Article pubs.acs.org/IECR
A Supercritical Fluid-Based Process for the Production of Fluorescein-Loaded Liposomes R. Campardelli, P. Trucillo, and E. Reverchon* Department of Industrial Engineering, University of Salerno, Via Giovanni Paolo II, 132, 84084 Fisciano, SA, Italy S Supporting Information *
ABSTRACT: Fluorescein is a synthetic dye used as a marker to monitor the absorption of liposomes into tissues, but its efficient entrapment is still challenging. In this work, we apply a supercritical fluid process that allows the production of liposomes with a high drug encapsulation efficiency. The effects of operating parameters such as the diameter of the injector, the concentration of phospholipids, and the concentration of fluorescein were studied. Liposomes with mean diameters down to ∼300 nm were obtained with fluorescein encapsulation efficiencies of ≤96%. In detail, the increase in the concentration of phospholipids produced a relative increase in liposome mean diameter and particle size distribution width. With an increase in fluorescein theoretical loading, instead, a reduction in liposome mean diameters and standard deviation was obtained. Smaller liposomes were produced using smaller injector diameters; however, the reduction of the injector diameter from 80 to 40 μm caused a reduction in the fluorescein encapsulation efficiency from 96 to 58%. Suspensions remained stable for more than four months, without drug leakage. has been encapsulated into liposomes as a model drug6 and as a marker to study permeation of liposomes through skin7,8 or to detect liposomes in ocular blood flow after administration.9 However, an efficient production of liposomes encapsulating fluorescein is still challenging. Indeed, conventional techniques used to prepare liposomes suffer from some common drawbacks, such as difficulties in controlling liposome size and distribution and low encapsulation efficiencies, especially for hydrophilic compounds. Results reported in the literature showed fluorescein encapsulation efficiencies of 80%). These results can be achieved thanks to the different approach to liposome production of this new process. The basic principle of SuperLip is to produce first the water-based droplets, and then, the liposomes are formed around them.38 Therefore, the aim of this work is to extend SuperLip application to the production of liposomes entrapping fluorescein, trying to increase its encapsulation efficiency while controlling liposome dimensions in the submicrometric range. SuperLip process parameters, such as the diameter of the injector used for water atomization, phospholipid concentration, and fluorescein concentration in the water solution, will be studied to obtain liposomes of a controlled size and distribution with a high fluorescein encapsulation efficiency. The stability during the time of liposomes loaded with fluorescein will be also investigated.
Figure 1. SuperLip apparatus layout: F, formation vessel; S1, saturator; S2, separator; R, suspension recovery.
The mixing between the ethanolic solution and CO2 produces an expanded liquid that is, then, delivered through a capillary tube (1/8 in. external diameter, 0.028 in. wall thickness) inside the high-pressure vessel (P). In the same formation vessel, a water solution is continuously sprayed through a nozzle. Water atomization produces small water droplets that are the basis for the formation of liposomes. The high-pressure vessel is a stainless steel tank with an internal volume of 0.5 dm3. A stainless steel separator (S2) is used to release CO2 through a decompression step, and the CO2 flow rate at the exit of the separator is measured using a rotameter (ASA, model N.52500). 2.3. Liposome Characterization: Morphology, Size Distribution, and Fluorescein Assay. The morphology of produced liposomes was studied using a field emission-scanning electron microscope (model LEO 1525, Carl Zeiss SMT AG, Oberkochen, Germany). Samples were prepared by spreading a drop of the liposome suspension over an adhesive carbon tab placed on an aluminum stub. The drop was left to dry in air overnight. The sample was then covered with gold, using a sputter coater (thickness of 250 Å, model B7341, Agar Scientific, Stansted, U.K.). A dynamic laser scattering (DLS) instrument (model Zetasizer Nano S) was used to measure the particle size distribution (PSD), mean diameter (MD), standard deviation (SD), and ζ potential of the liposomes loaded with fluorescein. The suspension was analyzed as produced, without any dilution step or addition of stabilizing agents. Three measurements of the same sample were performed for all produced suspensions. Nanoparticle tracking analysis (NTA) was also used to provide complementary information about the liposome size distribution. In particular, this technique, because of the possibility of on-line monitoring of liposomes in suspension, provides information about the liposome concentration in the sample. A laser beam is used to irradiate the sample. The particles in suspension scatter the light and can be visualized via a long working distance microscope onto which a video camera is mounted. The camera captures a video file of the particles moving under Brownian motion. In this way, the Nanoparticle Tracking Analysis (NTA) software tracks many particles
2. MATERIALS, APPARATUS, AND METHODS 2.1. Materials. L-α-Phosphatidylcholine from egg yolk (PC) was supplied by Sigma-Aldrich (99% pure, lyophilized powder). Ethanol used to dissolve phospholipids was purchased from Sigma-Aldrich (≥99.8%). Carbon dioxide (CO2) was provided by SON (>99.4% pure). Bidistilled water, used in all formulations, was provided by Carlo Erba Reagents. Fluorescein sodium salt powder used as a fluorescent tracer was provided by Sigma-Aldrich. All the compounds were used as received. 2.2. Description of the SuperLip Apparatus. The SuperLip apparatus is depicted in Figure 1. It consists of three different feed lines that deliver compressed CO2 and an ethanolic solution to the saturator (S1), and a water solution to the high-pressure vessel. CO2 is pumped using a Lewa Eco flow pump (model LDC-M-2, max). The ethanolic solution in which phospholipids are dissolved and the water/fluorescein solution are pumped using two high-pressure precision pumps (model 305, Gilson). CO2 and the ethanolic solution are delivered at a fixed gas-to-liquid ratio (GLR of 2.4 mass basis) to the saturator. The saturator is a high-pressure static mixer with an internal volume of ∼0.15 dm3, and it is filled with stainless steel perforated saddles and thermally heated by thin band heaters. 5360
DOI: 10.1021/acs.iecr.5b04885 Ind. Eng. Chem. Res. 2016, 55, 5359−5365
Article
Industrial & Engineering Chemistry Research individually, providing an evaluation of the concentration of the sample. NTA measurements were performed using a NanoSight LM20 instrument (NanoSight, Amesbury, U.K.), equipped with a sample vessel and a 640 nm laser. The samples were injected in the vessel using sterile syringes (BD Discardit II) until the liquid reached the tip of the nozzle. All measurements were performed at room temperature. Data were analyzed using NTA 2.0 Build 127 software. The samples were measured for 40 s with manual shutter and gain adjustments. Three measurements of the same sample were performed for all the produced suspensions. To determine encapsulation efficiency, the procedure for evaluation of the drug leakage was adopted, as reported in the literature.39 The liposome suspension was centrifuged at 6500 rpm for 45 min at 4 °C. Then, the concentration of fluorescein in the water supernatant (mgsup) was analyzed using UV−vis spectroscopy, at a wavelength of 515 nm. The encapsulation efficiency (EE) was calculated as the complement to 100% of the percentage of drug present in the supernatant, according to the following equation:
Figure 2. High-pressure vapor−liquid equilibria for the CO2/ethanol/ water system at 40 °C in the pressure range of 100−200 bar, adapted from ref 40. The operating point of the experiments is reported as a red dot.
⎛ mgsup ⎞ ⎟ EE = 100⎜⎜1 − mg loaded ⎟⎠ ⎝
mg/mL. Fluorescein was dissolved in 300 mL of water at a concentration of 0.017 mg/mL, to obtain a 1% by weight theoretical loading. An injector with a diameter of 80 μm was used for atomization of the water solution in the formation vessel. The experiment was successful, and liposomes with a nanometric diameter and a sharp PSD were produced. Indeed, the DLS-measured mean diameter (MD) of fluorescein-loaded liposomes was ∼289 ± 50 nm. These data are summarized in Table 1. A field emission-scanning electron microscopy (FESEM) image of the produced liposomes is shown in Figure 3; they are characterized by an irregular spherical shape, with a rough surface. Furthermore, it is possible to see that their mean diameter seems smaller than that measured by DLS (∼200 nm; see the reference bar in the SEM image). This result can be attributed to the shrinkage of the vesicles during the preparation of the sample for electronic microscopy. Fluorescein was entrapped successfully in these liposomes, with a high encapsulation efficiency (90%), as reported in Table 1. This result confirms that a water solution sprayed in the formation vessel was efficiently covered by phospholipids, leading to the formation of fluorescein-loaded liposomes. This result is significantly better when compared to those obtained for fluorescein encapsulation, reported in the literature,13,41 confirming the high potential of this process in the encapsulation of hydrophilic compounds. Considering the success of the first encapsulation test, a systematic study was performed to understand how SuperLip operating parameters affect the entrapment of the drug in the liposomes. Experiments with higher PC concentrations were performed, and the results are summarized in Table 1. The PC concentration was increased from 5 to 15 mg/mL; the other operating conditions were left unchanged (pressure of 100 bar, 40 °C, GLR of 2.4). A stable liposome suspension was produced at all PC concentrations, except for the test performed at 15 mg/mL. In this case, the experiment was unsuccessful because of the blockage of the injector. The explanation of this result is that this PC concentration caused the deposition of some of the phospholipids on the tip of the injector in the formation vessel, hindering the atomization of water. From the data reported in Table 1 for the other experiments, it is possible to observe that the increase in PC
where mgloaded is the theoretical fluorescein content dissolved in the water solution atomized in the formation vessel. Each encapsulation efficiency test was repeated in triplicate, and the results are the means over three different measurements.
3. RESULTS AND DISCUSSION The basic idea of SuperLip is to generate first the water droplets by atomization, i.e., the inner core of liposomes, and then, a lipid layer is formed around them in an expanded liquid environment.37 The expanded liquid is formed by supercritical CO2 and the ethanol/phospholipid solution and with respect with an ordinary liquid has a reduced viscosity and density and enhanced diffusivity. Droplets in contact with the expanded liquid, as previously discussed,37,38 are covered by phospholipids during their time in the formation vessel. Liposomes are formed because of favorable interactions between the expanded liquid phospholipid mixture and the atomized water-based droplets. At the end of this process, droplets fall in a continuous water pool located at the bottom of the vessel, in which they maintain their identity and can rearrange in the typical doublelayer structure of liposomes. The experiments discussed in this work were performed at 100 bar and 40 °C. The CO2 flow rate was set at 6.5 g/min, and the ethanol solution flow rate was set at 3.5 mL/min, to obtain a GLR in the saturator of 2.4 (mass basis). The water solution flow rate was set at 10 mL/min. Selecting these operating conditions and assuming that the presence of PC does not modify the high-pressure water−ethanol−CO2 ternary equilibrium, the operating point of the experiments in the ternary diagram of Figure 240 is located inside the miscibility hole, i.e., the area of the diagram in which the compounds are not miscible, ensuring that the water solution atomized in the vessel is not extracted by the expanded liquid. 3.1. Effect of Injector Diameter and of Fluorescein Theoretical Loading. In a first experiment, liposomes encapsulating fluorescein at a loading of 1% {weight of fluorescein/PC [% (w/w)]} were produced. Phospholipids were dissolved in 100 mL of ethanol at a concentration of 5 5361
DOI: 10.1021/acs.iecr.5b04885 Ind. Eng. Chem. Res. 2016, 55, 5359−5365
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Industrial & Engineering Chemistry Research
Table 1. Mean Diameters (MDs) and Encapsulation Efficiencies (EE%) of Liposomes Loaded with 1% Fluorescein, Using Injectors with Different Diameters and Different PC Concentrationsa injector diameter (μm) 80
60
40
a
MD ± SD (nm) PDI EE% MD ± SD (nm) PDI EE% MD ± SD (nm) PDI EE%
5 mg/mL PC
7.5 mg/mL PC
10 mg/mL PC
12 mg/mL PC
15 mg/mL PC
289 ± 50 0.17 90 260 ± 48 0.18 61 249 ± 46 0.18 58
283 ± 58 0.20 92 262 ± 50 0.19 58 −
305 ± 91 0.30 90 267 ± 53 0.20 60 −
298 ± 89 0.30 81 268 ± 69 0.26 56 −
− − − − − − −
−
−
−
−
Data are not reported for the experiments that could not be performed.
enlargement of the PSD curve, whereas the MD of the suspension was not significantly affected. A smaller injector diameter (40 μm) was used to confirm the trend observed previously. Only an experiment was possible using this injector (see Table 1); indeed, for PC concentrations of >5 mg/mL, the injector blockage systematically took place. Using this injector at 5 mg/mL, we produced liposomes with a mean diameter of 249 ± 46 nm, measured by DLS (Table 1). It is possible to compare the results obtained at the same PC concentration (5 mg/mL) using different injector diameters, as reported in Figure 4. In this figure, it is possible to see a Figure 3. FESEM image of liposomes loaded with fluorescein at a 1% theoretical loading.
concentration in the ethanol solution did not produce a significant effect on the mean diameter of liposomes. Indeed, with an increase in PC concentration from 5 to 12 mg/mL, the liposome mean diameter, measured by DLS, slightly increased from 289 ± 50 to 298 ± 89 nm; an enlargement of PSD can instead be noted (see standard deviations). Considering the encapsulation efficiency, fluorescein was entrapped for values larger than 80% (measured using a UV− vis spectrophotometer). This efficiency remained practically constant when the concentration of PC was increased from 5 to 10 mg/mL (see Table 1). The overall results suggest that a sufficient amount of lipid for droplet coverage is present in the formation vessel at 5 mg/mL, and therefore, a further increase to 7.5 and 10 mg/mL did not produce an improvement in EE. The reduction of EE to 81% is observed at 12 mg/mL, a condition very similar to the operability threshold of 15 mg/mL (injector blockage). Near this condition, the partial blockage of the injector may cause instability of the jet. Experiments using injectors of 60 and 40 μm were then performed. For each injector, the same set of experiments with different PC concentrations, from 5 to 15 mg/mL, was performed. The other operating parameters were kept constant (pressure 100 bar, temperature 40 °C, GLR = 2.4). These results are summarized in Table 1. Also for the case of 60 μm injector, at the PC concentration of 15 mg/mL the experiment was not successful and the explanation is the same as in previous case. Considering PC concentrations lower than 15 mg/mL, successful production of liposome suspensions was obtained. Data reported in Table 1 show that using the 60 μm injector, liposomes with a smaller mean diameter, around 260 nm, were obtained, according to DLS measurements. Also in this case the increase of PC concentration had an effect on the
Figure 4. Comparison of PSDs of liposomes produced using different injector diameters, at 5 mg/mL PC.
progressive reduction in liposome mean diameter when smaller injectors are used. In summary, the three injectors (80, 60, and 40 μm) produced liposomes with different mean diameters of ∼300, 260, and 240 nm, respectively. Furthermore, the smaller the injector, the narrower the PSDs. Results obtained previously confirm that, in this process, the diameter of liposomes does not depend on PC concentration but is related to the dimension of the injector used for water solution atomization; i.e., it influences the diameter of the atomized droplets in the formation vessel. Indeed, a reduction of the diameter of the injector can improve the efficiency of water atomization, allowing the generation of a water spray formed by smaller droplets. The fact that, generally, when the turbulence of the spray is increased, as in the case of the reduction of the injector diameter, better control of the PSD can be obtained also must be considered. Data reported in Table 1 show that the fluorescein encapsulation efficiency decreases using smaller injectors. Indeed, using the 60 μm injector, fluorescein was entrapped 5362
DOI: 10.1021/acs.iecr.5b04885 Ind. Eng. Chem. Res. 2016, 55, 5359−5365
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Industrial & Engineering Chemistry Research with an efficiency of ∼60%. As observed in the case of experiments performed using the 80 μm injector, at PC concentrations between 5 and 10 mg/mL, the encapsulation efficiency was practically constant; a reduction to 56% was instead obtained for a PC concentration near the threshold. Using the 40 μm injector, the encapsulation efficiency was 58%. These results can be explained considering that using an injector with a smaller diameter (or partially blocked injector) an increase in the velocity of the liquid jet exiting the nozzle is obtained; therefore, water droplets, falling along the formation vessel, have an impact on the surface of the water bulk at a higher velocity. This phenomenon may cause the disruption of the lipid layer and can produce a consequent leakage of the drug content. A set of experiments with different fluorescein loadings was also performed; the results are reported in Table 2. The Figure 5. Stability study of fluorescein-loaded liposomes.
Table 2. SuperLip Size Distribution Data and Encapsulation Efficiencies of Liposomes Loaded with 1−9% (w/w) Fluorescein fluorescein theoretical loading (%) 0 1 3 6 9
MD ± SD (nm)
PDI
real loading (%)
EE%
± ± ± ± ±
0.21 0.17 0.20 0.19 0.20
− 0.90 2.88 5.58 7.83
− 90 96 93 87
291 289 277 269 268
62 50 55 51 53
remained relatively constant for more than four months. Also, the liposome ζ potential was always approximately −20 mV, indicating a good suspension stability. Indeed, the general dividing line between stable and unstable suspensions is generally taken to be +30 or −30 mV with particles having ζ potentials outside of these limits normally considered stable.43 The ζ potential of liposomes produced in this paper is near the region of stability. The concentration of fluorescein in the external medium of the suspension was measured during storage; data collected confirmed that there was not significant leakage of fluorescein from liposomes. The NTA technique was used to measure the concentration of liposomes in the suspension and gain further indications about their diameter. The particles detected by the laser beam are counted by the NTA, yielding the sample concentration in terms of the number of particles per milliliter of suspension. A typical frame acquired for the sample obtained at 5 mg/mL with a fluorescein theoretical loading of 1% is proposed in Figure 6. The liposome population is represented by white
operating conditions for the encapsulation tests were selected considering previous experiments (5 mg/mL PC, using the 80 μm injector). These conditions were selected because they allowed the highest fluorescein encapsulation efficiency in the previous experiments (92%). Empty PC liposomes were also produced under the same process conditions, to verify the effect of the presence of a solute on liposome PSD. Liposomes produced using different fluorescein theoretical loadings were characterized by a mean diameter of ∼280 nm, which was not evidently affected by the different amounts of fluorescein dissolved in the water phase. The presence of the solute did not produce any valuable effect on liposome PSD. The possible explanation is that the presence of the solute did not alter the atomization efficiency. The fluorescein encapsulation efficiency was high in all cases. Indeed, encapsulation efficiencies between 87 and 96% were obtained with an increase in the theoretical fluorescein loading from 1 to 9%. This result is extremely relevant because SuperLip overcomes the drawback of traditional methods used for liposome preparation, in which, in most of the cases, the encapsulation efficiency is negatively affected by an increase in the theoretical loading.42 The liposome suspension produced using the SuperLip process still contains ethanol residues that can be removed using a supercritical process, as reported in a recent publication.6 3.2. Characterization of Liposome Suspensions. Liposome suspensions produced by SuperLip, under the optimized operative conditions (80 μm injector and 5 mg/mL PC), were also characterized in terms of liposome stability over time and concentration of liposomes in the final suspension. Liposome suspensions were stored at 4 °C, and PSD measurements were performed at fixed time intervals for more than 120 days using DLS. The results obtained are shown in Figure 5. The mean diameter of fluorescein-loaded liposomes
Figure 6. Example of a frame from NTA analysis of liposome suspensions produced at 5 mg/mL PC and a 1% fluorescein theoretical loading.
points on a dark background. Quantitative data showed that the suspension produced is characterized by a liposome concentration of ∼280 million vesicles per milliliter of suspension. The software can also calculate the liposome hydrodynamic diameters using the Stokes−Einstein equation smoothing the results and eliminating possible aggregates. NTA measurement can be even more accurate than DLS. The NTA-measured PSD of a SuperLip suspension is reported in Figure 7b, and a 5363
DOI: 10.1021/acs.iecr.5b04885 Ind. Eng. Chem. Res. 2016, 55, 5359−5365
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Figure 7. Comparison of PSD distribution data obtained with DLS as diameter vs intensity (a) and NTA as diameter vs concentration (b). SuperLip liposomes produced at 80 μm and 5 mg/mL PC.
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comparison of distribution data of the same sample, obtained using the DLS technique, is shown in Figure 7a. The mean size obtained by NTA was ∼166 ± 60 nm. Both techniques show that the suspension is characterized by a relatively narrow distribution; nevertheless, it is possible to observe a tail of the DLS size distribution toward larger sizes, which can be mostly due to the contribution of large particles to the overall scattering.44 Indeed, the DLS technique considers as single particles also the scattering produced by aggregates; therefore, the overall mean diameter measurement could be modified by the contribution of aggregates. Because of the accuracy of NTA measurement, it can be observed that liposome size distributions of the suspensions produced by SuperLip are even narrower than the data obtained using DLS, confirming the good control of PSD allowed by this process.
Corresponding Author
*E-mail:
[email protected]. Phone: +39089964116. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS The authors acknowledge Dr. Roberto Santoliquido of ALFATEST s.r.l. (Italy) for his kind help with NTA Nanosight measurements.
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REFERENCES
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4. CONCLUSIONS In this work, the efficient production of liposomes loaded with fluorescein characterized by a nanometric diameter and a narrow PSD was demonstrated using the SuperLip process. The analysis of the process parameters allowed us to obtain encapsulation efficiencies of ≤97%. The diameter of the injector had a significant effect on fluorescein encapsulation efficiency. The smaller the injector, the lower the encapsulation efficiency. The concentration of phospholipids in the supercritical solution and the concentration of fluorescein in the aqueous phase did not produce a significant effect on the fluorescein encapsulation efficiency, which remained constant and very high. Liposomes were stable during storage (4 months), retaining the drug in the inner compartment. Future developments of this work will regard the possibility of enlarging the applicability of this process to other hydrophilic drugs of interest in the nanomedical field.
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AUTHOR INFORMATION
ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.iecr.5b04885. This movie shows the sequence of NTA-frames (one of them is Figure 6) describing the live Brownian motion of liposomes in suspension, captured by the beam of the instrument. (AVI) 5364
DOI: 10.1021/acs.iecr.5b04885 Ind. Eng. Chem. Res. 2016, 55, 5359−5365
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Industrial & Engineering Chemistry Research
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DOI: 10.1021/acs.iecr.5b04885 Ind. Eng. Chem. Res. 2016, 55, 5359−5365
