Pharmaceutical Nanotechnology
LyP-1-conjugated nanoparticles for targeting drug delivery to lymphatic metastatic tumors

https://doi.org/10.1016/j.ijpharm.2009.10.014Get rights and content

Abstract

Active tumor targeting by biodegradable nanoparticles has been widely studied for cancer diagnosis and therapy. However, target-specific nanoparticles for drug delivery to lymphatic metastases have not been reported yet due to the lack of specific markers in the tumor lymphatics. Recently, peptide LyP-1 has been recognized for its specific home to tumors and their lymphatics. In this study, we tested the possibility of LyP-1 serving as a target-specific peptide of PEG–PLGA nanoparticles to tumor lymph metastases. LyP-1 was synthesized by using Boc-protected amino acids. The copolymers of maleimide–PEG–PLGA were formed by the conjugation of maleimide–PEG–NH2 to PLGA–COOH, which were applied to prepare pegylated nanoparticles with mPEG–PLGA by means of double emulsion/solvent evaporation technique. LyP-1 with sulfhydryl group was conjugated to the maleimide function located at the distal end of PEG surrounding the nanoparticle surface. LyP-1-conjugated PEG–PLGA nanoparticle (LyP-1-NPs) had a round and regular shape with a diameter around 90 nm. In vitro, cellular uptake of LyP-1-NPs was about four times of that of PEG–PLGA nanoparticles without LyP-1 (NPs). In vivo, the uptake of LyP-1-NPs in metastasis lymph nodes was about eight times of that of NPs. This study indicates that LyP-1-NP is a promising carrier for target-specific drug delivery to lymphatic metastatic tumors.

Introduction

As to many carcinomas, lymphatic metastasis occurs extensively, which leads to frequent tumor relapse even after extended lymphadenectomy. The inability to remove all the lymph metastasis remains the primary cause of cancer death (Pepper, 2001, Van Trappen and Pepper, 2002). Chemotherapy involves the use of powerful drugs to kill malignant cells. However, systemic delivery of chemotherapeutic agents often fails to make drug concentration high enough to eradicate all the metastatic lesions. Accordingly, targeting drug delivery specific to lymphatic metastatic lesions holds great promise in minimizing systemic drug exposure and maximizing the therapeutic efficacy of the drug (Reddy et al., 2006).

Liposomes have been proposed as carriers for the delivery of therapeutic and diagnostic agents to the lymphatic system (Oussoren and Storm, 2001). Nevertheless, researchers found that after a subcutaneous injection, approximately 50% of the injected dose of liposomes was cleared from the injection site into the lymphatic vessels, and only 1–2% of these liposomes were retained in each draining lymph node (Agarwal et al., 2008, Oussoren and Storm, 2001, Phillips et al., 2000). Thus, great attempts have been made on increasing the deposition of drug carriers into lymph nodes.

Recently, numerous investigations have been concentrated on biodegradable nanoparticles for anti-cancer drug delivery, which has great potential to revolutionise the future of cancer therapy (Brannon-Peppas and Blanchette, 2004, Cheng et al., 2007, Farokhzad et al., 2006, Garinot et al., 2007, Gu et al., 2008, Okada et al., 1988, Oussoren and Storm, 2001, Soppimath et al., 2001, Townsend et al., 2007). As effective drug delivery devices, biodegradable polymeric nanoparticles were known for their high efficiency, biocompatibility and controlled releasing ability (Okada et al., 1988, Soppimath et al., 2001). Active targeting nanoparticle, which involves the use of peripherally conjugated targeting moieties to enhance the ability of direct delivery a therapeutic agent to a targeting site, would minimize systemic drug exposure and provide the potential for increasing the therapeutic efficiency and reducing multi-drug resistance (Reddy et al., 2006). However, due to the relatively lagging development of specific markers of tumors lymphatics, active targeting by biodegradable nanoparticles to lymphatic metastases has not been reported yet.

LyP-1 is a 9-amino-acid cyclic peptide identified by in vivo phage display technology on the MDA-MB-435 human carcinoma xenograft tumors (Laakkonen et al., 2002). Fluorescein-labeled LyP-1 can accumulate in structures identified as lymphatic vessels and also in tumor cells within hypoxic areas, but not in the blood vessel after an intravenous injection (Laakkonen et al., 2002, Laakkonen et al., 2004). This peptide also homes to the metastatic lesions of tumors (Laakkonen et al., 2004). Therefore, it may serve as a good nanoparticle specific targeting moiety for lymphatic active drug delivery.

With these considerations, the purpose of this study is to synthesize LyP-1-NPs (Fig. 1) and examine the possibility of LyP-1-NP serving as a novel targeting drug carrier to lymphatic metastatic tumors.

Section snippets

Materials

Maleimide–PEG–NH2 (3500 MW) was obtained from Jenkem (Beijing, China); mPEG–PLGA (75:25, 34 kDa MW) and PLGA–COOH (34 kDa MW) were purchased from Daisheng (Ji’nan, China); N-hydroxysuccinimide (NHS) was from Medpep Co. Ltd. (Shanghai, China); dicyclohexylcarbodiimide (DCC) was from Sinopharm Chemcial Reagent Co. Ltd. (Shanghai, China); amino acids was purchased from Kabushiki Kaisha (Japan); peptide synthetic resin was from Fluka (Switzerland);

Examination of LyP-1s binding affinity to BxPC-3 cells

LyP-1 can recognize specific marker molecules on the surface of tumor lymphatic endothelial cells and home to tumor lymphatics and strongly accumulate in cancer xenografts and their metastases (Laakkonen et al., 2002, Laakkonen et al., 2004). However, the homing of Lyp-1 is tumor type-specific, despite the fact that nearly all tumors have been shown to contain lymphatic vessels (Laakkonen et al., 2008). In addition to the MDA-MB-435 tumors, it homes to a transgenic prostate tumor (TRAMP),

Conclusions

Because of the determining role of lymphatic metastasis on the prognosis of cancer patients, targeting drug delivery to lymphatic metastatic tumors is an area of significant research interest. In this study, we developed biodegradable nanoparticles from mPEG–PLGA and maleimide–PEG–PLGA polymers and used them as drug carriers to lymphatic metastatic tumors. LyP-1, which is well-known for its highly specific tumor and tumor lymphatics targeting, was firstly conjugated to biodegradable polymers

Acknowledgments

This work was supported by the National Natural Science Foundation of China (30500490) and Opening Foundation of Institute of Biomedical Sciences, Fudan University. We thank Dr. Cao Xie for expert technical assistance.

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