Dual Antimelanogenic Effect of Nicotinamide-Stabilized Phloretin Nanocrystals in Larval Zebrafish

Melanin is a kind of dark insoluble pigment that can cause pigmentation and free-radical clearance, inducing melasma, freckles, and chloasma, affecting the quality of life of patients. Due to poor water solubility and low safety, the absorption of poorly water-soluble drugs is limited by the hinderance of a skin barrier. Therefore, it is necessary to develop new, safe, and highly efficient drugs to improve their transdermal absorption efficiency and thus to inhibit the production of melanin. To address these issues, we developed a new nicotinamide (NIC)-stabilized phloretin nanocrystals (PHL-NCs). First, NC technology significantly increased the solubility of PHL. The in vitro release results indicated that at 6 h, the dissolution of the PHL-NIC-NCs was 101.39% ± 2.40% and of the PHL-NCs was 84.92% ± 4.30%, while that of the physical mixture of the two drugs was only 64.43% ± 0.02%. Second, NIC acted not only as a stabilizer to enlarge the storage time of PHL-NIC-NCs (improved to 10-day in vitro stability) but also as a melanin transfer inhibitor to inhibit melanin production. Finally, we verified the melanin inhibition effect of PHL-NIC-NCs evaluated by the zebrafish model. It showed that 0.38 mM/L PHL-NIC-NCs have a lower tyrosinase activity at 62.97% ± 0.52% and have less melanin at 36.57% ± 0.44%. The inhibition effect of PHL-NCs and PHL-NIC-NCs was stronger compared to the positive control arbutin. In conclusion, the combination of NIC and PHL achieves better inhibition of tyrosinase and inhibition of melanin production through synergism. This will provide a direction to the subsequent development of melanin-inhibiting drugs and the combined use of pharmaceutical agents.


Introduction
Melanin is a type of dark insoluble pigment that causes pigment precipitation and freeradical scavenging [1,2], inducing skin disorders, such as age spots, freckles, melasma, and malignant melanoma [3]. Existing drugs for treating hyperpigmentation can be divided into the following five categories [4]: cell receptor antagonists, inhibitors of melanocyte stimulation (retinoids), tyrosinase enzyme inhibitors (resveratrol) [5], inhibitors of melanosome transfer (niacinamide (NIC)) [6], and drugs that degrade melanin in keratinocytes (glycolic acid). However, there are a few problems with these drugs. First is the safety of the drugs itself. For example, kojic acid, hydroquinone, and corticosteroids lead to adverse reactions, such as dermatitis and skin irritation, ochronosis, cytotoxicity, and skin cancer [3]. Arbutin is degraded to toxic hydroquinone under high temperature, ultraviolet radiations, and a dilute acid [7]. L-ascorbic acid is heat-reactive and easy to degrade [8]. Second is low bioavailability [9]. Many melanogenesis inhibitors, including most flavonoids or polyphenols, such as ellagic acid [8], have excellent whitening and antioxidant effects. However, the brick-mud structure of the stratum corneum (SC) limits the transdermal absorption of these insoluble drugs. Thus, new, safe, and highly effective drugs are necessary, as well as

Investigation on the Stability of NCs
The particle size and polydispersity index (PDI) of PHL-NCs and PHL-NIC-NCs were measured by the Nano ® Zetasizer (Malvern Instruments, Worcestershire, UK) on days 1, 2, 3, 5, 7, 10, and 15. The suspensions were stored in a 4 • C refrigerator.

Characterization of NCs
The particle size and PDI of NCs were measured by the Nano ® Zetasizer (Malvern Instruments, Worcestershire, UK). The morphologies of the NCs were observed by a Nova Nano SEM (FEI, Hillsboro, OR, USA). The thermal properties of the raw medicine, stabilizer, physical mixture of the raw medicine and stabilizer, and three particle sizes of NCs were measured by a Diamond TG/DTA (Perkin Elmer, Waltham, MA, USA). The Raman spectra were obtained by a confocal Raman microscopy (inVia plus, Renishaw, UK). All these measurements were performed at three different times.

In Vitro Drug Release
The release of PHL-NCs and PHL-NIC-NCs in vitro was studied by the dialysis method [25]. A mixture of PBS (pH 6.8) and ethanol (7:3, w/w) [13] was selected as the release medium. Next, 10 mg of drugs (physical mixture of raw medicine as the control) was added to the dialysis bag (molecular interception of 12,000-14,000; Los Angeles Spectral Medical Industrial Corporation, Los Angeles, CA, USA). Each dialysis bag was suspended in 100 mL of the release medium and slowly stirred in a constant-temperature water bath at 37 ± 1 • C and a rotating speed of 100 r/min. During stirring, 1 mL of the release medium was absorbed at a predetermined time interval and supplemented with 1 mL of the same release medium. The collected samples were centrifuged at 12,000 rpm for 20 min and analyzed using high-performance liquid chromatography (HPLC). Measurements were performed in triplicate. The collected embryos (purchased from Feixi, Shanghai, China) were placed in a 24-well plate containing embryonic water and cultured in an artificial climate incubator (RGLC-P160A, Darth Carter, China) at 28 • C. After 24 h post-fertilization (hpf) [26], zebrafish embryos were transferred to embryonic water containing different concentrations of PHL-NCs. The solution was replaced every 12 h, with unscheduled stirring to ensure uniform distribution of drugs. After 72 hpf (48 h exposure) [26], the survival rate and effects of embryos were observed using a stereomicroscope (SRZ-7045DM, COSSim, China) and an inverted fluorescence microscope (Carl Zeiss AG, Oberkochen, Germany).

Transport of NCs in Embryonic Zebrafish In Vivo
The hybrid NCs were used to evaluate NC uptake in zebrafish. Embryos (from 24 hpf to 72 hpf) were exposed separately to PHL-NCs and PHL-NIC-NCs (0.38 mM/L). The embryos treated with embryonic water were set as the blank control group. The embryos were collected at 20 min, 2 h, 6 h, and 12 h, and larvae were collected at 48 h for imaging by an inverted fluorescence microscope (Carl Zeiss AG, Oberkochen, Germany).

Zebrafish Embryo Exposure Experiment
The preliminary experimental operation is shown in Section 2.6.1. After 24 hpf, zebrafish embryos were transferred to embryonic water containing α-arbutin (20 mM/L), PHL-NIC-NCs (0.38 mM/L), and PHL-NCs (0.38 mM/L). Arbutin is commonly used as a common positive control group for melanin inhibition experiments in zebrafish models [20,27,28]. It is a recognized tyrosinase inhibitor with an effective whitening effect [29]. The structure of arbutin is similar to that of tyrosine and competes for tyrosinase and produces competitive inhibition, thus inhibiting tyrosinase activity directly without affecting the mRNA expression of tyrosinase, achieving the effect of inhibiting melanin production [30]. In addition, we found that the optimal concentration of arbutin as a tyrosinase inhibitor was 20 mM/L and the inhibitory effect of α-arbutin was better than that of other arbutin types [19]. Therefore, we used 20 mM/L of α-arbutin as a positive control. After 72 hpf (48 h exposure), the embryos were detached with forceps for observation. The effects of different melanin inhibitors on zebrafish pigmentation were observed by a stereomicroscope (SRZ-7045DM, COSSim, China).

Determination of Tyrosinase Activity in Zebrafish Embryos
The tyrosinase activity of the samples was measured, as described previously with a slight modification [20]. After 48 h exposure, 30 embryos were washed twice with PBS and put into EP tubes containing 300 µL of cold lysate buffer. The tissues were ground and centrifuged at 10,000 r/min for 15 min at 4 • C. Next, 20 µL of the supernatant was added to 96-well plates containing 180 µL of L-DOPA. After incubation at 37 • C in the dark for 30 min, absorbance was measured at 492 nm using a microplate reader (SpectraMax ® , Molecular Devices, China). The relative tyrosinase activity of the blank control group was recognized as 100%, and the tyrosinase activity of the sample was expressed as the percentage of the blank control group. All the experiments and measurements were performed in triplicate.

Determination of Melanin Content in Zebrafish Embryos
The precipitate obtained by centrifugation in Section 2.6.4 was dissolved in 300 µL of NaOH (1 mol/L), and the EP orifice was sealed with a sealing film. The EP orifice was dried at 95 • C for 30 min, and absorbance was measured at 405 nm. The relative melanin content of the blank control group was considered 100%, and the melanin content of the sample was indicated as the percentage of the blank control group [20].

HPLC Analysis
PHL was detected using an HPLC system (Agilent 1100, Santa Clara, CA, USA) at 280 nm. Separation was performed on an XB-C18 column (Ultimate ® ; 150 × 4.6 mm, 5 µm) with acetonitrile (45:55 v/v) as the mobile phase, and the flow rate was set as 1 mL/min. In the range of 0.35 to 56 µg/mL, the PHL concentration (C) was linear, with its peak area (A) with a typical calibration curve of C = 0.0155 A-0.1451 and R2 = 0.9998.

Statistical Analysis
All data are presented as the mean ± standard deviation (SD). One-way analysis of variance (ANOVA) using IBM SPSS Statistics 26 (SPSS, Inc., Chicago, IL, USA) was used to compare the survival rate of zebrafish at different concentrations and tyrosinase activity and melanin production after culture with the blank control arbutin, PHL-NCs, and PHL-NIC-NCs. Significance is denoted in the figures as * p < 0.05, ** p < 0.01, and *** p < 0.001.

Characterization of NCs
PHL-NCs and PHL-NIC-NCs were successfully prepared by the anti-solvent precipitation method. The particle sizes of PHL-NCs were 91.8 ± 1.0 nm and of PHL-NIC-NCs were 94.6 ± 1.0 nm, while the PDIs were 0.085 ± 0.007 and 0.086 ± 0.003, respectively ( Figure 1a). PHL-HPS-NCs, PHL-NIC-HPS-NCs, and HPS-NCs were also prepared by the method described earlier. The particle sizes of PHL-HPS-NCs were 128.1 ± 2.0 nm, of PHL-NIC-HPS-NCs were 95.7 ± 1.0 nm, and of HPS-NCs were 353.3 ± 11.0 nm, while the PDIs were 0.171 ± 0.002, 0.121 ± 0.010, and 0.364 ± 0.046, respectively ( Figure 1b). The uncoated, pure NCs of HPS displayed bigger sizes than those displayed by PHL-NCs and PHL-NIC-NCs. The hybrid NCs showed bigger sizes than those showed by the uncoated, pure NCs of PHL and PHL-NIC. This indirectly proves the successful preparation of PHL-HPS-NCs and PHL-NIC-HPS-NCs [31]. The hybrid NCs were detected by an inverted fluorescence microscope (Carl Zeiss AG, Oberkochen, Germany), as shown in Figure 1c. Fluorescence images showed the uniform distribution of hybrid NCs. PHL-NCs displayed a spherical-like morphology (Figure 1d), and the sizes of NCs were comparable to the particle size measurement results. PHL-NCs are indicated by red arrows, and the precipitation after NIC drying is indicated by the yellow circle.
The DSC profiles of the PHL-NCs, PHL-NIC-NCs, physical mixtures, PHL, NIC, and HPMC are shown in Figure 1e. PHL displayed an endothermal peak at 269.73 • C. NIC showed two endothermal peaks at 129.21 • C and 248.19 • C, while HPMC did not display a typical endothermal peak. The first endothermal peak of the physical mixture was at 115.16 • C, indicating that it relates to metastable eutectic melting, and the second endothermal peak was at 189.17 • C, indicating that this is a characteristic of a mixture capable of cocrystal formation [32]. The endothermal peaks of PHL-NCs and PHL-NIC-NCs disappeared, indicating that the amorphous state was formed. The Raman spectra of the PHL-NCs, PHL-NIC-NCs, PHL, NIC, and HPMC are shown in Figure 1f. PHL [33] was bent as an O-C-C vibration at 855 cm −1 , a carbon-carbon tensile vibration at 1529 cm −1 and 1574 cm −1 , and a carbonyl C=O stretching vibration absorption peak at 1618 cm −1 . The NIC characteristic peaks were at 1042 cm −1 and 1596 cm −1 [34], which are the stretching vibration bands of the -CN amide group and the absorption peak of the deformation vibration of the in-plane ring of the pyridine molecule, respectively. For the PHL-NCs [35], the C=O stretching vibration absorption peak was indicated at 1631 cm −1 . The characteristic peaks still existed after the preparation of PHL-NIC-NCs. Compared to PHL, the peak of the PHL-NIC-NCs became wider and higher at 1596 cm −1 and split into two peaks for NIC at 1042 cm −1 , changing slightly. This proved that nanocrystals have no effect on the drug structure and do not influence the qualities of PHL and NIC [36]. stretching vibration absorption peak was indicated at 1631 cm −1 . The characteristic pea still existed after the preparation of PHL-NIC-NCs. Compared to PHL, the peak of t PHL-NIC-NCs became wider and higher at 1596 cm −1 and split into two peaks for NIC 1042 cm −1 , changing slightly. This proved that nanocrystals have no effect on the dr structure and do not influence the qualities of PHL and NIC [36].

The Stability of NCs
As shown in Figure 2a, according to the stability test, the PHL-NCs showed a lar amount of precipitation on the second day while the PHL-NIC-NCs showed stabil without any particle size change for 10 days and large precipitation in 15 days. The su pension color and clarity of the NCs over time are shown in Figure 2b. The PHL-N began to appear precipitated (red arrows indicate white precipitated particles) on the s ond day and gradually changed from a light-blue to a white-turbid suspension as the tim increased. The PHL-NIC-NCs did not precipitate until 10 days and changed from a lig blue to a colorless transparent suspension as the time increased and then to a white turb suspension, which was consistent with the previous particle size and PDI data.
Therefore, we found that PHL-NCs can be stabilized by NIC; the mechanism may that NIC can interact with PHL through noncovalent interaction and further connect w PHL through hydrogen bonds, while being stabilized by π-π interaction through the ph nyl ring between the layers [37].

The Stability of NCs
As shown in Figure 2a, according to the stability test, the PHL-NCs showed a large amount of precipitation on the second day while the PHL-NIC-NCs showed stability without any particle size change for 10 days and large precipitation in 15 days. The suspension color and clarity of the NCs over time are shown in Figure 2b. The PHL-NCs began to appear precipitated (red arrows indicate white precipitated particles) on the second day and gradually changed from a light-blue to a white-turbid suspension as the time increased. The PHL-NIC-NCs did not precipitate until 10 days and changed from a light-blue to a colorless transparent suspension as the time increased and then to a white turbid suspension, which was consistent with the previous particle size and PDI data.
Therefore, we found that PHL-NCs can be stabilized by NIC; the mechanism may be that NIC can interact with PHL through noncovalent interaction and further connect with PHL through hydrogen bonds, while being stabilized by π-π interaction through the phenyl ring between the layers [37].

In Vitro Drug Release Study
The release behaviors of PHL-NC and PHL-NIC-NC suspensions are shown in ure 2c. It shows that the release of PHL-NCs and PHL-NIC-NCs was faster than tha PHL+NIC powder and the release percentage was only 64.43% ± 0.02% at 6 h, which indicated that NCs could increase the dissolution rate of insoluble drugs. In addition, release of PHL-NIC-NCs was faster than that of PHL-NCs, indicating that PHL-NIC-N have more advantages in PHL dissolution performance. There may be two possible me anisms as previously reported, spring and parachute [37]. One is that PHL-NIC-NCs h weaker intermolecular interactions, which facilitates their dissolution [38], and the o is that PHL-NIC-NCs have weaker π-π interaction because the strength of the interac related to the bond distance between molecules, which can produce higher dissolu rates [39].

Screening of Drug Concentrations
In this section, we screened the concentrations of PHL-NCs and PHL-NIC-NC zebrafish. As shown in Figure 3a

In Vitro Drug Release Study
The release behaviors of PHL-NC and PHL-NIC-NC suspensions are shown in Figure 2c. It shows that the release of PHL-NCs and PHL-NIC-NCs was faster than that of PHL+NIC powder and the release percentage was only 64.43% ± 0.02% at 6 h, which also indicated that NCs could increase the dissolution rate of insoluble drugs. In addition, the release of PHL-NIC-NCs was faster than that of PHL-NCs, indicating that PHL-NIC-NCs have more advantages in PHL dissolution performance. There may be two possible mechanisms as previously reported, spring and parachute [37]. One is that PHL-NIC-NCs have weaker intermolecular interactions, which facilitates their dissolution [38], and the other is that PHL-NIC-NCs have weaker π-π interaction because the strength of the interaction related to the bond distance between molecules, which can produce higher dissolution rates [39].

Screening of Drug Concentrations
In this section, we screened the concentrations of PHL-NCs and PHL-NIC-NCs in zebrafish. As shown in Figure 3a melanin production in zebrafish in the molecular and low-and medium-concentration groups was observed. Figure 3c shows that the medium-concentration group had a better anti-melanogenesis effect than the molecular and low-concentration groups. Therefore, 0.38 mM/L of PHL-NCs and PHL-NIC-NCs was selected as the transport experimental concentration to evaluate the inhibition of melanin production in zebrafish. maceutics 2022, 14, x FOR PEER REVIEW 8 of anti-melanogenesis effect than the molecular and low-concentration groups. Therefo 0.38 mM/L of PHL-NCs and PHL-NIC-NCs was selected as the transport experimen concentration to evaluate the inhibition of melanin production in zebrafish.

Transport of NCs in Embryonic Zebrafish In Vivo
HSP-hybrid PHL-NCs and PHL-NIC-NCs were used to observe the uptake and d tribution of intact NCs in zebrafish embryos. As shown in Figure 4, the chorionic surfa fluorescence of zebrafish embryos gradually increased as the incubation time increase indicating the aggregation of intact NCs on the chorion. Fluorescence could also be o served in the inner mass of embryos (IME) and the yolk sac (YS) over time. Interesting after 48 h, fluorescence only appeared on the chorion surface and there was no obvio fluorescence in zebrafish larvae, indicating that the NCs entered the chorion in the for of molecules.

Transport of NCs in Embryonic Zebrafish In Vivo
HSP-hybrid PHL-NCs and PHL-NIC-NCs were used to observe the uptake and distribution of intact NCs in zebrafish embryos. As shown in Figure 4, the chorionic surface fluorescence of zebrafish embryos gradually increased as the incubation time increased, indicating the aggregation of intact NCs on the chorion. Fluorescence could also be observed in the inner mass of embryos (IME) and the yolk sac (YS) over time. Interestingly, after 48 h, fluorescence only appeared on the chorion surface and there was no obvious fluorescence in zebrafish larvae, indicating that the NCs entered the chorion in the form of molecules.

Influence of NCs on Tyrosinase Activity and Relative Melanin Content in Zebrafish Embryos
Embryos at 24 hpf were exposed to various melanogenic inhibitors for 48 h to assess melanogenic inhibitory activity. As shown in Figure 5a, in the blank control group without treatment by inhibitors, a large number of black spots, which were the melanin deposited in the zebrafish embryos, were clearly observed on the lateral and dorsal spine, eyes, and yolk sac of the zebrafish embryos. α-Arbutin was selected as a positive control at 20 mM/L [19]. The results showed that arbutin had little effect on retinal pigment epithelium (RPE) pigmentation but inhibited body pigmentation. Next, we studied the effects of PHL-NCs and PHL-NIC-NCs on zebrafish pigmentation. Two NCs had significant inhibitory effects on body pigmentation in zebrafish, and the inhibitory effect was much better compared to arbutin aqueous solution. This phenomenon may be explained by the fact that NCs increased the diffusion of PHL on the chorion by reducing the particle size of PHL so that the melanin inhibitor could bind more to the enzyme site [23], thereby improving bioavailability. The inhibitory effect of PHL-NIC-NCs on zebrafish melanin was better than that of PHL-NCs. For PHL-NCs, a small amount of lighter melanin was found in the yolk sac and on the top of the spine. For PHL-NIC-NCs, only a small amount of lighter melanin was deposited on the top of the spine and melanin inhibition in other parts was effective. In addition, we found that two NCs had an inhibitory effect on RPE pigmentation in zebrafish. The process of zebrafish pigmentation is different from that of RPE pigmentation, and its formation may be regulated by Tyr, Tyrp1, and Dct/Trp2 genes.

Influence of NCs on Tyrosinase Activity and Relative Melanin Content in Zebrafish Embryos
Embryos at 24 hpf were exposed to various melanogenic inhibitors for 48 h to assess melanogenic inhibitory activity. As shown in Figure 5a, in the blank control group without treatment by inhibitors, a large number of black spots, which were the melanin deposited in the zebrafish embryos, were clearly observed on the lateral and dorsal spine, eyes, and yolk sac of the zebrafish embryos. α-Arbutin was selected as a positive control at 20 mM/L [19]. The results showed that arbutin had little effect on retinal pigment epithelium (RPE) pigmentation but inhibited body pigmentation. Next, we studied the effects of PHL-NCs and PHL-NIC-NCs on zebrafish pigmentation. Two NCs had significant inhibitory effects on body pigmentation in zebrafish, and the inhibitory effect was much better compared to arbutin aqueous solution. This phenomenon may be explained by the fact that NCs increased the diffusion of PHL on the chorion by reducing the particle size of PHL so that the melanin inhibitor could bind more to the enzyme site [23], thereby improving bioavailability. The inhibitory effect of PHL-NIC-NCs on zebrafish melanin was better than that of PHL-NCs. For PHL-NCs, a small amount of lighter melanin was found in the yolk sac and on the top of the spine. For PHL-NIC-NCs, only a small amount of lighter melanin was deposited on the top of the spine and melanin inhibition in other parts was effective. In addition, we found that two NCs had an inhibitory effect on RPE pigmentation in zebrafish. The process of zebrafish pigmentation is different from that of RPE pigmentation, and its formation may be regulated by Tyr, Tyrp1, and Dct/Trp2 genes. The affinity of drugs or preparations to RPE or RPE-associated melanosomes may also impact their effects, but the effects of PHL-NCs and PHL-NIC-NCs on RPE pigmentation need further studies to prove. The affinity of drugs or preparations to RPE or RPE-associated melanosomes may also impact their effects, but the effects of PHL-NCs and PHL-NIC-NCs on RPE pigmentation need further studies to prove. To further evaluate the anti-melanogenic activity of PHL-NCs and PHL-NIC-NCs, their effects on the reduction in tyrosinase activity ( Figure 5b) and melanin content ( Figure  5c) in zebrafish embryos were investigated. The tyrosinase activity and melanin content decreased to 66.85% ± 0.87% and 47.04% ± 1.02%, respectively, after treatment with 0.38 mM/L of PHL-NCs. After treatment with 0.38 mM/L of PHL-NIC-NCs, the tyrosinase activity and melanin content decreased to 62.97% ± 0.52% and 36.57% ± 0.44%, respectively. It can be highlighted that PHL-NCs and PHL-NIC-NCs showed more significant inhibition of tyrosine activity (66.85% ± 0.87% and 62.97% ± 0.52%) than the positive control arbutin (89.04% ± 0.26%) and a more obvious anti-melanogenesis effect (47.04% ± 1.02% and 36.57% ± 0.44%) than arbutin (66.33% ± 1.03%). According to the experimental results of the tyrosinase activity and melanin content in zebrafish, there was no significant difference in the inhibitory effect of tyrosinase activity between PHL-NCs and PHL-NIC-NCs, indicating that NIC does not increase the inhibitory effect of PHL. However, for the inhibition of melanin content formation, the PHL-NIC-NC group showed significant improvement in anti-melanin performance, indicating that NIC reduces melanin content through synergy.
As shown in Figure 6, the melanin generation process is divided into three stages: proliferation of melanin, synthesis of tyrosine and melanin, and transfer of melanin from melanocytes to keratinocytes [40]. Tyrosinase activation is required when tyrosine is To further evaluate the anti-melanogenic activity of PHL-NCs and PHL-NIC-NCs, their effects on the reduction in tyrosinase activity ( Figure 5b) and melanin content (Figure 5c) in zebrafish embryos were investigated. The tyrosinase activity and melanin content decreased to 66.85% ± 0.87% and 47.04% ± 1.02%, respectively, after treatment with 0.38 mM/L of PHL-NCs. After treatment with 0.38 mM/L of PHL-NIC-NCs, the tyrosinase activity and melanin content decreased to 62.97% ± 0.52% and 36.57% ± 0.44%, respectively. It can be highlighted that PHL-NCs and PHL-NIC-NCs showed more significant inhibition of tyrosine activity (66.85% ± 0.87% and 62.97% ± 0.52%) than the positive control arbutin (89.04% ± 0.26%) and a more obvious anti-melanogenesis effect (47.04% ± 1.02% and 36.57% ± 0.44%) than arbutin (66.33% ± 1.03%). According to the experimental results of the tyrosinase activity and melanin content in zebrafish, there was no significant difference in the inhibitory effect of tyrosinase activity between PHL-NCs and PHL-NIC-NCs, indicating that NIC does not increase the inhibitory effect of PHL. However, for the inhibition of melanin content formation, the PHL-NIC-NC group showed significant improvement in anti-melanin performance, indicating that NIC reduces melanin content through synergy.
As shown in Figure 6, the melanin generation process is divided into three stages: proliferation of melanin, synthesis of tyrosine and melanin, and transfer of melanin from melanocytes to keratinocytes [40]. Tyrosinase activation is required when tyrosine is converted into melanin [41]. PHL and NIC act on stage 3 and stage 4, respectively. The mechanism of PHL is similar to that of arbutin, but PHL-NCs suppress the activity of tyrosinase in the first step of the rate-limiting enzyme in melanin synthesis, which causes conformational changes to the enzyme after binding to tyrosinase, thus reducing melanin production [10]. The mechanism of NIC is completely different with three main mechanisms [6, 42,43]: interfering with keratinocyte and melanocyte interactions, inhibiting the transfer of the generated melanin, and promoting melanin transfer to the cuticle and promoting cuticle shedding. maceutics 2022, 14, x FOR PEER REVIEW 11 o converted into melanin [41]. PHL and NIC act on stage 3 and stage 4, respectively. T mechanism of PHL is similar to that of arbutin, but PHL-NCs suppress the activity tyrosinase in the first step of the rate-limiting enzyme in melanin synthesis, which cau conformational changes to the enzyme after binding to tyrosinase, thus reducing mela production [10]. The mechanism of NIC is completely different with three main mec nisms [6, 42,43]: interfering with keratinocyte and melanocyte interactions, inhibiting transfer of the generated melanin, and promoting melanin transfer to the cuticle and p moting cuticle shedding. Figure 6. The mechanism of PHL and NIC inhibiting melanin production in zebrafish.

Conclusions
The formation of melanin involves mainly two pathways. We combined the inh tion of tyrosinase activity with the inhibition of melanin transfer by fabricated PHL-N NCs and evaluated the anti-hyperpigmentation effect in a larval zebrafish model. T study systematically investigated the preparation and characterization of PHL-NCs a PHL-NIC-NCs and administered them to zebrafish to evaluate the tyrosinase inhibito and anti-melanogenesis abilities of NCs. In addition, we studied PHL-NIC-NCs using HPS fluorescence marker to assess the uptake and distribution of NCs in zebrafish. T results indicated that PHL-NIC-NCs increase the inhibition effect of tyrosinase activ and melanogenesis. Furthermore, anti-melanin efficacy can be enhanced by a combinat of PHL-NCs and NIC.
NC solve many problems of existing preparations (including nanoemulsions, lip NPs, chitosan NPs, and polymer NPs), such as low drug loading, high production co and safety, and improved absorption efficiency and bioavailability. In addition, we fou that the combined use of two anti-melanin drugs, PHL and NIC, not only improves s bility and solubility but also improves bioavailability and absorption efficiency so as have more anti-melanin efficacy and produce higher value. This research will provid reference for the future development of anti-melanin efficacy and the combined use drug formulations.
However, this direction still needs further analysis, such as how to understa whether NIC increases the stability of PHL-NCs by increasing surface charge through z potential analysis so as to explore more mechanisms of NIC-stabilizing PHL-NCs. In dition, for this kind of preparation, we only hope that the drug can stay in the skin's ba layer and stay in the blood as little as possible. However, more animal models are s needed to verify this aspect, which will be the direction of future research.

Conclusions
The formation of melanin involves mainly two pathways. We combined the inhibition of tyrosinase activity with the inhibition of melanin transfer by fabricated PHL-NIC-NCs and evaluated the anti-hyperpigmentation effect in a larval zebrafish model. This study systematically investigated the preparation and characterization of PHL-NCs and PHL-NIC-NCs and administered them to zebrafish to evaluate the tyrosinase inhibitory and anti-melanogenesis abilities of NCs. In addition, we studied PHL-NIC-NCs using the HPS fluorescence marker to assess the uptake and distribution of NCs in zebrafish. The results indicated that PHL-NIC-NCs increase the inhibition effect of tyrosinase activity and melanogenesis. Furthermore, anti-melanin efficacy can be enhanced by a combination of PHL-NCs and NIC.
NC solve many problems of existing preparations (including nanoemulsions, lipid NPs, chitosan NPs, and polymer NPs), such as low drug loading, high production costs and safety, and improved absorption efficiency and bioavailability. In addition, we found that the combined use of two anti-melanin drugs, PHL and NIC, not only improves stability and solubility but also improves bioavailability and absorption efficiency so as to have more anti-melanin efficacy and produce higher value. This research will provide a reference for the future development of anti-melanin efficacy and the combined use of drug formulations.
However, this direction still needs further analysis, such as how to understand whether NIC increases the stability of PHL-NCs by increasing surface charge through zeta potential analysis so as to explore more mechanisms of NIC-stabilizing PHL-NCs. In addition, for this kind of preparation, we only hope that the drug can stay in the skin's basal layer and stay in the blood as little as possible. However, more animal models are still needed to verify this aspect, which will be the direction of future research.

Conflicts of Interest:
The authors declare no conflict of interest.