This document describes the design of hyaluronic acid-based nanosystems for targeted cancer drug delivery. Hyaluronic acid was functionalized with lipids, PEG, and thiols using click chemistry to form polymer blocks. These blocks were able to self-assemble into nanoparticles and encapsulate anticancer drugs. The nanoparticles were characterized and shown to effectively deliver drugs to cancer cells overexpressing CD44 receptors, demonstrating their potential as a targeted drug delivery platform.

1. Design of Customizable Self-Assembling Nanosystems Using ‘Clickable’ Hyaluronic Acid-Based Functional
Macrostructures for CD44 Target Mediated Drug Delivery
Arun K. Iyer, Ganesan Venkatesan, Faryal Mir, and Mansoor M. Amiji
Graduate
Category: Health Sciences
Degree Level: Masters
Abstract ID# 265
Figure 1: Copper mediated “click” synthesis of HA based functional
macrostructures. A 20 kDa HA was first reacted with an alkynyl moiety using
EDC/NHC coupling to form alkynyl-HA which further reacts with corresponding
azides using Cu+1 based “click” synthesis to form lipid modified HA, thiol modified
HA and PEG modified HA polymer blocks, respectively.
Polymer Characterization
Figure 2: 1H-NMR spectroscopy of unmodified HA and C12-lipid modified HA. The
appearance of additional peaks (methyl and methylene peaks) indicates successful conjugation of
the C12 lipid moiety .
TEM image of blank nanoparticles
Self-assembled Nanosystems
Cancer is a deadly disease and a major threat to mankind. The treatment of several
forms of cancer still remains a great challenge. Cytotoxic chemotherapy has been a
major arsenal against cancers, however it lacks selective tumor targeting. In this
study we have engineered hyaluronic acid (HA) based nanocarries for the targeted
treatment of cancers. Hyaluronic acid (HA) is a naturally occurring polysaccharide
present in the extracellular matrix and synovial fluids. It is biodegradable,
biocompatible, non-toxic, non immunogenic and non inflammatory, which makes it
ideal polymer for designing drug delivery systems. Interestingly, HA inherently acts
like a targeting moiety and specifically recognizes CD44 receptors that are
overexpressed on many tumors cell surfaces including tumor-initiating (stem) cells
that makes it an ideal polymer for targeted anticancer therapeutics. HA can self-
assemble into nanostructures when functionalized with lipophilic moieties. Based on a
customizable combinatorial library approach, we have synthesized a series of lipid
modified, PEGylated and thiol-functionalized HA macrostructures using copper (C+1)
catalyzed ‘click’ chemistry for efficient encapsulation of diverse payloads and
anticancer drugs that could be utilized as a platform technology for tumor targeting.
“Click” Synthesis of Functional HA Polymers
A series of lipid functionalized HA-derivatives with varying lipid chain lengths (C=4,
6, 8…18) were synthesized using copper catalyzed azide-alkyne “click” synthesis
methodology (Figure 1). A new homogenous reaction method was developed to
generate derivatives with high lipid modification that could facilitate efficient
incorporation of diverse anticancer drugs and promoted self-assembly to form
nanostructures in solution. Furthermore, in order to increase the stability of the HA
nanosystems and facilitate superior in vivo performance, PEGylated and thiol-
functionalized HA blocks were also synthesized using “click” methodology (Figure 1),
and incorporated into the nanosystems. The derivatives were purified and characterized
by 1H-NMR spectroscopy. The physicochemical properties of self-assembled
nanostructures were determined by dynamic light scattering (DLS) and the morphology
of the nanoparticles was visualized using transmission electron microscopy (TEM).
Introduction Characterization of Drug Loaded HA Nanosystems
Biological Activity of Drug Loaded HA Nanosystems
Table 1: Characterization of drug loaded HA nanosystems. The physicochemical properties
(size and charge) of HA based self-assembled nanostructures were determined by DLS . The drug
loading and encapsulation efficiency was determined using HPLC and spectrophotometric analysis.
a
b
Table 2: Comparison of cell killing ability of free
drug Vs drug loaded HA nanosystems in SKOV3
ovarian cancer cells.
Figure 5: Dose response curve. (a) Free
idarubicin Vs idarubicin NPs; (b) Free
taxol Vs taxol NPs
In vitro cytotoxicity (MTT) assays using SKOV3 ovarian cancer cells was performed to assess
targeted intracellular uptake and cell killing efficiency of the drug loaded HA nanosystems and
compared against corresponding free drug.
Conclusion
By judicious selection of the functionalized-HA polymer, its lipid chain length, charge, degree of
modification, molecular weight, log p value of the drug, drug class and other pertinent variables,
the HA derivatives can be tailored to encapsulate various drug payloads and inherently target
tumor cells expressing CD44 receptors. The HA-based macrostructures developed using “click”
synthesis thus demonstrated efficient drug encapsulation and comparable in vitro cell killing
efficiency and holds promising potentials for development of more robust nano-delivery systems.
Acknowledgments
This study was supported by the National Cancer Institute’s Alliance for
Nanotechnology in Cancer Center for Cancer Nanotechnology
Excellence (CCNE) grant U54- CA151881 and the Cancer
Nanotechnology Platform Partnership (CNPP) grant U01- CA151452.
Figure 4: Schematic representation of combinatorial approach in designing HA based self-
assembled nanosystems .
Figure 3: Transmission electron micrographs (TEM) of C12-HA blank nanoparticles
Department of Pharmaceutical Sciences, School of Pharmacy, Bouve College of Health Sciences, Northeastern University, Boston, MA 02115
(Email: venkatesan.g@husky.neu.edu)