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Nuclear-shell biopolymers initiated by telomere elongation for individual cancer cell imaging and drug delivery Zhen Zhang, Yuting Jiao, Mengting Zhu, and Shusheng Zhang Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b00591 • Publication Date (Web): 16 Mar 2017 Downloaded from http://pubs.acs.org on March 16, 2017

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Analytical Chemistry

Nuclear-shell biopolymers initiated by telomere elongation for individual cancer cell imaging and drug delivery Zhang Zhen1, Yuting Jiao2, Mengting Zhu3, Shusheng Zhang1* 1

Shandong Province Key Laboratory of Detection Technology for Tumor Makers, College of Chemistry and Chemical Engineering, Linyi University, Linyi 276000, P. R. China. 2 Collaborative Innovation Center of Functionalized Probes for Chemical Imaging in Universities of Shandong, Shandong Normal University, Jinan 250014, P. R. China 3 Shandong Province Key Laboratory of Life-Organic Analysis, College of Chemistry and Chemical Engineering, Qufu Normal University, Qufu 273165, P. R. of China. ABSTRACT: Here, we propose a strategy for unique nuclear-shell biopolymers initiated by telomere elongation for telomer-

ase activity detection and precise drug delivery to individual cancer cells. Telomerase-triggered DNA rolling-circle amplification (RCA) is used to assemble nuclear-shell biopolymers with signal molecules for selective cancer cell recognition and efficient drug delivery to targeted individual cells. This strategy not only should allow the creation of clustered 5carboxyfluorescein (FAM)-fluorescence spots in response to human-telomerase activity in individual cancer cells but also could efficiently deliver drugs to reduce the undesired death of healthy cells. These findings offer new opportunities to improve the efficacy of cancer cell imaging and therapy.

Human telomerase is a unique ribonucleoprotein that can increase the repeated DNA sequence TTAGGG at the 3’ end of telomeres.1-3 In normal cells, telomeres are progressively shortened after each replication cycle, and as a result, the cells undergo senescence and ultimately apoptosis.4-7 However, the telomere length can be retained in human cancer cells because of the upregulation or activation of telomerase in active cancer cells, allowing these cells to proliferate and survive uncontrollably.8-11 The elevated expression of telomerase and its high activity have been considered indicators in studies of cancer mechanisms and therapeutic research. Moreover, the distinct difference in telomerase activity between normal and tumor cells has been identified as a biological basis for designing telomerase-responsive drug carriers; there can deliver a precise and localized release of anti-tumor agents compared to some conventional stimuli-responsive drug delivery systems.12-15 Thus, the development of effective targeting platforms for telomerase monitoring and precise diagnosistreatment processes is urgently needed. Various methods are applicable to probe the telomerase activity, such as electrochemical detection,16-18 optical-based assays with nucleic acid beacons19 and the telomeric Gquadruplex structure20, scissor-like chiral metamolecules,21 polymerase chain reaction using the telomeric repeat amplification protocol,22, 23 the bio-barcode technique,24, 25 electroluminescence methods,26, 27 and nanomaterial-based methods.28, 29 These technologies require cell extraction for telomerase activity analysis and are not suitable for in situ detection or directing telomerase information in complex biological environments, particularly in individual cells. Although the monitoring of fragments in the telomerase reverse transcriptase promoter in vivo has been achieved, 11, 30, most of these meth-

ods do not directly reflect the actual telomerase activity in individual cells and are not sufficient for precise diagnosistreatment processes. Focusing on these limitations, our previous work has shown that mesoporous silica nanospheres with positively charged groups have the potential to construct selfassembled nucleic acid congeries based on a hybridization chain reaction.31, 32 However, we have not developed effective targeting platforms for precise diagnosis-treatment processes in cells until now. In this work, a new dual-functional nuclearshell biopolymer platform based on DNA rolling-circle amplification (RCA) and functionalized silica-nanoflowers (FSNFs) was proposed for telomerase activity detection and monitoring and to provide suitable drug delivery in individual cancer cells. Inspired by the function and nature of the telomerase primer and its extension product,17, 33, 34 we introduced new nuclearshell biopolymers initiated by telomere elongation, which could allow telomerase-triggered nuclear/RCA of signal molecules and thus lead to their accumulation in the cytoplasm, and thus drug release in response to telomerase activity for specific cancer treatments could be achieved (Scheme 1). Telomerase could elongate the telomere-primers, resulting in inner chain substitution followed by the release of RCA-primers and the trapped drug. This nuclear-shell self-assembly involves a core nanoball and an outer shell of RCA/product biopolymers with fluorescence labels attached. This complex is the nuclear-shell product that initiates upon the elongation of the telomere ends. The intracellular RCA/product biopolymers offer efficient signal amplification, fluorescence clustering and high sensitivity human telomerase activity monitoring. Furthermore, the in situ monitoring of telomerase in individual cells could provide greater clarity regarding the telomerase distribution and activity expression. Finally, the discrepancy between telomerase

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activity in normal and tumor cells could be utilized to target drug release and improve diagnosis-treatment processes. Scheme 1. Illustration of telomerase activity monitoring and drug delivery based on novel nuclear-shell biopolymers in individual cancer cells.

EXPERIMENTAL SECTION Materials and reagents. The human hepatocellular liver carcinoma cell line HepG2 was obtained from Shanghai Bioleaf Biotechnology Company (Shanghai, China). HeLa and MCF-7 cells were acquired from KeyGEN biotechnology Company (Nanjing, China), and normal human hepatocytes L02 were purchased from Silver Amethyst Biotech. Co. Ltd. (Beijing, China). The telomerase enzyme-linked immunosorbent assay (ELISA) kit was obtained from Innovation Beyond Limits (Germany) and included a bottle of telomerase standard solution (80 IU L−1). MgCl2, NaCl, Tris-HCl, ethylenediaminetetraacetic acid (EDTA), triethanolamine (TEAH3), tetraethylorthosilicate (TEOS) and 3-aminopropyltriethoxysilane (APTES) were acquired from Aladdin (Shanghai, China). The hybridization buffer (pH 7.4) contained 1 mM EDTA, 10 mM MgCl2, 50 mM NaCl, and 10 mM Tris-HCl. Dimethyl sulfoxide (DMSO) and 3-(4,5-dimethyl-thiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT) were purchased from Sigma Chemical Company. Cetyl-trimethylammonium tosylate (CTATos) was obtained from Merck. Ultrapure water was used throughout the experiments. All other reagents used in this study were of analytical grade and used without further purification. All oligonucleotides and the doxorubicin hydrochloride (DOX) used were from Sangon Biotech Co., Ltd. (Shanghai China). Their detailed sequences are listed in Table S1. Apparatus. Confocal fluorescence imaging was performed using a Leica TCS SP8 inverted confocal microscope (Leica, Germany). The cellular images were acquired using a 20× objective. 5-Carboxyfluorescein (FAM) fluorescence was excited at 488 nm and collected from 500 nm to 700 nm. The ultraviolet (UV)-visible spectra were recorded on a Cary 50 UV/Vis-NIR spectrophotometer. The transmission electron

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microscopy (TEM) images were acquired using a TEM instrument (JEM-2100, Hitachi, Japan). Fluorescence spectra were collected using a F-4600 Fluorescence Spectrophotometer (Hitachi, Japan). Preparation of FSNFs. FSNFs were synthesized as reported previously with the following modifications.35 In total, 20.8 g of TEAH3, 115.2 g of CTATos and 6.0 L of ultrapure water were mixed at 80 °C for 100 min. Then, 875.0 g of TEOS was added to the surfactant solution after being dissolved in a small organic amine (SOA). The resulting solution (molar composition, SiO2:CTATos:SOA:H2O = 1.0:0.06:0.026:85.0) was stirred at 65 °C for 2 h and then at 80 °C for 2 h. The silica-nanoflowers (SNFs) produced were filtered, washed, and dried in an oven at 90 °C for 24 h. Next, 5 g of the dried SNFs was suspended in 600 mL anhydrous ethanol, and then, APTES was added. The resulting solution was stirred for 9 h at 37 ºC. The final product was filtered, washed with ethanol and dried at 55 ºC to obtain FSNFs. Cell culture and telomerase extraction. HepG2, HeLa, MCF-7 and L02 cells were cultured in RPMI 1640 (penicillin, 100 U/mL; streptomycin, 100 µg/mL; and Hyclone) supplemented with 10% fetal bovine serum and maintained at 37 °C in a humidified atmosphere with 5% CO2, following the protocol of American Type Culture Collection. HeLa cells were selected as the representative cell line for intracellular telomerase activity analysis using the telomerase ELISA kit. HeLa cells were collected and centrifuged at 3000 rpm for 7 min in culture medium, washed once with hybridization buffer (pH 7.4), and then spun down at 3000 rpm for 6 min. The cell pellets were suspended in 800 µL of lysis solution. RESULTS AND DISCUSSION Design for telomerase activity monitoring and drug delivery. In this study, a strategy of using unique nuclear-shell biopolymers initiated by telomere elongation is proposed for telomerase activity detection and appropriate drug delivery to individual cancer cells (Scheme 1). In this strategy, intracellular telomerase activates the RCA reaction to generate the clustered FAM-fluorescence biopolymers in individual cancer cells and permit precise drug delivery to cancer cells, which could reduce the undesired death of healthy cells. The mechanism pathways are described as follows. First, the gold nanoparticles (AuNPs)-conjugated molecular probes with DOX molecules (Au-MPs-DOX) were designed with three functional regions. The first region is the detection region, which discerns the telomerase activity; the second is an amplification region that is responsible for initiating the RCA reaction; and the third region is the load region, which allows DOX molecules to intercalate into the prepared probes,11, 36, 37 and, thereby, quench the fluorescence and decrease the cytotoxicity of DOX. After incubating the probes with living cells, the presence of telomerase will elongate the primers, leading to inner chain substitution. Then, the RCA primer and trapped DOX molecules will be released from the probes and initiate the intracellular RCA reaction in the presence of circular DNA. Subsequently, the DOX molecules released in response to human telomerase activity will be directed to the cell cytoplasm and nucleus to initiate anticancer mechanisms and reduce the toxicity to normal organs. The results also indicate that the probes are efficient DOX loading and delivery systems. In addition, the RCA products and DNA6/FSNFs could produce the original nuclear-shell biopolymers by self-


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assembly on the surface of the nucleus through electrostatic interactions. This process could generate the telomerasetriggered nuclear/RCA of signal molecules assembling in the cytoplasm due to the fluorophore (FAM) that was relatively far away from the quencher (Black Hole Quencher [BHQ]) based on the fluorescence resonance energy transfer. The combination of nucleic acid biopolymers and SNF nanospheres modified with an electrostatic charge layer should provide a new nuclear/RCA self-assembly platform with potentially high efficiency for cellular delivery and fluorescence activation. Thus, telomerase activity detection and appropriate drug delivery to individual cancer cells were successfully achieved. Characterization of the FSNFs. TEM was performed (Figure 1A), and the average size of the FSNFs was approximately 90 nm. Although FSNFs and SNFs exhibited similar nitrogen adsorption-desorption dynamics, the nitrogen adsorption on the FSNF surface was lower than that on the SNFs. The FSNFs had a surface area of 215.4 m2 g-1, which is smaller than that of the SNFs, as measured using the BrunauerEmmett-Teller (BET) method. Additionally, the BarrettJoyner-Halenda (BJH) pore size distribution dynamics (Figure 1B) revealed that the SNF pores with a diameter of 3.5 nm had disappeared completely, whereas those with diameters below 25 nm diameter had partially diminished. These data suggest that positively charged aminopropyl groups were integrated into the SNF pores.

Figure 1. (A) TEM image of the synthesized FSNFs, (B) pore size distributions of FSNFs and SNFs,; and (C) nitrogen adsorption-desorption isotherms of FSNFs and SNFs. Preparation and cytotoxicity assay of FSNFs-assembled DNA6 probes. FSNFs were dissolved in 2 mL of hybridization buffer (pH 7.4) and stirred continuously at 37 °C for 90 min to generate an FSNF solution (10 mg/mL). The hairpin DNA6 probe (40 µL, 1.0 ×10 -5 M) was combined with FSNFs (10 µL, 10 mg/mL) and 100 µL of hybridization buffer and mixed in a 1.5-mL Eppendorf tube.After continuous mixing at 37 °C for 180 min, excess reagents were removed by centrifugation at 12000 rpm for 10 min. The sediment was repeatedly washed and centrifuged twice to obtain the FSNFs-DNA6 probes. The cytotoxicity of the FSNF-assembled DNA6 probes (FSNF-DNA6) was evaluated in HeLa cells by MTT assay (Figure 2). 33, 38 Briefly, cells were incubated in 200 µL of culture medium containing 15 µL of FSNF-DNA6 probes and washed once with 200 µL of hybridization buffer (pH 7.4) after incubation. MTT (0.5 mg mL-1, 100 µL) was seeded in the wells and incubated at 37 °C for 4 h before adding 150 µL of DMSO to each well to evenly dissolve the crystals produced by the live cells. The samples were then analyzed at 490 nm to measure the relative cell viability. The results revealed that the cell survival rate, as calculated by (Atest/Acontrol)×100%, was 89.2% after incubation with 30 µL of probes for 6 h, indicating that the probes had good biocompatibility.

Figure 2. Viability of HeLa cells (100 µL, 1.0×106 mL-1) after incubation with FSNF-DNA6 probes (20 mg/mL) for different durations. UV-visible spectra of the probes. Au-MPs for telomerase detection were successfully fabricated and characterized (Figure 3A). The average diameter of the Au-MPs was approximately 20 nm (Figure 3B). Unlike the absorption peak of AuNPs (curve a, Figure 3), which was observed at 520 nm, for the Au-MPs (curve c), a characteristic DNA peak was found at 260 nm, indicating that the DNA and AuNPs were successfully conjugated.

Figure 3. (A) UV-visible spectra of AuNPs (a), DNA (b) and Au-MPs (c); and (B) TEM image of Au-MPs. Drug release from the probes. Based on the fluorescence intensity, drug molecules could be successfully released from the prepared probes at room temperature within 3h (Figure 4). The DOX molecules intercalated into the prepared probes, quenching the fluorescence of the AuNPs (curve a).11 The presence of telomerase then elongated the primers, leading to inner chain substitution and DOX molecule release (curve b).

Figure 4. Fluorescence spectra of Au-MPs-DOX without telomerase (a) and with telomerase (b) after extraction of the HeLa cells.



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Fluorescence changes relating to nuclear-shell biopolymer dispersion and liquid supernatants and analysis of the RCA reaction. The changes in the fluorescence of the liquid supernatants were less intense than those of the nuclear-shell biopolymers (Figures 5A and 5B), suggesting that the nuclearshell biopolymers produced by RCA could be flexibly adsorbed on the surface of the nucleus. Fluorescence intensity of nuclear-shell biopolymer dispersion hardly changed in twentyfour hours at room temperature in dark, indicating that nuclear-shell biopolymers have good stability. The RCA reaction activated by telomerase activity was analyzed using polyacrylamide gel electrophoresis with ethidium bromide (EB) staining, as shown in Figure 5C.

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Monitoring capability for telomerase activity. To evaluate the validity of this method, we measured fluorescence changes in response to different concentrations of telomerase from cell extracts under the optimized conditions. The fluorescence intensity exhibited a linear relationship with the logarithm of the telomerase concentration in the range of 5.0×10-11 M to 5.0 ×10-8 M, as shown in Figure 7. The regression equation was F − F0 = 177.8 lgCTE + 1885.1, the correlation coefficient (R2) of the calibration curve was 0.996 (3σ), and the detection limit was calculated to be 1.6 × 10-11 M. The reproducibility of the nuclear-shell biopolymer system was examined by performing 11 replicates of 10-8 M telomerase measurements under optimal conditions. The relative standard deviation (RSD) was 12.1%, indicating that this method has good reproducibility.

Figure 6. (A) Effects of the amount of phi29 DNA polymerase and (B) the incubation time on the fluorescence intensity in response to 5.0 ×10-9 M telomerase.

Figure 5. Fluorescence responses of the nuclear-shell biopolymer dispersion (A) and liquid supernatants (B). F represents the fluorescence intensity of the amplification products in the presence of telomerase, F0 represents the fluorescence intensity in the absence of telomerase, and C represents the telomerase concentration. The telomerase concentrations were as follows: 0 M, 5.0 × 10-11 M, 10-10 M, 5.0 × 10-10 M, 10-9 M, 5.0 × 10-9 M, 10-8 M, and 5.0 × 10-8 M. (C) The RCA reaction was analyzed by 1.0% agarose gel electrophoresis: DNA5 (a), DNA2 (b), the RCA product (c), and the marker (d). Optimization of the reaction conditions. The amount of phi29 DNA polymerase was an important factor that influenced the fluorescence intensity. To improve the sensitivity of fluorescence detection, a series of control experiments were designed to optimize the amount of phi29 DNA polymerase. As shown in Figure 6A, the fluorescence intensity quickly increased as the amount of polymerase was increased from 0.05 to 0.3 UµL-1. However, at concentrations exceeding 0.3 UµL-1, only small changes in the fluorescence intensity were observed. Thus, 0.3-UµL-1 phi29 DNA polymerase was chosen as the optimal amount. To obtain the best detection performance, the incubation time was varied. As illustrated in Figure 6B, the fluorescence intensity rapidly increased as the incubation time was increased and plateaued after 180 min. Thus, the incubation time for the RCA reaction was chosen to be 180 min. Because of the physiological conditions in living cells, 37 ℃ and pH 7.4 were used in subsequent experiments.

Figure 7. Fluorescence spectra in response to varying concentrations of telomerase. The telomerase concentrations of the HeLa cell extracts were as follows: 0 M, 5.0 × 10-11 M, 10-10 M, 5.0 × 10-10 M, 10-9 M, 5.0 × 10-9 M, 10-8 M, and 5.0 × 10-8 M. Inset: The corresponding calibration curve of the fluorescence intensity versus telomerase concentration. Three spectra were acquired via different detections were averaged, and three repetitive experiments were performed. Error bars are the standard deviation of three experiments. The blank was deducted from each value. Cell imaging with nuclear/RCA-biopolymers and drug delivery. The response and efficient delivery of nuclear/RCA-biopolymer nanoassembly in vitro has the potential for the sensitive monitoring of telomerase activity and delivery of drugs to individual cancer cells. For performing the confo-


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cal fluorescence imaging experiments, four types of cells were cultured in 6-well slides. First, the fixed cells were incubated on the slides in culture medium containing Au-MPs-DOX, nuclear-DNA6 probes and Au-circular DNA for 3 h. These species could be rapidly adopted by the cells via targeted adsorption and endocytosis, and as a result, the nanoflowers were internalized in the cells within 2 h.31, 39 Next, the slides containing fixed cells were washed with phosphate-buffered saline (PBS, 0.01 M, pH 7.4) three times and then incubated with 2% formamide. The slides were successively dehydrated in ice-cold ethanol and air-dried. Subsequently, the nuclear/RCA reaction was performed in a humidified incubator at 37 °C for 90 min. The reaction solution contained 6 µL of dNTPs and 3 µL of phi29 DNA polymerase.39, 40 All cell imaging was performed at 37 °C and visualized using a confocal microscope.

could not penetrate the nucleopore. Most of the released DOX entered the nucleus, and some DOX remained in the cytoplasm of the individual cancer cells. The intensity of FAMfluorescence and DOX-fluorescence in individual cell were obtained the maximum at 4 hours, and their ﬂuorescence intensity could keep for 6 h. Three-dimensional confocal imaging further revealed the location of telomerase-activated FAMfluorescence in the cytoplasm and DOX in the cytoplasm and nucleus, as shown in Figure 9. However, these clustered FAM-fluorescence spots were not observed in normal human hepatocytes (L02). The scattered and small DOXfluorescence signal was very rare because the activity levels of telomerase in normal L02 cells were lower than that in MCF-7, HeLa and HepG2 cells. This difference should contribute to differentiating between cancer and normal cells and allow us inhibit the cancer while reducing the amount of toxicity to normal organs. Thus, this new nuclear/RCA method that is initiated by telomere elongation can be used to detect changes in telomerase activity and achieve appropriate drug delivery to individual cancer cells.

Figure 9. Three-dimensional fluorescence image of HeLa cells in which the nuclear-shell biopolymer method initiated by telomere elongation was performed (red: DOX; green: FAM; yellow: co-localization of red and green pixels). Figure 8. Evaluation of the nuclear/RCA-biopolymer method initiated by telomere elongation for telomerase activity monitoring and drug delivery in individual cells: HeLa, HepG2, MCF-7 and normal L02 cells. The intracellular target-telomerase generated primer extension and substitution hybridization by elongating the end of the telomeres. DOX molecules were released from the AuMPs-DOX complex, and the intracellular nuclear/RCA reaction was activated in the presence of circular DNA. As shown in Figure 8, the highlighted and clustered FAM-fluorescence spots in the cytoplasm accumulated, and DOX fluorescence could be clearly observed in the cell cytoplasm and nucleus of individual HepG2, HeLa and MCF-7 cells. Thus, telomerase activity monitoring and drug delivery to individual cancer cells are possible using this nuclear/RCA method that is initiated by telomere elongation. The reflection signal revealed the accumulation of nuclear-shell biopolymers in the cytoplasm because nano-biopolymers with a diameter of 15 nm

CONCLUSION In this study, novel nuclear-shell biopolymers initiated by telomere elongation that could allow for telomerase-triggered RCA to assemble and accumulate signal molecules for selective cancer cell identification and efficient drug delivery to targeted individual cells were presented. This strategy will not only aid in specific cancer cell recognition but also reduce the undesired death of healthy cells that commonly occurs in conventional chemotherapy. This method will provide new opportunities for intracellular biomolecule detection and targeted cancer cell therapy.

ASSOCIATED CONTENT Supporting Information The supporting information is available free of charge on the ACS Publications website.



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Sequences of DNA used in this study (Table S1), Preparation of AuNPs, Fluorescence intensity of cell imaging (Figure S1 and Figure S2).

AUTHOR INFORMATION Corresponding Author

*E-mail: [email protected]. Phone:86 05398766107. Fax:86 0539-8766107. Notes The authors declare no competing financial interest.

ACKNOWLEDGMENTS This work was supported by the National Science Foundation of China (21405071, 21535002, 21227008), Special Funds for the Construction of Taishan Scholars (grand No.tspd20150209), Shandong Provincal Natural Science Foundation (ZR2014BL021), Open Funds of the State Key Laboratory of Electroanalytical Chemistry (SKLEAC201605), PhD Research Foundation of Linyi University (LYDX2015BS013).

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