Preparation and Self-Assembly of pH-Responsive Hyperbranched Polymer Peptide Hybrid Materials

In recent years, the coupling of structurally and functionally controllable polymers with biologically active peptide materials to obtain polymer-peptide hybrids with excellent properties and biocompatibility has led to important research progress in the field of polymers. In this study, a pH-responsive hyperbranched polymer hPDPA was prepared by combining atom transfer radical polymerization (ATRP) with self-condensation vinyl polymerization (SCVP) using a three-component reaction of Passerini to obtain a monomeric initiator ABMA containing functional groups. The pH-responsive polymer peptide hybrids hPDPA/PArg/HA were obtained by using the molecular recognition of polyarginine (β-CD-PArg) peptide modified with β-cyclodextrin (β-CD) on the hyperbranched polymer, followed by the electrostatic adsorption of hyaluronic acid (HA). The two hybrid materials, h1PDPA/PArg12/HA and h2PDPA/PArg8/HA could self-assemble to form vesicles with narrow dispersion and nanoscale dimensions in phosphate-buffered (PB) at pH = 7.4. The assemblies exhibited low toxicity as drug carriers of β-lapachone (β-lapa), and the synergistic therapy based on ROS and NO generated by β-lapa had significant inhibitory effects on cancer cells.


Introduction
Compared with linear polymers, hyperbranched polymers with highly branched threedimensional spherical structures have specific properties, such as better solubility, higher rheology, and abundant modifiable terminal groups [1][2][3][4]. Three methods are commonly used for the synthesis of hyperbranched polymers: (1) the ABx-type monomer condensation method; (2) the SCVP method; and (3) the ring-opening polymerization method. The SCVP method is the most commonly used method [5][6][7][8][9][10][11][12][13][14][15][16]. The method is based on the polymerization of a monomer initiator (inimer) containing both a double bond and an initiator group to prepare hyperbranched polymers [17][18][19][20][21][22]. It is widely used for its advantages, such as simple operation and mild reaction conditions, but the SCVP method also has some problems, such as a wide molecular weight distribution of the polymer and difficulty in controlling the molecular weight. The SCVP method is usually combined with reactive polymerization, such as reversible addition-fragmentation transfer polymerization (RAFT) or ATRP, to obtain structurally controlled hyperbranched polymers with a large number of RAFT chain transfer agents or ATRP initiators at their ends; for example, Patrickios and colleagues [23] prepared hydrophobic, degradable hyperbranched polymers PMMA by combining the SCVP method with ATRP, and obtained amphiphilic hyperbranched multiarm polymers by using PMMA as a macromolecular initiator followed by polymerization of diaminoethyl methacrylate (DMAEMA). Subsequently, the ends can be modified to prepare functionalized polymers [24][25][26]. For example, Wais and colleagues [26] synthesized a series of star-shaped hyperbranched polymers by RAFT polymerization; these polymers
2.1. Synthesis of 1-Isocyanadamantane (Ad-NC) [37] Amantadine alcohol (658 mg, 4.33 mmol), ZnBr 2 (2.95 g, 13.1 mmol), and 25 mL CH 2 Cl 2 were added to a 100 mL round-bottom flask, the oxygen was removed by bubbling with argon gas, and TMSCN (1.60 mL, 13.2 mmol) was added rapidly under the atmosphere of argon gas. The reaction was confined at room temperature for 18 h. Then, 13 mL TBAF solution was added and stirred for 20 min. The reaction solution was washed three times with a saturated NaHCO 3 solution; then, the aqueous phase was extracted with ether. The combined organic phase was washed three times with a saturated NaCl solution and ultrapure water and finally dried with MgSO 4 ; then it was separated by column chromatography with petroleum ether: ethyl acetate = 16:1. The separation of Ad-NC was obtained by column chromatography (yield: 60%).

Synthesis of 4-Formylphenyl-2-bromo-2-methylpropionate (CHO-Br) [38]
P-hydroxybenzaldehyde (972 mg, 7.97 mmol) was dissolved in 15 mL CH 2 Cl 2 , triethylamine (TEA, 1.40 mL, 10.1 mmol) was added, and the reaction was placed in an ice-water bath. Dibromoisobutyryl bromide (1.20 mL, 9.71 mmol) was dissolved in 5 mL CH 2 Cl 2 and added dropwise to the above solution. The reaction was stirred for 12 h at room temperature. The reaction solution was washed three times with saturated NaHCO 3 solution and ultrapure water and dried with MgSO 4 ; then it was separated by column chromatography with petroleum ether: ethyl acetate = 6:1. The separation of CHO-Br was obtained by column chromatography (yield: 70%). Ad-NC (127 mg, 0.789 mmol), CHO-Br (107 mg, 0.399 mmol), and methacrylic acid (39.0 mg, 0.542 mmol) were dissolved in 1 mL CH 2 Cl 2 , and the reaction was stirred at room temperature for 48 h. The crude product was purified by silica gel column chromatography, petroleum ether: ethyl acetate = 12:1. (Yield: 70%) 2.4. Synthesis of Hyperbranched Poly(diisopropylaminoethyl Methacrylate) (hPDPA) 2,2 -bipyridine (bpy, 67.1 mg, 0.429 mmol) was dissolved in 0.6 mL DMF in a 5 mL Schlenk flask and degassed by three cycles of freeze-pump-thaw. CuBr (20.5 mg, 0.143 mmol) was added rapidly under argon atmosphere, the flask was degassed by three cycles of freeze-pump-thaw and was stirred under argon atmosphere at room temperature for 0.5 h. The monomer initiator ABMA (123 mg, 0.239 mmol) and DPA (300 mg, 1.41 mmol) were dissolved in 0.5 mL DMF in another 5 mL Schlenk flask and degassed by three cycles of freeze-pump-thaw. 0.5 mL CuBr solution was transferred into the polymer solution under argon protection, and the mixture was degassed by three cycles of freeze-pump-thaw. The polymerization was stirred at 60 • C for 5 h. After the polymerization reaction, DMF was removed from the reaction system, and the copper salts were removed by a neutral alumina column using CH 2 Cl 2 as a drenching agent; the polymer was dissolved in a small amount To synthesize β-CD-PArg, β-CD-dithiodipyridine (β-CD-s-s-py) was first synthesized by a reaction between β-CD-SH and 2,2 -dithiodipyridine (py-s-s-py). β-CD-SH (400 mg, 0.348 mmol) and py-s-s-py (230 mg, 1.05 mmol) were dissolved in 8 mL DMF, and the reaction was conducted at room temperature for 24 h. The product was purified by precipitation in acetone and centrifugation. The upper layer of yellow liquid was washed with acetone until the upper layer of liquid was colorless, and the precipitate was the target product.
β-CD-s-s-py (80.0 mg, 0.0651 mmol) and PArg (37.0 mg, 0.0270 mmol) were dissolved in 2.2 mL of DMF (containing 0.2 mL ultrapure water), and the reaction was conducted at room temperature for 24 h. The crude product was purified by precipitation in acetone, centrifuged, and the supernatant was taken to obtain β-CD-PArg.

Synthesis of Polymer Peptide Conjugates hPDPA/PArg/HA
The hyperbranched polymer hPDPA was dissolved in DMF, β-CD-PArg was dissolved in DMSO and mixed in a certain ratio, and HA was dissolved in 1 mL of pH = 7.4 phosphate buffer solution (PB, 10 mM), and 0.2 mL of the mixed solution was slowly added dropwise to the HA solution. The assemblies were obtained by dialysis after rapid stirring for 2 h. The details of the assembly of the two polymer peptide hybrids are shown in Table 2. The polymer peptide conjugates hPDPA/PArg/HA was first synthesized. A total of 2.0 mg β-lapa was dissolved in 0.5 mL DMSO, and 0.062 mL β-lapa solution was added dropwise to the above solution; stirring continued for 12 h. After the encapsulation process was completed, the unencapsulated β-lapa was removed by dialysis and centrifugation. The nanoparticle powder encapsulated with β-lapa was dissolved in DMSO and detected by UV-Vis spectrophotometer, and the encapsulation rate (EE) and drug loading rate (LC) of β-lapa were calculated according to the formula: where the m b is the mass of the encapsulated β-lapa, m o is the original β-lapa mass, and m p is the mass of the polymer hybrid material. dissolved in 1 mL DMSO every two hours, and the absorption intensity was measured by UV-Vis spectrophotometer to calculate the amount of released β-lapa according to the equation, and then supplemented with 1 mL PB to continue stirring.
2.9. In Vitro Cytotoxicity Assays h 2 PDPA/PArg 8 /HA and h 2 PDPA/PArg 8 /HA-β-lapa were cultured with Hela cells, respectively, and the cytotoxicity of hyperbranched polymer peptide hybrid materials was tested using the CCK-8 method. Hela cells were inoculated into 96-well plates with 5 × 10 3 cells per well. The cells were incubated in the medium of DMEM with 10% FBS at 37 • C and 5% CO 2 for 12 h until the cells were stable. h 2 PDPA/PArg 8 /HA and h 2 PDPA/PArg 8 /HA-β-lapa at different dilution concentrations were added to the medium and incubated for 24 h, respectively, and then the medium was carefully removed, and 100 µL of the medium was added to each well again (containing 10 µL CCK-8 reagent) and continued to incubate in a constant temperature incubator at 37 • C for 1 h. The absorbance at 450 nm was measured using an enzyme marker. Cell viability was calculated by using the following formula: Cell viability(%) = As − Ab Ac − Ab × 100% where As and Ac represent the absorbance of the cells treated by h 2 PDPA/PArg 8 /HA-βlapa and the control cells (untreated), respectively. Ab is the absorbance of CCK-8 regents without cells.

Intracellular NO, ROS, and ONOO − Release Assay
Hela cells were inoculated onto 35 mm 2 confocal-specific dishes with 1 × 10 5 cells per well. The cells were incubated at 37 • C with 5% CO 2 for 12 h. Diluted h 2 PDPA/PArg 8 /HA and h 2 PDPA/PArg 8 /HA-β-lapa were added, respectively, and the culture medium was removed, washed with PBS, and fixed with 4% cell tissue fixative for 10 min, then washed again with PBS, and the dilutions were provided according to the kit. The NO probe (DAF-FM DA probe) was diluted, 1 mL of diluted DAF-FM DA was added to each well, incubated in a 37 • C cell incubator for 20 min, then stained with DAPI for 5 min, observed by laser confocal microscopy (CLSM) and photographed. ROS were detected using the DCFH-DA probe and ONOO − using the O72 probe.

Results and Discussion
In this study, the monomer ABMA is a key compound for the synthesis of hyperbranched polymer hPDPA. It was synthesized by the Passerini three-component reaction of Ad-NC, CHO-Br, and methacrylic acid, providing the polymer branched chains with bromo groups to initiate the polymerization and adamantyl groups for molecular recognition. According to a previous report, Ad-NC was synthesized directly from adamantanol in one step. The synthesis of Ad-NC was confirmed by 1 H NMR and 13 C NMR spectra ( Figure S1, Supporting Information). The 13 C NMR spectrum of Ad-NC exhibits a peak at 150.66 ppm, belonging to the isocyanine group. CHO-Br was obtained by the esterification reaction of p-hydroxybenzaldehyde and 2-bromoisobutyryl bromide. The 1 H NMR spectrum of CHO-Br is shown in Figure S2. The synthesized ABMA was obtained in high yield (70.1%), and due to the high reactivity of the isocyanate, the reaction conditions were mild. As shown in the 1 H NMR spectrum of ABMA ( Figure S3, Supporting Information), the characteristic peaks of products of the Passerini reaction appear at 5.74 and 5.95 ppm, corresponding to the protons of the newly formed tertiary carbon atom and the amide group, respectively. In the 13 C NMR spectrum ( Figure S3), the peak at 74.03 ppm can be observed for the newly formed tertiary carbon atom. The NMR spectrum results can prove the successful preparation of monomer ABMA.
Two hyperbranched polymers, hPDPA with different branching degrees but similar molecular weights, were obtained by adjusting the feeding ratios of monomer initiator Nanomaterials 2023, 13, 1725 7 of 17 ABMA and copolymer monomer DPA. h 1 PDPA and h 2 PDPA were obtained with feeding ratios of 6 and 10 for DPA and ABMA, respectively. h 1 PDPA and h 2 PDPA were characterized by 1 H NMR and GPC.
The 1 H NMR spectrum of the hyperbranched polymer h 1 PDPA is shown in Figure 1a. As can be clearly seen, the peaks at 7.47 ppm and 7.20-6.91 ppm correspond to protons on the phenyl group of the monomer initiator ABMA, and the peaks at 6.20 ppm and 5.81 ppm correspond to protons on the tertiary carbon atom and on the amide, respectively, which are characteristic peaks formed by the Passerini reaction. The peaks at 3.85 ppm and 3.11 ppm correspond to methylene protons on the copolymer monomer DPA, and the peaks at 2.77 ppm and 1.20 ppm correspond to protons on the tertiary carbon atom and on the terminal methyl group of the copolymer monomer DPA, respectively. The peak intensity of the proton on adamantine at 2.10 ppm is higher due to the inclusion of protons on the main chain methylene group. The 1 H NMR spectra of the hyperbranched polymer h 2 PDPA are shown in Figure S4. Based on the 1 H NMR results, the molecular weight of the hyperbranched polymer, the degree of polymerization (DP n ) of the two monomers, and the ratio can be obtained by integrating the characteristic peaks. The DP n ratio of DPA to ABMA for h 1 PDPA and h 2 PDPA was calculated to be 5.8 and 11.2, and the obtained polymerization ratio was close to the feeding ratio. As the feeding ratio of DPA to ABMA increased, the percentage of monomer initiator ABMA decreased, and the branching degree gradually decreased. The GPC curve of h 1 PDPA is shown in Figure 1b. It can be seen that the GPC curve shows a single-peak distribution, as the hyperbranched polymers are all nitrogenous compounds, which will adsorb to the column, making the peak emergence time late and incomplete. The same phenomenon can be observed in the GPC curve of h 2 PDPA ( Figure S5). The structural data of the hyperbranched polymers based on 1 H NMR and GPC results are summarized in Table 3. for the newly formed tertiary carbon atom. The NMR spectrum results can prove the successful preparation of monomer ABMA. Two hyperbranched polymers, hPDPA with different branching degrees but similar molecular weights, were obtained by adjusting the feeding ratios of monomer initiator ABMA and copolymer monomer DPA. h1PDPA and h2PDPA were obtained with feeding ratios of 6 and 10 for DPA and ABMA, respectively. h1PDPA and h2PDPA were characterized by 1 H NMR and GPC.
The 1 H NMR spectrum of the hyperbranched polymer h1PDPA is shown in Figure  1a. As can be clearly seen, the peaks at 7.47 ppm and 7.20-6.91 ppm correspond to protons on the phenyl group of the monomer initiator ABMA, and the peaks at 6.20 ppm and 5.81 ppm correspond to protons on the tertiary carbon atom and on the amide, respectively, which are characteristic peaks formed by the Passerini reaction. The peaks at 3.85 ppm and 3.11 ppm correspond to methylene protons on the copolymer monomer DPA, and the peaks at 2.77 ppm and 1.20 ppm correspond to protons on the tertiary carbon atom and on the terminal methyl group of the copolymer monomer DPA, respectively. The peak intensity of the proton on adamantine at 2.10 ppm is higher due to the inclusion of protons on the main chain methylene group. The 1 H NMR spectra of the hyperbranched polymer h2PDPA are shown in Figure S4. Based on the 1 H NMR results, the molecular weight of the hyperbranched polymer, the degree of polymerization (DPn) of the two monomers, and the ratio can be obtained by integrating the characteristic peaks. The DPn ratio of DPA to ABMA for h1PDPA and h2PDPA was calculated to be 5.8 and 11.2, and the obtained polymerization ratio was close to the feeding ratio. As the feeding ratio of DPA to ABMA increased, the percentage of monomer initiator ABMA decreased, and the branching degree gradually decreased. The GPC curve of h1PDPA is shown in Figure 1b. It can be seen that the GPC curve shows a single-peak distribution, as the hyperbranched polymers are all nitrogenous compounds, which will adsorb to the column, making the peak emergence time late and incomplete. The same phenomenon can be observed in the GPC curve of h2PDPA ( Figure S5). The structural data of the hyperbranched polymers based on 1 H NMR and GPC results are summarized in Table 3.   According to Table 3, the number of adamantane groups of h 1 PDPA and h 2 PDPA can be obtained, which are 12 and 8, respectively. Therefore, it can be functionalized by recognition with molecules containing β-CD. In this study, β-CD-PArg was used as a model peptide for interaction with hyperbranched polymers. β-CD-s-s-py was prepared according to a previous report [39], and its 1 H NMR is shown in Figure S6. The β-CD-PArg was obtained by a disulfide bond exchange reaction between the sulfhydryl group and β-CD-s-s-Py, and the 1 H NMR of PArg before and after modification is shown in Figure S7. 2D NMR spectroscopy is a common method for analyzing the host-guest interaction because the H3 of cyclodextrin is located in the hydrophobic inner cavity, and when the adamantane group enters the inner cavity, the interaction with H3 is enhanced so that cross peaks appear on the 2D NMR spectra [40]. In this study, Host-guest recognition of According to Table 3, the number of adamantane groups of h1PDPA and h2PDPA can be obtained, which are 12 and 8, respectively. Therefore, it can be functionalized by recognition with molecules containing β-CD. In this study, β-CD-PArg was used as a model peptide for interaction with hyperbranched polymers. β-CD-s-s-py was prepared according to a previous report [39], and its 1 H NMR is shown in Figure S6. The β-CD-PArg was obtained by a disulfide bond exchange reaction between the sulfhydryl group and β-CDs-s-Py, and the 1 H NMR of PArg before and after modification is shown in Figure S7. 2D NMR spectroscopy is a common method for analyzing the host-guest interaction because the H3 of cyclodextrin is located in the hydrophobic inner cavity, and when the adamantane group enters the inner cavity, the interaction with H3 is enhanced so that cross peaks appear on the 2D NMR spectra [40]. In this study, Host-guest recognition of β-CD-PArg and hyperbranched polymer hPDPA (in molar amounts of adamantane groups) in a mixture of DMF and DMSO. The 2D NOESY NMR spectra of the hyperbranched polymer h1PDPA and β-CD-PArg are shown in Figure 2, with chemical shifts showing cross peaks at 1.80-2.05 ppm and 3.84-3.95 ppm, indicating that the adamantane group is incorporated into the hydrophobic cavity of β-CD. Due to the positively charged nature of peptides, they can adsorb HA by electrostatic interaction, and infrared spectroscopy is a common characterization method to study the formation of complexes between two substances by electrostatic interaction [41,42]. The infrared spectra of HA, h1PDPA/PArg12, and h1PDPA/PArg12/HA are shown in Figure 3, in the spectrum of HA, the absorption peak at 1732 cm −1 belongs to the stretching vibration absorption peak of C=O (COOH), while the spectrum of h1PDPA/PArg12/HA shows this Due to the positively charged nature of peptides, they can adsorb HA by electrostatic interaction, and infrared spectroscopy is a common characterization method to study the formation of complexes between two substances by electrostatic interaction [41,42]. The infrared spectra of HA, h 1 PDPA/PArg 12 , and h 1 PDPA/PArg 12 /HA are shown in Figure 3, in the spectrum of HA, the absorption peak at 1732 cm −1 belongs to the stretching vibration absorption peak of C=O (COOH), while the spectrum of h 1 PDPA/PArg 12 /HA shows this absorption peak shifted right to 1717 cm −1 and weakened in intensity, and a new absorption peak appears at 1650 cm −1 , which belongs to the N-H bending vibration absorption peak in h 1 PDPA/PArg 12 , and at 1610 cm −1 belongs to the anti-symmetric absorption peak of C=O (COOH), which is weaker than the intensity in the HA spectrum, due to the protonation of the amine in the polymer forming an ionic bond with the carboxyl group in HA, all of which can prove the electrostatic interaction between HA and h 1 PDPA/PArg 12 .
absorption peak shifted right to 1717 cm −1 and weakened in intensity, and a new absorption peak appears at 1650 cm −1 , which belongs to the N-H bending vibration absorption peak in h1PDPA/PArg12, and at 1610 cm −1 belongs to the anti-symmetric absorption peak of C=O (COOH), which is weaker than the intensity in the HA spectrum, due to the protonation of the amine in the polymer forming an ionic bond with the carboxyl group in HA, all of which can prove the electrostatic interaction between HA and h1PDPA/PArg12. Hyperbranched polymers and their conjugates are pH sensitive due to the presence of amino groups [43]. The polymer peptide conjugates hPDPA/PArg/HA and contains both hydrophobic group DPA and hydrophilic groups PArg and HA; thus, self-assembly can be performed in PB at pH = 7.4. The self-assembly behavior of hPDPA/PArg/HA was investigated by DLS, SEM, and TEM, and the hydrodynamic dimensions of the assemblies in an aqueous solution after dialysis were measured and summarized in Table 4. The DLS results are shown in Figure 4c,f. All the two hPDPA/PArg/HA self-assemble into narrowly dispersed assemblies. Although the two hybrids are similar in composition, the hydrodynamic dimensions are slightly different. The hyperbranched polymer h1PDPA contains more adamantane groups, which are then recognized with more PArg, and is therefore more charged, with more HA adsorbed by electrostatic interaction and a large hydrodynamic diameter. To further investigate the morphology and structure of the assemblies, the specific morphology was first observed by SEM, which samples were prepared by dropping the assemblies' solution on a silicon wafer and drying at 25 °C. The samples were treated with gold spray before measurement. It can be seen from the SEM images (Figure 4a,d) that both h1PDPA/PArg12/HA and h2PDPA/PArg8/HA are assembled into hollow structures with folds on the surface, indicating that they can self-assemble in an aqueous solution to form vesicles with slightly smaller sizes than those measured by DLS, suggesting that the assemblies shrink slightly during the drying process. At the same time, TEM was used to study morphology more visually. The samples were stained by OsO4, and it can be seen in the TEM images (Figure 4b,e) that both h1PDPA/PArg12/HA and h2PDPA/PArg8/HA self-assembled into spherical structures with low intermediate lining and high edge lining, and the sizes were in the range of 250-400 nm and 250-350 nm, respectively. The sizes were in the range of 250-400 nm and 250-350 nm, respectively, which were close to the results measured by DLS. Hyperbranched polymers and their conjugates are pH sensitive due to the presence of amino groups [43]. The polymer peptide conjugates hPDPA/PArg/HA and contains both hydrophobic group DPA and hydrophilic groups PArg and HA; thus, self-assembly can be performed in PB at pH = 7.4. The self-assembly behavior of hPDPA/PArg/HA was investigated by DLS, SEM, and TEM, and the hydrodynamic dimensions of the assemblies in an aqueous solution after dialysis were measured and summarized in Table 4. The DLS results are shown in Figure 4c,f. All the two hPDPA/PArg/HA self-assemble into narrowly dispersed assemblies. Although the two hybrids are similar in composition, the hydrodynamic dimensions are slightly different. The hyperbranched polymer h 1 PDPA contains more adamantane groups, which are then recognized with more PArg, and is therefore more charged, with more HA adsorbed by electrostatic interaction and a large hydrodynamic diameter. To further investigate the morphology and structure of the assemblies, the specific morphology was first observed by SEM, which samples were prepared by dropping the assemblies' solution on a silicon wafer and drying at 25 • C. The samples were treated with gold spray before measurement. It can be seen from the SEM images (Figure 4a,d) that both h 1 PDPA/PArg 12 /HA and h 2 PDPA/PArg 8 /HA are assembled into hollow structures with folds on the surface, indicating that they can self-assemble in an aqueous solution to form vesicles with slightly smaller sizes than those measured by DLS, suggesting that the assemblies shrink slightly during the drying process. At the same time, TEM was used to study morphology more visually. The samples were stained by OsO 4 , and it can be seen in the TEM images (Figure 4b,e) that both h 1 PDPA/PArg 12 /HA and h 2 PDPA/PArg 8 /HA self-assembled into spherical structures with low intermediate lining and high edge lining, and the sizes were in the range of 250-400 nm and 250-350 nm, respectively. The sizes were in the range of 250-400 nm and 250-350 nm, respectively, which were close to the results measured by DLS.   By using the hyperbranched polymer bioconjugates h2PDPA/PArg8/HA loaded with β-lapa. The UV absorption of a series of concentrations of β-lapa in DMSO was measured by a UV spectrophotometer, the standard curve was plotted ( Figure S8), and the encapsulation rate and drug loading rate were calculated. The size and PDI of the loaded nanoparticles were measured by DLS ( Figure S9). h1PDPA/PArg12/HA-β-lapa and h2PDPA/PArg8/HA-β-lapa loading data were summarized as shown in Table 5, both of which had narrower PDI, higher encapsulation rate, and lower loading due to the higher mass of adsorbed HA.  By using the hyperbranched polymer bioconjugates h 2 PDPA/PArg 8 /HA loaded with β-lapa. The UV absorption of a series of concentrations of β-lapa in DMSO was measured by a UV spectrophotometer, the standard curve was plotted ( Figure S8), and the encapsulation rate and drug loading rate were calculated. The size and PDI of the loaded nanoparticles were measured by DLS ( Figure S9). h 1 PDPA/PArg 12 /HA-β-lapa and h 2 PDPA/PArg 8 /HAβ-lapa loading data were summarized as shown in Table 5, both of which had narrower PDI, higher encapsulation rate, and lower loading due to the higher mass of adsorbed HA. To explore the release of β-lapa from h 2 PDPA/PArg 8 /HA-β-lapa in intracellular, two different pHs, which are 7.4 and 5.0, were selected to simulate the physiological environment of normal tissue cells and tumor cells in vitro to test the release of β-lapa under different pH conditions. As shown in Figure 5, it can be clearly seen that the release of β-lapa becomes faster and increases when the pH decreases from the physiological environment of 7.4 to the tumor microenvironment of 5.0. The cumulative release rates of the bioconjugates h 2 PDPA/PArg 8 /HA-β-lapa at pH 7.4 and 5.0 for 36 h were 28% and 48%, respectively. Its drug release rate was low in normal tissue pH conditions, while it was able to release drug molecules effectively in the low pH conditions of tumor cells. lapa becomes faster and increases when the pH decreases from the physiological e ment of 7.4 to the tumor microenvironment of 5.0. The cumulative release rate bioconjugates h2PDPA/PArg8/HA-β-lapa at pH 7.4 and 5.0 for 36 h were 28% an respectively. Its drug release rate was low in normal tissue pH conditions, whil able to release drug molecules effectively in the low pH conditions of tumor cells The carriers applied for intracellular drug delivery must be non-toxic or h toxicity, which is a necessary condition for the biological application of the asse The cytotoxicity of the carrier h2PDPA/PArg8/HA on Hela cells was evaluated by u CCK-8 method in the concentration range of 1.96-250 µg/mL and the same conce of the carrier containing β-lapa at 0.656-84 µg/mL. It is clear from Figure 6 that survival rate was above 90% at carrier concentrations up to 250 µg/mL, demon that the carrier h2PDPA/PArg8/HA has low toxicity to Hela cells. The drug β-lapa leased from h2PDPA/PArg8/HA-β-lapa when in a cancer cell environment with approximately 5.0, and when the β lapa at 84 µg/mL, the cell survival rate was on demonstrating that the oxidative stress induced by β-lapa has an inhibitory effect cer cells.
The h2PDPA/PArg8/HA and h2PDPA/PArg8/HA-β-lapa were co-cultured wi cells, respectively, and the release of ROS was observed by laser confocal mic (CLSM) at different time points. The nuclei were stained blue with DAPI, and R duction in Hela cells was detected by ROS probe (DCFH-DA), as shown in Figure  green fluorescence in h2PDPA/PArg8/HA and h2PDPA/PArg8/HA-β-lapa was sign enhanced with increasing time, demonstrating the production of ROS due to the the polymer hybrid material is biocompatible and targeted HA, making the nanop easily internalized by the cells. Since the hyperbranched polymer is pH-respons green fluorescence of h2PDPA/PArg8/HA-β-lapa is stronger than that of un h2PDPA/PArg8/HA due to the release of β-lapa in the tumor environment (acidic can generate large amounts of H2O2. PArg can be used as a NO donor with good patibility, and in the presence of large amounts of ROS conditions, NO was relea The carriers applied for intracellular drug delivery must be non-toxic or have low toxicity, which is a necessary condition for the biological application of the assemblies. The cytotoxicity of the carrier h 2 PDPA/PArg 8 /HA on Hela cells was evaluated by using the CCK-8 method in the concentration range of 1.96-250 µg/mL and the same concentration of the carrier containing β-lapa at 0.656-84 µg/mL. It is clear from Figure 6 that the cell survival rate was above 90% at carrier concentrations up to 250 µg/mL, demonstrating that the carrier h 2 PDPA/PArg 8 /HA has low toxicity to Hela cells. The drug β-lapa was released from h 2 PDPA/PArg 8 /HA-β-lapa when in a cancer cell environment with a pH of approximately 5.0, and when the β lapa at 84 µg/mL, the cell survival rate was only 10%, demonstrating that the oxidative stress induced by β-lapa has an inhibitory effect on cancer cells.
The h 2 PDPA/PArg 8 /HA and h 2 PDPA/PArg 8 /HA-β-lapa were co-cultured with Hela cells, respectively, and the release of ROS was observed by laser confocal microscopy (CLSM) at different time points. The nuclei were stained blue with DAPI, and ROS production in Hela cells was detected by ROS probe (DCFH-DA), as shown in Figure 7a. The green fluorescence in h 2 PDPA/PArg 8 /HA and h 2 PDPA/PArg 8 /HA-β-lapa was significantly enhanced with increasing time, demonstrating the production of ROS due to the shell of the polymer hybrid material is biocompatible and targeted HA, making the nanoparticles easily internalized by the cells. Since the hyperbranched polymer is pH-responsive, the green fluorescence of h 2 PDPA/PArg 8 /HA-β-lapa is stronger than that of unloaded h 2 PDPA/PArg 8 /HA due to the release of β-lapa in the tumor environment (acidic), which can generate large amounts of H 2 O 2 . PArg can be used as a NO donor with good biocompatibility, and in the presence of large amounts of ROS conditions, NO was released in a controlled manner. As shown in Figure 7b, the green fluorescence in h 2 PDPA/PArg 8 /HA and h 2 PDPA/PArg 8 /HA-β-lapa was significantly enhanced with increasing time, demonstrating the production of NO. The green fluorescence in h 2 PDPA/PArg 8 /HA-β-lapa was stronger than that in h 2 PDPA/PArg 8 /HA due to the large amount of ROS that can oxidize PArg to produce NO. Because H 2 O 2 can penetrate most cell membranes and generate hydroxyl radical ·OH with intracellular Fe, ·OH and NO can generate peroxynitrite anion (ONOO − ), and the generation of ONOO − in cells was detected by the O72 probe. The red fluorescence in h 2 PDPA/PArg 8 /HA and h 2 PDPA/PArg 8 /HA-β-lapa was significantly enhanced with increasing time, demonstrating the production of ONOO − (Figure 7c). The red fluorescence is stronger in h 2 PDPA/PArg 8 /HA-β-lapa than in h 2 PDPA/PArg 8 /HA because of the high production of ROS and thus more ONOO − . strating the production of NO. The green fluorescence in h2PDPA/PArg8/HA-β-lap stronger than that in h2PDPA/PArg8/HA due to the large amount of ROS that can o PArg to produce NO. Because H2O2 can penetrate most cell membranes and genera droxyl radical ·OH with intracellular Fe, ·OH and NO can generate peroxynitrite (ONOO − ), and the generation of ONOO − in cells was detected by the O72 probe. T fluorescence in h2PDPA/PArg8/HA and h2PDPA/PArg8/HA-β-lapa was significant hanced with increasing time, demonstrating the production of ONOO − (Figure 7c red fluorescence is stronger in h2PDPA/PArg8/HA-β-lapa than in h2PDPA/PArg8/H cause of the high production of ROS and thus more ONOO − .

Conclusions
First, a monomer initiator ABMA with double bonds, bromine groups, and adamantane groups was obtained by a Passerini three-component reaction using CHO-Br, Ad-NC, and methacrylic acid. The inimer was copolymerized with the monomer DPA, and pH-responsive hyperbranched polymer hPDPA with different DB were prepared by the ATRP-SCVP method. The pH-responsive hyperbranched polymer peptide conjugates hPDPA/PArg were obtained by the molecular recognition of adamantyl groups on hyperbranched polymers with β-CD-PArg, and then the pH-responsive polymer peptide hybrid material hPDPA/PArg/HA was obtained by the electrostatic interaction between hPDPA/PArg and HA. The self-assembly behavior of hPDPA/PArg/HA with different DB and the application of the assemblies as drug carriers for β-lapa were investigated. The two hybrid materials h1PDPA/PArg12/HA and h2PDPA/PArg8/HA were found to self-assemble to form vesicles in PB at pH = 7.4, and β-lapa can be effectively loaded by vesicles and released under acidic conditions (pH = 5.0). The carriers h2PDPA /PArg8/HA showed low cytotoxicity towards Hela cells. The release of β-lapa leads to an increase in reactive oxygen species, which can react with PArg to generate NO. The reaction between reactive oxygen species and NO leads to the production of ONOO − , and this synergistic therapy based on ROS and NO has a significant inhibitory effect on cancer cells.

Conclusions
First, a monomer initiator ABMA with double bonds, bromine groups, and adamantane groups was obtained by a Passerini three-component reaction using CHO-Br, Ad-NC, and methacrylic acid. The inimer was copolymerized with the monomer DPA, and pH-responsive hyperbranched polymer hPDPA with different DB were prepared by the ATRP-SCVP method. The pH-responsive hyperbranched polymer peptide conjugates hPDPA/PArg were obtained by the molecular recognition of adamantyl groups on hyperbranched polymers with β-CD-PArg, and then the pH-responsive polymer peptide hybrid material hPDPA/PArg/HA was obtained by the electrostatic interaction between hPDPA/PArg and HA. The self-assembly behavior of hPDPA/PArg/HA with different DB and the application of the assemblies as drug carriers for β-lapa were investigated. The two hybrid materials h 1 PDPA/PArg 12 /HA and h 2 PDPA/PArg 8 /HA were found to self-assemble to form vesicles in PB at pH = 7.4, and β-lapa can be effectively loaded by vesicles and released under acidic conditions (pH = 5.0). The carriers h 2 PDPA /PArg 8 /HA showed low cytotoxicity towards Hela cells. The release of β-lapa leads to an increase in reactive oxygen species, which can react with PArg to generate NO. The reaction between reactive oxygen species and NO leads to the production of ONOO − , and this synergistic therapy based on ROS and NO has a significant inhibitory effect on cancer cells.

Data Availability Statement:
The data presented in this study are available on request from the corresponding author.

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