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A supercritical fluid technology for liposome production and comparison with the film hydration method
International Journal of Pharmaceutics xxx (xxxx) xxx
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International Journal of Pharmaceutics journal homepage: www.elsevier.com/locate/ijpharm
A supercritical fluid technology for liposome production and comparison with the film hydration method No´emie Penoy a, Bruno Grignard b, Brigitte Evrard a, G´eraldine Piel a, * a
Laboratory of Pharmaceutical Technology and Biopharmacy, Nanomedicine Development, CIRM, University of Li`ege, Li`ege, Belgium Complex and Entangled Systems from Atoms to Materials (CESAM) Research Unit, Center for Education and Research on Macromolecules (CERM), University of Li`ege, Department of Chemistry, Li`ege, Belgium
b
A R T I C L E I N F O
A B S T R A C T
Keywords: Liposomes Drug delivery systems Nanoparticles Supercritical fluids Dense gas methods PGSS Quality by design
Liposomes were produced by an innovative method using supercritical carbon dioxide as a dispersing agent. A quality by design strategy was used to find optimal production conditions with specific parameters (lipid con centration, dispersion volume, agitation rate, temperature and pressure) allowing the production of liposomes with predicted physicochemical characteristics (particles size and PdI). Two conditions were determined with specific production parameters. It was shown that these two conditions allowed the production of liposomes of different compositions and that most of the liposome formulations had size and dispersity in accordance with the prediction values. The condition involving the higher lipid concentration showed a higher variability in terms of size and dispersity. However, this variability remained acceptable. This innovative supercritical method allowed the production of liposomes with physicochemical characteristics similar to those obtained by the conventional thin film hydration method. This new supercritical carbon dioxide method easily scalable in GMP conditions is a one-step production method contrarily to conventional methods which generally need an additional step as extrusion to homogenize the size of liposomes.
1. Introduction Liposomes are spherical nanoparticles composed of one or more lipid bilayers made of phospholipids surrounding an aqueous compartment. Thanks to these properties, these nanovectors are able to encapsulate both hydrophilic and hydrophobic active molecules with several ad vantages such as an increase of the stability, a prolongation of the drug effect or a targeting of pathological tissues with an improvement of the therapeutic index. Depending on their composition, these nanoparticles are structurally close to biological membranes and can be non-toxic, biocompatible and biodegradable (Akbarzadeh et al., 2013; Tikshdeep et al., 2012). Liposomes are thus widely studied as drug delivery systems and emerge as a promising tool for new pharmaceutical formulations as confirmed by the growing number of marketed products and clinical studies involving liposomes (Wagner et al., 2006). Indeed, even if few liposomal formulations are already approved by EMA and FDA such as
AmBisome®, Doxil®/Caelyx® or DaunoXome® many new liposome formulations are currently in Phase I, II or III clinical trials (Crommelin et al., 2020;318:256–63.). Liposomes are conventionally characterized by their size (small, large or giant vesicles), the number of bilayers (uni-, multi- or oligolamellar), the surface charge, the morphology, the degree of homoge neity and the lipid composition. Liposomes can also be categorized by their function such as conventional, PEGylated, ligand-targeted or multifunctional liposomes (Patil and Jadhav, 2014; Sercombe et al., 2015). Depending on their application, the lipid composition can vary and their physicochemical properties are tunable. For drug delivery applications, the desirable size of liposomes generally ranges between 50 and 200 nm (Patil and Jadhav, 2014; Woodle, 1995; Harashima et al., 1994). Different methods are described preparing liposomes at the labora tory scale. Thin film hydration, detergent removal, solvent injection,
Abbreviations: EMA, European Medicines Agency; FDA, Food and Drug Administration; R&D, Research and Development; GMP, Good Manufacturing Practices; SCF, Supercritical Fluid; CO2, Carbon dioxide; RESS, Rapid Expansion of a Supercritical Solution; GAS, Gas Antisolvent; SAS, Supercritical Antisolvent; ASES, Aerosol Solvent Extraction System; PGSS, Particles from Gas Saturated Solution; QbD, Quality by Design; TFH, Thin Film Hydration; CQA, Critical Quality Attributes; PDI, Polydispersity Index; DLS, Dynamic Light Scattering; DSPE PEG 2000, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000]. * Corresponding author. E-mail address: [email protected] (G. Piel). https://doi.org/10.1016/j.ijpharm.2020.120093 Received 11 September 2020; Received in revised form 9 November 2020; Accepted 10 November 2020 Available online 16 November 2020 0378-5173/© 2020 Elsevier B.V. All rights reserved.
Please cite this article as: Noémie Penoy, International Journal of Pharmaceutics, https://doi.org/10.1016/j.ijpharm.2020.120093
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reverse phase evaporation and emulsion method are recognized as conventional liposomes preparation methods (Karn et al., 2013; Samad et al., 2007). Among them, the thin film hydration is one of the most used in R&D. However, these conventional methods suffer from different disadvantages such as the small batch size, the difficulty of transferring product scale and the need of a large volume of organic solvents which can make the resulting liposomes toxic (Karn et al., 2013; Bridson et al., 2006). Moreover, all these methods are multiple steps methods and involve downstream steps such as sonication or extrusion to obtain liposomes with the desirable size with a good homogeneity. Finally, the lack of reproducibility and the non-GMP character of these methods are also important limitations. All these drawbacks hinder the manufacturing of liposomes at the industrial scale in GMP conditions (Wagner et al., 2006; Patil and Jadhav, 2014; Meure et al., 2008; Wagner and Vorauer-Uhl, 2011). Microfluidic methods have emerged as breakthrough technology to overcome these obstacles. This vigorous mixing of fluid technique at a nanoliter scale allows a continuous mode of operation at low volumes which guarantees a better reproducibility and a reduction of the number of steps involved in the preparation. Even if this technology can provide liposomes with regulated size and good encapsulation efficiency, the use of organic solvents still remains a process limitation (Patil and Jadhav, 2014; Shah et al., 2019). In order to bypass or minimize the use of organic solvents and to turn to a greener chemistry, the use of supercritical fluids (SCF) is booming in the pharmaceutical field. At the supercritical state, the fluid combines the physico-chemical properties of both liquids and gases such as a high density and a gas-like viscosity encouraging a good mass transfer. Moreover, the solvent strength may be finely adjusted by the appro priate choice of the operative conditions (pressure and temperature) (Parhi and Suresh, 2013). Carbon dioxide is the most frequently used SCF since it is inexpensive and nontoxic and has a low critical pressure (73.8 bar) and temperature (31.1 ◦ C) making this fluid safe and compatible with the handling of pharmaceutical compounds (Andonova and Chandra, 2016). In addition, upon depressurization, CO2 returns at its gaseous state, preventing any traces of residual solvents to contam inate the final product and causing toxicity issues. SCF-assisted processes are considered as a good alternative to the conventional methods in the fabrication of liposomes. Unlike conven tional methods, they allow a fast and simple one-step preparation pro cess with a better control of the physicochemical properties of the liposomes produced. Moreover, considering the properties of the SCF, the use of organic solvents can be reduced or completely avoided. The supercritical processes can be classified according to the role of the SCF for the lipid used (William et al., 2020; Kankala et al., 2017;6.). SCF can act as a solvent of lipid such as in the injection and decompression method developed by Castor et al. (1996a) where the compressed phase is sprayed into the aqueous phase (injection method) or where the aqueous phase is incorporated into the supercritical medium (decom pression method) (Karn et al., 2013). Another process is the Rapid Expansion of Supercritical Solution (RESS) where the lipid solution is rapidly expanded through an orifice (Karn et al., 2013; Girotra et al., 2013), however, as lipids have generally a limited solubility in SCF, an organic co-solvent is often added (William et al., 2020). SCF can also be used as an antisolvent in the GAS, SAS or ASES processes. An organic solvent, ethanol or chloroform, is first used to solubilize lipids which are then precipitated by the antisolvent effect of SCF (Lesoin et al., 2011; Lesoin et al., 2011). These methods are particularly used for lipids insoluble in SCF (Karn et al., 2013). A third possibility is to use the SCF as a dispersing agent. In these methods, the lipids are dispersed in pure water and then pressurized with SCF in order to be finely dispersed due to the collision and shear forces. In this process, the supercritical carbon dioxide (SC-CO2) is saturated in the hydrophobic chain of the lipids so that the phospholipids are well dispersed into the medium and lipo somes are formed during the depressurization step (Castor, 1996b). Zhao et al. (Zhao and Temelli, 2015), prepared liposomes using a
modified supercritical process via depressurization of the liquid phase. They investigated the effect of several parameters such as the pressure, the temperature or the depressurization rate and obtained particle’s size between 200 nm and 300 nm (Zhao and Temelli, 2015). The main advantage of the use of SCF as a dispersing agent is the avoidance of organic solvents. Moreover, having a reproducible and monitored pro cess in one step guarantee a better control of the finished product and make these processes suitable for industrial production under GMP conditions (Wagner et al., 2006; William et al., 2020; Castor, 1996b; Türeli and Türeli, 2020). Even if the number of nanomedicines in clin ical development continues to increase, the difficulties encountered in large-scale production with the quality and quantity required for these clinical trials remains a problem, the need for a large-scale production method is therefore more necessary than ever. The objective of this study was to develop a method using the SCCO2 as a dispersing agent with the PGSS process (Particles Gas from Saturated Solution) to produce different liposome formulations. A quality by design (QbD) strategy was used to determine optimal pro duction conditions. These conditions were then validated to appreciate the reproducibility of the method and applied to complex liposome formulations to appreciate the method transferability. Finally, the physicochemical characteristics were compared with the physicochem ical characteristics of liposomes produced by the conventional thin film hydration (TFH) method. 2. Materials and methods 2.1. Materials Soy phosphatidylcholine (SPC) was given by Lipoid AG (Switzerland), Egg L-α-phosphatidylcholine (EPC), 1,2-dioleoyl-3-trime thylammonium-propane (chloride salt) (DOTAP), 1,2-dioleoyl-sn-glyc N′ ero-3-phosphoethanolamine (DOPE), 3β – [N-(N′ , dimethylaminoethane)-carbamoyl] cholesterol hydrochloride (DCcholesterol), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N[methoxy (polyethylene glycol)-2000] (ammonium salt) (DSPE PEG 2000) and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N[methoxy (polyethylene glycol)-750] (ammonium salt) (DSPE PEG 750) were purchased from Avanti Polar Lipids, Inc. (Alabaster, AL, USA), and cholesteryl hemisuccinate (CHEMS) and cholesterol (CHOL) were pur chased from Sigma Aldrich (Belgium). 4-(2-Hydroxyethyl) piperazine-1-ethanesulfonic acid (HEPES) ≥ 99.5% was purchased from Sigma Aldrich (Belgium). Ultrapure water was produced by a Milli-Q system (Millipore, Bredford, MA, USA). Liquid CO2 with a purity of 99.7% was purchased from Air Liquide (Belgium). 2.2. Liposomes composition Five liposome formulations were prepared. Their composition is given in table 1.
Table 1 Liposomes composition. Formulation
Composition
% (w/w)
A B C
SPC/cholesterol SPC/cholesterol/DSPE PEG 2000 EPC/DC-cholesterol/cholesterol/DSPE PEG 2000 DOTAP/cholesterol/DOPE DOPE/CHEMS/cholesterol/DSPE PEG 750
70/30 65/30/5 50/29.4/0.6/ 20 44.5/33.3/22.2 43/21/30/6
D E
2
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2.3. Preparation of liposomes by the PGSS method
(400 nm, 200 nm and 100 nm) under pressure.
2.3.1. Equipment A schematic representation of the PGSS equipment (Separex, France) is presented in Fig. 1. This equipment is mainly composed of a highpressure reactor (G) of 50 mL closed by an electric stirrer (I). An elec tronic heating jacket (H) surrounds the high-pressure reactor to regulate and maintain a specific temperature. A pump (C) fitted with a refrigerant (D) allows the CO2 to be pumped in liquid form into the high-pressure reactor where it becomes supercritical thanks to the temperature and pressure. On/off valves (B, E and F) allow the circulation of the CO2 through the system. A pneumatic valve (J) allows the depressurization through a nozzle (K) of the supercritical content in the expansion tank (L). The sample is then collected in the recovery vial (N).
2.5. Characterization of liposomes
2.3.2. Preparation of the phospholipid dispersion A phospholipid dispersion was prepared by dispersing lipids in HEPES buffer (10 mM, pH value 7.4) at 65 ◦ C and stirred at 1200 rpm on a hot plate stirrer for 15 min. The total lipid concentration of the dispersion was fixed according to the experimental strategy.
2.5.2. pH value of phospholipid and liposome dispersions The pH value of the phospholipid dispersions and liposomes was measured with a pH meter (Mettler-Toledo®, Schwerzenbach, Swiss), previously calibrated.
2.5.1. Particles size, size distribution and zeta potential of liposomes The particles size of the liposomes (nm) and the polydispersity Index (PdI) were measured by the dynamic light scattering technique (DLS) using a Malvern Zetasizer® (Nano ZS, Malvern Instrument, UK) at 25 ◦ C with a fixed angle of 90◦ . The samples produced by the supercritical process were diluted in HEPES buffer (10 mM pH value 7.4) to obtain a final concentration of 0.45 mM. The samples prepared by the conven tional method were diluted in Milli-Q water or in HEPES buffer (10 mM pH value 7.4) 100 fold. The zeta potential (mV) was determined with the same instrument. All experiments were measured in triplicate (n = 3).
2.6. Statistics
2.3.3. Quality by design strategy A screening experimental plan was built with the JMP program taking into account the parameters involved in the process (tempera ture, pressure, agitation rate, volume of dispersion and contact time with the SCF) and the parameter involved in the formulation (lipid concentration). Preliminary tests were carried out taking into account the Critical Quality Attributes (CQA) of the liposomes (size and PdI). These preliminary tests allowed to set ranges of values to be varied and 2 blocks of experiments were determined (tables 2 and 3). Results were analyzed by the JMP program.
Under each processing working conditions, liposome samples were produced in triplicate (n = 3). The particles size and the size distribution of each sample were also measured in triplicate (n = 3). An ANOVA analyze was applied with a Tukey post-test to compare columns between them. The difference was considered as significant if p-value was infe rior to 0.05. 3. Results and discussions 3.1. QbD strategy
2.4. Liposomes preparation by the thin film hydration method
The QbD strategy aimed first at determining the parameters that influenced the physicochemical characteristics of liposomes with the ambition to fabricate liposomes with size around 200 nm and a poly dispersity index as weak as possible. After a risk analysis, process-related parameters such as the CO2 pressure, the temperature, the agitation rate, the contact time with the SC-CO2 and the dispersion volume as well as a formulation-related parameter, the lipid concentration, appeared to be the key parameters that could affect the characteristics of the liposomes. Preliminary tests regarding the pressure parameter were carried out. Liposomes (SPC/cholesterol 70/30 w/w, formulation A) were produced at 3 different CO2 pressures (pCO2 = 80, 175 and 250 bar) while main taining constant the temperature (65 ◦ C), the agitation rate (550 rpm) and the contact time (60 min). Liposomes produced at low CO2 pressure of 80 bar had a larger size (346 ± 72 nm) than the ones produced at 175 or 250 bar (111 ± 5 nm and 113 ± 34 nm respectively). To fabricate liposomes with size lower than 200 nm, the pressure range was fixed between 120 and 250 bar. Concerning the other parameters, the range of values was set around the value used in the preliminary tests. All the experiments of the block 1 of the experimental design were conducted with a simple liposome formulation composed of SPC and CHOL (70/30 w/w) (formulation A, table 1). The first analysis high lighted the parameters having an influence on the characteristics of the liposomes. As seen on Fig. 2, the higher the lipid concentration, the higher the size and the PdI of liposomes. It could also be observed that the volume of the dispersion and the contact time have no influence on the size and a low or even no impact on the PdI. An increase of the agitation rate slightly increases the size and especially the PdI of the liposomes. This study also highlights that a temperature higher than 50 ◦ C is necessary to obtain liposomes with size around 200 nm with a lower dispersity. The pressure has a weak influence on the size and the PdI but should not be too low. This first analysis helped us to construct the block 2 of the experi mental design (table 3). The range of the values of each influential
Liposomes were produced according to the formulation composition presented in table 1. Liposomes were prepared using the thin film hy dration method previously described by Bellefroid et al. (Bellefroid et al., 2019). In brief, the appropriate amount of lipids was dissolved in ethanol or chloroform in a round-bottomed flask. Then, the solution was dried at 30 ◦ C under vacuum using a rotavapor for one hour. The thin lipid film was then rehydrated with HEPES buffer (10 mM pH value 7.4) or ultrapure water. Then, dispersions were extruded through Nucleo pore® polycarbonate membranes (Whatman, UK) of a precise diameter
Fig. 1. Schematic representation of the PGSS equipment (A) CO2 bottle, (B, E, and F) on/off valves, (C) pump, (D) refrigerant, (G) high pressure reactor, (H) heating jacket, (I) Stirrer, (J) pneumatic valve, (K) nozzle, (L) expansion tank, (M) CO2 outlet (N) sample recovery vial, (P) pressure gauge, (T) thermostat. 3
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Table 2 Block 1 of the experimental design. Lipids concentration (mM)
Volume of dispersion (mL)
Contact time with SCF (h)
Agitation rate (rpm)
Temperature (◦ C)
Pressure (bar)
50 27,5 50 5 5 50 50 27,5 5 5 5 5 50 50 5 50 5 50 50 27,5
10 20 30 10 30 30 10 20 30 30 30 10 10 10 10 10 10 30 30 20
0,5 1 2 2 2 0,5 0,5 1 0,5 2 0,5 0,5 0,5 2 2 2 0,5 0,5 2 1,25
400 550 700 400 400 400 400 550 700 400 400 700 700 700 700 400 700 700 700 550
57,5 57,5 35 80 35 80 80 57,5 57,5 80 35 35 35 57,5 80 35 80 80 80 57,5
250 185 250 120 120 250 120 185 120 250 250 250 120 250 250 185 185 185 120 185
Table 3 Block 2 of the experimental design. Lipids concentration (mM)
Volume of dispersion (mL)
Contact time with SCF (h)
Agitation rate (rpm)
Temperature (◦ C)
Pressure (bar)
30 30 5 5 5 30 5 5 5 30 30 17,5 17,5 17,5
15 15 15 30 30 30 15 30 30 15 30 22,5 22,5 22,5
0,5 0,5 0,5 0,5 0,5 0,5 0,5 0,5 0,5 0,5 0,5 0,5 0,5 0,5
600 600 400 600 400 400 400 600 400 600 400 500 500 500
80 50 59 80 80 60,35 50 62,6 57,95 80 56,6 65 65 65
200 120 120 120 160 120 200 176,4 181,2 200 175,6 160 160 160
Fig. 2. Model effects visualisation of the block 1 of the experimental design.
parameter were reduced to be closer to values giving good results in terms of liposomes size and PdI. Regarding the contact time, this parameter was fixed to 30 min since it has no influence. The analysis of combined blocks 1 and 2 allowed to construct the
final experimental space (Fig. 3) with the lipid concentration ranging from 5 mM to 50 mM, the volume of the dispersion ranging from 10 mL to 30 mL, the temperature ranging from 35 ◦ C to 80 ◦ C and the pressure ranging from 120 to 250 bar. The probability to obtain the targeted 4
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Fig. 3. Experimental space resulting from the complete analysis of blocks 1 and 2 of the experimental design. Size and PdI: minimum probability of 0.56.
features of liposomes in terms of both size and PdI is 0.56. At the opposite of the red zone of the experimental space, the blue areas correspond to low defect rates enabling liposomes with the targeted size and low PdI to be reached. As illustrated in Fig. 3, two blues areas can be distinguished in terms of lipid concentration in the experimental space. These areas allowed us to define two working conditions with specific parameters where the targeted size and PdI should be reached. These working conditions and the expected features are defined in table 4. Due to the positive influence of the temperature, a temperature of 80 ◦ C was selected for both working conditions. Regarding the contact time and the agitation rate, given
their weak influence, they were fixed to 30 min and 500 rpm for the two conditions. Condition 1 is characterized by a higher lipid concentration and a higher pressure (C = 45 mM and PCO2 = 240 bar) than condition 2 (C = 5 mM and PCO2 156 bar). These two working conditions theoreti cally allow liposomes of expected size and PdI values given in table 4 to be obtained. 3.2. Validation of the process 3.2.1. Reproducibility of the two working conditions In order to appreciate the reproducibility of the process, the
Table 4 Expected size (nm) and PdI values obtained. Condition
Concentration (mM)
Volume (mL)
Temperature (◦ C)
Pressure (bars)
Expected size (nm)
Expected PdI
1 2
45 5
14 10
80 80
240 156
51 – 213 67 – 221
0,05 – 0,34 0,05 – 0,44
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0.02, COND 2:0.31 ± 0.08). The presence of the PEGylated lipid may increase the stability of the liposomes membrane by preventing the aggregation of the particles which can explain the more satisfying results obtained with this formulation in both working conditions (Edwards et al., 1997). Liposomes size and PdI obtained with formulation C composed of EPC, DC-CHOL, CHOL and DSPE PEG 2000 (50/29.4/0.6/20%) (table 1) produced with both conditions 1 or 2 also fit with the prediction values (Fig. 5.C). With this formulation, contrary to the formulation B, the size of liposomes obtained under condition 1 (Z-average size: 170 ± 41 nm, peak size: 217 ± 28 nm) is slightly higher but not significantly compared to the size of liposomes obtained under condition 2 (Z-average size: 135 ± 8 nm, peak size: 192 ± 9 nm) (p > 0.05). For formulation D composed of DOTAP, CHOL and DOPE (44.5/ 33.3/22.2%) and adapted for the gene therapy (Lechanteur et al., 2015), neither of the 2 working conditions allowed to produce liposomes whose size fell within the prediction values (Fig. 5.D). However, the size of liposomes obtained under condition 2 remains acceptable (Z-average size: 233 ± 23 nm, peak size: 272 ± 9 nm). Indeed, the size of liposomes obtained under condition 1 is higher than the size obtained under con dition 2, especially for the main peak size (492 ± 62 nm) which is significantly higher (p < 0.05) than other size results (COND 1 and 2). Regarding the PdI, liposomes produced under condition 2 have a good homogeneity (0.22 ± 0.04) which corresponds to the reproducible size of liposomes obtained under this condition. Liposomes produced under condition 1 have a higher PdI that remains acceptable (0.44 ± 0.13), just at the limit of the prediction values (0.05–0.44). A last formulation developed for peptide administration (Ducat et al., 2011) and composed of DOPE, CHEMS, CHOL and DSPE PEG 750 (43/ 21/30/6%) (formulation E, table 1) was produced and the results are presented in Fig. 5.E. Only the liposomes produced under condition 2 have size (Z-average size: 146 ± 27 nm, peak size: 211 ± 46 nm) included in the limits of the prediction values (51–221 nm). As previ ously, the size of liposomes obtained under condition 1 (Z-average: 315 ± 23 nm, peak size: 280 ± 16 nm) is higher than the size of liposomes produced under condition 2, particularly for the Z-average values (p < 0.05). As regards to the PdI, values fell perfectly within the limits and it was more or less identical for the 2 conditions. All liposome formulations produced under condition 2 have satis fying physicochemical features (size close to 200 nm and PdI < 0,4). This production condition allows the production of liposomes of varied composition. Liposomes produced under condition 1, characterized by a higher lipid concentration and a higher pressure; generally have higher size and variability than those produced under condition 2, particularly for formulation D and E. This may be due to the higher lipid concentration
formulation A, composed of SPC and CHOL (70/30%) was produced in triplicate in both production conditions defined in table 4. The size and the PdI were determined directly after the production. The results are presented in Fig. 4. The red dotted lines represent the lowest and the highest size and PdI values theoretically expected by the QbD strategy. The liposomes size is expressed in Z-average size which is defined as the harmonic intensity averaged particle diameter. This parameter, like the PdI, results from the cumulant analysis which is the simplest method to analyze the autocorrelation function generated by a DLS experiment. The Z-average size is especially representative when the sample is mono modal (Instruments, 2011). Size results can also be expressed in terms of main peak which comes from the analysis of the intensity distribution. The intensity distribution is naturally weighted according to the scattering intensity of each particle fraction or family (Instruments, 2011). It is important to note that the intensity distribu tion can be misleading as it is sensitive to the presence of large particles, aggregates or dust. For these reasons, in this study, the results are always presented both in terms of Z-average size and main peak size. Regarding the liposomes size, the values are in agreement with the predicted values (51 nm − 221 nm/0.05–0.44) excepted for the liposomes produced under condition 1 when size is expressed in Z-average size (310 ± 126 nm). However, the value of the main peak (215 ± 46 nm) fits into the predictions with a weak variability. This could mean that a small pro portion of aggregates or larger particles were considered in the zaverage value and that the main peak is more representative of the sample. This is confirmed by the PdI values. As for the size, liposomes produced under condition 1 are more polydispersed and the value (0.56 ± 0.22) doesn’t fit with the expected value. The polydispersity of lipo somes prepared under condition 2 is lower (0.36 ± 0.04). It therefore seems that the higher concentration of lipids could induce an increase in the size and a decrease in the homogeneity of the liposomes dispersion. 3.2.2. Transferability of the process to other liposome formulations More complex liposome formulations were selected to evaluate the transferability and versatility of the method with both working condi tions (Lechanteur et al., 2015; Ducat et al., 2011). First, DSPE PEG 2000 was added to formulation A. A formulation composed of SPC, CHOL and DSPE PEG 2000 (65/30/5%) (formulation B table 1) was produced in triplicate under both working conditions. The results are shown in Fig. 5 (formulation B). Liposomes have size between the expected limits (51 nm - 221 nm) whether in terms of Z-average size or in main peak size. The size of liposomes produced under condition 2 (Z-average size: 133 ± 46, peak size: 175 ± 62 nm) is, however, slightly higher but the differences are not significant (p > 0.05). Regarding the PdI, this parameter also fits with the predicted values (0.05–0.44) and is more or less the same for both production conditions (COND 1:0.32 ±
Fig. 4. (A) Size (nm) expressed in Z-average size and principal peak of the diffractogram and (B) PDI of SPC/CHOL 70/30% (w/w) liposomes produced under conditions 1 and 2. 6
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Fig. 5. Size (nm) expressed in Z-average size and principal peak of the diffractogram and PDI of liposomes formulations B, C, D and E produced under conditions 1 and 2.
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and the higher pressure as shown by Shaker et al. (Shaker et al., 2017) with an increase in the liposomes size with a higher lipid concentration and by Zhao et al. (Zhao and Temelli, 2015) with a larger particles size obtained with an increase in pressure. The formulation D contains a positively charged lipid (DOTAP) in a high proportion, which possibly could interfere with the formation of liposomes due to electrostatic repulsive forces. The particularity of the formulation E is the presence of two membranes stabilizers, CHEMS and CHOL, which could also affect the liposomes formation by increasing stiffness of the membrane (Ding et al., 2005); (Kaddah et al., 2018). The condition 1 is, however, the most interesting for the development of an industrial process as this condition offers the possibility to produce more concentrated liposomes with probably a higher encapsulation efficiency (Hamedinasab et al., 2019). It is important to note that the discussion of the results is based on the limits imposed by the design of experiments which are not necessarily the limits generally accepted for the administration of liposomes. Indeed, for some formulations, values obtained are slightly out of the limits imposed by the experimental design (213 or 221 nm) but remains acceptable for drug administration. Moreover, for the formulations which do not fit with the expected limits, an optimization of the pro duction parameters of condition 1 could be done in order to decrease the size and the PdI of liposomes.
attributed to the formation of carbonic acid by the reaction between the water of the aqueous buffer and the CO2. The maturation of the liposome dispersions of 48 h could be recommended to find back the initial pH value of the liposome dispersions. 3.4. Benchmarking with liposomes obtained by the conventional TFH method The physicochemical characteristics of the liposome formulations AE produced by the SC-CO2 method were benchmarked with those ob tained by the conventional TFH production method. Regarding the size (Fig. 7 A), liposomes produced by SC-CO2 under condition 2 have size similar to the size of the liposomes produced by the TFH method, regardless of the formulation. For condition 1, liposomes have size similar to the size obtained by the TFH method for formula tions B and C but greater for formulations A, D and E. Concerning the PdI (Fig. 7 B), liposomes produced by the supercritical process under both conditions have a higher PdI than liposomes prepared by the TFH method. The condition 2 allows the production of liposomes dispersion with a PdI close to 0.3 while the working condition 1 gives liposomes with a PdI higher than 0.3 for some formulations. The supercritical method of production allows to produce liposomes whose size are close to the size obtained by the conventional TFH method but with a higher polydispersity index. This can be explained by the fact that no additional homogenization step is used in the super critical process while the classical TFH method uses a final extrusion step in order to homogenize the size of liposomes around 200 nm. This explains the particularly low PdI values obtained by this production method (Fig. 7B).
3.3. Maturation of liposomes The size, the PdI and the pH value of the liposome dispersions was followed over the time (Fig. 6). The same trends were observed for all formulations. The size of the liposomes produced under condition 1 or 2 vary slightly over 1 week but not significantly (p > 0.05). Regarding the dispersity, the PdI of the liposomes does not evolve significantly (p > 0.05) over 1 week. For all formulations, a greater variability can be observed for liposomes produced under condition 1, may be due, once again, to the higher lipid concentration used. Finally, it was observed that the pH value of the liposome dispersions decreases significantly (<0.05) after production (T0) and increases after 2 days to reach its initial value. This initial fall in pH values just after production may be
4. Conclusion Using a QbD approach, this study allowed to determine two opera tive conditions for the production of liposomes by the PGSS process. A condition involving a low lipid concentration (C = 5 mM of lipids, v = 10 mL of dispersion, T = 80 ◦ C, pCO2 = 156 bar) which allows to produce liposomes of various compositions with physicochemical characteristics
Fig. 6. Evolution over a week of the (A) size (nm) expressed in Z-average size and principal peak of the diffractogram, (B) PDI and (C) pH of the liposomes dispersion of SPC/CHOL 70/30% (w/w) liposomes produced under conditions 1 and 2. 8
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Fig. 7. Comparison of the (A) size expressed in Z-average size and the (B) PdI of liposomes produced by the conventionnal TFH method and the supercritical method under conditions 1 and 2 for formulations A, B, C, D and E.
compatible with most of drug administrations (size close to 200 nm with PdI < 0.36). A second condition involving a high lipid concentration (C = 45 mM of lipids, V = 14 mL of dispersion, T = 80 ◦ C, PCO2 = 240 bar) was also determined by the experimental plan. This interesting condi tion from the industrial point of view (possibility of producing more concentrated batches with higher encapsulation efficiency) allows to produce liposomes of simple composition with satisfactory physico chemical characteristics. For the production of more complex formula tions like cationic liposomes with this production condition, liposomes of higher size and PdI were observed. However, unlike most conven tional methods, the supercritical process is a facile one-step method that does not require additional extrusion or ultrasonication steps. Therefore, the variability observed in these experiments remains entirely accept able. Moreover, for applications which require a lower dispersity, a final extrusion step can be added at the end of the process or an optimization of the production parameters remains possible. This supercritical CO2-assisted process is then a convenient and straightforward alternative to conventional methods to fabricate lipo somes with optimal and tunable physicochemical characteristics (size, PdI) while being adaptable to an industrial GMP process and suppressing the drawbacks associated to the use of organic solvents. This method will now be applied to the production of liposomes encapsulating hydrophobic and hydrophilic active molecules.
Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgments The authors gratefully acknowledge the FEDER Phare funds for the financial support in NANOPHARE project (884148-329407). Thanks to Dr. Pierre Lebrun and Hermane T. Avohou for the analysis of the experimental design as well as their precious help. References Akbarzadeh, A., Rezaei-Sadabady, R., Davaran, S., Joo, S.W., Zarghami, N., Hanifehpour, Y., et al., 2013. Liposome: Classification, preparation, and applications. Nanoscale Res Lett 8, 102. https://doi.org/10.1186/1556-276X-8-102. Tikshdeep, C., Sonia, A., Bharat, P., Abhishek, C., 2012. Liposome drug delivery: A review. Int J Pharm Chem Sci 1, 1103–1113. Wagner, A., Platzgummer, M., Kreismayr, G., Quendler, H., Stiegler, G., Ferko, B., et al., 2006. GMP production of liposomes – A new industral approach. J Liposome Res 16, 311–319. https://doi.org/10.1080/08982100600851086. Crommelin, D.J.A., van Hoogevest, P., Storm, G., 2020;318:256–63.. The role of liposomes in clinical nanomedicine development. What now? Now what? J Control Release. https://doi.org/10.1016/j.jconrel.2019.12.023. Patil, Y.P., Jadhav, S., 2014. Novel methods for liposome preparation. Chem Phys Lipids 177, 8–18. https://doi.org/10.1016/j.chemphyslip.2013.10.011. Sercombe, L., Veerati, T., Moheimani, F., Wu, S.Y., Hua, S., 2015. Advances and challenges of liposome assisted drug delivery. Front Pharmacol 6, 1–13. https://doi. org/10.3389/fphar.2015.00286. Woodle, M.C., 1995. Sterically stabilized liposome therapeutics. Adv Drug Deliv Rev 16, 249–265. https://doi.org/10.1016/0169-409X (95)00028-6. Harashima, H., Sakata, K., Funato, K., Kiwada, H., 1994. Enhanced hepatic uptake of liposomes through complement activation depending on the size of liposomes. Pharm Res An Off J Am Assoc Pharm Sci 11, 402–406. https://doi.org/10.1023/A: 1018965121222.
CRediT authorship contribution statement ´mie: Investigation, Visualization, Methodology, Writing Penoy Noe original draft. Grignard Bruno: Writing - review & editing. Evrard ´raldine: Brigitte: Supervision, Writing - review & editing. Piel Ge Validation, Supervision, Writing - review & editing.
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International Journal of Pharmaceutics xxx (xxxx) xxx Zhao, L., Temelli, F., 2015. Preparation of liposomes using a modified supercritical process via depressurization of liquid phase. J Supercrit Fluids 100, 110–120. https://doi.org/10.1016/j.supflu.2015.02.022. Türeli, N.G., Türeli, A.E., 2020. Upscaling and GMP production of pharmaceutical drug delivery systems. Drug Deliv Trends 3, 215–229. https://doi.org/10.1016/b978-012-817870-6.00011-0. Bellefroid, C., Lechanteur, A., Evrard, B., Mottet, D., Debacq-Chainiaux, F., Piel, G., 2019. In vitro skin penetration enhancement techniques: A combined approach of ethosomes and microneedles. Int J Pharm 572, 118793. https://doi.org/10.1016/j. ijpharm.2019.118793. Instruments M. Dynamic Light Scattering, common terms defined 2011:1–6. Lechanteur, A., Furst, T., Evrard, B., Delvenne, P., Hubert, P., Piel, G., 2015. Development of anti-E6 pegylated lipoplexes for mucosal application in the context of cervical preneoplastic lesions. Int J Pharm 483, 268–277. https://doi.org/ 10.1016/j.ijpharm.2015.02.041. Ducat, E., Deprez, J., Gillet, A., No¨ el, A., Evrard, B., Peulen, O., et al., 2011. Nuclear delivery of a therapeutic peptide by long circulating pH-sensitive liposomes: Benefits over classical vesicles. Int J Pharm 420, 319–332. https://doi.org/10.1016/j. ijpharm.2011.08.034. Edwards, K., Johnsson, M., Karlsson, G., Silvander, M., 1997. Effect of polyethyleneglycol-phospholipids on aggregate structure in preparations of small unilamellar liposomes. Biophys J 73, 258–266. https://doi.org/10.1016/S00063495 (97)78066-4. Shaker, S., Gardouh, A., Ghorab, M., 2017. Factors affecting liposomes particle size prepared by ethanol injection method. Res Pharm Sci 12, 346–352. https://doi.org/ 10.4103/1735-5362.213979. Zhao, L., Temelli, F., 2015. Preparation of liposomes using supercritical carbon dioxide via depressurization of the supercritical phase. J Food Eng 158, 104–112. https:// doi.org/10.1016/j.jfoodeng.2015.03.004. Ding, W.-X., Qi, X.R., Li, P., Maitani, Y., Nagai, T., 2005. Cholesteryl hemisuccinate as a membrane stabilizer in dipalmitoylphosphatidylcholine liposomes containing saikosaponin-d. Int J Pharm 300, 38–47. https://doi.org/10.1016/j. ijpharm.2005.05.005. Kaddah, S., Khreich, N., Kaddah, F., Charcosset, C., Greige-Gerges, H., 2018. Cholesterol modulates the liposome membrane fluidity and permeability for a hydrophilic molecule. Food Chem Toxicol 113, 40–48. https://doi.org/10.1016/j. fct.2018.01.017. Hamedinasab, H., Rezayan, A.H., Mellat, M., Mashreghi, M., Jaafari, M.R., 2019. Development of chitosan-coated liposome for pulmonary delivery of Nacetylcysteine. Int J Biol Macromol 156, 1455–1463. https://doi.org/10.1016/j. ijbiomac.2019.11.190.
Karn, P.R., Cho, W., Hwang, S.J., 2013. Liposomal drug products and recent advances in the synthesis of supercritical fluid-mediated liposomes. Nanomedicine 8, 1529–1548. https://doi.org/10.2217/nnm.13.131. Samad, A., Sultana, Y., Aqil, M., 2007. Liposomal drug delivery systems: An update review. Curr Drug Deliv 4, 297–305. https://doi.org/10.2174/ 156720107782151269. Bridson, R.H., Santos, R.C.D., Al-Duri, B., McAllister, S.M., Robertson, J., Alpar, H.O., 2006. The preparation of liposomes using compressed carbon dioxide: strategies, important considerations and comparison with conventional techniques. J Pharm Pharmacol 58, 775–785. https://doi.org/10.1211/jpp.58.6.0008. Meure, L.A., Foster, N.R., Dehghani, F., 2008. Conventional and dense gas techniques for the production of liposomes: A review. AAPS PharmSciTech 9, 798–809. https://doi. org/10.1208/s12249-008-9097-x. Wagner, A., Vorauer-Uhl, K., 2011. Liposome technology for industrial purposes. J Drug Deliv 2011, 1–9. https://doi.org/10.1155/2011/591325. Shah, V.M., Nguyen, D.X., Patel, P., Cote, B., Al-Fatease, A., Pham, Y., et al., 2019. Liposomes produced by microfluidics and extrusion: A comparison for scale-up purposes. Nanomed Nanotechnol 18, 146–156. https://doi.org/10.1016/j. nano.2019.02.019. Parhi, R., Suresh, P., 2013. Supercritical fluid technology: A review. J Adv Pharm Sci Technol 1, 13–36. https://doi.org/10.14302/issn.2328-0182.japst-12-145. Andonova, V., Chandra, Sekhar G., 2016. Supercritical fluid technology: A promising approach for preparation of nano-scale drug delivery systems. J Appl Sci Eng Methodol 2, 239–242. William, B., No´ emie, P., Brigitte, E., G´eraldine, P., 2020. Supercritical fluid methods: An alternative to conventional methods to prepare liposomes. Chem Eng J 383, 123106. https://doi.org/10.1016/j.cej.2019.123106. Kankala, R.K., Zhang, Y.S., Bin, Wang S, Lee, C.H., Chen, A.Z., 2017;6.. Supercritical fluid technology: An emphasis on drug delivery and related biomedical applications. Adv Healthc Mater. https://doi.org/10.1002/adhm.201700433. Castor TP. United States Patent US005554382A Methods and apparatus for making liposomes 1996;II:5–9. Girotra, P., Singh, S.K., Nagpal, K., 2013. Supercritical fluid technology: a promising approach in pharmaceutical research. Pharm Dev Technol 18, 22–38. https://doi. org/10.3109/10837450.2012.726998. Lesoin, L., Crampon, C., Boutin, O., Badens, E., 2011. Preparation of liposomes using the supercritical anti-solvent (SAS) process and comparison with a conventional method. J Supercrit Fluids 57, 162–174. https://doi.org/10.1016/j.supflu.2011.01.006. Lesoin, L., Crampon, C., Boutin, O., Badens, E., 2011. Development of a continuous dense gas process for the production of liposomes. J Supercrit Fluids 60, 51–62. https:// doi.org/10.1016/j.supflu.2011.04.018. Castor TP. Method and apparatus for making liposomes containing hydrophobic drugs – WO 96/15774 1996.
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International Journal of Pharmaceutics journal homepage: www.elsevier.com/locate/ijpharm
A supercritical fluid technology for liposome production and comparison with the film hydration method No´emie Penoy a, Bruno Grignard b, Brigitte Evrard a, G´eraldine Piel a, * a
Laboratory of Pharmaceutical Technology and Biopharmacy, Nanomedicine Development, CIRM, University of Li`ege, Li`ege, Belgium Complex and Entangled Systems from Atoms to Materials (CESAM) Research Unit, Center for Education and Research on Macromolecules (CERM), University of Li`ege, Department of Chemistry, Li`ege, Belgium
b
A R T I C L E I N F O
A B S T R A C T
Keywords: Liposomes Drug delivery systems Nanoparticles Supercritical fluids Dense gas methods PGSS Quality by design
Liposomes were produced by an innovative method using supercritical carbon dioxide as a dispersing agent. A quality by design strategy was used to find optimal production conditions with specific parameters (lipid con centration, dispersion volume, agitation rate, temperature and pressure) allowing the production of liposomes with predicted physicochemical characteristics (particles size and PdI). Two conditions were determined with specific production parameters. It was shown that these two conditions allowed the production of liposomes of different compositions and that most of the liposome formulations had size and dispersity in accordance with the prediction values. The condition involving the higher lipid concentration showed a higher variability in terms of size and dispersity. However, this variability remained acceptable. This innovative supercritical method allowed the production of liposomes with physicochemical characteristics similar to those obtained by the conventional thin film hydration method. This new supercritical carbon dioxide method easily scalable in GMP conditions is a one-step production method contrarily to conventional methods which generally need an additional step as extrusion to homogenize the size of liposomes.
1. Introduction Liposomes are spherical nanoparticles composed of one or more lipid bilayers made of phospholipids surrounding an aqueous compartment. Thanks to these properties, these nanovectors are able to encapsulate both hydrophilic and hydrophobic active molecules with several ad vantages such as an increase of the stability, a prolongation of the drug effect or a targeting of pathological tissues with an improvement of the therapeutic index. Depending on their composition, these nanoparticles are structurally close to biological membranes and can be non-toxic, biocompatible and biodegradable (Akbarzadeh et al., 2013; Tikshdeep et al., 2012). Liposomes are thus widely studied as drug delivery systems and emerge as a promising tool for new pharmaceutical formulations as confirmed by the growing number of marketed products and clinical studies involving liposomes (Wagner et al., 2006). Indeed, even if few liposomal formulations are already approved by EMA and FDA such as
AmBisome®, Doxil®/Caelyx® or DaunoXome® many new liposome formulations are currently in Phase I, II or III clinical trials (Crommelin et al., 2020;318:256–63.). Liposomes are conventionally characterized by their size (small, large or giant vesicles), the number of bilayers (uni-, multi- or oligolamellar), the surface charge, the morphology, the degree of homoge neity and the lipid composition. Liposomes can also be categorized by their function such as conventional, PEGylated, ligand-targeted or multifunctional liposomes (Patil and Jadhav, 2014; Sercombe et al., 2015). Depending on their application, the lipid composition can vary and their physicochemical properties are tunable. For drug delivery applications, the desirable size of liposomes generally ranges between 50 and 200 nm (Patil and Jadhav, 2014; Woodle, 1995; Harashima et al., 1994). Different methods are described preparing liposomes at the labora tory scale. Thin film hydration, detergent removal, solvent injection,
Abbreviations: EMA, European Medicines Agency; FDA, Food and Drug Administration; R&D, Research and Development; GMP, Good Manufacturing Practices; SCF, Supercritical Fluid; CO2, Carbon dioxide; RESS, Rapid Expansion of a Supercritical Solution; GAS, Gas Antisolvent; SAS, Supercritical Antisolvent; ASES, Aerosol Solvent Extraction System; PGSS, Particles from Gas Saturated Solution; QbD, Quality by Design; TFH, Thin Film Hydration; CQA, Critical Quality Attributes; PDI, Polydispersity Index; DLS, Dynamic Light Scattering; DSPE PEG 2000, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000]. * Corresponding author. E-mail address: [email protected] (G. Piel). https://doi.org/10.1016/j.ijpharm.2020.120093 Received 11 September 2020; Received in revised form 9 November 2020; Accepted 10 November 2020 Available online 16 November 2020 0378-5173/© 2020 Elsevier B.V. All rights reserved.
Please cite this article as: Noémie Penoy, International Journal of Pharmaceutics, https://doi.org/10.1016/j.ijpharm.2020.120093
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reverse phase evaporation and emulsion method are recognized as conventional liposomes preparation methods (Karn et al., 2013; Samad et al., 2007). Among them, the thin film hydration is one of the most used in R&D. However, these conventional methods suffer from different disadvantages such as the small batch size, the difficulty of transferring product scale and the need of a large volume of organic solvents which can make the resulting liposomes toxic (Karn et al., 2013; Bridson et al., 2006). Moreover, all these methods are multiple steps methods and involve downstream steps such as sonication or extrusion to obtain liposomes with the desirable size with a good homogeneity. Finally, the lack of reproducibility and the non-GMP character of these methods are also important limitations. All these drawbacks hinder the manufacturing of liposomes at the industrial scale in GMP conditions (Wagner et al., 2006; Patil and Jadhav, 2014; Meure et al., 2008; Wagner and Vorauer-Uhl, 2011). Microfluidic methods have emerged as breakthrough technology to overcome these obstacles. This vigorous mixing of fluid technique at a nanoliter scale allows a continuous mode of operation at low volumes which guarantees a better reproducibility and a reduction of the number of steps involved in the preparation. Even if this technology can provide liposomes with regulated size and good encapsulation efficiency, the use of organic solvents still remains a process limitation (Patil and Jadhav, 2014; Shah et al., 2019). In order to bypass or minimize the use of organic solvents and to turn to a greener chemistry, the use of supercritical fluids (SCF) is booming in the pharmaceutical field. At the supercritical state, the fluid combines the physico-chemical properties of both liquids and gases such as a high density and a gas-like viscosity encouraging a good mass transfer. Moreover, the solvent strength may be finely adjusted by the appro priate choice of the operative conditions (pressure and temperature) (Parhi and Suresh, 2013). Carbon dioxide is the most frequently used SCF since it is inexpensive and nontoxic and has a low critical pressure (73.8 bar) and temperature (31.1 ◦ C) making this fluid safe and compatible with the handling of pharmaceutical compounds (Andonova and Chandra, 2016). In addition, upon depressurization, CO2 returns at its gaseous state, preventing any traces of residual solvents to contam inate the final product and causing toxicity issues. SCF-assisted processes are considered as a good alternative to the conventional methods in the fabrication of liposomes. Unlike conven tional methods, they allow a fast and simple one-step preparation pro cess with a better control of the physicochemical properties of the liposomes produced. Moreover, considering the properties of the SCF, the use of organic solvents can be reduced or completely avoided. The supercritical processes can be classified according to the role of the SCF for the lipid used (William et al., 2020; Kankala et al., 2017;6.). SCF can act as a solvent of lipid such as in the injection and decompression method developed by Castor et al. (1996a) where the compressed phase is sprayed into the aqueous phase (injection method) or where the aqueous phase is incorporated into the supercritical medium (decom pression method) (Karn et al., 2013). Another process is the Rapid Expansion of Supercritical Solution (RESS) where the lipid solution is rapidly expanded through an orifice (Karn et al., 2013; Girotra et al., 2013), however, as lipids have generally a limited solubility in SCF, an organic co-solvent is often added (William et al., 2020). SCF can also be used as an antisolvent in the GAS, SAS or ASES processes. An organic solvent, ethanol or chloroform, is first used to solubilize lipids which are then precipitated by the antisolvent effect of SCF (Lesoin et al., 2011; Lesoin et al., 2011). These methods are particularly used for lipids insoluble in SCF (Karn et al., 2013). A third possibility is to use the SCF as a dispersing agent. In these methods, the lipids are dispersed in pure water and then pressurized with SCF in order to be finely dispersed due to the collision and shear forces. In this process, the supercritical carbon dioxide (SC-CO2) is saturated in the hydrophobic chain of the lipids so that the phospholipids are well dispersed into the medium and lipo somes are formed during the depressurization step (Castor, 1996b). Zhao et al. (Zhao and Temelli, 2015), prepared liposomes using a
modified supercritical process via depressurization of the liquid phase. They investigated the effect of several parameters such as the pressure, the temperature or the depressurization rate and obtained particle’s size between 200 nm and 300 nm (Zhao and Temelli, 2015). The main advantage of the use of SCF as a dispersing agent is the avoidance of organic solvents. Moreover, having a reproducible and monitored pro cess in one step guarantee a better control of the finished product and make these processes suitable for industrial production under GMP conditions (Wagner et al., 2006; William et al., 2020; Castor, 1996b; Türeli and Türeli, 2020). Even if the number of nanomedicines in clin ical development continues to increase, the difficulties encountered in large-scale production with the quality and quantity required for these clinical trials remains a problem, the need for a large-scale production method is therefore more necessary than ever. The objective of this study was to develop a method using the SCCO2 as a dispersing agent with the PGSS process (Particles Gas from Saturated Solution) to produce different liposome formulations. A quality by design (QbD) strategy was used to determine optimal pro duction conditions. These conditions were then validated to appreciate the reproducibility of the method and applied to complex liposome formulations to appreciate the method transferability. Finally, the physicochemical characteristics were compared with the physicochem ical characteristics of liposomes produced by the conventional thin film hydration (TFH) method. 2. Materials and methods 2.1. Materials Soy phosphatidylcholine (SPC) was given by Lipoid AG (Switzerland), Egg L-α-phosphatidylcholine (EPC), 1,2-dioleoyl-3-trime thylammonium-propane (chloride salt) (DOTAP), 1,2-dioleoyl-sn-glyc N′ ero-3-phosphoethanolamine (DOPE), 3β – [N-(N′ , dimethylaminoethane)-carbamoyl] cholesterol hydrochloride (DCcholesterol), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N[methoxy (polyethylene glycol)-2000] (ammonium salt) (DSPE PEG 2000) and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N[methoxy (polyethylene glycol)-750] (ammonium salt) (DSPE PEG 750) were purchased from Avanti Polar Lipids, Inc. (Alabaster, AL, USA), and cholesteryl hemisuccinate (CHEMS) and cholesterol (CHOL) were pur chased from Sigma Aldrich (Belgium). 4-(2-Hydroxyethyl) piperazine-1-ethanesulfonic acid (HEPES) ≥ 99.5% was purchased from Sigma Aldrich (Belgium). Ultrapure water was produced by a Milli-Q system (Millipore, Bredford, MA, USA). Liquid CO2 with a purity of 99.7% was purchased from Air Liquide (Belgium). 2.2. Liposomes composition Five liposome formulations were prepared. Their composition is given in table 1.
Table 1 Liposomes composition. Formulation
Composition
% (w/w)
A B C
SPC/cholesterol SPC/cholesterol/DSPE PEG 2000 EPC/DC-cholesterol/cholesterol/DSPE PEG 2000 DOTAP/cholesterol/DOPE DOPE/CHEMS/cholesterol/DSPE PEG 750
70/30 65/30/5 50/29.4/0.6/ 20 44.5/33.3/22.2 43/21/30/6
D E
2
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2.3. Preparation of liposomes by the PGSS method
(400 nm, 200 nm and 100 nm) under pressure.
2.3.1. Equipment A schematic representation of the PGSS equipment (Separex, France) is presented in Fig. 1. This equipment is mainly composed of a highpressure reactor (G) of 50 mL closed by an electric stirrer (I). An elec tronic heating jacket (H) surrounds the high-pressure reactor to regulate and maintain a specific temperature. A pump (C) fitted with a refrigerant (D) allows the CO2 to be pumped in liquid form into the high-pressure reactor where it becomes supercritical thanks to the temperature and pressure. On/off valves (B, E and F) allow the circulation of the CO2 through the system. A pneumatic valve (J) allows the depressurization through a nozzle (K) of the supercritical content in the expansion tank (L). The sample is then collected in the recovery vial (N).
2.5. Characterization of liposomes
2.3.2. Preparation of the phospholipid dispersion A phospholipid dispersion was prepared by dispersing lipids in HEPES buffer (10 mM, pH value 7.4) at 65 ◦ C and stirred at 1200 rpm on a hot plate stirrer for 15 min. The total lipid concentration of the dispersion was fixed according to the experimental strategy.
2.5.2. pH value of phospholipid and liposome dispersions The pH value of the phospholipid dispersions and liposomes was measured with a pH meter (Mettler-Toledo®, Schwerzenbach, Swiss), previously calibrated.
2.5.1. Particles size, size distribution and zeta potential of liposomes The particles size of the liposomes (nm) and the polydispersity Index (PdI) were measured by the dynamic light scattering technique (DLS) using a Malvern Zetasizer® (Nano ZS, Malvern Instrument, UK) at 25 ◦ C with a fixed angle of 90◦ . The samples produced by the supercritical process were diluted in HEPES buffer (10 mM pH value 7.4) to obtain a final concentration of 0.45 mM. The samples prepared by the conven tional method were diluted in Milli-Q water or in HEPES buffer (10 mM pH value 7.4) 100 fold. The zeta potential (mV) was determined with the same instrument. All experiments were measured in triplicate (n = 3).
2.6. Statistics
2.3.3. Quality by design strategy A screening experimental plan was built with the JMP program taking into account the parameters involved in the process (tempera ture, pressure, agitation rate, volume of dispersion and contact time with the SCF) and the parameter involved in the formulation (lipid concentration). Preliminary tests were carried out taking into account the Critical Quality Attributes (CQA) of the liposomes (size and PdI). These preliminary tests allowed to set ranges of values to be varied and 2 blocks of experiments were determined (tables 2 and 3). Results were analyzed by the JMP program.
Under each processing working conditions, liposome samples were produced in triplicate (n = 3). The particles size and the size distribution of each sample were also measured in triplicate (n = 3). An ANOVA analyze was applied with a Tukey post-test to compare columns between them. The difference was considered as significant if p-value was infe rior to 0.05. 3. Results and discussions 3.1. QbD strategy
2.4. Liposomes preparation by the thin film hydration method
The QbD strategy aimed first at determining the parameters that influenced the physicochemical characteristics of liposomes with the ambition to fabricate liposomes with size around 200 nm and a poly dispersity index as weak as possible. After a risk analysis, process-related parameters such as the CO2 pressure, the temperature, the agitation rate, the contact time with the SC-CO2 and the dispersion volume as well as a formulation-related parameter, the lipid concentration, appeared to be the key parameters that could affect the characteristics of the liposomes. Preliminary tests regarding the pressure parameter were carried out. Liposomes (SPC/cholesterol 70/30 w/w, formulation A) were produced at 3 different CO2 pressures (pCO2 = 80, 175 and 250 bar) while main taining constant the temperature (65 ◦ C), the agitation rate (550 rpm) and the contact time (60 min). Liposomes produced at low CO2 pressure of 80 bar had a larger size (346 ± 72 nm) than the ones produced at 175 or 250 bar (111 ± 5 nm and 113 ± 34 nm respectively). To fabricate liposomes with size lower than 200 nm, the pressure range was fixed between 120 and 250 bar. Concerning the other parameters, the range of values was set around the value used in the preliminary tests. All the experiments of the block 1 of the experimental design were conducted with a simple liposome formulation composed of SPC and CHOL (70/30 w/w) (formulation A, table 1). The first analysis high lighted the parameters having an influence on the characteristics of the liposomes. As seen on Fig. 2, the higher the lipid concentration, the higher the size and the PdI of liposomes. It could also be observed that the volume of the dispersion and the contact time have no influence on the size and a low or even no impact on the PdI. An increase of the agitation rate slightly increases the size and especially the PdI of the liposomes. This study also highlights that a temperature higher than 50 ◦ C is necessary to obtain liposomes with size around 200 nm with a lower dispersity. The pressure has a weak influence on the size and the PdI but should not be too low. This first analysis helped us to construct the block 2 of the experi mental design (table 3). The range of the values of each influential
Liposomes were produced according to the formulation composition presented in table 1. Liposomes were prepared using the thin film hy dration method previously described by Bellefroid et al. (Bellefroid et al., 2019). In brief, the appropriate amount of lipids was dissolved in ethanol or chloroform in a round-bottomed flask. Then, the solution was dried at 30 ◦ C under vacuum using a rotavapor for one hour. The thin lipid film was then rehydrated with HEPES buffer (10 mM pH value 7.4) or ultrapure water. Then, dispersions were extruded through Nucleo pore® polycarbonate membranes (Whatman, UK) of a precise diameter
Fig. 1. Schematic representation of the PGSS equipment (A) CO2 bottle, (B, E, and F) on/off valves, (C) pump, (D) refrigerant, (G) high pressure reactor, (H) heating jacket, (I) Stirrer, (J) pneumatic valve, (K) nozzle, (L) expansion tank, (M) CO2 outlet (N) sample recovery vial, (P) pressure gauge, (T) thermostat. 3
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Table 2 Block 1 of the experimental design. Lipids concentration (mM)
Volume of dispersion (mL)
Contact time with SCF (h)
Agitation rate (rpm)
Temperature (◦ C)
Pressure (bar)
50 27,5 50 5 5 50 50 27,5 5 5 5 5 50 50 5 50 5 50 50 27,5
10 20 30 10 30 30 10 20 30 30 30 10 10 10 10 10 10 30 30 20
0,5 1 2 2 2 0,5 0,5 1 0,5 2 0,5 0,5 0,5 2 2 2 0,5 0,5 2 1,25
400 550 700 400 400 400 400 550 700 400 400 700 700 700 700 400 700 700 700 550
57,5 57,5 35 80 35 80 80 57,5 57,5 80 35 35 35 57,5 80 35 80 80 80 57,5
250 185 250 120 120 250 120 185 120 250 250 250 120 250 250 185 185 185 120 185
Table 3 Block 2 of the experimental design. Lipids concentration (mM)
Volume of dispersion (mL)
Contact time with SCF (h)
Agitation rate (rpm)
Temperature (◦ C)
Pressure (bar)
30 30 5 5 5 30 5 5 5 30 30 17,5 17,5 17,5
15 15 15 30 30 30 15 30 30 15 30 22,5 22,5 22,5
0,5 0,5 0,5 0,5 0,5 0,5 0,5 0,5 0,5 0,5 0,5 0,5 0,5 0,5
600 600 400 600 400 400 400 600 400 600 400 500 500 500
80 50 59 80 80 60,35 50 62,6 57,95 80 56,6 65 65 65
200 120 120 120 160 120 200 176,4 181,2 200 175,6 160 160 160
Fig. 2. Model effects visualisation of the block 1 of the experimental design.
parameter were reduced to be closer to values giving good results in terms of liposomes size and PdI. Regarding the contact time, this parameter was fixed to 30 min since it has no influence. The analysis of combined blocks 1 and 2 allowed to construct the
final experimental space (Fig. 3) with the lipid concentration ranging from 5 mM to 50 mM, the volume of the dispersion ranging from 10 mL to 30 mL, the temperature ranging from 35 ◦ C to 80 ◦ C and the pressure ranging from 120 to 250 bar. The probability to obtain the targeted 4
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Fig. 3. Experimental space resulting from the complete analysis of blocks 1 and 2 of the experimental design. Size and PdI: minimum probability of 0.56.
features of liposomes in terms of both size and PdI is 0.56. At the opposite of the red zone of the experimental space, the blue areas correspond to low defect rates enabling liposomes with the targeted size and low PdI to be reached. As illustrated in Fig. 3, two blues areas can be distinguished in terms of lipid concentration in the experimental space. These areas allowed us to define two working conditions with specific parameters where the targeted size and PdI should be reached. These working conditions and the expected features are defined in table 4. Due to the positive influence of the temperature, a temperature of 80 ◦ C was selected for both working conditions. Regarding the contact time and the agitation rate, given
their weak influence, they were fixed to 30 min and 500 rpm for the two conditions. Condition 1 is characterized by a higher lipid concentration and a higher pressure (C = 45 mM and PCO2 = 240 bar) than condition 2 (C = 5 mM and PCO2 156 bar). These two working conditions theoreti cally allow liposomes of expected size and PdI values given in table 4 to be obtained. 3.2. Validation of the process 3.2.1. Reproducibility of the two working conditions In order to appreciate the reproducibility of the process, the
Table 4 Expected size (nm) and PdI values obtained. Condition
Concentration (mM)
Volume (mL)
Temperature (◦ C)
Pressure (bars)
Expected size (nm)
Expected PdI
1 2
45 5
14 10
80 80
240 156
51 – 213 67 – 221
0,05 – 0,34 0,05 – 0,44
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0.02, COND 2:0.31 ± 0.08). The presence of the PEGylated lipid may increase the stability of the liposomes membrane by preventing the aggregation of the particles which can explain the more satisfying results obtained with this formulation in both working conditions (Edwards et al., 1997). Liposomes size and PdI obtained with formulation C composed of EPC, DC-CHOL, CHOL and DSPE PEG 2000 (50/29.4/0.6/20%) (table 1) produced with both conditions 1 or 2 also fit with the prediction values (Fig. 5.C). With this formulation, contrary to the formulation B, the size of liposomes obtained under condition 1 (Z-average size: 170 ± 41 nm, peak size: 217 ± 28 nm) is slightly higher but not significantly compared to the size of liposomes obtained under condition 2 (Z-average size: 135 ± 8 nm, peak size: 192 ± 9 nm) (p > 0.05). For formulation D composed of DOTAP, CHOL and DOPE (44.5/ 33.3/22.2%) and adapted for the gene therapy (Lechanteur et al., 2015), neither of the 2 working conditions allowed to produce liposomes whose size fell within the prediction values (Fig. 5.D). However, the size of liposomes obtained under condition 2 remains acceptable (Z-average size: 233 ± 23 nm, peak size: 272 ± 9 nm). Indeed, the size of liposomes obtained under condition 1 is higher than the size obtained under con dition 2, especially for the main peak size (492 ± 62 nm) which is significantly higher (p < 0.05) than other size results (COND 1 and 2). Regarding the PdI, liposomes produced under condition 2 have a good homogeneity (0.22 ± 0.04) which corresponds to the reproducible size of liposomes obtained under this condition. Liposomes produced under condition 1 have a higher PdI that remains acceptable (0.44 ± 0.13), just at the limit of the prediction values (0.05–0.44). A last formulation developed for peptide administration (Ducat et al., 2011) and composed of DOPE, CHEMS, CHOL and DSPE PEG 750 (43/ 21/30/6%) (formulation E, table 1) was produced and the results are presented in Fig. 5.E. Only the liposomes produced under condition 2 have size (Z-average size: 146 ± 27 nm, peak size: 211 ± 46 nm) included in the limits of the prediction values (51–221 nm). As previ ously, the size of liposomes obtained under condition 1 (Z-average: 315 ± 23 nm, peak size: 280 ± 16 nm) is higher than the size of liposomes produced under condition 2, particularly for the Z-average values (p < 0.05). As regards to the PdI, values fell perfectly within the limits and it was more or less identical for the 2 conditions. All liposome formulations produced under condition 2 have satis fying physicochemical features (size close to 200 nm and PdI < 0,4). This production condition allows the production of liposomes of varied composition. Liposomes produced under condition 1, characterized by a higher lipid concentration and a higher pressure; generally have higher size and variability than those produced under condition 2, particularly for formulation D and E. This may be due to the higher lipid concentration
formulation A, composed of SPC and CHOL (70/30%) was produced in triplicate in both production conditions defined in table 4. The size and the PdI were determined directly after the production. The results are presented in Fig. 4. The red dotted lines represent the lowest and the highest size and PdI values theoretically expected by the QbD strategy. The liposomes size is expressed in Z-average size which is defined as the harmonic intensity averaged particle diameter. This parameter, like the PdI, results from the cumulant analysis which is the simplest method to analyze the autocorrelation function generated by a DLS experiment. The Z-average size is especially representative when the sample is mono modal (Instruments, 2011). Size results can also be expressed in terms of main peak which comes from the analysis of the intensity distribution. The intensity distribution is naturally weighted according to the scattering intensity of each particle fraction or family (Instruments, 2011). It is important to note that the intensity distribu tion can be misleading as it is sensitive to the presence of large particles, aggregates or dust. For these reasons, in this study, the results are always presented both in terms of Z-average size and main peak size. Regarding the liposomes size, the values are in agreement with the predicted values (51 nm − 221 nm/0.05–0.44) excepted for the liposomes produced under condition 1 when size is expressed in Z-average size (310 ± 126 nm). However, the value of the main peak (215 ± 46 nm) fits into the predictions with a weak variability. This could mean that a small pro portion of aggregates or larger particles were considered in the zaverage value and that the main peak is more representative of the sample. This is confirmed by the PdI values. As for the size, liposomes produced under condition 1 are more polydispersed and the value (0.56 ± 0.22) doesn’t fit with the expected value. The polydispersity of lipo somes prepared under condition 2 is lower (0.36 ± 0.04). It therefore seems that the higher concentration of lipids could induce an increase in the size and a decrease in the homogeneity of the liposomes dispersion. 3.2.2. Transferability of the process to other liposome formulations More complex liposome formulations were selected to evaluate the transferability and versatility of the method with both working condi tions (Lechanteur et al., 2015; Ducat et al., 2011). First, DSPE PEG 2000 was added to formulation A. A formulation composed of SPC, CHOL and DSPE PEG 2000 (65/30/5%) (formulation B table 1) was produced in triplicate under both working conditions. The results are shown in Fig. 5 (formulation B). Liposomes have size between the expected limits (51 nm - 221 nm) whether in terms of Z-average size or in main peak size. The size of liposomes produced under condition 2 (Z-average size: 133 ± 46, peak size: 175 ± 62 nm) is, however, slightly higher but the differences are not significant (p > 0.05). Regarding the PdI, this parameter also fits with the predicted values (0.05–0.44) and is more or less the same for both production conditions (COND 1:0.32 ±
Fig. 4. (A) Size (nm) expressed in Z-average size and principal peak of the diffractogram and (B) PDI of SPC/CHOL 70/30% (w/w) liposomes produced under conditions 1 and 2. 6
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Fig. 5. Size (nm) expressed in Z-average size and principal peak of the diffractogram and PDI of liposomes formulations B, C, D and E produced under conditions 1 and 2.
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and the higher pressure as shown by Shaker et al. (Shaker et al., 2017) with an increase in the liposomes size with a higher lipid concentration and by Zhao et al. (Zhao and Temelli, 2015) with a larger particles size obtained with an increase in pressure. The formulation D contains a positively charged lipid (DOTAP) in a high proportion, which possibly could interfere with the formation of liposomes due to electrostatic repulsive forces. The particularity of the formulation E is the presence of two membranes stabilizers, CHEMS and CHOL, which could also affect the liposomes formation by increasing stiffness of the membrane (Ding et al., 2005); (Kaddah et al., 2018). The condition 1 is, however, the most interesting for the development of an industrial process as this condition offers the possibility to produce more concentrated liposomes with probably a higher encapsulation efficiency (Hamedinasab et al., 2019). It is important to note that the discussion of the results is based on the limits imposed by the design of experiments which are not necessarily the limits generally accepted for the administration of liposomes. Indeed, for some formulations, values obtained are slightly out of the limits imposed by the experimental design (213 or 221 nm) but remains acceptable for drug administration. Moreover, for the formulations which do not fit with the expected limits, an optimization of the pro duction parameters of condition 1 could be done in order to decrease the size and the PdI of liposomes.
attributed to the formation of carbonic acid by the reaction between the water of the aqueous buffer and the CO2. The maturation of the liposome dispersions of 48 h could be recommended to find back the initial pH value of the liposome dispersions. 3.4. Benchmarking with liposomes obtained by the conventional TFH method The physicochemical characteristics of the liposome formulations AE produced by the SC-CO2 method were benchmarked with those ob tained by the conventional TFH production method. Regarding the size (Fig. 7 A), liposomes produced by SC-CO2 under condition 2 have size similar to the size of the liposomes produced by the TFH method, regardless of the formulation. For condition 1, liposomes have size similar to the size obtained by the TFH method for formula tions B and C but greater for formulations A, D and E. Concerning the PdI (Fig. 7 B), liposomes produced by the supercritical process under both conditions have a higher PdI than liposomes prepared by the TFH method. The condition 2 allows the production of liposomes dispersion with a PdI close to 0.3 while the working condition 1 gives liposomes with a PdI higher than 0.3 for some formulations. The supercritical method of production allows to produce liposomes whose size are close to the size obtained by the conventional TFH method but with a higher polydispersity index. This can be explained by the fact that no additional homogenization step is used in the super critical process while the classical TFH method uses a final extrusion step in order to homogenize the size of liposomes around 200 nm. This explains the particularly low PdI values obtained by this production method (Fig. 7B).
3.3. Maturation of liposomes The size, the PdI and the pH value of the liposome dispersions was followed over the time (Fig. 6). The same trends were observed for all formulations. The size of the liposomes produced under condition 1 or 2 vary slightly over 1 week but not significantly (p > 0.05). Regarding the dispersity, the PdI of the liposomes does not evolve significantly (p > 0.05) over 1 week. For all formulations, a greater variability can be observed for liposomes produced under condition 1, may be due, once again, to the higher lipid concentration used. Finally, it was observed that the pH value of the liposome dispersions decreases significantly (<0.05) after production (T0) and increases after 2 days to reach its initial value. This initial fall in pH values just after production may be
4. Conclusion Using a QbD approach, this study allowed to determine two opera tive conditions for the production of liposomes by the PGSS process. A condition involving a low lipid concentration (C = 5 mM of lipids, v = 10 mL of dispersion, T = 80 ◦ C, pCO2 = 156 bar) which allows to produce liposomes of various compositions with physicochemical characteristics
Fig. 6. Evolution over a week of the (A) size (nm) expressed in Z-average size and principal peak of the diffractogram, (B) PDI and (C) pH of the liposomes dispersion of SPC/CHOL 70/30% (w/w) liposomes produced under conditions 1 and 2. 8
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Fig. 7. Comparison of the (A) size expressed in Z-average size and the (B) PdI of liposomes produced by the conventionnal TFH method and the supercritical method under conditions 1 and 2 for formulations A, B, C, D and E.
compatible with most of drug administrations (size close to 200 nm with PdI < 0.36). A second condition involving a high lipid concentration (C = 45 mM of lipids, V = 14 mL of dispersion, T = 80 ◦ C, PCO2 = 240 bar) was also determined by the experimental plan. This interesting condi tion from the industrial point of view (possibility of producing more concentrated batches with higher encapsulation efficiency) allows to produce liposomes of simple composition with satisfactory physico chemical characteristics. For the production of more complex formula tions like cationic liposomes with this production condition, liposomes of higher size and PdI were observed. However, unlike most conven tional methods, the supercritical process is a facile one-step method that does not require additional extrusion or ultrasonication steps. Therefore, the variability observed in these experiments remains entirely accept able. Moreover, for applications which require a lower dispersity, a final extrusion step can be added at the end of the process or an optimization of the production parameters remains possible. This supercritical CO2-assisted process is then a convenient and straightforward alternative to conventional methods to fabricate lipo somes with optimal and tunable physicochemical characteristics (size, PdI) while being adaptable to an industrial GMP process and suppressing the drawbacks associated to the use of organic solvents. This method will now be applied to the production of liposomes encapsulating hydrophobic and hydrophilic active molecules.
Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgments The authors gratefully acknowledge the FEDER Phare funds for the financial support in NANOPHARE project (884148-329407). Thanks to Dr. Pierre Lebrun and Hermane T. Avohou for the analysis of the experimental design as well as their precious help. References Akbarzadeh, A., Rezaei-Sadabady, R., Davaran, S., Joo, S.W., Zarghami, N., Hanifehpour, Y., et al., 2013. Liposome: Classification, preparation, and applications. Nanoscale Res Lett 8, 102. https://doi.org/10.1186/1556-276X-8-102. Tikshdeep, C., Sonia, A., Bharat, P., Abhishek, C., 2012. Liposome drug delivery: A review. Int J Pharm Chem Sci 1, 1103–1113. Wagner, A., Platzgummer, M., Kreismayr, G., Quendler, H., Stiegler, G., Ferko, B., et al., 2006. GMP production of liposomes – A new industral approach. J Liposome Res 16, 311–319. https://doi.org/10.1080/08982100600851086. Crommelin, D.J.A., van Hoogevest, P., Storm, G., 2020;318:256–63.. The role of liposomes in clinical nanomedicine development. What now? Now what? J Control Release. https://doi.org/10.1016/j.jconrel.2019.12.023. Patil, Y.P., Jadhav, S., 2014. Novel methods for liposome preparation. Chem Phys Lipids 177, 8–18. https://doi.org/10.1016/j.chemphyslip.2013.10.011. Sercombe, L., Veerati, T., Moheimani, F., Wu, S.Y., Hua, S., 2015. Advances and challenges of liposome assisted drug delivery. Front Pharmacol 6, 1–13. https://doi. org/10.3389/fphar.2015.00286. Woodle, M.C., 1995. Sterically stabilized liposome therapeutics. Adv Drug Deliv Rev 16, 249–265. https://doi.org/10.1016/0169-409X (95)00028-6. Harashima, H., Sakata, K., Funato, K., Kiwada, H., 1994. Enhanced hepatic uptake of liposomes through complement activation depending on the size of liposomes. Pharm Res An Off J Am Assoc Pharm Sci 11, 402–406. https://doi.org/10.1023/A: 1018965121222.
CRediT authorship contribution statement ´mie: Investigation, Visualization, Methodology, Writing Penoy Noe original draft. Grignard Bruno: Writing - review & editing. Evrard ´raldine: Brigitte: Supervision, Writing - review & editing. Piel Ge Validation, Supervision, Writing - review & editing.
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International Journal of Pharmaceutics xxx (xxxx) xxx Zhao, L., Temelli, F., 2015. Preparation of liposomes using a modified supercritical process via depressurization of liquid phase. J Supercrit Fluids 100, 110–120. https://doi.org/10.1016/j.supflu.2015.02.022. Türeli, N.G., Türeli, A.E., 2020. Upscaling and GMP production of pharmaceutical drug delivery systems. Drug Deliv Trends 3, 215–229. https://doi.org/10.1016/b978-012-817870-6.00011-0. Bellefroid, C., Lechanteur, A., Evrard, B., Mottet, D., Debacq-Chainiaux, F., Piel, G., 2019. In vitro skin penetration enhancement techniques: A combined approach of ethosomes and microneedles. Int J Pharm 572, 118793. https://doi.org/10.1016/j. ijpharm.2019.118793. Instruments M. Dynamic Light Scattering, common terms defined 2011:1–6. Lechanteur, A., Furst, T., Evrard, B., Delvenne, P., Hubert, P., Piel, G., 2015. Development of anti-E6 pegylated lipoplexes for mucosal application in the context of cervical preneoplastic lesions. Int J Pharm 483, 268–277. https://doi.org/ 10.1016/j.ijpharm.2015.02.041. Ducat, E., Deprez, J., Gillet, A., No¨ el, A., Evrard, B., Peulen, O., et al., 2011. Nuclear delivery of a therapeutic peptide by long circulating pH-sensitive liposomes: Benefits over classical vesicles. Int J Pharm 420, 319–332. https://doi.org/10.1016/j. ijpharm.2011.08.034. Edwards, K., Johnsson, M., Karlsson, G., Silvander, M., 1997. Effect of polyethyleneglycol-phospholipids on aggregate structure in preparations of small unilamellar liposomes. Biophys J 73, 258–266. https://doi.org/10.1016/S00063495 (97)78066-4. Shaker, S., Gardouh, A., Ghorab, M., 2017. Factors affecting liposomes particle size prepared by ethanol injection method. Res Pharm Sci 12, 346–352. https://doi.org/ 10.4103/1735-5362.213979. Zhao, L., Temelli, F., 2015. Preparation of liposomes using supercritical carbon dioxide via depressurization of the supercritical phase. J Food Eng 158, 104–112. https:// doi.org/10.1016/j.jfoodeng.2015.03.004. Ding, W.-X., Qi, X.R., Li, P., Maitani, Y., Nagai, T., 2005. Cholesteryl hemisuccinate as a membrane stabilizer in dipalmitoylphosphatidylcholine liposomes containing saikosaponin-d. Int J Pharm 300, 38–47. https://doi.org/10.1016/j. ijpharm.2005.05.005. Kaddah, S., Khreich, N., Kaddah, F., Charcosset, C., Greige-Gerges, H., 2018. Cholesterol modulates the liposome membrane fluidity and permeability for a hydrophilic molecule. Food Chem Toxicol 113, 40–48. https://doi.org/10.1016/j. fct.2018.01.017. Hamedinasab, H., Rezayan, A.H., Mellat, M., Mashreghi, M., Jaafari, M.R., 2019. Development of chitosan-coated liposome for pulmonary delivery of Nacetylcysteine. Int J Biol Macromol 156, 1455–1463. https://doi.org/10.1016/j. ijbiomac.2019.11.190.
Karn, P.R., Cho, W., Hwang, S.J., 2013. Liposomal drug products and recent advances in the synthesis of supercritical fluid-mediated liposomes. Nanomedicine 8, 1529–1548. https://doi.org/10.2217/nnm.13.131. Samad, A., Sultana, Y., Aqil, M., 2007. Liposomal drug delivery systems: An update review. Curr Drug Deliv 4, 297–305. https://doi.org/10.2174/ 156720107782151269. Bridson, R.H., Santos, R.C.D., Al-Duri, B., McAllister, S.M., Robertson, J., Alpar, H.O., 2006. The preparation of liposomes using compressed carbon dioxide: strategies, important considerations and comparison with conventional techniques. J Pharm Pharmacol 58, 775–785. https://doi.org/10.1211/jpp.58.6.0008. Meure, L.A., Foster, N.R., Dehghani, F., 2008. Conventional and dense gas techniques for the production of liposomes: A review. AAPS PharmSciTech 9, 798–809. https://doi. org/10.1208/s12249-008-9097-x. Wagner, A., Vorauer-Uhl, K., 2011. Liposome technology for industrial purposes. J Drug Deliv 2011, 1–9. https://doi.org/10.1155/2011/591325. Shah, V.M., Nguyen, D.X., Patel, P., Cote, B., Al-Fatease, A., Pham, Y., et al., 2019. Liposomes produced by microfluidics and extrusion: A comparison for scale-up purposes. Nanomed Nanotechnol 18, 146–156. https://doi.org/10.1016/j. nano.2019.02.019. Parhi, R., Suresh, P., 2013. Supercritical fluid technology: A review. J Adv Pharm Sci Technol 1, 13–36. https://doi.org/10.14302/issn.2328-0182.japst-12-145. Andonova, V., Chandra, Sekhar G., 2016. Supercritical fluid technology: A promising approach for preparation of nano-scale drug delivery systems. J Appl Sci Eng Methodol 2, 239–242. William, B., No´ emie, P., Brigitte, E., G´eraldine, P., 2020. Supercritical fluid methods: An alternative to conventional methods to prepare liposomes. Chem Eng J 383, 123106. https://doi.org/10.1016/j.cej.2019.123106. Kankala, R.K., Zhang, Y.S., Bin, Wang S, Lee, C.H., Chen, A.Z., 2017;6.. Supercritical fluid technology: An emphasis on drug delivery and related biomedical applications. Adv Healthc Mater. https://doi.org/10.1002/adhm.201700433. Castor TP. United States Patent US005554382A Methods and apparatus for making liposomes 1996;II:5–9. Girotra, P., Singh, S.K., Nagpal, K., 2013. Supercritical fluid technology: a promising approach in pharmaceutical research. Pharm Dev Technol 18, 22–38. https://doi. org/10.3109/10837450.2012.726998. Lesoin, L., Crampon, C., Boutin, O., Badens, E., 2011. Preparation of liposomes using the supercritical anti-solvent (SAS) process and comparison with a conventional method. J Supercrit Fluids 57, 162–174. https://doi.org/10.1016/j.supflu.2011.01.006. Lesoin, L., Crampon, C., Boutin, O., Badens, E., 2011. Development of a continuous dense gas process for the production of liposomes. J Supercrit Fluids 60, 51–62. https:// doi.org/10.1016/j.supflu.2011.04.018. Castor TP. Method and apparatus for making liposomes containing hydrophobic drugs – WO 96/15774 1996.
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