Hyaluronate – Black Phosphorus Conjugates as a Copper Chelating Agent for Wilson Disease Treatment

Background: Wilson disease (WD) is a genetic disorder of copper storage, resulting in pathological accumulation of copper in the body. Because symptoms are generally related to the liver, chelating agents capable of capturing excess copper ions after targeted delivery to the liver are highly required for the treatment of WD. Methods: We developed hyaluronate - black phosphorus (HA-BP) conjugates for capturing copper ions accumulated in the liver for the treatment of WD. Results: HA-BP conjugates showed high hepatocyte-specific targeting efficiency, selective copper capturing capacity, excellent biocompatibility, and biodegradability. HA enhanced the stability of BP nanosheets and increased copper binding capacity. In vitro cellular uptake and competitive binding tests verified targeted delivery of HA-BP conjugates to liver cells via HA receptor mediated endocytosis. The cell viability test confirmed the high biocompatibility of HA-BP conjugates. Conclusion: HA-BP conjugates would be an efficient copper chelating agent to remove accumulated copper in the liver for the WD treatment.


Background
Wilson disease (WD) is an inborn disorder of copper metabolism and characterized by copper overload in the organs, especially liver and brain [1]. In patients with WD, copper cannot be eliminated properly, causing liver cirrhosis and liver transplantation in severe cases [2]. Accordingly, WD is one of the most challenging diseases in medicine. There are two available treatments to increase copper excretion and reduce copper absorption by using (1) chelators and (2) zinc salts [3]. Combination therapy using zinc salts and chelators leads to clearing copper overloaded tissues and blocking the pathological accumulation of copper in the organs [4,5]. However, the long-term use of the medication is limited because of the safety issues and severe adverse effects [6]. Chelators, such as D-penicillamine and trientine, have been reported to cause adverse events like marrow toxicity, lupus-like syndrome, and anemia [7,8]. As a suitable chelator, the agent should be able to efficiently capture excess transition-metal ions with excellent biocompatibility and biodegradability.
Recently, various nanomaterials have been widely investigated as novel chelators to form nontoxic metal complexes [9]. In addition, nanoparticlemediated drug delivery efficiently reduced several toxic metals in the body [10].
However, there are few in-depth studies focusing on targeted delivery of chelating agents to the liver for the treatment of WD.
Here, we developed a hyaluronate -black phosphorus (HA-BP) conjugate to remove accumulated copper, especially in the liver. Black phosphorus (BP) nanosheets are well known as a biodegradable 2D material composed of phosphorus atoms [11,12]. Due to its great biocompatibility, BP nanosheets have been used for biomedical applications including phototherapy, drug delivery, and biocatalysis [13]. Furthermore, phosphorus binds strongly with metal ions, especially Cu 2+ , making BP nanosheets a robust nanocaptor for copper ions [9]. To prevent the rapid degradation of BP nanosheets from oxidation, BP nanosheets were coated with HA [14]. HA is a natural biodegradable polymer with a high binding affinity toward liver cells [15,16,17,18]. After the physicochemical characterization of HA-BP conjugates, we investigated the copper capturing capability, biocompatibility and biodegradability via in vitro liver-specific targeted delivery of HA-BP conjugates for WD treatment. After centrifugation for 15 min at 12,500 rpm, the concentration of metal ions in the supernatants was measured using an atomic absorption spectrometer (TAS-990, Puxi, China). The binding capacity was calculated by the following equation as previously reported elsewhere [9]:

Materials
where C T represents the total concentration of metal ions in the mixture and Cs represents the concentration of metal ions in the supernatant.

Statistical analysis
Statistical comparison was performed using the software SigmaPlot 10.0 (Systat Software Inc. San Jose, CA). Values for *P < 0.05 and ***P < 0.001 were considered significant. Enhanced stability and biodegradation of HA-BP conjugates BP nanosheets are known to be decomposed into phosphoric acid (PA) in the presence of oxygen [19]. HA can prevent the rapid degradation of BP nanosheets under oxygen circumstance and aggregation with the adsorption of serum proteins [20,21]. To assess the effect of HA on the stability of BP nanosheets, BP nanosheets and HA-BP conjugates at the equal concentration were dispersed in DI water. As shown in Figure 2a, the absorbance of BP nanosheets was significantly decreased for 7 days and barely remained at day 6. In contrast, HA-BP conjugates were stable without precipitation and the absorbance spectra was nearly unchanged after 4 days (Figure 2b). In addition, the enhanced stability of HA-BP conjugates was assessed by the fluorescent intensity of FITC (Figure 2c). In vitro release of FITC indicated that FITC or HA-FITC was separated from BP-FITC or HA-BP-FITC conjugates by degradation.

Results
The amount of FITC released from HA-BP-FITC conjugates was relatively small with improved stability, and the concentration of FITC was consistent with the degradation rate (Figure 2a and b). The colour of BP nanosheets solution became thin after 7 days, compared to that of HA-BP conjugates (Figure 2d).
All these results confirmed that HA efficiently protected BP nanosheets from subsequent degradation.

Copper specific capturing of HA-BP conjugates
The Cu 2+ capturing capacity of BP nanosheets and HA-BP conjugates was investigated by mixing their solutions with Cu 2+ solution for 1 h, centrifugation, and dispersion in DI water. The TEM images showed that BP nanosheets and HA-BP conjugates maintained their structures after binding with Cu 2+ (Figure   3a). After 7 days, the degraded BP nanosheets and the degraded HA-BP conjugates interacted with Cu 2+ . The TEM images showed that only HA-BP conjugates still maintained the nanosheets structure binding with Cu 2+ (Figure   3a). The morphology of BP nanosheets disappeared and the spherical shape was obtained only due to copper [22]. To evaluate the effect of degradation on copper binding capacity, BP nanosheets and HA-BP conjugates were prepared at the desired degradation time point from day 0 to 6. After mixing the solution of BP nanosheets or HA-BP conjugates with Cu 2+ solution for 1 h, the mixture was centrifuged and the concentration of Cu 2+ in supernatants was analyzed with an atomic absorption spectrometer. As shown in Figure 3b, the Cu 2+ capturing capacity of BP nanosheets was dramatically decreased for 7 days. In contrast, the copper binding capacity of HA-BP conjugates was only slightly decreased owing to the HA coating, which was well matched with the degradation rate of HA-BP conjugates in Figure 2b.
Meanwhile, the copper specific binding of BP nanosheets and HA-BP conjugates was assessed with an atomic absorption spectrometer by comparing the intensity change of each metal ion before and after mixing for 1 h (Figure 3c). BP nanosheets and HA-BP conjugates selectively interacted with Cu 2+ , which indicated the excellent copper capturing specificity. The concentration of other metal ions, such as Ca 2+ , Mg 2+ , and Zn 2+ was nearly unchanged. The reason might be that the Gibbs free energy change (ΔG) of BP-Cu or HA-BP-Cu complex was much lower than that of BP-metal or HA-BPmetal complexes [9]. The binding capacity of HA-BP conjugates to Fe 2+ was slightly increased because of chelating effect of HA [23]. In addition, the copper capturing capacity of HA-BP conjugates was increased through the chelatormetal complex via amine groups of HA-DAH [6]. CD44 [20,24]. To study the cellular uptake mechanism of HA-BP-FITC conjugates, HepG2 cells were pre-treated with excess HA. The fluorescence of HA-BP-FITC conjugates was reduced in the cells pre-incubated with HA, which revealed that the uptake of HA-BP-FITC conjugates was mediated by HA receptor-mediated endocytosis. The in vitro biocompatibility of BP nanosheets and HA-BP conjugates was analyzed by CCK-8 assay. As shown in Figure 5b, the viability of HepG2 cells after treatment with HA-BP conjugates was slightly higher than that with BP nanosheets, which might be ascribed to the biocompatible HA coating [25,26].

Discussion
This study aimed to protect BP nanosheets from rapid degradation in oxygen circumstances and increase copper binding capacity for WD treatment.
Previous studies have reported limitations of BP nanosheets in biomedical applications due to the rapid degradation of BP nanosheets [13,19]. As shown in Figure 2 and 3, BP nanosheets were rapidly decomposed into phosphorus atoms and copper binding capacity also decreased with the degree of decomposition. To overcome this issue, BP nanosheets were coated with various polymers such as polyethylene glycol (PEG), HA, and poly lactic-coglycolic acid (PLGA) [27,28]. However, there are no previous studies of the change in the copper binding capacity of BP nanosheets after surface modification. Also, although the liver is a major organ where excess copper accumulates in WD, there are few in-depth studies on delivering chelating agents to the liver for the treatment of WD. In this research, BP nanosheets were modified with HA for improved stability and targeted delivery to the liver. HA has been widely investigated in the biomedical field due to its excellent biocompatibility and targeted delivery to the liver or tumor [17,18,20]. Our results demonstrated that HA successfully prevented BP nanosheets from rapid degradation and maintained the Cu 2+ capturing capacity of BP nanosheets after degradation for 7 days (Figure 2 and 3). The most significant implication was that the copper binding capacity of HA-BP conjugates was higher than that of BP nanosheets, as shown in Figure 3b and c. Amine groups of HA-DAH can react with transition metals via chelator-metal complex, such as Fe 2+ and Cu 2+ , and may increase copper binding capacity [6]. Moreover, the cellular uptake of HA-BP conjugates into the liver cancer cells, HepG2, demonstrated the liver cell targeted delivery of HA-BP conjugates (Figure 5a). Although the exact mechanism underlying the improved copper capturing capacity of HA-BP conjugates was not evaluated in this study, our results sufficiently showed enhanced stability, copper-specific binding capacity, and hepatic targeted delivery of HA-BP conjugates. This study could be further processed for in vivo animal studies of WD treatment, as schematically shown in Figure 6.

Conclusion
We       Schematic illustration of HA-BP conjugates as a copper chelating agent to remove copper ions accumulated in the liver for WD treatment.