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Particle size analyses of polydisperse liposome formulations with a novel multispectral advanced nanoparticle tracking technology
International Journal of Pharmaceutics 566 (2019) 680–686
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International Journal of Pharmaceutics journal homepage: www.elsevier.com/locate/ijpharm
Particle size analyses of polydisperse liposome formulations with a novel multispectral advanced nanoparticle tracking technology
T
Pushpendra Singha,b, Jeffrey Bodycombc, Bill Traversd, Kuba Tatarkiewicze, Sean Traversd, ⁎ Gary R. Matyasb, Zoltan Becka,b, a
U.S. Military HIV Research Program, Henry M. Jackson Foundation for the Advancement of Military Medicine, 6720A Rockledge Drive, Bethesda, MD, USA Laboratory of Adjuvant and Antigen Research, U S Military HIV Research Program, Walter Reed Army Institute of Research, 503 Robert Grant Avenue, Silver Spring, MD, USA c HORIBA Instruments Inc, 20 Knightsbridge Rd, Piscataway Township, NJ, USA d Anatom Technology Inc, 22803 Shady Grove Ct, Baldwin, MD, USA e HORIBA Scientific, 9755 Research Dr, Irvine, CA, USA b
A R T I C LE I N FO
A B S T R A C T
Keywords: Dynamic light scattering Laser diffraction Nanoparticle tracking analysis MANTA
Liposomes are potent adjuvant constituents for licensed vaccines and vaccine candidates and carriers for drug delivery. Depending on the method of preparation, liposomes vary in size distribution, either forming uniform small size vesicles or a heterogeneous mixture of small to large vesicles. Importantly, differences in liposomal size have been demonstrated to induce differential immune responses. Determination of particle size distribution could therefore be crucial for the efficacy and stability of vaccine formulations. We compared the techniques of dynamic light scattering, laser diffraction, and conventional nanoparticle tracking analysis with a novel multispectral advanced nanoparticle tracking analysis (MANTA) for particle size determination of mono- and polydisperse liposomes. MANTA reported an average 146 nm size of monodisperse liposomes but showed a multimodal distribution of polydisperse liposomes with continuous sizes from 50 to 2000 nm. However, approximately 95% of particles were in the size range of 50–1500 nm and only few particles were identified in the 1500–2000 nm range for the investigated volume. Based on our results, we conclude that MANTA is the most suitable approach and can serve as stand-alone technique for particle size characterization of heterogeneous liposome samples in the 50–2000 nm size range.
1. Introduction Liposome are membrane vesicles that have been used as vehicles for therapeutic drugs and as adjuvant systems. They are constituents of licensed drugs and vaccines, which have been used to prevent and cure human diseases (Allen and Cullis, 2013; Alving et al., 2016a, 2012a,b; Bulbake et al., 2017; De Serrano and Burkhart, 2017; Rao and Alving, 2016). Recently, a shingles vaccine containing a liposomal adjuvant, AS01b (Shingrix®, GlaxoSmithKline) has been licensed by the U.S. Food and Drug Administration (Cohen, 2015; Lal et al., 2015). A critical component of the development of liposomal drug and vaccine formulations is the characterization of the liposome size (FDA, 2018). The currently developed sizing technologies, dynamic light scattering and laser diffraction are excellent technologies for the determination of
small monodisperse liposome formulations. However, these techniques have significant deficiencies in the determination of polydisperse liposomes formulations. Army Liposome Formulation (ALF) is a liposome formulation that has been used for multiple preclinical and phase I and II clinical trials against various infectious diseases and cancer (Alving et al., 2016a,b). The ALF-type liposomes contain saturated phospholipids, dimyristoyl phosphatidylcholine (DMPC) and dimyristoyl phosphatidylglycerol (DMPG), cholesterol and monophosphoryl lipid A (MPLA) as an immunostimulant (Beck et al., 2015a,b, 2018; Hanson et al., 2015; Singh et al., 2018). ALF-type liposomes vary in membrane architecture and particle size distribution owing to their molecular constituents and the method of preparation. The aqueous form of liposomes (Lwet) contains nanoparticles with a size < 200 nm, whereas the reconstituted
Abbreviations: ALF, Army Liposome Formulation; MPLA, monophosphoryl lipid A; DMPC, dimyristoyl phosphatidylcholine; DMPG, dimyristoyl phosphatidylglycerol; PSL, polystyrene latex beads; SUV, small unilamellar vesicles; DLS, dynamic light scattering; LD, laser diffraction; NTA, nanoparticle tracking analysis; MANTA, multispectral advanced nanoparticle tracking analysis; PdI, polydispersity index ⁎ Corresponding author at: Walter Reed Army Institute of Research, Room 2N79, 503 Robert Grant Ave, Silver Spring, MD 20910, USA. E-mail address: [email protected] (Z. Beck). https://doi.org/10.1016/j.ijpharm.2019.06.013 Received 5 March 2019; Received in revised form 24 May 2019; Accepted 6 June 2019 Available online 06 June 2019 0378-5173/ © 2019 Elsevier B.V. All rights reserved.
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lyophilized-liposomes in aqueous suspension (Llyo) is a mixture of particles with wide size distribution in the range of nanometer to micrometer (Beck et al., 2015b, 2018). Lwet contains uniformly distributed (monodisperse) small unilamellar vesicles (SUVs), whereas Llyo contains heterogeneous mixture (polydisperse) of liposomes of nanometer to micrometer sizes. Particle size is shown to impact adjuvanticity of the liposomes by influencing the immune responses (Brewer et al., 2004, 1998; Goya et al., 2008; Mann et al., 2009; Singh et al., 2018; Tandrup Schmidt et al., 2016). Small size (< 200 nm) liposomes were reported to enhance Th2 responses, whereas large (> 500 nm) liposome particles promoted Th1 responses (Brewer et al., 1998; Mann et al., 2009). Vaccines containing either ALFwet or ALFlyo with identical amount of lipids and antigen exhibited different immunological outcomes, suggesting that the adjuvant activity of ALFwet and ALFlyo could be attributed to their size (Beck et al., 2018). Determination of particle size distribution is therefore crucial for both stability and immunogenicity of vaccine formulation. Dynamic light scattering (DLS), laser diffraction (LD), and conventional nanoparticle tracking analysis (NTA) are the most commonly used techniques for the particle size analyses of nanoparticles such as liposomes (Beck et al., 2018). However, none of these techniques alone is able to accurately determine the entire range of particle sizes in Llyo. Upon investigation with DLS and LD on monodisperse polystyrene latex beads (PSLs) of different sizes or their mixtures, we observed that these techniques were not capable of determining the accurate particle size distribution in a heterogeneous population. The NTA measured particle size and their abundance by tracking each individual particle in both the monodisperse and polydisperse samples, but was not capable of detecting particles larger than 1000 nm. Here, we describe a novel multispectral advanced nanoparticle tracking analysis (MANTA) technology that accurately measured particle size in polydisperse samples ranging from 50 to 2000 nm. We demonstrate that MANTA is superior to the existing techniques in determining the particle size distribution for polydisperse liposomes.
2.3. Size analysis of PSLs and liposome formulations using DLS, LD, NTA and MANTA
2. Materials and methods
2.3.4. MANTA ViewSizer® 3000 (MANTA Instruments Inc., San Diego, CA, recently acquired by HORIBA Scientific, Irvine, CA) uses advanced multispectral NTA for measurement of particle number, size, and concentration. Similar to NanoSight, ViewSizer® 3000 also utilizes Brownian motion detection to determine the number and size of individual particles. It is equipped with blue (450 nm), green (532 nm), and red (635 nm) lasers that create a polarized, narrow and bright light sheet allowing for precise determination of investigated volume. The built-in multispectral detection system tracks the scattered light of different wavelengths simultaneously from each individual particle. ViewSizer® 3000 provides proper conversion of mean squared distance into size without using any fitting of particle size distribution data, unlike the finite track length adjustment (FTLA) method employed by NanoSight (van der Pol et al., 2014). The sample cell (cuvette with a special insert) does not have ‘O’ rings or narrow channels allowing for better mixing of polydisperse colloids without any deposition of particles or cross-contamination of samples. Since the MANTA is more sensitive and can detect particle number about an order of magnitude less than NTA, nanoparticle-free water or buffer were used for sample preparation. Software version 1.8.0.3417/1.0.7.2617 was used for the particle size analysis and calculation of density of Particle Size Distribution and accompanying parameters.
2.3.1. DLS All measurements were performed with Malvern Zetasizer Nano S (Malvern, Worcestershire, United Kingdom) equipped with a 633-nm He-Ne laser at 25 °C. Dispersion Technology Software version 6.01 (Malvern) was used to collect and analyze the data. Three runs of 60 s measurements were performed with a refractive index of 1.59 and 1.45 for PSLs and liposomes, respectively. The intensity, size distribution, the hydrodynamic diameter as Z-average, and the polydispersity index (PdI) were obtained from the autocorrelation fit of the data. 2.3.2. LD All measurements were performed with HORIBA LA-960 (HORIBA Instruments Inc, Irvine CA) equipped with red (650 nm) and blue (405 nm) solid state laser diodes, which allowed for the detection of particles in the size range of 10 nm to 3 mm. PSL standards and liposome samples were diluted in deionized water and dPBS, respectively. Particles were measured in a 15 ml glass cuvette while mixing continuously with a magnetic stir-bar at maximum speed. Software version 8.1 was used for the particle size analysis and calculation of D10, D50, and D90 distribution values. 2.3.3. NTA NanoSight NS300 (Malvern) uses conventional NTA for analyses of particle number, size, and concentration. NanoSight was demonstrated to measure size distribution of particles reliably between 30 and 1000 nm (Filipe et al., 2010). NanoSight used for this study was equipped with a 633 nm laser. Both monodisperse and polydisperse samples were injected in the sample chamber with sterile syringes. At least three measurements of each sample were performed at room temperature for 60 s with a manual shutter and gain adjustments. The NTA 3.2 software was used for capturing and analyzing the data. The mean and SD were obtained from arithmetic values of all the particle sizes.
2.1. Lipids and polystyrene beads DMPC, DMPG, cholesterol (plant derived), and synthetic monophosphoryl MPLA (3D-PHAD®) were purchased from Avanti Polar Lipids (Alabaster, AL). Polystyrene latex beads (PSLs) of 70, 200, 600 and 1300 nm sizes were from ThermoFisher Scientific (Asheville, NC). Although the exact concentration of the individual particles in the PSL mix were not provided by the manufacturer, PSL mix was prepared by mixing 25, 75, 200 and 450 µl of 70, 200, 600 and 1300 nm PSL standards, respectively, in 10 ml volume. All individual PSLs and bead mixture (PSLmix) were diluted in deionized water to the concentration of 107–109 particles/ml.
2.2. Preparation of liposomes Small unilamellar liposome formulation (Lwet) consisting of saturated zwitterionic DMPC, cholesterol, and anionic DMPG phospholipids in a molar ratio of 9:7.5:1 were prepared in Dulbecco’s phosphate buffered saline pH 7.2 (dPBS) as described previously (Beck et al., 2015a). The 43 mol% cholesterol concentration with respect to the total lipids was selected to match that of the human erythrocyte membrane. Cholesterol concentration was quantified by colorimetric assay (Beck et al., 2015b). For preparation of polydisperse liposomes (Llyo), Lwet was lyophilized and then rehydrated in sterile deionized water and kept on a roller for 1 h at room temperature. Both Lwet and Llyo formulations were stored at +4 °C.
3. Results 3.1. Analyses of size standard polystyrene latex beads Monodisperse samples have an extremely narrow particle size 681
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both the monodisperse PSLs and polydisperse PSLmix with an additional low intensity peak in the range of 660–710 nm. The NTA reported average D10, D50 and D90 as 255, 428, and 645 nm for PSLmix. MANTA Instruments has developed a multispectral advanced technology which uses NTA with significant improvements enabling accurate measurement of the number and size of particles. Fig. 1G shows that MANTA detected not only 70, 200 and 600 nm size particles but also 1300 nm size standard in monodisperse PSLs with unprecedented accuracy. Importantly, MANTA detected the small peak of 1300 nm size also in PSLmix, which could not be discerned by another techniques (Fig. 1H). MANTA detected peaks of 70 and 600 nm size distinctly, but did not resolve the 70 and 200 nm size PSLs. The MANTA determined average D10, D50 and D90 values to be 90, 550 and 1130 nm in PSLmix. Taken together, these results demonstrate that MANTA is capable in detecting particle size beyond 1000 nm. 3.2. Size analyses of Lwet and Llyo After examining the performance of different techniques for size measurement on polydisperse PSLmix, we employed the DLS, LD, NTA, and MANTA techniques on liposomes. Liposomes were made and stored either in aqueous form (Lwet) or Lwet was lyophilized and reconstituted in aqueous solution (Llyo). Although Lwet contained small unilamellar liposomes (SUVs) with uniform size distribution in the range of 30–150 nm, Llyo contained heterogeneous mixture of liposomes of different sizes ranging from nanometer to micrometer (Beck et al., 2018). Also, the particle size distribution of Llyo is continuous, as opposed to discrete sizes in PSLmix. DLS analyses reported a normal distribution in Lwet with average liposome size of 56 nm and a low polydispersity index, PdI < 0.2 (Fig. 2A). In contrast, Llyo showed a multimodal distribution of particles in the range of 500 to 1600 nm with high particle size variation (PdI > 0.5). Much like PSLmix (Fig. 1B), DLS did not detect any small size particle in the polydisperse Llyo. Similar to DLS, LD analyses reported a normal distribution with a mean liposome size of 120 nm in Lwet and D10, D50, and D90 values as 78, 123, and 194 nm (Fig. 2B). In contrast, LD detected the large (500 nm–40 µm), but not the small size liposomes (< 500 nm) in Llyo. LD analyses reported the average D10, D50 and D90 values as 7.49, 12.42, and 18.52 µm for Llyo. The NTA analyses of Lwet also reported a single peak of the liposomes with an average size of 114 nm (Fig. 2C). The average D10, D50, and D90 values were 89, 118, and 188 nm, respectively. In contrast, multiple overlapping peaks were identified in the range of 30–1000 nm in Llyo. The NTA did not detect particles > 1000 nm, and the average D10, D50 and D90 values were 254, 409, and 996 nm in Llyo. Because NTA did not detect particles larger than 1000 nm, we employed MANTA for Llyo, which contains a heterogeneous mixture of the liposomes. As expected, MANTA displayed unimodal distribution of Lwet. The average liposome size of Lwet was reported to be 146 nm (Fig. 2D). The MANTA showed a multimodal distribution of Llyo from 50 nm to 2 µm, where ∼95% of particles were smaller than 1500 nm size and only very few particles were present beyond 1500 nm as shown in Fig. 2D. This suggests that MANTA covered almost the entire size range of particle sizes in Llyo. Similar to NTA, MANTA recognized several peaks in Llyo. However, these peaks were more continuous. Fig. 3A–C provide a visual representation of the particle distribution for PSLmix, Lwet and Llyo, respectively. These images are taken from a video of the particle scattering at 90°. Intensity of scattering depends on particle size, but the relationship is not linear (Bohren and Huffman, 1983). Therefore, accurate size determination requires particle tracking. The particle sizes in Lwet were uniform with the average D10, D50 and D90 values reported by MANTA as 70, 150 and 370 nm, respectively (Fig. 4). The average D10, D50 and D90 values were 150, 670 and 1550 nm for Llyo. These results were in agreement with the other techniques that Llyo had much wider particle size distribution
Fig. 1. Particle size distribution of 70, 200, 600, and 1300 nm polystyrene beads. Size analyses of 70 (······), 200 ( ), 600 ( ) and 1300 ( ) nm polystyrene latex (PSL) beads by (A and B) dynamic light scattering, (C and D) laser diffraction, (E and F) conventional NTA, and (G and H) multispectral advanced NTA. Panels A,C,E,F represent size of individual beads whereas panels B,D,F,H show particle size distribution for PSLmix.
distributions, whereas polydisperse samples contain particles in a broad size range. We compared the standard available techniques of dynamic light scattering, laser diffraction, conventional and multispectral advanced NTA for particle size measurements of monodisperse PSLs corresponding to 70, 200, 600 and 1300 nm diameters and polydisperse samples created by mixing of the individual beads. Fig. 1A shows that DLS accurately detected PSLs of different standard sizes with unimodal distribution and low polydispersity indexes (PdI < 0.2) when they were measured individually. Although, as shown in Fig. 1B, DLS reported a high polydispersity index (PdI > 0.4) for the PSLmix, it did not detect the individual PSLs of corresponding sizes and displayed a unimodal size distribution with a mean diameter of 600 nm. We then employed LD, which has a wide dynamic range for the detection of particles from the nanometer to millimeter size range. Fig. 1C shows that LD displayed a single sharp peak for the individual PSLs. Similar to DLS, LD did not detect the individual beads of the corresponding sizes and reported a unimodal average size distribution of the PSLmix (Fig. 1D). Unlike DLS, LD does not measure the polydispersity index of the samples, but provides particle size distribution as D10, D50 and D90 values. LD reported D10, D50 and D90 of 134, 562, and 1394 nm, respectively for the PSLmix. Conventional NTA tracks individual particles undergoing Brownian motion and deduces the size based on the diffusion of individual particles in the dispersant. Fig. 1E and F show that NTA reported sizes of 72, 203, and 605 nm in the monodisperse PSLs and identified peaks at 68, 207, and 602 nm in PSLmix for the size standards of 70, 200 and 600 nm. However, 1300 nm size standard was detected as 1000 nm in 682
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Fig. 3. Multispectral advanced NTA video frames for (A) mix of 70, 200, 600 and 1300 nm PSLs, (B) Lwet and, (C) Llyo. Data acquisition and analyses were from 50 frames of each sample.
4. Discussion Our laboratory recently showed that HIV-1 gp140 adjuvanted with either ALFwet or ALFlyo gave significantly different immune response in mice suggesting that adjuvanticity is determined by the number and size of liposome particles (Beck et al., 2018). Based on size, liposomal particles were trafficked differently from the injection site: they were directly moved either to the local lymph nodes or stayed at the injection sites for some time before the uptake by antigen presenting cells (Mann et al., 2009; Richards et al., 1995). When phagocytosed by the immune cells, antigens associated with large liposomes show better processing and antigen presentation as compared to small liposomes (Brewer et al., 2004). Dendritic cells were shown to uptake small-size particles without size preference in the range of 10–200 nm, but uptake of beads larger than 280 nm was significantly reduced in DC compared to monocytes (Goya et al., 2008; Reece et al., 2001). Further, liposome size was demonstrated to influence the pharmacokinetics and biodistribution of antigen, thereby, impacting the stimulation of the immune response (Manolova et al., 2008). It was most likely a result of the immune cell’s size preference for the uptake of the particles (Goya et al., 2008; Reece et al., 2001). For example, dendritic cells more efficiently process small size liposomes, whereas other immune cells, such as
Fig. 2. Particle size distribution of Lwet ( ) and Llyo ( ) determined by (A) dynamic light scattering, (B) laser diffraction, (C) conventional NTA, and (D) multispectral advanced NTA. The graphs shown for each technique are representatives of three independent measurements for each sample.
than Lwet. Taken together, our results demonstrate that MANTA is superior to other particle sizing techniques reported here for measuring particles in samples with wide size range between 50 and 2000 nm.
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polydisperse samples (e.g., PSLmix and Llyo) was not determined accurately by DLS analyses. This technique could not provide reliable data on polydisperse samples with high PdI values (> 0.4) (Hassan et al., 2015). Since DLS performs an intensity-based measurement, it could not detect small size particles in Llyo formulations as they were masked by the strongly scattered light from large particles. Laser diffraction (LD) infers the size and number of particles by measuring the intensity of scattered light as a function of angle (Bohren and Huffman, 1998; Jillavenkatesa et al., 2001). Small particles scatter light at wider angles with low intensity, whereas large particles scatter light at narrower angles with high intensity. LD detected size and intensity of monodisperse PSLs and gave a wide size distribution for PSLmix, which was different from the sizes of the individual components. LD gives a volume-based particle size distribution, and due to dependence on the cube of diameter, measurements obtained from LD were dominated by the larger particles in the sample. Furthermore, since laser diffraction is an ensemble technique, results for mixtures were inherently low-resolution. Consequently, LD could not detect the individual populations with closely spaced sizes in the PSLmix and polydisperse liposome samples. Due to this low resolution, LD analysis, shown in Fig. 1D, showed a broad peak in the size distribution, rather than the expected distinct peaks. LD described particle size distributions as D10, D50 and D90 values that represent particle size comprising < 10, 50, and 90% of the total population. Despite the substantial number of the small particles present in Llyo, which represented only a small fraction of the liposome volume, LD reported a high D10 value (7.49 µm). Nanoparticle tracking analysis determines the particle size in a sample from the speed of the individual particle undergoing Brownian motion (Einstein, 1905). In conventional NTA, each particle is tracked through the multiple images in a video produced by light scattered from the freely moving particles over time. Since each particle is analyzed independently, resolution of mixtures has much higher resolution than that of LD or DLS. Based on the mean squared distance travelled by a particle in a given time frame, individual particle size is calculated. NanoSight uses conventional NTA that employs a single laser to illuminate the sample volume and detects the light scattered from freely diffusing particles. This technology has been successfully utilized in measuring the particle size distribution between 30 and 1000 nm (Filipe et al., 2010; van der Pol et al., 2014). We employed PSL beads of 70, 200, 600, and 1300 nm size for NTA. It detected the particle size accurately for individual PSLs and PSLmix up to 600 nm. However, it did not detect accurately the 1300 nm PSL either alone or in the PSLmix. These observations were consistent to the limitation of the NTA for measurement of particle sizes beyond 1000 nm (Filipe et al., 2010). This was also the case for Llyo, where particles larger than 1000 nm were not detected and D90 value was reported as 996 nm. In conventional NTA, detection limit is imposed by the hardware and the capabilities of the FTLA model-based software. The absorption of the light by particles with large surface area increases the temperature resulting in higher movement and this makes them appear to be of smaller size. In addition to DLS, LD, and NTA, tunable resistive pulse sensing (TRPS), asymmetric flow field-flow fractionation coupled with multiangle light scattering (AF4-MALS) and transmission electron cryomicroscopy (CryoTEM) are other most widely used techniques for particle characterization (Fatouros et al., 2007; Fuentes et al., 2018; Willmott, 2018). TRPS works on the Coulter principle where resistivity of the particle enables measurements of size and concentration of particles in the wide size range (Willmott, 2018). AF4 performs size-based physical separation of biomolecules prior to their size measurement by MALS set up (Fuentes et al., 2018). Cryo-TEM can visualize liposomes at their native state and do not need any sample fixation (Fatouros et al., 2007; Ferreira et al., 2006). Despite the above-mentioned advantages, all of these techniques have certain limitations toward particle size measurement of polydisperse liposomes. For example, TRPS does not provide consistent results on liposome size, as it gave significantly different
Fig. 4. Representative particle size distribution by multispectral advanced NTA. D10, D50 and D90 show the mean ± SD values of 5 measurements in (A) Lwet and Llyo, and (B) three different manufactures of Llyo.
macrophages phagocytose large size particle (Goya et al., 2008; Reece et al., 2001). Therefore, determining the particle size distribution of liposomes could help predicting their adjuvanticity. However, due to wide range and inherent size variation, determining size distribution of polydisperse samples such as ALFlyo by a single technique has been an arduous task. In this study, we reported the utility of MANTA technology compared to other popularly used particle size determining techniques, such as DLS, LD and NTA for mono- and polydisperse liposomes. DLS captures the fluctuations in intensity of scattered light from an ensemble of particles undergoing Brownian motion in the solvent on a microsecond time scale (Pecora, 1985). Intensity fluctuations are then statistically autocorrelated to infer the diffusion coefficient. Hydrodynamic diameter is then determined from the diffusion coefficient. Typically, the method of cumulants is used to fit the autocorrelation function (22412, 2017; Koppel, 1972) and average hydrodynamic diameter and polydispersity index of the sample are determined. The polydispersity index is a useful quantitative indicator of sample heterogeneity for sufficiently narrow particle size distributions with a polydispersity index of < ∼0.3 (Hassan et al., 2015; Xu et al., 1987). For broader distributions, Laplace inversion is used to determine particle size distribution (22412, 2017; Hassan et al., 2015). As shown in Fig. 1B, DLS worked well for the monodisperse sample, i.e., the average size of the particles was tight with PdI < 0.2 displayed correctly. However, DLS failed to detect the individual particles of different sizes in the polydisperse PSLmix. As a result, the particle size distribution of 684
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References
size estimations at different applied pressure (Maas et al., 2015). AF4 offers separation of macromolecules and nanoparticles from 1 nm to 1000 nm, and it is not suitable technique for particles larger than 1000 nm in polydisperse samples. In addition, besides its cumbersome design, applied fluid shear might induce re-sizing of soft liposome assemblies during the process of size-based physical separation of particles. Lastly, Cryo-TEM is not a quantitative method and need to be integrated with another quantitative biophysical analysis techniques for detailed structural and morphological characterization of self-assembled nanostructures (Fatouros et al., 2007). ViewSizer® 3000 employs three lasers that are used together to create polarized, narrow bright light sheet to visualize particles. The MANTA is an elegant and absolute method that does not require calibration standards or knowledge of particle material properties such as refractive index. Because of the usage of three lasers together with sensitive color charge-coupled device (CCD) camera, it enables the detection of different wavelengths simultaneously. The power of each laser can be individually set for each size population of the polydispersed sample to maximize sensitivity (Maguire et al., 2018). Consequently, the measurements using MANTA are more sensitive than NTA and cover wider particle size range (50 nm to 2 µm). Although the MANTA could not differentiate well the 70 and 200 nm beads in the PSLmix, it was able to detect the continuum of all sizes in Llyo. As apparent from the particle size distribution in Llyo, MANTA covered almost the entire particle size range.
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Hanson, M.C., Abraham, W., Crespo, M.P., Chen, S.H., Liu, H., Szeto, G.L., Kim, M., Reinherz, E.L., Irvine, D.J., 2015. Liposomal vaccines incorporating molecular adjuvants and intrastructural T-cell help promote the immunogenicity of HIV membrane-proximal external region peptides. Vaccine 33, 861–868. Hassan, P.A., Rana, S., Verma, G., 2015. Making sense of Brownian motion: colloid characterization by dynamic light scattering. Langmuir 31, 3–12. Jillavenkatesa, A., Dapkunas, S.J., Lum, L.H., 2001. NIST Practice Guide: Particle Size Characterization NIST Special Publication 960–1. U.S. government printing office, Washington. Koppel, D.E., 1972. Analysis of macromolecular polydispersity in intensity correlation spectroscopy: the method of cumulants. J. Phys. Chem. 57, 4814–4820. Lal, H., Cunningham, A.L., Heineman, T.C., 2015. Adjuvanted herpes zoster subunit vaccine in older adults. N. Engl. J. Med. 373, 1576–1577. Maas, S.L., de Vrij, J., van der Vlist, E.J., Geragousian, B., van Bloois, L., Mastrobattista, E., Schiffelers, R.M., Wauben, M.H., Broekman, M.L., Nolte-'t Hoen, E.N., 2015. Possibilities and limitations of current technologies for quantification of biological extracellular vesicles and synthetic mimics. J. Control. Release 200, 87–96. Maguire, C.M., Rosslein, M., Wick, P., Prina-Mello, A., 2018. Characterisation of particles in solution – a perspective on light scattering and comparative technologies. Sci. Technol. Adv. Mater. 19, 732–745. Mann, J.F., Shakir, E., Carter, K.C., Mullen, A.B., Alexander, J., Ferro, V.A., 2009. Lipid vesicle size of an oral influenza vaccine delivery vehicle influences the Th1/Th2 bias in the immune response and protection against infection. Vaccine 27, 3643–3649. Manolova, V., Flace, A., Bauer, M., Schwarz, K., Saudan, P., Bachmann, M.F., 2008. Nanoparticles target distinct dendritic cell populations according to their size. Eur. J.
5. Conclusion Liposome-based product development for human clinical trials require a method to compare particle sizes. MANTA can measure size of polydisperse samples between 50 and 2000 nm. To determine particle size distribution of the Llyo formulations containing particles in this range, MANTA was found to be the best stand-alone approach among the popularly utilized particle size measurement methods. MANTA will be a strong contender as the preferred method of choice to characterize nanoparticle formulations with polydisperse size distribution for human use. Since ViewSizer® 3000 has been only recently launched on the market, further study and validation of the system on polydisperse liposomal based adjuvants such as ALF and ALFQ is required. Declaration of Competing Interest None. Acknowledgements This work was supported by a cooperative agreement (W81XWH18-2-0040) between the Henry M. Jackson Foundation for the Advancement of Military Medicine, Inc., and the U.S. Department of Defense. Disclosures The views expressed are those of the authors and should not be construed to represent the positions of the U.S. Army or the Department of Defense. Jeffrey Bodycomb and Kuba Tatarkiewicz work for HORIBA Instruments. Kuba Tatarkiewicz was VP of Engineering of MANTA Instruments that have patents issued on the technologies described in this manuscript. The other authors declare no competing interests. Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.ijpharm.2019.06.013. 685
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Tandrup Schmidt, S., Foged, C., Korsholm, K.S., Rades, T., Christensen, D., 2016. Liposome-based adjuvants for subunit vaccines: formulation strategies for subunit antigens and immunostimulators. Pharmaceutics 8, E7. van der Pol, E., Coumans, F.A., Grootemaat, A.E., Gardiner, C., Sargent, I.L., Harrison, P., Sturk, A., van Leeuwen, T.G., Nieuwland, R., 2014. Particle size distribution of exosomes and microvesicles determined by transmission electron microscopy, flow cytometry, nanoparticle tracking analysis, and resistive pulse sensing. J. Thromb. Haemost. 12, 1182–1192. Willmott, G.R., 2018. Tunable resistive pulse sensing: better size and charge measurements for submicrometer colloids. Anal. Chem. 90, 2987–2995. Xu, R., Ford, J.R., Chu, B., 1987. Photon correlation spectroscopy, transient electric birefringence, and characterization of particle size distributions in colloidal suspensions, Particle Size Distribution. ACS Sympos. Series 115–132.
Immunol. 38, 1404–1413. Pecora, R., 1985. Dynamic Light Scattering. Springer US, Boston, MA. Rao, M., Alving, C.R., 2016. Adjuvants for HIV vaccines. Curr. Opin. HIV AIDS 11, 585–592. Reece, J.C., Vardaxis, N.J., Marshall, J.A., Crowe, S.M., Cameron, P.U., 2001. Uptake of HIV and latex particles by fresh and cultured dendritic cells and monocytes. Immunol. Cell Biol. 79, 255–263. Richards, R.L., Hailey, J.R., Egan, J.E., Gordon, D.M., Jonhnson, A.J., Alving, C.R.A., Wassef, K.M., 1995. Intramuscular fate and local effects of liposomes as carriers for drugs and vaccines. In: Shek, P.N. (Ed.), Liposomes in Biomedical Applications. Dovepress, pp. 167–178. Singh, P., Beck, Z., Matyas, G.R., Alving, C.R., 2018. Saturated phospholipids are required for nano- to micron-size transformation of cholesterol-containing liposomes upon QS21 addition. J. Liposome Res. 1–16.
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International Journal of Pharmaceutics journal homepage: www.elsevier.com/locate/ijpharm
Particle size analyses of polydisperse liposome formulations with a novel multispectral advanced nanoparticle tracking technology
T
Pushpendra Singha,b, Jeffrey Bodycombc, Bill Traversd, Kuba Tatarkiewicze, Sean Traversd, ⁎ Gary R. Matyasb, Zoltan Becka,b, a
U.S. Military HIV Research Program, Henry M. Jackson Foundation for the Advancement of Military Medicine, 6720A Rockledge Drive, Bethesda, MD, USA Laboratory of Adjuvant and Antigen Research, U S Military HIV Research Program, Walter Reed Army Institute of Research, 503 Robert Grant Avenue, Silver Spring, MD, USA c HORIBA Instruments Inc, 20 Knightsbridge Rd, Piscataway Township, NJ, USA d Anatom Technology Inc, 22803 Shady Grove Ct, Baldwin, MD, USA e HORIBA Scientific, 9755 Research Dr, Irvine, CA, USA b
A R T I C LE I N FO
A B S T R A C T
Keywords: Dynamic light scattering Laser diffraction Nanoparticle tracking analysis MANTA
Liposomes are potent adjuvant constituents for licensed vaccines and vaccine candidates and carriers for drug delivery. Depending on the method of preparation, liposomes vary in size distribution, either forming uniform small size vesicles or a heterogeneous mixture of small to large vesicles. Importantly, differences in liposomal size have been demonstrated to induce differential immune responses. Determination of particle size distribution could therefore be crucial for the efficacy and stability of vaccine formulations. We compared the techniques of dynamic light scattering, laser diffraction, and conventional nanoparticle tracking analysis with a novel multispectral advanced nanoparticle tracking analysis (MANTA) for particle size determination of mono- and polydisperse liposomes. MANTA reported an average 146 nm size of monodisperse liposomes but showed a multimodal distribution of polydisperse liposomes with continuous sizes from 50 to 2000 nm. However, approximately 95% of particles were in the size range of 50–1500 nm and only few particles were identified in the 1500–2000 nm range for the investigated volume. Based on our results, we conclude that MANTA is the most suitable approach and can serve as stand-alone technique for particle size characterization of heterogeneous liposome samples in the 50–2000 nm size range.
1. Introduction Liposome are membrane vesicles that have been used as vehicles for therapeutic drugs and as adjuvant systems. They are constituents of licensed drugs and vaccines, which have been used to prevent and cure human diseases (Allen and Cullis, 2013; Alving et al., 2016a, 2012a,b; Bulbake et al., 2017; De Serrano and Burkhart, 2017; Rao and Alving, 2016). Recently, a shingles vaccine containing a liposomal adjuvant, AS01b (Shingrix®, GlaxoSmithKline) has been licensed by the U.S. Food and Drug Administration (Cohen, 2015; Lal et al., 2015). A critical component of the development of liposomal drug and vaccine formulations is the characterization of the liposome size (FDA, 2018). The currently developed sizing technologies, dynamic light scattering and laser diffraction are excellent technologies for the determination of
small monodisperse liposome formulations. However, these techniques have significant deficiencies in the determination of polydisperse liposomes formulations. Army Liposome Formulation (ALF) is a liposome formulation that has been used for multiple preclinical and phase I and II clinical trials against various infectious diseases and cancer (Alving et al., 2016a,b). The ALF-type liposomes contain saturated phospholipids, dimyristoyl phosphatidylcholine (DMPC) and dimyristoyl phosphatidylglycerol (DMPG), cholesterol and monophosphoryl lipid A (MPLA) as an immunostimulant (Beck et al., 2015a,b, 2018; Hanson et al., 2015; Singh et al., 2018). ALF-type liposomes vary in membrane architecture and particle size distribution owing to their molecular constituents and the method of preparation. The aqueous form of liposomes (Lwet) contains nanoparticles with a size < 200 nm, whereas the reconstituted
Abbreviations: ALF, Army Liposome Formulation; MPLA, monophosphoryl lipid A; DMPC, dimyristoyl phosphatidylcholine; DMPG, dimyristoyl phosphatidylglycerol; PSL, polystyrene latex beads; SUV, small unilamellar vesicles; DLS, dynamic light scattering; LD, laser diffraction; NTA, nanoparticle tracking analysis; MANTA, multispectral advanced nanoparticle tracking analysis; PdI, polydispersity index ⁎ Corresponding author at: Walter Reed Army Institute of Research, Room 2N79, 503 Robert Grant Ave, Silver Spring, MD 20910, USA. E-mail address: [email protected] (Z. Beck). https://doi.org/10.1016/j.ijpharm.2019.06.013 Received 5 March 2019; Received in revised form 24 May 2019; Accepted 6 June 2019 Available online 06 June 2019 0378-5173/ © 2019 Elsevier B.V. All rights reserved.
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lyophilized-liposomes in aqueous suspension (Llyo) is a mixture of particles with wide size distribution in the range of nanometer to micrometer (Beck et al., 2015b, 2018). Lwet contains uniformly distributed (monodisperse) small unilamellar vesicles (SUVs), whereas Llyo contains heterogeneous mixture (polydisperse) of liposomes of nanometer to micrometer sizes. Particle size is shown to impact adjuvanticity of the liposomes by influencing the immune responses (Brewer et al., 2004, 1998; Goya et al., 2008; Mann et al., 2009; Singh et al., 2018; Tandrup Schmidt et al., 2016). Small size (< 200 nm) liposomes were reported to enhance Th2 responses, whereas large (> 500 nm) liposome particles promoted Th1 responses (Brewer et al., 1998; Mann et al., 2009). Vaccines containing either ALFwet or ALFlyo with identical amount of lipids and antigen exhibited different immunological outcomes, suggesting that the adjuvant activity of ALFwet and ALFlyo could be attributed to their size (Beck et al., 2018). Determination of particle size distribution is therefore crucial for both stability and immunogenicity of vaccine formulation. Dynamic light scattering (DLS), laser diffraction (LD), and conventional nanoparticle tracking analysis (NTA) are the most commonly used techniques for the particle size analyses of nanoparticles such as liposomes (Beck et al., 2018). However, none of these techniques alone is able to accurately determine the entire range of particle sizes in Llyo. Upon investigation with DLS and LD on monodisperse polystyrene latex beads (PSLs) of different sizes or their mixtures, we observed that these techniques were not capable of determining the accurate particle size distribution in a heterogeneous population. The NTA measured particle size and their abundance by tracking each individual particle in both the monodisperse and polydisperse samples, but was not capable of detecting particles larger than 1000 nm. Here, we describe a novel multispectral advanced nanoparticle tracking analysis (MANTA) technology that accurately measured particle size in polydisperse samples ranging from 50 to 2000 nm. We demonstrate that MANTA is superior to the existing techniques in determining the particle size distribution for polydisperse liposomes.
2.3. Size analysis of PSLs and liposome formulations using DLS, LD, NTA and MANTA
2. Materials and methods
2.3.4. MANTA ViewSizer® 3000 (MANTA Instruments Inc., San Diego, CA, recently acquired by HORIBA Scientific, Irvine, CA) uses advanced multispectral NTA for measurement of particle number, size, and concentration. Similar to NanoSight, ViewSizer® 3000 also utilizes Brownian motion detection to determine the number and size of individual particles. It is equipped with blue (450 nm), green (532 nm), and red (635 nm) lasers that create a polarized, narrow and bright light sheet allowing for precise determination of investigated volume. The built-in multispectral detection system tracks the scattered light of different wavelengths simultaneously from each individual particle. ViewSizer® 3000 provides proper conversion of mean squared distance into size without using any fitting of particle size distribution data, unlike the finite track length adjustment (FTLA) method employed by NanoSight (van der Pol et al., 2014). The sample cell (cuvette with a special insert) does not have ‘O’ rings or narrow channels allowing for better mixing of polydisperse colloids without any deposition of particles or cross-contamination of samples. Since the MANTA is more sensitive and can detect particle number about an order of magnitude less than NTA, nanoparticle-free water or buffer were used for sample preparation. Software version 1.8.0.3417/1.0.7.2617 was used for the particle size analysis and calculation of density of Particle Size Distribution and accompanying parameters.
2.3.1. DLS All measurements were performed with Malvern Zetasizer Nano S (Malvern, Worcestershire, United Kingdom) equipped with a 633-nm He-Ne laser at 25 °C. Dispersion Technology Software version 6.01 (Malvern) was used to collect and analyze the data. Three runs of 60 s measurements were performed with a refractive index of 1.59 and 1.45 for PSLs and liposomes, respectively. The intensity, size distribution, the hydrodynamic diameter as Z-average, and the polydispersity index (PdI) were obtained from the autocorrelation fit of the data. 2.3.2. LD All measurements were performed with HORIBA LA-960 (HORIBA Instruments Inc, Irvine CA) equipped with red (650 nm) and blue (405 nm) solid state laser diodes, which allowed for the detection of particles in the size range of 10 nm to 3 mm. PSL standards and liposome samples were diluted in deionized water and dPBS, respectively. Particles were measured in a 15 ml glass cuvette while mixing continuously with a magnetic stir-bar at maximum speed. Software version 8.1 was used for the particle size analysis and calculation of D10, D50, and D90 distribution values. 2.3.3. NTA NanoSight NS300 (Malvern) uses conventional NTA for analyses of particle number, size, and concentration. NanoSight was demonstrated to measure size distribution of particles reliably between 30 and 1000 nm (Filipe et al., 2010). NanoSight used for this study was equipped with a 633 nm laser. Both monodisperse and polydisperse samples were injected in the sample chamber with sterile syringes. At least three measurements of each sample were performed at room temperature for 60 s with a manual shutter and gain adjustments. The NTA 3.2 software was used for capturing and analyzing the data. The mean and SD were obtained from arithmetic values of all the particle sizes.
2.1. Lipids and polystyrene beads DMPC, DMPG, cholesterol (plant derived), and synthetic monophosphoryl MPLA (3D-PHAD®) were purchased from Avanti Polar Lipids (Alabaster, AL). Polystyrene latex beads (PSLs) of 70, 200, 600 and 1300 nm sizes were from ThermoFisher Scientific (Asheville, NC). Although the exact concentration of the individual particles in the PSL mix were not provided by the manufacturer, PSL mix was prepared by mixing 25, 75, 200 and 450 µl of 70, 200, 600 and 1300 nm PSL standards, respectively, in 10 ml volume. All individual PSLs and bead mixture (PSLmix) were diluted in deionized water to the concentration of 107–109 particles/ml.
2.2. Preparation of liposomes Small unilamellar liposome formulation (Lwet) consisting of saturated zwitterionic DMPC, cholesterol, and anionic DMPG phospholipids in a molar ratio of 9:7.5:1 were prepared in Dulbecco’s phosphate buffered saline pH 7.2 (dPBS) as described previously (Beck et al., 2015a). The 43 mol% cholesterol concentration with respect to the total lipids was selected to match that of the human erythrocyte membrane. Cholesterol concentration was quantified by colorimetric assay (Beck et al., 2015b). For preparation of polydisperse liposomes (Llyo), Lwet was lyophilized and then rehydrated in sterile deionized water and kept on a roller for 1 h at room temperature. Both Lwet and Llyo formulations were stored at +4 °C.
3. Results 3.1. Analyses of size standard polystyrene latex beads Monodisperse samples have an extremely narrow particle size 681
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both the monodisperse PSLs and polydisperse PSLmix with an additional low intensity peak in the range of 660–710 nm. The NTA reported average D10, D50 and D90 as 255, 428, and 645 nm for PSLmix. MANTA Instruments has developed a multispectral advanced technology which uses NTA with significant improvements enabling accurate measurement of the number and size of particles. Fig. 1G shows that MANTA detected not only 70, 200 and 600 nm size particles but also 1300 nm size standard in monodisperse PSLs with unprecedented accuracy. Importantly, MANTA detected the small peak of 1300 nm size also in PSLmix, which could not be discerned by another techniques (Fig. 1H). MANTA detected peaks of 70 and 600 nm size distinctly, but did not resolve the 70 and 200 nm size PSLs. The MANTA determined average D10, D50 and D90 values to be 90, 550 and 1130 nm in PSLmix. Taken together, these results demonstrate that MANTA is capable in detecting particle size beyond 1000 nm. 3.2. Size analyses of Lwet and Llyo After examining the performance of different techniques for size measurement on polydisperse PSLmix, we employed the DLS, LD, NTA, and MANTA techniques on liposomes. Liposomes were made and stored either in aqueous form (Lwet) or Lwet was lyophilized and reconstituted in aqueous solution (Llyo). Although Lwet contained small unilamellar liposomes (SUVs) with uniform size distribution in the range of 30–150 nm, Llyo contained heterogeneous mixture of liposomes of different sizes ranging from nanometer to micrometer (Beck et al., 2018). Also, the particle size distribution of Llyo is continuous, as opposed to discrete sizes in PSLmix. DLS analyses reported a normal distribution in Lwet with average liposome size of 56 nm and a low polydispersity index, PdI < 0.2 (Fig. 2A). In contrast, Llyo showed a multimodal distribution of particles in the range of 500 to 1600 nm with high particle size variation (PdI > 0.5). Much like PSLmix (Fig. 1B), DLS did not detect any small size particle in the polydisperse Llyo. Similar to DLS, LD analyses reported a normal distribution with a mean liposome size of 120 nm in Lwet and D10, D50, and D90 values as 78, 123, and 194 nm (Fig. 2B). In contrast, LD detected the large (500 nm–40 µm), but not the small size liposomes (< 500 nm) in Llyo. LD analyses reported the average D10, D50 and D90 values as 7.49, 12.42, and 18.52 µm for Llyo. The NTA analyses of Lwet also reported a single peak of the liposomes with an average size of 114 nm (Fig. 2C). The average D10, D50, and D90 values were 89, 118, and 188 nm, respectively. In contrast, multiple overlapping peaks were identified in the range of 30–1000 nm in Llyo. The NTA did not detect particles > 1000 nm, and the average D10, D50 and D90 values were 254, 409, and 996 nm in Llyo. Because NTA did not detect particles larger than 1000 nm, we employed MANTA for Llyo, which contains a heterogeneous mixture of the liposomes. As expected, MANTA displayed unimodal distribution of Lwet. The average liposome size of Lwet was reported to be 146 nm (Fig. 2D). The MANTA showed a multimodal distribution of Llyo from 50 nm to 2 µm, where ∼95% of particles were smaller than 1500 nm size and only very few particles were present beyond 1500 nm as shown in Fig. 2D. This suggests that MANTA covered almost the entire size range of particle sizes in Llyo. Similar to NTA, MANTA recognized several peaks in Llyo. However, these peaks were more continuous. Fig. 3A–C provide a visual representation of the particle distribution for PSLmix, Lwet and Llyo, respectively. These images are taken from a video of the particle scattering at 90°. Intensity of scattering depends on particle size, but the relationship is not linear (Bohren and Huffman, 1983). Therefore, accurate size determination requires particle tracking. The particle sizes in Lwet were uniform with the average D10, D50 and D90 values reported by MANTA as 70, 150 and 370 nm, respectively (Fig. 4). The average D10, D50 and D90 values were 150, 670 and 1550 nm for Llyo. These results were in agreement with the other techniques that Llyo had much wider particle size distribution
Fig. 1. Particle size distribution of 70, 200, 600, and 1300 nm polystyrene beads. Size analyses of 70 (······), 200 ( ), 600 ( ) and 1300 ( ) nm polystyrene latex (PSL) beads by (A and B) dynamic light scattering, (C and D) laser diffraction, (E and F) conventional NTA, and (G and H) multispectral advanced NTA. Panels A,C,E,F represent size of individual beads whereas panels B,D,F,H show particle size distribution for PSLmix.
distributions, whereas polydisperse samples contain particles in a broad size range. We compared the standard available techniques of dynamic light scattering, laser diffraction, conventional and multispectral advanced NTA for particle size measurements of monodisperse PSLs corresponding to 70, 200, 600 and 1300 nm diameters and polydisperse samples created by mixing of the individual beads. Fig. 1A shows that DLS accurately detected PSLs of different standard sizes with unimodal distribution and low polydispersity indexes (PdI < 0.2) when they were measured individually. Although, as shown in Fig. 1B, DLS reported a high polydispersity index (PdI > 0.4) for the PSLmix, it did not detect the individual PSLs of corresponding sizes and displayed a unimodal size distribution with a mean diameter of 600 nm. We then employed LD, which has a wide dynamic range for the detection of particles from the nanometer to millimeter size range. Fig. 1C shows that LD displayed a single sharp peak for the individual PSLs. Similar to DLS, LD did not detect the individual beads of the corresponding sizes and reported a unimodal average size distribution of the PSLmix (Fig. 1D). Unlike DLS, LD does not measure the polydispersity index of the samples, but provides particle size distribution as D10, D50 and D90 values. LD reported D10, D50 and D90 of 134, 562, and 1394 nm, respectively for the PSLmix. Conventional NTA tracks individual particles undergoing Brownian motion and deduces the size based on the diffusion of individual particles in the dispersant. Fig. 1E and F show that NTA reported sizes of 72, 203, and 605 nm in the monodisperse PSLs and identified peaks at 68, 207, and 602 nm in PSLmix for the size standards of 70, 200 and 600 nm. However, 1300 nm size standard was detected as 1000 nm in 682
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Fig. 3. Multispectral advanced NTA video frames for (A) mix of 70, 200, 600 and 1300 nm PSLs, (B) Lwet and, (C) Llyo. Data acquisition and analyses were from 50 frames of each sample.
4. Discussion Our laboratory recently showed that HIV-1 gp140 adjuvanted with either ALFwet or ALFlyo gave significantly different immune response in mice suggesting that adjuvanticity is determined by the number and size of liposome particles (Beck et al., 2018). Based on size, liposomal particles were trafficked differently from the injection site: they were directly moved either to the local lymph nodes or stayed at the injection sites for some time before the uptake by antigen presenting cells (Mann et al., 2009; Richards et al., 1995). When phagocytosed by the immune cells, antigens associated with large liposomes show better processing and antigen presentation as compared to small liposomes (Brewer et al., 2004). Dendritic cells were shown to uptake small-size particles without size preference in the range of 10–200 nm, but uptake of beads larger than 280 nm was significantly reduced in DC compared to monocytes (Goya et al., 2008; Reece et al., 2001). Further, liposome size was demonstrated to influence the pharmacokinetics and biodistribution of antigen, thereby, impacting the stimulation of the immune response (Manolova et al., 2008). It was most likely a result of the immune cell’s size preference for the uptake of the particles (Goya et al., 2008; Reece et al., 2001). For example, dendritic cells more efficiently process small size liposomes, whereas other immune cells, such as
Fig. 2. Particle size distribution of Lwet ( ) and Llyo ( ) determined by (A) dynamic light scattering, (B) laser diffraction, (C) conventional NTA, and (D) multispectral advanced NTA. The graphs shown for each technique are representatives of three independent measurements for each sample.
than Lwet. Taken together, our results demonstrate that MANTA is superior to other particle sizing techniques reported here for measuring particles in samples with wide size range between 50 and 2000 nm.
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polydisperse samples (e.g., PSLmix and Llyo) was not determined accurately by DLS analyses. This technique could not provide reliable data on polydisperse samples with high PdI values (> 0.4) (Hassan et al., 2015). Since DLS performs an intensity-based measurement, it could not detect small size particles in Llyo formulations as they were masked by the strongly scattered light from large particles. Laser diffraction (LD) infers the size and number of particles by measuring the intensity of scattered light as a function of angle (Bohren and Huffman, 1998; Jillavenkatesa et al., 2001). Small particles scatter light at wider angles with low intensity, whereas large particles scatter light at narrower angles with high intensity. LD detected size and intensity of monodisperse PSLs and gave a wide size distribution for PSLmix, which was different from the sizes of the individual components. LD gives a volume-based particle size distribution, and due to dependence on the cube of diameter, measurements obtained from LD were dominated by the larger particles in the sample. Furthermore, since laser diffraction is an ensemble technique, results for mixtures were inherently low-resolution. Consequently, LD could not detect the individual populations with closely spaced sizes in the PSLmix and polydisperse liposome samples. Due to this low resolution, LD analysis, shown in Fig. 1D, showed a broad peak in the size distribution, rather than the expected distinct peaks. LD described particle size distributions as D10, D50 and D90 values that represent particle size comprising < 10, 50, and 90% of the total population. Despite the substantial number of the small particles present in Llyo, which represented only a small fraction of the liposome volume, LD reported a high D10 value (7.49 µm). Nanoparticle tracking analysis determines the particle size in a sample from the speed of the individual particle undergoing Brownian motion (Einstein, 1905). In conventional NTA, each particle is tracked through the multiple images in a video produced by light scattered from the freely moving particles over time. Since each particle is analyzed independently, resolution of mixtures has much higher resolution than that of LD or DLS. Based on the mean squared distance travelled by a particle in a given time frame, individual particle size is calculated. NanoSight uses conventional NTA that employs a single laser to illuminate the sample volume and detects the light scattered from freely diffusing particles. This technology has been successfully utilized in measuring the particle size distribution between 30 and 1000 nm (Filipe et al., 2010; van der Pol et al., 2014). We employed PSL beads of 70, 200, 600, and 1300 nm size for NTA. It detected the particle size accurately for individual PSLs and PSLmix up to 600 nm. However, it did not detect accurately the 1300 nm PSL either alone or in the PSLmix. These observations were consistent to the limitation of the NTA for measurement of particle sizes beyond 1000 nm (Filipe et al., 2010). This was also the case for Llyo, where particles larger than 1000 nm were not detected and D90 value was reported as 996 nm. In conventional NTA, detection limit is imposed by the hardware and the capabilities of the FTLA model-based software. The absorption of the light by particles with large surface area increases the temperature resulting in higher movement and this makes them appear to be of smaller size. In addition to DLS, LD, and NTA, tunable resistive pulse sensing (TRPS), asymmetric flow field-flow fractionation coupled with multiangle light scattering (AF4-MALS) and transmission electron cryomicroscopy (CryoTEM) are other most widely used techniques for particle characterization (Fatouros et al., 2007; Fuentes et al., 2018; Willmott, 2018). TRPS works on the Coulter principle where resistivity of the particle enables measurements of size and concentration of particles in the wide size range (Willmott, 2018). AF4 performs size-based physical separation of biomolecules prior to their size measurement by MALS set up (Fuentes et al., 2018). Cryo-TEM can visualize liposomes at their native state and do not need any sample fixation (Fatouros et al., 2007; Ferreira et al., 2006). Despite the above-mentioned advantages, all of these techniques have certain limitations toward particle size measurement of polydisperse liposomes. For example, TRPS does not provide consistent results on liposome size, as it gave significantly different
Fig. 4. Representative particle size distribution by multispectral advanced NTA. D10, D50 and D90 show the mean ± SD values of 5 measurements in (A) Lwet and Llyo, and (B) three different manufactures of Llyo.
macrophages phagocytose large size particle (Goya et al., 2008; Reece et al., 2001). Therefore, determining the particle size distribution of liposomes could help predicting their adjuvanticity. However, due to wide range and inherent size variation, determining size distribution of polydisperse samples such as ALFlyo by a single technique has been an arduous task. In this study, we reported the utility of MANTA technology compared to other popularly used particle size determining techniques, such as DLS, LD and NTA for mono- and polydisperse liposomes. DLS captures the fluctuations in intensity of scattered light from an ensemble of particles undergoing Brownian motion in the solvent on a microsecond time scale (Pecora, 1985). Intensity fluctuations are then statistically autocorrelated to infer the diffusion coefficient. Hydrodynamic diameter is then determined from the diffusion coefficient. Typically, the method of cumulants is used to fit the autocorrelation function (22412, 2017; Koppel, 1972) and average hydrodynamic diameter and polydispersity index of the sample are determined. The polydispersity index is a useful quantitative indicator of sample heterogeneity for sufficiently narrow particle size distributions with a polydispersity index of < ∼0.3 (Hassan et al., 2015; Xu et al., 1987). For broader distributions, Laplace inversion is used to determine particle size distribution (22412, 2017; Hassan et al., 2015). As shown in Fig. 1B, DLS worked well for the monodisperse sample, i.e., the average size of the particles was tight with PdI < 0.2 displayed correctly. However, DLS failed to detect the individual particles of different sizes in the polydisperse PSLmix. As a result, the particle size distribution of 684
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References
size estimations at different applied pressure (Maas et al., 2015). AF4 offers separation of macromolecules and nanoparticles from 1 nm to 1000 nm, and it is not suitable technique for particles larger than 1000 nm in polydisperse samples. In addition, besides its cumbersome design, applied fluid shear might induce re-sizing of soft liposome assemblies during the process of size-based physical separation of particles. Lastly, Cryo-TEM is not a quantitative method and need to be integrated with another quantitative biophysical analysis techniques for detailed structural and morphological characterization of self-assembled nanostructures (Fatouros et al., 2007). ViewSizer® 3000 employs three lasers that are used together to create polarized, narrow bright light sheet to visualize particles. The MANTA is an elegant and absolute method that does not require calibration standards or knowledge of particle material properties such as refractive index. Because of the usage of three lasers together with sensitive color charge-coupled device (CCD) camera, it enables the detection of different wavelengths simultaneously. The power of each laser can be individually set for each size population of the polydispersed sample to maximize sensitivity (Maguire et al., 2018). Consequently, the measurements using MANTA are more sensitive than NTA and cover wider particle size range (50 nm to 2 µm). Although the MANTA could not differentiate well the 70 and 200 nm beads in the PSLmix, it was able to detect the continuum of all sizes in Llyo. As apparent from the particle size distribution in Llyo, MANTA covered almost the entire particle size range.
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5. Conclusion Liposome-based product development for human clinical trials require a method to compare particle sizes. MANTA can measure size of polydisperse samples between 50 and 2000 nm. To determine particle size distribution of the Llyo formulations containing particles in this range, MANTA was found to be the best stand-alone approach among the popularly utilized particle size measurement methods. MANTA will be a strong contender as the preferred method of choice to characterize nanoparticle formulations with polydisperse size distribution for human use. Since ViewSizer® 3000 has been only recently launched on the market, further study and validation of the system on polydisperse liposomal based adjuvants such as ALF and ALFQ is required. Declaration of Competing Interest None. Acknowledgements This work was supported by a cooperative agreement (W81XWH18-2-0040) between the Henry M. Jackson Foundation for the Advancement of Military Medicine, Inc., and the U.S. Department of Defense. Disclosures The views expressed are those of the authors and should not be construed to represent the positions of the U.S. Army or the Department of Defense. Jeffrey Bodycomb and Kuba Tatarkiewicz work for HORIBA Instruments. Kuba Tatarkiewicz was VP of Engineering of MANTA Instruments that have patents issued on the technologies described in this manuscript. The other authors declare no competing interests. Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.ijpharm.2019.06.013. 685
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