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

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

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

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

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

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. 2017.

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

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

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

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

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

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

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