1. Testing the Importance of Conjugation Chemistry on Antibody Loaded Nanoparticle Platforms
Ellie M. Papoutsakis, Rachel S. Riley, and Emily S. Day
University of Delaware
Summer Fellows 2017

2. Introduction
Antibody conjugation to nanoparticles provides several therapeutic benefits:
Higher binding affinity to cells compared to free antibodies
Active nanoparticle targeting
Reduced antibody dosages
Gold nanoparticle
Antibodies

3. Antibody Conjugation Chemistry is Important for Therapeutic Efficacy
Directional Conjugation
Non-Directional Conjugation
Gold nanoparticle
versus
PEG backfill
Antibodies
Unorganized antibody orientations
Blocks functional parts of some antibodies
Most common conjugation method
Outwardly presented antibodies
Maximal antibody functionality
Less common conjugation method

4. Hypothesis
Directional conjugation of antibodies to nanoparticles will yield higher binding affinity to cells than non-directional conjugation.
Goal: Develop conjugation protocols to achieve similar antibody loading density with both directional and non-directional chemistries

5. Nanoshells (NS) as a Model Nanoparticle for Antibody Conjugation
Nanoshells offer:
High surface area-to-volume ratio
Unique optical properties for imaging and secondary therapies
Good biocompatibility and safety profile
Nanoshell synthesis procedure:
Silica cores
(~120 nm)
Aminate silica cores
Add gold colloid
Add additional gold and formaldehyde

6. Nanoshell Characterization
150 nm
Scanning electron micrograph reveals NS have a homogenous size distribution and complete gold shells.
UV-Vis scan of NS showing a narrow peak at ~800 nm.
Riley RS, Day ES. Small

7. Add PEGylated antibody
Non-Directional Antibody Conjugation Results in Unorganized Antibody Orientation
Step 1: Attach OPSS-PEG-NHS linker to form PEGylated antibody
Step 2: Add PEGylated antibodies to nanoshells
-NH2 + OPSS-PEG-NHS
-NH-PEG-OPSS
NHS
Add PEGylated antibody
Add mPEG-SH
tor stability
Silica core
Gold shell
Rock for 4 hours
Rock overnight

8. Directional Conjugation Chemistry Results in Outwardly Presented Antibodies
Step 1: Combine antibodies and hydrazide-PEG-OPSS linkers
Step 2: Conjugate PEGylated antibody to nanoshells
Add sodium periodate
Add Hydrazide-PEG-OPSS linker
- OH
- CH=O
Add PEGylated antibody
Add mPEG-SH
Rock for 1 hour
Rock overnight

9. In-Solution Enzyme-Linked Immunosorbent Assay (ELISA) to Quantify Antibody Loading on Nanoshells
Used to colorimetrically detect and quantify the number of antibodies on NS
Antibody-loaded NS
HRP-conjugated secondary antibodies bind primary antibodies
Color-changing substrate reacts with HRP to produce blue
Sulfuric acid changes solution to yellow, and the absorbance at
450 nm is recorded
HRP = Horseradish peroxidase

10. Quantification of Antibodies on Non-Directionally Conjugated Nanoparticles
Trial 1
Trial 2
Trial 3
PEG-NS
IgG-NS
PEG-NS
IgG-NS
PEG-NS
IgG-NS
Quantification of antibody loading shows an average of ~86 IgG antibodies/NS

11. IgG Antibodies per Nanoshell
ELISA Results from Directionally Conjugated Antibody-Nanoparticle Conjugates
Trial 1
Trial 2
Trial 3
IgG Antibodies per Nanoshell
IgG-NS
PEG-NS
IgG-NS
PEG-NS
IgG-NS
PEG-NS
NS coated with antibodies using directional conjugation have an average of ~77 IgG antibodies/NS, which is comparable to non-directional antibody conjugation

12. Directional antibody conjugation Non-directional antibody conjugation
Conclusions
Similar antibody loading on NS can be achieved for both directional and non-directional conjugation methods
Future work will investigate binding affinity of antibody-loaded nanoparticles for each conjugation chemistry
vs.
Directional antibody conjugation
Non-directional antibody conjugation

13. for their support this summer
Acknowledgements
I would like to thank the University of Delaware Summer Fellows Program and the members of the Day lab
for their support this summer