varying different laser light intensity from scattering, the size distribution of nano-carriers can be measured due to the Brownian motion of particles in suspensions. The hydrodynamic diameter size of particles was measured based on spherical particle correlation. Nano-carriers samples were measured over 5 runs where each run was completed in 30 s at room temperature (25°C) with a dust cutoff of 30%. The details has been reported elsewhere [37].
3.3 Encapsulation Efficiency Study The encapsulation efficiency of INH within the vesicles was determined gravimetrically. Ultra-centrifuation filtering device with a 10 kDa membrane cutoff (supplied by the Millipore Company [52]) was used to separate the unencapsulated free INH by
12
centrifugation. The details of sample preparation and assumptions have been previously reported [37]. The encapsulation efficiency of INH is defined as
(%) =
× 100%
3.4 Characterization of Residual Solvent Gas Chromatography (Shimadzu GC-2010) with a flame ionization detector (FID) was used in determining the residual solvent of nano-carriers products. The selected GC column for this study was polyethylene glycol (SGE, BP20, 25 m length, 0.53 mm inner diameter, and 1 µm film thickness). The carrier gas used was helium, while hydrogen and air were supplied to the detector. Triton X-100 was added to the nano-carriers samples to break the nanocarriers. The suspension was then filtered with a Maxi-Clean cartridge with C8 sorbent (supplied by the Millipore Company) to eliminate the solutes – phospholipids for liposomes and block copolymers for polymersomes and micelles. More details have been described elsewhere [37].
4.
Results
A summary of the experimental operating conditions and results is given in Table 1.
13
Table 1: Summary of the experimental operating conditions and results Type of Nano-Carrier Systems
Materials Type of Organic Solvent Used Concentration of DSPC in Ethanol (mg/mL) Concentration of Cholesterol in Ethanol (mg/mL) Concentration of PBD36-PEO20 in DCM (mg/mL) Concentration of PBD406-PEO286 in DCM (mg/mL) Number of Batch Sample Collected Operating Temperature (K) Operating Pressure (MPa) Flowrate of H2O (mL/min) Flowrate of Organic Solution (mL/min) 1 Flowrate of CO2 (g/min) Amount of CO2 Used (g) Ratio of Organic Solvent : H2O Morphology Average Hydrodynamic Diameter (nm) Polydispersity Index (PDI) Residual Solvent % v/v
Liposomes
Polymersomes
Polymeric Micelles
DSPC*, Cholesterol
PBD36-PEO20
PBD406-PEO286
Ethanol
Dichloromethane
Dichloromethane
1.26
-
-
0.38
-
-
-
1.24
-
-
-
1.22
4
4
5
333 14 0.50 0.25 17 1020 0.50
328 3.5 0.50 0.25 7 210 0.50 Spherical, Stomatocyte, Prolate [53] 480 460 590 545
328 3.5 0.50 0.25 7 210 0.50
Spherical 135
145
150
160
145
140
160
190
210
0.093 0.089 0.151 0.119 0.314 0.260 0.295 0.243 0.275 0.243 0.139 0.032 0.149 Not detected
Not detected
*DSPC: 1,2-distearoyl-sn-glycero-3-phosphatidylcholine (Phospholipids)
1
Spherical
Note: Volume of CO2 was measured at the operating pressure of 14MPa and 279K.
14
Not detected
4.1. Formation of Nano-Carriers The liposomes, polymersomes, and polymeric micelles that were produced using the NADEG process were spherical as were the nano-carriers produced by the DESAM process [23]. In each experiment, 4 to 5 aliquots were collected in sequence. The TEM images of liposomes, polymersomes and micelles are shown in Figure 4, Figure 5 and Figure 6 respectively. The average hydrodynamic diameter of nano-carriers at every collection was measured by PCS.
Figure 7 shows that hydrodynamic diameters of liposomes and micelles in the fractions collected have a tendency to grow with the number of fractions collected. Polymersomes
presented no trend. The hydrodynamic diameters of polymersomes as determined by PCS were bigger than the size observed by TEM. Such observation can be explained based on the agglomeration of vesicles in suspension. The difference in hydrodynamic diameter of nanocarriers from different batches varied from 8% for liposomes and micelles to 17% for
polymersomes. A comparison with the PCS results obtained for the nano-carriers produced by the DESAM
process is illustrated in Figure 8: liposomes and polymeric micelles produced by the NADEG process had lower hydrodynamic diameter than the DESAM products; tthe he difference being 18% and 25% for liposomes and micelles, respectively. On the other hand, the average hydrodynamic diameter of polymersomes produced by the NADEG process appeared to be higher than the polymersomes produced by the DESAM process. However, comparison of the
TEM images indicated that this may be due to agglomeration of the polymersomes in suspension rather than major differences in the actual size of the vesicles.
Figure 4: Liposomes produced by NADEG process at 14MPa, 333K
Figure 5: Polymersomes produced by NADEG process at 3.5MPa, 328K
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Figure 6: Micelles produced by NADEG process at 3.5MPa, 328K
Hydrodynamic Diameter of Nano-Carriers by NADEG 700
Average Hydrodynamic Diameter (nm)
600
500
400
300
200
100
0 1st
2nd
3rd
4th
Liposomes
1st
2nd
3rd
4th
Polymersomes
1st
2nd
3rd
4th
5th
Micelles
Figure 7: Average hydrodynamic diameter of liposomes, polymersomes, and polymeric micelles produced by the NADEG process
16
Hydrodynamic Diameter of Nano-Carriers: NADEG-DESAM Processes
Average Hydrodynamic Diameter (nm)
600
500
400
300
200
100
0 NADEG
DESAM
NADEG
Liposomes
DESAM Polymersomes
NADEG
DESAM
Micelles
Figure 8: Comparison of average hydrodynamic diameters of nano-carriers produced by the NADEG and the DESAM processes with CO2
Discrepancies between the size of nano-carriers obtained by the TEM and PCS can be ascribed to different factors. Transmission electron microscopy was used to analyse the
morphology and size of nano-carriers while PCS was used to measure measure their hydrodynamic diameter in suspension based on Brownian motion. In TEM, sample preparation involved
drying of nano-carriers with filter paper and applying of negative staining agent (uranyl acetate). Hence, solid samples were imaged under TEM and and estimations of projected area diameter were made. In PCS, thin layers of solvent can adhere to the surface of the
suspended nano-carriers affecting Brownian motion and thus size estimation. Hydrodynamic diameters observed in this work were larger than the the dimensions registered by TEM, this may be due to the solvation and aggregation of nano-carriers in suspension. Solvation and
aggregation of nano-carriers in suspension can affect morphology and falsely induced intra batch variability [37, 54-58].
17
4.2. Encapsulation of Hydrophilic Compound (Isoniazid) The encapsulation of isoniazid (INH) in liposomes and polymersomes was investigated using the NADEG process. Liposomes encapsulating INH were produced at 14 MPa and 333 K, while polymersomes encapsulating INH were produced at 3.5 MPa and 328 K. A summary of the results and experimental conditions is provided in Table 2.
Table 2: Summary results of INH encapsulation by the NADEG process Vesicles Type Liposomes Polymersomes Polymersomes
Raw materials in suitable organic solvent, mg/mL
Isoniazid in deionised water, mg/mL
Average Encapsulation Efficiency, Mass %
4.0 2.1 6.2
1.0 1.1 1.0
14.7 ± 1.4 9.7 ± 0.3 9.3 ± 0.4
The vesicles encapsulating INH produced by using the NADEG process with CO2 were spherical as observed from TEM imaging. Figure 9 shows the average hydrodynamic diameter of each batch of vesicles encapsulating INH produced. The liposomes encapsulating INH samples collected have similar hydrodynamic diameter measurement of 135nm while the polymersomes encapsulating INH samples have average hydrodynamic diameter of 590±100nm. Figure 10 provides a comparison of the average hydrodynamic diameter of the empty vesicles and vesicles encapsulating INH produced by the DESAM process and the NADEG process. The difference in hydrodynamic diameter between the empty liposomes and the liposomes encapsulating INH generated by the DESAM process and the NADEG process was 3.4% and 9.4%, respectively. The difference in hydrodynamic diameter between empty polymersomes and polymersomes encapsulating INH that were produced by DESAM and the NADEG processes was 11.2% and 12.1%, respectively. The encapsulation of INH in polymersomes was further investigated by using different concentrations of block copolymer (PBD36-PEO20) in the starting solutions: 2mg/mL and 6mg/mL, as shown in Table 2. The results indicated that the starting concentration of block copolymer did not affect encapsulation efficiency. The results obtained suggested that the encapsulation efficiencies achieved when using the NADEG process and the DESAM process are similar. Study on drug encapsulation by the 18
NADEG process is worth further investigation. Results from this work demonstrated that the NADEG process is able to produce nano-carriers with lower hydrodynamic diameter for liposomes and micelles than the DESAM process. Furthermore, NADEG process achieved
similar encapsulation efficiency of INH in vesicles than the conventional methods and DESAM process, which was approximately 10-12% [7, 32, 37, 54]. Having to achieve similar encapsulation efficiency, the newly developed NADEG process could be a better process for production of nano-carriers due to the elimination of residual sol solvent vent in products.
Hydrodynamic Diameter of INH Loaded Vesicles
Average Hydrodynamic Diameter (nm)
800 700 600 500 400 300 200 100 0 1st
2nd
1st
2nd
3rd
4th
Polymersomes
Liposomes
Figure 9: Average hydrodynamic diameters of vesicles encapsulating INH produced by the NADEG process. In each run multiple batches were collected and numbered in series. The numbers on the x axis indicates the sequential number of the batch.
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Average Hydrodynamic Diameter (nm)
Comparison of Hydrodynamic Diameter of Empty Vesicles and INH Loaded Vesicles 700 600 500 400 300
100
E
L
DESAM
DESAM
E
L
NADEG
NADEG
E
L
L
DESAM
DESAM
NADEG
NADEG
0 Liposomes
E
E 200
Empty Vesicles
L
Polymersomes
INH Loaded Vesicles
Figure 10: Comparison of average hydrodynamic diameters of empty and INH loaded vesicles produced by the NADEG and the DESAM processes
The maximum production rates of liposomes and polymersomes/micelles by the DESAM
process are 2.5 g/day and 0.6 g/day, respectively with consideration of the time needed for experiment preparation and cleaning up after the experiment. The maximum production rates of liposomes and polymersomes/micelles by the NADEG process are 10.7 g/day and
2.6 g/day, respectively. The NADEG process is effectively a 4 times scale up of the batch DESAM process, which is a leapt forward to process intensifications. The NADEG process
also involves less labour while increasing the product output rate, in comparison to the DESAM process. The controllable mixing using a low volume mixing device in the NADEG process has allowed the process to have reproducible results and hence, a more feasible process to be scaled up to a continuous process.
It is important to overcome the residual ethanol removal step in the production of liposomes using the NADEG process. The use of a longer or larger vessel with ceramic packings is worth further investigation because it would allow a more effective contact
20
between the scCO2 and liquid ethanol. As a consequence, the extraction process efficiency should increase [44, 55].
5.
Conclusion
The newly developed NADEG process was proven to be a successful alternative method for producing nano-carriers. The new process overcame limitations that were experienced in the batch DESAM process [1, 28, 56]. The NADEG process has the ability to reduce the residual solvent in the nano-carrier systems products to below undetectable levels. In addition, the encapsulation efficiency of INH in vesicles produced in the NADEG process was higher than the encapsulation efficiency achieved in the batch DESAM process [56].
6.
Reference
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[28] L.A. Meure, R. Knott, N.R. Foster, F. Dehghani, The depressurization of an expanded solution into aqueous media for the bulk production of liposomes, Langmuir, 25 (2009) 326337. [29] N. Ventosa, S. Sala, J. Veciana, DELOS process: a crystallization technique using compressed fluids: 1. Comparison to the GAS crystallization method, The Journal of Supercritical Fluids, 26 (2003) 33-45. [30] M. Cano-Sarabia, N. Ventosa, S. Sala, C. Patiño, R. Arranz, J. Veciana, Preparation of uniform rich cholesterol unilamellar nanovesicles using CO2-expanded solvents, Langmuir, 24 (2008) 2433-2437. [31] T.P. Castor, Phospholipid nanosomes, Current Drug Delivery, 2 (2005) 329-340. [32] L. Frederiksen, K. Anton, P. Van Hoogevest, H.R. Keller, H. Leuenberger, Preparation of liposomes encapsulating water-soluble compounds using supercritical carbon dioxide, Journal of Pharmaceutical Sciences, 86 (1997) 921-928. [33] P. Chattopadhyay, B.Y. Shekunov, D. Yim, D. Cipolla, B. Boyd, S. Farr, Production of solid lipid nanoparticle suspensions using supercritical fluid extraction of emulsions (SFEE) for pulmonary delivery using the AERx system, Advanced Drug Delivery Reviews, 59 (2007) 444453. [34] D.E. Discher, F. Ahmed, Polymersomes, Annual review of biomedical engineering, 8 (2006) 323-341. [35] L. Lesoin, C. Crampon, O. Boutin, E. Badens, Development of a continuous dense gas process for the production of liposomes, The Journal of Supercritical Fluids, 60 (2011) 51-62. [36] I.E. Santo, R. Campardelli, E.C. Albuquerque, S.V. de Melo, G. Della Porta, E. Reverchon, Liposomes preparation using a supercritical fluid assisted continuous process, Chemical Engineering Journal, 249 (2014) 153-159. [37] C.C. Beh, R. Mammucari, N.R. Foster, Formation of Nanocarrier Systems by Dense Gas Processing, Langmuir, 30 (2014) 11046-11054. [38] M.L. Gilbert, M.E. Paulaitis, Gas-liquid equilibrium for ethanol-water-carbon dioxide mixtures at elevated pressures, Journal of Chemical & Engineering Data, 31 (1986) 296-298. [39] G. Brunner, N.T. Machado, Process design methodology for fractionation of fatty acids from palm fatty acid distillates in countercurrent packed columns with supercritical CO2, The Journal of Supercritical Fluids, 66 (2012) 96-110. [40] N.T. Machado, G. Brunner, Separation of satured and unsaturated fatty acids from palm fatty acids distillates in continuous multistage countercurrent columns with supercritical carbon dioxide as solvent: A process design methodology, Ciência e Tecnologia de Alimentos, 17 (1997) 361-370. [41] E.M. Martin Del Valle, M.A. Galan, Supercritical fluid technique for particle engineering: Drug delivery applications, Reviews in Chemical Engineering, 21 (2005) 33-69. [42] M. McHugh, V. Krukonis, Supercritical Fluid Extraction: Principles and Practice, 2nd ed., Butterworth-Heinemann, 1994. [43] J.-H. Yoon, H. Lee, B.H. Chung, High pressure three-phase equilibria for the carbon dioxide-ethanol-water system, Fluid Phase Equilibria, 102 (1994) 287-292. [44] M. Budich, G. Brunner, Supercritical fluid extraction of ethanol from aqueous solutions, Journal of Supercritical Fluids, 25 (2003) 45-55. [45] A. Güvenç, Ü. Mehmetoǧlu, A. Çalimli, Supercritical CO2 extraction of ethanol, Turkish Journal of Chemistry, 23 (1999) 285-291.
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[46] A. Ruiz-Rodríguez, T. Fornari, L. Jaime, E. Vázquez, B. Amador, J.A. Nieto, M. Yuste, M. Mercader, G. Reglero, Supercritical CO2 extraction applied toward the production of a functional beverage from wine, The Journal of Supercritical Fluids, 61 (2012) 92-100. [47] H. Inomata, A. Kondo, K. Arai, S. Saito, Phase equilibrium study of the separation of ethanol-water solution with supercritical CO2, Journal of Chemical Engineering of Japan, 23 (1990) 199-207. [48] L.S. Daintree, A. Kordikowski, P. York, Separation processes for organic molecules using SCF Technologies, Advanced Drug Delivery Reviews, 60 (2008) 351-372. [49] J.L. Hedrick, L.J. Mulcahey, L.T. Taylor, Supercritical fluid extraction, Mikrochimica Acta, 108 (1992) 115-132. [50] G. Brunner, Industrial process development: Countercurrent multistage gas extraction (SFE) processes, The Journal of Supercritical Fluids, 13 (1998) 283-301. [51] FDA, Guidance for Industry: Q3C — Tables and List, in: U.S.D.o.H.a.H. Services, F.a.D. Administration, C.f.D.E.a.R. (CDER), C.f.B.E.a.R. (CBER) (Eds.), 2012, pp. 10. [52] Millipore, Amicon Ultra-0.5 Centrifugal Filter Unit with Ultracel-10 membrane, in, 2013. [53] H.-G. Döbereiner, Fluctuating Vesicle Shapes, in: Perspectives in Supramolecular Chemistry, John Wiley & Sons, Ltd., 2007, pp. 149-167. [54] R. Pandey, S. Sharma, G.K. Khuller, Liposome-based antitubercular drug therapy in a guinea pig model of tuberculosis, International Journal of Antimicrobial Agents, 23 (2004) 414-415. [55] C. Perakis, E. Voutsas, K. Magoulas, D. Tassios, Thermodynamic modeling of the vapor– liquid equilibrium of the water/ethanol/CO2 system, Fluid Phase Equilibria, 243 (2006) 142150. [56] C.C. Beh, Formation of Nano-carrier systems by dense gas processing, in: Chemical Sciences and Engineering, The University of New South Wales: Sydney, 2013.
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Highlights • • • • •
Process intensification by transforming a batch to a continuous dense gas process A new continuous dense gas process, known as the NADEG process, was developed Encapsulation of hydrophilic compounds in vesicles was achieved NADEG process is able to enhance production output Nano-carriers free of harmful residual organic solvent produced by NADEG process
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S1385-8947(14)01695-7 http://dx.doi.org/10.1016/j.cej.2014.12.072 CEJ 13073
To appear in:
Chemical Engineering Journal
Received Date: Revised Date: Accepted Date:
18 August 2014 8 December 2014 18 December 2014
Please cite this article as: C.C. Beh, R. Mammucari, N.R. Foster, Process Intensification: Nano-Carrier Formation by a Continuous Dense Gas Process, Chemical Engineering Journal (2015), doi: http://dx.doi.org/10.1016/j.cej. 2014.12.072
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Process Intensification: Nano-Carrier Formation by a Continuous Dense Gas Process Chau Chun Beh, Raffaella Mammucari*, Neil R. Foster* School of Chemical Engineering, UNSW Australia *Authors to whom correspondence should be addressed; Tel: +61 2 9385 4341; Fax: +61 2 93855966; Email: [email protected]; Tel: +61 2 9385 5575; Email: [email protected]
Abstract Formation of nano-carriers such as vesicles and micelles using dense gas processing has been under extensive research for decades. Several dense gas processes have been developed to produce nano-carriers, most of them being batch processes. In the present study, a novel continuous dense gas, known as Nano-carrier by a Continuous Dense Gas (NADEG) process was developed as an evolution of a dense gas batch process known as the Depressurization of an Expanded Solution into Aqueous Media (DESAM) process. Transforming a batch process into a continuous process is a main aspect of process intensification. The NADEG process developed in this work enhances the production output of the batch process while producing nano-carriers free of harmful residual organic solvent. The NADEG process is a one-step process for the production of nano-carriers with lower size and higher encapsulation efficiency than the nanocarriers produced by other batch processes. Encapsulation efficiencies as high as 15% were achieved using liposomes to encapsulate a model hydrophilic compound (isoniazid) while encapsulation efficiencies of 10% were achieved in polymersomes for the same model compound. Keywords: Liposomes; Polymersomes; Micelles; Dense Gas Technology; Supercritical Fluid Technology; Continuous process 1
1.
Introduction
Nano-carriers such as vesicles and micelles are widely used as delivery systems, especially for pharmaceutical applications. Production of nano-carriers using different techniques has been under extensive research for decades. Several conventional production methods including the Bangham method (also known as the hand-shaken or thin film hydration method), the detergent depletion process, the ethanol and ether injection technique, the reverse-phase evaporation vesicles (REV) method and the emulsion method have been discussed in the literature [1-6]. The large amount of organic solvents required by conventional techniques has always been a limitation in pharmaceutical applications. Post-removal of organic solvents after the processes is essential. The additional purification and waste disposal steps greatly contribute to production costs, especially on a large scale production plant. Dense gas technologies are among the techniques that have been under extensive research recently to improve the processing of nano-carriers, especially control of morphologies and sizes of nano-carriers and minimisation of residue organic solvent [7]. Dense gas techniques are capable of reducing the use of organic solvents in the production of nano-carriers. In addition, dense gas processes are conducted under sterile operating conditions and involved simple production steps, both advantages in scale up and critical aspects to commercial application. Several dense gas processes have been investigated for the production of nano-carriers in the last decade, such as: 1. Rapid Expansion of Supercritical Solutions (RESS) [8-14] 2. Supercritical Anti-Solvent (SAS) [8, 14-20] 3. Gas Antisolvent Precipitation (GAS) [10, 21] 4. Particles from Gas-Saturated Solutions (PGSS) [22, 23] 5. Supercritical Reverse Phase Evaporation Method (scRPE) [24-27] 6. Depressurization of an Expanded Solution into Aqueous Media (DESAM) [1, 28] 7. Depressurization of an Expanded Liquid Organic Solution (DELOS) [29, 30] 8. SuperFluids (SFS-CFN) [31] 9. Supercritical Liposome Method [32] 2
10. Supercritical Fluid Extraction of Emulsions (SFEE) [33] The dense gas processes listed above are mostly batch processes that generally involve intensive labor, multiple steps, limited production output and limited control of the formation of nano-carriers. The major advantage of continuous processing over batch operation is the increase in production rate while lowering labour demand. The feasibility of scaling up a process is ultimately based on economic evaluations such as the cost of materials, equipment associated with the process and its control. Generally, scaling up reduces production costs and thus brings economic advantage. In addition, process scale up increases the production load, which leads to faster entry into the market place or better product distribution or faster response to the market demands and correspondingly larger market-share [34]. There are two continuous dense gas processes for liposomes production by Lesoin et al. [35] and Santo et al. [36]. However, neither publication included residual solvent analysis. Given the substantial amount of organic solvent used, the assessment of residual solvent in the product can be crucial especially for pharmaceutical applications. Among the above mentioned batch dense gas processes, the DESAM process was developed in the Supercritical Fluid Research Group at UNSW Australia [1, 28]. The DESAM process has been successfully used to manufacture nano-carriers with minimal residual solvent at milder operating conditions than the other dense gas processes listed above [1, 7, 28, 37]. Polymersomes and polymeric micelles produced using the DESAM process had undetectable residual organic solvent while liposomes produced by the DESAM process had about 2% v/v residual ethanol, which is equivalent to 1.6x104 ppm in the products [1, 7, 28]. Despite the successful production of nano-carriers by the DESAM process, the process has relatively poor control over the formation of nano-carriers. In this study, the NADEG process was developed as an evolution of the batch DESAM process to overcome the limitations of the DESAM process in the production of nano-carrier systems (liposomes, polymersomes and polymeric micelles), with a better control over the formation of the nano-carriers and reduction of residual solvent levels in products. The NADEG process is an intermediate step to develop a continuous process for production of nano-carriers based on the 3
same process mechanism. The results obtained from the NADEG process were compared to the DESAM process. In the present work, the design of the NADEG process for the manufacturing and purification of nano-carriers suspensions is presented. Supercritical fluid (SCF) is used as an extraction tool in the NADEG process as discussed in detail in the following section. The implications of further developing the NADEG process are also discussed.
1.1. Supercritical Fluids as Extraction Media Supercritical fluids have the ability to remove organic residual solvents, which is essential in the manufacturing of any product for pharmaceutical application because of the toxicity of most organic solvents. Supercritical fluids (SCFs) are well-known for being able to extract organic compounds with which they are miscible or partially soluble [38, 39]. There are three main advantages of using supercritical fluids in pharmaceutical applications: 1. Removal of organic solvent contaminants from biodegradable polymers, labile compounds and from aqueous solutions [38, 39]. 2. Operation in inert environments at moderate temperatures, which is particularly advantageous for labile pharmaceutical ingredients [40]. 3. Total or partial replacement of toxic solvents with environmentally benign non-toxic dense gas solvents for processing [41]. The range of SCF solvents that can be used in extraction processes includes carbon dioxide (CO2), ethylene, ethane, nitrous oxide, propane, butane, pentane, and hexane. Among all the SCF solvents, CO2 is the most commonly used because it is non-toxic, non-flammable, economical, non-corrosive and environmentally acceptable. Removal of organic solvents from pharmaceutical product by dense gas CO2 was demonstrated recently and the removal of organic solvents could be performed without damaging the characteristics of drug carrier systems [40].
4
Several studies have demonstrated that ethanol can be extracted from aqueous solutions using scCO2 [38, 42-48]. The DESAM process utilised ethanol to dissolve the raw material (phospholipids) in the production of liposomes. Liposomes were formed by self-assembly in water. Supercritical CO2 (scCO2) was selected as the extraction tool in the NADEG process to remove the residual ethanol.
1.2. Multistage Extraction Multistage counter-current contacting is generally the most effective operation typology in an extraction process [49], hence multistage gas extraction in counter-current columns has also received attention in the pharmaceutical industry [7, 10, 16, 40, 42, 50]. Supercritical fluid extraction can be conducted in a multistage counter-current column operated similarly to liquid-liquid extraction methods. Multistage counter-current separation by SCF has been widely applied. Examples of application in the food industry are deacidification and deodorization of vegetable oils, separation or enhancement of flavors and fat-soluble vitamins, and organic components of aqueous solutions [39]. A counter-current contact extraction design maximizes mass transfer between phases compared to co-current flow exchange. Extraction occurs when there is a difference in the solubilities of the organic components between the two phases. When the feed and scCO2 come in contact, mass transfer of the organic compound takes place from the feed solution to the scCO2 phase and the organic compounds are extracted from the feed solution. The use of a packed column can help to increase the contacting surface between the feed solution and the scCO2. A sufficient residence time must be ensured to allow efficient extraction of the organic compounds. In this study, a 316L stainless steel sampling cylinder with double openings with a volume of 150 cm3 from Swagelok (Part number 316L-50DF4-150) was filled with ceramic packing and was used as the extraction column. A preliminary investigation was conducted to determine the experimental operating conditions for an efficient removal of residual organic solvent.
5
1.3. The NADEG Process The DESAM process firstly pre-expands a solution of raw materials (phospholipids and block copolymers) in a pressurization vessel then the expanded solution is delivered via a nozzle into a vessel containing aqueous media. The delivery of the expanded solution is guided by a pressure gradient. The formation of nano-carriers occurs by self-assembly when the raw materials come in contact with the aqueous media. The mixing of the raw materials is facilitated by a continuous flow of dense gas passing through the suspension. In the NADEG process, a Low Volume Mixing Tee (LVMT), as illustrated in Figure 1, was used to achieve a better mixing during the formation of nano-carriers when the raw materials solution came in contact with an aqueous solution, in comparison to the DESAM process [7, 37]. The production of nano-carriers using the NADEG process occurred in a LVMT, in a temperature controlled water bath. An organic solution containing the raw materials and a deionized water solution were fed simultaneously into the LVMT in a pressurized system. Nano-carriers formed by self-assembly when feed inlets came into contact. The resulting suspension of nano-carriers was then delivered into a packed vessel that was filled with raschig rings. The suspension was delivered to the packed bed from the top by a nozzle. A flow of CO2 was introduced into the system from the bottom of the vessel to remove the organic solvent. Figure 1 shows the LVMT with a 25 µL column mixer. The purpose of using a LVMT is to generate consistent mixing conditions when the nanocarriers self-assemble, which overcame the limitation of the DESAM process. A nozzle was used to deliver the mixture suspension into the vessel where the extraction took place, preventing the suspension from escaping through the exit vent that was placed at the top of the packed vessel. A counter-current flow of CO2 was introduced from the bottom of the vessel to extract the organic solvent as the newly formed suspension of nano-carriers dripped from the top of the vessel. Ceramic raschig rings were used as packings in the vessel to maximize the contact between the CO2 and the suspension thus increasing the efficiency of organic solvent extraction. A filter stone was connected to the CO2 inlet as a sparger to help homogeneity of CO2 distribution in the vessel.
6
The LVMT was a micro volume housing device purchased from Grace Davison Discovery Sciences (Part Number 30168) with 25µL mixer cartridge (Part Number 30166). The device is depicted in Figure 1.
Figure 1: The Low Volume Mixing Tee (housing) with a 25 µL column mixer
2.
Experimental Section
2.1. Materials 2.1.1. For liposome production: Absolute ethanol (gradient HPLC grade) was purchased from Scharlau, cholesterol with 99% minimum purity was purchased from Sigma Aldrich, and 1,2-distearoyl-sn-glycero-3phosphatidylcholine (DSPC) with 99% minimum purity was purchased from Avanti. All compounds were used as received.
2.1.2. For polymersome and micelle production: Dichloromethane (DCM) (99% minimum purity) was purchased from Ajax Chemicals and used as received. Poly(butadiene)-block-poly(ethylene oxide) (PBD-PEO) with 2 different numbers of block unit (PBD36-PEO20 and PBD406-PEO286) were purchased from Polymer Source, Inc. (Canada) and used as received.
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2.1.3. For encapsulation study: Isoniazid (isonicotinic hydroxide, INH) with 99% minimum purity was purchased from Sigma Aldrich and used as received.
2.2. Experimental Operating Conditions The NADEG process was developed based on the operating conditions used for the batch DESAM process. The formation of polymersomes and micelles using the DESAM process has been successful with undetectable residual solvent [7, 37]. Hence, the operating conditions to produce polymersomes and micelles using the NADEG process were the same as the DESAM process, specifically 3.5MPa and 328K. The formation of liposomes using the DESAM process at 3.5MPa and 348K generated the product with 2% residual solvent (which is equivalent to 1.6x104 ppm (g/mL) [7, 37]. Ethanol is classified as low risk to human health. Ethanol residual level of 5x103 ppm ppm (0.5%) or below is considered acceptable without justification [51]. The operating condition for producing the liposomes using the NADEG process has been selected within the supercritical range for CO2 to improve solvent removal. The operating temperature was chosen between the phase transition temperature of the raw materials and the boiling point of the aqueous phase in the process. Operating above the phase transition temperature of the raw materials ensured better formation of nano-carriers because the materials were in the liquid-crystal state, thus molecules were mobile enough to ensure re-arrangement in the aqueous phase instead of solid state. The liposomes production by the NADEG process was set at 333K. Carbon dioxide was selected as the processing supercritical fluid. A preliminary study was conducted to determine the operating pressure for production of liposomes to ensure efficient removal of ethanol. The study was conducted using the NADEG process. Removal of ethanol was carried out at a constant temperature of 333K, and at pressures of 12MPa and 14MPa. Figure 2 summarizes the average residual ethanol and the volume ratio of CO2 to ethanol at 333K, and pressures of 12MPa and 14MPa. From Figure 2, it can be observed that working at 14MPa could allow for a more effective extraction of ethanol. Hence, the production of liposomes was conducted using the NADEG 8
process at 333K, and at pressure of 14MPa with approximately 121 of CO2:ethanol ratio To achieve complete ethanol removal a purification step was designed that consistent passing of CO2 at the working condition through the suspension for an additional 30 minutes before product collection. The production of polymersomes and polymeric micelles required the use of dichloromethane to solubilize the raw materials before feeding into the system. As no residual solvent was detected by GC analysis in the final products, the purification step was not required.
Ethanol Extraction by NADEG at 333K 25.00
Residual Ethanol %
20.00 15.00 12Mpa
10.00
14Mpa 5.00 0.00 0.00
50.00
100.00
150.00
200.00
250.00
300.00
350.00
CO2 used : Ethanol feed
Figure 2: Graph of residual ethanol against the volume ratio of CO2 used to ethanol feed at 333K (STD 0.1-0.5%).
2.3 Experimental Method Firstly, the water bath was set at the working temperature. The system was then pressurized with CO2 to the desired working pressure, via a sparger, from the bottom of the high pressure vessel that was pre-filled with raschig rings packings. Before being introduced into the vessel, CO2 was passed through a heating coil, so that it was at the same temperature as the water 9
bath. The system was allowed to run at constant pressure and temperature and with a constant CO2 flowrate. Carbon dioxide flowrate was regulated by valve V3 on the vent line. Raw materials for the nano-carrier were dissolved in a suitable organic solvent and introduced into the system to one side of the LVMT. Deionized water, or an aqueous solution of a hydrophilic guest compound was introduced into the system to the other side of the inlets of LVMT simultaneously to the organic solution. The water solution was passed through a heating coil before entering the LVMT so that the operating temperature could be reached. When the inlet feeds came in contact, nano-carrier was formed by self-assembly. The hydrophilic guest compound was encapsulated in the vesicles in the process. A continuous flow of CO2 was allowed to pass through the vessel counter-currently from the bottom of the vessel as droplets of nano-carrier suspension were dripping from the top of the vessel via a nozzle, as illustrated in Figure 3. After 30 minutes, both feed inlets of the solution of raw materials and deionized water were stopped. Carbon dioxide was allowed to flow through the system continuously from the bottom of the vessel for additional 30 minutes, to ensure complete removal of residual solvent. Then the CO2 flux was stopped and V3 was closed, and the product was collected by opening valve V4 (Figure 3). Product collection did not require the depressurization of the system and the process was resumed by simply reactivating the inputs and regulating the opening of V3 (Figure 3). The procedure was repeated for the collection of additional product fractions. In the present study, a total of 4 to 5 collections of product were made in each experiment. The operating pressure, temperature, CO2 flowrate and time required for extraction were determined by the preliminary study, as described in the previous section. The experimental conditions and results are tabulated in Table 1 (Section 4).
10
Figure 3: Schematic diagram of the NADEG process
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3.
Product Characterization
The morphology and size of the nano-carriers were analysed by Transmission Electron Microscopy (TEM). The particle size distribution of the nano-carriers in suspension was investigated by Photon Correlation Spectroscopy (PCS). The residual solvent was quantified by Gas Chromatography (GC). Lastly, encapsulation of isoniazid in vesicles (liposomes and polymersomes) was determined gravimetrically aided by ultra-centrifugation filtering device.
3.1 Characterization of Nano-Carriers Morphology Nano-carriers are transparent under TEM, hence, a negative staining agent was required to give contrast to electron-transparent samples [18]. The negative staining agent used in this study was uranyl acetate. Transmission Electron Microscopy (JEOL 1400, 100 kV accelerating voltage) was used with negative staining to investigate the morphology and size of the nano-carriers. The details of the sample preparation has been reported elsewhere [37].
3.2 Particle Size Distribution Analysis Photon Correlation Spectroscopy (PCS) was used to analyse the particle size distribution of nano-carriers population. By varying different laser light intensity from scattering, the size distribution of nano-carriers can be measured due to the Brownian motion of particles in suspensions. The hydrodynamic diameter size of particles was measured based on spherical particle correlation. Nano-carriers samples were measured over 5 runs where each run was completed in 30 s at room temperature (25°C) with a dust cutoff of 30%. The details has been reported elsewhere [37].
3.3 Encapsulation Efficiency Study The encapsulation efficiency of INH within the vesicles was determined gravimetrically. Ultra-centrifuation filtering device with a 10 kDa membrane cutoff (supplied by the Millipore Company [52]) was used to separate the unencapsulated free INH by
12
centrifugation. The details of sample preparation and assumptions have been previously reported [37]. The encapsulation efficiency of INH is defined as
(%) =
× 100%
3.4 Characterization of Residual Solvent Gas Chromatography (Shimadzu GC-2010) with a flame ionization detector (FID) was used in determining the residual solvent of nano-carriers products. The selected GC column for this study was polyethylene glycol (SGE, BP20, 25 m length, 0.53 mm inner diameter, and 1 µm film thickness). The carrier gas used was helium, while hydrogen and air were supplied to the detector. Triton X-100 was added to the nano-carriers samples to break the nanocarriers. The suspension was then filtered with a Maxi-Clean cartridge with C8 sorbent (supplied by the Millipore Company) to eliminate the solutes – phospholipids for liposomes and block copolymers for polymersomes and micelles. More details have been described elsewhere [37].
4.
Results
A summary of the experimental operating conditions and results is given in Table 1.
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Table 1: Summary of the experimental operating conditions and results Type of Nano-Carrier Systems
Materials Type of Organic Solvent Used Concentration of DSPC in Ethanol (mg/mL) Concentration of Cholesterol in Ethanol (mg/mL) Concentration of PBD36-PEO20 in DCM (mg/mL) Concentration of PBD406-PEO286 in DCM (mg/mL) Number of Batch Sample Collected Operating Temperature (K) Operating Pressure (MPa) Flowrate of H2O (mL/min) Flowrate of Organic Solution (mL/min) 1 Flowrate of CO2 (g/min) Amount of CO2 Used (g) Ratio of Organic Solvent : H2O Morphology Average Hydrodynamic Diameter (nm) Polydispersity Index (PDI) Residual Solvent % v/v
Liposomes
Polymersomes
Polymeric Micelles
DSPC*, Cholesterol
PBD36-PEO20
PBD406-PEO286
Ethanol
Dichloromethane
Dichloromethane
1.26
-
-
0.38
-
-
-
1.24
-
-
-
1.22
4
4
5
333 14 0.50 0.25 17 1020 0.50
328 3.5 0.50 0.25 7 210 0.50 Spherical, Stomatocyte, Prolate [53] 480 460 590 545
328 3.5 0.50 0.25 7 210 0.50
Spherical 135
145
150
160
145
140
160
190
210
0.093 0.089 0.151 0.119 0.314 0.260 0.295 0.243 0.275 0.243 0.139 0.032 0.149 Not detected
Not detected
*DSPC: 1,2-distearoyl-sn-glycero-3-phosphatidylcholine (Phospholipids)
1
Spherical
Note: Volume of CO2 was measured at the operating pressure of 14MPa and 279K.
14
Not detected
4.1. Formation of Nano-Carriers The liposomes, polymersomes, and polymeric micelles that were produced using the NADEG process were spherical as were the nano-carriers produced by the DESAM process [23]. In each experiment, 4 to 5 aliquots were collected in sequence. The TEM images of liposomes, polymersomes and micelles are shown in Figure 4, Figure 5 and Figure 6 respectively. The average hydrodynamic diameter of nano-carriers at every collection was measured by PCS.
Figure 7 shows that hydrodynamic diameters of liposomes and micelles in the fractions collected have a tendency to grow with the number of fractions collected. Polymersomes
presented no trend. The hydrodynamic diameters of polymersomes as determined by PCS were bigger than the size observed by TEM. Such observation can be explained based on the agglomeration of vesicles in suspension. The difference in hydrodynamic diameter of nanocarriers from different batches varied from 8% for liposomes and micelles to 17% for
polymersomes. A comparison with the PCS results obtained for the nano-carriers produced by the DESAM
process is illustrated in Figure 8: liposomes and polymeric micelles produced by the NADEG process had lower hydrodynamic diameter than the DESAM products; tthe he difference being 18% and 25% for liposomes and micelles, respectively. On the other hand, the average hydrodynamic diameter of polymersomes produced by the NADEG process appeared to be higher than the polymersomes produced by the DESAM process. However, comparison of the
TEM images indicated that this may be due to agglomeration of the polymersomes in suspension rather than major differences in the actual size of the vesicles.
Figure 4: Liposomes produced by NADEG process at 14MPa, 333K
Figure 5: Polymersomes produced by NADEG process at 3.5MPa, 328K
15
Figure 6: Micelles produced by NADEG process at 3.5MPa, 328K
Hydrodynamic Diameter of Nano-Carriers by NADEG 700
Average Hydrodynamic Diameter (nm)
600
500
400
300
200
100
0 1st
2nd
3rd
4th
Liposomes
1st
2nd
3rd
4th
Polymersomes
1st
2nd
3rd
4th
5th
Micelles
Figure 7: Average hydrodynamic diameter of liposomes, polymersomes, and polymeric micelles produced by the NADEG process
16
Hydrodynamic Diameter of Nano-Carriers: NADEG-DESAM Processes
Average Hydrodynamic Diameter (nm)
600
500
400
300
200
100
0 NADEG
DESAM
NADEG
Liposomes
DESAM Polymersomes
NADEG
DESAM
Micelles
Figure 8: Comparison of average hydrodynamic diameters of nano-carriers produced by the NADEG and the DESAM processes with CO2
Discrepancies between the size of nano-carriers obtained by the TEM and PCS can be ascribed to different factors. Transmission electron microscopy was used to analyse the
morphology and size of nano-carriers while PCS was used to measure measure their hydrodynamic diameter in suspension based on Brownian motion. In TEM, sample preparation involved
drying of nano-carriers with filter paper and applying of negative staining agent (uranyl acetate). Hence, solid samples were imaged under TEM and and estimations of projected area diameter were made. In PCS, thin layers of solvent can adhere to the surface of the
suspended nano-carriers affecting Brownian motion and thus size estimation. Hydrodynamic diameters observed in this work were larger than the the dimensions registered by TEM, this may be due to the solvation and aggregation of nano-carriers in suspension. Solvation and
aggregation of nano-carriers in suspension can affect morphology and falsely induced intra batch variability [37, 54-58].
17
4.2. Encapsulation of Hydrophilic Compound (Isoniazid) The encapsulation of isoniazid (INH) in liposomes and polymersomes was investigated using the NADEG process. Liposomes encapsulating INH were produced at 14 MPa and 333 K, while polymersomes encapsulating INH were produced at 3.5 MPa and 328 K. A summary of the results and experimental conditions is provided in Table 2.
Table 2: Summary results of INH encapsulation by the NADEG process Vesicles Type Liposomes Polymersomes Polymersomes
Raw materials in suitable organic solvent, mg/mL
Isoniazid in deionised water, mg/mL
Average Encapsulation Efficiency, Mass %
4.0 2.1 6.2
1.0 1.1 1.0
14.7 ± 1.4 9.7 ± 0.3 9.3 ± 0.4
The vesicles encapsulating INH produced by using the NADEG process with CO2 were spherical as observed from TEM imaging. Figure 9 shows the average hydrodynamic diameter of each batch of vesicles encapsulating INH produced. The liposomes encapsulating INH samples collected have similar hydrodynamic diameter measurement of 135nm while the polymersomes encapsulating INH samples have average hydrodynamic diameter of 590±100nm. Figure 10 provides a comparison of the average hydrodynamic diameter of the empty vesicles and vesicles encapsulating INH produced by the DESAM process and the NADEG process. The difference in hydrodynamic diameter between the empty liposomes and the liposomes encapsulating INH generated by the DESAM process and the NADEG process was 3.4% and 9.4%, respectively. The difference in hydrodynamic diameter between empty polymersomes and polymersomes encapsulating INH that were produced by DESAM and the NADEG processes was 11.2% and 12.1%, respectively. The encapsulation of INH in polymersomes was further investigated by using different concentrations of block copolymer (PBD36-PEO20) in the starting solutions: 2mg/mL and 6mg/mL, as shown in Table 2. The results indicated that the starting concentration of block copolymer did not affect encapsulation efficiency. The results obtained suggested that the encapsulation efficiencies achieved when using the NADEG process and the DESAM process are similar. Study on drug encapsulation by the 18
NADEG process is worth further investigation. Results from this work demonstrated that the NADEG process is able to produce nano-carriers with lower hydrodynamic diameter for liposomes and micelles than the DESAM process. Furthermore, NADEG process achieved
similar encapsulation efficiency of INH in vesicles than the conventional methods and DESAM process, which was approximately 10-12% [7, 32, 37, 54]. Having to achieve similar encapsulation efficiency, the newly developed NADEG process could be a better process for production of nano-carriers due to the elimination of residual sol solvent vent in products.
Hydrodynamic Diameter of INH Loaded Vesicles
Average Hydrodynamic Diameter (nm)
800 700 600 500 400 300 200 100 0 1st
2nd
1st
2nd
3rd
4th
Polymersomes
Liposomes
Figure 9: Average hydrodynamic diameters of vesicles encapsulating INH produced by the NADEG process. In each run multiple batches were collected and numbered in series. The numbers on the x axis indicates the sequential number of the batch.
19
Average Hydrodynamic Diameter (nm)
Comparison of Hydrodynamic Diameter of Empty Vesicles and INH Loaded Vesicles 700 600 500 400 300
100
E
L
DESAM
DESAM
E
L
NADEG
NADEG
E
L
L
DESAM
DESAM
NADEG
NADEG
0 Liposomes
E
E 200
Empty Vesicles
L
Polymersomes
INH Loaded Vesicles
Figure 10: Comparison of average hydrodynamic diameters of empty and INH loaded vesicles produced by the NADEG and the DESAM processes
The maximum production rates of liposomes and polymersomes/micelles by the DESAM
process are 2.5 g/day and 0.6 g/day, respectively with consideration of the time needed for experiment preparation and cleaning up after the experiment. The maximum production rates of liposomes and polymersomes/micelles by the NADEG process are 10.7 g/day and
2.6 g/day, respectively. The NADEG process is effectively a 4 times scale up of the batch DESAM process, which is a leapt forward to process intensifications. The NADEG process
also involves less labour while increasing the product output rate, in comparison to the DESAM process. The controllable mixing using a low volume mixing device in the NADEG process has allowed the process to have reproducible results and hence, a more feasible process to be scaled up to a continuous process.
It is important to overcome the residual ethanol removal step in the production of liposomes using the NADEG process. The use of a longer or larger vessel with ceramic packings is worth further investigation because it would allow a more effective contact
20
between the scCO2 and liquid ethanol. As a consequence, the extraction process efficiency should increase [44, 55].
5.
Conclusion
The newly developed NADEG process was proven to be a successful alternative method for producing nano-carriers. The new process overcame limitations that were experienced in the batch DESAM process [1, 28, 56]. The NADEG process has the ability to reduce the residual solvent in the nano-carrier systems products to below undetectable levels. In addition, the encapsulation efficiency of INH in vesicles produced in the NADEG process was higher than the encapsulation efficiency achieved in the batch DESAM process [56].
6.
Reference
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Highlights • • • • •
Process intensification by transforming a batch to a continuous dense gas process A new continuous dense gas process, known as the NADEG process, was developed Encapsulation of hydrophilic compounds in vesicles was achieved NADEG process is able to enhance production output Nano-carriers free of harmful residual organic solvent produced by NADEG process
25