Facilitated Excretion of Gold Nanoparticles by Copper Sulfide Nanoparticles Through the ATP7B Transporter

Scientific areas have utilized the exclusive qualities of nanomaterials for medicine, diagnostics, drug delivery, tissue engineering and environmental protection. Inorganic metal nanoparticles such as gold and copper have been widely studied in the past decades. Due to the strong and tunable surface plasmon resonance (SPR), nanostructures including nanoshells, nanorods, nanocages, and hollow nanospheres exhibit strong optical absorption at near-infrared (NIR) wavelengths (650–900 nm), resulting in resonance and transfer of thermal energies to the surrounding tissue to raise the temperature. The absorbance of NIR light is desirable because it minimizes thermal injury to normal tissues while providing optimal light penetration. The efficient photothermal energy transfer effect by inorganic metallic nanoparticles such as gold has been widely used for photothermal ablation of tumor tissues, as well as drug delivery system for small molecules like protein, antibodies, DNAs, and small interfering RNAs by NIR laser triggered-release. Even though different kinds of gold nanoparticles have a great advantage on the photothermal transaction and are promising for clinical applications, they are nonbiodegradable, raising concerns regarding their short/long-term metabolism and safety. Tail vein injections of polyethylene glycol (PEG)-coated gold nanoparticles have been reported to induce two phases of toxicity concerning inflammation in the liver. The acute phase occurred immediately after administration of nanoparticles. The second phase happened at 7 days post injection when the nanoparticles become localized in the tissues, mostly in the liver and spleen after circulation in the blood. Due to multiple valences of gold, redox reaction of gold within cells can increase the levels of


Introduction
Metal nanoparticles were widely used in making paints and glass panel staining long before their properties have been discovered and understood. [1] Precious metal, such as gold, has extensively been used and played crucial roles in ancient China and Arabic medicine to treat rheumatoid arthritis. [2] Colloidal gold has been used to treat alcoholism in the United States since the nineteenth century. [3] Silver, another noble metal, also has been used as eye drops to prevent gonorrhea in newborns from 1880s to the late 1900s [4]. After the successful synthesis of the platinum compound in the 1960s, cisplatin has become one of the most extensively used anticancer drugs, especially in the treatment of ovarian and testicular cancers. [5,6] Since then, a plethora of literatures have been published to explain the recent advances in nanotechnology [7][8][9][10][11]. Nanoparticles demonstrate a useful platform with enormous potential in cancer therapy and other biological applications. [12] Some noble metal nanoparticles including gold have received increased attention from the scientific community due to their distinctive properties and wide-range of applications.
The major current and most promising applications of metal nanoparticles include in vivo and in vivo imaging, diagnostics, therapeutics, drug carrier and delivery, biomaterials, biosensor and tissue engineering (

Methods for Preparation of Metal Nanoparticles
In order to prepare metallic nanoparticles with the preferred sizes and morphologies, many methods including physical, chemical and green biological approaches are used (some important methods are listed in Figure 2). Every method has its advantages and drawbacks, which include using toxic solvents, expensive materials, intensive labor and many others. 5 In chemical reduction method, different kinds of reducing agents including sodium borohydride, ascorbate, sodium citrate, dimethylformamide, etc., are used to reduce metal ions. [13][14][15] It is very important to use stabilizing agents throughout the reduction process to protect the metallic nanoparticles from agglomeration and sedimentation. [16] Polymeric compounds such as polyvinylpyrrolidone (PVP), polyethylene glycol (PEG), and polymethacrylic acid, which comprise thiols, amines, alcohols groups interact with particle surfaces to stabilize the final nanoparticles and prevent them from growing and losing their surface properties. 6 High-energy mechanical milling is a very effective physical method for metallic nanoparticle synthesis. Fe−Cu nanoparticles have been successfully synthesized using a ball-milling process that utilizes FeCl 3 and CuCl 2 [17]. Huang and Yang reported that silver nanoparticles were prepared by using UV-initiated photoreduction of silver nitrate in inorganic lay suspension at room temperature. [18] Metallic nanoparticles prepared in the two-phase aqueous-organic system was reported for silver nanoparticle synthesis. [19] The major disadvantage of this method is the tremendous toxic organic solvents. Therefore, large amounts of surfactants and organic solvents need to be separated and removed from the nanoparticles. In the electrochemical synthetic method, the homogeneity and particle length of a nanoparticle is easier to be controlled by modifying the composition of the electrolytic solution and adjusting the electrolysis parameters. In one study, spherical silver nanoparticles with narrow length distributions were prepared by this method, and poly N-vinylpyrrolidone was used to protect nanoparticles from agglomeration [20].

Green Biological Methods
There is always a demand for using green biological methods to synthesize metallic nanoparticles. Advantages of these methods include i) non-toxic and inexpensive materials and solvents ii) less hazardous chemical process and waste iii) cost-effective and iv) procedures can be done at room temperature. However, disadvantages include i) limitations to repeat and scale up the massive production ii) more studies are needed to elucidate the exact synthesis mechanism and iii) difficulty producing the appropriate size and morphologies of the nanoparticles. 7 Instead of organic solvents, polysaccharides (e.g., corn starch, dextran, cellulose) and chitosan have been used for the green preparation of metallic nanoparticles. It is reported that silver NPs were synthesized using polysaccharides as capping agents and water as an eco-friendly solvent [21]. Polysaccharides have hydroxyl groups and a hemiacetal-reducing end that can be used as a reducer. The oxidation of hydroxyl groups in polysaccharides to carbonyl groups performs a crucial role in reducing gold salts to form nanoparticles. Furthermore, the hemiacetalreducing end of polysaccharides can be used to stabilize metallic nanoparticles and prevent sedimentation [22,23]. For example, starch-stabilized and glucose-reduced silver NPs with a mean diameter of 5.3 nm were prepared through the incubation of silver salt with starch-stabilization and glucose-reduction at 40 °C for 20 h. [24].

Application of Metallic Nanoparticles
Nanoparticles have different shapes and a large surface to volume ratio. These Besides nanoparticles with LSPRs property, other heavy metals and quantum dots also have the potential to be used in diagnostic tests and as cell trackers and contrasting agents. [25] Moreover, some of the metallic nanoparticles, such as gold and copper have shown the extraordinary ability to be used in drug delivery and photothermal therapy [26]. The application of metallic nanoparticles can be sorted into three 8 interrelated areas: diagnosis, drug delivery and regenerative therapies.

Metallic Nanoparticles in Diagnostics
The application of metallic nanoparticles in diagnostics can be further categorized into i) in vitro diagnostic tool by using nanoparticles as a probe for disease-related biomarker detection and ii) in vivo imaging for in situ disease monitoring.
Metallic nanoparticles made with semiconductor material have proved to better bioprobes compared to conventional fluorescent probes. They are more photochemically stable and have a more controllable symmetric emission spectrum.
[27] Gold nanoparticles have been used as a color marker for rapid pregnancy testing since the 1980s. Collagen has been used as a biocoating to increase the biocompatibility and improve the interaction with biomolecules, while also reducing the degradation rate of nanoparticles. Metallic nanoparticles with localized surface plasmon resonance (SPR) properties exhibit enhanced scattering and absorption light at specific light wavelengths. [28] Gold nanoparticles have very good SPR property.
Within a protein-rich environment, the surface of gold nanoparticles can be conjugated with different proteins. This binding process will increase the size of gold nanoparticles and shift the SPR towards the longer wavelengths. This process can be used for protein qualification and quantification. [29] As an application, specific antibody-coated gold nanoparticles are applied to detect antigen at extremely low concentrations in the nanomolar range, which cannot be achieved with current diagnostic tools. This will identify subtypes of cancer earlier and promote the 9 diagnosis and prognosis of cancers. [29] In vivo imaging, such as magnetic resonance imaging (MRI), computed tomography (CT), positron-emission tomography (PET), and single-photon-emission computed tomography (SPECT) are widely used imaging instruments for monitoring and diagnosing complex diseases, including cancer metastasis, and for monitoring the different stages of cardiovascular disease. [30] Gold nanoparticles are superparamagnetic compared to its bulk counterparts. It can be used as an MRI contrast agent. Also, gold nanoparticles are high-resolution candidates for X-ray contrast agents. They are reported to monitor the tumor-related microvasculature progress in cancer metastasis. The leaky capillaries produced during metastasis allow the gold nanoparticles to invade into the extravascular region of the newly malignant tissue. [31]. Also, Zuo and his colleagues reported that the iron oxide nanoparticles loaded with iRGD peptide nanocomplex can be successfully used as ultrasensitive MRI contrast agent for the detection of pancreatic cancer. [32] 3.2 Metallic Nanoparticles in Advanced Drug Delivery Drug delivery systems are designed to bring therapeutic agents to the target site efficiently with minimum toxicity to healthy surrounding tissue. [33,34] In regards to the application of metal nanoparticles as a drug delivery system, they have to overcome the reticuloendothelial system barriers such as macrophages. The phagocytic removal of nanoparticles could limit the range of their application as delivery tools in human biology and medicine. However, these nanocarriers surface can be modified with polyethylene glycol to the surface. This coating will form a 10 hydrated layer between proteins and nanoparticles, which reduces the risk of opsonization by serum proteins and internalization of nanoparticles by phagocytes. [35] Conventionally, chemotherapy target cancer cells by stopping cell division and inducing cell apoptosis/necrosis. The healthy cells, however, will also be impacted by chemotherapy, and as a result, the quality of life for patients is impaired significantly.
In this context, highly selective targeted delivery of chemotherapy is a key to efficient cancer therapy and improving cancer patients' quality of life. There are two strategies used to reduce toxicity to healthy tissue during the targeted delivery of metal nanoparticles to the cancer site: active and passive.
In active targeting, the surfaces of metal nanoparticles are conjugated with ligands, whose receptors are expressed on the tumor cell surface. The high affinity binding of the ligand and receptor will enhance the rapid internalization of the nanoparticles. In passive targeting, blood vessels in tumor tissue have a high degree of porosity because they lack angiotensin II receptors and pericyte coverage within vessels, and this leads to the enhanced permeability and retention effect (EPR) [36].
The EPR effect allows nanoparticles conjugated with anticancer therapeutics to penetrate into the extracellular tumor matrix from the tumor capillaries [37].
Besides being applied as drug delivery tools, nanoparticles are also emerging as relatively simple and effective vectors for gene delivery due to their wide-range of sizes, easier surface modification, higher loading capacity and better biocompatibility.
[38] Viral and non-viral gene transfer techniques have been widely used for gene transfer. These methods, however, have innate drawbacks such as less efficient transfer, higher toxicity and higher immunogenicity that limit their applications. As a 11 gene delivery tool, metal nanoparticles protect DNA from degradation by nucleases and facilitate the internalization of DNA into the nucleus. Guo and his colleagues reported the feasibility of applying the charge-reversal functional gold nanoparticles as a means of improving the siRNA delivery efficiency, and the final knockdown efficiency was better than that of commercial Lipofectamine 2000. [39] Other nanoparticles such as ion oxide, carbon nanotubes, magnesium phosphate and other metal nanoparticles are also candidates for DNA delivery vehicles. [40] Polyethyleneimine (PEI)-PEG-chitosan copolymer coated superparamagnetic ion oxide nanoparticles (SPIO) were also reported to deliver the plasmid DNA into the nucleus successfully and safely. [41,42] 3

.3 Metal Nanoparticles in Regenerative Therapies
Using stem cells to regenerate tissues and organs have revolutionized the current techniques in organ transplantations. This better developed technique will be a promising option to meet the needs of organ transplantation and save more lives.
Regenerative therapies using nanoparticles form a new area combining nanotechnology and engineering. Tissues and organs developed by using stem cells from an individual can be cultured in vitro and then transplanted back into the same individual using a scaffold that the body's immune system cannot distinguish to avoid rejection [43,44]. Superparamagnetic ion oxide nanoparticles (SPIO) as described above, are not only used to improve the safety and efficiency of gene delivery, but also used to improve the detection sensitivity for MRI imaging. [45] The SPIO used for MRI does not alter the function, proliferation or differentiation of hematopoietic stem 12

cells (HSC) and mesenchymal stem cells (MSC) while being nontoxic and
biodegradable. [46] Magnetic nanoparticles can be conjugated with specific antibodies for selectively targeting stem cells, which express specific receptors for antibodies. The stem cells then can be isolated and recovered by applying a magnetic field. Using this "fishing technique", Lui et al. reported the successful extraction and isolation of stem/progenitor cells from the brain of rats by using antibodies conjugated to magnetic nanoparticles (Ab-MNPs). These magnetically isolated stem cells are functional and can be differentiated into different types of cells in vitro. This technique of nanoparticle-mediated isolation and retransplantation of stem cells is now well adopted for the treatment of leukemia and several cardiac diseases. [47] Recently, immense attention has been placed on metallic nanoparticles as scaffolds in bone regeneration. [48] The nanostructure size of bones is around 1 to 100 nm. Therefore, coating the artificial implants with nano-scaled materials before transplantation would increase the biocompatibility and reduce graft rejection by the immune system. Some bone morphogenic proteins (BMPs) have been reported to play a crucial role in bone regeneration, especially in periodontal tissue [49]. They however cannot be used directly due to inherent limitations such as local inflammation. In recent years, gold nanoparticles have been used as a new generation of osteogenic agents for bone. Heo and colleagues have developed a new approach for bone tissue regeneration utilizing biodegradable hydrogel loaded with gold nanoparticles (GNPs).
The in vitro results show that GNPs loaded with hydrogels promote the differentiation of human adipose-derived stem cells (ADSCs) into osteoblast cells. Moreover, the in 13 vivo results show that the hydrogel nanostructure has a significant influence on the new bone formation and can be useful for bone tissue engineering. [50]

Pharmacokinetics and Toxicity of Metal Nanoparticles
Recently, physiologically based pharmacokinetic (PBPK) modeling is widely used by researchers to investigate the absorption, distribution, metabolism, and excretion (ADME) of metal nanoparticles. The Organization for Economic Cooperation and Development (OECD) and the new European Union regulatory framework REACH (Registration, Evaluation and Authorization of Chemicals) have published guidance, standards and quantitative tools for metal nanoparticle toxicity tests, quantitative structure-activity relationship (QSAR) and PBPK models. [51] The toxicological aspects of metal nanoparticles highly depend on its administration routes, concentrations and exposure times. Exposure to high doses and high levels of metal nanoparticles always induce toxicity. [52] Different routes of administration lead to different organ accumulation of metal nanoparticles. Also, the blood supply amount also plays an important role. After oral administration, nanoparticles may be digested by gastrointestinal (GI) tract and excreted in feces. [53]. If the nanoparticles are administrated through intraperitoneal (IP) and other injection routes, the nanoparticles will be mostly absorbed into lymph nodes [54]. No matter which route nanoparticles are administrated, most of them will be trapped in the reticuloendothelial system (RES), especially in the liver and spleen. This can be reduced or decreased however by coating the surface of nanoparticles with hydrophilic polymers such as PEG. [55]. In addition to the liver and spleen, metal nanoparticles are also found in the 14 GI tract, kidney, lung, heart, and brain. Metal nanoparticles also encounter different proteins including the complement system in the blood. After coating with these hydrophilic polymers, some of the metal nanoparticles such as copper sulfide can be metabolized and excreted in the urine and feces. [56] Although toxicity studies or be used for clinical applications directly. In the future, establishing a precise toxicological study platform for evaluating metal nanoparticles in medicine is essential and required for improving its safety on human life.

Summary
The recent development of nanotechnology and material sciences has created a 15 plethora of studies in metal nanoparticles. The results of these studies are widely applicable in our daily lives, especially in the medical field for use in high contrast imaging, drug delivery, and therapeutics. In the future, applications of metal nanotechnology show great promise in improving our health and daily lives. The attractive properties of metal nanoparticles such as their ability to be circulated in the blood and translocated successfully into the nucleus due to their small size also make them very dangerous. Diseases from the exposure to metal nanoparticles may not be diagnosed immediately, but will manifest in the future. Many metal nanoparticles also have low metabolic rates compared to their bulk counterparts. In short, metal nanoparticles will have a huge impact on our lives in the future. Therefore, standards and regulations should be created to protect us from the toxicity and adverse effects of metal nanoparticles. 16 Cu nanoparticles by mechanochemical processing using a ball mill. Mater.   Semiconductor copper sulfide (CuS) nanoparticles, a class of new nanomaterials, have been widely reported for their biomedical applications in recent years. The intriguing optical and physicochemical properties of CuS nanoparticles endue them with great potential in cancer photothermal ablation therapy, 1-4 drug delivery, 5-7 photoacoustic imaging, 8 nuclear medicine [9][10][11][12] and their combinations. 7,[9][10][11][12][13][14][15][16] Although Cu is an essential element in the body, excess free Cu ions in excess are toxic. 17 A recent study has shown that CuS nanoparticles are much less cytotoxic than copper oxide nanoparticles because of the extremely low solubility of CuS. 18 Through histological and biochemical examinations, we found that pegylated hollow CuS nanoparticles (PEG-HCuSNPs) do not induce significant liver toxicity during the 3month study period. 19 PEG-HCuSNPs exhibit a reversible change in the proteomic profile in liver, offering long-term safety benefit. 19 With the above evidence, we believe that the slow dissociation rate of Cu ions from CuS nanoparticles, along with effective mechanisms of Cu elimination, may allow physiological homeostasis to be reached at an optimized dosing schedule without drastically increased toxicity.
Metabolism of CuS nanoparticles, accordingly, is critical to Cu homeostasis and toxicity.
Existing pharmacokinetic analyses have shown that CuS nanoparticles can be metabolized. 11,19 CuS nanoparticles, if smaller than the effective cut-off size for glomerular filtration (~10 nm), are mainly excreted from the kidney. 11 Ultra-small CuS nanoparticles with hydrodynamic diameters less than 6 nm have minimally nonspecific uptake by the mononuclear phagocyte system and highly efficient clearance via the renal-urinary system. 11 Comparatively, clearance of CuS 27 nanoparticles larger than 10 nm is slower than that of the ultra-small CuS nanoparticles. 11,19 We have previously demonstrated that about 67% of the injected dose is eliminated from the liver within one month postinjection of PEG-HCuSNPs of ~70 nm in diameter. 19 Therefore, liver is the major organ metabolizing these relatively "large" CuS nanoparticles. 11,19 However, the mechanism of hepatic metabolism of CuS nanoparticles remains unknown.
Even though different kinds of gold nanoparticles with bioinert properties have a great advantage in the photothermal transaction and are promising for clinical applications, 20, 21 they are non-biodegradable, raising concerns regarding their longterm metabolism and safety. 22,24 Tail vein injections of polyethylene glycol (PEG)coated gold nanoparticles have been reported to induce two phases of toxicity due to inflammation in the liver. The acute phase occurred immediately after administration of nanoparticles. The second phase happened at 7 days when the particles become localized in the tissues, mostly in the liver and spleen after circulation in the blood. 25 Due to the multiple valences of gold, redox reaction of gold within cells can increase the levels of reactive oxygen species, which interferes with the mitochondrial membrane potential. Disturbance of the mitochondrial membrane potential after exposure to gold nanoparticles initiates an apoptotic cascade in cells. 26,27 In this study, we use PEG-HCuSNPs as models to investigate their trafficking in hepatocytes and Kupffer cells in vivo, and to elucidate their metabolic pathway in mouse liver. It is generally believed that nanoparticles predominantly interact with and are engulfed by cells of the mononuclear phagocyte system, such as Kupffer cells in the liver, once these nanoparticles enter the blood. 28 Distinctively, we find that the 28 The swift disintegration of PEG-HCuSNPs may be ascribed to the interaction between the nanoparticles and cellular components, facilitating the dissociation of Cu ions. To prove this hypothesis, we prepared various media to investigate their effects on Cu 2+ release from PEG-HCuSNPs. As shown in Figure S1A, there was low dissociation of Cu 2+ dissociated from PEG-HCuSNPs in phosphate buffered saline (PBS) due to the extremely low solubility of CuS. 18 Addition of amino acids to the medium significantly increased Cu 2+ release, due to the formation of copper/amino acid complexes. 29 Interestingly, in PBS supplemented with cysteine (Cys) or Cys-HCl, the Cu ion concentration at 24 h was lower than at 1 h or 4 h. This was because Cu 2+ can induce the oxidation of Cys to cystine, thus precipitating the Cu. 30 In addition, glutathione (GSH) or metallothionine, considered to be part of the Cu storage system, 31 enhanced Cu ion dissolution (Fig. S1B). TEM confirmed that with GSHtreatment, PEG-HCuSNPs collapsed and transformed from hollow structures to solid particles with small CuS particle dissociation (Fig. S1C, arrowheads). A similar phenomenon was observed in PEG-HCuSNPs treated with metallothionine (Fig. S1D, arrowheads). Also, media at lysosomal pH (pH 4.9) 32 significantly increased 32 dissolution of Cu ions compared to the physiological pH (Fig. S1E). These findings supported the finding that cellular components such as amino acids and Cu storage systems interacted with PEG-HCuSNPs to facilitate their dissociation and further release of Cu ions. Acidic lysosomal conditions may also increase Cu dissolution from the particles. In late endosomes with multivesicular structures, the nanoparticles also broke down into small dots but with smaller amounts (Fig. 3C, white, pink and orange arrows).

Efficient excretion of PEG-HCuSNPs by hepatocytes and
Some decomposed CuS nanoparticles were in vacuoles inside the late endosome (Fig.   3C, orange arrows), suggesting that decomposition of nanoparticles occurred during early-to-late endosomal maturation. Figure 3D illustrated that the nanoparticles were excreted from hepatocytes through exocytosis. The nanoparticles either collapsed or disintegrated in the exosomes (Fig. 3D, arrowheads). In the exosomes released directly from the plasma membrane, the nanoparticles were indistinguishable because of light electron dense (Fig. 3D, arrows). However, a great number of CuS nanoparticleloaded vesicles (Fig. 3E, arrows) appeared inside the multivesicular bodies fused to the plasma membrane (Fig. 3E, the enlarged image with yellow margin) and excreted out of the cell (Fig. 3E, the enlarged image with red margin). These findings proved that the decomposed CuS nanoparticles were released from hepatocytes through exocytosis by multivesicular bodies such as late endosomes or lysosomes. finding showed that the liver quickly launched endogenous detoxification mechanism in hepatocytes in the presence of high loading of PEG-HCuSNPs. Remarkably, elevation of ATP7B on plasma membrane was substantially more than that in cytosol, indicating that more ATP7B trafficked to the apical side membrane in order to accelerate Cu ion excretion for homeostasis. 33,39 Nevertheless, the cytoplasmic ATP7B declined to 71% at 4 h and further to 66% at 24 h compared to basal levels, while membrane ATP7B returned to baseline. One possible explanation was that the cellular storage of ATP7B was exhausted and production of ATP7B did not meet its demand. Reduction of ATP7B would decrease the cellular capability of removing Cu in hepatocytes, subsequently raising intracellular Cu ion level and causing potential toxicity. This hypothesis was supported by the mRNA analysis that the Atp7b levels declined to ~50% of baseline at 4 h postinjection (Fig. 4D). However, the mRNA transcription restored at 24 h, indicating that hepatocytes quickly recovered from the toxicity and regained ATP7B productivity. Concomitantly, protein expression of cytoplasmic ATP7B was restored to baseline at 2 d postinjection, which was 1 d after recovery of mRNA transcription (Fig. S3). Our previous study demonstrated a reversible change in the proteomic profile of liver tissues in mice receiving PEG-HCuSNPs through matrix-assisted laser desorption ionization-time-of-flight imaging mass spectrometry (MALDI-TOF). 19 The current results from ATP7B analysis, similar to the previous result with proteomic profiling, confirmed that PEG-HCuSNPsinduced toxicity was transient and reversible at a therapeutic dose of 20 mg/kg of Cu.
Cuillel et al. disclosed the metabolic mechanism of CuO nanoparticles in hepatocytes. 40 The CuO nanoparticles underwent fast dissolution once it is delivered 36 to endo/lysosomes after endocytosis. The burst release of Cu ions caused destabilization/disruption of lysosomes, resulting in Cu 2+ overload in the cytoplasm.
ATP7B played a key role in binding and removing Cu ions in cytosol. 40 In contrast, because of the extremely low solubility of CuS, dissolution of Cu ions from CuS nanoparticles was slow. 18  receptors. 46 Complement C3b and iC3b are products of C3 activation, while complement C5a is a fragment of C5. In order to confirm C3-coating on the nanoparticles, we incubated PEG-HCuSNPs with 10% mouse serum followed by centrifugation of the nanoparticles. The nanoparticle-bound proteins were then isolated and analyzed by Western blot. Figure 5A illustrated that C3 and its fragment Enhanced excretion of gold nanoparticles by HCuSNPs conjugation through ATP7B. We isolated primary hepatocytes from BALB/c mice by our modified two-step collagenase digestion method 19,37,38 with high purity (Fig. 3A). It is reported that functional bile canaliculi can be detected by fluorescence microscopy following 30 min incubation with 5 (6)-carboxy-2',7'-dichlorofluorescein diacetate (CDFDA), which can passively diffuses into hepatocytes, where it is hydrolyzed to fluorescent carboxy-dichlorofluorescein (CDF) only by functional polarized hepatocytes. After that, CDF is excreted into bile canaliculi. 49 After 48 h incubation of the seeded hepatocytes on collagen gel, much more CDF was formed and excreted into bile canaliculi compared to the cells only incubated for 24 h. (Fig. 6A) The ATP7B also correlative formed more after 48 h incubation compared to the 24 h incubation, and stayed around the nucleus. (Fig. 6B) The above data denoted that the perfused hepatocytes need 48 h to form ATP7B and for the polarized anatomy structure-bile canaliculi to function properly. After that, we treated cells with PEG-42 HCuSNPs-RITC and CDFDA together and found that HCuSNPs colocalized with CDF, which can be excreted into bile canaliculi. It suggested HCuSNPs were excreted into bile canaliculi at last. Meanwhile, PEG-HCuSNPs-FITC was also colocalized with ATP7B ( Fig. 6C and 6D), denoted that HCuSNPs were excreted into bile canaliculus facilitated by ATP7B.
With this finding, we then want to find out whether the excretion of HCuSNPs can be used to enhance the gold nanoparticles excretion. Gold nanosphere (~40nm) conjugated to the HCuSNPs (PEG-HCuSNPs@Au) (Fig. 7A) were used to treat primary cultured hepatocytes with CDFDA. After 30 min incubation, PEG-HCuSNPs@Au was colocalized with CDF and ATP7B, (Fig. 7B and 7C) however, in PEG-AuNP group, CDF was excreted into the bile canaliculi without the PEG-AuNP (Fig. 7B). Moreover, PEG-AuNPs after endocytosis were not colocalized with ATP7B in the cultured hepatocytes and still stayed in cells. (Fig. 7C) The quantitative analysis of gold exocytosis rate between PEG-AuNP and PEG-HCuSNPs@Au was also studied using transwells seeded with mouse and human primary hepatocyte to mimic the in vivo environment. In mouse primary hepatocytes, 36.10% of PEG-AuNPs were excreted out in the first 30 min and kept at around 15% after 60 min exocytosis process, however, 55.10% of PEG-HCuSNPs@Au were excreted in the first 30 min and keep at around 8% after 60 min exocytosis. (Fig. 8A and 8B) In human primary hepatocyte, 36.64% of PEG-AuNPs were excreted in the first 10 min and kept at around 35% after 60 min exocytosis. PEG-HCuSNPs@Au excreted 54.47% in the first 10 min and kept at around 17% after 60 min exocytosis. (Fig. 8C and 8D) It suggested from the mouse and human primary hepatocytes transwell studies that PEG-43 HCuSNPs@Au had a much faster excretion rate than the PEG-AuNPs alone.
Meanwhile, less gold was left after 60 min excretion in the PEG-HCuSNPs@Au treated group, which denoted that HCuSNPs not only increased the gold excretion rate, also enhanced the clearance of gold from the hepatocytes significantly and had less Besides the gold sphere, we also synthesized gold nanorod (AuNR) (~40nm) and AuNR conjugated with CuSNPs(~5nm) (PEG-AuNR@CuS) (Fig. 10A) to compare the excretion process between gold nanorod and PEG-AuNR@CuS. At 5 min after the CuSNPs treatment, PEG-CuSNPs already colocalized with ATP7B, which moved from cytosol to along with the bile canaliculi in a fast rate. The same observation happened in the PEG-AuNR@CuS treated group. (Fig. 10B and D) However, after 35 min treated with PEG-AuNR alone, the nanorods were still within the hepatocytes and not overlap with ATP7B. (Fig. 10C) After 35 min incubation with CDFDA, PEG-CuSNPs and AuNR@CuS were excreted into bile canaliculi together with CDF. However, PEG-AuNRs were still within hepatocytes. (Fig. 10E) It suggested that conjugating PEG-AuNR@CuS facilitated the excretion of PEG-AuNR into bile canaliculi. Meanwhile, the exocytosis rate of gold was compared between PEG-AuNR and PEG-AuNR@CuS using transwells seeded with primary cultured 44 mouse and human hepatocytes to mimic the in vivo environment. In mouse primary hepatocyte, in the PEG-AuNR treated group, 38.3% of gold were excreted out in the first 10 min and still 10% left after 60 min exocytosis, However, 87.7% of gold in the PEG-AuNR@CuS treated group were drained in first 10 min, and less than 1% left after 60 min exocytosis. (Fig. 11A and 11B) The fast excretion rate of PEG-AuNR@CuS also happens in human primary hepatocytes. In the PEG-AuNR treated group, 59.6% of gold were excreted out in the first 10 min and still 1.5% left after 60 min exocytosis, However, in the PEG-AuNR@CuS treat group, 80.5% of gold in the AuNR@CuS treated group were drained in first 10 min, and less than 0.7% left after 60min exocytosis. (Fig. 11C and 11D). Based on the transwell endo/exocytosis study, even though we synthesized gold and copper sulfide nanoparticles in different sizes and shapes, the exocytosis rate of gold will increase dramatically after conjugation of gold and copper sulfide nanoparticles compared to gold nanoparticles only. 45

CONCLUSION
In conclusion, we clearly elucidated the liver metabolic pathway of PEG-HCuSNPs in which hepatocytes played a major role in taking up, degrading and eliminating the nanoparticles through efficient hepatobiliary excretion. Cu-ATPase lysate was collected and transferred into a 1.5 mL Eppendorf tubes. Then, the lysate was centrifuged at 13,523 g for 10 min and the supernatant was removed from the pellet and stored at 4 o C separately. 2) Exocytosis: Endocytosis is the same process as described above. After 5 min incubation at 37 o C, media with nanoparticles in the upper chamber side was aspirated and quickly washed with 500 uL warm WME once, and then 500 µL of fresh cell culture media was added into the upper chamber side.
The cell culture insert was transferred to another well with 1.5 mL of cell culture media. After 10 min, the upper chamber media was aspirated and washed with 500 µL of warm WME on both sides. Then 100 µL CelLytic M cell lysis reagent was added to the upper chamber and cell lysate was transferred into a 1.5 mL Eppendorf tubes.
After another 30 min and 1 h, the above exocytosis step was repeated. The collected cell lysates were centrifuged at 13,523 g for 10 min, and the pellet was moved from the supernatant for the ICP-MS analysis.      with 10% freshly collected mouse serum from two mice, followed by centrifugation to separate nanoparticles (pellet) from the serum (supernatant). Samples were loaded for Western blotting analysis using rat anti-mouse C3 mAb or rat anti-mouse C5a mAb as first antibody. Lanes (1) and (7), supernatant from PEG-HAuNS-incubated serum;