Elsevier

Biomaterials

Volume 33, Issue 29, October 2012, Pages 7182-7193
Biomaterials

A Poly(γ, l-glutamic acid)-citric acid based nanoconjugate for cisplatin delivery

https://doi.org/10.1016/j.biomaterials.2012.06.071Get rights and content

Abstract

A cisplatin-loaded nanoconjugate, poly(γ, l-glutamic acid)-citric acid-cisplatin [γ-PGA-CA-CDDP], as a tumor-targeted drug delivery system with sustained release capacity was successfully synthesized and characterized, and its antitumor activity was evaluated. The particle size (107 ± 6.3 nm) and average molecular weight (66 kDa) were determined by dynamic light scattering (DLS) and gel permeation chromatography (GPC), respectively. The nanoconjugate delivery system released platinum in a sustained manner in PBS at 37 °C with an initial burst release during the first 8 h and 50% cumulative release within 48 h. Both in-vitro and in-vivo studies showed that the toxicity of γ-PGA-CA-CDDP nanoconjugate significantly decreased by comparison to that of unconjugated CDDP. The maximum tolerated dose (MTD) of γ-PGA-CA-CDDP nanoconjugate was about 38 mg/kg versus 8 mg/kg for CDDP. No apathy or acute adverse reactions were observed in γ-PGA-CA-CDDP nanoconjugate groups while mice expressed apathy at all dose levels with CDDP treatment. In ICR mice, the area under the curve and total body clearance values for γ-PGA-CA-CDDP nanoconjugate were 9-fold and one-twentieth of the values for CDDP, respectively. With the aid of near-infrared fluorescence (NIRF) imaging system, it was demonstrated that γ-PGA-CA-CDDP nanoconjugate gradually accumulated at the tumor site within 15 min postinjection and exhibited prolonged retention for more than 8 h. In H22-implanted mice, γ-PGA-CA-CDDP showed a significantly higher antitumor activity versus CDDP. These results reveal that γ-PGA-CA-CDDP nanoconjugate with improved stability, reduced toxicity and prolonged in-vivo retention time holds great potential in terms of clinical application to cancer therapy.

Introduction

The cis-dichlorodiammineplatinum (II) (CDDP or cisplatin) was first reported to display anticancer properties by Rosenberg et al. in 1965 [1]. So far, CDDP is being one of the most widely used antitumor drugs for the clinical treatment of many malignancies including breast, liver, lung, neck, ovarian, testicular, bladder, small-cell and non-small-cell lung cancers because of its wide spectrum of anti-tumor activity [2], [3]. It can be used alone or in combination with other antitumor drugs to treat human tumors [4]. However, a major obstacle to its use is the associated severe toxic side effects including acute nephrotoxicity, myelosuppression and chronic neurotoxicity [5], [6], [7]. Additionally, another major issue is intrinsic or acquired tumor resistance [8]. Therefore, it is urgent to develop an efficient drug delivery system (DDS) for cisplatin to overcome these shortcomings. One such method is the discovery and development of new simple platinum complexes such as carboplatin and oxaliplatin [9], [10], [11], which have been the standard drugs for ovarian cancer [12]and colon cancer [13]. The other method is based on the observations of the enhanced permeability and retention (EPR) effect which ensures that macromolecular carriers can potentially exhibit prolonged blood circulation with reduced nonspecific accumulation in normal tissues and preferential tumor accumulation [14]. The macromolecular carriers, including polymeric micelles [15], [16], long-circulating liposomes [17], [18], and water-soluble polymers [19], [20], which have been reported for clinical use or being studied currently in clinical trials [21], [22], [23]. Despite the aforementioned advantages of macromolecular carriers, the poor stability, poor water solubility and none significant sustained release of the drug may hamper the development of useful macromolecular carriers. Therefore, more efforts are needed to be devoted to their development.

Gamma-Polyglutamicacid (γ-PGA) was produced through biosynthesis by Graciela et al. [24], and it was regarded as an ideal carrier to approach sustained release of drugs and specificity for carcinoma-targeted drug delivery with good solubility in water, low toxicity and minimal side effects because of its biological compatibility and biodegradability [25]. Furthermore, Haifeng Ye et al. reported that γ-PGA-CDDP was successfully synthesized and showed better performance than CDDP both in vitro and in vivo [26], [27]. However, some barriers were also observed in the development of γ-PGA, such as its large molecular weight, poor drug-loading capacity and low release rate of conjugated drugs.

To overcome these drawbacks, a hydrosoluble polymer, γ-Glutamyl citrate (γ-PGA-CA), was designed and synthesized by modification of γ-PGA with citrate acid, and CDDP was then conjugated to γ-PGA-CA to yield γ-PGA-CA-CDDP nanoconjugate. On one hand, citrate acid-modified γ-PGA presented more carboxyl groups (-COOH) on which CDDP is bound. On the other hand, the lateral chains of γ-PGA were extended by modification with citrate acid to reduce the stereospecific blockade when reacted with CDDP. The aforementioned two reasons render γ-PGA-CA capable of higher drug-loading than γ-PGA.

In the present study, the properties of the synthesized nanoconjugate, γ-PGA-CA-CDDP, such as structure, particle size and molecular weight, have been investigated in detail. The release profile of platinum from γ-PGA-CA-CDDP nanoconjugate was carried out simply by a dialysis method and analysis with HPLC. Human tumor cell lines of the breast (BcaP-37) and liver (Bel-7402) were cultured to evaluate the cytotoxicity of γ-PGA-CA-CDDP nanoconjugate as well as CDDP for comparison.

In order to systematically assess its potential for clinical carcinoma therapy, we further investigated the in-vivo antitumor activity and toxicity of γ-PGA-CA-CDDP nanoconjugate and unconjugated CDDP under the same experimental conditions. The tissue distribution study of γ-PGA-CA-CDDP nanoconjugate was carried out with the aid of in-vivo fluorescence imaging system.

Section snippets

Materials

γ-PGA (MW ≈ 66 kDa) was a gift from Nanjing University of Technology. CDDP (purity≥99.9%) was purchased from Platinum Energy Co., Ltd. (Shandong, china). Citric acid monohydrate was purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Cy7 acid, mono-NHS ester (Cy7, SE) was obtained from Fanbo Biochemical Co. 1-Ethyl-3-(3-dimethyllaminopropyl) carbodiimide hydrochloride (EDCI), N-Hydroxysulfosuccinimide sodium salt (NHSS), Sodium diethyldithiocarbamatre (DETC), and 3-(4,

Identification of γ-PGA-CA and γ-PGA-CA-CDDP nanoconjugate

The γ-PGA-CA was prepared from γ-PGA with the modification by citric acid, while the γ-PGA-CA-CDDP nanoconjugate was synthesized from γ-PGA-CA and CDDP, through the displacement of chlorine ions on CDDP by hydrogen of carboxyl groups on γ-PGA-CA side-chains. The preparation steps and structures of γ-PGA-CA and γ-PGA-CA-CDDP nanoconjugate were shown in Figs. 1 and 2, respectively. There may be many possible structures of γ-PGA-CA-CDDP nanoconjugate because of the different conjugated manners of

Discussion

γ-PGA is water-soluble and biodegradable. It can be used as a thickener, humectant, sustained release material or drug carrier with biodegradability in the fields of food, cosmetics or medicine [38]. In order to improve its drug loading ability, interest in the modification of γ-PGA has meant much research being done on its various chemical reactions. One of the best modifications is the esterification of carboxyl groups [39], [40]. In our research, the γ-PGA-CA was successfully synthesized

Conclusion

In summary, the γ-PGA-CA-CDDP nanoconjugate was demonstrated to be a formulation with a sustained drug release. The nanoconjugate showed effective antitumor activity in-vitro as well as in-vivo, while it decreased side effects. Therefore γ-PGA-CA may be used as an ideal drug carrier for CDDP and γ-PGA-CA-CDDP nanoconjugate may have a potential application in clinical treatment of cancer.

Acknowledgments

This work was financially supported by the National Natural Science Foundation of China (No. 81072588) and the Fundamental Research Funds for the Central Universities (No. JKQ2011018). The authors also acknowledge Xueming Li (Nanjing University of Technology) for providing γ-PGA as a gift.

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