Nucleoside analogue based supramolecular nanodrugs driven by

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Nucleoside analogue based supramolecular nanodrugs driven by molecular recognition for synergistic cancer therapy Dali Wang, Chunyang Yu, Li Xu, Leilei Shi, Gangsheng Tong, Jieli Wu, Hong Liu, Deyue Yan, and Xinyuan Zhu J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.8b04556 • Publication Date (Web): 25 Jun 2018 Downloaded from http://pubs.acs.org on June 25, 2018

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Journal of the American Chemical Society

Nucleoside analogue based supramolecular nanodrugs driven by molecular recognition for synergistic cancer therapy Dali Wang,† Chunyang Yu,† Li Xu,† Leilei Shi,† Gangsheng Tong,† Jieli Wu,† Hong Liu,‡ Deyue Yan† 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, P. R. China ‡ Institute of Theoretical Chemistry, State Key Laboratory of Supramolecular Structure and Materials, Jilin University, Changchun 130021, P. R. China Supporting Information Placeholder ABSTRACT: The utilization of nanotechnology for the delivery of a wide range of anticancer drugs has the potential to reduce adverse effects of free drugs and improve the anticancer efficacy. However, carrier materials and/or chemical modifications associated with drug delivery make it difficult for nanodrugs to achieve clinical translation and final Food and Drug Administration (FDA) approvals. We’ve discovered a molecular recognition strategy to directly assemble two FDA-approved small-molecular hydrophobic and hydrophilic anticancer drugs into well-defined, stable nanostructures with high and quantitative drug loading. Molecular dynamics simulations demonstrate that purine nucleoside analogue clofarabine and folate analogue raltitrexed can self-assemble into stable nanoparticles through molecular recognition. In vitro studies exemplify how the clofarabine:raltitrexed nanoparticles could greatly improve synergistic combination effects by arresting more G1 phase of the cell cycle and reducing intracellular deoxynucleotides pools. More importantly, the nanodrugs increase blood retention half-life of the free drugs, improve the accumulation of drugs in tumor sites and promote the synergistic tumor suppression property in vivo.

INTRODUCTION Nanotechnology-based chemotherapy has potential for treating a variety of neoplastic diseases due to its ability of delivering anticancer drugs and biologics, and preferentially targeting capacities to the disease1-5. Various nanocarrier drug delivery systems, including liposomes6,7, vesicles8,9, polymeric nanoparticles10-14, nanoemulsions15,16 and inorganic particles17, have been widely explored to improve the safety and efficacy of chemotherapeutic agents. With the help of nanocarriers, chemotherapeutic agents can be delivered to the sites of disease through physical entrapment or chemical conjugation18-20, which demonstrates efficient protection of bioactive drugs and high therapeutic indexes21,22. Although there is a large amount of nanodrug delivery systems reported, only few have been approved by the FDA and applied in the clinic23-25. This can be attributed to the fact that current nanodrug delivery systems often need new carrier materials or chemical modifications to drug molecules26-30. The synthetic materials may cause short-term and long-term toxicities in the processes of degradation, metabolism, and excretion within the human body31,32. Furthermore, the clinical translation of these nanodrugs can only happen if these carriers and/or new drug molecules are systematically evaluated as new excipients, such as pharmacokinetics, in vivo biodistribution, drug release behavior, clearance route and toxicity. Also, most of these nanodrugs are not easily prepared and generally require complicated synthesis. This could

potentially alter the physical and chemical properties of certain parent drugs thus preventing them from practical pharmaceutical development33,34. The exploration of simple, efficient and robust methods to prepare carrier-free drug nanoparticles with desirable bioactivity and without any chemical modifications remains an enormous challenge in biomedical science and is of great significance in clinical medicine35. To resolve this challenge, we’ve proposed and constructed supramolecular self-delivery nanodrugs using non-covalent molecular recognition to link hydrophobic and hydrophilic drugs together. Inspired by natural Watson-Crick base parings in DNA and RNA36,37, in our new strategy, nanodrugs are fabricated by self-assembly of hydrophobic and hydrophilic nucleoside analogues approved by the FDA through molecular recognition, which would avoid using non-clinically proven materials to facilitate clinical translation. As proof of concept, we’ve selected the water-soluble anticancer drug clofarabine (purine nucleoside analogue, CA) and the water-insoluble anticancer drug raltitrexed (quinazoline-based folate analogue, RT). Moreover, CA acts by inhibiting ribonucleotide reductase and DNA polymerase, while RT exerts its cytotoxic activity by the specific inhibition of thymidylate synthase38,39. The combination of CA and RT may induce the synergistic cytotoxicity based on their different mechanisms of action40-42. To evaluate the possibility, the nanodrugs were constructed using an easy mixing procedure.

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Figure 1. Chemical characterization of self-assembled CA:RT nanoparticles. (a) 1H NMR spectra of CA, RT and CA:RT in D2O/dimethylsulfoxide-d6 mixed solvent (10:1). The left figure is the full spectrum and the right figure is a partial enlarged spectrum in the 7.3-8.3 ppm region. (b) Representative TEM image of supramolecular CA:RT nanoparticles. (c) Representative DLS curve of CA:RT nanoparticles. (d) Representative SEM image of supramolecular CA:RT nanoparticles. (e) Relationship of the absorbance and the concentration of CA:RT in aqueous solutions. (λ = 313 nm, 25 °C). (f) Influence of storage on diameter of CA:RT nanoparticles. The solution of CA:RT nanoparticles was stored at 4 °C in refrigerator for 15 days. At different time intervals (0, 3, 6, 9, 12 and 15 d), the average size was determined by DLS. Samples were measured in triplicates. The values are the mean ± SD. (g) In vitro CA release kinetics from CA:RT nanoparticles at different pH values (5.0 and 7.4) containing 10% FBS (or not) at 37 °C. Unexpectedly, we observed that they were able to and considerably enhance anticancer efficacy as shown by in self-organize in aqueous solution into spherical nanoparticles vitro and in vivo studies. with high stability. The computer simulations further RESULTS AND DISCUSSION confirmed the molecular recognition between CA and RT as well as the feasibility of their self-assembly. These nanodrugs Synthesis, morphological characterization, and demonstrate the ability to decrease side effects of free drugs physicochemical properties of CA:RT nanoparticles.


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Nucleoside analogs are chemically modified analogs of natural nucleosides, nucleotides, and bases and have been in the clinic for decades to treat both viral pathogens and neoplasms43. Currently, there are 16 FDA-approved nucleoside analogs used to treat various cancers, which account for nearly 20% of all chemotherapeutic drugs that are used to treat cancer44,45. In addition, more than 50 nucleoside analogs are currently being investigated in clinical trials as monotherapy or combination therapy for multiple cancers. Despite the clinical success, their short plasma half-life and adverse toxicity greatly limit their chemotherapeutic efficacy. To increase their chemotherapeutic index, it would be desirable to develop a new strategy, for example, carriers to protect the drug from renal clearance and prolong their circulation half-life. When designing drug carriers, the drug-to-carrier ratio and/or chemical modification of drugs require crucial consideration since the use of high quantities of carriers may cause high toxicity due to poor metabolism and elimination and the structural changes may alter their properties. However, these issues would be of less concern if both the drug and carrier had therapeutic effects. We hypothesize that nucleoside analogs could assemble into nanoparticles that can be used in the delivery of both of them through molecular recognition, which would address above mentioned issues. To verify this hypothesis, the hydrophilic anticancer drug CA and hydrophobic anticancer drug RT were used to construct supramolecular nanodrug in aqueous solutions by an easy mixing procedure followed by sonication (See supporting information). Firstly, to prove the hydrogen bonding interactions between CA and RT, 1H NMR was measured after blending the two drugs with an equivalent molarity in D2O and dimethylsulfoxide-d6 (10:1), and then compared with that of free CA and RT under identical conditions. Figure 1a depicts the chemical structures and 1H NMR spectra of free CA, free RT and CA:RT. It is found that the chemical shift of the CH resonance moves up-field systematically from 8.21 to 8.14 ppm after CA formed CA:RT nanoparticles. The chemical shifts of three CH peaks in the RT also change apparently from 7.89 ppm, 7.60 ppm, and 7.44 ppm to 7.84 ppm, 7.54 ppm, and 7.39 ppm respectively, after the formation of CA:RT nanoparticles. This can be attributed to the formation of hydrogen bond interactions between CA and RT46,47. The CA:RT nanodrugs were also characterized by ultraviolet-visible spectrophotometer (UV-vis) (Figure S1), which demonstrates that CA:RT nanodrugs were synthesized successfully. The size and morphology of CA:RT nanodrugs were then characterized by dynamic light scattering (DLS), transmission electron microscopy (TEM) and scanning electron microscopy (SEM). The TEM image in Figure 1b shows spherical nanoparticles with an average size of approximately 30 nm. Figure 1c presents a monomodal size distribution with a Z-average diameter of 32 nm and a polydispersity index of 0.164. The size determined by TEM is slightly smaller than that measured by DLS due to the shrinkage of nanoparticles in the drying state. The SEM image of the CA:RT aggregates shows almost uniform spherical particles, and the average diameter of around 30 nm is consistent with that observed by TEM (Figure 1d).

To further evaluate the self-assembly ability of CA:RT in aqueous solution, the critical aggregation concentration (CAC) was measured by UV-vis spectra with 1,6-diphenyl-1,3,5-hexatriene (DPH) as a hydrophobic probe. The relationship of the intensity of absorbance of DPH with the CA:RT concentration is present in Figure 1e. The absorption intensity remains nearly unchanged at low CA:RT concentration but increases sharply when the CA:RT concentration reaches a certain value, indicating the characteristic level of DPH in a hydrophobic environment48. According to the inflection of the curve, the CAC value of the CA:RT is about 12 µg/mL. In addition, we tested the stability of CA:RT nanoparticles by DLS measurement. The CA:RT nanoparticles showed excellent physical and chemical stability during storage for two weeks (at 4 °C in the dark) (Figure 1f), whereas sensitivity to increased ionic strength was observed. The diameter of CA:RT nanoparticles decreases a little after adding PBS and 10% FBS (from 32 nm to 29 nm). However, no momentous change in diameter is observed within 72 h (Figure S3). Afterwards, we investigated the in vitro release behavior of CA:RT nanoparticles under neutral conditions (PBS, pH 7.4) both with or without 10% FBS, and in an acidic environment (acetate buffer, pH 5.0) at 37 °C. The cumulative release curves show that the concentration of released drugs is much faster in the acidic condition (pH 5.0) than that in PBS (pH 7.4) with or without 10% FBS, as shown in Figure 1g. More precisely, more than 80% of drugs are released in pH 5.0 after 13 h, whereas under the neutral PBS condition about 45% of drugs are released. This indicates that the drug release of CA:RT nanoparticles can be triggered and controlled by acidic conditions. Potentially, this could be due to the protonation of the amino group within a specific drug that disrupts the interactions between CA and RT in the nanoparticles. Molecular simulations disclose the noncovalent interactions and self-assembly mechanisms between CA and RT. To verify the experimental results and unveil the detailed mechanism for nanoparticle formation, quantum chemical calculations and dissipative particle dynamics (DPD) simulations were performed to explore the hydrogen bond interaction and the self-assembly process of CA:RT. We can see from Figure 2a that two stable hydrogen bonds are observed between CA and RT. The distances of O…H and N…H are 1.763 Å and 2.106 Å, respectively. Meanwhile, the density functional theory (DFT) calculation shows that the binding energy of CA and RT is 10.46 kcal/mol. Furthermore, to capture the essential features of the CA:RT complex, a model molecule CA1-CA2…RT1-RT2 is constructed in DPD simulations and the corresponding coarse-grain mapping is shown in Figure 2b. The details of the simulation model and method are described in the Methods Section. Figures 2c-2h display snapshots of the self-assembly process at different time intervals of the CA:RT complex calculated through DPD simulations. Starting from a random state (Fig. 2c), the amphiphilic CA:RT complexes aggregate into many small micelles (Figure 2d). Thereafter, these small micelles gradually fuse to form several medium sized micelles (Figures 2e-2g), eventually fusing into one large complex micelle



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Figure 2. Molecular simulations disclose the noncovalent interactions and self-assembly mechanisms between CA and RT. (a) The optimized structure of CA:RT complex and binding energy by DFT calculations; (b) Coarse-grained models of CA, RT and water (W). DPD simulations on the self-assembly of CA:RT complex in solution. (c) The initial state; (d) 2.0×104 time steps; (e) 1.5×105 time steps; (f) 3.5×105 time steps; (g) 5.0×105 time steps; (h) 9.0×105 time steps; (i) The final structure and the cross-sectional view of one micelle. Water beads are omitted for clarity. The color codes are the same as those in Figure 2b.


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Figure 3. Effect of CA:RT nanoparticles on cancer cellular uptake, cell proliferation, cell apoptosis, cell apoptosis-related protein expression, and cell cycle in vitro. (a) Time-dependent profiles of CA:RT nanoparticles fluorescence intensity in the HeLa cells by flow cytometry analysis. Insert: representative flow cytometry histogram profiles of HeLa cells treated with NR-loaded CA:RT nanoparticles for 4 h, the cells without any treatment are used as a control. (b) Representative CLSM image of HeLa cells treated with NR-loaded CA:RT nanoparticles for 1 h. Cell nuclei were stained with Hoechst 33342. The red fluorescence is from NR and the blue fluorescence is from Hoechst 33342. (c) Cell viability of HeLa cells against the RT, CA, CA/RT mixture and CA:RT nanoparticles after incubation for 72 h with different drug concentrations determined by MTT assay. The data are presented as average ± standard error (n = 6). (d) The combination index (CI) plots for HeLa cells calculated with the CompuSyn software. (additive effect, CI = 1; synergism, CI1). (e) Flow cytometry analysis for apoptosis of HeLa cells induced by RT, CA, CA/RT mixture and CA:RT nanoparticles at the same drug concentration (50 µM) for 24 h. Lower left: living cells; Lower right: early apoptotic cells; Upper right: late apoptotic cells; Upper left: necrotic cells. Inserted numbers in the profiles indicate the percentage of the cells present in this area. (f) The expression levels of caspase-3 in HeLa cells induced by RT, CA, CA/RT mixture and CA:RT nanoparticles at the same drug concentration (50 µM) for 24 h, analyzed by Western blotting. The β-actin is the loading control. (g) The cell cycle distribution histograms of HeLa cells treated with RT, CA, CA/RT mixture and CA:RT nanoparticles at the same drug concentration (50 µM) for 24 h.



(Figure 2h). Moreover, the self-assembly morphologies of the CA:RT micelles were not displayed as the classical core-shell structure (Figure 2i). Instead, the hydrophilic segments RT1 and CA2 were also distributed in the micelle core, forming a small microphase domain. This morphology explains why this small amphiphilic complex can self-assemble into a large nanoparticle. In vitro cell internalization and anticancer capability. To confirm whether the CA:RT nanoparticles could effectively be transported into tumor cells, both flow cytometry and confocal laser scanning microscopy (CLSM) were performed to monitor the cell internalization in HeLa cells (a human cervical carcinoma cell line). The hydrophobic nile red (NR) was used as a fluorescent probe and encapsulated into the CA:RT nanoparticles since CA and RT show no obvious fluorescence (Figure S4). The time-dependent cellular adhesion of the NR-loaded CA:RT nanoparticles was estimated from the fluorescence intensities of HeLa cells via flow cytometry. As clearly shown in Figure 3a and Figure S5, the fluorescence intensity of HeLa cells treated with NR-loaded CA:RT nanoparticles gradually increase with increasing time. The enhancement of fluorescence signals suggests high cellular adhesion of nanoparticles by HeLa cells, which facilitates the occurrence of cellular internalization. The ability of CA:RT nanoparticles to enter HeLa cells was further investigated by CLSM. Representative images show that NR-loaded CA:RT nanoparticles (red) rapidly localize into the cell cytoplasm after 0.5 h of incubation and give much stronger signals after 1 h under the identical imaging settings (Figure 3b and Figure S6), indicating the successful internalization of the CA:RT nanoparticles. After having validated effective intracellular uptake of these nanodrugs, we further examined the antitumor efficacy of CA:RT nanoparticles on the growth of HeLa cells, comparing with free CA, RT, and CA/RT mixture. As shown in Figure 3c, RT exhibits lower cytotoxicity to HeLa cells when compared with the other three drug formulations, which is most probably due to the poor cellular uptake of hydrophobic RT by tumor cells. Although CA/RT mixture displays a slightly better antiproliferative effect than CA (IC50 of 78 and 94 µM, respectively), a much more efficient suppression of the tumor cell invasion is observed with CA:RT nanoparticles (IC50 of 36 µM). Incubation with CA:RT nanoparticles has reduced the cell proliferation in a concentration-dependent manner. Remarkably, the self-assembled CA:RT nanodrugs show much better proliferation inhibition efficacy than CA/RT mixture when the concentration is higher than the CAC value. The higher anticancer efficacy can be attributed to the promoted cellular uptake of self-assembled CA:RT nanoparticles and the possible synergistic action of the released free CA and RT (Figure 3d). It has been reported CA and RT induce mitochondrial-mediated apoptosis in human cancer cells38,39. To further confirm the nanodrug-induced apoptosis, HeLa cells were treated with CA:RT nanoparticles at a concentration of 50 µM and compared with free CA, RT, and CA/RT mixture under identical conditions. After 24 h of incubation, FITC-Annexin V/propidium iodide (PI) staining of the HeLa cells revealed a higher induction of apoptosis in CA:RT nanoparticle-treated cells compared with the other drug

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formulation-treated cells at the same molar concentration (Figure 3e). Indeed, treatment of the cells with CA:RT nanoparticles results in 67.4% of cells in the apoptosis phase; more precisely, 51.5% of cells are in the early apoptosis phase and 15.9% cells in the late apoptosis phase, whereas treatment with free CA or RT results in only 31.8% and 45.4% of cells in the apoptosis phase, respectively. The difference is still more pronounced compared with CA/RT mixture, where the mixture treatment leads to 44.5% of cells in the early apoptosis phase and 13% in the late apoptosis phase. To further confirm these effects, we analyzed caspase-3 activation, which has been considered as a hallmark of the cell apoptosis induction. HeLa cells were treated with the above four drug formulations at the same concentration of 50 µM for 24 h, and the expression levels of caspase-3 was detected by Western blot analysis. As shown in Figure 3f, caspase-3 protein expression is up-regulated slightly by RT, CA, and CA/RT mixture compared to the untreated control, whereas the expression of caspase-3 protein is significantly enhanced by CA:RT nanoparticles. These results suggest that the antiproliferative and cytotoxic effects of CA:RT nanoparticles could be attributed to the activation of the apoptosis mediator caspase-3. We next studied the effect of CA:RT nanoparticles on cell cycle by measuring DNA content using flow cytometry. The cells treated with RT display a similar cell cycle to that of the control cells while those exposed to CA and CA/RT mixtures show an increase in the G1 phase (Figure 3g). Notably, cell-cycle analysis revealed that after treatment with CA:RT nanoparticles, a significant portion of the HeLa cells accumulate in G1 phase: the percentage of G1 phase increases to 71.76%. Thus, CA:RT nanoparticles induce a G1 cell cycle arrest in HeLa cells in vitro and exhibit a much higher delay in cell cycle progression through S-phase than free drugs. Anticancer mechanism of CA:RT nanoparticles. Having demonstrated the excellent anticancer efficacy of the CA:RT nanodrugs, we next investigated its anticancer mechanism by analyzing intracellular deoxynucleotide (dNTP) pools (Figures 4a-4d) including: deoxyadenosine triphosphate (dATP), deoxythymidine triphosphate (dTTP), deoxycytidine triphosphate (dCTP), and deoxyguanosine triphosphate (dGTP)49. CA is a new-generation purine nucleoside analogue, which is thought to work via three mechanisms: incorporation into DNA; inhibition of ribonucleotide reductase; and induction of apoptosis38. Upon entry into cells, CA is phosphorylated to the nucleotide analogues clofarabine 5’-mono-, di- and triphosphate by cytosolic kinases in a stepwise manner. Among them, clofarabine triphosphate is the active form. It is a potent competitor with dATP for DNA polymerase-α and -ε, which then incorporates clofarabine-monophosphate into internal and terminal DNA sites, causing the impairment of DNA elongation and/or repair. In addition, clofarabine triphosphate is an inhibitor of ribonucleotide reductase, presumably by binding to the allosteric site on the regulatory subunit. These effects deplete the dNTP pool primarily of dATP, dCTP, and dGTP, but not dTTP. RT is a quinazoline-based folate analogue that produces cytotoxic activity by the specific inhibition of thymidylate


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Figure 4. Mechanisms of action and metabolism of CA:RT nanodrugs. Effects of CA, RT, CA/RT mixture, and CA:RT nanoparticles on (a) dATP, (b) dTTP, (c) dCTP, and (d) dGTP pools in HeLa cells. Significantly different from control levels with *P < 0.05, **P < 0.01, and ***P < 0.001. (e) Schematic of anticancer mechanism of CA:RT nanodrugs. CA:RT nanoparticles are transported into cells and release the CA and RT. They inhibit ribonucleotide reductase with reduction of dNTP pools and act to terminate DNA chain elongation. Also, the CA triphosphate can inhibit DNA repair through incorporation into the DNA chain. Abbreviations: RAL-(glu)3-5 = polyglutamated raltitrexed species; dAMP = deoxyadenosine monophosphate; dTMP = thymidine monophosphate; dCMP = deoxycytidine monophosphate; dGMP = deoxyguanosine monophosphate.



synthase40. After it is actively transported into cells, RT undergoes rapid, extensive metabolism by folylpolyglutamate synthetase to a series of polyglutamates. These metabolites result in potent inhibitors of thymidylate synthase and prolonged intracellular retention of RT. Thymidylate synthase catalyzes the reductive methylation of 2’-deoxyuridine-5’-monophosphate (dUMP) to 2’-deoxythymidine-5’-monophosphate (dTMP). dTMP is phosphorylated to the triphosphate form (dTTP), which is an essential component for DNA replication and repair. Inhibition of thymidylate synthase leads to breakages in single- and double-stranded DNA and ultimately produces the cytotoxicity. Therefore, the combination of CA and RT may have a better effect on the impairment of the synthesis and repair of DNA by indirectly reducing the levels of dATP, dTTP, dCTP, and dGTP. Decreased dATP, dTTP, dCTP, and dGTP pools could explain the improved synergistic combination of CA and RT in cancer therapy38,40,42. As expected, incubation of HeLa cells with CA resulted in decreased of dATP and dCTP pools (Figures 4a and 4c), quantitatively followed by the dGTP pool (Figure 4d). However, there was very little effect in the dTTP pool (Figure 4b). In contrast, the dTTP pool is markedly reduced when the HeLa cells are treated with RT (Figure 4b). Notably, the HeLa cells treated with CA:RT nanoparticles could reduce the levels of dATP, dTTP, dCTP, and dGTP to the minimum and result in an additive effect on the reduction of dNTP levels, which limits DNA synthesis and inhibits DNA repair. The mechanism of action for CA:RT nanoparticles is summarized in Figure 4e. These data further indicate that CA:RT nanoparticles provide a synergistic therapeutic effect to reduce the side effect of free drugs and improve anticancer efficacy. Blood retention time and biodistribution in vivo. Generally, nanodrugs with a suitable size exhibit a longer retention time than free small-molecule drugs in blood circulation in vivo50. To confirm this hypothesis, a pharmacokinetic study was undertaken by administration of intravenous injections of free CA, RT, and CA:RT nanoparticles to Sprague-Dawley (SD) rats. The drug concentration-time profiles of free CA, RT, and CA:RT nanoparticles in plasma are illustrated in Figure 5a. Free CA or RT rapidly disappears from blood circulation with a more than 75% reduction within 4 h. In contrast, the drug concentration in the blood of mice treated with CA:RT nanoparticles is significantly higher and can be retained for 12 h after injection. These data suggest that the nanodrugs facilitate the accumulation of anticancer drugs at the tumor tissue and enhance drug delivery. To assess the effectiveness of drug delivery, the biodistribution of CA:RT nanoparticles and free CA or RT in normal organs and a tumor were examined in HeLa tumor-bearing mice after intravenous injection at different time intervals. As displayed in Figure 5b, free CA or RT mainly accumulates in the kidney, followed by the liver, spleen, lung and heart within the first 1 h. In contrast, the CA:RT nanoparticles are predominately localized in liver, spleen, kidney and lung. After 8 h post-injection, the content of CA:RT nanoparticles obviously decreases in liver, spleen, kidney and lung, whereas an upward trend in the tumor is

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apparent. Compared to that of CA:RT nanoparticles, the concentration of free CA or RT is remarkably low in the tumor. To confirm the aforementioned result, in vivo animal imaging in tumor-bearing mice was performed using free Cy5.5 and Cy5.5-loaded CA:RT nanoparticles. As shown in Figure 5c, at 1 h after intravenous injection, the Cy5.5 fluorescence is detectable over the whole animal body for these two groups. More importantly, stronger fluorescence in the tumor area is observable at 4 h only for mice treated with Cy5.5-loaded CA:RT nanoparticles, indicating preferential accumulation of the nanodrugs in tumors. The fluorescence in the tumor site in mice treated with Cy5.5-loaded CA:RT nanoparticles remains almost unchanged at 8 h. By contrast, no fluorescence was detectable at 4 h or later in the tumor of mice treated with free Cy5.5. Furthermore, the Cy5.5 fluorescence could still be detected at 24 h in the tumor of mice treated with the CA:RT nanoparticles. These data demonstrate that the CA:RT nanoparticles have a prolonged half-life in vivo, a desirable property of nanoparticles aimed for enhancing drug delivery. In vivo antitumor activity in tumor xenograft models. The anticancer activity of the CA:RT nanoparticles was investigated using xenograft models of human cervical carcinoma and compared to that of free CA, RT, and CA/RT mixture. All of the drug formulations were intravenously injected into HeLa tumor-bearing mice, with saline as the control. As indicated in Figures 5d and 5e, the treatment with free CA or RT reduces the tumor volume to some extent. The CA/RT mixture group exhibits a slightly better effect than the free drug group, whereas mice treated with CA:RT nanoparticles show a more drastic tumor growth inhibition. After 28 days of treatment, the control group of mice experiences a weight gain due to rapid increases of tumor volumes (Figure 5f). The absolute weight-loss differences in the free CA or RT and CA:RT nanoparticles-treated groups are modest in mice bearing HeLa xenograft. In contrast, a drastic weight loss is observed in the CA/RT mixture-treated mice bearing HeLa tumors, whereas a slight weight decrease is observed after CA:RT nanoparticles treatment, thus demonstrating the superiority of CA:RT nanodrugs. The therapeutic effect of CA:RT nanodrugs was further confirmed by hematoxylin-eosin (H&E) staining of pathological sections and immunofluorescence analysis of HeLa tumors. As shown in Figure 5g, the control group shows apparent histologic characteristics of malignant tumors, such as hyperchromatic nuclei, scant cytoplasm, more nuclear pleomorphism and mitoses, indicating a rapid growth trend for the tumor. Compared with the control group, the drug-treated groups exhibit obvious differences in tissue morphology, including shrinkage of tumor cells, cell separation from surrounding cells (named apoptotic body) and large necrotic areas, especially for the CA:RT nanoparticles group. The proliferating cell nuclear antigen (PCNA) was used to analyze cell proliferation in the tumor tissues after systemic administration of various drug formulations. The PCNA results clearly revealed that the percentage of PCNA-positive (red) tumor cells apparently decreased in these drug-treated groups compared to that of the PBS groups (Figure S7).


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Figure 5. Antitumor efficacy of CA:RT nanoparticles in HeLa tumor-bearing Balb-c/nude mice. (a) Representative plasma concentration-time profiles of CA, RT and CA:RT nanoparticles after intravenous administration in rats; drug equivalent doses are 131 µmol/kg. Data are presented as the average ± standard deviation (n = 4). (b) Biodistribution of CA, RT and CA:RT nanoparticles administrated intravenous injection to mice. Data are presented as average ± standard error (n = 4), and the statistical significance level is *P < 0.05. (c) In vivo non-invasive NIR images of time-dependent whole body imaging of HeLa tumor-bearing nude mice after intravenous injection of free Cy5.5 and Cy5.5-loaded CA:RT nanoparticles. Solid arrows indicate the tumors. (d) Tumor volume changes with increasing time in Balb-c/nude mice after intravenous administration of PBS, CA, RT, CA/RT mixture and CA:RT nanoparticles at equivalent molar concentrations (131 µmol/kg) every 4th day. Data are represented as average ± standard error (n = 6). Statistical significance: *P < 0.05; **P < 0.005. (e) Tumor dissection photographs through intravenous administration. (f) Body weight changes of HeLa tumor-bearing mice after treatment with PBS, CA, RT, CA/RT mixture and CA:RT nanoparticles. (g) H&E histology of tumors from mice after treatment. Particularly, drastic suppression and replacement of the tumor cells by normal tissue were observed in the CA:RT nanodrug-treated group, consistent with its best inhibition of tumor growth in HeLa tumor-bearing mice. Hence, both H&E and immunofluorescence results confirm the superior in vivo antitumor efficacy of CA:RT nanodrugs. Our finding has numerous advantages over currently existing nanotechnology as a delivery vehicle. A large number of carrier materials, including organic and inorganic delivery systems, have been developed to experimentally evaluate their properties for treatment of diseases51-56. Each of these carrier materials has advantages. However, potential systematic toxicity, unclear metabolism related to the use of nanocarriers and large-scale economical production are challenging issues confronting these technologies. Our approach using FDA approved hydrophobic and hydrophilic drugs to fabricate

nanomedicines has the advantage of avoiding the use of non-clinically proven materials. Also, it has the ability to be produced economically and thus facilitate their clinical translation. Because our method does not involve covalent modification of drugs or specialized chemistry, it has the advantage of flexibility, allowing easy preparation and evaluation of various nucleoside analog mixtures with molecular recognition. The method can be readily adapted to almost any tumor cell type. Moreover, the method is readily accessible for in vivo testing by academic laboratories. Our use of sonication allowed for large-scale production of CA:RT nanodrugs, which is another major advantage over the multiple steps and cumbersome techniques required for nanomedicines with carriers or chemical modification of parent drugs.



CONCLUSIONS In summary, we have put forward and developed a nucleoside analogue-based nanodrug self-delivery system for cancer therapy where the carrier itself displayed anticancer effects. This nanodrug system was formulated by the molecular recognition of FDA-approved hydrophobic and hydrophilic anticancer drugs, which avoids chemical modification of parent drugs or introduction of any new materials. Both the computer simulations and experiments confirmed that the CA:RT formed stable nanoparticles in aqueous solution, which shows a synergistic therapeutic effect by arresting the cell population in G1-phase and reducing the intracellular levels of dATP, dTTP, dCTP, and dGTP. The nanoscale characteristics of CA:RT nanoparticles improved pharmacokinetics, bioavailability and therapeutic efficacy but drastically reduced side effects of free drugs. This supramolecular strategy can be extended to construct nanostructures of other types of anticancer drugs and thus provides new opportunities for the development of self-delivering drugs for cancer therapeutics.

ASSOCIATED CONTENT Supporting Information. Materials, experimental procedures, instrumentation and supplemental figures. This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Author X.Z.: [email protected]

Notes The authors declare no competing financial interests.

ACKNOWLEDGMENT This work was supported by the Center for High Performance Computing, Shanghai Jiao Tong University. This work was financially supported by the National Natural Science Foundation of China (51690151, 51473093) and National Basic Research Program (2015CB931801).

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