LHRH-targeted nanogels as a delivery system for cisplatin to ovarian cancer

Abstract

Targeted drug delivery using multifunctional polymeric nanocarriers is a modern approach for cancer therapy. Our purpose was to prepare targeted nanogels for selective delivery of chemotherapeutic agent cisplatin to luteinizing hormone-releasing hormone (LHRH) receptor overexpressing tumor in vivo. Building blocks of such delivery systems consisted of innovative soft block copolymer nanogels with ionic cores serving as a reservoir for cisplatin (loading 35%) and a synthetic analogue of LHRH conjugated to the nanogels via poly(ethylene glycol) spacer. Covalent attachment of (D-Lys6)-LHRH to nanogels was shown to be possible without loss in either the ligand binding affinity or the nanogel drug incorporation ability. LHRH-nanogel accumulation was specific to the LHRH-receptor positive A2780 ovarian cancer cells and not toward LHRH-receptor negative SKOV-3 cells. The LHRH-nanogel cisplatin formulation was more effective and less toxic than equimolar doses of free cisplatin or untargeted nanogels in the treatment of receptor-positive ovarian cancer xenografts in mice. Collectively, the study indicates that LHRH mediated nanogel-cisplatin delivery is a promising formulation strategy for therapy of tumors that express the LHRH receptor.

Figure 1

Scheme for preparation of LHRH-targeted nanogels. a) Synthesis of LHRH-PEG-NH₂ and b) conjugation of PEGylated LHRH to nanogels. The abbreviations of chemical reagents are summarized in Section 2.2.

Figure 2

Characterization of PEGylated peptide and nanogels. a) ¹H-NMR spectra of the LHRH-PEG-NH₂ in D₂O. b) 4-20% Tris-HCl gel followed by Coomassie Blue staining. Lanes: 1. protein ladder, 2. free LHRH, 3. free NHS-PEG-Fmoc, 4. LHRH-PEG modified using 5-fold excess of NHS-PEG-Fmoc, 5. LHRH-PEG synthesized using the equimolar amount of protein and PEG, 6. mixture of LHRH and NHS-PEG-Fmoc. c) UV spectra for LHRH and PEGylated LHRH (0.5 mg polymer/ml, PBS). d) Tapping-mode AFM images of nanogels deposited from aqueous solutions on the APS-mica. Scan size is 1.8 Zm.

Figure 3

Drug release and stability of nanogels. a) Drug release profiles for CDDP in targeted and untargeted nanogels in PBS, pH 7.4 or ABS, pH 5.5 at 37°C. b) Stability of CDDP-loaded untargeted and targeted nanogels in plasma (20% v/v, 37°C) and PBS over time. Data are means ± SD (n = 3).

Figure 4

a) Uptake of FITC-labeled nanogel and LHRH-nanogels by A2780 and SKOV-3 cells (3 h, 37°C, 0.1 mg/ml). b) Whole-cell Pt accumulation in A2780 and SKOV-3 cells as measured by ICP-MS (24 h, 37°C, > 0.5 mg/ml). c) Confocal micrographs (×63) of nanogel and LHRH-nanogel in A2780 cells. The cells were treated for 3 h with the FITC-labeled nanogels (green), stained with Hoechst 33342 (blue) for nucleus for 10 min at 37°C, followed by live cell imaging. Data are mean ± SD, n = 3, *p < 0.05, NS is not significant.

Figure 5

In vivo antitumor efficacy of CDDP-loaded nanogels and Pt accumulation in A2780 human ovarian cancer xenograft-bearing female nude mice. a) Tumor growth, b) body weight (calculated as body weight subtract tumor) and c) survival time after administration of 5% dextrose (1, control), CDDP alone (2), nanogel/CDDP (3), LHRH-nanogel/CDDP (4) at a dose of 4 mg CDDP equivalents/kg body weight every 4^th day. d) Concentration of Pt in organs and tumor by ICP-MS. Mice were sacrificed in a week after receiving four i.v. injections of free CDDP, nanogel/CDDP and LHRH-nanogel/ CDDP. Data are mean ± SEM, n = 7-8, *p < 0.05, **p < 0.01, NS is not significant.