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One-step, Rapid and Green Synthesis of Multifunctional Gold Nanoparticles for Tumor-Targeted Imaging and Therapy

One-step, Rapid and Green Synthesis of Multifunctional Gold Nanoparticles for Tumor-Targeted... Gold nanoparticles (GNPs) have always been used as doxorubicin (DOX) transport vectors for tumor diagnosis and therapy; however, the synthesis process of these vectors is to prepare GNPs via chemical reduction method firstly, followed by conjugation with DOX or specific peptides, so these meth� ods faced some common problems including multiple steps, high cost, time consuming, complicated preparation, and post-processing. Here, we present a one-step strategy to prepare the DOX-conjugated GNPs on the basis of DOX’s chemical constitution for the first time. Moreover, we prepare a multifunctional GNPs (DRN-GNPs) with a one-step method by the aid of the reductive functional groups possessed by DOX, RGD peptides, and nuclear localization peptides (NLS), which only needs 30 min. The results of scattering images and cell TEM studies indicated that the DRN-GNPs could target the Hela cells’ nucleus. The tumor inhibition rates of DRN-GNPs via tumor and tail vein injection of nude mice were 66.7% and 57.7%, respectively, which were significantly enhanced compared to control groups. One step synthesis of multifunctional GNPs not only saves time, materials, but also it is in line with the development direction of green chemistry, and it would lay the foundation for large-scale applications within the near future. Our results suggested that the fabrication strategy is efficient, and our prepared DRN-GNPs possess good colloidal stability in the physiological system; they are a potentially contrast agent and an efficient DOX transport vector for cervical cancer diagnosis and therapy. Keywords: One-step, Green, Multifunctional, Tumor imaging, Tumor therapy Introduction nanomaterials [18–20], fluorescent nanoparticles [21– Doxorubicin (DOX) is a commonly used anticancer drug 24], and metal nanomaterials [25–30] were developed as which has been widely used in a variety of cancer DOX transport vectors for tumor diagnosis and therapy. chemotherapy, such as blood malignant tumor [1], a Gold nanomaterials have been widely used due to their variety of adenocarcinoma [2–5], soft tissue sarcoma [6], unique chemical and optical properties, low toxicity, and so on. However, long term and high dosages of good biocompatibility, and surface modification of con- DOX will lead to drug resistance [7], nausea [8], hair trol ability [31, 32] among these nanomaterials. So far, loss [9], and acute and chronic toxicity [10], which may there are three kinds of approaches for the conjugation lead to congestive heart failure [11]. Therefore, it is very of DOX onto GNPs. The first is to conjugate DOX onto necessary to develop a drug carrier with good biocom- the surface of GNPs with the aid of hydrazine [25], 1- patibility and high drug loading ability. In recent years, a ethyl-3-[3-dimethylaminopropyl] carbodiimide hydro- lot of nanomaterials, such as quantum dots [12, 13], chi- chloride (EDC) [26], pH-sensitive agent [27], DCC/NHS tosan [14, 15] silicon nanomaterials [16, 17], polymeric system [28]. The second is to incubate GNPs with DOX for at least 24 h [29]. The third is to replace citrate con- jugated on the surface of gold nanoparticles (GNPs) by * Correspondence: cesgf@mail.sysu.edu.cn; dr.yegang@126.com DOX [30]. Although these DOX-conjugated GNPs have School of Chemistry, Sun Yat-Sen University, Guangzhou 510275, People’s been used in tumor therapy, the application is still Republic of China Department of Gastroenterology, the First Affiliated Hospital of Jinan limited due to the lack of sufficient specificity. Recently, University, Guangzhou 510630, People’s Republic of China © The Author(s). 2020 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. Yin et al. Nanoscale Research Letters (2020) 15:29 Page 2 of 15 El-sayed’s[27] group functionalized GNPs with DOX, same as ref. [35]. Doxorubicin was purchased from San- RGD peptides, and nuclear localizing signal (NLS) pep- gon Biotech (Shanghai) Co., Ltd. CCYNLS peptides (the tides. The highlight of their work is that the GNPs enter sequence of amino acid is cysteine-cysteine-tyrosine- tumor cells via receptor-mediated endocytosis of RGD proline-proline-lysine-lysine-lysine-arginine-lysine-val- peptides and then enter nucleus by the aid of NLS pep- ine, CCYPPKKKRKV) were supplied by China Peptides tides, which makes DOX effectively interfere with DNA Co., Ltd (Shanghai, China). The Hela cell line and the synthesis. Unfortunately, it needed at least 120 h by MCF-7 cell line were provided by Guangzhou Youdi coupling peptides or drugs layer by layer on the GNPs, Biological Technology Co. Ltd. Fetal bovine serum (FBS) and each process needed at least 24 h. Similarly, all of and Dulbecco modified Eagle’s medium (DMEM) were the methods mentioned above faced some common purchased from GibcoBRL Life Technologies. The problems such as multiple steps, high cost, complicated Proliferation and Cytotoxicity Assay Kit (MTT) was pur- preparation, and post-processing. So it is necessary to chased from Beijing Dingguo Changsheng Biotechnology develop a simple and rapid method for synthesis of Co. Ltd. multifunctional gold nanomaterials. In this paper, we presented a one-step strategy to pre- Synthesis of DOX-GNPs, NLS-GNPs, DR-GNPs, DN-GNPs, pare the DOX-conjugated GNPs on the basis of DOX’s DRN-GNPs chemical constitution for the first time. Moreover, we For the preparation of the DOX-GNPs, DOX solution presented a convenient synthetic method to prepare a (0.2 mg/mL, 1.0 mL) was added into the chloroauric acid multifunctional DOX transport vector to make DOX solution (0.86 mM, 1.0 mL) under magnetic stirring, work better, which can be used in tumor diagnosis and then NaOH (2 M, 5 μL) was added to adjust pH. For the therapy. The key difference between our work from preparation of NLS-GNPs, CCYNLS peptides solution others is that we use the DOX, RGD peptides, and NLS (0.5 mM, 1.0 mL) was added into the chloroauric acid peptides as reducing reagents and stabilizing reagents to solution (3.44 mM, 1.0 mL) under magnetic stirring, fabricate the GNPs and make all of these three sub- then NaOH (2 M, 20 μL) was added to adjust pH. For stances conjugate on the surface of the GNPs to form the preparation of DR-GNPs, DOX solution (0.2 mg/mL, DRN-GNPs in the meantime. Our previous works [33– 1.0 mL) and RGD peptides solution (1.0 mM, 1.0 mL) 35] had proved that the RGD and other peptides can be was added into the chloroauric acid solution (1.72 mM, used to reduce gold ions to fabricate gold nanomaterials. 2.0 mL) under magnetic stirring, then NaOH (2 M, 15 The N-terminal of the NLS can be modified by using μL) was added to adjust pH. For the preparation of DN- cysteine cysteine tyrosine (CCY) sequence to construct GNPs, DOX solution (0.2 mg/mL, 1.0 mL) and CCYNLS CCYNLS, which can reduce gold ions while still keep peptides solution (0.5 mM, 1.0 mL) was added into the NLS’s targeting effect [36]. We also found that DOX chloroauric acid solution (1.72 mM, 2.0 mL) under mag- possesses two phenolic hydroxyl groups with strong netic stirring, then NaOH (2 M, 20 μL) was added to ad- reducibility under basic condition [37, 38], so it may just pH. For the preparation of DRN-GNPs, solutions of reduce gold ions to form the GNPs too. Moreover, the DOX (0.2 mg/mL, 1.0 mL), CCYNLS peptides (0.5 mM, anti-tumor effect of DOX might not be influenced be- 1.0 mL), and RGD peptides (1.0 mM, 1.0 mL) was added cause it embeds into DNA by the amino group of carbon into the chloroauric acid solution (1.72 mM, 3.0 mL) 7 and the hydroxyl group of carbon 9 [39]. In the mean- under magnetic stirring, then NaOH (2 M, 35 μL) was time, the sulfur, oxygen, and nitrogen on the peptides added to adjust pH. All of the reactions continued for 30 and DOX can be combined with the GNPs to keep the min at 100 °C in a reflux system and the pH value was colloidal stable [33]. The results show that the DRN- 10–12. All the GNPs were purified by centrifugation at GNPs were successfully fabricated and worked well in 55,000 rpm for 30 min (Optima MAX-TL, Beckman tumor imaging, diagnosis, and therapy (Scheme 1). To Coulter). After taking away the supernatant, the GNPs our knowledge, this is the first report for synthesis of were redispersed in ultrapure water. DOX, RGD, and NLS peptides conjugated multifunc- tional GNPs with a one-step method, and their applica- tion in tumor diagnosis and therapy. Characterization The characterizations of the GNPs’ spectra were imple- Materials and Methods mented by using a UV 3150 Spectrophotometer (Shi- Materials madzu, Japan), a Nicolet Avatar FTIR model 330 HAuCl 4H O was purchased from Sinopharm Chemical spectrometer (Thermo, America), and a ESCALab250 X- 4 2 Reagent Co., Ltd, cyclic RGD peptides (the sequence of ray photoelectron spectroscopy (XPS) with the same amino acid is arginine-glycine-aspartate-tyrosine-glu- analytical conditions as ref. [35]. The transmission elec- tamic acid (c(RGDyE))) and sodium hydroxide were the tron microscope (TEM) images of the GNPs were Yin et al. Nanoscale Research Letters (2020) 15:29 Page 3 of 15 Scheme 1 Schematic illustration of the synthesis of DRN-GNPs, the uptake by Hela cells and the tumor diagnosis of DRN-GNPs obtained from a TEM (JEM-2100F, JEOL, Japan) oper- Cytotoxicity Assay of Free DOX and DRN-GNPs ated at an accelerating voltage of 200 kV. The Hela and MCF-7 cells in the exponential phase were added to the wells of 96-well plates (about 5 × 10 cells/ well) and incubated for 24 h, respectively. Then the Dynamic Light Scattering and Zeta-Potential DRN-GNPs of concentrations 0, 20, 40, 60, 80, and 100 Measurements μg/mL (the corresponding DOX’s concentration was 0.0, A Zeta PALS Zeta Potential Analyzer (Brookhaven 7.8, 15.6, 23.4, 31.2, and 39.0 μg/mL) were incubated Instrument Corporation) was used to measure the with each well and incubated for 24 h at 37 °C, respect- dynamic light scatterings (DLS) of the DOX–GNPs, NLS- ively. The control group was set by adding only PBS buf- GNPs, DR-GNPs, DN-GNPs, and DRN-GNPs, which fer solution, and its viability was set as 100%. The MTT worked at 90° angle with a solid-state laser (λ = 670 nm) (20 μL, 5 mg/mL) was added to each well and incubated at room temperature. When equipped with an AQ-827 at 37 °C for about 4 h. Then the culture medium was electrode, the analyzer was used to measure the carefully suck out, and 150 μL of two dimethyl sulfoxide GNPs’ zeta-potentials. was added into per well for 10 min until the crystal was fully dissolved. A microplate reader (Thermo Multiskan ICP Analysis Mk3) was used to measure each well’s OD value at 490 The quantitative analysis of Au in the GNPs was the nm. Three wells for each group were prepared and the same as ref. [35]. calculated values were expressed as mean ± S.D. Flow cytometry experiment The Stability of DR-GNPs, DN-GNPs, and DRN-GNPs The Hela and MCF-7 cells in the exponential phase were Dispersed in PBS, HCl, NaOH, NaCl Solution added to each well of two 12-well plates (about 5 × 10 We mixed 100 μL of the precipitated GNPs and 500 μL cells/well) and incubated for 24 h, respectively. Then, of high concentration of 0.2 M PBS, 0.5 M HCl, 0.5 M the DRN-GNPs solution was added into each of the NaOH, and 1.0 M NaCl solution, respectively. Twelve wells (final concentrations with 0, 20, 40, 60, 80, and 100 hours later, we took their photos and tested their UV μg/mL) and incubated at 37 °C for 24 h, respectively. absorption spectra. Removing the culture medium and adding Trypsin (con- taining EDTA) solution into the wells to digest the cells. Cell Culture Then removing the Trypsin solution from the wells, The Hela and MCF-7 cells were cultured in an incubator adding the culture medium in the wells, and transferring (humidified with 5% CO balanced air) at 37 °C for 24 h. the cells into centrifuge tubes. After centrifugating at The culture medium was DMEM with 10% FBS. The 1000 g for 5 min, the supernatant was removed and the number of live cells are counted by a cell-count board. cells in each tube were collected and counted in PBS Yin et al. Nanoscale Research Letters (2020) 15:29 Page 4 of 15 solution. About 5–10 × 10 of the cells were taken into of the People’s Republic of China (Document NO. 55, another centrifugate tubes and were centrifugated at 2001) and the guidelines for the Care and Use of 1000 g for 5 min again. Removing the supernatant and Laboratory Animals of China Pharmaceutical University. adding 100 μL AnnexinV buffer solution into the tubes. Adding 5 μL of AnnexinV-FITC and 5 μL of propidium Therapeutic Efficacy of Free DOX and Its Conjugates in iodide into the tubes and mixing them with the cells Tumor-Bearing Mice gently. The tubes were incubated at room temperature Briefly, 36 nude mice (aged 5–6 weeks, weighed 16–18 (20–25 °C) for 15 min in the dark and then tested by a g) were divided randomly into six groups. They were flow cytometer (BD Accuri C6). subcutaneously injected of Hela cells (5 × 10 cells in PBS) into the right axillary fossa. Five days later, mice in Optical Dark-Field Scattering Imaging and Transmission the control group were injected by tail vein with saline Electron Microscopy Studies on Cell Culture solution (0.154 mol/L, 0.2 mL). Mice of free DOX, DR- The Hela and MCF-7 cells were implanted onto the 8- GNPs, DN-GNPs, and DRN-GNPs groups were injected hole cell culture slide about 7 × 10 cells for each well, by tail vein too. In order to compare the DRN-GNPs’ respectively. The DRN-GNPs were added into each well anti-tumor efficacy between the intravenous injection with the final concentration of 35 μg/mL. After being and tumor injection, we set a group to be injected with incubated for 24 h, we removed physically and free equal amount of DRN-GNPs on the tumor site. These absorbed GNPs by rinsing these cells with PBS solution five groups maintained a dose of 0.3 mg/kg equivalent for three times. Then, we fixed them with 4% para- free or conjugated DOX in each mouse. Each mouse formaldehyde for 20 min, coated with a few drops of underwent tail vein or tumor injection once every day. glycerol, and sealed these 8-hole cell culture slides with The body weight and the tumor volume of each mouse another coverslips. A dark field microscope (Zeiss was monitored every day over 14 days. To further inves- Imager Z1) was used to assess the cells in × 200 magnifi- tigate the therapeutic effects of DOX, DR-GNPs, DN- cation and reflective mode. The exposure time for GNPs, and DRN-GNPs, the tumors of the six groups bright-field and dark-field images at a voltage of 5 V was were excised for pathological analysis after 14 days of 1 ms and 400 ms, respectively. treatment. Tumors and the internal organs of heart, For TEM studies, Hela and MCF-7 cells were incu- liver, spleen, lung, and kidney of the mice were isolated bated with the DRN-GNPs (35 μg/mL) for 24 h in a cell from the mice, fixed with 10% neutral-buffered formalin, culture flask, respectively. After incubating and sucking and embedded in paraffin. The sliced tumor tissues were out the mixed solution of medium and DRN-GNPs, we stained with Hematoxylin and Eosin (H&E) and exam- rinsed the cells with PBS solution for three times and ined by Olympus optical microscope. then digested them with trypsin. The cells were trans- ferred in a centrifugal tube with a little volume of the Results and Discussion DMEM medium. The cells were centrifugated (1500 Synthesis and Characterization of DOX-GNPs, NLS-GNPs, rpm) for 10 min, the DMEM medium was sucked out DN-GNPs, DR-GNPs, DRN-GNPs carefully, and the 3% glutaraldehyde solution (usually 40 In the synthesis of the DOX-GNPs, when the NaOH so- times the volume of materials) was added carefully into lution was added into the mixed solution of DOX and the tube to fix the cells in the bottom of the tube for chloroauric acid solution, the color of the solution im- more than 1 h. The cells were fixed further by adding mediately changed from orange to shallow purple, it 1% osmium tetroxide for 1 h, dehydrated with ethanol, then became wine red at 100 °C in a reflux system under and then embedded in Spurr (Sigma-Aldrich Co., USA). magnetic stirring in one minute, indicating the forma- The cells were took out from the tube, cut into sections, tion of the DOX-GNPs. The result indicated that the re- stained with aqueous uranyl acetate and lead citrate, and duction ability of DOX is very strong because DOX then mounted on a copper grid (300 mesh). The speci- possesses two phenolic hydroxyl groups (the No. 6 and mens were measured using a FEI TECNAI-12 transmis- No. 11 carbon) with strong reducibility under basic con- sion electron microscope (working voltage 120 kV). dition. The reaction continued for 30 min until the color do not change. Animal Experiments In order to avoid confusing the DOX’s orange red Animal Subjects color and the GNPs’ wine red color, we put forward Athymic female nude mice were purchased from Animal more evidence by charactering their UV absorption Experimental Center of Southern Medical University spectra (as shown in Fig. 1a). DOX has a characteristic (Guangzhou, China) for in vivo tumor therapy test. All absorption peak near 485 nm and a peak at 530 nm; animal experiments were carried out in compliance with however, the peak at 480 nm disappeared in the the Animal Management Rules of the Ministry of Health spectrum of the product’s solution and a characteristic Yin et al. Nanoscale Research Letters (2020) 15:29 Page 5 of 15 Fig. 1 a UV-Visible absorption spectra of DOX and the as-prepared DOX–GNPs, the inset of (a) is the images of DOX (1, 2) and DOX-GNPs (3, 4) before (1, 3) and after centrifugation (2, 4). b TEM image of DOX–GNPs. c FTIR spectra of DOX and the as-prepared DOX–GNPs. d XPS spectrum of DOX–GNPs peak at 520 nm characteristic surface plasmon resonance As shown in Fig. 1c, although the number of the DOX- (SPR) absorption peak of the GNPs arose. Even so, the GNPs’ infrared absorption peak is significantly less than formation of GNPs is still not completely determined that of the DOX, many of the characteristics of the DOX between the 520 nm of the GNPs and 530 nm of the have been still retained, such as the characteristic IR ab- −1 peak of DOX. Taking into account the GNPs will be sorption peaks at 2910 cm (C-H, stretch vibration) −1 precipitated with high-speed centrifugation, and DOX [42], 1631 cm (carbonyl stretch vibration) [43], 1114 −1 −1 molecules will not, we can see purple precipitate in the cm (C-O, C-N stretch vibration) [44], and 867 cm product of the bottom of the centrifuge tube (inset of (N-H, C-H, out-of-plane bending vibration) [45], re- Fig. 1a(3-4)) and DOX do not have either (inset of spectively. Those representative peaks of DOX were also Fig. 1a(1-2)). displayed in the spectra of the DOX-GNPs, indicated DOX has red fluorescence under the irradiation of 365 that the DOX was successfully bind to the DOX-GNPs. −1 nm ultraviolet light; however, the fluorescence of DOX Interestingly, the peak at 1214 cm characteristic peak disappeared after the synthesis of GNPs. There are two for C-O stretch vibration of the phenolic hydroxyl group possible reasons; the first is that the GNPs’ extinction [46] disappeared in the spectra of DOX-GNPs. The coefficient is very strong, they tend to quench the fluor- phenolic group can reduce Au ions into GNPs, and then escence of molecules when they are closely located to it was transported to carbonyl group. We speculate that the surface of the GNPs (< 5 nm) [40]. If the distance in- the effect of DOX is not affected, because it embeds into creases to 20 nm or more, the nanoparticles’ plasmon DNA by the amino group of carbon 7 and the hydroxyl field is too far to quench their fluorescence signal [41]. group of carbon 9 [39]. The other reason is that the fluorescent group of DOX We can observe that the DOX-GNPs’ particle size is was destroyed after playing the role of reduction agent. about 6 nm from the TEM results (Fig. 1b). The calculated Yin et al. Nanoscale Research Letters (2020) 15:29 Page 6 of 15 ratio of Au (I) was 31.93% from the XPS spectrum of the DRN-GNPs; the characteristics of their surface plasmon DOX-GNPs (Fig. 1d), which is high enough to maintain absorption peak was 520–530 nm [33]. One can also see the stability of the colloid. However, when the deposit of from the inset that the GNPs showed a bright red wine DOX-GNPs dispersed in 0.2 M PBS buffer solution with color. TEM images (Additional file 1: Figure S1) show that high-speed centrifugation after removing supernatant, the the particle size of NLS-GNPs and DRN-GNPs was about color of this solution become purple black, and there was 10 nm and 5 nm, and the GNPs are distributed uniformly, visible insoluble floc. It indicated that the DOX-GNPs’ sta- no obvious coagulation was found. bility was not very well in the environment of the high salt In order to validate the synthesis of NLS-GNPs and concentration. It can be explained that DOX possesses DRN-GNPs, we characterized the FTIR spectra of NLS only one amino group but does not possess sulfhydryl peptides and the GNPs in Fig. 2c. Some FTIR peaks of group, which is not enough for DOX to connect the sur- NLS peptides were found in the spectra of NLS-GNPs face of GNPs via Au-S and Au-N bond. So more efforts and DRN-GNPs, such as C-H stretching vibration ab- −1 are needed if DOX-GNPs is used as contrast agent and sorption peak (2840–2972 cm )[47], C=O stretching −1 anti-tumor material for tumor imaging and therapy. vibration absorption peak (1630–1700 cm )[48], and = −1 We synthesized DRN-GNPs with DOX, the RGD pep- C-H in-plane bending vibration peak (1300–1475 cm ) tides, and NLS peptides as reducing and stabilizing agents. [49]; it indicated that the CCYNLS peptides were The RGD peptides can specifically target some tumor cells successfully conjugated on the surface of GNPs. We also overexpressed the α β integrain, and the NLS peptides found that the benzene skeleton vibration peak of v 3 −1 can specifically target the nucleus. Figure 2a shows the tyrosine terminal in the NLS peptide at 1529 cm and UV-Vis spectra of the fabricated DOX-GNPs, NLS-GNPs, the phenolic hydroxyl C-O stretching vibration peak at Fig. 2 a UV-Visible absorption spectra and the photo images of DOX–GNPs (a), NLS-GNPs (b), and DRN-GNPs (c). b UV-Visible absorption spectra of basic DOX, RGD peptides, NLS peptides, and the supernatant of DRN-GNPs. c FTIR spectra of NLS peptides, NLS-GNPs, and DRN-GNPs. The distance between the GNPs’ spectrum and NLS peptides’ spectrum are enlarged for the contrast distinctly Yin et al. Nanoscale Research Letters (2020) 15:29 Page 7 of 15 Fig. 3 XPS spectrum of the Au 4f of the (a) NLS-GNPs, (b) DRN-GNPs. The Au 4f7/2 binding energy of the original spectra (black) and fitted results (red) could be deconvoluted into two components, Au(0)-blue curve and Au(I)-purple curve. UV-Visible absorption spectra and images of DRN-GNPs dispersed in PBS, NaCl, NaOH, and HCl, respectively (c). −1 1193 cm [49] disappeared in the infrared spectra of found in the DRN-GNPs’ supernatant because its color NLS-GNPs and DRN-GNPs, that means the benzene was light brown but the color of DOX under basic con- skeleton of tyrosine terminal was transported into a dition was transparent light purple. It can be concluded quinone structure after playing its role of reduction agent. that the three materials almost react completely. We centrifuge the DRN-GNPs with high speed, col- Similarly, the DR-GNPs and DN-GNPs are also lected the supernatant fluid, which was almost colorless, explained in this way, see Additional file 1: Figure S2. and then tested whether there were left reactants via UV-Vis spectra in Fig. 2b. The UV absorption peaks of RGD and NLS peptides are derived from tyrosine resi- Table 1 The zeta-potential, hydrodynamic size, and polydispersity index of DOX-GNPs, NLS-GNPs, DN-GNPs, DR- dues, the absorption peak was located at 275 nm. How- GNPs, and DRN-GNPs. Data are provided with mean ± S.D. (n = ever, the phenolic hydroxyl group became negative 5) oxygen ions under alkaline conditions, which increases Zeta potential Hydrodynamic size Polydispersity index the electron density of benzene ring, the peak was red DOX-GNPs − 31.5 ± 6.2 46.1 ± 1.9 0.341 ± 0.031 shifted to 292 nm. We did not see the characteristic peak in the UV-Vis spectrum of the DRN-GNPs’ super- NLS-GNPs − 41.8 ± 2.9 88.8 ± 4.1 0.341 ± 0.018 natant, which means that both peptides took part in the DN-GNPs − 33.6 ± 2.5 77.4 ± 3.2 0.352 ± 0.010 reaction. DOX possesses three absorption peaks in the DR-GNPs − 36.7 ± 5.8 17.2 ± 0.4 0.232 ± 0.013 range of 450–600 nm, but the DRN-GNPs’ supernatant DRN-GNPs − 38.4±3.2 61.0 ± 5.3 0.339 ± 0.007 do not have those peaks. Besides that, no DOX was Yin et al. Nanoscale Research Letters (2020) 15:29 Page 8 of 15 We used XPS spectroscopy to examine the valence of 57% and 43%, respectively. Similarly, for the DRN-GNPs, Au for the NLS-GNPs and DRN-GNPs. As shown in it can be deconvoluted into Au (0) (83.6 eV) and Au (I) Fig. 3a, b, two peaks with binding energies of around (84.4 eV), the peak area ratios are 62% and 38%, respect- 83.6 eV and 87.0 eV are consistent with the emission of ively, thus they have a higher proportion of Au (I). The Au 4f7/2 and 4f 5/2 photoelectrons [33]. Their Au 4f7/2 charge can form Au (I)-S complexes, which help to binding energy position of both GNPs is nearly 84.0 eV. maintain the GNPs’ colloidal stability. Interestingly, we For the NLS-GNPs, it can be deconvoluted into Au (0) identified three types of S in the XPS spectra of NLS- (83.5 eV) and Au (I) (84.1 eV), their peak area ratio is GNPs, DN-GNPs, and DRN-GNPs in Additional file 1: Fig. 4 The contents of the rows are listed as follows: “PBS buffer” represents the cells incubate with PBS buffer, “DRN-GNPs” represents the cells incubate with DRN-GNPs. The subscript of “1” represents the bright field images of cells, “2” represents the dark field images of cells, and “3” represents the overlapping images (bright-field and dark-field) of cells. All of the scale bars are 20 μm. The picture of E is a small region chosen from B in Fig. 4, the picture of F is a small region chosen from D in Fig. 4, the scale bars are 10 μm 3 Yin et al. Nanoscale Research Letters (2020) 15:29 Page 9 of 15 Table 2 Comparison of DRN-GNPs taken up by Hela and MCF-7 Stability of DRN-GNPs cells. The ratio of the value of the cells’ densitometric mean Dynamic light scattering (DLS) and zeta potential data (grey) to that of the background in the same image of these five GNPs were displayed in Table 1; their Ratio of mean grey (cell / background) hydrodynamic size is larger than that characterized by Hela (PBS) 1.05 TEM, which can be attributed to a certain amount of hy- drated molecules around the core of the water-soluble Hela (DRN-GNPs) 4.80 GNPs. Their zeta potentials were negative because of the MCF-7 (PBS) 1.12 carboxyl group conjugated on the surface of GNPs. The MCF-7 (DRN-GNPs) 1.01 GNPs solution shows good colloidal stability when the absolute value of zeta potential is greater than 30 mV; Figure S3. The blue and black curves represent the oxida- the greater the absolute value, the better the stability. tion states of sulfur, purple curves represent the S-H bond, One can see that they all possess good colloidal stability and the green curve represents the Au-S bond. The forma- from Table 1. In comparison, the NLS-GNPs’ value of tion of Au-S bond illustrated that the NLS peptides were zeta potential was the highest, that may be because their successfully conjugated on the surface of these three GNPs. surface are full of NLS peptides which can form strong Moreover, the peaks of N 1s, C 1s, and O 1s of XPS Au-S bond via the thiol group. The absolute value of spectra demonstrated that the peptides and DOX were DOX-GNPs’ zeta potential was close to 30 mV; it was attached to GNPs (Additional file 1: Figure S4). consistent with the previously mentioned that the DOX- The XRD spectra (Additional file 1: Figure S5) shown GNPs were not very stable when they were dispersed in that the crystal structure of these GNPs are face- PBS buffer solution. The spectra data were displayed in centered-cubic crystalline structure, because all of their Additional file 1: Figure S6. XRD spectra contained four peaks at 38.2°, 44.5°, 64.7°, The changes of the GNPs’ characteristic surface plas- and 77.8° corresponding to the (111), (200), (220), and mon resonance (SPR) bands can be used to determine the (311) planes [33]. aggregation of GNPs by utilizing UV-Vis spectrometry Fig. 5 TEM images of DRN-GNPs incubated in Hela and MCF-7 cells for 24 h. A ,A , and A are corresponding to HeLa cells while B and B are 1 2 3 1 2 to MCF-7 cells Yin et al. Nanoscale Research Letters (2020) 15:29 Page 10 of 15 [49]. We measured their UV-Vis spectra and take the Plasmonic Dark-Field Scattering Imaging of Tumor Cells photographs of the DRN-GNPs (Fig. 3c) when they and Cell TEM Microscopy were dispersed in high salt (1 M NaCl and 0.2 M PBS), For the specific targeted imaging of tumor cells with strong acid (0.5 M HCl), and strong base (0.5 M DRN-GNPs, we selected the Hela cells as test group and NaOH) solutions. Neither solution color change nor the MCF-7 cells as the control group. The reason is that UV-vis spectra shift was observed under various harsh the Hela cells overexpress the integrin α β [50] but the v 3 conditions except in the HCl solution, in which their MCF-7 cells do not, so it has no specific binding of color changed to slight purple and the ultraviolet RGD; it is a good control group [51]. The uptake of absorption peak red shift about 20 nm, which means DRN-GNPs by these two cell lines after incubation for GNPs coagulated slightly under strong acid condition. 24 h with a final concentration of 35 μg/mL was The reason might be that the negatively charged GNPs assessed by an upright fluorescence microscopy in the colloidal stability was affected under strong acidic con- dark-field model. The cells being treated without GNPs dition, but it was not influenced in the neutral PBS buf- do not show plasmonic light scattering (Fig. 4 A-Hela fer and alkali environment. We can conclude that the cells, 5C-MCF-7 cells). Images of Hela cells being DRN-GNPs are highly stable under high salt and alkali treated with the DRN-GNPs show that scattering signals conditions, indicating that our synthesized DRN-GNPs from the GNPs is heavily localized at the nucleus (Figs. were expected to be used for in vitro cell imaging and 4b and 5e). However, we could hardly see the scattering in vivo anti-tumor therapy. light from the MCF-7 cells after they were incubated Fig. 6 MTT assay was used to qualitatively display in vitro anti-tumor activity of DOX and DRN-GNPs on Hela (a) and MCF-7 cells (b) for 24 h (100%). The concentrations of C -C are 0, 20, 40, 60, 80, 100 μg/mL for DRN-GNPs; the corresponding DOX’s concentrations are 0, 7.8, 15.6, 23.4, 0 5 31.2, 39.0 μg/mL. Data are represented as means ± standard deviations of triplicate samples (n = 3). Images of Hela cells incubated with different concentration of DRN-GNPs (c) Yin et al. Nanoscale Research Letters (2020) 15:29 Page 11 of 15 with the DRN-GNPs, even though little amount of GNPs taken up amounts of DRN-GNPs by these two cell lines. might have entered the tumor cells through the tumor The ratios of the cells incubated with PBS buffer and the cells’ passive enhanced permeation and retention effect MCF-7 cells were nearly 1.0, the brightness of cells was (EPR effect) (Figs. 4d and 5f), the enhanced permeability nearly close to the background. The ratio of Hela cells and retention (EPR) effect is a unique phenomenon of incubated with DRN-GNPs was 4.80, that means the solid tumors related to their anatomical and patho- synthesized DRN-GNPs could be specifically targeted physiological differences from normal tissues. For and entered the tumor cells overexpressed the integrin example, angiogenesis leads to high vascular density in α β . v 3 solid tumors, large gaps exist between endothelial cells To confirm receptor-specific internalization of the in tumor blood vessels, and tumor tissues show selective DRN-GNPs, we investigated the cellular uptake of the extravasation and retention of macromolecular drugs DRN-GNPs by Hela and MCF-7 cells utilizing the TEM [52]. So they might not be clearly detected under the technique (see Fig. 5). There was a large amount of same experimental condition. The results demonstrated DRN-GNPs localized in the nucleus and cytoplasm that the endocytosis of Hela cells via receptor-mediated regions of α β -positive Hela cells (Fig. 5(A -A )). On v 3 1 3 endocytosis was facilitated by the RGD peptides on the the other hand, only a negligible amount of particles was GNPs’ surface. The NLS peptides could also maintain found inside the cytoplasm regions of α β -negative v 3 their activity to specifically targeted nucleus. So our MCF-7 cells (Fig. 5(B -B )), they might be accumulated 1 2 synthesized DRN-GNPs could be a very potential contrast in the MCF-7 cells by means of the passive targeting agent for tumor targeted imaging. EPR effect. We found that there were high contrast Table 2 shows the ratio values between the cells’ black continuous regions in the MCF-7 cells’ nucleus; densitometric mean grey and the background in the they were uranyl acetate, which stained the nucleus, and same image, which has a positive correlation with the they were different from the granular particles in the Fig. 7 Representative two-dimensional contour density plots to determine fractions of live, apoptotic, and necrotic cells, when exposed to DRN- GNPs at different concentrations (0–100 μg/mL) for 24 h, respectively. Cell necrosis and apoptosis were measured by using PI and Annexinv-FITC dyes Yin et al. Nanoscale Research Letters (2020) 15:29 Page 12 of 15 nucleus of Hela cells. The TEM images are consistent the number and state of the Hela and MCF-7 cells were with the dark-field imaging results, indicating that the relatively good, so the concentration of 35 μg/mL was RGD and NLS peptides on the surface of GNPs facili- the suitable concentration for imaging. We observed that tated the DRN-GNPs targeting Hela cells’ nucleus. So when the DRN-GNPs’ concentration was high to 60 μg/ our synthesized DRN-GNPs could be used as a perfect mL (Fig. 6(C )), the Hela cells became necrosis ob- contrast agent for tumor targeted imaging and diagnosis. viously. When the concentration of DRN-GNPs goes high to 100 μg/mL, salient reduction of the number cells Cellular Therapeutic Efficacy Evaluation can be observed and clear cellular morphological change To evaluate the therapeutic efficacy of DOX and DRN- as the characteristic of necrosis was observed. The same GNPs at cell level, MTT assay was firstly carried out to concentration of conjugated DOX still exhibited nearly study cell viability of Hela and MCF-7 cells under the the same anti-tumor efficacy compared with the free treatment of these two samples for 24 h. DRN-GNPs DOX. These results indicated that our synthesized demonstrated almost the same inhibition effect by killing DRN-GNPs could be used not only as a contrast agent nearly 70% of cells at the same DOX’s concentration but also as a good drug delivery system. (39.0 μg/mL, Fig. 6(A, B)). In line with the MTT results, The result detected by the flow cytometry in Fig. 7 also when the concentration of DRN-GNPs was 40 μg/mL, demonstrates the anti-tumor efficacy of DRN-GNPs. Fig. 8 a Tumor images isolated from tumor-bearing mice after treatment of saline, free DOX, DN-GNPs, DR-GNPs, and DRN-GNPs (tumor injection and intravenous injection). b The histological images of tumors of the mice after treated with saline, free DOX, DN-GNPs, DR-GNPs, and DRN- GNPs (tumor injection and intravenous injection). c Body weight, tumor volume (d), tumor inhabitation rate (e), and tumor’s weight (f) of tumor- bearing mice during 14 days treatment; data are represented as means ± standard deviations (n =6) Yin et al. Nanoscale Research Letters (2020) 15:29 Page 13 of 15 Viability of Hela cells when exposed to DRN-GNPs of dif- conjugated DOX was the same as that of the free DOX. ferent concentrations was measured by flow cytometry We observed that the tumor sizes of DRN-GNPs-treated based assays. Two modes of cell death, apoptosis and ne- groups were obviously smaller than that of the saline, crosis, were measured using Annexinv-FITC and propi- DOX, DN-GNPs, and DR-GNPs-treated groups (Fig. 8a). dium iodide (PI) dyes, respectively (Fig. 7). Similar to the The tumor volume and body weight of the mice were results of MTT assay, the data showed that DRN-GNPs monitored every 2 days. Significant variation of tumor vol- induced cell necrosis was dose-dependent. The proportion ume and tumor weight among all of the treated groups is of necrotic cells was 8% without incubating with DRN- shown in Fig. 8d, f; the tumor volumes and tumor weight GNPs; when the concentration was high to 100 μg/mL, of DN-GNPs and DR-GNPs-treated groups were smaller the proportion of necrotic cells was 23.84%; that means than that of the free DOX-treated group, but still larger our synthesized DRN-GNPs could kill Hela cells effi- than that of the DRN-GNPs-treated groups because of the ciently. The difference between the results of MTT and less targeted activity. That might be because DOX mole- flow cytometry can be explained that the difference of cell cules are small, lack targeting, and they have a short half- number and the methods. life in vivo; therefore, they might be cleared out quickly. However, DRN-GNPs possess of strong stability in the Therapy Evaluation of DRN-GNPs in Tumor-Bearing Mice blood system, long half-life, and highly targeted ability, so To further determine whether DRN-GNPs induced a they can be targeted to the tumor site, and then play the combined therapeutic effect on tumor cells in vivo, we anti-tumor effect of DOX. Between the DRN-GNPs intra- evaluated the anti-tumor effect of DRN-GNPs in tumor- venously injected and tumor-injected groups, the anti- bearing mice for long-term intravenously injection of tumor efficacy of the latter group is better than that of the drugs. The saline group and the drugs of free DOX, DR- former group because the drugs do not need a complex GNPs, and DN-GNPs intravenously injected groups were system of blood circulation into the focus. The tumor in- set as the control groups, in which the concentration of hibition rate of DOX is less than 50%, and the rates of all Fig. 9 The histological images of the main organs (heart, liver, spleen, lung, and kidney) of the mice after treated with saline, free DOX, DR-GNPs, DN-GNPs, and DRN-GNPs (tumor injection and intravenous injection) Yin et al. Nanoscale Research Letters (2020) 15:29 Page 14 of 15 of the DOX-conjugated GNPs are larger than 50%; they glutamic acid; DOX: Doxorubicin; DR-GNPs: DOX-RGD-GNPs, DN-GNPs: DOX-NLS-GNPs, DRN-GNPs: DOX-RGD-NLS GNPs; GNPs: Gold nanoparticles can be high to 66.7% and 57.7%, respectively. The HE stain results showed that there was a certain degree of Acknowledgments damage for liver organs in the intravenous injected DRN- This work was supported by the National Natural Science Foundation of China (20875106), Guangdong Natural Science Found Committee GNPs group (Fig. 9). Furthermore, the bio-toxicity of (9151027501000003). The Science and Technology Planning Project of DOX, DR-GNPs, and DN-GNPs on tumor-bearing mice Guangdong Province (2015A010105013). The Science and Technology were also assessed by histological analysis. As shown in Planning Project of Guangzhou City (201607010349). The Fundamental Research Funds for the Central Universities (NO.21617497). Fig. 8b, tumor cell volume of saline group is comparatively larger than that of treatment groups, and tumor cells dem- Authors’ Contributions onstrate with more mitotic figures. All the results showed HQY participated in the design of the experiments, participated in all of the experiments, and drafted the manuscript. FG participated in the design of that the tumor inhibition effect of DRN-GNPs was the the experiments. GS participated in the experiments of Characterization. GY best among the GNPs. Thus, our synthesized DRN-GNPs participated in the experiments related to cell and animals studies. All could be used as a perfect DOX delivery system for tumor authors read and approved the final manuscript. therapy. Funding This work was supported by the National Natural Science Foundation of Conclusion China (20875106), Guangdong Natural Science Found Committee (9151027501000003). The Science and Technology Planning Project of In this study, we have successfully developed a one-step Guangdong Province (2015A010105013). The Science and Technology method to prepare the multifunctional DRN-GNPs in Planning Project of Guangzhou City (201607010349). The Fundamental about half an hour. The GNPs have also been successfully Research Funds for the Central Universities (NO.21617497). applied to the tumor targeted imaging and tumor therapy Availability of Data and Materials both in vitro and in vivo. The success shows that it is pos- All datasets are presented in the main paper or in the additional supporting sible to make a fully use of the reducing and stabilizing files. ability of the DOX, RGD peptides, and CCYNLS peptides, Competing Interests which will make the GNPs’ synthesis process simple and The authors declare that they have no competing interests. efficient. More importantly, the one-step synthesized DRN-GNPs still possess good colloidal stability in the Received: 2 April 2019 Accepted: 15 December 2019 physiological system, and the peptides conjugated on the surface remained the targeted ability and DOX still pos- References sesses its anti-tumor ability. To our knowledge, this is the 1. Lipshultz SE, Colan SD, Gelber RD, Perezatayde AR (1991) Late cardiac effects of doxorubicin therapy for acute lymphoblastic leukemia in first report for synthesis of DOX, RGD and CCYNLS pep- childhood. N Engl J Med 324:808–815 tides conjugated multifunctional GNPs with a one-step 2. 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One-step, Rapid and Green Synthesis of Multifunctional Gold Nanoparticles for Tumor-Targeted Imaging and Therapy

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Springer Journals
Copyright
Copyright © The Author(s). 2020
ISSN
1931-7573
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1556-276X
DOI
10.1186/s11671-019-3232-3
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Abstract

Gold nanoparticles (GNPs) have always been used as doxorubicin (DOX) transport vectors for tumor diagnosis and therapy; however, the synthesis process of these vectors is to prepare GNPs via chemical reduction method firstly, followed by conjugation with DOX or specific peptides, so these meth� ods faced some common problems including multiple steps, high cost, time consuming, complicated preparation, and post-processing. Here, we present a one-step strategy to prepare the DOX-conjugated GNPs on the basis of DOX’s chemical constitution for the first time. Moreover, we prepare a multifunctional GNPs (DRN-GNPs) with a one-step method by the aid of the reductive functional groups possessed by DOX, RGD peptides, and nuclear localization peptides (NLS), which only needs 30 min. The results of scattering images and cell TEM studies indicated that the DRN-GNPs could target the Hela cells’ nucleus. The tumor inhibition rates of DRN-GNPs via tumor and tail vein injection of nude mice were 66.7% and 57.7%, respectively, which were significantly enhanced compared to control groups. One step synthesis of multifunctional GNPs not only saves time, materials, but also it is in line with the development direction of green chemistry, and it would lay the foundation for large-scale applications within the near future. Our results suggested that the fabrication strategy is efficient, and our prepared DRN-GNPs possess good colloidal stability in the physiological system; they are a potentially contrast agent and an efficient DOX transport vector for cervical cancer diagnosis and therapy. Keywords: One-step, Green, Multifunctional, Tumor imaging, Tumor therapy Introduction nanomaterials [18–20], fluorescent nanoparticles [21– Doxorubicin (DOX) is a commonly used anticancer drug 24], and metal nanomaterials [25–30] were developed as which has been widely used in a variety of cancer DOX transport vectors for tumor diagnosis and therapy. chemotherapy, such as blood malignant tumor [1], a Gold nanomaterials have been widely used due to their variety of adenocarcinoma [2–5], soft tissue sarcoma [6], unique chemical and optical properties, low toxicity, and so on. However, long term and high dosages of good biocompatibility, and surface modification of con- DOX will lead to drug resistance [7], nausea [8], hair trol ability [31, 32] among these nanomaterials. So far, loss [9], and acute and chronic toxicity [10], which may there are three kinds of approaches for the conjugation lead to congestive heart failure [11]. Therefore, it is very of DOX onto GNPs. The first is to conjugate DOX onto necessary to develop a drug carrier with good biocom- the surface of GNPs with the aid of hydrazine [25], 1- patibility and high drug loading ability. In recent years, a ethyl-3-[3-dimethylaminopropyl] carbodiimide hydro- lot of nanomaterials, such as quantum dots [12, 13], chi- chloride (EDC) [26], pH-sensitive agent [27], DCC/NHS tosan [14, 15] silicon nanomaterials [16, 17], polymeric system [28]. The second is to incubate GNPs with DOX for at least 24 h [29]. The third is to replace citrate con- jugated on the surface of gold nanoparticles (GNPs) by * Correspondence: cesgf@mail.sysu.edu.cn; dr.yegang@126.com DOX [30]. Although these DOX-conjugated GNPs have School of Chemistry, Sun Yat-Sen University, Guangzhou 510275, People’s been used in tumor therapy, the application is still Republic of China Department of Gastroenterology, the First Affiliated Hospital of Jinan limited due to the lack of sufficient specificity. Recently, University, Guangzhou 510630, People’s Republic of China © The Author(s). 2020 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. Yin et al. Nanoscale Research Letters (2020) 15:29 Page 2 of 15 El-sayed’s[27] group functionalized GNPs with DOX, same as ref. [35]. Doxorubicin was purchased from San- RGD peptides, and nuclear localizing signal (NLS) pep- gon Biotech (Shanghai) Co., Ltd. CCYNLS peptides (the tides. The highlight of their work is that the GNPs enter sequence of amino acid is cysteine-cysteine-tyrosine- tumor cells via receptor-mediated endocytosis of RGD proline-proline-lysine-lysine-lysine-arginine-lysine-val- peptides and then enter nucleus by the aid of NLS pep- ine, CCYPPKKKRKV) were supplied by China Peptides tides, which makes DOX effectively interfere with DNA Co., Ltd (Shanghai, China). The Hela cell line and the synthesis. Unfortunately, it needed at least 120 h by MCF-7 cell line were provided by Guangzhou Youdi coupling peptides or drugs layer by layer on the GNPs, Biological Technology Co. Ltd. Fetal bovine serum (FBS) and each process needed at least 24 h. Similarly, all of and Dulbecco modified Eagle’s medium (DMEM) were the methods mentioned above faced some common purchased from GibcoBRL Life Technologies. The problems such as multiple steps, high cost, complicated Proliferation and Cytotoxicity Assay Kit (MTT) was pur- preparation, and post-processing. So it is necessary to chased from Beijing Dingguo Changsheng Biotechnology develop a simple and rapid method for synthesis of Co. Ltd. multifunctional gold nanomaterials. In this paper, we presented a one-step strategy to pre- Synthesis of DOX-GNPs, NLS-GNPs, DR-GNPs, DN-GNPs, pare the DOX-conjugated GNPs on the basis of DOX’s DRN-GNPs chemical constitution for the first time. Moreover, we For the preparation of the DOX-GNPs, DOX solution presented a convenient synthetic method to prepare a (0.2 mg/mL, 1.0 mL) was added into the chloroauric acid multifunctional DOX transport vector to make DOX solution (0.86 mM, 1.0 mL) under magnetic stirring, work better, which can be used in tumor diagnosis and then NaOH (2 M, 5 μL) was added to adjust pH. For the therapy. The key difference between our work from preparation of NLS-GNPs, CCYNLS peptides solution others is that we use the DOX, RGD peptides, and NLS (0.5 mM, 1.0 mL) was added into the chloroauric acid peptides as reducing reagents and stabilizing reagents to solution (3.44 mM, 1.0 mL) under magnetic stirring, fabricate the GNPs and make all of these three sub- then NaOH (2 M, 20 μL) was added to adjust pH. For stances conjugate on the surface of the GNPs to form the preparation of DR-GNPs, DOX solution (0.2 mg/mL, DRN-GNPs in the meantime. Our previous works [33– 1.0 mL) and RGD peptides solution (1.0 mM, 1.0 mL) 35] had proved that the RGD and other peptides can be was added into the chloroauric acid solution (1.72 mM, used to reduce gold ions to fabricate gold nanomaterials. 2.0 mL) under magnetic stirring, then NaOH (2 M, 15 The N-terminal of the NLS can be modified by using μL) was added to adjust pH. For the preparation of DN- cysteine cysteine tyrosine (CCY) sequence to construct GNPs, DOX solution (0.2 mg/mL, 1.0 mL) and CCYNLS CCYNLS, which can reduce gold ions while still keep peptides solution (0.5 mM, 1.0 mL) was added into the NLS’s targeting effect [36]. We also found that DOX chloroauric acid solution (1.72 mM, 2.0 mL) under mag- possesses two phenolic hydroxyl groups with strong netic stirring, then NaOH (2 M, 20 μL) was added to ad- reducibility under basic condition [37, 38], so it may just pH. For the preparation of DRN-GNPs, solutions of reduce gold ions to form the GNPs too. Moreover, the DOX (0.2 mg/mL, 1.0 mL), CCYNLS peptides (0.5 mM, anti-tumor effect of DOX might not be influenced be- 1.0 mL), and RGD peptides (1.0 mM, 1.0 mL) was added cause it embeds into DNA by the amino group of carbon into the chloroauric acid solution (1.72 mM, 3.0 mL) 7 and the hydroxyl group of carbon 9 [39]. In the mean- under magnetic stirring, then NaOH (2 M, 35 μL) was time, the sulfur, oxygen, and nitrogen on the peptides added to adjust pH. All of the reactions continued for 30 and DOX can be combined with the GNPs to keep the min at 100 °C in a reflux system and the pH value was colloidal stable [33]. The results show that the DRN- 10–12. All the GNPs were purified by centrifugation at GNPs were successfully fabricated and worked well in 55,000 rpm for 30 min (Optima MAX-TL, Beckman tumor imaging, diagnosis, and therapy (Scheme 1). To Coulter). After taking away the supernatant, the GNPs our knowledge, this is the first report for synthesis of were redispersed in ultrapure water. DOX, RGD, and NLS peptides conjugated multifunc- tional GNPs with a one-step method, and their applica- tion in tumor diagnosis and therapy. Characterization The characterizations of the GNPs’ spectra were imple- Materials and Methods mented by using a UV 3150 Spectrophotometer (Shi- Materials madzu, Japan), a Nicolet Avatar FTIR model 330 HAuCl 4H O was purchased from Sinopharm Chemical spectrometer (Thermo, America), and a ESCALab250 X- 4 2 Reagent Co., Ltd, cyclic RGD peptides (the sequence of ray photoelectron spectroscopy (XPS) with the same amino acid is arginine-glycine-aspartate-tyrosine-glu- analytical conditions as ref. [35]. The transmission elec- tamic acid (c(RGDyE))) and sodium hydroxide were the tron microscope (TEM) images of the GNPs were Yin et al. Nanoscale Research Letters (2020) 15:29 Page 3 of 15 Scheme 1 Schematic illustration of the synthesis of DRN-GNPs, the uptake by Hela cells and the tumor diagnosis of DRN-GNPs obtained from a TEM (JEM-2100F, JEOL, Japan) oper- Cytotoxicity Assay of Free DOX and DRN-GNPs ated at an accelerating voltage of 200 kV. The Hela and MCF-7 cells in the exponential phase were added to the wells of 96-well plates (about 5 × 10 cells/ well) and incubated for 24 h, respectively. Then the Dynamic Light Scattering and Zeta-Potential DRN-GNPs of concentrations 0, 20, 40, 60, 80, and 100 Measurements μg/mL (the corresponding DOX’s concentration was 0.0, A Zeta PALS Zeta Potential Analyzer (Brookhaven 7.8, 15.6, 23.4, 31.2, and 39.0 μg/mL) were incubated Instrument Corporation) was used to measure the with each well and incubated for 24 h at 37 °C, respect- dynamic light scatterings (DLS) of the DOX–GNPs, NLS- ively. The control group was set by adding only PBS buf- GNPs, DR-GNPs, DN-GNPs, and DRN-GNPs, which fer solution, and its viability was set as 100%. The MTT worked at 90° angle with a solid-state laser (λ = 670 nm) (20 μL, 5 mg/mL) was added to each well and incubated at room temperature. When equipped with an AQ-827 at 37 °C for about 4 h. Then the culture medium was electrode, the analyzer was used to measure the carefully suck out, and 150 μL of two dimethyl sulfoxide GNPs’ zeta-potentials. was added into per well for 10 min until the crystal was fully dissolved. A microplate reader (Thermo Multiskan ICP Analysis Mk3) was used to measure each well’s OD value at 490 The quantitative analysis of Au in the GNPs was the nm. Three wells for each group were prepared and the same as ref. [35]. calculated values were expressed as mean ± S.D. Flow cytometry experiment The Stability of DR-GNPs, DN-GNPs, and DRN-GNPs The Hela and MCF-7 cells in the exponential phase were Dispersed in PBS, HCl, NaOH, NaCl Solution added to each well of two 12-well plates (about 5 × 10 We mixed 100 μL of the precipitated GNPs and 500 μL cells/well) and incubated for 24 h, respectively. Then, of high concentration of 0.2 M PBS, 0.5 M HCl, 0.5 M the DRN-GNPs solution was added into each of the NaOH, and 1.0 M NaCl solution, respectively. Twelve wells (final concentrations with 0, 20, 40, 60, 80, and 100 hours later, we took their photos and tested their UV μg/mL) and incubated at 37 °C for 24 h, respectively. absorption spectra. Removing the culture medium and adding Trypsin (con- taining EDTA) solution into the wells to digest the cells. Cell Culture Then removing the Trypsin solution from the wells, The Hela and MCF-7 cells were cultured in an incubator adding the culture medium in the wells, and transferring (humidified with 5% CO balanced air) at 37 °C for 24 h. the cells into centrifuge tubes. After centrifugating at The culture medium was DMEM with 10% FBS. The 1000 g for 5 min, the supernatant was removed and the number of live cells are counted by a cell-count board. cells in each tube were collected and counted in PBS Yin et al. Nanoscale Research Letters (2020) 15:29 Page 4 of 15 solution. About 5–10 × 10 of the cells were taken into of the People’s Republic of China (Document NO. 55, another centrifugate tubes and were centrifugated at 2001) and the guidelines for the Care and Use of 1000 g for 5 min again. Removing the supernatant and Laboratory Animals of China Pharmaceutical University. adding 100 μL AnnexinV buffer solution into the tubes. Adding 5 μL of AnnexinV-FITC and 5 μL of propidium Therapeutic Efficacy of Free DOX and Its Conjugates in iodide into the tubes and mixing them with the cells Tumor-Bearing Mice gently. The tubes were incubated at room temperature Briefly, 36 nude mice (aged 5–6 weeks, weighed 16–18 (20–25 °C) for 15 min in the dark and then tested by a g) were divided randomly into six groups. They were flow cytometer (BD Accuri C6). subcutaneously injected of Hela cells (5 × 10 cells in PBS) into the right axillary fossa. Five days later, mice in Optical Dark-Field Scattering Imaging and Transmission the control group were injected by tail vein with saline Electron Microscopy Studies on Cell Culture solution (0.154 mol/L, 0.2 mL). Mice of free DOX, DR- The Hela and MCF-7 cells were implanted onto the 8- GNPs, DN-GNPs, and DRN-GNPs groups were injected hole cell culture slide about 7 × 10 cells for each well, by tail vein too. In order to compare the DRN-GNPs’ respectively. The DRN-GNPs were added into each well anti-tumor efficacy between the intravenous injection with the final concentration of 35 μg/mL. After being and tumor injection, we set a group to be injected with incubated for 24 h, we removed physically and free equal amount of DRN-GNPs on the tumor site. These absorbed GNPs by rinsing these cells with PBS solution five groups maintained a dose of 0.3 mg/kg equivalent for three times. Then, we fixed them with 4% para- free or conjugated DOX in each mouse. Each mouse formaldehyde for 20 min, coated with a few drops of underwent tail vein or tumor injection once every day. glycerol, and sealed these 8-hole cell culture slides with The body weight and the tumor volume of each mouse another coverslips. A dark field microscope (Zeiss was monitored every day over 14 days. To further inves- Imager Z1) was used to assess the cells in × 200 magnifi- tigate the therapeutic effects of DOX, DR-GNPs, DN- cation and reflective mode. The exposure time for GNPs, and DRN-GNPs, the tumors of the six groups bright-field and dark-field images at a voltage of 5 V was were excised for pathological analysis after 14 days of 1 ms and 400 ms, respectively. treatment. Tumors and the internal organs of heart, For TEM studies, Hela and MCF-7 cells were incu- liver, spleen, lung, and kidney of the mice were isolated bated with the DRN-GNPs (35 μg/mL) for 24 h in a cell from the mice, fixed with 10% neutral-buffered formalin, culture flask, respectively. After incubating and sucking and embedded in paraffin. The sliced tumor tissues were out the mixed solution of medium and DRN-GNPs, we stained with Hematoxylin and Eosin (H&E) and exam- rinsed the cells with PBS solution for three times and ined by Olympus optical microscope. then digested them with trypsin. The cells were trans- ferred in a centrifugal tube with a little volume of the Results and Discussion DMEM medium. The cells were centrifugated (1500 Synthesis and Characterization of DOX-GNPs, NLS-GNPs, rpm) for 10 min, the DMEM medium was sucked out DN-GNPs, DR-GNPs, DRN-GNPs carefully, and the 3% glutaraldehyde solution (usually 40 In the synthesis of the DOX-GNPs, when the NaOH so- times the volume of materials) was added carefully into lution was added into the mixed solution of DOX and the tube to fix the cells in the bottom of the tube for chloroauric acid solution, the color of the solution im- more than 1 h. The cells were fixed further by adding mediately changed from orange to shallow purple, it 1% osmium tetroxide for 1 h, dehydrated with ethanol, then became wine red at 100 °C in a reflux system under and then embedded in Spurr (Sigma-Aldrich Co., USA). magnetic stirring in one minute, indicating the forma- The cells were took out from the tube, cut into sections, tion of the DOX-GNPs. The result indicated that the re- stained with aqueous uranyl acetate and lead citrate, and duction ability of DOX is very strong because DOX then mounted on a copper grid (300 mesh). The speci- possesses two phenolic hydroxyl groups (the No. 6 and mens were measured using a FEI TECNAI-12 transmis- No. 11 carbon) with strong reducibility under basic con- sion electron microscope (working voltage 120 kV). dition. The reaction continued for 30 min until the color do not change. Animal Experiments In order to avoid confusing the DOX’s orange red Animal Subjects color and the GNPs’ wine red color, we put forward Athymic female nude mice were purchased from Animal more evidence by charactering their UV absorption Experimental Center of Southern Medical University spectra (as shown in Fig. 1a). DOX has a characteristic (Guangzhou, China) for in vivo tumor therapy test. All absorption peak near 485 nm and a peak at 530 nm; animal experiments were carried out in compliance with however, the peak at 480 nm disappeared in the the Animal Management Rules of the Ministry of Health spectrum of the product’s solution and a characteristic Yin et al. Nanoscale Research Letters (2020) 15:29 Page 5 of 15 Fig. 1 a UV-Visible absorption spectra of DOX and the as-prepared DOX–GNPs, the inset of (a) is the images of DOX (1, 2) and DOX-GNPs (3, 4) before (1, 3) and after centrifugation (2, 4). b TEM image of DOX–GNPs. c FTIR spectra of DOX and the as-prepared DOX–GNPs. d XPS spectrum of DOX–GNPs peak at 520 nm characteristic surface plasmon resonance As shown in Fig. 1c, although the number of the DOX- (SPR) absorption peak of the GNPs arose. Even so, the GNPs’ infrared absorption peak is significantly less than formation of GNPs is still not completely determined that of the DOX, many of the characteristics of the DOX between the 520 nm of the GNPs and 530 nm of the have been still retained, such as the characteristic IR ab- −1 peak of DOX. Taking into account the GNPs will be sorption peaks at 2910 cm (C-H, stretch vibration) −1 precipitated with high-speed centrifugation, and DOX [42], 1631 cm (carbonyl stretch vibration) [43], 1114 −1 −1 molecules will not, we can see purple precipitate in the cm (C-O, C-N stretch vibration) [44], and 867 cm product of the bottom of the centrifuge tube (inset of (N-H, C-H, out-of-plane bending vibration) [45], re- Fig. 1a(3-4)) and DOX do not have either (inset of spectively. Those representative peaks of DOX were also Fig. 1a(1-2)). displayed in the spectra of the DOX-GNPs, indicated DOX has red fluorescence under the irradiation of 365 that the DOX was successfully bind to the DOX-GNPs. −1 nm ultraviolet light; however, the fluorescence of DOX Interestingly, the peak at 1214 cm characteristic peak disappeared after the synthesis of GNPs. There are two for C-O stretch vibration of the phenolic hydroxyl group possible reasons; the first is that the GNPs’ extinction [46] disappeared in the spectra of DOX-GNPs. The coefficient is very strong, they tend to quench the fluor- phenolic group can reduce Au ions into GNPs, and then escence of molecules when they are closely located to it was transported to carbonyl group. We speculate that the surface of the GNPs (< 5 nm) [40]. If the distance in- the effect of DOX is not affected, because it embeds into creases to 20 nm or more, the nanoparticles’ plasmon DNA by the amino group of carbon 7 and the hydroxyl field is too far to quench their fluorescence signal [41]. group of carbon 9 [39]. The other reason is that the fluorescent group of DOX We can observe that the DOX-GNPs’ particle size is was destroyed after playing the role of reduction agent. about 6 nm from the TEM results (Fig. 1b). The calculated Yin et al. Nanoscale Research Letters (2020) 15:29 Page 6 of 15 ratio of Au (I) was 31.93% from the XPS spectrum of the DRN-GNPs; the characteristics of their surface plasmon DOX-GNPs (Fig. 1d), which is high enough to maintain absorption peak was 520–530 nm [33]. One can also see the stability of the colloid. However, when the deposit of from the inset that the GNPs showed a bright red wine DOX-GNPs dispersed in 0.2 M PBS buffer solution with color. TEM images (Additional file 1: Figure S1) show that high-speed centrifugation after removing supernatant, the the particle size of NLS-GNPs and DRN-GNPs was about color of this solution become purple black, and there was 10 nm and 5 nm, and the GNPs are distributed uniformly, visible insoluble floc. It indicated that the DOX-GNPs’ sta- no obvious coagulation was found. bility was not very well in the environment of the high salt In order to validate the synthesis of NLS-GNPs and concentration. It can be explained that DOX possesses DRN-GNPs, we characterized the FTIR spectra of NLS only one amino group but does not possess sulfhydryl peptides and the GNPs in Fig. 2c. Some FTIR peaks of group, which is not enough for DOX to connect the sur- NLS peptides were found in the spectra of NLS-GNPs face of GNPs via Au-S and Au-N bond. So more efforts and DRN-GNPs, such as C-H stretching vibration ab- −1 are needed if DOX-GNPs is used as contrast agent and sorption peak (2840–2972 cm )[47], C=O stretching −1 anti-tumor material for tumor imaging and therapy. vibration absorption peak (1630–1700 cm )[48], and = −1 We synthesized DRN-GNPs with DOX, the RGD pep- C-H in-plane bending vibration peak (1300–1475 cm ) tides, and NLS peptides as reducing and stabilizing agents. [49]; it indicated that the CCYNLS peptides were The RGD peptides can specifically target some tumor cells successfully conjugated on the surface of GNPs. We also overexpressed the α β integrain, and the NLS peptides found that the benzene skeleton vibration peak of v 3 −1 can specifically target the nucleus. Figure 2a shows the tyrosine terminal in the NLS peptide at 1529 cm and UV-Vis spectra of the fabricated DOX-GNPs, NLS-GNPs, the phenolic hydroxyl C-O stretching vibration peak at Fig. 2 a UV-Visible absorption spectra and the photo images of DOX–GNPs (a), NLS-GNPs (b), and DRN-GNPs (c). b UV-Visible absorption spectra of basic DOX, RGD peptides, NLS peptides, and the supernatant of DRN-GNPs. c FTIR spectra of NLS peptides, NLS-GNPs, and DRN-GNPs. The distance between the GNPs’ spectrum and NLS peptides’ spectrum are enlarged for the contrast distinctly Yin et al. Nanoscale Research Letters (2020) 15:29 Page 7 of 15 Fig. 3 XPS spectrum of the Au 4f of the (a) NLS-GNPs, (b) DRN-GNPs. The Au 4f7/2 binding energy of the original spectra (black) and fitted results (red) could be deconvoluted into two components, Au(0)-blue curve and Au(I)-purple curve. UV-Visible absorption spectra and images of DRN-GNPs dispersed in PBS, NaCl, NaOH, and HCl, respectively (c). −1 1193 cm [49] disappeared in the infrared spectra of found in the DRN-GNPs’ supernatant because its color NLS-GNPs and DRN-GNPs, that means the benzene was light brown but the color of DOX under basic con- skeleton of tyrosine terminal was transported into a dition was transparent light purple. It can be concluded quinone structure after playing its role of reduction agent. that the three materials almost react completely. We centrifuge the DRN-GNPs with high speed, col- Similarly, the DR-GNPs and DN-GNPs are also lected the supernatant fluid, which was almost colorless, explained in this way, see Additional file 1: Figure S2. and then tested whether there were left reactants via UV-Vis spectra in Fig. 2b. The UV absorption peaks of RGD and NLS peptides are derived from tyrosine resi- Table 1 The zeta-potential, hydrodynamic size, and polydispersity index of DOX-GNPs, NLS-GNPs, DN-GNPs, DR- dues, the absorption peak was located at 275 nm. How- GNPs, and DRN-GNPs. Data are provided with mean ± S.D. (n = ever, the phenolic hydroxyl group became negative 5) oxygen ions under alkaline conditions, which increases Zeta potential Hydrodynamic size Polydispersity index the electron density of benzene ring, the peak was red DOX-GNPs − 31.5 ± 6.2 46.1 ± 1.9 0.341 ± 0.031 shifted to 292 nm. We did not see the characteristic peak in the UV-Vis spectrum of the DRN-GNPs’ super- NLS-GNPs − 41.8 ± 2.9 88.8 ± 4.1 0.341 ± 0.018 natant, which means that both peptides took part in the DN-GNPs − 33.6 ± 2.5 77.4 ± 3.2 0.352 ± 0.010 reaction. DOX possesses three absorption peaks in the DR-GNPs − 36.7 ± 5.8 17.2 ± 0.4 0.232 ± 0.013 range of 450–600 nm, but the DRN-GNPs’ supernatant DRN-GNPs − 38.4±3.2 61.0 ± 5.3 0.339 ± 0.007 do not have those peaks. Besides that, no DOX was Yin et al. Nanoscale Research Letters (2020) 15:29 Page 8 of 15 We used XPS spectroscopy to examine the valence of 57% and 43%, respectively. Similarly, for the DRN-GNPs, Au for the NLS-GNPs and DRN-GNPs. As shown in it can be deconvoluted into Au (0) (83.6 eV) and Au (I) Fig. 3a, b, two peaks with binding energies of around (84.4 eV), the peak area ratios are 62% and 38%, respect- 83.6 eV and 87.0 eV are consistent with the emission of ively, thus they have a higher proportion of Au (I). The Au 4f7/2 and 4f 5/2 photoelectrons [33]. Their Au 4f7/2 charge can form Au (I)-S complexes, which help to binding energy position of both GNPs is nearly 84.0 eV. maintain the GNPs’ colloidal stability. Interestingly, we For the NLS-GNPs, it can be deconvoluted into Au (0) identified three types of S in the XPS spectra of NLS- (83.5 eV) and Au (I) (84.1 eV), their peak area ratio is GNPs, DN-GNPs, and DRN-GNPs in Additional file 1: Fig. 4 The contents of the rows are listed as follows: “PBS buffer” represents the cells incubate with PBS buffer, “DRN-GNPs” represents the cells incubate with DRN-GNPs. The subscript of “1” represents the bright field images of cells, “2” represents the dark field images of cells, and “3” represents the overlapping images (bright-field and dark-field) of cells. All of the scale bars are 20 μm. The picture of E is a small region chosen from B in Fig. 4, the picture of F is a small region chosen from D in Fig. 4, the scale bars are 10 μm 3 Yin et al. Nanoscale Research Letters (2020) 15:29 Page 9 of 15 Table 2 Comparison of DRN-GNPs taken up by Hela and MCF-7 Stability of DRN-GNPs cells. The ratio of the value of the cells’ densitometric mean Dynamic light scattering (DLS) and zeta potential data (grey) to that of the background in the same image of these five GNPs were displayed in Table 1; their Ratio of mean grey (cell / background) hydrodynamic size is larger than that characterized by Hela (PBS) 1.05 TEM, which can be attributed to a certain amount of hy- drated molecules around the core of the water-soluble Hela (DRN-GNPs) 4.80 GNPs. Their zeta potentials were negative because of the MCF-7 (PBS) 1.12 carboxyl group conjugated on the surface of GNPs. The MCF-7 (DRN-GNPs) 1.01 GNPs solution shows good colloidal stability when the absolute value of zeta potential is greater than 30 mV; Figure S3. The blue and black curves represent the oxida- the greater the absolute value, the better the stability. tion states of sulfur, purple curves represent the S-H bond, One can see that they all possess good colloidal stability and the green curve represents the Au-S bond. The forma- from Table 1. In comparison, the NLS-GNPs’ value of tion of Au-S bond illustrated that the NLS peptides were zeta potential was the highest, that may be because their successfully conjugated on the surface of these three GNPs. surface are full of NLS peptides which can form strong Moreover, the peaks of N 1s, C 1s, and O 1s of XPS Au-S bond via the thiol group. The absolute value of spectra demonstrated that the peptides and DOX were DOX-GNPs’ zeta potential was close to 30 mV; it was attached to GNPs (Additional file 1: Figure S4). consistent with the previously mentioned that the DOX- The XRD spectra (Additional file 1: Figure S5) shown GNPs were not very stable when they were dispersed in that the crystal structure of these GNPs are face- PBS buffer solution. The spectra data were displayed in centered-cubic crystalline structure, because all of their Additional file 1: Figure S6. XRD spectra contained four peaks at 38.2°, 44.5°, 64.7°, The changes of the GNPs’ characteristic surface plas- and 77.8° corresponding to the (111), (200), (220), and mon resonance (SPR) bands can be used to determine the (311) planes [33]. aggregation of GNPs by utilizing UV-Vis spectrometry Fig. 5 TEM images of DRN-GNPs incubated in Hela and MCF-7 cells for 24 h. A ,A , and A are corresponding to HeLa cells while B and B are 1 2 3 1 2 to MCF-7 cells Yin et al. Nanoscale Research Letters (2020) 15:29 Page 10 of 15 [49]. We measured their UV-Vis spectra and take the Plasmonic Dark-Field Scattering Imaging of Tumor Cells photographs of the DRN-GNPs (Fig. 3c) when they and Cell TEM Microscopy were dispersed in high salt (1 M NaCl and 0.2 M PBS), For the specific targeted imaging of tumor cells with strong acid (0.5 M HCl), and strong base (0.5 M DRN-GNPs, we selected the Hela cells as test group and NaOH) solutions. Neither solution color change nor the MCF-7 cells as the control group. The reason is that UV-vis spectra shift was observed under various harsh the Hela cells overexpress the integrin α β [50] but the v 3 conditions except in the HCl solution, in which their MCF-7 cells do not, so it has no specific binding of color changed to slight purple and the ultraviolet RGD; it is a good control group [51]. The uptake of absorption peak red shift about 20 nm, which means DRN-GNPs by these two cell lines after incubation for GNPs coagulated slightly under strong acid condition. 24 h with a final concentration of 35 μg/mL was The reason might be that the negatively charged GNPs assessed by an upright fluorescence microscopy in the colloidal stability was affected under strong acidic con- dark-field model. The cells being treated without GNPs dition, but it was not influenced in the neutral PBS buf- do not show plasmonic light scattering (Fig. 4 A-Hela fer and alkali environment. We can conclude that the cells, 5C-MCF-7 cells). Images of Hela cells being DRN-GNPs are highly stable under high salt and alkali treated with the DRN-GNPs show that scattering signals conditions, indicating that our synthesized DRN-GNPs from the GNPs is heavily localized at the nucleus (Figs. were expected to be used for in vitro cell imaging and 4b and 5e). However, we could hardly see the scattering in vivo anti-tumor therapy. light from the MCF-7 cells after they were incubated Fig. 6 MTT assay was used to qualitatively display in vitro anti-tumor activity of DOX and DRN-GNPs on Hela (a) and MCF-7 cells (b) for 24 h (100%). The concentrations of C -C are 0, 20, 40, 60, 80, 100 μg/mL for DRN-GNPs; the corresponding DOX’s concentrations are 0, 7.8, 15.6, 23.4, 0 5 31.2, 39.0 μg/mL. Data are represented as means ± standard deviations of triplicate samples (n = 3). Images of Hela cells incubated with different concentration of DRN-GNPs (c) Yin et al. Nanoscale Research Letters (2020) 15:29 Page 11 of 15 with the DRN-GNPs, even though little amount of GNPs taken up amounts of DRN-GNPs by these two cell lines. might have entered the tumor cells through the tumor The ratios of the cells incubated with PBS buffer and the cells’ passive enhanced permeation and retention effect MCF-7 cells were nearly 1.0, the brightness of cells was (EPR effect) (Figs. 4d and 5f), the enhanced permeability nearly close to the background. The ratio of Hela cells and retention (EPR) effect is a unique phenomenon of incubated with DRN-GNPs was 4.80, that means the solid tumors related to their anatomical and patho- synthesized DRN-GNPs could be specifically targeted physiological differences from normal tissues. For and entered the tumor cells overexpressed the integrin example, angiogenesis leads to high vascular density in α β . v 3 solid tumors, large gaps exist between endothelial cells To confirm receptor-specific internalization of the in tumor blood vessels, and tumor tissues show selective DRN-GNPs, we investigated the cellular uptake of the extravasation and retention of macromolecular drugs DRN-GNPs by Hela and MCF-7 cells utilizing the TEM [52]. So they might not be clearly detected under the technique (see Fig. 5). There was a large amount of same experimental condition. The results demonstrated DRN-GNPs localized in the nucleus and cytoplasm that the endocytosis of Hela cells via receptor-mediated regions of α β -positive Hela cells (Fig. 5(A -A )). On v 3 1 3 endocytosis was facilitated by the RGD peptides on the the other hand, only a negligible amount of particles was GNPs’ surface. The NLS peptides could also maintain found inside the cytoplasm regions of α β -negative v 3 their activity to specifically targeted nucleus. So our MCF-7 cells (Fig. 5(B -B )), they might be accumulated 1 2 synthesized DRN-GNPs could be a very potential contrast in the MCF-7 cells by means of the passive targeting agent for tumor targeted imaging. EPR effect. We found that there were high contrast Table 2 shows the ratio values between the cells’ black continuous regions in the MCF-7 cells’ nucleus; densitometric mean grey and the background in the they were uranyl acetate, which stained the nucleus, and same image, which has a positive correlation with the they were different from the granular particles in the Fig. 7 Representative two-dimensional contour density plots to determine fractions of live, apoptotic, and necrotic cells, when exposed to DRN- GNPs at different concentrations (0–100 μg/mL) for 24 h, respectively. Cell necrosis and apoptosis were measured by using PI and Annexinv-FITC dyes Yin et al. Nanoscale Research Letters (2020) 15:29 Page 12 of 15 nucleus of Hela cells. The TEM images are consistent the number and state of the Hela and MCF-7 cells were with the dark-field imaging results, indicating that the relatively good, so the concentration of 35 μg/mL was RGD and NLS peptides on the surface of GNPs facili- the suitable concentration for imaging. We observed that tated the DRN-GNPs targeting Hela cells’ nucleus. So when the DRN-GNPs’ concentration was high to 60 μg/ our synthesized DRN-GNPs could be used as a perfect mL (Fig. 6(C )), the Hela cells became necrosis ob- contrast agent for tumor targeted imaging and diagnosis. viously. When the concentration of DRN-GNPs goes high to 100 μg/mL, salient reduction of the number cells Cellular Therapeutic Efficacy Evaluation can be observed and clear cellular morphological change To evaluate the therapeutic efficacy of DOX and DRN- as the characteristic of necrosis was observed. The same GNPs at cell level, MTT assay was firstly carried out to concentration of conjugated DOX still exhibited nearly study cell viability of Hela and MCF-7 cells under the the same anti-tumor efficacy compared with the free treatment of these two samples for 24 h. DRN-GNPs DOX. These results indicated that our synthesized demonstrated almost the same inhibition effect by killing DRN-GNPs could be used not only as a contrast agent nearly 70% of cells at the same DOX’s concentration but also as a good drug delivery system. (39.0 μg/mL, Fig. 6(A, B)). In line with the MTT results, The result detected by the flow cytometry in Fig. 7 also when the concentration of DRN-GNPs was 40 μg/mL, demonstrates the anti-tumor efficacy of DRN-GNPs. Fig. 8 a Tumor images isolated from tumor-bearing mice after treatment of saline, free DOX, DN-GNPs, DR-GNPs, and DRN-GNPs (tumor injection and intravenous injection). b The histological images of tumors of the mice after treated with saline, free DOX, DN-GNPs, DR-GNPs, and DRN- GNPs (tumor injection and intravenous injection). c Body weight, tumor volume (d), tumor inhabitation rate (e), and tumor’s weight (f) of tumor- bearing mice during 14 days treatment; data are represented as means ± standard deviations (n =6) Yin et al. Nanoscale Research Letters (2020) 15:29 Page 13 of 15 Viability of Hela cells when exposed to DRN-GNPs of dif- conjugated DOX was the same as that of the free DOX. ferent concentrations was measured by flow cytometry We observed that the tumor sizes of DRN-GNPs-treated based assays. Two modes of cell death, apoptosis and ne- groups were obviously smaller than that of the saline, crosis, were measured using Annexinv-FITC and propi- DOX, DN-GNPs, and DR-GNPs-treated groups (Fig. 8a). dium iodide (PI) dyes, respectively (Fig. 7). Similar to the The tumor volume and body weight of the mice were results of MTT assay, the data showed that DRN-GNPs monitored every 2 days. Significant variation of tumor vol- induced cell necrosis was dose-dependent. The proportion ume and tumor weight among all of the treated groups is of necrotic cells was 8% without incubating with DRN- shown in Fig. 8d, f; the tumor volumes and tumor weight GNPs; when the concentration was high to 100 μg/mL, of DN-GNPs and DR-GNPs-treated groups were smaller the proportion of necrotic cells was 23.84%; that means than that of the free DOX-treated group, but still larger our synthesized DRN-GNPs could kill Hela cells effi- than that of the DRN-GNPs-treated groups because of the ciently. The difference between the results of MTT and less targeted activity. That might be because DOX mole- flow cytometry can be explained that the difference of cell cules are small, lack targeting, and they have a short half- number and the methods. life in vivo; therefore, they might be cleared out quickly. However, DRN-GNPs possess of strong stability in the Therapy Evaluation of DRN-GNPs in Tumor-Bearing Mice blood system, long half-life, and highly targeted ability, so To further determine whether DRN-GNPs induced a they can be targeted to the tumor site, and then play the combined therapeutic effect on tumor cells in vivo, we anti-tumor effect of DOX. Between the DRN-GNPs intra- evaluated the anti-tumor effect of DRN-GNPs in tumor- venously injected and tumor-injected groups, the anti- bearing mice for long-term intravenously injection of tumor efficacy of the latter group is better than that of the drugs. The saline group and the drugs of free DOX, DR- former group because the drugs do not need a complex GNPs, and DN-GNPs intravenously injected groups were system of blood circulation into the focus. The tumor in- set as the control groups, in which the concentration of hibition rate of DOX is less than 50%, and the rates of all Fig. 9 The histological images of the main organs (heart, liver, spleen, lung, and kidney) of the mice after treated with saline, free DOX, DR-GNPs, DN-GNPs, and DRN-GNPs (tumor injection and intravenous injection) Yin et al. Nanoscale Research Letters (2020) 15:29 Page 14 of 15 of the DOX-conjugated GNPs are larger than 50%; they glutamic acid; DOX: Doxorubicin; DR-GNPs: DOX-RGD-GNPs, DN-GNPs: DOX-NLS-GNPs, DRN-GNPs: DOX-RGD-NLS GNPs; GNPs: Gold nanoparticles can be high to 66.7% and 57.7%, respectively. The HE stain results showed that there was a certain degree of Acknowledgments damage for liver organs in the intravenous injected DRN- This work was supported by the National Natural Science Foundation of China (20875106), Guangdong Natural Science Found Committee GNPs group (Fig. 9). Furthermore, the bio-toxicity of (9151027501000003). The Science and Technology Planning Project of DOX, DR-GNPs, and DN-GNPs on tumor-bearing mice Guangdong Province (2015A010105013). The Science and Technology were also assessed by histological analysis. As shown in Planning Project of Guangzhou City (201607010349). The Fundamental Research Funds for the Central Universities (NO.21617497). Fig. 8b, tumor cell volume of saline group is comparatively larger than that of treatment groups, and tumor cells dem- Authors’ Contributions onstrate with more mitotic figures. All the results showed HQY participated in the design of the experiments, participated in all of the experiments, and drafted the manuscript. FG participated in the design of that the tumor inhibition effect of DRN-GNPs was the the experiments. GS participated in the experiments of Characterization. GY best among the GNPs. Thus, our synthesized DRN-GNPs participated in the experiments related to cell and animals studies. All could be used as a perfect DOX delivery system for tumor authors read and approved the final manuscript. therapy. Funding This work was supported by the National Natural Science Foundation of Conclusion China (20875106), Guangdong Natural Science Found Committee (9151027501000003). The Science and Technology Planning Project of In this study, we have successfully developed a one-step Guangdong Province (2015A010105013). The Science and Technology method to prepare the multifunctional DRN-GNPs in Planning Project of Guangzhou City (201607010349). The Fundamental about half an hour. The GNPs have also been successfully Research Funds for the Central Universities (NO.21617497). applied to the tumor targeted imaging and tumor therapy Availability of Data and Materials both in vitro and in vivo. 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Published: Jan 31, 2020

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