Elsevier

Biomaterials

Volume 32, Issue 23, August 2011, Pages 5417-5426
Biomaterials

Folate-decorated nanogels for targeted therapy of ovarian cancer

https://doi.org/10.1016/j.biomaterials.2011.04.006Get rights and content

Abstract

Nanogels are comprised of swollen polymer networks and nearly 95% water and can entrap diverse chemical and biological agents for cancer therapy with very high loading capacities. Here we use diblock copolymer poly(ethylene oxide)-b-poly(methacrylic acid) (PEO-b-PMA) to form nanogels with the desired degree of cross-linking. The nanogels are further conjugated to folic acid (FA) and loaded with different types of drugs (cisplatin, doxorubicin). For the first time we demonstrate a tumor-specific delivery and superior anti-tumor effect in vivo of an anti-cancer drug using these polyelectrolyte nanogels decorated with folate-targeting groups. This reinforces the use of nanogels for the therapy of ovarian and other cancers, where folate receptor (FR) is overexpressed.

Introduction

Targeted delivery of drugs to cancer cells has attracted considerable attention in developing new chemotherapeutic modalities. In this field folic acid (FA) has recently emerged as a prominent targeting moiety capable of specific interaction with cells expressing the folate receptor (FR) [1]. FR consists of a high affinity (Kd ∼10−9–10−10 M) folate binding protein (FBP) attached to the membrane through a glycosylphosphatidyl-inositol anchor [2]. It is overexpressed in ovarian carcinomas and other human tumors and has little expression in normal tissues [3], [4]. This provides tumor cells with increased amounts of the FA essential for DNA synthesis and seems to aid in aggressive tumor growth. In patients diagnosed with epithelial ovarian cancer the overexpression of FR isoform α correlates with a higher histological grade and more advanced stage of the disease [5]. The differential expression of FR in ovarian and other cancers makes it an attractive marker and target molecule for diagnosis and therapy of the disease [6]. Several folate-conjugated drugs and imaging agents have reached clinical evaluation stage [7].

The site-specific delivery of drugs to the tumors using FR can be enhanced using high capacity carriers that can simultaneously incorporate multiple drug molecules into one particle and target them to the disease sites. One recent type of high capacity carriers is nanogels composed of water-soluble polymer chains cross-linked within a nanoscale volume [8], [9]. Such nanogels are highly swollen and can incorporate 30% wt. and more drug molecules through covalent or electrostatic bonding with the nanogel chains. These loading capacities are unusually high and exceed those of liposomes and polymeric micelles [10]. Furthermore, nanogels do not have a dense core or a defined surface and can undergo dramatic volume transitions upon environmental changes. As a result, nanogels have unprecedented capacity for steric stabilization and decreased non-specific interactions, which can be used for example to stabilize colloidal microemulsions [11]. The very same properties could be beneficial for the targeted delivery of nanogels in the body. So far, however, nanogels were not used for this purpose, because extreme softness and flexibility of their hydrated chains have presented a challenge for attachment of targeting groups. This now becomes possible due a controlled template synthesis of nanogels by polyion complexation and cross-linking of doubly hydrophilic block ionomers, such as PEO-b-PMA. Using this synthetic approach [12], here we evaluate the drug therapy efficacy of FA decorated nanogels loaded with drugs (cis-dichlorodiamminoplatinum (II) (cisplatin, CDDP) and doxorubicin (DOX)) in an animal model of ovarian cancer.

Section snippets

Materials

PEO-b-PMA with terminal hydroxyl group in PEO block was from Polymer Source Inc., Canada (Mw/Mn = 1.16, PEO 5.5 kDa, PMA 15.5 kDa). Disposable PD-10 desalting columns, Amicon YM-30 centrifugal filters (MWCO 30 kDa, Millipore), CaCl2, 1,2-ethylenediamine, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), ethylenediaminetetraacetic acid (EDTA), folic acid (FA), divinyl sulfone (DVS), folate binding protein (FBP), fluorescein isothiocyanate (FITC),

Results

The synthesis of FA-conjugated nanogels (FA-nanogels) involved three steps: 1) preparation of nanogels with free OH groups at the PEO termini; 2) synthesis of stable intermediate with terminal amino groups; 3) conjugation of the intermediate with activated FA. Each of these steps produced stable intermediates, which can be isolated, characterized and stored (Scheme 1, Fig. S1). The preparation of active FA-nanogels was confirmed by surface plasmon resonance using FBP immobilized onto a

Discussion

Pharmaceutical drug delivery has experienced explosive growth in the last decade due to introduction of novel nanoformulations. Following development of liposomes and polymeric micelles, which are approved for clinical use or undergo clinical trials [24], numerous other materials based on polymer-coated drug nanocrystals, insoluble polymer nanoparticles, dendrimers, and polyion complexes have been explored as drug carriers [25]. These materials often contain water-soluble polymer brushes (such

Conclusions

This study demonstrated possibility of delivery of FR-targeted nanogels and their therapeutic cargo to the cancer cells in vivo. Here we use diblock copolymer poly(ethylene oxide)-b-poly(methacrylic acid) (PEO-b-PMA) for controlled template synthesis of nanogels by polyion complexation and cross-linking of doubly hydrophilic block ionomer. An optimal number of folate molecules were conjugated to nanogels to maintain good anti-cancer drug loading, stability and cellular uptake. Such optimized

Author contributions

N.V.N. synthesized all materials, performed the research, analyzed the data, and wrote the manuscript, H.S.O. participated as equal contributor in cell and animal studies and data analysis; A.V.K. designed the research, analyzed data and wrote manuscript; T.K.B. designed the research, analyzed data and wrote the manuscript.

Acknowledgments

This work was supported by the grants from U.S.A. National Institute of Health CA116590 (T.K.B.) and Department of Defense USA MRMC 06108004 (A.V.K.). We thank Dr. Frederic C. Laquer for ICP-MS Pt measurements and Dr. Larisa Poluektova for her help with preparation of tissue sections. We acknowledge the assistance of the Nanomaterials Core facility of the Center for Biomedical Research Excellence (CoBRE) Nebraska Center for Nanomedicine supported by the NIH grant RR021937. We also thank

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    NVN and HSO contributed equally to this work.

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