Design of Dual-Targeted pH-Sensitive Hybrid Polymer Micelles for Breast Cancer Treatment: Three Birds with One Stone

Breast cancer has a high prevalence in the world and creates a substantial socio-economic impact. Polymer micelles used as nano-sized polymer therapeutics have shown great advantages in treating breast cancer. Here, we aim to develop a dual-targeted pH-sensitive hybrid polymer (HPPF) micelles for improving the stability, controlled-release ability and targeting ability of the breast cancer treatment options. The HPPF micelles were constructed using the hyaluronic acid modified polyhistidine (HA-PHis) and folic acid modified Plannick (PF127-FA), which were characterized via 1H NMR. The optimized mixing ratio (HA-PHis:PF127-FA) was 8:2 according to the change of particle size and zeta potential. The stability of HPPF micelles were enhanced with the higher zeta potential and lower critical micelle concentration compared with HA-PHis and PF127-FA. The drug release percents significantly increased from 45% to 90% with the decrease in pH, which illustrated that HPPF micelles were pH-sensitive owing to the protonation of PHis. The cytotoxicity, in vitro cellular uptake and in vivo fluorescence imaging experiments showed that HPPF micelles had the highest targeting ability utilizing FA and HA, compared with HA-PHis and PF127-FA. Thus, this study constructs an innovative nano-scaled drug delivery system, which provides a new strategy for the treatment of breast cancer.


Introduction
Breast cancer has become the most commonly diagnosed cancer in 2020, and the incidence is rising year after year. According to a report, the numbers of new breast cancer cases and deaths in the United States in 2021 were 284,200 and 43,600, respectively [1]. Breast cancer is mainly caused by malignant changes in the epithelium of breast ducts, which seriously affects the physical and mental health of female patients [2]. At present, there are a variety of treatment methods, such as surgery, radiotherapy, chemotherapy, and molecular targeted therapy. Among them, chemotherapy is an active treatment for all stages of breast cancer, which significantly prolongs the median survival of patients [3]. However, chemotherapeutic drugs along with killing the cancer cells, bring serious damages to the normal cells as well, thereby causing systemic toxicity [4]. Therefore, the development of a novel drug delivery system for targeted and controlled release of chemotherapeutic drugs to tumor sites has attracted widespread attention.
Nanocarriers are often applied for treating breast cancer [5]. An enzymatically transformable polymer-based nanotherapeutic approach containing colchicine and marimastat is developed to prevent malignant progression of metastatic breast cancer [6]. The exosome membrane coated nanoparticles containing cationic bovine serum albumin conjugated siS100A4 are designed, which significantly inhibits the growth of malignant breast cancer

Cell Lines and Animals
HepG2 (human liver cancer cells) and MCF-7 (human breast cancer cells) were pu chased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). All cel were cultured in DMEM medium (Gibco, Thermal Fisher, Lenexa, TX, USA) suppl mented with 8% fetal bovine serum (Gibco, Thermal Fisher, Lenexa, TX, USA) and 1 penicillin-streptomycin in a humidified atmosphere of 95% air and 5% CO2 at 37 °C, r spectively.
Female BALB/c mice (18 ± 2 g) were purchased from the laboratory animal center Shantou University Medical College (Shantou, China). All operational processes were ca ried out according to the NIH Guidelines for the Care and Use of Laboratory Animals an were approved by the Animal Ethics Committee of Shantou University Medical Colleg (SUMC2022-152).

Cell Lines and Animals
HepG2 (human liver cancer cells) and MCF-7 (human breast cancer cells) were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). All cells were cultured in DMEM medium (Gibco, Thermal Fisher, Lenexa, TX, USA) supplemented with 8% fetal bovine serum (Gibco, Thermal Fisher, Lenexa, TX, USA) and 1% penicillin-streptomycin in a humidified atmosphere of 95% air and 5% CO 2 at 37 • C, respectively.
Female BALB/c mice (18 ± 2 g) were purchased from the laboratory animal center of Shantou University Medical College (Shantou, China). All operational processes were carried out according to the NIH Guidelines for the Care and Use of Laboratory Animals and were approved by the Animal Ethics Committee of Shantou University Medical College (SUMC2022-152).

Synthesis and Characterization of Nim-DNP-L-Histidine
Briefly, Nα-CBZ-Nim-DNP-L-histidine was dissolved in anhydrous tetrahydrofuran, then thionyl chloride was added to react for 5 h. Finally, the products were obtained by filtration and recrystallization, and the structure was characterized using 1 H NMR. Nim-DNP-L-histidine was dissolved in DMF containing isopropylamine, and the solution was reacted under N 2 at room temperature for 4 days. Next, the solution precipitated in the cold diethyl ether. Finally, the poly (Nim-DNP-L-histidine) was obtained by solvent evaporation and characterized using 1 H NMR.

Synthesis and Characterization of HA-PHis
HA was dissolved in anhydrous formamide at 55 • C, then cooled to room temperature, then NHS and EDC were added to react for 2 h on ice. Subsequently, poly (Nim-DNP-L-histidine) was dissolved in DMF and added to HA solution to react for 48 h at room temperature. The mixture was dialyzed with distilled water for 3 days and lyophilized under vacuum. Next, the mixture was dissolved in anhydrous formamide containing mercaptoethanol to react for 48 h at room temperature for removing 2, 4-dinitrophenyl from poly (Nim-DNP-L-histidine). Finally, the HA-PHis were dialyzed with distilled water for 3 days and lyophilized. The structure of HA-PHis was characterized using 1 H NMR.

Synthesis and Characterization of CDI-PF127
An appropriate amount of PF127 was dissolved in acetone and precipitated by precooled n-hexane. The purified PF127 was obtained by vacuum drying, then dissolved in anhydrous acetonitrile. In addition, the CDI was dissolved in anhydrous acetonitrile, then slowly dripped into PF127 anhydrous acetonitrile solution within 2 h under nitrogen, for 4 h. Afterwards, it was concentrated by rotary evaporation and washed three times with precooled ether. The CDI-PF127 was collected by vacuum drying, and characterized using 1 H NMR.

Synthesis and Characterization of NH 2 -PF127
CDI-PF127 was dissolved in anhydrous acetonitrile. The ethylenediamine was slowly dripped into the above solution within 3 h and stirred overnight at room temperature. The excess ethylenediamine was removed by rotary evaporation and washed with precooled ether three times. The white crystalline powder (NH 2 -PF127) was obtained by vacuum drying, and characterized using 1 H NMR.

Synthesis and Characterization of PF127-FA
NH 2 -PF127 was dissolved in anhydrous DMSO, then added to triethylamine as the liquid A. FA, NHS and DCC were dissolved in DMSO, and triethylamine was added and reacted for 10 h under magnetic stirring at room temperature (liquid B). Liquid B was slowly added to liquid A under the protection of nitrogen and stirred overnight at room temperature. The deionized water was slowly dripped into the reaction solution to remove the unreacted FA. The supernatant was dialyzed with deionized water for 3 days. The yellowish solid powder (PF127-FA) was obtained by freeze-drying, and characterized using 1 H NMR.

Preparation and Characterization of Micelles
HPPF micelles were prepared using the film dispersion method [24]. The copolymers were dissolved in acetonitrile, then dried. The mixing ratios of HA-PHis and PF127-FA were shown in Table 1. The optimized prescription of HPPF micelles was determined according to particle size and zeta potential. The particle size and zeta potential of HPPF micelles were determined via the Malvern particle size analyzer (Malvern, UK). The morphology of micelles was observed using transmission electron microscope (TEM). The entrapment efficiency (EE%) and drug loading (DL%) of HPPF micelles were determined according to Formulas (1) and (2).
where, C free was the concentration of free DTX (µg/mL); C total was the total concentration of DTX in the suspension (µg/mL); W drug was the amount of drugs encapsulated in HPPF micelles (mg); and W lipid was the weight of mixed carrier material in the prescription (mg).
Pyrene was used to determine the critical micelle concentration (CMC) of HPPF micelles. When the polymer concentration was greater than a certain value, the excitation wavelength shifted from 334 nm to 336 nm. The different volumes of polymer solution were added to the pyrene, and the polymer concentration range was 10 −4 −10 −1 g/L.

In Vitro Drug Release
The drug-loaded micelles were added into the dialysis bag (interception of molecular weight: 12,000 Da), then placed in the PBS release medium. The medium was removed and the equal amount of fresh-release medium was replenished. The drug content in the release medium was determined via HPLC, and the cumulative release percent was calculated according to the Formula (3).
where, E r was cumulative drug release amount (%); V e was replacement volume of PBS (mL); V 0 was total volume of release medium (mL); C i was concentration of release solution during the i h displacement sampling (µg/mL); m drug was total mass of drugs carried (mg); and n was number of replacement PBS.
Cell survival rate% = OD experimenta group OD control group (4) where, OD was optical density.

In Vitro Cellular Uptake
HepG2 and MCF-7 cells in the logarithmic phase were inoculated at a concentration of 1 × 10 5 cells·mL −1 . Next, 100 µg·mL −1 of HA-PHis, PF127-FA, and HPPF containing coumarin-6 were added. DAPI was added for nucleus staining, and the cell uptake was observed using the laser confocal microscope.

In Vivo Fluorescence Imaging and Tissue Distribution
MCF-7/ADR tumor-bearing mice were injected with 200 µL HA-PHis, HPPF, and Dir fluorescence markers, respectively. At 0.5, 6, 12, 24, and 48 h, the fluorescence intensity of tumor site in mice was monitored using fluorescence imaging. After 48 h, the mice were killed and main organs (heart, liver, spleen, lung, kidney, and tumor) were washed with normal saline three times. Then, the fluorescence intensity of organ was measured.

Statistical Analysis
Results were expressed as mean ± S.D. The data were subjected to analysis of variance (ANOVA) using SPSS 21.0 software. p < 0.05 was taken as a significant level.
HepG2 and MCF-7 cells in the logarithmic phase were inoculated at a concentration of 1 × 10 5 cells·mL −1 . Next, 100 µg·mL −1 of HA-PHis, PF127-FA, and HPPF containing coumarin-6 were added. DAPI was added for nucleus staining, and the cell uptake was observed using the laser confocal microscope.

In Vivo Fluorescence Imaging and Tissue Distribution
MCF-7/ADR tumor-bearing mice were injected with 200 µL HA-PHis, HPPF, and Dir fluorescence markers, respectively. At 0.5, 6, 12, 24, and 48 h, the fluorescence intensity of tumor site in mice was monitored using fluorescence imaging. After 48 h, the mice were killed and main organs (heart, liver, spleen, lung, kidney, and tumor) were washed with normal saline three times. Then, the fluorescence intensity of organ was measured.

Statistical Analysis
Results were expressed as mean ± S.D. The data were subjected to analysis of variance (ANOVA) using SPSS 21.0 software. p < 0.05 was taken as a significant level.
The 1 H-NMR spectrum of PF127, FA, physical mixture of PF127 and FA, and PF127-FA were shown in Figure 3. The characteristic peaks of PF127 were δA 3.38 ppm (CH2CH(CH3)O), and δB 3.51 ppm (CH2CH(CH3)O). The characteristic peak shift of FA was δA 11.48 ppm (OH) [28]. The characteristic peak shift of physical mixture of PF127 and FA was 11.61 ppm, which illustrated that FA was covalently bound to PF127 [28]. In addition, the characteristic peak shift of OH (FA) disappeared, which proved that the COOH of FA interacted with the PF127 through the covalent bond. It proved that PF127-FA was synthesized. The 1 H-NMR spectrum of PF127, FA, physical mixture of PF127 and FA, and PF127-FA were shown in Figure 3. The characteristic peaks of PF127 were δ A 3.38 ppm (CH 2 CH(CH 3 )O), and δ B 3.51 ppm (CH 2 CH(CH 3 )O). The characteristic peak shift of FA was δ A 11.48 ppm (OH) [28]. The characteristic peak shift of physical mixture of PF127 and FA was 11.61 ppm, which illustrated that FA was covalently bound to PF127 [28]. In addition, the characteristic peak shift of OH (FA) disappeared, which proved that the COOH of FA interacted with the PF127 through the covalent bond. It proved that PF127-FA was synthesized.

Particle Size and Zeta Potential
The HPPF micelles were prepared using HA-PHis and PF127-FA, and particle size varied with the mass ratio of two block copolymers (Table 2). When the mass ratio was 5:5 and 6:4, two block polymers existed separately as single-component micelles. It demonstrated that they were not well assembled into hybrid polymer micelles [29]. When the mass ratio was 8:2 and 9:1, the hybrid polymer micelles with uniform particle size and good dispersion were formed. When the mass ratio was 9:1, the value of zeta potential was lower than that of 8:2. When the absolute value of zeta potential was higher, the electrostatic repulsive force between the particles was greater [30]. Therefore, the mixed micelles with a mass ratio of 8:2 was selected as the optimized prescription for next studies (PDI: 0.19 ± 0.06). In addition, the stability of HPPF (−17.4 ± 0.9 mV) was significantly enhanced compared with HA-PHis (−13.2 ± 7.8 mV) and PF127-FA (−8.5 ± 1.1 mV) (p < 0.05), which proved that the strategy using hybrid polymer micelles was successful. The shape of HPPF/DTX and HA-PHis was observed using TEM and shown in Figure  4. The shape of HPPF/DTX was spherical and the distribution was uniform. The particle size of HPPF micelles was slightly larger than that of HA-PHis micelle. The reason was that PF127-FA and HA-PHis were self-assembled into HPPF micelles in an embedded

Particle Size and Zeta Potential
The HPPF micelles were prepared using HA-PHis and PF127-FA, and particle size varied with the mass ratio of two block copolymers (Table 2). When the mass ratio was 5:5 and 6:4, two block polymers existed separately as single-component micelles. It demonstrated that they were not well assembled into hybrid polymer micelles [29]. When the mass ratio was 8:2 and 9:1, the hybrid polymer micelles with uniform particle size and good dispersion were formed. When the mass ratio was 9:1, the value of zeta potential was lower than that of 8:2. When the absolute value of zeta potential was higher, the electrostatic repulsive force between the particles was greater [30]. Therefore, the mixed micelles with a mass ratio of 8:2 was selected as the optimized prescription for next studies (PDI: 0.19 ± 0.06). In addition, the stability of HPPF (−17.4 ± 0.9 mV) was significantly enhanced compared with HA-PHis (−13.2 ± 7.8 mV) and PF127-FA (−8.5 ± 1.1 mV) (p < 0.05), which proved that the strategy using hybrid polymer micelles was successful.

Morphological Observation
The shape of HPPF/DTX and HA-PHis was observed using TEM and shown in Figure 4. The shape of HPPF/DTX was spherical and the distribution was uniform. The particle size of HPPF micelles was slightly larger than that of HA-PHis micelle. The reason was that PF127-FA and HA-PHis were self-assembled into HPPF micelles in an embedded form. The hydrophilic chain of HA-PHis was exposed owing to the long chain of PF127-FA, which caused the larger particle size [31]. form. The hydrophilic chain of HA-PHis was exposed owing to the long chain of PF127-FA, which caused the larger particle size [31].

Entrapment Efficiency and Drug Loading
The entrapment efficiency and drug loading of HPPF micelles were 87.2 ± 1.9% and 6.0 ± 0.1%, respectively, which were higher than HA-PHis (84.8 ± 2.1% and 4.2 ± 0.1%). The PF127-FA increased the proportion of hydrophobic blocks of the micelle core, which was beneficial to the loading of hydrophobic drugs (DTX). This study showed that the length of the hydrophobic blocks was closely related to drug loading [32]. Thus, HPPF micelles improved the poor solubility of DTX.

Determination of Critical Micelle Concentration
The aggregation behavior of HPPF micelles was investigated by measuring the fluorescence spectral curve of pyrene (Figure 5a). The critical micelle concentration of HPPF micelles was 0.04 mg·mL −1 . The lower critical micelle concentration was beneficial for the stability of micelles in vivo [33]. This study showed that the CMC value of micelles was an important factor that signified the stability, and that a lower CMC value provided greater solubilization of loaded payload [34].

In Vitro Drug Release
The in vitro drug release experiments were performed to investigate the pH-sensitive release of HPPF micelles in phosphate buffers with different pH values (7.4 and 5.0). As expected, more than 90% of the free drugs were released from DTX solution within 8 h at pH 7.4 (Figure 5b). However, within 72 h, only 45% of DTX was released from the HPPF micelles, which indicated that HPPF micelles ensured long-term stability in the

Entrapment Efficiency and Drug Loading
The entrapment efficiency and drug loading of HPPF micelles were 87.2 ± 1.9% and 6.0 ± 0.1%, respectively, which were higher than HA-PHis (84.8 ± 2.1% and 4.2 ± 0.1%). The PF127-FA increased the proportion of hydrophobic blocks of the micelle core, which was beneficial to the loading of hydrophobic drugs (DTX). This study showed that the length of the hydrophobic blocks was closely related to drug loading [32]. Thus, HPPF micelles improved the poor solubility of DTX.

Determination of Critical Micelle Concentration
The aggregation behavior of HPPF micelles was investigated by measuring the fluorescence spectral curve of pyrene (Figure 5a). The critical micelle concentration of HPPF micelles was 0.04 mg·mL −1 . The lower critical micelle concentration was beneficial for the stability of micelles in vivo [33]. This study showed that the CMC value of micelles was an important factor that signified the stability, and that a lower CMC value provided greater solubilization of loaded payload [34].
form. The hydrophilic chain of HA-PHis was exposed owing to the long chain of PF127-FA, which caused the larger particle size [31].

Entrapment Efficiency and Drug Loading
The entrapment efficiency and drug loading of HPPF micelles were 87.2 ± 1.9% and 6.0 ± 0.1%, respectively, which were higher than HA-PHis (84.8 ± 2.1% and 4.2 ± 0.1%). The PF127-FA increased the proportion of hydrophobic blocks of the micelle core, which was beneficial to the loading of hydrophobic drugs (DTX). This study showed that the length of the hydrophobic blocks was closely related to drug loading [32]. Thus, HPPF micelles improved the poor solubility of DTX.

Determination of Critical Micelle Concentration
The aggregation behavior of HPPF micelles was investigated by measuring the fluorescence spectral curve of pyrene (Figure 5a). The critical micelle concentration of HPPF micelles was 0.04 mg·mL −1 . The lower critical micelle concentration was beneficial for the stability of micelles in vivo [33]. This study showed that the CMC value of micelles was an important factor that signified the stability, and that a lower CMC value provided greater solubilization of loaded payload [34].

In Vitro Drug Release
The in vitro drug release experiments were performed to investigate the pH-sensitive release of HPPF micelles in phosphate buffers with different pH values (7.4 and 5.0). As expected, more than 90% of the free drugs were released from DTX solution within 8 h at pH 7.4 ( Figure 5b). However, within 72 h, only 45% of DTX was released from the HPPF micelles, which indicated that HPPF micelles ensured long-term stability in the

In Vitro Drug Release
The in vitro drug release experiments were performed to investigate the pH-sensitive release of HPPF micelles in phosphate buffers with different pH values (7.4 and 5.0). As expected, more than 90% of the free drugs were released from DTX solution within 8 h at pH 7.4 ( Figure 5b). However, within 72 h, only 45% of DTX was released from the HPPF micelles, which indicated that HPPF micelles ensured long-term stability in the bloodstream and prolonged the circulation time [35]. At pH 5.0, nearly 90% of DTX was liberated from the HPPF micelles within 8 h of incubation, which was in good agreement with previous studies [36]. The pKa value of histidine was close to the tumor site acidic environment, which caused protonation and soluble transformation [37]. Hence, the pH sensitivity of PHis in HPPF micelles was confirmed. In addition, in vitro drug release behaviors were all consistent with the Higuchi model (r = 0.9545, r = 0.9573, and r = 0.9521), indicating that drugs were released through diffusion from the micelles [38]. In summary, the experiments proved the pH-sensitive behavior of the HPPF micelles.

Cytotoxicity
The effects of blank HA-PHis, PF127-FA and HPPF on the growth of HepG2 and MCF-7 cells were determined via MTT (Figure 6a,b). With the increase in the concentration, the survival rates of HepG2 and MCF-7 cells did not change significantly (p > 0.05), indicating that blank HA-PHis, PF127-FA and HPPF had no obvious cytotoxic effect on HepG2 and MCF-7 cells.
tion, the survival rates of HepG2 and MCF-7 cells did not change significantly (p > 0.05), indicating that blank HA-PHis, PF127-FA and HPPF had no obvious cytotoxic effect on HepG2 and MCF-7 cells.
Then, effects of micelles containing DTX on the cell survival rate in HepG2 and MCF-7 cells were evaluated and results were shown in Figure 6c,d. It was found that toxic effects were dependent on the concentration of micelles. For the HepG2 cells, the cytotoxicity of the micelles was ranked as follows: HPPF/DTX (IC50: 1.7 µg/mL) > PF127-FA/DTX (IC50: 2.5 µg/mL) > HA-PHis/DTX (IC50: 4.6 µg/mL). This is because FA was specifically targeted on the surface of tumor cells, and FA receptor was highly expressed on the surface of tumor cells [39,40]. However, HA-PHis had no targeting ability to HepG2 cells owing to low expression of the CD44 receptor [41]. For the MCF-7 cells, the cytotoxicity of the micelles was ranked as follows: HPPF/DTX (IC50: 4.2 µg/mL) > HA-PHis/DTX (IC50: 7.7 µg/mL) > PF127-FA/DTX (IC50: 10.3 µg/mL). HA-PHis had targeting ability to MCF-7 cells, because the CD44 receptor were overexpressed on the surface of the MCF-7 tumor [42]. Hence, the HPPF owned the highest targeting ability utilizing the FA and HA, which formed more DTX and killed tumor cells.  Then, effects of micelles containing DTX on the cell survival rate in HepG2 and MCF-7 cells were evaluated and results were shown in Figure 6c,d. It was found that toxic effects were dependent on the concentration of micelles. For the HepG2 cells, the cytotoxicity of the micelles was ranked as follows: HPPF/DTX (IC 50 : 1.7 µg/mL) > PF127-FA/DTX (IC 50 : 2.5 µg/mL) > HA-PHis/DTX (IC 50 : 4.6 µg/mL). This is because FA was specifically targeted on the surface of tumor cells, and FA receptor was highly expressed on the surface of tumor cells [39,40]. However, HA-PHis had no targeting ability to HepG2 cells owing to low expression of the CD44 receptor [41]. For the MCF-7 cells, the cytotoxicity of the micelles was ranked as follows: HPPF/DTX (IC 50 : 4.2 µg/mL) > HA-PHis/DTX (IC 50 : 7.7 µg/mL) > PF127-FA/DTX (IC 50 : 10.3 µg/mL). HA-PHis had targeting ability to MCF-7 cells, because the CD44 receptor were overexpressed on the surface of the MCF-7 tumor [42]. Hence, the HPPF owned the highest targeting ability utilizing the FA and HA, which formed more DTX and killed tumor cells.

In Vitro Cellular Uptake
Cellular uptake of HPPF micelles were observed via laser confocal localization using HepG2 and MCF-7 cells. Coumarin-6 carrier was chosen as the probe. After incubation for 2 h, the fluorescence intensity of HepG2 cells was very dark, and the HPPF micelles were distributed in the cytoplasm of the cells, but not in the nucleus (Figure 7a). It was suggested that HPPF was swallowed into the cytoplasm by cells, but the uptake was very small [43]. The fluorescence intensity of MCF-7 cells was much stronger than that of HepG2 cells (Figure 7b). The HPPF micelles was mainly distributed in the cytoplasm, but not in the nucleus. The results showed that HPPF micelles were effectively swallowed endocytosis into the cytoplasm by MCF-7 cells. Additionally, the uptake was significantly higher than that of HepG2, which was in good agreement with the results of cytotoxicity. It also proved that only FA did not ensure that the prepared micelles were targeted to the tumor site. The previous study also proved that the conjugation of mesoporous silica nanoparticles with FA increased the efficiency of nanoparticles entering the cell and localization in the close vicinity of the nucleus [44]. The results of confocal microscopy proved that the HA-receptor mediated cellular uptake of redox-sensitive chitosan-based nanoparticle [45].
Cellular uptake of HPPF micelles were observed via laser confocal localization using HepG2 and MCF-7 cells. Coumarin-6 carrier was chosen as the probe. After incubation for 2 h, the fluorescence intensity of HepG2 cells was very dark, and the HPPF micelles were distributed in the cytoplasm of the cells, but not in the nucleus (Figure 7a). It was suggested that HPPF was swallowed into the cytoplasm by cells, but the uptake was very small [43]. The fluorescence intensity of MCF-7 cells was much stronger than that of HepG2 cells (Figure 7b). The HPPF micelles was mainly distributed in the cytoplasm, but not in the nucleus. The results showed that HPPF micelles were effectively swallowed endocytosis into the cytoplasm by MCF-7 cells. Additionally, the uptake was significantly higher than that of HepG2, which was in good agreement with the results of cytotoxicity. It also proved that only FA did not ensure that the prepared micelles were targeted to the tumor site. The previous study also proved that the conjugation of mesoporous silica nanoparticles with FA increased the efficiency of nanoparticles entering the cell and localization in the close vicinity of the nucleus [44]. The results of confocal microscopy proved that the HA-receptor mediated cellular uptake of redox-sensitive chitosan-based nanoparticle [45].

In Vivo Fluorescence Imaging and Tissue Distribution
The targeting ability of HPPF micelles to tumors in mice was observed via in vivo fluorescence imaging. The HPPF micelles were distributed all over the body after injection for 0.5 h (Figure 7c). With the extension of time, the Dir fluorescence were transferred to the liver, spleen, and other organs. Additionally, the enhancement of fluorescence intensity indicated the accumulation of HPPF. After 12 h, the fluorescence intensity of tumor reached the peak, which was significantly higher than other tissues and organs. In addition, the HPPF fluorescence intensity of the tumor site was significantly higher than that in other organs, indicating good tumor targeting (Figure 7d). Compared with HPPF, the fluorescence intensity of HA-PHis was significantly weakened, indicating that HPPF increased the drug accumulation in tumor site and prolonged the accumulation time of drug in the tumor site. This is mainly attributed to the dual-targeted action of HA and FA, which effectively solved the off-target phenomenon. For example, the study developed high-efficiency dual-targeted nanoflowers containing ferroferric oxide and HA, which improved the specific uptake of drugs at tumor site by the dual action of CD44 ligand HA and magnetic nanoparticles guided by magnetic force [46].

In Vivo Fluorescence Imaging and Tissue Distribution
The targeting ability of HPPF micelles to tumors in mice was observed via in vivo fluorescence imaging. The HPPF micelles were distributed all over the body after injection for 0.5 h (Figure 7c). With the extension of time, the Dir fluorescence were transferred to the liver, spleen, and other organs. Additionally, the enhancement of fluorescence intensity indicated the accumulation of HPPF. After 12 h, the fluorescence intensity of tumor reached the peak, which was significantly higher than other tissues and organs. In addition, the HPPF fluorescence intensity of the tumor site was significantly higher than that in other organs, indicating good tumor targeting (Figure 7d). Compared with HPPF, the fluorescence intensity of HA-PHis was significantly weakened, indicating that HPPF increased the drug accumulation in tumor site and prolonged the accumulation time of drug in the tumor site. This is mainly attributed to the dual-targeted action of HA and FA, which effectively solved the off-target phenomenon. For example, the study developed high-efficiency dual-targeted nanoflowers containing ferroferric oxide and HA, which improved the specific uptake of drugs at tumor site by the dual action of CD44 ligand HA and magnetic nanoparticles guided by magnetic force [46].

Conclusions
In this study, a novel dual-target pH-sensitive HPPF hybrid micelle was successfully constructed. The optimal mixing ratio (HA-PHis: PF127-FA = 8:2) was obtained according to particle size and zeta potential. The HPPF micelles improved the stability with higher zeta potential and lower critical micelle concentration. The pH-sensitive release of HPPF micelles was demonstrated owing to histidine protonation. In vivo image demonstrated that the targeting ability of HPPF micelles was higher than FA and HA. In conclusion, this study provided a new strategy for the development of polymer micelle, which reduced the side effects of chemotherapeutic drugs and improved the treatment of breast cancer. Institutional Review Board Statement: The animal study protocol was approved by the Animal Ethics Committee of Shantou University Medical College (protocol code SUMC2022-152 and 2022.08). for studies involving animals.