Biosynthetic Mechanism of Luminescent ZnO Nanocrystals in the

Research Article Cite This: ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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Biosynthetic Mechanism of Luminescent ZnO Nanocrystals in the Mammalian Blood Circulation and Their Functionalization for Tumor Therapy Yi-Zhou Wu,† Jie Sun,‡,§ Haowen Yang,∥ Xiaohui Zhao,⊥ Dacheng He,# Maomao Pu,† Gen Zhang,*,† Nongyue He,*,∇ and Xin Zeng*,○ †

Department of Cell Biology, School of Basic Medicine, ‡The Center for Hygienic Analysis and Detection, School of Public Health, and §Safety Assessment and Research Center for Drug, Pesticide and Veterinary Drug of Jiangsu Province, School of Public Health, Nanjing Medical University, Nanjing 211166, China ∥ Department of Chemistry and Applied Biosciences, Institute for Chemical and Bioengineering, ETH Zürich, Zurich 8093, Switzerland ⊥ Key Laboratory of Tibetan Medicine Research, Northwest Institute of Plateau Biology, Chinese Academy of Sciences, Xining 810001, China # Department of Cell Biology, College of Life Science, Beijing Normal University, Beijing 100875, China ∇ The State Key Laboratory of Bioelectronics, Department of Biological Science and Medical Engineering, Southeast University, Nanjing 210096, China ○ Nanjing Maternity and Child Health Medical Institute, Obstetrics and Gynecology Hospital Affiliated to Nanjing Medical University, Nanjing 210004, China S Supporting Information *

ABSTRACT: The biosynthesis of nanoparticles in bioreactors using microbial, plant, or animal cells is at the forefront of nanotechnology. We demonstrated for the first time that luminescent, water-soluble ZnO nanocrystals (bio-ZnO NCs) can be spontaneously biosynthesized in the mammalian blood circulation, not in cells, when animals were fed with Zn(CH3COO)2 aqueous solution. Serum albumin, rather than metallothioneins or glutathione, proved to play the pivotal role in biosynthesis. The bio-ZnO NCs were gradually taken up in the liver and degraded and excreted in the urine. Thus, we propose that in mammals such as rodents, bovinae, and humans, excess metal ions absorbed into the cardiovascular system via the intestine can be transformed into nanoparticles by binding to serum albumin, forming a “provisional metal-pool”, to reduce the toxicity of free metal ions at high concentration and regulate metal homeostasis in the body. Furthermore, the bio-ZnO NCs, which showed favorable biocompatibility, were functionalized with the anticancer drug daunorubicin and effectively achieved controlled drug release mediated by intracellular glutathione in tumor xenograft mice. KEYWORDS: nanocrystal, albumin, biosynthesis, cardiovascular system, mammal

1. INTRODUCTION The use of bacteria, viruses, fungi, and other lower organisms with metal ions to biosynthesize nanoparticles has several advantages, including cost-effectiveness, low toxicity/nontoxic properties, and structural control.1−7 Several studies have also demonstrated that biosynthetic nanoparticles could exhibit some peculiar properties, such as favorable biocompatibility and near-infrared fluorescence.6,8 Therefore, these bionanoparticles are attracting considerable interest from scientists. However, to date, the biosynthetic mechanism is poorly understood, especially in mammals. For example, how are the nanoparticles formed? Where does the nanoparticle biosynthesis occur in cells, or are they initially synthesized outside cells, such as in the © XXXX American Chemical Society

plasma, and subsequently transferred into cells? Here, as a step toward filling this knowledge gap, we designed an in vivo and in vitro zinc treatment, synthesized bio-ZnO nanocrystals (bioZnO NCs) in mouse plasma, and revealed the underlying molecular mechanism. Because of their favorable biocompatibility, these bio-ZnO NCs were further functionalized with daunorubicin (DNR), and the resulting species showed feasibility and effectiveness in anticancer treatment. Received: September 9, 2017 Accepted: November 30, 2017

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DOI: 10.1021/acsami.7b13691 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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ACS Applied Materials & Interfaces

Figure 1. Characterization of the bio-ZnO NCs. (a) TEM image of the bio-ZnO NCs. (b) HRTEM image of the lattice fringes. (c) SAED pattern of the bio-ZnO NCs and assigned Miller indices. (d) EDS spectrum of the bio-ZnO NCs. (e) UV−vis absorption spectrum of the bio-ZnO NCs. (f) Photoluminescence spectrum of the bio-ZnO NCs in water with excitation at 302 nm. (g) HEK293 cells were treated with 10 mg/L bio-ZnO NCs for 2 h. The fluorescence images (blue) were obtained with excitation at 300 nm. Cell morphology was visualized by differential interference contrast (DIC). Scale bar: 100 μm.

2. RESULTS AND DISCUSSION 2.1. Synthesis, Purification, and Characterization of the Bio-ZnO NCs in Mammals. As one of the micronutrients, zinc is involved in bodily metabolic and cellular functions, maintaining the metal homeostasis network. We chose Zn2+ as the preferred material for the synthesis of bionanoparticles in this study. First, the ICR mice were fed with a 10 mM aqueous solution of Zn(CH3COO)2 for 48 h. Each mouse was equally administered 50 μmol of Zn2+ per day. Then, the plasma was collected for hemolysis treatment in water, and the preconcentration of the bio-ZnO NCs was performed via differential centrifugation. The precipitate containing the raw extracts of the bio-ZnO NCs was still mixed with a large amount of biological macromolecules, such as proteins, lipids, and subcellular fractionations. To obtain the purified bio-ZnO NCs, we developed a time-lapse collection method using highperformance liquid chromatography (HPLC) with a C18 column. Generally, we divided and collected the eluent separately every 5 min. The whole running time was 40 min, which was long enough for the bio-ZnO NCs to flow through the HPLC column. The solvents in each collection were

removed by freeze-drying, and then the dried contents were redissolved in deionized water. Based on the transmission electron microscopy (TEM) analysis, the retention time of the bio-ZnO NCs was determined to be approximately 29.828 min (Figure S1a). After 48 h of biosynthesis, the yield of the bioZnO NCs was approximately 1.5 ± 0.8% (w/w) in dried serum. The bio-ZnO NCs were characterized by TEM. The average diameter of the bio-ZnO NCs was 6.0 ± 0.5 nm (Figure 1a). Without the above purification procedure, obtaining a large number of clear bio-ZnO NCs was difficult (Figure S1b). Highresolution transmission electron microscopy (HRTEM) showed clear lattice fringes and confirmed the presence of crystalline nanoparticles with a d-spacing of 0.268 nm corresponding to the (002) reflection (Figure 1b), which was consistent with previous reports.9,10 Meanwhile, selective area electron diffraction (SAED) showed diffuse rings that were consistent with ZnO (100), (102), and (103) reflections from ZnO nanoparticles (Figure 1c). Furthermore, the Raman spectrum exhibited a similar peak (wave range: 1300−1500 nm) to previous reports (Figure S1c), which indicated that the obtained NCs could be oxide NCs.11,12 Energy-dispersive X-ray B


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Figure 2. Study on the proteins in biosynthesis of ZnO NCs. (a) Western blot analysis of the expression of the MTs in K562 cells that were treated with Zn2+ at different concentrations for 48 h. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was detected as an internal standard. (b) The activity of GSH in untreated K562 cells and K562 cells treated with 10 mM Zn2+ for 48 h. *Significant difference compared with the control group (P < 0.05). (c) K562 cells with or without treatment with 10 mM Zn2+ for 48 h and then stained with Alexa 594-conjugated anti-MT antibody. Scale bar: 10 μm. (d) Dot blot assay of albumin in mouse plasma, bio-ZnO NCs, and chem-ZnO NCs. (e) Identification of albumin in bio-ZnO NCs by MALDI-TOF/TOF. The tandem mass spectrometry (MS/MS) spectra of two typical precursor ions (m/z were 1479.967 and 1567.751) and the resulting peaks were searched against the Swiss-Prot protein sequence database using the MASCOT search engine. The corresponding peptides were identified as LGEYGFQNALIVR and DAFLGSFLYEYSR.

chloroauric acid.1 These reports indicate the importance of considering the function of the MTs and GSH, both of which belong to the family of sulfur-containing proteins/peptides. Considering that the bio-ZnO NCs mainly exist in mouse blood, we chose human leukemia K562 cells for use in subsequent assays. The K562 cells were incubated with different concentrations of Zn2+. First, we used a messenger RNA microarray to explore the expression levels of different MTs in K562 cells treated with Zn. The mRNAs of the MTs were found to be significantly elevated in K562 cells treated with Zn (Table S1). The protein levels of the MTs were further analyzed by Western blot. The results showed that the expression levels of the MTs were upregulated in Zn-treated K562 cells in a concentration-dependent manner (Figure 2a). Furthermore, the activity of GSH in K562 cells was increased after Zn treatment (Figure 2b). These results suggested that both the MTs and GSH were involved in the synthesis of the bio-ZnO NCs. The MTs are a class of cysteine-rich proteins with a higher affinity for zinc ions than other metals.17,18 As our findings implied that the MTs may directly participate in the synthesis of the bio-ZnO NCs, we performed an immunofluorescent assay to analyze the intracellular location of the MTs. After Zn treatment, the expression levels of the MTs was significantly elevated in K562 cells. The upregulated MT proteins showed a distinct aggregation in sparklike areas (Figure 2c). The same results were also confirmed by an immunocytochemistry assay (Figure S2a). Up to this point, the results suggested that the excess zinc ions possibly were sequestered by MTs and converted into the bio-ZnO NCs in the MT-aggregation regions. However, there was a contradiction. If numerous cysteine-rich proteins, such as MTs, were

spectroscopy (EDS) analysis revealed remarkable Zn and oxygen contents in the bio-ZnO NCs. The elemental ratio between Zn and oxygen was approximately 1:1.07 (Figure 1d). Additionally, the UV−vis absorption spectrum showed the characteristic band of bio-ZnO NCs at 340 nm (Figure 1e), with a slight blue shift compared with that of ordinary ZnO NCs,13,14 whereas no such band was observed in an unpurified sample. The bio-ZnO NCs displayed a blue emission peak at 389 nm when excited at 302 nm (Figure 1f). The emission quantum yield of bio-ZnO NCs was calculated to be 10.6% (against 9,10-diphenylanthracene). For visualization, HEK293 cells were exposed to the bio-ZnO NCs and imaged by a confocal fluorescence microscope.15,16 The bright blue fluorescence observed indicated that the bio-ZnO NCs may be useful for superficial tumor imaging in vivo (Figure 1g). 2.2. In Vivo Synthetic Mechanism of the Bio-ZnO NCs. Although bionanoparticles have been investigated for many years, there are few reports on how the nanoparticles are synthesized in the biological specimens, especially in mammals. To reveal the synthetic mechanism of the bio-ZnO NCs, we first considered several hypotheses from previous studies.1,4 By utilizing the biosynthetic pathway in earthworms, Green and colleagues obtained the CdTe quantum dots from tissues near the earthworm gut and, more importantly, proposed the first comprehensive biosynthesis theory.4 They suggested that the metallothioneins (MTs) played an important role in the biosynthesis of CdTe quantum dots in guts cells of earthworm treated with Cd2+ and TeO32−. Wang and colleagues suggested that as the main reducing agents in cells, glutathione (GSH) played an important role in the biosynthesis of gold nanoclusters in human hepatoma HepG2 cells treated with C


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identified the presence of albumin in the bio-ZnO NCs, whereas no albumin was observed in the chemically synthesized ZnO NCs (chem-ZnO NCs) (Figure S2g). Moreover, in addition to albumin, oxygen was another essential factor in the synthesis of the ZnO NCs. The oxygen involved in the synthesis may have originated from the dissolved oxygen in an aqueous solution. Therefore, we measured the content of oxygen during the bio-ZnO NC formation. The results demonstrated that oxygen was consumed in a sustained and time-dependent manner (Figure S2h). According to the results mentioned above regarding the synthetic mechanism, we proposed that the excess zinc ions absorbed into the blood vessels of mice through the intestine can be transformed into bio-ZnO NCs. After the bio-ZnO NCs were separated from mouse plasma and purification via HPLC, the dot blot assay using albumin antibody revealed the presence of albumin on the bio-ZnO NCs (Figure 2d). Mass spectrometry identified that the albumin (Mus musculus) was bound on the surface of the bio-ZnO NCs (Figure 2e). These results confirmed our hypothesis regarding the in vivo synthetic mechanism of the bio-ZnO NCs: zinc ions bound to albumin along with oxygen to form the bio-ZnO NCs in mouse blood vessels. Moreover, the bio-ZnO NCs were further characterized by a 120 kV biological electron microscope (Figure S2i). The images revealed that bio-ZnO NCs had a homogeneous distribution and an average size of approximately 22 nm, in contrast to the 6 nm naked NCs observed under a 200 kV TEM. The hydrodynamic diameter of bio-ZnO NCs was measured by a nanoparticle tracking analysis technique. The average particle size of the bio-ZnO NCSs in water was approximately 76 nm (Figure S2j). 2.3. Metabolic Pathway of the Bio-ZnO NCs. With blood circulation, the bio-ZnO NCs were dispersed in the liver and kidney (Figure S3a). However, we did not observe the bioZnO NCs in the brain, either because there were no NCs present or because the quantity was too low for detection. We presumed that the main reason was related to the blood−brain barrier, which comprises a layer of endothelial cells that are very tightly bound each other and thus block the transport of nanoparticles from the blood to the brain.22,23 Furthermore, pathological analysis found that mice fed with 0.1 M or less Zn2+ showed a minor damage to the liver and kidneys, whereas those fed with 0.3 M Zn2+ showed a significant enlargement of the hepatocytes and numerous protein casts in the tubular lumen (Figure S3b). Nevertheless, we were also interested in the fate of the bio-ZnO NCs in vivo. Are they metabolized and excreted, or do they remain in the animal body for a long time? Interestingly, we found that the bio-ZnO NCs did not permanently remain in the mouse plasma or organs. One week after the Zn2+ treatment was ended, the amount of bioZnO NCs was significantly decreased, and after 2 weeks, the NCs could hardly be observed (data not shown). We proposed that the bio-ZnO NCs could be acidified and degraded in the cellular lysosome, and then the soluble zinc ions were excreted through the circulatory system. Therefore, we determined the urinary concentrations of Zn2+ by atomic absorption spectroscopy, which showed that between 48 and 72 h after the Zn2+ treatment was halted, the urinary Zn content still markedly increased (Figure S3c). This finding demonstrated that the metabolic Zn in the circulatory system was further excreted via urine, suggesting that the bio-ZnO NCs function as a provisional “Zn-pool” in the body. When the amount of Zn2+ increased, the serum albumin transformed Zn2+ into bio-ZnO

involved in the synthesis of the bio-ZnO NCs, then the NCs should have a high content of sulfur atoms, similar to ZnS NCs or derivatives of ZnS NCs. However, the characterization results indicated that the NCs consisted of ZnO rather than ZnS. This led to the question of whether the MTs were essential for the synthesis of the bio-ZnO NCs. To determine the role of the MTs in the synthesis of the bioZnO NCs, we designed the following experiments. First, the expression of MT in K562 cells was knocked down using siRNA. Then, the cells were cultured for 48 h at 37 °C in medium supplemented with 10% fetal bovine serum and Zn2+ (1 mM). We observed almost the same amount of bio-ZnO NCs in the subsequent TEM analysis (Figure S2b). However, when Zn2+ (1 mM) was directly incubated with either purified MTs at 3 μg/mL, which is equal to the intracellular concentration of MTs,19 or purified GSH at 2 mM, which is equal to the intracellular concentration of GSH,20 for 48 h at 37 °C, no bio-ZnO NCs were observed by TEM (data not shown). Conversely, when we used a high concentration of GSH (100 mM), a high temperature (80 °C), and continuous stirring, a small amount of bio-NCs was synthesized (Figure S2c). However, this concentration of GSH is far greater than that found in mammals. Taken together, the above results demonstrated that the synthesis of the bio-ZnO NCs was not dependent on the MTs or GSH. Consequently, the upregulated expression of the MTs and GSH after Zn exposure could be a stress response of cells. This result was contrary to the current view of bio-NCs synthesized in vivo.1,4 Thus, the following question remained unanswered: What is the essential factor leading to the synthesis of the bio-ZnO NCs? When K562 cells were cultured in a serum-free medium and treated with different concentrations of Zn2+, we did not observe bio-ZnO NCs via TEM analysis, either because no ZnO NCs were formed or because the quantity of bio-ZnO NCs obtained was too low for detection. This result further indicated that in the presence of only intracellular biological macromolecules, the bio-NCs are barely synthesized. We hypothesized that the materials of cell culture medium, such as serum, rather than the cells were more likely to play the pivotal role in the bio-ZnO NC synthesis. As expected, the bioZnO NCs could be synthesized when Zn2+ (1 mM) was incubated with aqueous solutions of bovine serum, rat serum, or even human serum, but were not obtained when serum-free culture medium was used (Figure S2d). The above results fully indicated that the serum played a key role in the biosynthetic process. Not only bovine serum but also other mammalian serum could be applied to the synthesis of bio-ZnO NCs. Thus, which exact substance in serum governed the biosynthesis? In mammalian organisms, serum albumin is the most abundant protein in plasma. The normal concentration of albumin is approximately 40−55 g/L, accounting for 50−60% of the total serum proteins.21 This also explains why intracellular biomolecules such as MTs, GSH, or other substances could not affect the synthesis of the ZnO NCs in cells: The cellular concentrations of these molecules are too low compared with albumin in plasma.19,20 Thus, we proposed that the Zn2+ ions were bound by albumin, and that the ZnO NCs were subsequently formed. To corroborate this theory, an aqueous solution of bovine serum albumin was incubated with Zn2+ for 48 h at 37 °C. Interestingly, many ZnO NCs were produced by this process (Figure S2e). A typical Xray diffraction pattern of ZnO NCs was presented (Figure S2f). Using a dot blot assay with albumin monoclonal antibody, we D


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Figure 3. Assay of the biocompatibility of the bio-ZnO NCs. (a) The cytotoxic effects of the bio-ZnO NCs, chem-ZnO NCs, or zinc ions in HEK293 cells were analyzed by MTT assay. The concentrations ranged from 0.1 to 1000 mg/L. (b) Mouse erythrocyte suspension was treated with bio-ZnO NCs or chem-ZnO NCs for 2 h. The hemolysis rate was assayed by counting the remanent erythrocytes. The concentrations ranged from 1 to 100 mg/L. (c) Mice were treated with 10 mg/kg bio-ZnO NCs or chem-ZnO NCs by tail intravenous injection. The serum was obtained after 24 h. The protein levels of IL-2, IL-4, IL-6, IL-10, TNF-α, and IFN-γ were measured by flow cytometry using the Mouse Th1/Th2/Th17 Cytokine Kit. *Significant difference compared with the untreated group (P < 0.05).

leukin (IL)-2, IL-6, tumor necrosis factor (TNF)-α, and interferon (IFN)-γ were significantly increased after chemZnO NC treatment (Figure 3c), whereas bio-ZnO NC treatment did not induce such an increase, which indicated that the bio-ZnO NCs hardly activated the immune system compared to the chem-ZnO NCs. The protein levels of immunosuppressive cytokines including IL-4 and IL-10 did not show significant changes after bio-ZnO NC treatment (Figure 3c), indicating these NCs possessed almost no immunosuppressive effect. All of these results demonstrated that the bioZnO NCs possessed little immunogenicity to mice and indicated them as a good biocompatible nanomaterial. 2.5. Application of Targeted Drug Delivery with the Bio-ZnO NCs. Albumin−paclitaxel nanoparticles have been shown to accumulate in tumors through the well-established enhanced permeability and retention effect. Albumin can bind to SPARC (secreted protein, acidic, and rich in cysteine) and subsequently endocytose by gp60 receptor and caveolin-1 in the tumor cells.25,27,28 Considering that the structures on the surface of the bio-Zn NCs and those of albumin-bound paclitaxel nanoparticles were similar, we speculated that the bioZnO NCs could achieve the targeted drug delivery by taking advantage of albumin. To determine this, we conducted an immunofluorescence assay to analyze the colocalization of SPARC and the bio-ZnO NCs. First, we found the regions in HepG2 cells that were rich in SPARC also contained a larger number of bio-ZnO NCs (Figure 4a). Such a close proximity could enable the bio-ZnO NCs to target a tumor site. Then, we established a K562-bearing nude mouse model and treated the mice with bio-ZnO NCs. The results showed that the bio-ZnO

NCs and stored them temporarily. When the amount of external Zn2+ decreased, the bio-ZnO NCs slowly degraded into metabolic Zn2+. We presumed that the animal body catalyzes the conversion of metal ions into NC to reduce the toxicity of high metal ion concentration as a matter of expediency. 2.4. Cytotoxicity and Immunogenicity of the Bio-ZnO NCs. As a carrier with favorable biocompatibility, albumin has been reported for the synthesis of paclitaxel nanoparticles, such as Abraxane, which was approved by the FDA for treating malignancies.24,25 In this study, we proposed that the metallic toxicity of Zn was greatly reduced by the conversion of zinc ions into bio-ZnO NCs via albumin. Therefore, coating bioZnO NCs with albumin may have the potential to increase the biocompatibility of Zn nanomaterials for clinical applications. First, we assessed the cytotoxicity of the bio-ZnO NCs by MTT assay, in which HEK293 cells were incubated with Zn2+, the bio-ZnO NCs, or the chem-ZnO NCs. The results demonstrated that the bio-ZnO NCs, even at high concentration, were significantly less cytotoxic than Zn2+ or chem-ZnO NCs (Figure 3a). Second, we assessed the hemolytic effect of the bio-ZnO NCs on mouse erythrocytes. The results showed that only a small number of cells had hemolytic reaction to incubation with 100 mg/L bio-ZnO NCs. The hemolysis rate of bio-ZnO NCs was significantly lower than chem-ZnO NCs (Figure 3b). Third, we assessed the immune response of mice after the treatment with bio-ZnO NCs. Specifically, the Mouse Th1/Th2/Th17 Cytokine Kit was used to assay several pivotal cytokines related to immunogenicity in plasma samples.26 The protein levels of proinflammatory cytokines including interE


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Figure 4. Application of DNR-loaded bio-ZnO NCs to tumor growth suppression in vivo. (a) HepG2 cells were treated with 10 mg/L bio-ZnO NCs (blue) for 2 h and then stained with Alexa 488-conjugated anti-SPARC antibody (green). Scale bar: 100 μm. (b) Scheme of the synthesis of DNRloaded bio-ZnO NCs. (c) K562 xenograft nude mice were treated (1) without or with (2) 3.2 mg/kg DNR (equivalent to the 32% loading rate on the bio-ZnO NCs), (3) 10 mg/kg DNR-loaded chem-ZnO NCs, or (4) 10 mg/kg DNR-loaded bio-ZnO NCs. The apoptotic cells were assayed by TUNEL staining. The nuclei of apoptotic cells (brown) and viable cells (blue) were stained. Scale bar: 100 μm. The bars in the histogram represent the results from five independent experiments. *Significant difference compared with the untreated group (P < 0.05). (d) The protein levels of cleaved caspase-3, -8, and -9 in tumor samples were analyzed by Western blot. GAPDH was detected as an internal standard.

pressure homogenization and minimized energy consumption (Figure 4b). Generally, at a high concentration of GSH (10 mM) and under reducing conditions, noncovalent interactions such as hydrogen bonding and the hydrophobic effect are diminished, and the disulfide bonds in albumin are cleaved, leading to albumin unfolding into a linear structure. DNR interacted with the hydrophobic domains and induced the selfassembly of the bio-ZnO NCs. Moreover, DNR (ζ potential, 4.9 mV) was adsorbed onto the negatively charged bio-ZnO NCs (ζ potential, −10.7 mV) with high efficiency. After centrifugation, the supernatant containing GSH was discarded, and the precipitate was redispersed in aqueous solution. The subsequent recovery of disulfide bridges further stabilized the

NCs accumulated in the tumor site, suggesting that these NCs may be useful for superficial tumor imaging in vivo (Figure S4a). Subsequent cryo-sectioning and immunofluorescence analysis further verified that the bio-ZnO NCs were localized closely with SPARC in the tumor samples (Figure 4a). These results indicated that SPARC was involved in the tumortargeting ability of the bio-ZnO NCs, which was similar to the transport mechanism of albumin−paclitaxel nanoparticles. To functionalize the bio-ZnO NCs with the anticancer drug daunorubicin (DNR) and achieve controlled drug release, we developed a green self-assembly approach based on the GSH denaturation for drug loading that avoided the use of crosslinking agents or emulsification processes mediated by highF


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Figure 5. Illustration of the synthesis of the bio-ZnO NCs in a mammal and their functionalization with DNR for tumor treatment.

initiated by external “death signal” stimulation, leading to caspase-8 activation and apoptosis, and another, the “mitochondria pathway”, induced by caspase-9 activation.15,32 As caspase3 is a known downstream target of cleaved caspase-8 and -9, the DNR-loaded bio-ZnO NCs likely induced K562 tumor cell apoptosis by activating the caspase pathway. Moreover, the results also suggested that the bio-ZnO NCs might play a critical role in enhancing the apoptotic induction ability of DNR.

bio-ZnO NCs. HPLC analysis showed that the DNR loading rate on the bio-ZnO NCs was approximately 32.6% (Figure S4b). The overwhelming majority of GSH in the human body is found in the cytoplasm,20,29 as the concentration of GSH ranges from 1 to 11 mM in the cytosol of different human cells compared with only approximately 0.002−0.01 mM in human plasma and the extracellular environment.29−31 Thus, when the DNR-loaded bio-ZnO NCs entered cells, the high concentration of intracellular GSH (e.g., 2 mM in K562 cells) automatically caused the opening of the disulfide bonds in albumin and effectively released DNR from the bio-ZnO NCs (Figure S4c). To determine the anticancer potential of the bio-ZnO NCs, BALB/c nude mice were xenografted with K562 cells as previously described32 and then treated with different agents (Figure 4c). Compared with the untreated group, DNR treatment alone inhibited tumor growth, leading to a decrease in tumor volumes to approximately 64.10%. When DNR was loaded onto the chem-ZnO NCs, treatment with this nanocomposite showed a significant suppressive effect, with the tumor volume reduced to approximately 56.4%. The efficient antitumor activity was further improved when DNR was loaded onto the bio-ZnO NCs, as the tumor volume was reduced to approximately 38.5%. The above results indicated that the bio-ZnO NCs were an effective drug delivery tool for inhibiting tumor growth. Furthermore, we explored the induction of apoptosis in the tumor samples by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining.33 Compared with the untreated group, the apoptotic rates in the groups treated with DNR alone and the DNR-loaded chem-ZnO NCs were increased to approximately 20.8 and 31.4%, respectively (Figure 4c). As expected, the apoptotic rate was drastically increased to approximately 63.5% when applying the DNR-loaded bio-ZnO NCs. To further investigate the anticancer signaling of DNRloaded bio-ZnO NCs in tumor-xenografted nude mice, we performed Western blot to analyze the protein extracts from the tumor samples. Among the experimental groups, the DNRloaded bio-ZnO NCs were found to induce the greatest activation of caspase-8, -9, and -3 (cleaved forms) (Figure 4d). There are two major pathways of caspase activation: one

3. CONCLUSIONS In this study, we investigated the hypothesis that Zn2+ could react in mouse blood in vivo to form bio-ZnO NCs and then successfully purified and characterized the bio-ZnO NCs. Next, we sought to answer the following question: How are the bioZnO NCs synthesized in the mice blood? In contrast to previous reports about the intracellular role of metal ionbinding proteins (e.g., MTs) or small molecules (e.g., GSH), we found that albumin exerted the strongest effect on the biosynthesis. The albumin-coated bio-ZnO NCs showed good biocompatibility, including low toxicity and immunogenicity. Bio-ZnO NCs functionalized with the anticancer drug DNR can be used as a tumor-targeting agent through the SPARC pathway, and then can induce cell apoptosis through caspase signaling. Generally, it comprises four steps in antitumor application (Figure 5). (1) Synthesis. The mammal is fed with Zn solution, and the bio-ZnO NCs are spontaneously synthesized in cardiovascular circulation. (2) Purif ication. The bio-ZnO NCs are separated by differential centrifugation, followed by purification through HPLC. (3) Drug loading. The bio-ZnO NCs are incubated with GSH to unfold the spatial conformation of albumin, which could facilitate the drug molecules to bind to the surface of ZnO by electrostatic adsorption. Further, the GSH is removed through centrifugation and the spatial conformation of albumin is recovered. (4) Treatment. The drug-loaded bio-ZnO NCs are injected into the tumor xenograft mouse through intravenous injection. Then, the tumor cells absorb the ZnO mainly through albumin/ SPARC interaction. The drug molecules are further released by intracellular GSH and finally induce cell apoptosis. G


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ACS Applied Materials & Interfaces Several earlier reports have indicated that large ZnO nanoparticles (several tens of nanometers) and ZnO rods could be synthesized in low-level organisms or its extracts (e.g., bacteria, plant).34,35 However, these processes were technically considered chemical synthesis instead of biosynthesis because they always required extreme conditions, including strong alkaline solutions, high temperature, high speed mixing, and an additional dispersant, such as thiol, ethanol, or GSH.36−38 Here, we reported a mild and more advanced synthetic procedure for bio-ZnO NCs in mammals that did not require such extreme conditions and showed unique features. The cardiovascular system of mammal is closed, meaning that the blood never leaves the network of blood vessels. The circulating blood provided sufficient oxygen, albumin, and continuous mixing of the zinc ions to improve the synthetic efficiency. As a warmblooded animal, the mouse can provide a stable temperature for biosynthesis. Therefore, compared to previous techniques in plant or animal cells, the biosynthesis of bio-ZnO NCs within a mammalian bioreactor represents an advanced landmark in nanotechnology.

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drug loading analysis, and mRNA analysis of the MT variant types (PDF)

AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected] (G.Z.). *E-mail: [email protected] (N.H.). *E-mail: [email protected] (X.Z.). ORCID

Yi-Zhou Wu: 0000-0001-9768-8144 Jie Sun: 0000-0001-7823-2897 Gen Zhang: 0000-0001-9067-4786 Author Contributions

Y.-Z.W. and J.S. contributed equally to this work. Y.-Z.W. and G.Z. conceived, designed, and performed the experiments. Y.Z.W. and J.S. wrote and revised the article. X.Z. and N.H. analyzed the data of mRNA chips and nanocrystals. All of the authors discussed the results. Notes

The authors declare no competing financial interest.

4. EXPERIMENTAL SECTION

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4.1. Synthesis and Purification of Bio-ZnO NCs in Vivo. The ICR mice, BALB/c nude mice, and Sprague-Dawley rats were purchased from Charles River Laboratories (Beijing, China), maintained in a controlled environment at 24 ± 2 °C with a 12 h light/dark cycle, and received food and water ad libitum. All of the experiments were conducted according to the guidelines from the Animal Research Ethics Board of Nanjing Medical University. For the synthesis of the bio-ZnO NCs in vivo, animals were fed with a 10 mM aqueous solution of Zn(CH3COO)2 (Sigma) for 48 h. Then, the animals were sacrificed, and the plasma was collected. To separate the bio-ZnO NCs, differential centrifugation was performed. Generally, the sample solutions were first centrifuged at 1000g for 20 min, the supernatant was collected and recentrifuged at 9000g for 30 min, and then the supernatant was collected and recentrifuged at 100 000g for 2 h. The final precipitate containing the water-soluble bio-ZnO NCs was dissolved in 1 mL of deionized water. Then, the bio-ZnO NCs were further purified by HPLC (Hewlett-Packard Agilent 1100) equipped with an ultimate XB-C18 column (5 μm, 300 Å, 4.6 mm × 250 mm, Welth). The composition of the optimal mobile phase was 50% H2O, 45% methanol, and 5% isopropyl alcohol (pH 8.0), and the flow rate was 0.1 mL/min. The eluent for each chromatographic peak was collected separately, diluted, and transferred for characterization. More details on the reagents, instruments, and methods are provided in the Supporting Information. 4.2. Preparation of DNR-Loaded Bio-ZnO NCs. The ζ potentials of the bio-ZnO NCs and DNR were determined. A volume of DNR (1 mg, 1 mg/mL) solution was added to an aqueous suspension of the bio-ZnO NCs (1 mg, 1 mg/mL) in a solution with a high GSH concentration (10 mM) under mixing conditions. The resulting solution was kept overnight in the dark to allow the construction of the DNR-loaded bio-ZnO NCs. To eliminate the residual GSH, the solution was centrifuged at 100 000g for 2 h. The precipitate containing the DNR-loaded bio-ZnO NCs was collected and washed three times with water.

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ACKNOWLEDGMENTS This study was supported by Jiangsu Provincial Key Research and Development Program (BE2016620), Natural Science Foundation of Jiangsu Province (BK20171050), Natural Science Foundation of the Jiangsu Higher Education Institutions (17KJB310006), Jiangsu Planned Projects for Postdoctoral Research Funds (1601010C), Key Project Supported by Medical Science and Technology Development Foundation of Nanjing Department of Health (JQX15010), and Nanjing Medical Science and Technique Development Foundation (QRX17072).

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REFERENCES

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S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.7b13691. Detailed materials and methods, including reagents, instruments, and analytical procedures; experimental data for separation and characterization of the bio-ZnO NCs, biological effect and metabolic pathway analysis, H


Research Article


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