Proof of Concept Single IL-4 Conjugation

  • IL-4 conjugated silk nanoparticles had significantly higher antibody concentrations than controls
Figure 1. Antibody concentrations of blank nanoparticles, blank nanoparticles incubated with secondary antibody, and IL4 conjugated nanoparticles incubated with secondary antibody. (n=3; **** p<0.0001; *** p = 0.0002). 

Conclusion: Successful IL-4 conjugation to silk nanoparticles

Single and Dual PSTAT3 and anti-rVEGF Silk Nanoparticles

  • Successful EDC/NHS single conjugation of PSTAT3 and anti-rVEGF with significantly higher antibody concentrations than controls (Figure 4B and 4D)
  • EDC/NHS dual conjugation of PSTAT3 and anti-rVEGF had significantly higher antibody concentrations than controls (Figure 4A and 4C)
  • Keyence Microscope images showed high levels of colocalization of anti-rVEGF and PSTAT3 (Figure 3 and Table 1)

Conclusion: High levels of colocalization predict dual antibody conjugation. Successful single conjugation of PSTAT3 and anti-rVEGF to silk nanoparticles

Figure 2. Green anti-rabbit AlexaFluor 488 binds to PSTAT3, and red anti-rat AlexaFluor 594 binds to anti-rVEGF. (A) Dual PSTAT3/anti-rVEGF conjugated nanoparticles tagged with AlexaFluor 488 and AlexaFluor 594; (B) single PSTAT3 conjugated nanoparticles tagged with AlexaFluor 488; (C) single anti-rVEGF conjugated nanoparticles tagged with AlexaFluor 594 were imaged using Keyence fluorescence microscopy. Blank nanoparticles were tagged with (D) AlexaFluor 488 and AlexaFluor 594; (E) AlexaFluor 488; (F) AlexaFluor 594
Figure 3. Fluorescence images of nanoparticles dually conjugated with PSTAT3 and anti-rVEGF. Red signifies the presence of anti-rVEGF and green signifies the presence of PSTAT3. Image processing with ImageJ superimposed white where red and green signals overlap. (A) shows the overlay of PSTAT3 and anti-rVEGF presence on the NPs, and (B) and (C) show the PSTAT3 and anti-rVEGF layers separated from each other. (D-F) illustrate the areas in white in which PSTAT3 and anti-rVEGF overlap
Area of PSTAT339.326
Area of anti-rVEGF38.826
Area of overlap31.842
overlap/PSTAT380.969%
overlap/anti-rVEGF82.012%

Table 1. ImageJ Analysis of images in Figure 3 calculated relative areas of overlap as well as green and red fluorescent signals for PSTAT3 and anti-rVEGF respectively. 80.969% of PSTAT3 was colocalized with anti-rVEGF, and 82.012% of anti-rVEGF was colocalized with PSTAT3.

Figure 4. Antibody concentrations of (A) dual conjugated PSTAT3 and anti-rVEGF nanoparticles tagged with green AlexaFluor 488, (B) single conjugated PSTAT3 nanoparticles tagged with green AlexaFluor 488, (C) dual conjugated PSTAT3 and anti-rVEGF nanoparticles tagged with red AlexaFluor 594, (D) single conjugated anti-rVEGF nanoparticles tagged with red AlexaFluor 594 compared to blank nanoparticles and blank nanoparticles incubated with their respective secondary antibodies. Tukey’s multiple comparisons test on ordinary one-way ANOVA was used to statistically analyze the data. (A, C, D n=3; B n=9; ** p=0.0006; ***p=0.0002;****p<0.0001) 

How did you determine successful antibody binding?

We determined that primary antibodies successfully conjugated in the correct orientation when secondary antibodies were able to bind to the antigen site on the primary antibody and fluoresce. The fluorescence was then checked against blank nanoparticles and blank nanoparticles incubated with secondary antibodies to confirm that any fluorescence was only due to proper secondary binding and not influenced by silk autofluorescence or non-specific secondary coating respectively. Antibodies that had improperly conjugated would not have their binding site available for secondary binding thus they would not fluoresce.

What are some limitations to this work?

White arrows point out aggregated nanoparticles
  1. Nanoparticles observed during imaging were still aggregated despite several rounds of sonification, hindering the characterization of singular nanoparticles.
  2. Clinical relevance was unable to be established during this round of experimentation as timing did not permit for cell uptake studies.
  3. Future replicate studies are needed for stronger data

However, both of these points can be ameliorated with future work!

Read a more thorough explanation of the results in our Technical Report:

BME 8 Final Technical ReportDownload