Optimal LCNP Formulations
3.2.1 Optimising the Sonication Method
Although the characterisation of LCNPs is consistently reported in the literature, the optimal parameters affecting quality of a drug delivery system are not routinely investigated. Thus, it is important perform a range of tests on preparation techniques to justify the choice of parameters and ensure optimal conditions that are suited for their intended application. LCNPs were prepared from different ratios of SPC:citrem ratios (1:1 and 1:4) with the aim of creating LCNP dispersions with a particle size <200 nm to avoid rapid immune clearance.
Optimisation of the sonication method was achieved by varying sonication conditions on 5% SPC:citrem (1:1) in PBS. The effects of varying sonication power and time on particle size, size distribution and polydispersity were then assessed. Incremental increases by 10% in sonication power produced smaller particle sizes with increasing power, up to a threshold of 50%, in a near-linear fashion (R2=0.9161) (Figure 3.2). A mean size of approximately 342 ± 20 nm (PDI=0.37) was obtained using 10% power while a mean size of approximately 132 ± 1 nm (PDI=0.24) at 50% power was observed. The coefficient of determination (R2) signifies the correlation between x and y data fitted to a regression line, where a value of 0 indicates no linearity and 1 perfect linearity. Thus, the R2 value of 0.9161 suggests a strong linear relationship between increasing sonication time and decrease in LCNP size. Additionally, standard deviation and PDI values also decreased with increasing power. PDI decreased from 0.37 at 10% to 0.24 at 50%, which indicates that a higher power reduces polydispersity. Beyond a power of 50%, splashing and foaming occurred which resulted in incomplete dispersion of the lipids and in turn increased polydispersity. Dispersions were then sonicated at 50% power in pulse mode in order to limit the introduction of heat. Sonication modes of 5-s/5-s (on:off) and 10-s/5-s (on:off) for 10 minutes produced similar results on particle size and PDI, although samples sonicated in the 10-s/5-s (on:off) mode were warmer to the touch by the end of the run.
99 Thus, pulsed sonication was employed in a 5-s/5-s (on:off) mode for the preparation of the LCNP dispersions in this project.
Additionally, the amount of time the dispersions were subjected to sonication significantly affected particle size (Figure 3.3). A decrease in particle size by about 41 nm was observed when sonication time was increased from 1 minute (203 ± 25 nm; PDI=0.27) to 3 minutes (162 ± 11 nm; PDI=0.25). Extending the sonication time to 5 minutes (146 ± 9 nm; PDI=0.22) resulted in a further decrease in size by approximately 16 nm. At 10 minutes (132 ± 2nm; PDI=0.13) sonication time, LCNPs were 14 nm smaller and were at the smallest obtainable size for these preparations, and standard deviation and PDI values were lower compared to shorter sonication times. A longer sonication time of 12 minutes (138 ±3 nm; PDI=0.12) and 15 minutes (148 ± 9 nm; PDI=0.09) had a reverse effect, resulting in an increase in particle size by about 6 and 16 nm respectively.
In relation to formulation volume, sonication power did not proportionally change with volume (data not shown). This study was undertaken to assess the potential of producing smaller volumes to avoid excess waste. For 1 ml dispersions, a sonication power of 20%
produced the particles with the smallest size (~159 nm) whereas for 2 ml formulations a power of 50% was ideal (~132 nm). Thus, halving the volume did not translate into halving the sonication power to attain the same size LCNPs. Taking these data into account, a sonication power of 50% and a sonication time of 10 minutes were used for the preparation of all subsequent 2 ml formulations. A pulse sonication mode 5-s/5-s (on:off) did not affect particle size and was employed to reduce the introduction of heat during formulation.
100
% Power Size (nm) Average PDI
10 342.2 ± 19.9 0.37
20 235.7 ± 5.9 0.32
30 199.2 ± 15.1 0.35
40 147 ± 1.2 0.22
50 131.9 ± 1.1 0.24
Figure 3.2: The effect of sonication power on particle size and dispersity.
Formulations of 5% SPC:citrem (1:1) in PBS were sonicated for 3 minutes and the particle size and PDI were measured by DLS. Data given as mean ± standard deviation (n=3). R² = 0.9161
0 10 20 30 40 50 60
0 100 200 300 400
% Power
S iz e ( n m )
101
1 3 5 10 12 15
100 120 140 160 180 200 220 240 260
Time (minutes)
S iz e ( n m )
Time (minutes) Size (nm) Average PDI
1 203.0 ± 25.4 0.27
3 162.4 ± 10.9 0.25
5 146.8 ± 8.8 0.22
10 132.2 ± 1.7 0.13
12 138.4 ± 3.5 0.12
15 148.0 ± 8.9 0.09
Figure 3.3: The effect of sonication time on particle size. Formulations of 5%
SPC:citrem (1:1) in PBS were sonicated at 50% power for varying lengths of time and the particle size was measured by DLS. Data given as mean ± standard deviation (n=4-5).
102
3.2.2 LCNP Dispersion Stability
Assessment of the stability of the SPC: citrem (1:1; 5%) dispersions by DLS revealed significant differences between storage at room temperature (21 °C) and in the fridge (4
°C). In both cases, there was a gradual increase in mean particle size (Figure 3.4).
SPC:citrem (1:1) dispersions stored at 4 °C were colloidally stable throughout the 6 month testing period. However, samples stored at 21 °C showed visual signs of instability within 3 months, where lipids had sedimented and were stuck to the sides of the vial.
Formulations stored at room temperature increased in size at a faster rate than refrigerated formulations with statistically significant differences in particle size observed as early as 1 day (169.2 nm vs 146.7 nm; p<0.001). After 1, 3 and 7 days, the mean size of dispersed particles stored at room temperature increased by approximately 22, 46 and 70 nm while the mean size of refrigerated formulations increased by only 3, 8 and 17 nm respectively.
At 6 weeks, the last measured timepoint where room temperature formulations were stable, refrigerated particle size increased by a mean value of 56 nm (193.7 ± 2.1 nm), while formulations stored at room temperature increased by a mean value of 252 nm (399 ± 54 nm) (p=0.003). At 3 months and 6 months, a sharp drop in mean particle size was detected for dispersions stored at room temperature. However, this observation was due to significant aggregation and sedimentation since solid lipid deposits were observed at the bottom and sides of the vials. The remaining dispersion was almost clear at room temperature, and thus only the smallest particles remained suspended. Conversely, dispersions stored in the fridge remained as a fluid milky liquid with no visible signs of aggregation.
In order to further support the above assessment of the dispersions, the PDI and DCR values were also obtained by DLS and analysed. PDI is a unitless value used to indicate size uniformity, where lower PDI values suggest a more monodisperse formulation and higher values indicate polydispersity. PDI values of both sets of formulations remained comparable for the first 2 weeks (Figure 3.5-A). At 3 weeks to 6 months, PDI values were noticeably higher for formulations stored at 21 °C. The higher PDI values coupled with the larger particle sizes observed at the higher storage temperature suggests more pronounced aggregation occurring at a faster rate at the higher storage temperature.
103 The DCR is expressed in kilo counts per second (kcps) where higher values suggest a higher concentration of dispersed NPs (Wallace et al., 2010; Wallace et al., 2012; Tilley et al., 2013). The DCR represents the actual intensity of scattered light in DLS measurements by accounting for signal inhibition by the attenuator (Nobbmann, 2015). An initial drop in DCR was observed at both storage temperatures between day 0 and day 1, which may be indicative of the need for an equilibration period of 24 hours following sonication (Figure 3.5-B). Differences in DCR values became apparent in as early as 1 week between the two groups (p<0.05). Beyond the 1 week timepoint, an even more statistically significant number of LCNPs remained dispersed at 4 °C from week 2 up to 6 months at all measured time points (p<0.001). In addition, refrigerated dispersions maintained a relatively consistent DCR while dispersions stored at room temperature showed a decreasing trend with time. Thus, the SPC:citrem LCNPs (1:1) appear to remain in dispersion more effectively at 4 °C than at 21 °C where significant sedimentation occurred.
Formulations of SPC:citrem:DSPE-PEG2000-maleimide (1:1:0.072) were prepared to allow for surface-conjugation of peptides on LCNPs. After demonstrating the superior stability of our SPC:citrem (1:1) LCNPs in the fridge, SPC:citrem:DSPE-PEG2000 -maleimide (1:1:0.072) formulations, with and without surface-bound peptides, were stored in the fridge for 4 weeks (Figure 3.6). Particle size and DCR remained stable over the 28-day period suggesting good colloidal stability in all formulations. However, to avoid degradation of the fluorescent labels, all formulations were prepared fresh at the beginning of the week prior to experiments.
104 Figure 3.4: Graph of particle size against time over a 6 month period comparing between storage at room temperature (21 °C) and storage in the fridge (4 °C).
Formulations of 5% SPC:citrem (1:1) were sonicated at 50% power for 10 minutes using a 5-s/5-s (on/off) pulse mode giving a particle size of approximately 132 nm. The formulations were then covered in aluminium foil and stored in a cupboard at room temperature or refrigerated.
0 30 60 90 120 150 180 100
200 300 400 500
Time (days)
S iz e ( n m )
Room (21°C)
Fridge (4°C)
105