Particle shape analysis

48 the mass weighted size distribution, calculated by the Astra software. The particle size distribution of MCTCurc-NE and TMCurc-NE was calculated from the LD raw data by applying a spherical model. For the calculation of the size distribution of TMCurc-NS and TSCurc-NS, a spherical and a non-spherical calculation model were applied. Interestingly, no difference in size distribution and characteristic diameters, like d10, d50 and d90, between spherical and non-spherical model was found for TMCurc-NS and TSCurc-NS.

Unfortunately the mathematical basics of the applied models are not accessible to the software user. Thus, the reasons of the aforementioned discrepancies of the characteristic particle sizes d10, d50 and d90 between LD and AF4/MALLS could not be clarified.

AF4/MALLS was seen as a valuable tool for the determination of the particle size distribution. The setup of suitable separation methods was more time-consuming at the beginning, compared to other applied particle sizing techniques. However, when an adequate method was found, the samples could be examined quickly and reproducible results with an acceptable standard deviation were obtained. One advantage of the applied analytical software was the direct access to the raw data of the particular measurement. A more or less individual fit of the raw data was possible. The evaluation of the MALLS data was therefore more comprehensible, compared to the result evaluation of LD and PCS measurements. However, the free choice of parameters during evaluation of the MALLS data can easily result in false interpretation and incorrect results. One further has to keep in mind that larger particles may be overestimated by MALLS and especially the mean diameters dRMSz and dRMSw should be interpreted carefully.

49 0.15 for all samples. For TMCurc-NE, the dH(PCS) complies well with the dGEOM-curve obtained from the MALLS data (Fig. 4.2-6). The ratio dRMS/dH(PCS) was between 0.8 and 0.87 and the supercooled particles of TMCurc-NE were confirmed to be of spherical shape. The dH(PCS) of the TMCurc-NS fractions was smaller than the dRMS values obtained by AF4/MALLS (Fig. 4.2-6). The dRMS/dH(PCS) ratio for the crystalline TMCurc-NS particles was between 1 and 1.4, indicating a non-spherical particle shape, which was already demonstrated by the TEM micrographs (chapter 4.2.3).

Figure 4.2-6 AF4/MALLS elution profiles of a sample of (A) TMCurc-NE and (B) TMCurc-NS.

(●) dRMS, (●) dGEOM, (●) scattered light intensity at a detector angle (θ) of 90°, (o) hydrodynamic diameter dH (z-average), determined by PCS. The (o) dots represent the mean of three runs of one sample and the error bars their respective standard deviation.

The aforementioned experiments with the injection of a high lipid concentration (0.1 mg absolute) into the channel resulted in some cases in a blockade of the system. It was suggested that the particles might form aggregates within the machine, probably causing the blockade. A similar agglomeration phenomenon was sometimes observed during LD measurements of the SLN preparations, too. Though, the nanoparticles did not tend to form aggregates during long-time storage at 8 °C or 22 °C respectively. The dilution of the nanoparticles with bidistilled water, as it was done for the PCS measurements, did also not result in a detectable agglomeration. One potential explanation for the observations in LD and AF4 is that a part of the emulsifier layer on the particle surface is washed off due to the high shear forces and dilution in these systems. It is reasonable, that the partial removal of the emulsifier makes the particles more prone to aggregation.

As a consequence of the AF4 system blockade, the eluent was changed to an aqueous 0.1 % (m/v) poloxamer 188 solution and a smaller lipid amount (0.05 mg absolute) was injected. Indeed, the changes of the experimental setup reduced the incidence of system blockades. Though, the decreasing width and the overall appearance of the elution peak

50 indicated a decreasing separation quality when poloxamer 188 was added to the eluent (Fig. 4.2-7).

Figure 4.2-7 AF4/MALLS elution profiles of a sample of (A) MCTCurc-NE, (B) TMCurc-NE, (C) TMCurc-NS and (D) TSCurc-NS. (●) d_RMS, (●) d_H, (●) scattered light intensity at a detector angle (θ) of 90°, (o) z-average determined by PCS. The (o) dots represent the mean of three runs of one sample and the error bars their respective standard deviation.

The progression of d-vs-t curves was also different from the curves obtained in poloxamer-free eluent. The curves showed a rather flat slope at the beginning, followed by a steep increase of the curve after 50 minutes, indicating a rapid increase of the particle size.

Thus, a good fractionation of the particles was probably not provided in the latter stage of the experiment. However, the characteristic particle sizes of the investigated formulations were thought to be reliable (Tab.4.2-5). The differences of the particle sizes between the formulations examined with an aqueous eluent and the formulations examined with a poloxamer-based eluent were attributed to normal batch-to-batch fluctuations (Tab.4.2-3 and Tab.4.2-5). Thus, the presence of poloxamer 188 in separation method 3 was thought to alter the fractionation quality, but the calculated particle sizes were overall comparable to the poloxamer-free methods.

51 Table 4.2-5 Characteristic diameters of the curcuminoid-loaded preparations, determined by AF4/MALLS. dGEOMz/dRMSz: z-average diameter. dGEOMw/dRMSw: mass weighted diameter.

Given MALLS values were calculated from elution profile of one sample.

Sample dGEOMz [nm] dGEOMw [nm] d10 [nm] d50 [nm] d90 [nm]

MCTCurc-NE 267.6 259.8 95.8 138.5 241.8

TMCurc-NE 264.2 238.4 79.6 136 239.8

Sample dRMSz [nm] dRMSw [nm] d10 [nm] d50 [nm] d90 [nm]

TMCurc-NS 332.2 308.4 105.4 243.6 421

TSCurc-NS 377.2 341 179.7 345.8 458.6

As it was described for TMCurc-NE and TMCurc-NS before, the fractions of the investigated samples were again collected and subsequently examined with PCS. The hydrodynamic diameter dH(PCS) of the collected fractions increased with collection time. However, the PDI of the samples was always above 0.18, pointing to a broad distribution of the single fraction. Moreover, the dH(PCS) did not fit the dGEOM or dRMS curves, respectively, as it was expected from the previous experiments conducted with poloxamer-free eluent (Fig. 4.2-7 and Fig. 4.2-6). Generally, the dH(PCS) was concluded to be too small in comparison to the dGEOM or dRMS, respectively.

As a consequence, the determined ratios dRMS/dH(PCS) were tending to higher values, indicating to an erroneous particle shape. For example, the dRMS/dH(PCS) ratio of MCTCurc-NE was between 0.9 and 1.1, which pointed rather to anisometric particles, than to compact spheres, which are actually present in the MCT-based nanoemulsion. Moreover, the estimated particle shapes of TMCurc-NE and TMCurc-NS were found to be distinctly different for the experiments conducted with a poloxamer-containing eluent compared to the data obtained with a poloxamer-free eluent. Therefore, an appropriate estimation of the particle shape by AF4/MALLS in combination with PCS was concluded to be not possible, when the eluent contained higher amounts of poloxamer 188. The fractionation quality of AF4 was shown to be decreased by the presence of poloxamer 188, but the gained particle sizes were altogether seen as correct values. Hence, the presence of poloxamer 188 in the samples was thought to influence the PCS measurement in an unfavorable way. One possible reason might be the changed viscosity of the samples, containing poloxamer, although this was regarded by adjusting the samples viscosity within the PCS software (see chapter 3.2.2). Another reason might be the formation of ordered structures by excessive poloxamer, which would also have influenced the light scattering of the sample. Moreover, the injected amount of lipid was thought to be too

52 low, resulting in a low particle concentration within the single fractions. The high PDI of the fractionated samples was seen as the consequence of the low particle concentration and of the disturbing effect of poloxamer 188. Thus, the results, obtained by PCS, were thought to be inappropriate for the comparison with the MALLS data and for an assessment of the of the particle shape. It was concluded that the injected lipid amount has to be increased in future experiments, to achieve better conditions for the PCS measurements. Furthermore, the influence of poloxamer 188 on the PCS measurement has to be evaluated thoroughly.