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Hyaluronan Oligomers-HPMA Copolymer Conjugates for Targeting Paclitaxel to CD44-Overexpressing Ovarian Carcinoma

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Abstract

Purpose

To evaluate the effect of the size of low molecular weight hyaluronan (LMW-HA) oligomers on the targeting ability of the HA-containing copolymers to the CD44-overexpressing cells for delivering Paclitaxel (PTX) to ovarian cancer.

Methods

LMW-HA oligosaccharides of 4, 6, 8, 10, 12 and 14 sugar residues were attained by digestion of HMW-HA using hyaluronate lyase at different incubation times and then attached to FITC-labeled HPMA copolymer precursor. The binding and uptake of the HA-modified HPMA-copolymer into CD44-expressing cells was studied by flow cytometry and confocal microscopy. PTX was further attached to HPMA-copolymer precursor bearing HA oligosaccharide at the size of 34 monosaccharides, through an acid-sensitive hydrazone linker. The cytotoxicity of the polymer was tested using cell viability assay.

Results

Polymer conjugates bearing HA oligomers at the size of 10 oligosaccharides and above (HA10–14) bind actively and profoundly to CD44-overexpressing ovarian cancer cells (SK-OV-3) and internalize to the greatest extent relative to HA-polymer conjugates of 8 oligomers and below (HA4–8). The HA-modified HPMA-copolymer PTX conjugate (P-(HA)34-PTX) exhibited 50-times higher cytotoxicity towards CD44-overexpressing cells relative to the control, non-targeted, HPMA-copolymer PTX conjugate (P-PTX).

Conclusions

P-(HA)34-PTX was significantly more toxic than the non-targeted P-PTX in cells expressing high levels of CD44

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Abbreviations

2AB:

2-amino benzamide

AIBN:

2,2′-azobis(isobutyronitrile)

BSA:

bovine serum albumin

DCC:

N,N’-dicyclohexyl-carbodiimide

DCU:

N,N’-dicyclohexylurea

DMAP:

4-dimethylaminopyridine

DOX:

Doxorubicin

EPR:

enhanced permeability and retention

FITC:

fluorescein-5-isothiocyanate

GlcNAc:

N-acetyl-D-glucosamine

GlcUA:

D-glucuronic acid

HA:

hyaluronic acid hyaluronan

HA-TBA:

hyaluronic acid-tetrabutylammonium bisulfate

HMW-HA:

high molecular weight HA

HPMA:

N-(2-hydroxypropyl)methacrylamide

I:

polydispersity

LEV:

levulinic acid

LMW-HA:

low molecular weight HA

MA-AP:

N-(3-aminopropyl)methacrylamide

MA-AP(Boc):

N-(tert-butyloxycarbonyl-aminopropyl)methacrylamide

MA-AP-FITC:

methacryloyl-aminopropyl-fluorescein-5-isothiocyanate

MA-GG-HZBoc:

methacryloyl-glycylglycine hydrazide-Boc

MA-GG-OH:

methacryloyl-glycylglycine

MA-GG-ONp:

methacryloyl-glycylglycine p-nitrophenyl ester

Mn:

number average molecular weight

MTT:

3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolinium bromide

MW:

molecular weight

Mw:

weight average molecular weight

PFA:

paraformaldehyde

PGA:

Poly(L-glutamic acid)

PTX:

Paclitaxel

RHAMM:

receptor for hyaluronic acid-mediated motility

SEC:

size-exclusion chromatography

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Acknowledgments & Disclosures

This study was supported by a research grant from the US−Israel Binational Science Foundation (BSF) (2007319). We thank Ms. Mazal Rubin for her valuable assistance during this project.

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Authors and Affiliations

  1. Department of Pharmacology, Faculty of Health Sciences, Ben-Gurion University of the Negev, Beer-Sheva, 84105, Israel

    Gal Journo-Gershfeld, Dana Kapp, Yosi Shamay & Ayelet David

  2. Department of Pharmaceutics and Pharmaceutical Chemistry, University of Utah, Salt Lake City, Utah, 84112-9452, USA

    Jindřich Kopeček

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  1. Gal Journo-Gershfeld
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  2. Dana Kapp
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  3. Yosi Shamay
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  4. Jindřich Kopeček
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Correspondence to Ayelet David.

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Gal Journo-Gershfeld and Dana Kapp contributed equally.

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Journo-Gershfeld, G., Kapp, D., Shamay, Y. et al. Hyaluronan Oligomers-HPMA Copolymer Conjugates for Targeting Paclitaxel to CD44-Overexpressing Ovarian Carcinoma. Pharm Res 29, 1121–1133 (2012). https://doi.org/10.1007/s11095-012-0672-1

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