English

• Malignancy is one of the most serious public health problems in the world^[1-2]. People have conducted a large number of studies on cancer treatment, such as surgery, chemotherapy, and radiotherapy^[3-5]. Due to the lack of specificity and targeting aimed at chemotherapy, drug therapy inevitably incurs side effects such as damage to healthy cells, drug resistance, risk of tumor recurrence, and even destruction of the body′s own system^[6-9]. It is urgent to develop drug carrier systems for achieving high-resolution diagnosis and effective tumor treatment. Melanin, as an endogenous natural biopolymer in the body, has attracted more and more attention because of its excellent physical and chemical properties^[10-12]. The melanin nanoparticles (MNP) have good water solubility, biological safety, strong metal ion chelating ability, near-infrared light absorption, anti- inflammatory, and free radical scavenging functions, and can be used for single/multi-mode imaging such as magnetic resonance imaging (MRI), positron emission tomography (PET), and photoacoustic imaging^[13-15]. However, the small size of MNP (< 10 nm) is easy to be cleared through kidney metabolism, and the MNP accumulation at the tumor region is relatively limited. To overcome this obstacle, there is an urgent need to develop an MNP with a targeting ability that can improve tumor accumulation for achieving accurate detection and effective treatment.

  Hyaluronic acid (HA) and (4-carboxybutyl) triphenylphosphonium bromide (TPP) have been widely studied in tumor therapy because of their excellent active targeting ability^[16-19]. HA can interact with overexpressed receptors in cancer cells, such as cluster determinant 44 (CD44) and HA motility receptor (RHAMM), and can be degraded by a family of enzymes called hyaluronidase (HAdase) to release drugs or molecules^[20]. HA-based nanomaterials allow enhanced targeted cancer therapy through interaction with receptors or enzymatic degradation within cancer cells^[21-23]. As a drug delivery system, HA has been combined with various chemical drugs such as paclitaxel (PTX) and doxorubicin (Dox), as well as other biopharmaceuticals. Therefore, HA as a target ligand has been widely studied in the field of drug delivery^[24]. TPP is a lipophilic cationic substance that is currently being extensively studied for mitochondrial targeting^[25-28]. Due to the electrostatic attraction between the positively charged TPP part and the negatively charged mitochondrial membrane, the TPP can be effectively attached to the mitochondria.

  To improve the targeting ability of melanin, we constructed a dual-target nanoparticle (MNP-TPP-HA) synthesized by 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxy succinimide (NHS) reaction based on activated carboxyl groups on HA and TPP and amine groups on MNP-polyethylene glycol-amino (PEG) (Scheme 1). In comparison with the MNP-HA and MNP-TPP nanoparticles (MNP-TPP NPs) group with single-target features, dual-target MNP-TPP-HA promotes the enrichment and retention of melanin at the tumor site through the combination of cell membrane targeting of HA and mitochondrial targeting of TPP. In conclusion, MNP-TPP-HA may be a promising mitochondrial delivery system, laying a foundation for melanin drug delivery in the treatment of tumors.

  Scheme 1

  

  Scheme 1.  (a) Schematic representation of the preparation of MNP-TPP-HA and (b) dual-targeted tumor photoacoustic imaging

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  1.   Experimental

  1.1   Materials

  Melanin was obtained from Sigma-Aldrich. Sodium hydroxide (NaOH) and hydrochloric acid (37% HCl) were obtained from Sinopharm Chemical Reagent Beijing Co., Ltd. NH₂-PEG-COOH (M_w=5 kDa) was purchased from Beijing Solar Energy Biotechnology Co., Ltd. NHS, EDC, HA, and TPP were provided by Energy Chemical (Shanghai, China). The Cell Counting Kit-8 (CCK-8) was purchased from Dojindo Molecular Technologies, Inc. (Shanghai, China). Dulbecco′s modified Eagle′s medium (DMEM), Dulbecco′s phosphate buffered saline (PBS), fetal bovine serum (FBS), and trypsin were purchased from Invitrogen Corp. All reagents were used directly without further purification.

  1.2   Characterization

  The surface morphology and size of NPs were investigated by JEM-2100 transmission electron microscopy (TEM, 80-300 kV Hitachi, Japan). ζ potential was measured by a Zetasizer (Nano-Z, Malvern, UK). Ultraviolet (UV) absorption spectra were obtained using a Cary 5000 UV-Vis-NIR spectrophotometer (Varian, USA). The absorbance of the plates was read at the corresponding wavelength using a microplate reader (Multiskan FC, Thermo Fisher Scientific). Cell fluorescence images were taken by confocal laser scanning microscopy (CLSM, HP Apo TIRF 100X N.A. 1.49, Nikon, Ti-E-A1R, Japan).

  1.3   Synthesis of MNP-TPP, MNP-HA, and MNP-TPP-HA

  The MNP were modified with NH₂-PEG-COOH according to the previous work^[29]. MNP-PEG was modified with HA and TPP molecules by an amidation reaction. To be specific, adding 10.00 mg EDC (0.05 mmol) and 10.00 mg NHS (0.09 mmol) to a 2 mL solution containing TPP (0.005 mmol) and HA (0.005 mmol), the carboxyl groups of TPP and HA are activated^[30]. After 20 min, the carboxy-activated TPP and HA solution were added to 4 mL MNP-PEG solution, and then the solution was stirred for 4 h and centrifuged by ultrafiltration to obtain MNP-TPP, MNP-HA, and MNP-TPP-HA.

  1.4   Cell culture

  Murine mammary carcinoma (4T1) cells were cultured in DMEM medium with 10% FBS and 1% antibiotics at 37 ℃ in a humidified atmosphere containing 5% CO₂.

  1.5   Flow cytometric analysis on sample uptake

  4T1 cells were seeded into 6-well plates at an initial cell density of 1×10⁵ units per well. After incubating overnight, the old media was replaced with new ones containing MNP-PEG, MNP-HA, MNP-TPP, and MNP-TPP-HA, and further incubated for 12 h. The sample concentrations were maintained at an equivalent level of 100 μg·mL^-1. The media was then removed, and the cells were washed once with PBS, digested with trypsin without Ethylenediaminetetraacetic acid disodium salt (EDTA-Na), and again washed twice before centrifugation. The fluorescence intensity of the internalized samples in each group was quantified by flow cytometry (CytoFLEX, Beckman Coulter).

  1.6   Cell viability test

  The in vitro cytotoxicity of NPs was determined using a CCK-8 assay. 4T1 cells were cultured in a complete medium with Roswell Park Memorial Institute (RPMI) 1640 (89%) + FBS (10%) + penicillin/streptomycin (1%) at 37 ℃ in CO₂ (5%) atmosphere. After preparing 4T1 cells as a cell suspension, they were seeded in 96-well plates at a density of 1×10⁴ units per well, cells with log growth were grown to 80% with different concentrations (25, 50, 100, 200, 400, 800, and 1 200 μg·mL^-1) of samples (MNP-PEG, MNP-HA, MNP-TPP, and MNP-TPP-HA) and blank control group was set with 6 compound wells in each group. After further incubation for 24 h, cells were washed by PBS three times. Finally, 100 μL fresh medium containing 10 μL of CCK-8 was added to each well, followed by another 1 h of incubation. The cell viability was measured by CCK-8 assay.

  1.7   Penetration behavior of different samples under three-dimensional MTSs condition

  4T1 cells (2×10³) were inoculated on Corning′s ultra-low adsorption 96-well plate and the medium was replaced every 2 d with fresh DMEM containing 10% fetal bovine serum^[31-32]. After 7 d, the three-dimensional (3D) multicellular tumor spheroids (MTSs) model was established. To demonstrate our system capable of penetrating tumor tissue, the MTSs were treated with MNP-PEG, MNP-HA, MNP-TPP, and MNP-TPP-HA with different concentrations. Afterwards, the MTSs were observed by CLSM.

  1.8   Animals and tumor model

  Balb/c nude mice (8 weeks old, 18-20 g) were obtained from Weitong Lihua Experimental Animal Technology Co., Ltd (Beijing). All animal experiments were carried out under the permission by Shanxi Medical University Laboratory Animal Center. 4T1 cells were subcutaneously injected into the right thigh of each nude mice to establish a 4T1 tumor-bearing mice model. Tumor-bearing mice were used when the tumor volume reached 100 mm³.

  1.9   In vivo photoacoustic imaging

  Using the tumor-bearing mice model established above, the mice were treated with MNP-PEG (200 μL, 2 mg·mL^-1) and MNP-TPP-HA (200 μL, 2 mg·mL^-1) via intravenous injection and imaged after 0, 2, 5, 7, 12, and 24 h on the multispectral optoacoustic tomography (MSOT) imaging system.

  2.   Results and discussion

  2.1   Synthesis and characterization

  It is commonly accepted that melanin is a heterogeneous biopolymer composed of 5, 6-dihydroxyindole (DHI) and 5, 6-dihydroxyindole-2-carboxylic acid (DHCIA) units^[10]. To further prolong the circulation time in blood and enhance the in vivo interaction, NH₂-PEG5000‐NH₂ was introduced to the surface of ultrasmall MNP. After conjugation with PEG chains, the MNP possessed excellent solubility and biosafety, and the average size of MNP-PEG was around 7 nm (Fig. 1a). Subsequently, the MNP-PEG was modified by HA and TPP, in which the carboxyl groups of HA and TPP could be readily coupled to the amine group of MNP-PEG via the EDC/NHS reaction. As shown in Fig. 1d, MNP-TPP-HA poddessed a well-defined spherical shape with uniform size. TEM images also revealed that MNP-TPP-HA were slightly aggregated, which could be ascribed to the weak interaction between ligands.

  Figure 1

  

  Figure 1.  (a) DLS analysis, (b) ζ potential, (c) UV-Vis absorption spectra of MNP-PEG, MNP-HA, MNP-TPP and MNP-TPP-HA; (d) TEM image, (e) particle size stability of MNP-TPP-HA

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  The dynamic light scattering (DLS) results showed that the diameter of MNP-TPP and MNP-HA were close to 10 and 15 nm respectively, which is slightly larger than that of MNP-PEG (Fig. 1a). The particle size of MNP-TPP-HA was about 20 nm larger than the size observed by TEM (Fig. 1a and 1d), mainly due to the interaction of the surrounding water molecules with the nanoparticles. The size diameter of nanoparticle had been investigated in PBS solution for 72 h. There was no obvious change in particle size (Fig. 1e), indicating that the nanoparticle remains stable in the physiological environment. The ζ potential results revealed that the four kinds of nanoparticles showed negative ζ potential and remained stable in the physiological environment. Compared with MNP-PEG, the ζ potential increased from -9.6 mV to -10.6 mV for MNP-HA and -4.5 mV for MNP-TPP, verifying the successful conjugation of HA or TPP. The ζ potential of MNP-TPP-HA was -6.3 mV, considering that TPP could neutralize the negative charges of MNP-HA (Fig. 1b). As shown in Fig. 1c, the characteristic absorption peak of TPP centered at 260 nm was observed, confirming the successful modification of TPP onto MNP-HA. These experiment results all confirmed the successful synthesis of MNP-TPP-HA. The mass ratio of MNP-PEG, TPP, and HA was 10∶9∶3 by calculation.

  2.2   In vitro cellular uptake

  We investigated cellular uptake to assess the uptake efficiency of four kinds of different samples. The results showed that the average fluorescence intensity of MNP-HA and MNP-TPP was 3.2 times and 2.8 times higher than that of MNP-PEG group, respectively (Fig. 2a). Significantly, flow cytometric analysis showed the highest uptake ability from MNP-TPP-HA. As known to all, HA-modified MNP can specifically recognize CD44 receptors on the surface of tumor cells and enter the cells through receptor-mediated pathways. Moreover, TPP can effectively attach to the mitochondria by the electrostatic attraction between the positively charged TPP and the negatively charged mitochondrial membrane, improving the targeting ability of nanoparticles. For these reasons, TPP and HA reinforce synergistically the target ability of MNP, achieving perfectly double targeting performance of MNP-TPP-HA and further improving the cell uptake efficiency of nanoparticles.

  Figure 2

  

  Figure 2.  (a) Flow cytometric uptake analysis; (b) Viability of the cells treated with MNP-PEG, MNP-HA, MNP-TPP, and MNP-TPP-HA respectively

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  2.3   Cytotoxicity assay

  After 4T1 cells treated with MNP-PEG, MNP-HA, MNP-TPP, and MNP-TPP-HA, CCK-8 was used to detect the survival rate of the cells. As shown in Fig. 2b, there was no obvious toxicity in the cells after treated with four kinds of samples in the concentration range of 25-1 200 μg·mL^-1, indicating that MNP-PEG, MNP-HA, MNP-TPP, and MNP-TPP-HA have good biocompatibility.

  2.4   In vitro three-dimensional tumor model

  To further verify the enhanced cell penetration effect of dual-targeted MNP-TPP-HA NPs, we established a 3D 4T1 tumor globule model and incubated it with fluorescein isothiocyanate (FITC)-labeled nanomaterials of four kinds of samples. Fluorescence imaging in tumor spheroids was observed at different depths using CLSM. From the fluorescence imaging results and quantitative analysis shown in Fig. 3, MNP-PEG was mainly localized at the edge of the spheroids with a penetration depth of < 80 μm. MNP-TPP and MNP-HA have slight cell penetration capacity due to their respective mitochondrial targeting and cell membrane targeting, with penetration depths of 120 μm and 140 μm respectively, but still do not penetrate the center of the tumor spheroids. Notably, the penetration depth of MNP-TPP-HA group could reach 180 μm and the green fluorescence had penetrated the tumor pellet, and it was completely evenly distributed throughout the pellet model (Fig. 3a). The experimental results were consistent with the fluorescence uptake results obtained by flow cytometry (Fig. 3b). In conclusion, our experiment results indicate that TPP and HA play a key role in enhancing the exocytosis and deep tumor penetration of MNP, helping to improve the drug administration efficiency and achieve precise and efficient cancer therapy by melanin-based nanocarrier.

  Figure 3

  

  Figure 3.  (a) 3D cell penetration of MNP-PEG, MNP-HA, MNP-TPP, and MNP-TPP-HA and (b) the corresponding semi-quantitative analysis of MNP-TPP-HA

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  2.5   In vivo photoacoustic imaging in tumor site

  A tumor-bearing mouse model was established by subcutaneously injecting 4T1 cells into the hind leg region of female Balb/c mice. Since melanin has a good advantage in one-zone photoacoustic imaging, we evaluated the targeted aggregation ability of MNP-TPP-HA in tumor sites through a photoacoustic imaging system. As shown in Fig. 4a, after intravenous injection of MNP-PEG or MNP-TPP-HA with the same concentration of MNP respectively, the photoacoustic signal of MNP-PEG weakly appeared at the peripheral region of the tumor in all periods, while the photoacoustic signal of MNP-TPP-HA reached its maximum at 7 h and was about 3 times higher than that of the initial signal. The signal intensity in tumor regions can continue for 12 h, subsequently disappear, and metabolize after 24 h post-injection (Fig. 4b). These results show that MNP-TPP-HA NPs have the highest tumor-specific accumulation and cellular uptake compared with MNP-PEG. This can be attributed to the synergistic effect of targeted accumulation mediated by TPP and HA. Similar results can be observed by corresponding relative mean photoacoustic intensity analysis (Fig. 4c and 4d).

  Figure 4

  

  Figure 4.  In vivo two-dimensional (2D) and 3D photoacoustic images before and after intravenous injection of (a) MNP-PEG and (b) MNP-TPP-HA, (c, d) the corresponding photoacoustic signal intensity

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  3.   Conclusions

  In summary, we have developed a tumor-double-targeted melanin nanocarriers (MNP-TPP-HA) in which the carboxyl groups of HA and TPP can be conveniently coupled with the amine groups on MNP‐PEG through EDC and NHS activation. Through a series of experiments, such as cell uptake, 3D cell sphere penetration, and in vivo photoacoustic imaging, it has been proved that the modification of HA and TPP not only improves the biocompatibility of MNP but also enhances the preferential tumor accumulation and the specific cell uptake. These results show that MNP modified by HA and TPP ligands has a promising future in improving targeted cancer therapy. We hope that future research based on MNP-TPP-HA will provide innovative results and insights into the development of tumor therapy.

  
  Acknowledgments: This project was supported by the National Natural Science Foundation of China (Grants No.82120108016, 82071987), the National Ten Thousand Talents Program (Grant No.SQ2022RA2A300118), Research Project Supported by Shanxi Scholarship Council of China (Grant No.2020-177), Fund Program for the Scientific Activities of Selected Returned Overseas Professionals in Shanxi Province (Grant No.20200006), Four Batches of Scientific Research Projects of Shanxi Provincial Health Commission (Grants No.2020TD11, 2020SYS15, 2020XM10), Key Laboratory of Nano-imaging and Drug-loaded Preparation of Shanxi Province (Grant No.202104010910010). Declaration of competing interest: All authors of this paper declare that they have no competing interests.
  
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  Scheme 1  (a) Schematic representation of the preparation of MNP-TPP-HA and (b) dual-targeted tumor photoacoustic imaging

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  Figure 1  (a) DLS analysis, (b) ζ potential, (c) UV-Vis absorption spectra of MNP-PEG, MNP-HA, MNP-TPP and MNP-TPP-HA; (d) TEM image, (e) particle size stability of MNP-TPP-HA

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  Figure 2  (a) Flow cytometric uptake analysis; (b) Viability of the cells treated with MNP-PEG, MNP-HA, MNP-TPP, and MNP-TPP-HA respectively

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  Figure 3  (a) 3D cell penetration of MNP-PEG, MNP-HA, MNP-TPP, and MNP-TPP-HA and (b) the corresponding semi-quantitative analysis of MNP-TPP-HA

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  Figure 4  In vivo two-dimensional (2D) and 3D photoacoustic images before and after intravenous injection of (a) MNP-PEG and (b) MNP-TPP-HA, (c, d) the corresponding photoacoustic signal intensity

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