Real-time near-infrared bioimaging of a receptor-targeted cytotoxic dendritic theranostic agent

Abstract

Efficient and site-specific delivery of anticancer drugs to tumors is important in the development of effective cancer chemotherapy. As an undecapeptide of the tachykinin neuropeptide family, the substance P (SP)/neurokinin-1 receptor (NK1R) system has been identified as a promising ligand-receptor pair in tumor-specific drug delivery. However, the rational design of suitable theranostic agents with high drug loading capacity and tumor targeting for cancer patients remains a great challenge. Herein, we report a dendritic strategy that utilizes the two amine functionalities of lysine to create branch points that allow conjugation of the anticancer drug 5-fluorouracil (5-FU) to the tumor-targeting ligand substance P, along with an additional near-infrared (NIR) squaraine dye, to construct a theranostic dendritic agent, P-FU 4. This cytotoxic theranostic agent, containing four carboxyl-modified 5-FU molecules, has several desirable advantages: i) the ability to self-assemble into nanoparticles; ii) enhanced cytotoxicity with high drug loading capacity (16%) and a specific receptor-targeted interaction with NK1R through the SP moiety; and iii) a high NIR squaraine fluorescence efficiency due to the specific dendron isolation, avoiding aggregation-mediated quenching. As demonstrated in this report, the cytotoxic activity of P-FU 4 is dose-dependent against the tested cancer cells. The improved drug loading capacity with dendritic branching distinctly enhanced cytotoxicity to tumor cells but had little effect on the viability of normal cells. P-FU 4 was preferentially taken up by tumor cells through a receptor-mediated interaction, which was monitored by effective NIR fluorescence with high tissue penetration. Studies using a mouse model revealed that P-FU 4 can significantly inhibit tumor progression, with a tumor-inhibition rate of 60.2%. The receptor-targeted cytotoxic dendritic theranostic agent is highly preferable to standard chemotherapeutic treatments and decreases the negative side effects of medications on healthy cells, which establishes its utility in drug delivery and cancer chemotherapy.

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.