Interrogating the Interaction of CD52 Functionalised Metallic Nanoparticles with Malignant B Lymphocytes
Authors: Kickham Laura Christine , McElligott Anthony M. , Prina-Mello Adriele , Vandenberghe Elisabeth A. , Volkov Yuri , Browne Paul 
Published: October 2019

Abstract
Chronic Lymphocytic Leukaemia (CLL) is a common B-lymphoid malignancy with over 200,000 people affected annually in Europe and the US. The aim of therapy is to increase the quality and duration of life using well tolerated treatment. Novel intracellular drug delivery systems such as functionalised nanoparticles (NPs), conjugated to antibodies such as anti-CD20 or anti-CD52 directed at cell surface markers may help address this need. We have explored the feasibility of targeting nanoparticles in CLL using a microfluidics based adhesion assay, anti CD52 cell targeting and Fludarabine therapy.
Methods
B cells were isolated from the peripheral blood of normal healthy donors and CLL patients. The CLL cell line, I-83, was maintained under standard conditions. Epifluorescent, laser scanning confocal and electron microscopy was utilised for imaging the interaction of metallic NPs with cells. The metallic NPs were polymer-coated for biocompatibility and cellular toxicity was assessed using flow cytometric analysis based on changes in light scattering. NPs distribution on the surface of the cells was visualized using epifluorescent and Helium ion microscopy, cellular uptake and alterations in cell morphology after NP treatment was imaged by confocal microscopy.
Cell adhesion and migration behaviour under fluid shear flow conditions mimicking CLL cells in vivo was investigated using a microfluidics system utilising biochips coated with VCAM-1 and seeded with Human Umbilical Vein Endothelial Cells (HUVEC) or Human Dermal Lymphatic Endothelial Cells.
CD52-Alexa Fluor® 633 was conjugated to the surface of silanized NPs (NP1) using standard carbo-diimide cross linker chemistry techniques and successful functionalisation of NPs was validated using flow cytometric analysis, monitoring a shift in fluorescent population.
I-83 cells and patient-derived malignant B cells were treated using pH sensitive dye doped NPs and pH sensitive dye doped NP1 in order to assess interaction of nanoparticles with cells. Uptake measurements were performed through quantification of the fluorescence of the pH sensitive dye. As proof of concept, Fludarabine was then incorporated on to the surface of NPs in order to investigate its potential as a nanotherapeutic. Cytotoxicity studies were performed using flow cytometric analysis mentioned above following a 24 hour incubation.
Results and Conclusions
Quantitative and qualitative analysis identified uptake of NPs by normal and malignant B-lymphocytes with optimal NPs concentration for uptake determined at 25 μg/ml. Non-functionalised NPs in the range of 15-50nm were internalised by cells.
There was a notable decrease in the interaction of NPs with cells under physiologically relevant fluid shear flow in comparison to static conditions, resulting in a corresponding decrease in uptake, highlighting the rationale for a CLL cell-targeted NP. The results of the adhesion experiment using I-83 cells and patient derived CLL cells to the HUVEC monolayer in a micro-fluidics system showed that patient CLL adhesion decreased after NP treatment (p=0.01, n=3).
Cytotoxicity studies show that exposure to uncoated Fe2O3 nanoparticles yields an IC50 value of 23μg/mL +/- 5 μg/mL in comparison to coated, stabilized Fe2O3 nanoparticles with an IC50 of 49μg/mL +/- 5 μg/mL. Functionalisation of NPs with CD52 antibody (NP-1) resulted in significantly increased uptake (p<0.0001, n=3) and cytotoxicity. Preparation of these nanoparticles was reproducible and the particles remained stable in suspension for over 4 weeks. Cells treated with NPs bound Fludarabine were found to have significantly increased cytotoxicity in comparison to stabilized NPs (IC50 of 21μg/mL +/- 1μg/mL.
In summary, this work provides proof of concept of efficacy for a targeted nanotherapeutic in haematological malignancies.

Disclosures
No relevant conflicts of interest to declare.