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Effect of the concentration process on unloaded and doxorubicin loaded liposomal dispersions
Accepted Manuscript Effect of the concentration process on unloaded and doxorubicin loaded liposomal dispersions Diego Romano Perinelli, Marco Cespi, Filippo Rendina, Giulia Bonacucina, Giovanni Filippo Palmieri PII: DOI: Reference:
S0378-5173(19)30144-9 https://doi.org/10.1016/j.ijpharm.2019.02.021 IJP 18159
To appear in:
International Journal of Pharmaceutics
Received Date: Revised Date: Accepted Date:
22 January 2019 12 February 2019 13 February 2019
Please cite this article as: D.R. Perinelli, M. Cespi, F. Rendina, G. Bonacucina, G.F. Palmieri, Effect of the concentration process on unloaded and doxorubicin loaded liposomal dispersions, International Journal of Pharmaceutics (2019), doi: https://doi.org/10.1016/j.ijpharm.2019.02.021
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Effect of the concentration process on unloaded and doxorubicin loaded liposomal dispersions Diego Romano Perinelli 1, Marco Cespi 1, Filippo Rendina 2, Giulia Bonacucina Giovanni Filippo Palmieri 1,
1,
* and
1
School of Pharmacy, Via Gentile III da Varano, University of Camerino, 62032 Camerino, Italy Janssen-Pharmaceutical Company of Johnson and Jonhson, via C. Janssen, Borgo S. Michele, Latina, Italy * Correspondence: [email protected]; Tel.: +39-0737402289 2
Abstract Liposomes are unilamellar nanovesicles made of phospholipids of great interest as drug delivery carriers, able to encapsulate both hydrophilic and lipophilic compounds. Some liposomal formulations have reached the market, including the doxorubicin loaded PEGylated liposomal dispersion Doxil®. The aim of the work was to investigate the possibility of concentrating liposomes through the ultrafiltration process under nitrogen pressure, using Doxil® formulation as a model. The concentrated liposomal dispersions (4x and 8x) obtained from Doxil® were characterised in terms of size evolution (dynamic light scattering), morphology (cryo-TEM) and thermal behaviour (microcalorimetry, mDSC and high-resolution ultrasonic spectroscopy, HR-US) and compared to the unloaded liposomes of the same composition. The ultrafiltration process resulted to be effective in concentrating both loaded and unloaded liposomal dispersions, which showed a particle size and thermal properties comparable to those of the non concentrated ones. Moreover, all liposomal dispersions did not show any remarkable variation in term of particle size distribution and morphology for at least 8 weeks after concentration. Altogether, results demonstrated the effectiveness in using ultrafiltration as methodology to concentrate both loaded and unloaded liposomes without affecting the quality of the processed product. Keywords: PEGylated liposomes, Doxil®, microcalorimetry, high-resolution ultrasound spectroscopy, dynamic light scattering, thermal analysis
1. Introduction Liposomes are vesicles of size generally between 50-500 nm and made of a single or multi lipid bilayer, mainly composed of phospholipids (Zylberberg and Matosevic, 2016). In the last decades, they have been widely investigated as versatile drug delivery carriers thanks to their ability to incorporate hydrophilic drugs and biotherapeutics (nucleic acid, proteins, peptides and vaccines) inside their inner aqueous cavity as well as hydrophobic drugs intercalated in the lipid bilayer (Allen and Cullis, 2013; Fan and Zhang, 2013). Liposomes showed some advantages as drug delivery nanocarriers since they increase the stability and reduce the systemic toxicity of the encapsulated compounds, especially after parental administration (Sercombe et al., 2015). Several modifications have been performed on liposomal vesicles such as PEGylation to avoid the recognition by the immune cells, thus, prolonging their residential time into the circulation (stealth liposomes) (Lasic and Martin, 2018; Milla et al., 2012) and/or functionalization with ligands to improve their delivery properties at the targeted site (Palankar et al., 2008). These modifications led to the development of “the so-called” second and third generation of liposomes and they have been amply characterised in the last decades, especially for the delivery of several antitumor drugs (Belfiore et al., 2018; Riaz et al., 2018). Among them, some liposomal formulations have reached the market such as those containing doxorubicin (Doxil®, DaunoXome®, Myocet®), cytarabine/AraC (Depocyt®), vincristine (Marquibo®), mifarmurtide (Mepact®) and irinotecan (Onivyde®) (Bulbake et al., 2017). Particularly, the first approved liposomal dispersion approved for the clinical use was Doxil®, a formulation containing doxorubicin entrapped in liposomes for the treatment of the ovarian and breast cancer, and Kaposi’s sarcoma. This liposomal formulation has a stable loading of doxorubicin via a transmembrane ammonium sulfate pH-dependent gradient, responsible also for drug release at the tumor site (Barenholz, 2012). Recently, after the introduction on the market of follow-on products (sometimes referred to as ‘nanosimilars’), the scientific interest on Doxil® has been renewed and some papers, aimed at clarifying some chemical-physical aspects on this liposomal formulation, have been published. These studies have been focused on the investigation of the crystalline nature of the entrapped doxorubicin sulfate nanocrystals (Schilt et al., 2016), on the thermal behaviour (Perinelli et al., 2017; Wei et al., 2016) and morphology and size of liposomes (Wibroe et al., 2016). Doxil is also a suitable model of a PEGylated liposomal dispersion to study technological processes through which liposomes could be processed. One of the less investigated is the concentration. Concentration of liposomal dispersions can be achieved by different methods such as: ultracentrifugation, ultrafiltration and reconstitution in a smaller volume after freeze-drying (Magin and Chan, 1987). Despite, there is no an optimal procedure for liposomes concentration, ultracentrifugation is the most common but, event at the lowest effective
speed, it could induce vesicle fusion and changes in the morphology (Devi et al., 2010; Heeremans et al., 1995). Ultrafiltration has been largely used in liposomal drug delivery formulations to remove the un-encapsulated drug (Li et al., 2009; Zalba et al., 2012; Zhong et al., 2012), but no study, according to our knowledge, was aimed at investigating the effect of the concentration process by ultrafiltration on unloaded and drug loaded liposomal dispersions in terms of size, stability and thermal behaviour. The aim of the work was to explore the possibility of using the ultrafiltration under nitrogen pressure process as a strategy to concentrate a doxorubicin loaded PEGylated liposomal dispersion (Doxil®) and to characterise the concentrated liposomal dispersions in terms of size evolution, morphology and thermal behaviour. Results obtained from doxorubicin loaded dispersions were compared to those from unloaded liposomes of the same composition as reference. 2. Materials and Methods 2.1. Materials Loaded liposomes (Doxil®) and unloaded liposomes have been supplied by Janssen Pharmaceutical Companies of Johnson & Johnson. Doxil® is a marketed liposomal formulation of doxorubicin (2 mg/ml) composed of a mixture of hydrogenated soy phospholipids, cholesterol and methyldiasteroyl-phosphoethanolamine-polyethylenglicole 2kDa (DSPE-PEG) sodium salt at the weight ratio 3:1:1. Unloaded liposomes have the same qualitative and quantitative composition of loaded liposomes, except for the encapsulation of doxorubicin. All liposomal dispersions are prepared in 9.4% w/w sucrose (as main component) aqueous solution. 2.2. Liposomal formulation concentration by ultrafiltration Unloaded liposomes and loaded liposomes were concentrated by ultrafiltration performed by applying a nitrogen positive pressure, which forced the dispersion medium to pass through a polyether sulfone membrane 100 kDa (Sartorius, Gottingen, Germany). Liposomes were retained and separated from the filtrated passed through the membrane. The ultrafiltration was performed under magnetic stirring using the ultrafiltration stirred cell (Amicon ®, USA), as shown in Figure 1, in a temperature controlled chamber (10 °C), at different nitrogen operating pressures (1.5, 2, and 5 bar) to select the best condition to be applied for this process. The ultrafiltration was performed starting from 40 ml until the final volume was reduced to 10 ml or 5 ml corresponding to a 4x and 8x liposomes concentration in the final dispersion.
2.3. Liposomal formulation characterization by dynamic light scattering (DLS) DLS analyses were performed using a Malvern Zetasizer nanoS (Malvern instrument Worcestershire, UK), detecting scattered light at 173°. The scattered signals (counts) were recorded at 25 °C after a thermal equilibration of 180 s. Results are reported as Z-average (diameter, nm) and polydispersity index (PDI) as the average of 3 measurements of 11 runs. All analyses were performed in triplicate. To study the possible effect exerted by concentration process on liposome dimensions, particle size of concentrated liposomal dispersion (4x and 8x) was measured before and after dilution with different aqueous media (9.4% sucrose, 0.9% NaCl and ultrapure water) and compared with that obtained from non concentrated. For each media, different dilution ratios were performed (1:2; 1:5; 1:10 1:25 and 1:50) and samples were analysed after 5 minutes. The viscosity of the aqueous dispersion media used for each analysis was adjusted on the base of the effective concentration of the solute as provided by Malvern software. A summary of the viscosity of the dispersant used for each analysis can be found in Table S1. In addition, DLS was employed to assess the particle size and particle size distribution stability over time of the concentrated liposomal dispersions. Liposomal concentrated samples were stored at 4 °C up to 2 months and analysed by DLS at different timepoints (1 week, 2 week, 3 week, 4 week and 8 weeks).
2.4. Liposomal characterization by microcalorimetry (mDSC) 0.750 g of concentrated liposomal dispersion and an equal amount of 9.4% w/v sucrose solution as reference were filled in a Hallostey calorimetric cells and equilibrated at 5 °C for 20 min. Then, a heating and a cooling ramp from 5 °C to 80 °C at 1 °C /min were performed. Calorimetric analyses were carried out using a microDSC III (Setaram, France). The temperature (Tm, °C) and enthalpy (ΔH, J/g of solution) were calculated from the peak and the area of the transition using the software of the instrument (Setsoft2000, Setaram) according to the tangent method. Moreover, for loaded liposomes temperature and enthalpy of the transition were calculated after performing a peak deconvolution using a multi-peaks non-linear modelling by fitting the thermograms with a Gaussian function (OriginPro8, software). All measurements were performed in triplicates.
2.5. Liposomal characterization by high-resolution ultrasonic spectroscopy
Differential relative ultrasonic velocity and attenuation for concentrated liposomal dispersion were recorded as a function of temperature using a HR-US 102 high-resolution spectrometer (Ultrasonic Scientific, Ireland) at the frequency of 5.4 MHz, preliminarily determined by a broad amplitude frequency scan. Around 2 ml of samples and reference (9.4% w/v sucrose solution in water) were filled in the ultrasonic cells and left at 5 °C for at least 20 min for temperature equilibration and, then, subject to the same thermal programme used for mDSC analyses (from 5 °C to 80 °C at 1 °C/min). Temperature was controlled using a HAAKE C25P thermostat. Ultrasonic attenuation and sound speed are reported as differential values, obtained by subtracting the contribution of the reference from the value recorded in the sample cell. Sample transitions were calculated by the peak value from the attenuation profile or from the first-derivate of the signal in the case of sound speed (Perinelli et al., 2013). The area of the attenuation signal, the slopes and the height of the first derivative peak of sound speed traces were determined using OriginPro8 software. All measurements were performed in triplicate.
2.6. Drug content determination The eventual changes of drug content after the concentration process by ultrafiltration has been evaluated by UV-vis spectroscopy (UV-1800, Shimadzu) by measuring the absorbance of doxorubicin in the permeate at 485 nm. The amount of the free doxorubicin in the permeate was quantified by a calibration curve (y = 12.679 x + 0.001, r2 = 0.9997). Analyses were performed in triplicates.
2.7. Cryo-TEM analysis Sample of the 4X concentrated liposomal solution dispersion was applied onto continuous carbon grid, the excess of solution was blotted off and the specimen was plunged-frozen in liquid ethane under controlled temperature (16 °C) and humidity (100% RH) using Leica EM GP2. Subsequently, the grid was transferred in liquid nitrogen prior to EM imaging. Imaging was performed under cryogenic condition using a Talos L120C G2 transmission electron microscope run at 120kV accelerating voltage. Both low and high magnification images were acquired in representative areas using a BM-Ceta camera.
3. Results and discussion 3.1. Optimization of the ultrafiltration process to obtain concentrated liposomal dispersion DLS was firstly used for the optimization of the concentration process by ultrafiltration in term of operating pressure. Indeed, the operating pressure is the main parameter to be controlled, which markedly affects the concentration process by ultrafiltration. A too high operating pressure would force liposomes through the pore of the filter, determining the passage of liposomes in the filtrate, thereby decreasing the liposomal concentration of the samples. Thus, the optimal operating condition was considered the maximum pressure, assuring the retaining of liposomes by the filtration membrane during the concentration process. To assess this, samples of unloaded liposomes were processed by ultrafiltration at different conditions of pressures (1.5, 2.0 and 3.0 bar) and the corresponding retentates and permeates were measured by DLS (Figure 2A and Figure 2B). We observed that the retentates obtained at different operative pressures maintained a similar mean hydrodynamic diameter when compared to the control (non concentrated sample). However, a broader particle size distribution and higher PDI values were recorded (PDI was 0.052 ± 0.001 for non concentrated sample and 0.203 ± 0.010 after concentration at 1.5 bar). As regard the analysis of the permeates (Figure 2B), particle size distributions similar to those of retentates were observed at an operating pressure of 2 bar and 3 bar, indicating that these pressure values were too high for the concentration process, thereby liposomes partly passed the membrane. On the contrary, the permeate collected at the operating pressure of 1.5 bar showed a clear particle size distribution at around 1 nm and no particles size distribution around 70 nm, which can be attributed to that of liposomal dispersion, suggesting that liposome did not pass through the membrane. The absence of liposomes in the permeate obtained at 1.5 bar is confirmed by the low scattered intensity (kilocounts per second; 230 ± 8 kCps) measured at the detector, close to that of a pure dispersion medium. Thus, 1.5 bar pressure represents the boundary value, above which liposomes are forced to pass through the ultrafiltration membrane. According to these results, 1.2 bar was selected as the maximum operating pressure to be applied for the ultrafiltration process, maintaining a margin from the validated 1.5 bar value. Doxorubicin in the loaded liposomal dispersions is present into two forms as encapsulated inside the core of the liposomes and as “free doxorubicin”. The free doxorubicin represents about 2% of
the total drug content (2 mg/mL). The doxorubicin content in the eluate was found to be 0.019±0.002 mg/mL and 0.021±0.002 mg/mL, respectively for the 4x and 8x concentrated liposomes dispersions, amount well below the 2% of free doxorubicin present in the loaded liposome sample. The above is an additional element suggesting liposomes remain intact during the concentration process. The amount of doxorubicin trapped in the ultrafiltration membrane has not been investigated and included in the scope of this work. Liposomal particle size of non concentrated and concentrated samples (loaded and unloaded) was investigated in different media (9.4% sucrose, 0.9% NaCl and ultrapure water) and dilution ratios (1:2, 1:5, 1:10, 1:25 and 1:50) (Figure 3). Liposomes are particularly sensitive to the ionic strength and osmolality of the media, determining a variation on liposomal size (L-S Mui et al., 1993; Moon et al., 1998). All liposomal dispersions tested showed a typical and comparable profile, correlated to the dilution in different media. Particularly, a tendency of increasing particles size with the dilution ratio was observed for all media used. This effect was less pronounced when 9.4% sucrose solution was used to dilute the samples, since 9.4% sucrose solution is the same medium used for the preparation of liposomal dispersion. The higher effect on particle size was, instead, observed when samples were diluted in ultrapure water, probably due to the consequent variation in osmolarity, determining a swelling of the liposomes. Dilution with 0.9% of NaCl solution had an intermediated effect in increasing particles size of liposomes. Nevertheless, both unloaded and loaded liposomes revealed a not marked size variation over the investigated dilution ratios, since, even in ultrapure water, the increasing of particle size was 10-15 nm when compared with the non concentrated sample. The not marked variation in particle size over dilution in different media of concentrated samples and their behaviour similar to that of not concentrated ones can be considered as an indirect proof that liposomal bilayer is not strongly affected by the concentration process by ultrafiltration. DLS traces for non diluted samples (loaded and unloaded) before and after the concentration process are reported in Figure S1. Moreover, PDI values of non concentrated and concentrated samples before and after dilution were analysed (Figure S2). Despite after concentration PDI slightly increased (up to 0.2 for 4x samples and 0.25 for 8x samples), values comparable to those of non concentrated samples (between 0.05 and 0.1) were recorded after dilution. Particularly, the reduction of PDI to values comparable to those of non concentrated samples was reached at a dilution ratio of 1:5 for 4x concentrated samples and 1:10 for 8x concentrated samples. No appreciable changes in PDI was observed as a function of the different dilution media. These results highlighted the reversibility of the concentrated liposomal dispersions in term of particle size distribution after dilution.
3.2. Thermal behaviour of the concentrated liposomal dispersions 3.2.1. Microcalorimetry (mDSC) Figure 4 shows the thermograms obtained from the analysis of concentrated unloaded and loaded liposomes compared to the non concentrated samples. This analysis was performed to investigate whether ultrafiltration can affect the thermal behaviour of liposomal dispersions. Liposomal dispersion after concentration showed the same transitions at the same temperature with the respect to non concentrated samples. Specifically, a broad endothermic transition in the range 15 °C - 65 °C can be observed for both unloaded and loaded liposomes, attributed to the sol-gel transition of phospholipids forming liposomes. In addition, only in the case of loaded liposomes, a sharper transition centred at around 70 °C was detected and related to the melting of doxorubicin encapsulated in the liposomes. All calculated transition temperatures are reported in Table 1. The area as well as the enthalpy (J/g of solution) associated to each transition was proportional to the concentration of the samples. In fact, phospholipid sol-gel transition in concentrated unloaded and loaded liposomal dispersions and doxorubicin melting in concentrated loaded liposomes have an enthalpy value approximately of 4 or 8 times higher than the non concentrated ones (Table 1).
3.2.2. High resolution ultrasonic spectroscopy (HR-US) Thermal behaviour of concentrated unloaded and loaded liposomal dispersion was also studied using HR-US technique (Figure 5). Using this technique, thermal transitions can be detected from ultrasonic parameters such as ultrasonic velocity and ultrasonic sound speed. Sound speed is sensitive to any change in compressibility or density of the material, resulting from a physical transition of the material. Generally, it has a linear decreasing trend as a function of temperature and thermal transitions can be highlighted as a deviation from the linearity of sound speed signal. The differential sound speed traces, obtained testing all samples of liposomes dispersion, showed a deviation from linearity in a range of temperatures corresponding to those at which a variation in heat flow from mDSC analysis was observed. This deviation from linearity of the signal is much more pronounced for loaded liposomes with the respect to unloaded ones. Moreover, it shows a clear dependence on liposomes concentration, as observed from the first-derivate plot (Figure S3), which has been used to calculate the transition temperature (Table 1). The effect of concentration
was not only evident from the transition itself but from the overall sound speed trace. Indeed, as a function of liposomal concentration, ultrasonic velocity traces has a different slope, indicating a marked effect of temperature variation on the compressibility of the system. The differences in the values of the slopes of the signal before and after the transition were proportional to the extent of concentration (Table 2). Similarly, at the same temperature range, a deviation from the baseline was observed for the attenuation parameter, for which the maximum value measured can be assumed as the transition temperature (Table 1). As for sound speed, variation in attenuation was much more pronounced for loaded liposomes as a function of concentration. Indeed, the attenuation for the loaded liposomal sample 8x concentrated was over scale, and it cannot be measured in the range of temperature 50 °C - 70 °C. Moreover, the values of attenuation of the baseline below and above the transition temperatures were also proportional to the concentration. The transition temperatures calculated from attenuation are reported in Table 1.
3.3. Cryo-TEM analysis Figure 6 shows the pictures obtained by cryo-TEM imaging for the 4x concentrated samples both loaded and unloaded. After a 4x concentration process by ultrafiltration, loaded sample maintained the characteristic “coffee bean” morphology of Doxil, several times reported in the literature (Schilt et al., 2016; Wei et al., 2016; Wibroe et al., 2016). Particularly, loaded liposomes appeared as spherical or slightly prolate ellipsoidal unilamellar vesicles, in which are clearly observable the presence of more electron dense needle-shaped structures, attributed to the doxorubicin sulfate nanocrystals, self-organised in stacked rods (Figure 6A and 6B). These structures are not present in the images captured form the observation of unloaded liposomes (Figure 6C and 6D).
3.4. Size evolution of liposomal dispersions The size evolution of liposomal dispersions over time was evaluated through particle size analysis. Both unloaded and loaded liposomal dispersion displayed a particle size (Z-average) for all timepoints in the range of 75-80 nm for the 4x concentrated samples and in the range of 85-90 nm for the 8x concentrated samples over time. No remarkable variation was observed in term of particle size distribution as revealed by PDI values, which remains around 0.2-0.25 for 4x concentrated
samples and around 0.25-0.3 for 8x concentrated samples during all the 8 weeks investigated period (Figure 7).
4. Conclusions The ultrafiltration method revealed to be effective for the concentration of PEGylated liposomal dispersions unloaded or loaded with doxorubicin. The obtained 4x and 8x concentrated liposomal dispersions showed properties comparable to those of the not concentrated ones in terms of particles size after dilution with different media. The comparable behaviour on particle size changes, when non concentrated and concentrated liposomal solution are tested, as is or diluted with different aqueous media, suggests that no modification of the liposomes membrane takes place during the ultrafiltration process. All formulations were stable after dilution since a not marked variation in particle size was recorded. Moreover, the same thermal behaviour was observed between concentrated and non concentrated liposomal dispersions as evidenced by the comparable transition temperatures. In addition, both the techniques used (mDSC and HR-US) highlighted the effect exerted by liposomal concentration on the recorded signals. Particularly, this effect is evident from the area associated to each transition (mDSC analysis) and from both ultrasound parameters calculated from HR-US analysis. In the case of attenuation, the variation of the signal from the baseline at temperatures close to the transition was directly related to liposome concentration as well as sound speed traces. The slope values calculated from sound speed signal and the high of the peak of the first derivative were proportional to the concentration of liposomal dispersions. In addition, cryo-TEM images confirmed the maintenance of the characteristic “coffee bean” morphology for the 4x concentrated loaded liposomes as reported in the literature for Doxil®. All liposomal formulations showed no remarkable changes in term of particle size and particle size distribution for at least for 8 weeks after concentration. In conclusion, concentration by ultrafiltration method has proven to be effective and not to affect the quality of the processed liposomal dispersions and their stability over a 8 weeks period.
Acknowledgments Cryo_EM method and images: Courtesy of “Vironova AB” Gävlegatan 22, 113 30 Stockholm, Sweden.
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Figure 1 Ultrafiltration apparatus used for the concentration process of liposomal dispersions Figure 2 DLS traces obtained by analysing the unloaded liposomal dispersion retentates (A) and the permeates (B) after ultrafiltration at 1.5, 2 and 3 bar compared to the not concentrated sample. Figure 3 Variation of particle size (Z-average) for not concentrated and concentrated liposomal dispersions at different dilution ratios. Unloaded liposomal dispersions not concentrated (A), 4x concentrated (C) and 8x concentrated (E). Loaded liposomal dispersions not concentrated (B), 4x concentrated (D) and 8x concentrated (F). ND stands for non-diluted samples; 1:2, 1:5 1:10 1:25 and 1:50 indicate the dilution ratio. Figure 4 mDSC traces of unloaded liposomes (A) and loaded liposomes (B) before and after concentration (4x and 8x). Traces for non concentrated samples are reported from (Perinelli et al., 2017). Figure 5 Variation of the ultrasound parameters differential sound speed and differential attenuation as a function of temperature for unloaded (A and B) and loaded liposomes (C and D). Signals for non concentrated samples are reported from (Perinelli et al., 2017). Figure 6 Cryo-TEM images of 4x concentrated samples (loaded, A and B; unloaded, C and D) at low and high magnifications. Figure 7 Size evolution (Z-average, nm and PDI) over time up to 8 weeks for unloaded (A and B) and loaded (C and D) concentrated (4x and 8x) liposomal dispersions.
Table 1 Thermodynamic parameters (temperature, °C and enthalpy of the transition, J/g) for unloaded and loaded liposomes calculated from microcalorimetry and high-resolution ultrasonic spectroscopy. For microcalorimetry the 1° transition is related to the phase transition of phospholipid membrane and the second transition is related to the doxorubicin sulfate nanocrystals melting.
Microcalorimetry 1° transition
HR-US 2° transition
Attenuation
Sound speed
Enthalpy Enthalpy Temperature (J/g of (J/g of Temperature (°C) (°C) solution) solution) Non concentrated* 52.42±0.31 0.265±0.011 55.86±0.48 * Unloaded 4X concentrated 53.38±0.78 0.923±0.020 53.50±0.32 53.58±0.59 liposomes 8X concentrated 53.22±0.15 1.822±0.093 52.71±0.42 51.26±0.61 Non concentrated* 52.20±0.55 0.233±0.021 69.89±0.11 0.021±0.003 57.38±0.55 51.40±0.63 Loaded Concentrated 4X 54.64±0.69 0.897±0.052 69.11±0.55 0.078±0.003 56.88±0.44 55.53±0.71 liposomes Concentrated 8X 55.04±0.78 1.749±0.089 69.13±0.66 0.153±0.008 ** 54.36±0.53 Values for non concentrated samples are reported from ref (Perinelli et al., 2017). *No transition values can be calculated from the first-derivative peak of sound speed in the case unloaded liposomes. ** Attenuation signal for 8X concentrated loaded liposomes was overscale, not allowing the calculation of the transition temperature. Temperature (°C)
Table 2 Slope values and height of the first derivative for sound speed parameter and the area associated to the variation of attenuation parameter (related to the transition) calculated for nonconcentrated and concentrated unloaded and loaded liposomal dispersions.
HR-US Sound speed 1° Slope (m/s °C) Unloaded liposomes Loaded liposomes
Non-concentrated* 4X concentrated 8X concentrated Non-concentrated* Concentrated 4X Concentrated 8X
-0.09±0.00 -0.34±0.01 -0.59±0.01 -0.11±0.01 -0.42±0.01 -0.87±0.02
2° slope (m/s °C) -0.11±0.00 -0.35±0.01 -0.54±0.01 -0.10±0.00 -0.39±0.01 -0.88±0.01
Attenuation 1° derivative peak height (m/s °C) -0.04±0.00 -0.14±0.01 -0.15±0.01 -0.14±0.01 -0.72±0.01 -0.90±0.02
Area (°C/m) 23.44±1.05 71.70±3.45 83.99±3.93 229.60±10.87 983.54±14.23 -
Declaration of interests
☐ 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.
☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:
S0378-5173(19)30144-9 https://doi.org/10.1016/j.ijpharm.2019.02.021 IJP 18159
To appear in:
International Journal of Pharmaceutics
Received Date: Revised Date: Accepted Date:
22 January 2019 12 February 2019 13 February 2019
Please cite this article as: D.R. Perinelli, M. Cespi, F. Rendina, G. Bonacucina, G.F. Palmieri, Effect of the concentration process on unloaded and doxorubicin loaded liposomal dispersions, International Journal of Pharmaceutics (2019), doi: https://doi.org/10.1016/j.ijpharm.2019.02.021
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Effect of the concentration process on unloaded and doxorubicin loaded liposomal dispersions Diego Romano Perinelli 1, Marco Cespi 1, Filippo Rendina 2, Giulia Bonacucina Giovanni Filippo Palmieri 1,
1,
* and
1
School of Pharmacy, Via Gentile III da Varano, University of Camerino, 62032 Camerino, Italy Janssen-Pharmaceutical Company of Johnson and Jonhson, via C. Janssen, Borgo S. Michele, Latina, Italy * Correspondence: [email protected]; Tel.: +39-0737402289 2
Abstract Liposomes are unilamellar nanovesicles made of phospholipids of great interest as drug delivery carriers, able to encapsulate both hydrophilic and lipophilic compounds. Some liposomal formulations have reached the market, including the doxorubicin loaded PEGylated liposomal dispersion Doxil®. The aim of the work was to investigate the possibility of concentrating liposomes through the ultrafiltration process under nitrogen pressure, using Doxil® formulation as a model. The concentrated liposomal dispersions (4x and 8x) obtained from Doxil® were characterised in terms of size evolution (dynamic light scattering), morphology (cryo-TEM) and thermal behaviour (microcalorimetry, mDSC and high-resolution ultrasonic spectroscopy, HR-US) and compared to the unloaded liposomes of the same composition. The ultrafiltration process resulted to be effective in concentrating both loaded and unloaded liposomal dispersions, which showed a particle size and thermal properties comparable to those of the non concentrated ones. Moreover, all liposomal dispersions did not show any remarkable variation in term of particle size distribution and morphology for at least 8 weeks after concentration. Altogether, results demonstrated the effectiveness in using ultrafiltration as methodology to concentrate both loaded and unloaded liposomes without affecting the quality of the processed product. Keywords: PEGylated liposomes, Doxil®, microcalorimetry, high-resolution ultrasound spectroscopy, dynamic light scattering, thermal analysis
1. Introduction Liposomes are vesicles of size generally between 50-500 nm and made of a single or multi lipid bilayer, mainly composed of phospholipids (Zylberberg and Matosevic, 2016). In the last decades, they have been widely investigated as versatile drug delivery carriers thanks to their ability to incorporate hydrophilic drugs and biotherapeutics (nucleic acid, proteins, peptides and vaccines) inside their inner aqueous cavity as well as hydrophobic drugs intercalated in the lipid bilayer (Allen and Cullis, 2013; Fan and Zhang, 2013). Liposomes showed some advantages as drug delivery nanocarriers since they increase the stability and reduce the systemic toxicity of the encapsulated compounds, especially after parental administration (Sercombe et al., 2015). Several modifications have been performed on liposomal vesicles such as PEGylation to avoid the recognition by the immune cells, thus, prolonging their residential time into the circulation (stealth liposomes) (Lasic and Martin, 2018; Milla et al., 2012) and/or functionalization with ligands to improve their delivery properties at the targeted site (Palankar et al., 2008). These modifications led to the development of “the so-called” second and third generation of liposomes and they have been amply characterised in the last decades, especially for the delivery of several antitumor drugs (Belfiore et al., 2018; Riaz et al., 2018). Among them, some liposomal formulations have reached the market such as those containing doxorubicin (Doxil®, DaunoXome®, Myocet®), cytarabine/AraC (Depocyt®), vincristine (Marquibo®), mifarmurtide (Mepact®) and irinotecan (Onivyde®) (Bulbake et al., 2017). Particularly, the first approved liposomal dispersion approved for the clinical use was Doxil®, a formulation containing doxorubicin entrapped in liposomes for the treatment of the ovarian and breast cancer, and Kaposi’s sarcoma. This liposomal formulation has a stable loading of doxorubicin via a transmembrane ammonium sulfate pH-dependent gradient, responsible also for drug release at the tumor site (Barenholz, 2012). Recently, after the introduction on the market of follow-on products (sometimes referred to as ‘nanosimilars’), the scientific interest on Doxil® has been renewed and some papers, aimed at clarifying some chemical-physical aspects on this liposomal formulation, have been published. These studies have been focused on the investigation of the crystalline nature of the entrapped doxorubicin sulfate nanocrystals (Schilt et al., 2016), on the thermal behaviour (Perinelli et al., 2017; Wei et al., 2016) and morphology and size of liposomes (Wibroe et al., 2016). Doxil is also a suitable model of a PEGylated liposomal dispersion to study technological processes through which liposomes could be processed. One of the less investigated is the concentration. Concentration of liposomal dispersions can be achieved by different methods such as: ultracentrifugation, ultrafiltration and reconstitution in a smaller volume after freeze-drying (Magin and Chan, 1987). Despite, there is no an optimal procedure for liposomes concentration, ultracentrifugation is the most common but, event at the lowest effective
speed, it could induce vesicle fusion and changes in the morphology (Devi et al., 2010; Heeremans et al., 1995). Ultrafiltration has been largely used in liposomal drug delivery formulations to remove the un-encapsulated drug (Li et al., 2009; Zalba et al., 2012; Zhong et al., 2012), but no study, according to our knowledge, was aimed at investigating the effect of the concentration process by ultrafiltration on unloaded and drug loaded liposomal dispersions in terms of size, stability and thermal behaviour. The aim of the work was to explore the possibility of using the ultrafiltration under nitrogen pressure process as a strategy to concentrate a doxorubicin loaded PEGylated liposomal dispersion (Doxil®) and to characterise the concentrated liposomal dispersions in terms of size evolution, morphology and thermal behaviour. Results obtained from doxorubicin loaded dispersions were compared to those from unloaded liposomes of the same composition as reference. 2. Materials and Methods 2.1. Materials Loaded liposomes (Doxil®) and unloaded liposomes have been supplied by Janssen Pharmaceutical Companies of Johnson & Johnson. Doxil® is a marketed liposomal formulation of doxorubicin (2 mg/ml) composed of a mixture of hydrogenated soy phospholipids, cholesterol and methyldiasteroyl-phosphoethanolamine-polyethylenglicole 2kDa (DSPE-PEG) sodium salt at the weight ratio 3:1:1. Unloaded liposomes have the same qualitative and quantitative composition of loaded liposomes, except for the encapsulation of doxorubicin. All liposomal dispersions are prepared in 9.4% w/w sucrose (as main component) aqueous solution. 2.2. Liposomal formulation concentration by ultrafiltration Unloaded liposomes and loaded liposomes were concentrated by ultrafiltration performed by applying a nitrogen positive pressure, which forced the dispersion medium to pass through a polyether sulfone membrane 100 kDa (Sartorius, Gottingen, Germany). Liposomes were retained and separated from the filtrated passed through the membrane. The ultrafiltration was performed under magnetic stirring using the ultrafiltration stirred cell (Amicon ®, USA), as shown in Figure 1, in a temperature controlled chamber (10 °C), at different nitrogen operating pressures (1.5, 2, and 5 bar) to select the best condition to be applied for this process. The ultrafiltration was performed starting from 40 ml until the final volume was reduced to 10 ml or 5 ml corresponding to a 4x and 8x liposomes concentration in the final dispersion.
2.3. Liposomal formulation characterization by dynamic light scattering (DLS) DLS analyses were performed using a Malvern Zetasizer nanoS (Malvern instrument Worcestershire, UK), detecting scattered light at 173°. The scattered signals (counts) were recorded at 25 °C after a thermal equilibration of 180 s. Results are reported as Z-average (diameter, nm) and polydispersity index (PDI) as the average of 3 measurements of 11 runs. All analyses were performed in triplicate. To study the possible effect exerted by concentration process on liposome dimensions, particle size of concentrated liposomal dispersion (4x and 8x) was measured before and after dilution with different aqueous media (9.4% sucrose, 0.9% NaCl and ultrapure water) and compared with that obtained from non concentrated. For each media, different dilution ratios were performed (1:2; 1:5; 1:10 1:25 and 1:50) and samples were analysed after 5 minutes. The viscosity of the aqueous dispersion media used for each analysis was adjusted on the base of the effective concentration of the solute as provided by Malvern software. A summary of the viscosity of the dispersant used for each analysis can be found in Table S1. In addition, DLS was employed to assess the particle size and particle size distribution stability over time of the concentrated liposomal dispersions. Liposomal concentrated samples were stored at 4 °C up to 2 months and analysed by DLS at different timepoints (1 week, 2 week, 3 week, 4 week and 8 weeks).
2.4. Liposomal characterization by microcalorimetry (mDSC) 0.750 g of concentrated liposomal dispersion and an equal amount of 9.4% w/v sucrose solution as reference were filled in a Hallostey calorimetric cells and equilibrated at 5 °C for 20 min. Then, a heating and a cooling ramp from 5 °C to 80 °C at 1 °C /min were performed. Calorimetric analyses were carried out using a microDSC III (Setaram, France). The temperature (Tm, °C) and enthalpy (ΔH, J/g of solution) were calculated from the peak and the area of the transition using the software of the instrument (Setsoft2000, Setaram) according to the tangent method. Moreover, for loaded liposomes temperature and enthalpy of the transition were calculated after performing a peak deconvolution using a multi-peaks non-linear modelling by fitting the thermograms with a Gaussian function (OriginPro8, software). All measurements were performed in triplicates.
2.5. Liposomal characterization by high-resolution ultrasonic spectroscopy
Differential relative ultrasonic velocity and attenuation for concentrated liposomal dispersion were recorded as a function of temperature using a HR-US 102 high-resolution spectrometer (Ultrasonic Scientific, Ireland) at the frequency of 5.4 MHz, preliminarily determined by a broad amplitude frequency scan. Around 2 ml of samples and reference (9.4% w/v sucrose solution in water) were filled in the ultrasonic cells and left at 5 °C for at least 20 min for temperature equilibration and, then, subject to the same thermal programme used for mDSC analyses (from 5 °C to 80 °C at 1 °C/min). Temperature was controlled using a HAAKE C25P thermostat. Ultrasonic attenuation and sound speed are reported as differential values, obtained by subtracting the contribution of the reference from the value recorded in the sample cell. Sample transitions were calculated by the peak value from the attenuation profile or from the first-derivate of the signal in the case of sound speed (Perinelli et al., 2013). The area of the attenuation signal, the slopes and the height of the first derivative peak of sound speed traces were determined using OriginPro8 software. All measurements were performed in triplicate.
2.6. Drug content determination The eventual changes of drug content after the concentration process by ultrafiltration has been evaluated by UV-vis spectroscopy (UV-1800, Shimadzu) by measuring the absorbance of doxorubicin in the permeate at 485 nm. The amount of the free doxorubicin in the permeate was quantified by a calibration curve (y = 12.679 x + 0.001, r2 = 0.9997). Analyses were performed in triplicates.
2.7. Cryo-TEM analysis Sample of the 4X concentrated liposomal solution dispersion was applied onto continuous carbon grid, the excess of solution was blotted off and the specimen was plunged-frozen in liquid ethane under controlled temperature (16 °C) and humidity (100% RH) using Leica EM GP2. Subsequently, the grid was transferred in liquid nitrogen prior to EM imaging. Imaging was performed under cryogenic condition using a Talos L120C G2 transmission electron microscope run at 120kV accelerating voltage. Both low and high magnification images were acquired in representative areas using a BM-Ceta camera.
3. Results and discussion 3.1. Optimization of the ultrafiltration process to obtain concentrated liposomal dispersion DLS was firstly used for the optimization of the concentration process by ultrafiltration in term of operating pressure. Indeed, the operating pressure is the main parameter to be controlled, which markedly affects the concentration process by ultrafiltration. A too high operating pressure would force liposomes through the pore of the filter, determining the passage of liposomes in the filtrate, thereby decreasing the liposomal concentration of the samples. Thus, the optimal operating condition was considered the maximum pressure, assuring the retaining of liposomes by the filtration membrane during the concentration process. To assess this, samples of unloaded liposomes were processed by ultrafiltration at different conditions of pressures (1.5, 2.0 and 3.0 bar) and the corresponding retentates and permeates were measured by DLS (Figure 2A and Figure 2B). We observed that the retentates obtained at different operative pressures maintained a similar mean hydrodynamic diameter when compared to the control (non concentrated sample). However, a broader particle size distribution and higher PDI values were recorded (PDI was 0.052 ± 0.001 for non concentrated sample and 0.203 ± 0.010 after concentration at 1.5 bar). As regard the analysis of the permeates (Figure 2B), particle size distributions similar to those of retentates were observed at an operating pressure of 2 bar and 3 bar, indicating that these pressure values were too high for the concentration process, thereby liposomes partly passed the membrane. On the contrary, the permeate collected at the operating pressure of 1.5 bar showed a clear particle size distribution at around 1 nm and no particles size distribution around 70 nm, which can be attributed to that of liposomal dispersion, suggesting that liposome did not pass through the membrane. The absence of liposomes in the permeate obtained at 1.5 bar is confirmed by the low scattered intensity (kilocounts per second; 230 ± 8 kCps) measured at the detector, close to that of a pure dispersion medium. Thus, 1.5 bar pressure represents the boundary value, above which liposomes are forced to pass through the ultrafiltration membrane. According to these results, 1.2 bar was selected as the maximum operating pressure to be applied for the ultrafiltration process, maintaining a margin from the validated 1.5 bar value. Doxorubicin in the loaded liposomal dispersions is present into two forms as encapsulated inside the core of the liposomes and as “free doxorubicin”. The free doxorubicin represents about 2% of
the total drug content (2 mg/mL). The doxorubicin content in the eluate was found to be 0.019±0.002 mg/mL and 0.021±0.002 mg/mL, respectively for the 4x and 8x concentrated liposomes dispersions, amount well below the 2% of free doxorubicin present in the loaded liposome sample. The above is an additional element suggesting liposomes remain intact during the concentration process. The amount of doxorubicin trapped in the ultrafiltration membrane has not been investigated and included in the scope of this work. Liposomal particle size of non concentrated and concentrated samples (loaded and unloaded) was investigated in different media (9.4% sucrose, 0.9% NaCl and ultrapure water) and dilution ratios (1:2, 1:5, 1:10, 1:25 and 1:50) (Figure 3). Liposomes are particularly sensitive to the ionic strength and osmolality of the media, determining a variation on liposomal size (L-S Mui et al., 1993; Moon et al., 1998). All liposomal dispersions tested showed a typical and comparable profile, correlated to the dilution in different media. Particularly, a tendency of increasing particles size with the dilution ratio was observed for all media used. This effect was less pronounced when 9.4% sucrose solution was used to dilute the samples, since 9.4% sucrose solution is the same medium used for the preparation of liposomal dispersion. The higher effect on particle size was, instead, observed when samples were diluted in ultrapure water, probably due to the consequent variation in osmolarity, determining a swelling of the liposomes. Dilution with 0.9% of NaCl solution had an intermediated effect in increasing particles size of liposomes. Nevertheless, both unloaded and loaded liposomes revealed a not marked size variation over the investigated dilution ratios, since, even in ultrapure water, the increasing of particle size was 10-15 nm when compared with the non concentrated sample. The not marked variation in particle size over dilution in different media of concentrated samples and their behaviour similar to that of not concentrated ones can be considered as an indirect proof that liposomal bilayer is not strongly affected by the concentration process by ultrafiltration. DLS traces for non diluted samples (loaded and unloaded) before and after the concentration process are reported in Figure S1. Moreover, PDI values of non concentrated and concentrated samples before and after dilution were analysed (Figure S2). Despite after concentration PDI slightly increased (up to 0.2 for 4x samples and 0.25 for 8x samples), values comparable to those of non concentrated samples (between 0.05 and 0.1) were recorded after dilution. Particularly, the reduction of PDI to values comparable to those of non concentrated samples was reached at a dilution ratio of 1:5 for 4x concentrated samples and 1:10 for 8x concentrated samples. No appreciable changes in PDI was observed as a function of the different dilution media. These results highlighted the reversibility of the concentrated liposomal dispersions in term of particle size distribution after dilution.
3.2. Thermal behaviour of the concentrated liposomal dispersions 3.2.1. Microcalorimetry (mDSC) Figure 4 shows the thermograms obtained from the analysis of concentrated unloaded and loaded liposomes compared to the non concentrated samples. This analysis was performed to investigate whether ultrafiltration can affect the thermal behaviour of liposomal dispersions. Liposomal dispersion after concentration showed the same transitions at the same temperature with the respect to non concentrated samples. Specifically, a broad endothermic transition in the range 15 °C - 65 °C can be observed for both unloaded and loaded liposomes, attributed to the sol-gel transition of phospholipids forming liposomes. In addition, only in the case of loaded liposomes, a sharper transition centred at around 70 °C was detected and related to the melting of doxorubicin encapsulated in the liposomes. All calculated transition temperatures are reported in Table 1. The area as well as the enthalpy (J/g of solution) associated to each transition was proportional to the concentration of the samples. In fact, phospholipid sol-gel transition in concentrated unloaded and loaded liposomal dispersions and doxorubicin melting in concentrated loaded liposomes have an enthalpy value approximately of 4 or 8 times higher than the non concentrated ones (Table 1).
3.2.2. High resolution ultrasonic spectroscopy (HR-US) Thermal behaviour of concentrated unloaded and loaded liposomal dispersion was also studied using HR-US technique (Figure 5). Using this technique, thermal transitions can be detected from ultrasonic parameters such as ultrasonic velocity and ultrasonic sound speed. Sound speed is sensitive to any change in compressibility or density of the material, resulting from a physical transition of the material. Generally, it has a linear decreasing trend as a function of temperature and thermal transitions can be highlighted as a deviation from the linearity of sound speed signal. The differential sound speed traces, obtained testing all samples of liposomes dispersion, showed a deviation from linearity in a range of temperatures corresponding to those at which a variation in heat flow from mDSC analysis was observed. This deviation from linearity of the signal is much more pronounced for loaded liposomes with the respect to unloaded ones. Moreover, it shows a clear dependence on liposomes concentration, as observed from the first-derivate plot (Figure S3), which has been used to calculate the transition temperature (Table 1). The effect of concentration
was not only evident from the transition itself but from the overall sound speed trace. Indeed, as a function of liposomal concentration, ultrasonic velocity traces has a different slope, indicating a marked effect of temperature variation on the compressibility of the system. The differences in the values of the slopes of the signal before and after the transition were proportional to the extent of concentration (Table 2). Similarly, at the same temperature range, a deviation from the baseline was observed for the attenuation parameter, for which the maximum value measured can be assumed as the transition temperature (Table 1). As for sound speed, variation in attenuation was much more pronounced for loaded liposomes as a function of concentration. Indeed, the attenuation for the loaded liposomal sample 8x concentrated was over scale, and it cannot be measured in the range of temperature 50 °C - 70 °C. Moreover, the values of attenuation of the baseline below and above the transition temperatures were also proportional to the concentration. The transition temperatures calculated from attenuation are reported in Table 1.
3.3. Cryo-TEM analysis Figure 6 shows the pictures obtained by cryo-TEM imaging for the 4x concentrated samples both loaded and unloaded. After a 4x concentration process by ultrafiltration, loaded sample maintained the characteristic “coffee bean” morphology of Doxil, several times reported in the literature (Schilt et al., 2016; Wei et al., 2016; Wibroe et al., 2016). Particularly, loaded liposomes appeared as spherical or slightly prolate ellipsoidal unilamellar vesicles, in which are clearly observable the presence of more electron dense needle-shaped structures, attributed to the doxorubicin sulfate nanocrystals, self-organised in stacked rods (Figure 6A and 6B). These structures are not present in the images captured form the observation of unloaded liposomes (Figure 6C and 6D).
3.4. Size evolution of liposomal dispersions The size evolution of liposomal dispersions over time was evaluated through particle size analysis. Both unloaded and loaded liposomal dispersion displayed a particle size (Z-average) for all timepoints in the range of 75-80 nm for the 4x concentrated samples and in the range of 85-90 nm for the 8x concentrated samples over time. No remarkable variation was observed in term of particle size distribution as revealed by PDI values, which remains around 0.2-0.25 for 4x concentrated
samples and around 0.25-0.3 for 8x concentrated samples during all the 8 weeks investigated period (Figure 7).
4. Conclusions The ultrafiltration method revealed to be effective for the concentration of PEGylated liposomal dispersions unloaded or loaded with doxorubicin. The obtained 4x and 8x concentrated liposomal dispersions showed properties comparable to those of the not concentrated ones in terms of particles size after dilution with different media. The comparable behaviour on particle size changes, when non concentrated and concentrated liposomal solution are tested, as is or diluted with different aqueous media, suggests that no modification of the liposomes membrane takes place during the ultrafiltration process. All formulations were stable after dilution since a not marked variation in particle size was recorded. Moreover, the same thermal behaviour was observed between concentrated and non concentrated liposomal dispersions as evidenced by the comparable transition temperatures. In addition, both the techniques used (mDSC and HR-US) highlighted the effect exerted by liposomal concentration on the recorded signals. Particularly, this effect is evident from the area associated to each transition (mDSC analysis) and from both ultrasound parameters calculated from HR-US analysis. In the case of attenuation, the variation of the signal from the baseline at temperatures close to the transition was directly related to liposome concentration as well as sound speed traces. The slope values calculated from sound speed signal and the high of the peak of the first derivative were proportional to the concentration of liposomal dispersions. In addition, cryo-TEM images confirmed the maintenance of the characteristic “coffee bean” morphology for the 4x concentrated loaded liposomes as reported in the literature for Doxil®. All liposomal formulations showed no remarkable changes in term of particle size and particle size distribution for at least for 8 weeks after concentration. In conclusion, concentration by ultrafiltration method has proven to be effective and not to affect the quality of the processed liposomal dispersions and their stability over a 8 weeks period.
Acknowledgments Cryo_EM method and images: Courtesy of “Vironova AB” Gävlegatan 22, 113 30 Stockholm, Sweden.
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Figure 1 Ultrafiltration apparatus used for the concentration process of liposomal dispersions Figure 2 DLS traces obtained by analysing the unloaded liposomal dispersion retentates (A) and the permeates (B) after ultrafiltration at 1.5, 2 and 3 bar compared to the not concentrated sample. Figure 3 Variation of particle size (Z-average) for not concentrated and concentrated liposomal dispersions at different dilution ratios. Unloaded liposomal dispersions not concentrated (A), 4x concentrated (C) and 8x concentrated (E). Loaded liposomal dispersions not concentrated (B), 4x concentrated (D) and 8x concentrated (F). ND stands for non-diluted samples; 1:2, 1:5 1:10 1:25 and 1:50 indicate the dilution ratio. Figure 4 mDSC traces of unloaded liposomes (A) and loaded liposomes (B) before and after concentration (4x and 8x). Traces for non concentrated samples are reported from (Perinelli et al., 2017). Figure 5 Variation of the ultrasound parameters differential sound speed and differential attenuation as a function of temperature for unloaded (A and B) and loaded liposomes (C and D). Signals for non concentrated samples are reported from (Perinelli et al., 2017). Figure 6 Cryo-TEM images of 4x concentrated samples (loaded, A and B; unloaded, C and D) at low and high magnifications. Figure 7 Size evolution (Z-average, nm and PDI) over time up to 8 weeks for unloaded (A and B) and loaded (C and D) concentrated (4x and 8x) liposomal dispersions.
Table 1 Thermodynamic parameters (temperature, °C and enthalpy of the transition, J/g) for unloaded and loaded liposomes calculated from microcalorimetry and high-resolution ultrasonic spectroscopy. For microcalorimetry the 1° transition is related to the phase transition of phospholipid membrane and the second transition is related to the doxorubicin sulfate nanocrystals melting.
Microcalorimetry 1° transition
HR-US 2° transition
Attenuation
Sound speed
Enthalpy Enthalpy Temperature (J/g of (J/g of Temperature (°C) (°C) solution) solution) Non concentrated* 52.42±0.31 0.265±0.011 55.86±0.48 * Unloaded 4X concentrated 53.38±0.78 0.923±0.020 53.50±0.32 53.58±0.59 liposomes 8X concentrated 53.22±0.15 1.822±0.093 52.71±0.42 51.26±0.61 Non concentrated* 52.20±0.55 0.233±0.021 69.89±0.11 0.021±0.003 57.38±0.55 51.40±0.63 Loaded Concentrated 4X 54.64±0.69 0.897±0.052 69.11±0.55 0.078±0.003 56.88±0.44 55.53±0.71 liposomes Concentrated 8X 55.04±0.78 1.749±0.089 69.13±0.66 0.153±0.008 ** 54.36±0.53 Values for non concentrated samples are reported from ref (Perinelli et al., 2017). *No transition values can be calculated from the first-derivative peak of sound speed in the case unloaded liposomes. ** Attenuation signal for 8X concentrated loaded liposomes was overscale, not allowing the calculation of the transition temperature. Temperature (°C)
Table 2 Slope values and height of the first derivative for sound speed parameter and the area associated to the variation of attenuation parameter (related to the transition) calculated for nonconcentrated and concentrated unloaded and loaded liposomal dispersions.
HR-US Sound speed 1° Slope (m/s °C) Unloaded liposomes Loaded liposomes
Non-concentrated* 4X concentrated 8X concentrated Non-concentrated* Concentrated 4X Concentrated 8X
-0.09±0.00 -0.34±0.01 -0.59±0.01 -0.11±0.01 -0.42±0.01 -0.87±0.02
2° slope (m/s °C) -0.11±0.00 -0.35±0.01 -0.54±0.01 -0.10±0.00 -0.39±0.01 -0.88±0.01
Attenuation 1° derivative peak height (m/s °C) -0.04±0.00 -0.14±0.01 -0.15±0.01 -0.14±0.01 -0.72±0.01 -0.90±0.02
Area (°C/m) 23.44±1.05 71.70±3.45 83.99±3.93 229.60±10.87 983.54±14.23 -
Declaration of interests
☐ 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.
☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: