Particle Characterization Using Dynamic Light Scattering and the Coulter Principle

Liposomes are special lipid vesicles ranging from 50 nm to 5 µm in diameter. Liposomes have a broad application range in a number of industries that include cosmetics, textiles, the medical industry and biological research.

Determining the exact liposome concentration from both a mass perspective (grams/liter) and a count perspective (particles/liter) can aid in precision dosing of therapeutic compounds in the medical industries. For biomimetic systems, determining the total number of liposomes can be critical for efficient and functional reconstitution of membrane proteins into liposomes. Ensemble techniques include sedimentation, sieving, and light scattering (dynamic and static).

Discrete characterizing techniques include electron microscopy, optical microscopy, and the Coulter Principle. Sedimentation and sieving techniques have very limited accuracy and require long periods of time for each measurement.

DLS helps measure the average diameter and polydispersity of the sample, however as an ensemble technique, concentration information is fundamentally unavailable. Discrete techniques, such as electron and optical microscopy, give accurate size and shape for particles. Using the Coulter Principle with DLS provides additional details along with concentration, at the same time verifying the size information derived from DLS on a rigorous, particle-by-particle basis.

Method

DelsaMax CORE Analysis

Librede, a leading company in lipid development purchased the liposomes. Within three weeks of production, the liposomes were analyzed. The liposomes were made of lipid type DPhPC (1,2-diphytanoyl-sn-glycero-3-phosphocholine); the suspension buffer comprised 150 mM NaCl, 5mM CaCl2, 10mM HEPES, and pH8.0. The initial concentration was 0.8 mg/mL.

For DLS analysis on the DelsaMax CORE, the liposomes were diluted 1:100 in deionized, submicron filtered water resulting in a final concentration of 0.008 mg/mL, and analyzed in the disposable cyclic polyolefin cuvette using 20 µl of sample. The liposome sample was run five times; each run consisted of three acquisitions that lasted five seconds/acquisition. The set cell temperature was 25°C.

The Sum-of-Squares (SOS) fit for all samples were less than 10, indicating a clean sample without any contamination from dust or bubbles. Diameter and polydispersity reported are from cumulant analysis, which assumes a monomodal sample, regularization analysis (representative data shown in Figure 2) indicated that the sample is monomodal.

The analysis on the DelsaMax CORE takes as little as one second and can easily verify the sizing data from the MS4e, as shown in Figure 2. The average diameter of the liposomes in the DelsaMax CORE is reported at 320.2 ± 19.8nm, very close to the mean diameter of the liposomes from the MS4e (343 nm) and much larger than the median size (292 nm) from the MS4e is shown in Table 1.

In Figure 1, particle size is plotted in bins with width less than 10 nm in size. There are 400 total bins across the diameter range (x-axis). Table 1. The two reported mean diameters are within 10%, supporting the veracity of each particle characterization method

Differential number count of liposomes.

Figure 1. Differential number count of liposomes.

Dynamic light scattering measurement of the average diameter of the DPhPC liposomes.

Figure 2. Dynamic light scattering measurement of the average diameter of the DPhPC liposomes.

DelsaMax PRO Analysis

The DPhPC liposomes from Librede were also analyzed on the DelsaMax PRO, which offers simultaneous size and zeta potential. For comparison, generic liposomes from Lypo-Spheric™ were also diluted in water and run.

The DPhPC liposomes were run in the same conditions as above (0.008 mg/mL), while the generic liposomes were run at ~0.05 mg/mL in water. The samples were injected into the flow cells (~200 pl/sample) and each sample was analyzed a total of five times. The zeta potential collection period was 20 s, during which DLS data was collected as well (10 runs, 2 s/run). All samples were run at 25°C. Figure 3 shows the representative size and zeta potential while the quantitative data is shown in Table 2.

Table 1. The two reported mean diameters are within 10%, supporting the veracity of each particle characterization method.

Comparison of Data from Coulter Principle (MS4E) and Dynamic Light Scattering (DelsaMax CORE)
Instrument Technology Mean Diameter Median Diameter Polydispersity
DelsaMax CORE Dynamic Light Scattering 320.2 + 19.8nm N/A 42.0 + 8.2nm
MS4e Coulter Principle 343 + 174nm 292nm N/A

Table 2. Note that the extruded liposomes from Librede have a much smaller polydispersity, indicating that the sample is much more uniform in size.

Comparison of Data from Generic Liposomes from Lypo-Spheric™ and Extruded Liposomes Provided by Librede
Type Mean Diameter Polydisperity Zeta Potential
DPhPC Liposomes 307.4 + 2.6nm 47.8 + 16.2nm -68.01 + 3.1mV
Generic Liposomes 494.8 + 60.4nm 141.2 + 17.2nm -99.94 + 3.03mV

Comparison of generic liposomes vs. DPhPC-Extruded liposomes on the DelsaMax PRO. (a) The raw mobility data from all 31 zeta potential detectors for the DPhPC liposomes; and (b) the raw mobility data from all 31 zeta potential detectors for the generic liposomes (c) Diameter comparison of the two types of liposomes. Note that larger particles are clearly present in the generic liposomes.

Comparison of generic liposomes vs. DPhPC-Extruded liposomes on the DelsaMax PRO. (a) The raw mobility data from all 31 zeta potential detectors for the DPhPC liposomes; and (b) the raw mobility data from all 31 zeta potential detectors for the generic liposomes (c) Diameter comparison of the two types of liposomes. Note that larger particles are clearly present in the generic liposomes.

Figure 3. Comparison of generic liposomes vs. DPhPC-Extruded liposomes on the DelsaMax PRO. (a) The raw mobility data from all 31 zeta potential detectors for the DPhPC liposomes; and (b) the raw mobility data from all 31 zeta potential detectors for the generic liposomes (c) Diameter comparison of the two types of liposomes. Note that larger particles are clearly present in the generic liposomes.

Multisizer 4e COULTER COUNTER Analysis

The same stock liposome solution used in the DelsaMax CORE analysis was used for the Multisizer 4e analysis. ISOTON II Diluent (Beckman Coulter, Inc.) was used to dilute the liposomes and the same were run on a Multisizer 4e instrument equipped with 10 µm aperture.

5 µl of stock liposome solution (0.8 mg/mL) was diluted with 19.995 mL of ISOTON II (1:4,000 dilution). The 10µm aperture was installed for the analysis and the analytical range was 0.2-6 µm with 400 size bins logarithmically spaced. For determining liposome molarity, the analysis was run in volumetric mode and 10 µl of sample were analyzed in total. Ten separate runs were conducted.

Discussion

The Multisizer 4e COULTER COUNTER (MS4e) has the largest dynamic range of any instrument based on the Coulter Principle, spanning from 200 nm up to 1,600 µm. By analyzing the entire sample particle-by-particle, any outlier can be easily detected and quantified.

As the MS4e digitally acquires and saves pulse height and width data, reanalysis is possible with any width of analysis. Thus the user can zoom in on an area of interest and have particle bin sizes less than 0.1nm in diameter, as seen in Figure 4.

A zoomed-in view of the MS4e Data. Because the pulse data is saved digitally, particle size bin width can be recalculated to have any width that is needed, with minimum-width size smaller than 0.1nm/bin. In this plot, the percent of liposome particles from 235 nm to 240 nm is displayed.

Figure 4. A zoomed-in view of the MS4e Data. Because the pulse data is saved digitally, particle size bin width can be recalculated to have any width that is needed, with minimum-width size smaller than 0.1nm/bin. In this plot, the percent of liposome particles from 235 nm to 240 nm is displayed.

This information has been sourced, reviewed and adapted from materials provided by Beckman Coulter, Inc. - Particle Characterization.

For more information on this source, please visit Beckman Coulter, Inc. - Particle Size Characterization.

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