Real-time near-infrared bioimaging of a receptor-targeted cytotoxic dendritic theranostic agent
Introduction
The development of cutting-edge theranostic strategies, which can help to concurrently diagnose early stage cancer, deliver therapeutic drugs to tumors, and enable real-time monitoring of the drug release process, is paving the way for cancer patients to obtain effective and personalized medicine [1], [2]. By combining diagnosis and therapy, as well as other aspects of cancer treatment, in a single dose, theranostic agents have the potential to target specific tumor sites, minimize side effects, and enhance the overall efficacy of existing drugs [3], [4], [5], [6], [7], [8]. Ligand-receptor interactions have attracted increasing attention as important factors in the development of selective anticancer agents. Current approaches focus on conjugating theranostic agents with target molecules to exploit the over-expression of certain receptors by various cancer cells [9], [10]. Among these target molecules, substance P (SP), an undecapeptide of the tachykinin neuropeptide family, has been identified as a promising candidate [11]. In many types of cancers, including breast carcinomas, astrocytomas and glioblastomas, SP is rapidly internalized through specific interaction with the neurokinin-1 receptor (NK1R), a G-protein coupled receptor that is highly over-expressed in these malignant cells [12], [13]. In addition, SP is easy to synthesize and to modify with various reactive or linker groups, facilitating conjugation to many different drug molecules [14]. Therefore, owing to the unique and essential role of the SP/NK1R ligand-receptor system, conjugating SP with anticancer drugs may provide a promising platform for developing chemotherapeutic agents that target cancer cells with high selectivity.
5-fluorouracil (5-FU), an antimetabolite drug that disrupts DNA and RNA synthesis and causes cell death, is widely used in the treatment of a variety of tumors. Nevertheless, the clinical use of 5-FU is limited by severe side-effects and drug resistance [15], [16]. Novel delivery strategies can be used to improve the antitumor efficacy of 5-FU, but there are many inherent difficulties, such as achieving sufficiently high drug loading in the carrier [17]. Dendron-based delivery of anticancer drugs to tumors has provided a potential alternative strategy to overcome the challenge brought by drug loading in the development of drug delivery [18]. A useful feature of SP is that only its C-terminal amino acids are essential for binding NK1R and subsequent internalization [19]. Therefore, incorporation of linker groups at the N-terminus to create branch points for drug attachment with high loading capacity would not interfere with the binding of SP to NK1R.
Herein, we report the design and synthesis of an anticancer theranostic agent, P-FU 4, for cancer diagnosis, therapy, and real-time monitoring (Scheme 1). We assembled a dendrimer that contained 5-FU as the anticancer agent, SP as the targeting moiety, and an unsymmetrical squaraine dye, which we reported in previous work [20], as the near-infrared (NIR) fluorescence tag for in vivo and real-time monitoring. Significant advances have been made in the development of lung cancer imaging techniques [21], [22], and NIR fluorescence bioimaging technology has emerged as a powerful supporting tool for real-time detection during the drug treatment process [23], [24], [25], [26]. Squaraine dyes are a promising class of NIR fluorophores [27], [28], [29], [30] because of their excellent photophysical properties in the NIR region, and they are highly desirable for in vivo imaging because of their high tissue penetration, low auto-fluorescence background, good sensitivity, excellent selectivity, minimal invasiveness, and rapid response [31], [32], [33], [34]. The drug conjugate contained four branched 5-FU molecules and showed a significantly enhanced cytotoxicity due to the high drug loading content and the specific interaction between SP and NK1R. Accordingly, we were able to improve the safety profile and overcome the multidrug resistance associated with existing anticancer drugs that restricts their clinical use. As demonstrated by fluorescence confocal microscopy images and flow cytometry analysis, P-FU 4 was preferentially taken up by tumor cells through a receptor-targeted interaction. A remarkable suppression of tumor growth was realized upon treatment with P-FU 4 in a xenograft mouse model.
Section snippets
Synthesis of carboxyl-modified 5-FU
5-FU was dissolved in aqueous sodium hydroxide (15 mL, 0.82 g, 114.6 mmol), and the solution was slowly heated to 40 °C. Bromoacetic acid (0.78 g, 5.7 mmol) was added dropwise over 15 min. The reaction was stirred for approximately 2 h and then cooled to ambient temperature. Hydrochloric acid (20%) was slowly added to the solution until a precipitate appeared at pH 1-2. The precipitate was isolated by filtration, washed with water, and dried to yield 0.51 g (71.8%) of carboxyl-modified 5-FU as
Receptor-targeted theranostic agent with increased drug loading capacity via dendron conjugation
Chemotherapy, as a dominant treatment modality against cancer, is hindered by its severe side-effects to normal cells and tissues because of its nonselectivity and high toxicity. Our main strategy for improving the therapeutic efficacy is to exploit specific ligand-receptor interactions for drug targeting and to increase the drug loading content via dendron conjugation. As an undecapeptide of the tachykinin neuropeptide family, we chose the ligand SP, which can be taken up preferentially by
Conclusions
For the precise treatment of cancer, we have developed a primary strategy for improving therapeutic efficacy through exploiting specific ligand-receptor interactions for drug targeting and increasing the drug loading content via dendron conjugation. The carboxylic-conjugated hydrophilic dendron configuration can distinctly increase the drug-loading amount by as much as 16%, and it exhibits specific dendron isolation that avoids fluorescence quenching. These characteristics of P-FU 4, in
Acknowledgments
We thank the National Basic Research 973 Program (2013CB733700), and NSFC for Creative Research Groups (21421004) and Distinguished Young Scholars (21325625), NSFC/China, Shanghai Sci. & Tech. and Edu. Committee (15XD1501400), the Fundamental Research Funds for the Central Universities (WJ1213007 and WJ1416005), and Programme of Introducing Talents of Discipline to Universities (B16017) for financial support.
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