A Molecular Recognition Approach To Synthesize Nucleoside

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A Molecular Recognition Approach To Synthesize Nucleoside Analogue Based Multifunctional Nanoparticles for Targeted Cancer Therapy Dali Wang,†,∥ Bing Liu,‡,§,∥ Yuan Ma,† Chenwei Wu,† Quanbing Mou,† Hongping Deng,† Ruibin Wang,† Deyue Yan,† Chuan Zhang,*,† and Xinyuan Zhu*,† †

School of Chemistry and Chemical Engineering, State Key Laboratory of Metal Matrix Composites, Shanghai Jiao Tong University, 800 Dongchuan Road, Shanghai 200240, China ‡ Translational Medicine Research and Cooperation Center of Northern China, Heilongjiang Academy of Medical Sciences, Heilongjiang, China § Department of Oral and Maxillofacial Surgery, The First Affiliated Hospital of Harbin Medical University, 23 Youzheng Street, Nangang District, Harbin 150001, China S Supporting Information *

become the new fashion in recent years.4 The introduction of each new functionality to multifunctional nanoparticle-based platforms has specific beneficial impact on their therapeutic outcome.5 For example, nanocarriers can be designed to tailor the drug release kinetics using microenvironment sensors (pH, hypoxia, esterase).6 The targeting moieties are utilized for improving the specificity and accumulation of drugs at the tumor sites.7 The imaging agents could be empolyed to track the delivered drug in vivo, monitor treatment responses, and provide other theranostic information.8 Therefore, multifunctional nanoparticle-based therapeutic systems have drawn great interest for innovative therapies and provided significant advantages over conventional nanoparticle-based therapeutic modalities. To date, some multifunctional nanoparticles for anticancer drug delivery have been designed and synthesized for both in vitro and in vivo therapeutic evaluation.4,5 However, most of them are prepared based on covalently conjugations and the introduction of each new functionality elevates the complexity and cost (e.g., tedious syntheses, purification of the intermediates, characterizations, and low overall yields).9 Thus, there is a strong need to develop novel multifunctional nanoparticles for comprehensive antitumor therapy. Inspired by Watson−Crick base parings in natural DNA and RNA,10 in our new strategy, nucleoside analogue prodrugs and functional DNA strands are used and assembled together through molecular recognition of nucleobases to form multifunctional nanoparticles (Scheme 1 and Figure 1A). As widely used chemotherapeutics, nucleoside analogues have performed as cornerstones of anticancer chemotherapy for decades.11 Thus far, there are at least 15 FDA-approved nucleoside analogues used to treat various cancers, which account for nearly 20% of the current cancer chemotherapeutic arsenal.12 Meanwhile, recent studies have shown that functional DNA macromolecules (e.g., aptamers) have great potential for cancer therapy.13 As a proof of concept, we first synthesized an amphiphilic prodrug 3′,5′dioleoyl clofarabine (DOC) by conjugating the oleic acids with a

ABSTRACT: Tumor-targeted drug delivery with simultaneous cancer imaging is highly desirable for personalized medicine. Herein, we report a supramolecular approach to design a promising class of multifunctional nanoparticles based on molecular recognition of nucleobases, which combine excellent tumor-targeting capability via aptamer, controlled drug release, and efficient fluorescent imaging for cancer-specific therapy. First, an amphiphilic prodrug dioleoyl clofarabine was self-assembled into micellar nanoparticles with hydrophilic nucleoside analogue clofarabine on their surface. Thereafter, two types of singlestranded DNAs that contain the aptamer motif and fluorescent probe Cy5.5, respectively, were introduced onto the surface of the nanoparticles via molecular recognition between the clofarabine and the thymine on DNA. These drug-containing multifunctional nanoparticles exhibit good capabilities of targeted clofarabine delivery to the tumor site and intracellular controlled drug release, leading to a robust and effective antitumor effect in vivo.


anoparticle-based drug delivery systems have been extensively exploited for cancer therapy since they can enhance the pharmacokinetic properties, reduce the systemic toxicity, and improve therapeutic index of a myriad of drugs.1 Despite great advances, nanoscale anticancer agents generate only a modest improvement in anticancer activity because of their limited targeting ability (in general, passive targeting effect generated by the enhanced permeability and retention (EPR)).2 Additionally, for a better cancer therapy, the ability of visualizing tumor tissues and monitoring the therapeutic responses are also of great significance.3 Unfortunately, conventional nanoparticlebased drug delivery systems could not easily support these requirements. To address these challenges and ultimately achieve a better therapeutic efficacy for patients, multifunctional nanoparticles that combine different moieties such as antitumor therapeutics, tumor targeting, and imaging agents in an all-in-one system have © 2017 American Chemical Society

Received: August 5, 2017 Published: September 25, 2017 14021

DOI: 10.1021/jacs.7b08303 J. Am. Chem. Soc. 2017, 139, 14021−14024


Journal of the American Chemical Society

Owing to the presence of the hydrophobic fatty acid chains and hydrophilic clofarabine, the DOC could self-assemble into nanoparticles in aqueous solution (Scheme 1A). The dynamic light scattering (DLS) measurement revealed the average diameter of nanoparticle was ∼97 nm with a polydispersity index (PDI) of 0.089 (Figure S3A). In TEM image, spherical DOC nanoparticles can be clearly observed with an average size of approximate 90 nm (Figure S3B), which is consistent with the DLS result. Moreover, the critical aggregation concentration of DOC was determined as 13 μg/mL in aqueous solution (Figure S4), revealing the high stability of DOC nanoparticle.17 To systematically probe the recognition ability between DOC nanoparticles and DNAs, six DNA strands with different lengths and pyrimidines (T10, T20, T30, C10, C20, and C30) were mixed with DOC nanoparticles, respectively. Agarose gel electrophoresis showed that all poly dT oligonucleotides could complex with DOC very well at 4 °C, while only the DOC/T20 and DOC/T30 maintained excellent stability at 37 °C (Figures 1B and S5A). In contrast, poly dC oligonucleotides could not complex with DOC naoparticles even when increasing the length up to 30-mer at 37 °C (Figure S5B), indicating the specificity of nucleobase recognition. Considering the high yield and stability, T30 was selected to complex with DOC nanoparticles. The DOC/AS1411 nanoparticles were then prepared by complexing the DOC nanoparticles with AS1411 aptamercontaining strands, which is a 56-mer oligonucleotide composed of (1) the 26-mer DNA aptamer (G-rich segment in green color) that has high binding affinity to nucleolin normally seen overexpressed on the tumor cells18 and (2) a 30-mer poly dT oligonucleotide, a programmable recognition region, which recognizes clofarabine on the surface of the DOC nanoparticles. The noncovalent interaction between the DOC nanoparticles and AS1411-containing strands was investigated using the 2D diffusion-ordered spectroscopy (DOSY) NMR technique. As shown in Figure 1C, clear proof of the DOC/AS1411 nanoparticles formation was demonstrated by comparing the diffusion coefficients (D) of DOC nanoparticles, AS1411, and DOC/AS1411 nanoparticles. The D value of AS1411 was much higher than that of DOC nanoparticles because of its high hydrophilicity. Interestingly, the D value of DOC/AS1411 nanoparticles falled somewhere in between, indicating the molecular complexing between DOC nanoparticles and hydrophilic DNA strands. To further endow the functional nanoparticles with a fluorescent property, a Cy5.5 tag was incorporated at the 5′ end of T30 and then introduced to the surface of DOC/AS1411 nanoparticles with the same procedure. The gel electrophoresis analysis indicated the successful preparation of DOC/AS1411/ Cy5.5 nanoparticles (Figure S6). The DLS measurment revealed the average diameter of DOC/AS1411/Cy5.5 nanoparticles was around 107 nm with a PDI of 0.171 (Figure 1D), which was slightly larger than that of DOC nanoparticles owing to the introduction of DNA strands on the nanoparticle surface. In a TEM image, again spherical nanoparticles with an average diameter of ∼90 nm could be found (Figure 1E), indicating that the DNA complexing would not significantly change the morphology of these multifunctional nanoparticles. Moreover, the time-dependent changes in the hydrodynamic diameter of DOC/AS1411 nanoparticles confirmed the good stability of nanoparticles DOC/AS1411 under neutral conditions even in the presence of serum (Figure S7). As drug carriers, the in vitro release behavior of DOC/AS1411 nanoparticles was evaluated under simulated physiological

Scheme 1. Multifunctional DOC/AS1411/Cy5.5 Nanoparticles for Targeted Drug Delivery to Tumor with Simultaneous Fluorescent Imaging

Figure 1. (A) Schematic illustration for the molecular recognition of multifunctional DOC/AS1411/Cy5.5 nanoparticles. (B) Agarose gel electrophoresis of free DNAs and DOC/DNA nanoparticles at 37 °C. (C) 2D-DOSY spectra of AS1411, DOC and DOC/AS1411 nanoparticles. (D) Representative DLS profile and (E) TEM image of DOC/ AS1411/Cy5.5 nanoparticles.

second-generation adenosine analogue clofarabine,14 which could self-assemble into micellar nanoparticles with hydrophilic clofarabine on their surface in aqueous solution. Then the designed aptamer AS1411 with targeting capability and Cy5.5labeled fluorescent DNA were further employed to complex with DOC nanoparticles through the molecular recognition15 between an adenosine (A) analogue and thymidine (T) to form multifunctional nanoparticles DOC/AS1411/Cy5.5 via a simple mixing procedure. Upon being equipped with funtional DNA strands, the resulting multifunctional nanoparticles can exhibit excellent targeting and imaging capacity to enhance the antitumor efficacy, as well as overcoming the downsides of free clofarabine, such as poor stability, short half-life, and lack of specificity toward tumor tissues.16 The synthesis of amphiphilic DOC was illustrated in Scheme 1A, and the chemical structure of DOC was confirmed by 1H NMR spectroscopy. Based on the integral ratio of the characteristic peaks at 8.04 ppm (methine protons in Nheterocycle) and 0.86 ppm (methyl protons in oleic acid), two fatty acid chains that were successfully coupled to the hydroxyl groups of clofarabine could be verified (Figure S1). Also, the synthesis of DOC was confirmed by high resolution mass spectroscopy with a molecular weight of 832.6 (Figure S2). 14022

DOI: 10.1021/jacs.7b08303 J. Am. Chem. Soc. 2017, 139, 14021−14024


Journal of the American Chemical Society conditions (PBS, pH 7.4) and in an acidic environment (acetate buffer, pH = 5.0) with (or without) esterase (30 U/mL) at 37 °C (Figure S8). Less than 20% of clofarabine was released in neutral PBS solution over a period of 48 h, indicating the relatively high stability of DOC/AS1411 nanoparticles in normal conditions. In contrast, more than 60% of clofarabine was released from nanoparticles in an acidic environment containing esterase, as the esterase and acidic pH both can facilitate the break of the ester bond. To confirm whether the DOC/AS1411/Cy5.5 nanoparticles could effectively target and internalize the tumor cells, both flow cytometry analysis and fluorescence microscopy were employed to evaluate their cellular uptake. Flow cytometry revealed that the fluorescence intensity of MCF-7 cells treated with DOC/ AS1411/Cy5.5 or DOC/Cy5.5 nanoparticles gradually increased along with the incubation time (Figures 2A and S9). Apparently,

L929 cells, near-equipotent cytotoxicity could be observed for the DOC/AS1411 and DOC nanoparticles (Figure 2D), indicating nonselectivity of these nanoparticles to normal cells. Moreover, apoptosis results demonstrated that DOC/AS1411 nanoparticles-induced apoptosis was much higher than that of DOC nanoparticles (53.5% vs 39.4%) (Figure S11). Compared to free clofarabine, DOC/AS1411 nanoparticles resulted in nearequipotent apoptosis against MCF-7 cells (53.5% vs 52.6%). To further verify this result, we analyzed activation and expression of caspase-3, which has been considered as a key effector of cell apoptosis. The expression of caspase-3 protein was remarkably upregulated and comparable to that of free clofarabine when treating the cells with DOC/AS1411 nanoparticles (Figure S12), indicating a better apoptosis-inducing effect of the DOC/ AS1411 nanoparticles over the nontargeting DOC nanoparticles. The in vivo imaging of breast cancer xenografts in mice was performed to evaluate the tumor-specific targeting capability of DOC/AS1411/Cy5.5 nanoparticles. As shown in Figure 3A, the

Figure 2. (A) Relative geometrical mean fluorescence intensities of Cy5.5-labeled nanoparticles treated cells. (B) Representative fluorescence images of MCF-7 cells treated with Cy5.5-labled samples. Scale bar: 30 μm. Cell viability of (C) MCF-7 and (D) L929 against the different drug formulations.

Figure 3. (A) In vivo fluorescence imaging of the tumor-bearing nude mice after intravenous administration. Arrows indicate the sites of tumors. (B) Tumor dissection photographs through intravenous administration. (C) Tumor volume changes in tumor-bearing mice during the treatments. (D) Hematoxylin/eosin (H and E) histology of tumors after treatment. Statistical significance: *P < 0.05; **P < 0.005.

the DOC/AS1411/Cy5.5 group exhibited much higher fluorescence signals compared to the group of DOC/Cy5.5 without targeting capability. Representative fluorescence images also demonstrated the successful internalization of these nanoparticles in MCF-7 cells after 1 and 4 h incubation (Figures 2B and S10). Remarkably, the red fluorescent intensity in the cytoplasm of MCF-7 cells treated with DOC/AS1411/Cy5.5 nanoparticles was much higher than that treated with DOC/ Cy5.5 nanoparticles. These results suggested that the more effective cellular uptake could be achieved once these nanoparticles were equipped with the aptamers. Next, we investigated whether the targeted delivery of the DOC/AS1411 nanoparticles can be used to achieve selective inhibition of cancer cells (MCF-7 cells) over the normal cells (L929 cells). Figure 2C showed that DOC/AS1411 nanoparticles exhibited slightly lower cytotoxicity to MCF-7 cancer cells when compared with free clofarabine, which can be attributed to the time-consuming process of drug release from nanoparticles. Notably, DOC/AS1411 nanoparticles showed much higher cytotoxicity than DOC nanoparticles without the aptamer in all doses against MCF-7 cells. In contrast, when incubating the three above-mentioned drug formulations with

fluorescence signal of free Cy5.5 in the whole body was relatively weak at 12 h postinjection, suggesting that the free small molecules were quickly cleared under physical conditions. In contrast, the fluorescence signal at the tumor site increased when treating the mice with DOC/Cy5.5 nanoparticles, which could be attributed to the accumulation of anticancer drugs through EPR effect. Upon being loaded with AS1411, nanoparticles could effectively accumulate at the tumor site as evidenced by the significant strong fluorescence after 12 h postinjection, indicating that the aptamer indeed enhanced the targeting capability of DOC/AS1411/Cy5.5 nanoparticles. Finally, we evaluate the in vivo anticancer potential of the multifunctional nanoparticles in MCF-7 human breast tumorbearing mice. Both DOC and DOC/AS1411 nanoparticles presented remarkably higher inhibition efficacy toward tumor growth than the free clofarabine (Figure 3B and 3C), which may primarily result from the passive or active tumor targeting capability and the controlled drug release. A noticeable difference in tumor-size inhibition between DOC and DOC/AS1411 nanoparticles was observed, suggesting that the active targeted delivery of clofarabine by DOC/AS1411 nanoparticles is of great 14023

DOI: 10.1021/jacs.7b08303 J. Am. Chem. Soc. 2017, 139, 14021−14024


Journal of the American Chemical Society ORCID

importance to enhance the antitumor activity. Additionally, there was no significant decrease regarding the body weights of mice during the treatment with DOC/AS1411 nanoparticles. In contrast, mice treated with free clofarabine exhibited a drastic weight loss (more than 20%) within 21 days (Figure S13), indicating that the new formulation could remarkably reduce clofarabine toxicity to normal tissues. The antitumor effects of multifunctional nanoparticles were further analyzed by histological examination and immunohistochemistry. The DOC and DOC/AS1411 groups showed neither obvious pathological changes in heart, lung, kidney, and spleen, nor obvious liver damage while the free clofarabine caused obvious kidney damage after treatment (Figure S14A), indicating the advantages of nanodrugs for anticancer therapy. Additionally, there were clear differences in tumor tissue morphology between the PBS and nanodrug-treated groups. The PBS group showed typically histologic characteristics of malignant tumors, including hyperchromatic nuclei, scant cytoplasm, and more nuclear pleomorphism. In contrast, extensive nuclear shrinkage and fragmentation were observed in the clofarabine, DOC nanoparticle, and DOC/AS1411 nanoparticle-treated groups, especially for the multifunctional nanoparticle-treated one (Figure 3D). Furthermore, the proliferating cell nuclear antigen (PCNA) results clearly revealed that the percentage of PCNA-positive (brown) tumor cells apparently decreased in the multifunctional nanoparticle-treated groups compared to that of the PBS group (Figure S14B). Particularly, drastic suppression and replacement of the tumor cells by normal tissue were observed in the DOC/AS1411 nanoparticle-treated group, consistent with its best inhibition of tumor growth in MCF-7 tumor-bearing mice. In conclusion, we successfully designed and synthesized a novel kind of multifunctional nanoparticles through the molecular recognition of nucleobases for tumor-targeted drug delivery and imaging at the tumor site. In vitro and in vivo studies demonstrated that the supramolecular multifunctional nanoparticles could significantly improve the drug accumulation at the tumor site, enable the tumor be visualized, controllably release the anticancer drug in response to tumor environment, and finally inhibit the tumor growth efficiently. This supramolecular approach provides a significant advantage over the conventional syntheses of multifunctional drug-delivery vehicles which usually involve cumbersome, multistep covalent conjugations. Moreover, the supramolecular strategy can be readily adopted for other nucleoside analogues and functional nucleic acids. We believe that our multifunctional nanoparticle delivery platform engineered based on a combination of nanotechnology and functional nucleic acids has great potential for cancer diagnostics and treatment.

Chuan Zhang: 0000-0002-9311-0799 Xinyuan Zhu: 0000-0002-2891-837X Author Contributions ∥


The authors declare no competing financial interest.

ACKNOWLEDGMENTS This work was financially supported by the National Basic Research Program (2015CB931801), National Natural Science Foundation of China (21504053, 21661162001, 21374062, 51690151, 51473093).


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S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/jacs.7b08303.

D.W. and B.L. contributed equally.

Materials, experimental procedures, instrumentation and supplemental figures (PDF)


Corresponding Authors

*[email protected] *[email protected] 14024

DOI: 10.1021/jacs.7b08303 J. Am. Chem. Soc. 2017, 139, 14021−14024