Fig 1.
Mean particle size of secretoneurin loaded proticles obtained by different mass ratios and preparation methods.
The size of particles is given as mean hydrodynamic diameter (ZAve d.nm) separated by the applied preparation method (red bars: single protamine addition, blue bars: titration process) and mass ratio (1 = 100 μg/ml). (A) shows particles with 300 μg/ml protamine and (B) with 150 μg/ml. The results are given as mean values +/- standard deviation, each sample was measured at least in duplicate (number of samples are listed in Table 1). Statistical significance between preparation methods is marked as * (p<0.05), ** (p<0.01) and *** (p<0.001).
Table 1.
PdI values of secretoneurin loaded proticles.
Fig 2.
Secretoneurin (SN) loading of proticles obtained by different mass ratios and preparation methods.
Secretoneurin (SN) loading is shown as relative percentage of applied SN and as absolute concentration (shaded bars). Red bars are referred to particles prepared by a single protamine addition and blue bars to the titration process. (A) shows the formulations with 300 μg/ml protamine and (B) with 150 μg/ml. The results are given as mean values incl. standard deviation, each sample was measured in duplicate (number of samples are listed in Table 1).
Fig 3.
Correlation of mean particle size and derived count rate throughout protamine titration process.
Development of mean particle size (ZAve, blue lines) and derived count rate (red lines) for each mass ratio (captioned on the top left of each graph). Horizontal axis indicates the steps of protamine addition along the titration process. The results are given as mean values + standard deviation (dashed error bars refer to particle size).
Table 2.
Derived count rate (DCR) of the initial ODN/SN mixtures.
Fig 4.
Development of zeta potential throughout protamine titration.
Particles were assembled without SN (blank marks) or 100 μg/ml SN (filled marks), as well as with 150 μg/ml protamine (blue lines) or 300 μg/ml (red lines). The ODN concentration was set to 100 μg/ml, each undiluted sample was measured at least in duplicate.
Table 3.
DLS measurements of SN loaded particles before and after lyophilization.
Fig 5.
AFM images of secretoneurin loaded proticles.
Images recorded by atomic force microscopy showed spherical secretoneurin loaded proticles for mass ratio of 1 : 1 : 1.5 prepared by titration process (A) as well as by single protamine addition (B). Triangular clustered proticles were recorded for mass ratio 1 : 0.25 : 1.5 prepared by protamine addition at once (C).
Fig 6.
In-vivo biodistribution scan of fluorescently labeled secretoneurin in solution and assembled within proticles.
Biodistribution of SN after intramuscular injection (30 μl) into hind limb of partly shaved C57BL/6 mice. Fluorescently labeled SN was applied in solution (50 μg/ml, upper panels) and assembled within the proticle matrix (mass ratio 1:0.25:1.5, lower panels).