Victor U.
Weiss†
a,
Carlos
Urey†
b,
Andreas
Gondikas
c,
Monika
Golesne
a,
Gernot
Friedbacher
a,
Frank
von der Kammer
c,
Thilo
Hofmann
c,
Roland
Andersson
b,
György
Marko-Varga
d,
Martina
Marchetti-Deschmann
a and
Günter
Allmaier
*a
aInstitute of Chemical Technologies and Analytics, TU Wien, Vienna, Austria. E-mail: guenter.allmaier@tuwien.ac.at; Fax: +43 1 58801 16199; Tel: +43 1 58801 15160
bDepartment of Surgery, University of Lund, Lund, Sweden
cDepartment of Environmental Geosciences and Environmental Science Research Network, University of Vienna, Vienna, Austria
dBiomedical Center, University of Lund, Lund, Sweden
First published on 3rd August 2016
Liposomes are biodegradable nanoparticle vesicles consisting of a lipid bilayer encapsulating an aqueous core. Entrapped cargo material is shielded from the extra-vesicular medium and sustained release of encapsulated material can be achieved. However, application of liposomes as nano-carriers demands their characterization concerning size and size distribution, particle-number concentration, occurrence of vesicle building blocks in solution and determination of the resulting vesicle encapsulation capacity. These questions can be targeted via gas-phase electrophoretic mobility molecular analysis (GEMMA) based on a nano electrospray (nES) charge-reduction source. This instrument separates single-charged nanoparticles in the gas-phase according to size in a high-laminar sheath-flow by means of an orthogonal, tunable electric field. nES GEMMA analysis enables to confirm liposome integrity after passage through the instrument (in combination with atomic force microscopy) as well as to exclude vesicle aggregation. Additionally, nanoparticle diameters at peak apexes and size distribution data are obtained. Differences of hydrodynamic and dry particle diameter values, as well as the effect of number- and mass-based concentration data analysis on obtained liposome diameters are shown. Furthermore, the repeatability of liposome preparation is studied, especially upon incorporation of PEGylated lipids in the bilayer. Finally, the instruments applicability to monitor mechanical stress applied to vesicles is demonstrated.
To date, various analytical setups for liposome characterization have been described. These include, imaging techniques like atomic force microscopy (AFM),4 transmission electron (TEM) or cryo electron microscopy (cryo EM)5 for visualization of particles. However, as a large number of particle images (1000 and upwards) are needed to allow for the determination of accurate particle shape and size distributions with good statistics, these methods are time consuming even with adequate software support. Furthermore, the application of high vacuum, e.g. in TEM or cryo EM, might lead to liposome shape distortion due to excessive interactions of analytes with sample carrier materials. Moreover, preferential enrichment of certain sample constituents of the final liposome preparation on the carrier material employed for analyte imaging has to be avoided. Nevertheless, imaging techniques yield number-concentrations of nanoparticles as recommended by the European Commission in 2011 (2011/696/EU from October 18th, 2011). Other analytical setups for nanoparticle characterization are liquid phase-based, e.g. electrophoretic separations6,7 or field-flow fractionations.8 Especially, dynamic light scattering (DLS) analysis is very popular due to its straightforward manner for size characterization of liposomes in solution.1 However, due to preferential detection of larger nanoparticles, information on smaller-sized sample components is often lost completely. Therefore, DLS is well-suitable to detect monodisperse nanoparticles within a sample, but is biased when nanoparticles cover a broad size range, i.e. are of higher polydispersity or show a multimodal size distribution.
Within this manuscript, we want to present nano electrospray gas-phase electrophoretic mobility molecular analysis (nES GEMMA) as a valuable alternative to the described established analytical methods for liposome vesicle characterization. nES GEMMA separates single-charged analytes in the gas-phase at ambient pressure according to their electrophoretic mobility diameter (EM diameter).9,10 In the case of spherical particles, the EM diameter corresponds to the analyte size. Single-charged analytes are obtained from a nES process with subsequent drying of nanoparticle-containing droplets and charge conditioning. The latter occurs in a bipolar atmosphere induced by a 210Po α-particle emitter. Depending on the size of analytes, in large part neutral analytes are obtained (which are not considered further) as well as a certain percentage of single- and, considerably less, multiple-charged particles (positive and negative, respectively).11 nES GEMMA results are based on data obtained from single-charged analytes. Size separation of particles occurs in the nano differential mobility analyzer (nDMA) part of the instrument in a high laminar flow of filtered (i.e. particle free), compressed air and an orthogonal, tunable electric field (scanning a certain voltage range). EM diameter values between 2.0 and 64.4 nm are separable at a high laminar flow value of 15 liters per minute (Lpm), i.e. at the maximum resolving power of the applied device. Application of lower Lpm values results in a larger EM diameter size range accessible with the used instrumental set-up. Variations in the field strength (voltage scanning) lead to deviation of charged particles from their high laminar flow imposed trajectory. Hence, only nanoparticles of a given EM diameter corresponding to an applied field strength are able to pass through the nDMA and enter the detection unit of the instrument, a condensation particle counter (CPC). In the CPC, singly-charged particles act as condensation nuclei in a supersaturated atmosphere of n-butanol and are subsequently counted as they pass a focused laser beam. This detection setup allows single particle and number-concentration based detection in accordance with the EC recommendation concerning nanoparticle analysis (2011/696/EU). Furthermore, the nanoparticle detection is completely independent of the chemical nature of the nanoparticles. However, it is worth mentioning that volatile electrolyte solutions for electrospraying of the nanoparticles are required as high amounts of non-volatile sample components (e.g. from employed buffers) are interfering with detection of the target nanoparticles.9,12
Several synonyms for nES GEMMA instruments can be found in the literature: macro ion mobility spectrometer (macroIMS),13 LiquiScan-ES (official manufacturing-company-given name of the instrument for a short time period), ES-DMA14 or scanning mobility particle sizer (SMPS) spectrometer.15 However, for matters of consistency with previous publications of others (e.g.16–19) and our group (e.g.10,20,21) we stick to the term nES GEMMA.
To date, several reports have been presented, targeting the analysis of liposomes with nES GEMMA-like instruments. Epstein and colleagues described in an innovative work the number-concentration measurement of liposomes in water and the comparison of their measurements to calculated values based on theoretical considerations.22 Their instrument focused on the detection of larger liposome particles. Hence, a so-called long DMA (lDMA) was used which allows separation of particles from approximately 15 nm up to 900 nm EM diameter at very low resolving power. Thus, in order to generate analyte containing aerosol a combination of an atomizer (different to the applied nES GEMMA system) and a diffusion dryer was employed and the integrity of liposomes was assessed finally via TEM. The group of Biswas on the other hand concentrated on the preparation and characterization of 100 nm liposome containing aerosol for pulmonary drug delivery.23,24 Either an atomizer/diffusion dryer combination was employed (comparable to the instrument of Epstein and colleagues) or a nES setup without additional drying of the nES spray gas. For the latter, 40 μm inner diameter spray capillaries were used. Liposome aggregation was observed in subsequent TEM analyses, which was suggested to be related to liposome aggregation already during the nES process. Additionally, the same group attributed an observed bimodal nano-vesicle distribution in nES GEMMA likewise to liposome aggregation during the nES process.
In addition to liposome analyses, individual samples of purified (from human blood) high density, low density and very low density lipoprotein particles (lipids as for example cholesterinester, triglycerides and protein containing macromolecule assemblies without an aqueous lumen and containing only lipid monolayers) have been size-analyzed by nES GEMMA25 or sera from volunteers were employed to determine lipoprotein particle sizes and concentrations in blood.26 Likewise, nanolipoprotein (NLP) particles,27 including additional membrane proteins, could be separated from bare NLPs via nES GEMMA.28
It was the aim of our work to (i) determine whether indeed single liposome vesicles are detected via the nES GEMMA, and not unspecific lipid aggregates or aggregates of smaller vesicles, and (ii) to answer the question of liposome integrity and aggregation after nES GEMMA passage by including subsequent AFM experiments (a non-vacuum device in contrast to TEM) after collection. Additionally, (iii) we wanted to highlight the importance of dry air application for the nES process. (iv) Moreover, ultrasonication was employed to subject vesicles to mechanical stress, which leads to liposome disruption as demonstrated from nES GEMMA measurements. Based on our data, we furthermore present (v) obtained particle size values concerning mass- and number based data evaluation. Differences between dry particle diameter values and hydrodynamic size values are shown and critically discussed. Finally, (vi) we targeted the question of liposome preparation repeatability, especially when PEGylated lipids are included in the lipid bilayer. Resulting experimental data can be employed to calculate the theoretical vesicle drug encapsulation capacity.
AFM measurements were performed with a NanoScope 8 scanning probe microscope (Bruker, Santa Barbara, CA, USA) operated in tapping mode using single crystal silicon cantilevers (NCH, Bruker).
The intensity-, volume-, and number-weighted average hydrodynamic diameter of the liposome suspension was determined by DLS using a Malvern Zetasizer instrument (Malvern Instruments, Malvern, United Kingdom) using incident light (λ = 633 nm) scattered at 173°. Mean size was expressed as Z-average from cumulate fit analysis.
Additional experiments focused on the formation of unspecific aggregates in solution and during the nES process. As demonstrated in Fig. 1B and C, results from dilution experiments are comparable when spectra are plotted as relative data: no differences concerning peak apexes or particle size distributions are detected. Hence, no unspecific aggregates are formed in solution. Based on this data, also liposome aggregate formation during the nES process seems highly unlikely. It is to be noted that components detected with approx. 20 nm EM diameter might originate from micelles or other lipid aggregates.
Subsequently, we employed AFM to investigate vesicle integrity after GEMMA analysis. AFM, in contrast to TEM as previously reported,22 works at ambient pressure and hence we expected a reduced impact of the applied imaging method on liposome particle integrity. Fig. 2A shows a corresponding nES GEMMA spectrum of the investigated sample indicating the EM diameter at which sampling occurred. Liposomes were collected as previously described in detail20 for 150 min at 86 nm EM diameter on a silicon wafer surface. Initial experiments had demonstrated a sufficiently even surface of the applied silicon wafer (data not shown). Collection of liposomes via nES GEMMA relates spherical shaped particles exhibiting a diameter in the size range of the EM diameter at which particles were sampled (Fig. 2B). However, the height of the collected particles is significantly reduced (max. 20 nm) in comparison to their diameter (approx. 100 nm). We concluded already in a previous study30 that this is most likely caused by force application of the AFM tip or interaction of particles with the wafer surface, or a combination of both.
To further corroborate vesicle integrity during nES GEMMA measurements, sonication experiments for intended vesicle disruption are presented. As depicted in Fig. 3, sonication indeed eliminates the particle peak at approx. 80 nm EM diameter peak apex. Concomitantly, peaks at lower EM diameter values gain in their intensity as these peaks probably originate from lipid molecular aggregates, disrupted liposome bilayers or smaller vesicles. For future measurements these results indicate that nES GEMMA is an appropriate analytical technique to investigate the impact of mechanical stress on liposomes.
Secondly, it has to be considered that nES GEMMA measurements relate to dry particle diameters whereas liquid phase methods, e.g. DLS, obtain hydrodynamic diameter values in a corresponding solvent. Differences between these two values can be significant as was shown for the characterization of gelatin or silica based nanoparticles.31–33 Similar observations were recently reported for the size determination of exosomes in solution or after nanoparticle drying.34Fig. 4 likewise demonstrates the significant size difference between surface-dry particles (nES GEMMA) and measurements in solution yielding the liposome's hydrodynamic diameter in NH4OAc. For the given analyte the DLS derived hydrodynamic diameter (based on calculated number-concentrations) exceeded measured number-based nES GEMMA data by about 23%. To conclude, the question of liposome particle size and size distribution can only be answered, if the analytical measurement technique is provided and if the physical condition in which nanoparticle size determination occurs is clearly described.32,35
Subsequently, we inquired whether it is possible to deduce further parameters of liposome batches from nES GEMMA measurements besides (i) the particle size values for dry vesicles and (ii) size distributions of liposome preparations revealing the amount of undesired smaller sized sample components. Indeed, we suggest that nES GEMMA data allows (iii) to calculate the relative theoretical encapsulation capacities (EC) of vesicle preparations via (iv) calculation of liposomal vesicle volumes V = (1/6) × π × (EM diameter)3, deduced from the EM diameter of particles at the peak apex assuming spherical shape as proven by AFM, and (v) vesicle particle numbers (PN) as deduced from number concentration data, as well at the peak apex. EC is obtained as product V × PN. Resulting values for liposome preparations shown in Fig. 5A are given in Fig. 5B.
We are aware of the fact that the EM diameter is determined by the diameter of the lumen and the thickness of the bilayer alike, the latter being dependent on the phospholipid composition of the bilayer. However, in order to set up a generalizable and easily applicable model we applied a simplification disregarding the contribution of the bilayer to the liposome EM diameter in our approach to calculate the theoretical EC. Likewise, our simplified model neither considers multilamellarity nor the possibility of slight differences in charge conditioning between preparations as these factors were not suggested to impact the observed general trend. However, it has to be kept in mind that for a detailed model of a given vesicle batch these numbers have to be determined by orthogonal and time-consuming techniques, e.g. cryo EM.
Based on our generalizable as well as easily applicable model and as expected from nES GEMMA data, also the theoretical encapsulation capacity of the four liposome preparation batches was of very good repeatability. Keep in mind that the smaller-sized sample components of the bimodal size distribution (small liposomes, micelles, liposome building blocks or similar) were neglected for the calculation of the theoretical vesicle encapsulation capacity as their aqueous lumen (if any in case of micelles and unspecific aggregates) is negligible when compared to full-sized liposomes.
PEGylation of liposomes is today a recognized method to increase blood circulation (i.e. half-life) of vesicles during drug delivery. Therefore, in a final step, we made an attempt to perform the analysis of vesicles in which phosphoethanolamine (PE) of original liposomes was substituted with its PEGylated form, DSPE-PEG2000. Resulting GEMMA spectra of n = 4 preparations (likewise duplicates from single – 1st and 2nd – and serial extrusion experiments – 3rd and 4th) are shown in Fig. 6A. Surprisingly, the very good repeatability of liposome preparation was lost upon application of PE in its PEGylated form. This finding is also reflected in the theoretical encapsulation capacity of vesicles (Fig. 6B), which was calculated as described above. From our experiments it appears that PEGylation greatly influences the repeatability of vesicle preparation probably due to (i) interaction of PEG side chains during lipid film and liposome formation, (ii) possibly (at least partially) disoriented PEG side chains, (iii) heterogeneity of PEG side chain length upon DSPE-PEG2000 conjugate formation and (iv) influence of PEGylation on vesicle lamellarity. Therefore, especially for liposomal carriers including PEGylated lipids the exact characterization of vesicles appears beneficial to assess the drug encapsulation capacity of carriers for which nES GEMMA seems to be a well suited analytical platform.
Fig. 6 Repeatability of liposome preparation and nES GEMMA measurement of vesicle batches upon DSPE-PEG2000 incorporation in lipid bilayers; DPPC:Chol:DSPE-PEG2000 (6:3:1 molar ratio) liposomes, extruded to 100 nm diameter (either in a single extrusion step or after serial extrusions) are compared. In contrast to non-PEGylated liposomes as shown in Fig. 5, the excellent repeatability of vesicle batch preparation is lost as can be deduced from nES GEMMA spectra (A) and resulting theoretical encapsulation capacities of liposomes (B). |
Furthermore, we closely inspected differences between two data evaluation modes of nES GEMMA spectra: number- and mass-based. Only number-based data allowed the detection of a bimodal size distribution, showing smaller and larger sized compounds within one sample. In case of mass-based data analysis, results were biased towards the detection of larger particles. Hence the latter is better suited for analyte aggregate determination. We also matched nES GEMMA to DLS data to compare EM diameter values of dry particles to hydrodynamic particle diameters. We could clearly point out that there is a significant difference between these two methods and we therefore highly recommend the combination of various independent analytical methods to allow for a most comprehensive nanoparticle characterization.
Finally, we demonstrated the repeatability of liposome preparation and nES GEMMA measurements and showed that theoretical encapsulation capacities can be calculated from particle size data and vesicle numbers in a simplified, generalized and easily applicable model. Yet we also demonstrated that vesicles carrying PEG chains exhibited a significantly lower sample preparation repeatability which was also reflected in their calculated drug encapsulation capacity scattering significantly higher around an average value than for non-PEGylated liposomes.
To summarize, we were able to demonstrate that nES GEMMA is an exceptionally well-suited method for liposome characterization. Additionally, this method allows us to assess the degree of vesicle alteration in response to mechanical stress. Likewise, batch reproducibility of vesicle preparations can be easily accessed in addition to the cargo encapsulation capacities, which can be calculated from nES GEMMA data. Especially for pharmaceutical applications, the latter is suggested to give highly valuable information on a biodegradable carrier material.
Footnote |
† Equal contribution to this work. |
This journal is © The Royal Society of Chemistry 2016 |