A Star-Shaped Copolymer with Tetra-Hydroxy-Phenylporphyrin Core and Four PNIPAM-b-PMAGA Arms for Targeted Photodynamic Therapy

The novel thermosensitive star-shaped tetra-hydroxy-phenylporphyrin-cored (THPP) double hydrophilic poly(N-isopropylacrylamide)-b-poly(methylacrylamide glucose) block copolymers (THPP-(PNIPAM-b-PMAGA)4) were synthesized via the reversible addition-fragmentation chain transfer (RAFT) polymerization. Notably, the low critical solution temperatures (LCSTs) of THPP-(PNIPAM-b-PMAGA)4 were above normal body temperature (37 °C) which depended on the hydrophilic PMAGA contents of copolymers. When the temperature was higher than the LCST of the copolymer, the copolymer could be neutralized into micelles in aqueous and could be coated with antitumor drugs and released around tumor cells. The MTT study indicated that THPP-(PNIPAM-b-PMAGA)4 had a low toxicity to L929 and HeLa cells in the absence of light. However, THPP-(PNIPAM-b-PMAGA)4 showed a high toxicity with HeLa cells under light irradiation which could be used as a potential photosensitizer for photodynamic therapy (PDT). In addition, THPP-(PNIPAM-b-PMAGA)4 showed specific a recognition function with Concanavalin A (Con A) to achieve active targeted drug delivery. This work provides a new approach for the development of tumor targeting and chemotherapy/PDT.


Characterization
The structures of the products were characterized by Fourier Transform infrared (FT-IR) spectra recorded on Shimadzu-8400S (Shimadzu Corporation, Tokyo, Japan)infrared Scheme 1. Schematic diagram of PDT mechanism and synthesis of THPP-(PNIPAM-b-PMAGA) 4 -1(M n = 18,700)/paclitaxel. When the temperature was higher than LCST, the PNIPAM segment of THPP-(PNIPAMb-PMAGA) 4 changed from hydrophilic to hydrophobic, while the PMAGA segment remained hydrophilic and soluble, resulting in the formation of micelles in the aqueous solution of THPP-(PNIPAM-b-PMAGA) 4 . By adjusting the LCST of the copolymer, the hydrophilic PNIPAM chain segment became hydrophobic and released drugs in the tumor tissue. Consequently, it provided not only a specific sugar recognition and temperaturetargeting block copolymer, but also a potential porphyrin-cored star-shaped PNIPAM-b-PMAGA copolymers for combination chemotherapy/PDT strategies.
In short, a certain amount of THPP-(DMP) 4 was added into DMF (2 mL) solution of 226 mg NIPAM and stirred to deoxygenate. Then    D-(+) -glucosamine hydrochloride (5.00 g, 28 mmol) and potassium carbonate (4.2 g, 30 mmol) were added to 125 mL of methanol and cooled to −15 • C. The methylacrylyl chloride (3.12 g, 30 mmol) drop by drop to the reactor and the reaction was performed at −15 • C for 30 min. The reaction was continued at 25 • C for 3 h. After the reaction, the filtrate was filtered and spun to the suspension. The suspension was purified by silica gel column chromatography (the V CH2Cl2 :V CH3OH = 4:1) and the white solid was obtained.  Using pyrene as a fluorescent probe, a certain amount of pyrene was added into acetone solvent, and 0.1 mL pyrene-acetone solution was added into 10 mL volumetric bottles respectively [46]. The acetone solvent was volatilized at room temperature. Then 10 mL of polymer aqueous solution with different concentrations were added to above volumetric bottles, and the final concentration of pyrene reached 2 × 10 −6 mol/L. The emission spectrum of the copolymer in the range of 340~600 nm was measured at an excitation wavelength of 335 nm. The CMC was taken as the cross-point when extrapolating the intensity ratio I383/I372 at low-and high-concentration regions.
The Φ ∆ was calculated according to Equation (1): Among them, Φ Std ∆ is 0.62 of the 1 O 2 quantum yield for TPP. The DPBF photobleaching rates of copolymers and TPP are R and R Std , respectively, while I abs and I Std abs are their photoabsorption rates.
At 37 • C, the cells were incubated for 48 h, washed with PBS 3 times, incubated for 6 h, added 20 µL MTT solution containing micelle solution of different concentrations for 4 h, and then returned to the incubator. Carefully, the MTT solution was taken out, 200 µL DMSO was added and gently stirred for 10 min and the formazan product was extracted. Absorbance at 492 nm was read with an enzyme-linked immunosorbent assay (ELISA) microplate reader (Bio-Rad). According to Formula (2), cell viability (%) can be calculated.
At the same time, the phototoxicity of THPP-(PNIPAM-b-PMAGA) 4 micelles on cells were measured by red light irradiation at 670 nm for 5 min according to the above method.
HeLa cells were cultured in 96-well plates containing 2 mL medium for 24 h with a cell density of 1 × 10 6 cells per well. The cells were then treated with samples for a certain period of time and washed with phosphate buffered saline (PBS). Finally, the expected precipitation was analyzed by BD FACS Calibur flow cytometry. The data were analyzed by FlowJo software.

Lectin Recognition
The Con A lectin recognition behavior of the sugar-containing copolymer solution was determined by UV-VIS method at 360 nm. In addition, the concentration of Con A was set as 0.5 mg·mL −1 , and different concentrations of aggregate solution were added to the Con A solution.

Analysis of Copolymers Architecture
The architecture of the THPP-(PNIPAM-b-PMAGA)4 was authenticated by FTIR spectra and 1 HNMR. Figure S1 shows O-H absorbance and N-H tensile vibration in THPP at 3430 cm −1 and 3350 cm −1 , respectively. From Figure 2a, the chemical shift of 9.96, 8.65, and −2.86 ppm were the characteristic signals of THPP's hydroxyl group, benzene ring, and amino group ( Figure S1 and Figure 2a). The absorbance at 1760 cm −1 was ascribed to the ester group of THPP-(DMP) 4 ( Figure S2). In Figure S3, 1 HNMR spectra of THPP-(DMP) 4 showed that (δ~3.4) and (δ~0.98) were assigned to the proton of -CH 2 S and -CH 3 . The FTIR and 1 HNMR spectra of Methylacrylamide glucose (MAGA) are shown in Figures 1 and 2a. The peak at 2937 cm −1 was the methyl antisymmetric stretching vibration peak, 2920 cm −1 and 2955 cm −1 were the methylene C-H stretching vibration peak, the peaks at 1664 cm −1 and 1542 cm −1 were vested in the stretching vibration peak of the carbon oxygen double bond on MAGA, which were the characteristic absorption peaks of amide I band and the characteristic absorption peaks of N-H bending vibration coupling with C-N stretching vibration in CONH, namely the amide II band ( Figure 1). In Figure 2b, two peaks at about 1.6 ppm, 5.5 ppm, and 5.7 ppm were attributed to protons of the methyl group attached to the double bond, and the MAGA double bond and N-H, respectively. The target product MAGA was successfully characterized by FTIR and 1 HNMR.
The FTIR spectrum of THPP-(PNIPAM-b-PMAGA) 4 was shown in Figure 1. The absorption peaks of the amide I and II bands in PNIPAM were at 1643 cm −1 and 1552 cm −1 , respectively. After copolymerization with MAGA, the presence of a large number of hydroxyl groups in the glucose molecule resulted in a broad and strong hydroxyl absorption peak near 3320 cm −1 . From the 1 HNMR of Figure 2c, the acylamino groups and methenyl adjacent to acylamino of PNIPAM were located at 6.0-7.0 ppm and 4.0 ppm. In Figure 2d, the new signal at 3.0-5.0 ppm was corresponding to protons in PMGA. Figure S4 shows the GPC curve of the polymer. The polymerization data of THPP-(PNIPAM) 4 and THPP-(PNIPAM-b-PMAGA) 4 is shown in Table 1. All the above test results confirmed that THPP-(PNIPAM-b-PMAGA) 4 copolymers were synthesized successfully [47,48].  Fluorescence emission spectra of THPP and THPP-(PNIPAM-b-PMAGA)4 were illustrated in Figure 3b. At the excitation wavelength of 380 nm, the maximum emission peak was around 650 nm, but the charge-transfer effect of THPP-(PNIPAM-b-PMAGA)4 was reduced due to the presence of long polymer chains, and therefore, the emission band was enhanced and potentially split [49,50].   4 with the introduction of hydrophilic MAGA segment was higher than that of THPP-PNIPAM 4 [51,52] (Table 2). The LCST increased with the increase in hydrophilic MAGA segments. Figure 3c It was shown that the particle size of a THPP-(PNIPAM-b-PMAGA)4 micelle was 43.6 nm when the temperature was 35 °C, but the particle size increased to 181.6 nm when the temperature was increased to 45 °C in Figure 4. The results revealed that when the temperature was higher than the LCST of the copolymer, the PNIPAM segment changed from hydrophilic to hydrophobic and the completed collapse of hydrogen bonds led to an increase in the particle size of the formed micelles.  It was shown that the particle size of a THPP-(PNIPAM-b-PMAGA) 4 micelle was 43.6 nm when the temperature was 35 • C, but the particle size increased to 181.6 nm when the temperature was increased to 45 • C in Figure 4. The results revealed that when the Polymers 2023, 15, 509 8 of 14 temperature was higher than the LCST of the copolymer, the PNIPAM segment changed from hydrophilic to hydrophobic and the completed collapse of hydrogen bonds led to an increase in the particle size of the formed micelles.

CMC of THPP-(PNIPAM-b-PMAGA) 4
The CMC of THPP-(PNIPAM-b-PMAGA) 4 are shown in Figure 4b. With the increasing of THPP-(PNIPAM-b-PMAGA) 4 concentration, the ratio of I383/I372 remained unchanged, but it increased sharply when the copolymer concentration gradually increased to a certain value. It indicated that hydrophobic pyrene accumulates to the micelle core, which was a sign of micelle formation. The CMC value of THPP-(PNIPAM-b-PMAGA) 4 copolymer increased from 0.0005 mg·mL −1 to 0.0025 mg·mL −1 with the increasing of the hydrophilic PMAGA segment. It was confirmed that the polymer micelle might be formed with the increase in the length of hydrophobic block.

Singlet Oxygen Quantum Yields of THPP-(PNIPAM-b-PMAGA) 4
The absorbance intensity of DPBF and the THPP-(PNIPAM-b-PMAGA) 4 mixture in DMF determined by a UV-UV spectrophotometer is shown in Figure 5. The Φ ∆ of THPP-(PNIPAM-b-PMAGA) 4  copolymer increased from 0.0005 mg·mL −1 to 0.0025 mg·mL −1 with the increasing of the hydrophilic PMAGA segment. It was confirmed that the polymer micelle might be formed with the increase in the length of hydrophobic block.

Singlet Oxygen Quantum Yields of THPP-(PNIPAM-b-PMAGA)4
The absorbance intensity of DPBF and the THPP-(PNIPAM-b-PMAGA)4 mixture in DMF determined by a UV-UV spectrophotometer is shown in Figure 5. The     PDT was evaluated by investigating the cytotoxicity of porphyrin derivate at varyin concentrations (0 to 0.25 mg·mL −1 ) against L929 cells and HeLa cells under red light i radiation (Figure 6b). The results revealed that THPP and THPP-(PNIPAM-b-PMAGA have very low phototoxicity to L929 cells. However, for the phototoxicity of HeLa cel the THPP-(PNIPAM-b-PMAGA)4 was higher than THPP which may be due to π-π stac ing and the poor water solubility of THPP. All the decreasing effects of PDT were in aqu ous solution to trigger quenching. Figure 6c shows that the toxicity of paclitaxe coated THPP-(PNIPAM-b-PMAGA)4 micelles to HeLa cells under light was high than that of uncoated micelles, which might be due to the death of HeLa cells cause by the release of paclitaxel.

Cellular Uptake of THPP-(PNIPAM-b-PMAGA)4 Micelles
The PDT was evaluated by investigating the cytotoxicity of porphyrin derivate at varying concentrations (0 to 0.25 mg·mL −1 ) against L929 cells and HeLa cells under red light irradiation (Figure 6b). The results revealed that THPP and THPP-(PNIPAM-b-PMAGA) 4 have very low phototoxicity to L929 cells. However, for the phototoxicity of HeLa cells, the THPP-(PNIPAM-b-PMAGA) 4 was higher than THPP which may be due to π-π stacking and the poor water solubility of THPP. All the decreasing effects of PDT were in aqueous solution to trigger quenching. Figure 6c shows that the toxicity of paclitaxel-coated THPP-(PNIPAM-b-PMAGA) 4 micelles to HeLa cells under light was higher than that of uncoated micelles, which might be due to the death of HeLa cells caused by the release of paclitaxel.

Recognition Properties of Star-Shaped THPP-(PNIPAM-b-PMAGA) 4 Copolymers
The mutual sugar-protein recognition and specific binding in the living body was of great significance for drug discovery and biomaterial application. Con A could specifically recognize that D-glucopyranoside contains free 3-, 4-, and 6-hydroxyl groups which usually combine with sugar copolymers form Con A cross-linked aggregation. Therefore, the interaction between Con A and THPP-(PNIPAM-b-PMAGA) 4 was studied in an aqueous solution at room temperature. It was found that the turbidity of THPP-(PNIPAM-b-PMAGA) 4 samples increased slightly with the increase in copolymer concentration and there was no precipitation in the solution. It indicated that THPP-(PNIPAM-b-PMAGA) 4 copolymer may combine with Con A to form aggregates. There were no significant changes of Con A aggregation in aqueous which was measured by UV-vis solution turbidity (Figure 7). The specific binding of THPP-(PNIPAM-b-PMAGA) 4 copolymer to ConA in aqueous solution indicated that the sugar copolymer with the core of porphyrin could be used for targeted drug delivery.

Recognition Properties of Star-Shaped THPP-(PNIPAM-b-PMAGA)4 Copolymers
The mutual sugar-protein recognition and specific binding in the living body was of great significance for drug discovery and biomaterial application. Con A could specifically recognize that D-glucopyranoside contains free 3-, 4-, and 6-hydroxyl groups which usually combine with sugar copolymers form Con A cross-linked aggregation. Therefore, the interaction between Con A and THPP-(PNIPAM-b-PMAGA)4 was studied in an aqueous solution at room temperature. It was found that the turbidity of THPP-(PNIPAM-b-PMAGA)4 samples increased slightly with the increase in copolymer concentration and there was no precipitation in the solution. It indicated that THPP-(PNIPAM-b-PMAGA)4 copolymer may combine with Con A to form aggregates. There were no significant changes of Con A aggregation in aqueous which was measured by UV-vis solution turbidity (Figure 7). The specific binding of THPP-(PNIPAM-b-PMAGA)4 copolymer to ConA in aqueous solution indicated that the sugar copolymer with the core of porphyrin could be used for targeted drug delivery.

Exploration of Paclitaxel Release In Vitro
The sustained-release behavior of THPP-(PNIPAM-b-PMAGA)4 micelles was evaluated using paclitaxel as the model drug. The encapsulation rate (EE) of the copolymer was about 42.7% by HPLC.The results indicated that the copolymer with a star structure was suitable for drug encapsulation. The two-phase release curves in free paclitaxel and paclitaxel-loaded nanoparticles buffer solutions were observed (Figure 8). It could be found that micelles release of paclitaxel was significantly slower than the free paclitaxel release, followed by a sustained and slower release over a prolonged period of time (72 h, 85.9%). Therefore, the continuous release of paclitaxel-coated nano-micelles could improve its bioavailability and induce cancer cell death to achieve better therapeutic effect.

Exploration of Paclitaxel Release In Vitro
The sustained-release behavior of THPP-(PNIPAM-b-PMAGA) 4 micelles was evaluated using paclitaxel as the model drug. The encapsulation rate (EE) of the copolymer was about 42.7% by HPLC.The results indicated that the copolymer with a star structure was suitable for drug encapsulation. The two-phase release curves in free paclitaxel and paclitaxel-loaded nanoparticles buffer solutions were observed (Figure 8). It could be found that micelles release of paclitaxel was significantly slower than the free paclitaxel release, followed by a sustained and slower release over a prolonged period of time (72 h, 85.9%). Therefore, the continuous release of paclitaxel-coated nano-micelles could improve its bioavailability and induce cancer cell death to achieve better therapeutic effect.

Conclusions
The star-shaped double hydrophilic THPP-(PNIPAM-b-PMAGA)4 with thermo-sensitivity was synthesized by RAFT using porphyrin-cored as a chain transfer agent. The LCST of THPP-(PNIPAM-b-PMAGA)4 increased with the increasing of the hydrophilic chain segment PMAGA and which could self-assemble into micelles in an aqueous above 37 °C. In vitro experiments of THPP-(PNIPAM-b-PMAGA)4 showed good biocompatibility and high phototoxicity to HeLa which might be a potential application prospect. In addition, these THPP-(PNIPAM-b-PMAGA)4 copolymers had a stable Con A aggregation in aqueous solution, so it had a specific ability to recognize Con A. In addition, these block copolymers could be used as nanoscale photosensitizers to further encapsulate hydrophobic paclitaxel.

Conclusions
The star-shaped double hydrophilic THPP-(PNIPAM-b-PMAGA) 4 with thermo-sensitivity was synthesized by RAFT using porphyrin-cored as a chain transfer agent. The LCST of THPP-(PNIPAM-b-PMAGA) 4 increased with the increasing of the hydrophilic chain segment PMAGA and which could self-assemble into micelles in an aqueous above 37 • C.
In vitro experiments of THPP-(PNIPAM-b-PMAGA) 4 showed good biocompatibility and high phototoxicity to HeLa which might be a potential application prospect. In addition, these THPP-(PNIPAM-b-PMAGA) 4 copolymers had a stable Con A aggregation in aqueous solution, so it had a specific ability to recognize Con A. In addition, these block copolymers could be used as nanoscale photosensitizers to further encapsulate hydrophobic paclitaxel.