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)