Elsevier

Biomaterials

Volume 30, Issue 20, July 2009, Pages 3476-3485
Biomaterials

The use of biotinylated-EGF-modified gelatin nanoparticle carrier to enhance cisplatin accumulation in cancerous lungs via inhalation

https://doi.org/10.1016/j.biomaterials.2009.03.010Get rights and content

Abstract

To develop a polymer-anticancer drug conjugate, we employed gelatin nanoparticles (GPs) as carriers of cisplatin (CDDP) with anticipated improved therapeutic effect and reduced side effects. The anticancer activities of CDDP-incorporated in GPs (GP–Pt) with biotinylated-EGF (bEGF) modification (GP–Pt–bEGF) were studied. GP–Pt–bEGF with EGFR affinity produced much higher Pt concentrations in A549 cells (high EGFR expression) than in HFL1 cells (low EGFR expression). An in vitro anticancer study showed that GP–Pt–bEGF was more potent than free CDDP or GP–Pt because of its rapid effect on the cell cycle as well as a lower IC50 (1.2 μg/ml) that inhibits A549 cell growth. PI staining showed that cells treated with GP–Pt-bEGF for only 4 h had the highest sub-G1 population.

The CDDP formulations – free CDDP, GP–Pt, and GP–Pt–bEGF – were given by intratumorous injections to SCID mice in a subcutaneous model. This treatment showed that GP–Pt–bEGF had stronger anti-tumor activity and was less toxic than free CDDP in vivo. Mice treated with GP–Pt–bEGF showed slight body weight loss, whereas free CDDP treatment at the same dose caused a body weight loss of 20–30%. Furthermore, these formulations were given to mice with lung cancer via aerosol delivery. This treatment showed that inhaled GP–Pt–bEGF could target EGFR-overexpressing cells to achieve high cisplatin dosage in cancerous lungs.

To summarize, gelatin nanoparticles loaded with CDDP and decorated with EGF tumor-specific ligand were successfully developed. Their in vitro and in vivo targeting ability and anticancer effect were confirmed. The aerosol delivery of the nanodrug carrier was demonstrated. Simple aerosol delivery of targeted drug carriers may prove useful for the clinical treatment of lung cancer patients.

Introduction

Lung cancer is one of the most harmful forms of cancer. The long-term survival rate of lung cancer patients treated by conventional modalities, such as surgical resection, radiation, and chemotherapy remains far from satisfactory. Systemic drug delivery is rarely successful because only a limited dosage of the chemotherapeutic drugs target lung tumor sites, even when administered at a high dose [1]. Most chemotherapeutic drugs act on normal cells, inhibiting their growth; this makes the patient extremely weak and can even result in death.

Cisplatin (cis-dichlorodiammineplatinum (II); CDDP) is a key drug used in the chemotherapy of cancers, including gastrointestinal, genitourinary and lung cancer; cisplatinum-based chemotherapy is accepted as a standard first-line treatment for advanced non-small cell lung carcinoma (NSCLC) [2]. Chemotherapy with cisplatin is associated with various secondary effects, such as anemia, nausea, vomiting, neurotoxicity and nephrotoxicity [3], which impair the patient's quality of life and can even be life-threatening in preexisting conditions. Due to these side effects, alternative methods of administering toxic cisplatin are needed.

For the reduction of side effects, specific drug delivery systems (DDSs) have been investigated. Several polymers, of natural as well as synthetic origin, have been used as reservoirs for delivery of cisplatin, such as polymeric micelles [3], poly(γ,l-glutamic acid)(γ-PGA) [4], polylactic acid (PLA) [5], etc. In this study, gelatin was selected as the raw material to prepare the drug carrier because of its biocompatibility and biodegradability [6], [7]. Its degradation rate can be regulated by the degree of crosslinking to control drug release rate. Konishi et al. showed that a gelatin hydrogel containing CDDP could release the drug over a long period, resulting in a superior anticancer effect [8]. However, there have been few published studies on the formulation of gelatin nanoparticles (GPs) with cisplatin. In this study, we have successfully prepared gelatin nanoparticles complexed with CDDP (GP–Pt).

Another way to reduce the side effects of drugs is active targeting. A tumor-specific ligand can be employed for more specific recognition of and interaction with cancer cells, reducing the effect on normal cells. The overexpression of epidermal growth factor receptor (EGFR) on human tumors, especially on NSCLC, has been reported [9]. In the present study, epidermal growth factor (EGF) was conjugated with the gelatin–cisplatin nanocomplex as a targeting vehicle for lung cancer treatment.

The objective of the present work was to evaluate the anticancer efficiency and specific targeting ability of a cisplatin–gelatin nanomedicine with affinity for EGFR. We developed a gelatin nanoparticle (GP) complex with CDDP (GP–Pt) surface-modified with NeutrAvidinFITC-biotinylated epidermal growth factor (bEGF), abbreviated as GP–Pt–bEGF, to target the tumor site and be taken up by EGFR-mediated endocytosis in tumor cells overexpressing EGFR. In vitro anticancer efficiency was examined by the MTT assay. The in vivo anticancer effect was evaluated by intratumor injection in a mouse subcutaneous model. Aerosol delivery was adopted to allow inhaled chemotherapy for metastatic lung cancer.

Section snippets

Reagent and chemicals

Gelatin type A (derived from porcine skin, bloom 175), cisplatin, Ham's F12k medium, Bicinchoninic Acid (BCA) protein assay kit, bovine serum albumin (BSA), Ethylenediaminetetraacetic acid (EDTA), 1-(4,5-Dimethylthiazol-2-yl)-3,5-diphenyl- formazan (MTT), hematoxylin and eosin were purchased from Sigma–Aldrich (Saint Louis, MO, USA). Fetal calf serum (FCS) was from Biological industries (Kibbutz Beit Haemek, Israel). Trypsin–EDTA, penicillin/streptomycin, and phosphate-buffered saline (PBS)

Results

A schematic representation of the cisplatin with different formulations is represented in Fig. 1. Gelatin nanoparticles (GPs) were prepared by the two desolvation method through acetone addition (Fig. 1(a)). Cisplatin loaded GPs (GP–Pt) were formed via a ligand exchange reaction of Pt(II) from the chloride to the carboxyl group in the GPs (Fig. 1(b)). Finally, surface modification of GP–Pt was performed by NeutrAvidinFITC-biotinylated-EGF conjugation. The EGFR-targeted formulation of CDDP is

Discussion

Many research reports about CDDP delivery systems with polymer materials (such as PGA [4], and PEG-P(Glu) block copolymers [19]) have indicated possible interactions between the carboxyl groups of the polymer and CDDP. The functional groups of the natural polymer gelatin, such as carboxyl, hydroxyl, and amino groups, are available for chemical reaction. Based on their nature, it is conceivable for gelatin to interact with CDDP because it contains many carboxyl groups. The FT-IR spectrum (Fig. 2

Conclusion

A polymer-anticancer drug conjugate, gelatin nanoparticle (GPs), was developed as a carrier of CDDP to enhance its therapeutic effects and reduce its side effects. GP–Pt–bEGF with EGFR affinity produced Pt concentration in cells highly expressing EGFR. The in vitro anticancer study showed that GP–Pt–bEGF was more potent than free CDDP or GP–Pt, due to a rapid onset of action on the cell cycle and a lower IC50 for the inhibition of A549 cell growth. The in vivo anticancer experiment showed that

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