Effect of tertiary amino groups in the hydrophobic segment of an amphiphilic block copolymer on zinc phthalocyanine encapsulation and photodynamic activity

Polymer micelles are promising nanocarriers for hydrophobic photosensitizers of photodynamic therapy (PDT). Poly(styrene-co-(2-(N,N-dimethylamino)ethyl acrylate))-block-poly(polyethylene glycol monomethyl ether acrylate) (P(St-co-DMAEA)-b-PPEGA; 1) was prepared via reversible addition and fragmentation chain transfer (RAFT) polymerization as a carrier for a zinc phthalocyanine (ZnPc) photosensitizer to be used in PDT. The DMAEA-unit composition in the P(St-co-DMAEA) segment was adjusted to 0.40 molar ratio, which caused a sharp increase in water-solubility when the pH decreased from 7.4 to 5.0. The polymer 1 micelle size distribution also shifted to lower when the pH decreased, whereas this change was not observed in PSt-co-PPEGA (2), which was previously reported. The UV-vis spectrum of the ZnPc-loaded micelles of polymer 1 exhibited relatively sharp Q bands, comparable to those measured in DMSO, indicating good compatibility of the condensed core with ZnPc. ZnPc-loaded micelles of polymer 1 exerted excellent photocytotoxicity in the MNNG-induced mutant of the rat murine RGM-1 gastric epithelial cell line (RGK-1). In contrast, the ZnPc-loaded micelles of polymer 2 were completely inactive under the same conditions. Fluorescence from the RGK-1 cells treated with ZnPc-loaded micelles of polymer 1 was observed after 4 h of co-incubation, while no fluorescence was observed in cells treated with ZnPc-loaded micelles of polymer 2. These results indicate that the pH-responsive nature and good compatibility with ZnPc exhibited by the polymer 1 micelles are essential characteristics of ZnPc carriers for efficient photodynamic therapy.


Introduction
Photodynamic therapy (PDT) is a minimally-invasive cancer therapy using photosensitizers and light that is currently applied to esophageal cancer and non-small cell lung cancer. [1][2][3][4][5][6][7][8] Aer a photosensitizer is administered to a patient and accumulates at the tumor site, photoirradiation is used to generate a cytotoxic reactive oxygen species (ROS). Several mechanisms have been suggested to kill tumor cells, such as initiating the apoptosis cascade and vascular shutdown. In addition to being precisely controlled by photoirradiation, tumor-accumulating photosensitizers regulate legions to eliminate malignancies. Limited light availability in the deeper tissue is a major drawback in PDT treatment. Effective photosensitizers must absorb light in the near-infrared (NIR) region, [9][10][11] due to the fact that longer wavelength increases tissue transparency.
Another promising approach is the use of drug delivery system (DDS) technology. This allows us to design the photosensitizer and carrier separately to meet photophysical and physiological requirements, respectively. Among the many types of carriers for PDT photosensitizers, polymer micelles are very promising nanomaterials because of their biocompatibility and synthetic availability. Several ZnPc-loaded polymer micelles have been prepared using amphiphilic block copolymers such as poly(ethylene glycol)-b-poly(L-lactide) (PEG-b-PLLA), [49][50][51] poly(ethylene glycol)-b-poly(3-caprolacton) (PEG-b-PCL), 52 and more highly sophisticated polymers. [53][54][55] ZnPc can be physically trapped in the hydrophobic core of the polymer micelle. However, most ZnPc-loaded polymer micelles show severely broadened UV-vis spectra, indicating the formation of ZnPc aggregates. Aggregation changes the photophysical properties of ZnPc molecules, and deactivates the photodynamic effect. 56,57 Previously, we demonstrated the synthesis of polystyrene-bpoly(polyethylene glycol monomethyl ether acrylate) (PSt-b-PPEGA) and the preparation of ZnPc-loaded polymer micelles. 58 The efficiency of encapsulating ZnPc depended on the molecular architecture of PSt-b-PPEGA. Disappointingly, the photocytotoxicity of the resulting ZnPc-loaded polymer micelles was very low in HeLa cells. Similar to their behavior in other polymer micelles, ZnPc molecules are strongly aggregated in the polymer micelles, as evidenced by the UV-vis spectra. In addition, the ZnPc-loaded polymer micelles were kinetically frozen and lacked the ability to release ZnPc. Hence, the ZnPc molecules trapped in the polymer micelles were photochemically inactivated. This might have caused the low photocytotoxicity of ZnPc-loaded polymer micelles. Therefore to achieve successful PDT treatment, it is crucial that carriers physically encapsulate ZnPc and prevent its aggregation.
Recently, Borovkov et al. reported that a mixture of a paccepting solvent such as chlorobenzene and a nitrogenous base such as triethylamine dissolves ZnPc to make a clear solution with molecular dispersion. 59 In addition, we demonstrated that the side group of the hydrophobic chain affects the aggregating behavior of ZnPc in the polymer micelles. 60 These two ndings inspired us to investigate the molecular dispersion of ZnPc in polymer micelles with the hydrophobic chains consisting of repeating units with aromatic rings and tertiary amines. In addition, tertiary amines, with pK a values approximately 6.5-7.0, can be used as a pH-sensitive element between the extracellular physiological environment (pH ¼ 7.4) and acidic organelles (pH ¼ 5.0) during cellular internalization. In this study, we synthesized a pH-responsive amphiphilic block copolymer using 2-(N,Ndimethylamino)ethyl acrylate (DMAEA) as a pH-sensitive element, and preparation of the ZnPc-loaded polymer micelles. The pHresponsive properties, aggregating behavior of ZnPc, and photocytotoxicity of the polymer micelles are discussed.

Design of pH-responsive hydrophobic chain
To provide a pH-response ability, we designed a copolymer of styrene and DMAEA as a hydrophobic segment. First, an appropriate molar fraction of DMAEA units in the copolymer, Scheme 1 Synthesis of P(St-co-DMAEA) for solubility test via RAFT polymerization.   Table 1. The F DMAEA values varied from 0.12 to 0.64, while the M n values were relatively constant at approximately 10 4 , except for the case of F DMAEA ¼ 0.64. The relative water solubility was examined in phosphatebuffered saline (PBS), and PBS adjusted to pH 5.0, by adding HCl. The amount of the copolymer dissolved in the buffered solution was quantied by the absorbance at 264 nm and divided by the weight fraction of styrene units to determine the water solubility of the copolymer. Fig. 1 shows the plots of the relative water solubility as a function of the F DMAEA value of the copolymer. The relative water solubility gradually increased with an increase in the F DMAEA value at pH 7.4. In contrast, the    relative water solubility suddenly increased at an F DMAEA of approximately 0.3 at pH 5.0. This suggests that the copolymer F DMAEA of approximately 0.3 exerts a large water-solubility change between the physiological environment (pH ¼ 7.4) and acidic intracellular organelles such as endosomes and lysosomes (pH ¼ 5.0). Therefore, we choose the F DMAEA value of 0.4 for the hydrophobic P(St-co-DMAEA) segment for the pHresponsive amphiphilic block copolymer.

Synthesis of P(St-co-DMAEA)-b-PPEGA
The amphiphilic block copolymer, P(St-co-DMAEA)-b-PPEGA, was synthesized by RAFT polymerization of styrene with   19 F NMR spectra (right panel) of 1 in acetone-d 6 . The asterisks indicate the peaks from CF 3 CH 2 OTTMS as an internal cross reference for peak area normalization between the 1 H and 19 F NMR spectra.  It should be noted that we used triuoromethyl-labeled CPADB (CF 3 -CPADB) as a chain transfer agent to determine the degree of polymerization (DP n ) of each repeating unit by the combination of 1 H and 19 F NMR spectroscopies. First, a statistical copolymer, P(St-co-DMAEA) macroCTA, was synthesized by bulk RAFT polymerization of styrene with DMAEA using CF 3 -CPADB and azobisisobutyronitrile (AIBN) as a chain transfer agent and initiator, respectively, at 60 C for 24 h. The DP n values of styrene and DMAEA of the resulting polymer were 16 and 12, respectively, indicating the F DMAEA value was 0.43 (Table 2).
For comparison, a homopolymer, PSt macroCTA, was also synthesized by a similar procedure. The resulting macroCTA was extended with PEGA using AIBN as an initiator in 1,4dioxane at 60 C for 24 h to produce block copolymer P(St-co-DMAEA)-b-PPEGA and PSt-b-PPEGA. Fig. 2 shows the SEC traces of P(St-co-DMAEA) macroCTA and P(St-co-DMAEA)-b-PPEGA. The SEC trace of P(St-co-DMAEA)-b-PPEGA clearly shied toward a higher molecular mass than that of P(St-co-DMAEA) macroCTA without signicant broadening. Fig. 3 shows the 1 H DOSY spectrum of P(St-co-DMAEA)-b-PPEGA, in which the styrene units exhibit a diffusion coefficient identical to that of the PEGA units. These results indicate the formation of a block copolymer, P(St-co-DMAEA)-b-PPEGA. Fig. 4 shows the 1 H and 19 F NMR spectra of P(St-co-DMAEA)b-PPEGA in acetone-d 6 mixed with tris(trimethylsilyl)-2,2,2-triuoroethoxysilane (CF 3 CH 2 OTTMS) as an internal crossreference for peak area normalization between these two spectra. The triuoromethyl group at the initiating end was unequivocally identied in the 19 F NMR spectrum owing to the nuclear specicity of NMR spectroscopy. The 19 F NMR spectrum affords the ratio of polymer to CF 3 CH 2 OTTMS, while the 1 H NMR spectrum affords the ratio of each repeating unit to CF 3 -CH 2 OTTMS. Therefore, the combination of these two spectra afforded DP n values for each repeating unit. This 1 H-19 F crossreference technique is helpful for determining the DP n and M n values by end group analysis, especially in the case that the polymer affords very intense peaks in the 1 H NMR spectrum, such as a PPEGA segment. Table 3 summarizes the DP n values for each repeating unit of the block copolymers. Finally, we obtained a pH-responsive block copolymer, P(St 17 -co-DMAEA 13 )-b-PPEGA 90 (hereinaer denoted as 1), and the block copolymer, PSt 37 -b-PPEGA 82 (hereinaer denoted as 2), as shown in Scheme 2. The pHresponsive nature of the resulting block copolymer was examined by acid-base titration. Fig. 5 shows the titration curves of polymers 1 and 2. The curve for polymer 1 had an Chart 1 Flow chart for preparation of polymer micelle solution. inection point at pH of approximate 7 due to the buffering capacity of DMAEA units, while polymer 2 showed no buffering nature.

Preparation and characterization of polymer micelles
Polymer micelles were prepared by the dialysis method (Chart 1). Briey, polymer 1 or 2 was dissolved in DMF and slowly added to the same amount of PBS, then DMF was removed by dialysis to afford a clear solution without any precipitates. Aer adjusting the polymer concentration to 2 mg mL À1 , the solution was ltered through a membrane lter (pore size of 0.22 mm). The critical micelle concentration (CMC) in PBS (pH ¼ 7.4) and acetate buffered saline (ABS, pH ¼ 5.0) was estimated by the uorometric method using pyrene as a hydrophobic uorescent probe. Fig. 6 shows plots of the uorescence intensity ratio (I 338 / I 333 ) as a function of the logarithm of the polymer concentration.
Interestingly, the plots of polymer 1 in ABS slightly shied toward higher concentrations than those measured in PBS, while no such shi was observed for polymer 2. The CMC value was determined by the crossing point of the baseline and the tangent at the inection point of the best-tted sigmoidal curve. The CMC value of polymer 1 was determined to be 40 mg L À1 in PBS and changed to 56 mg L À1 in ABS. Signicant changes due  to the pH switch were also observed in dynamic light scattering (DLS) measurements. Fig. 7 shows the size distribution of the micelles of polymers 1 and 2 in PBS and ABS at a polymer concentration of 1 mg mL À1 . The size distribution of the micelles of polymer 1 shied to lower when the pH changed from 7.4 to 5.0. In contrast, no changes were observed for polymer 2. These results suggest that DMAEA units act as pHresponsive moieties to destabilize the polymeric micelles under acidic conditions. Zinc phthalocyanine (ZnPc), a highly hydrophobic photosensitizer, was used as the payload of these polymer micelles. ZnPc-loaded micelles were prepared by a procedure similar to that of free micelles except for the use of a DMF solution of ZnPc instead of pure DMF. Aer dialysis and adjustment of the polymer concentration, insoluble ZnPc precipitates were removed by ltration with a membrane lter (pore size 0.22 mm). The concentrations of ZnPc in the micelle solutions were determined spectrouorometrically and are listed in Table 4.
Triplicate experiments suggest that the encapsulation efficiency (E.E.) did not depend on the presence of DMAEA units, and ranged from 28% to 73%. Fig. 8 shows the UV-vis spectra of ZnPc-loaded polymer micelles in PBS. The ZnPc-loaded micelles of polymer 2 showed a broadened absorption band, indicating that ZnPc molecules were tightly aggregated in the hydrophobic core. In contrast, the ZnPc-loaded micelles of polymer 1 showed Q bands similar to those recorded in dimethyl sulfoxide (DMSO). Therefore, the micelles of polymer 1 afford the microenvironment to dissolve ZnPc in a monomeric form. The difference in the microenvironment of the polymer micelles crucially affects the photophysical and photodynamic effects of ZnPc.

In vitro photocytotoxicity of ZnPc-loaded polymer micelles
The in vitro photocytotoxicity of ZnPc-loaded micelles was examined in MNNG-induced mutant of the rat murine RGM-1 gastric epithelial cell line (RGK-1). 61 RGK-1 cells were coincubated with the micelles in a mixture of PBS and culture medium (1/1, v/v) for 24 h. Aer removing the excess amount of the drug, the cells were photoirradiated with a 100 W halogen lamp equipped with a Y-50 cut lter. The cell survival rate was determined by WST-8 assay and normalized by the value for no drug treatment with photoirradiation. Fig. 9a shows the cell survival rate aer treatment with the payload-free micelles of polymers 1 and 2 at a concentration of 1 mg mL À1 as a function of the light dose. Fig. 9a indicates that the micelles of 1 and 2 essentially have no cytotoxicity regardless of photoirradiation. The cell survival rates aer the ZnPc-loaded polymer micelle treatment are shown in Fig. 9b. ZnPc-loaded micelles of 1 and 2 did not show cytotoxicity without photoirradiation (light dose of 0 J cm À2 ). ZnPc-loaded micelles of 2 did not show cytotoxicity even upon photoirradiation at a light dose of 20 J cm À2 . This can be expected because of the very low ZnPc concentration in comparison to that used in our previous study. 58 In contrast, ZnPc-loaded micelles of 1 exerted excellent photocytotoxicity under the same condition; almost complete annihilation was achieved upon photoirradiation of 20 J cm À2 with a ZnPc dose of only 0.05 mM. Fig. 10 shows the images of RGK-1 cells co-incubated with ZnPc-loaded polymer micelles captured by uorescence microscopy. The red uorescence from the cells treated with ZnPc-loaded micelles of 1 gradually increased with coincubation time. In contrast, almost no uorescence was observed in the cells treated with 2. This distinct difference in the photophysical behavior in the cellular microenvironment affected the photocytotoxicity of the ZnPc-loaded micelles.
The photodynamic behavior of ZnPc-loaded micelles of 1 and 2 should be related to the fate of polymer micelles coincubated with cells, that is, cellular uptake via endocytosis, endosome escape, and ZnPc release or conservation in polymer micelles. The pH-responsive nature of 1 enhances endosome escape and facilitates ZnPc release. However, another mechanism is also possible. If the polymer micelle is stable enough in the cellular microenvironment, ZnPc encapsulated in the micelles of 1 must be photochemically active rather than that in 2. The rst hypothesis is a line along with the usual drug Table 4 Preparation of the micelle solutions of polymers 1 and 2 a Run no.

In preparation
Micelle solution delivery regime, in which polymer micelles act as simple carriers for photosensitizers. However, the latter hypothesis is beyond the simple drug delivery regime; polymer micelles provide a suitable microenvironment for photosensitizers. The key is the kinetic properties of polymer micelles, which are not yet fully understood, especially in very complicated microenvironments such as the cytosol. To address this issue, a study on the behavior of micelles of 1 is in progress in our laboratory using Förster resonance energy transfer (FRET) and aggregation-induced emission (AIE) dye labeling techniques.

Conclusions
Amphiphilic block copolymers, P(St-co-DMAEA)-b-PPEGA (1) and PSt-b-PPEGA (2), were successfully prepared by successive RAFT polymerizations. The mole fraction of styrene units in P(St-co-DMAEA) of 1 was 0.4, which caused the water solubility to increase when the pH decreased from 7.4 to 5.0. Polymers 1 and 2 both formed micelles in PBS. The unique nature of 1 was evidenced by DLS and the UV-vis measurements. DLS results revealed the pH sensitivity of the micelles of 1: the size distribution of the micelle of 1 dramatically shied lower with a decrease in the pH, while that of 2 did not change. The UV-vis spectrum of ZnPc-loaded micelles of 1 indicated the excellent compatibility of the core components of the micelle with ZnPc: the Q bands of ZnPc-loaded micelle of 1 were very similar to those observed in DMF, while those of 2 were severely broadened. ZnPc-loaded micelle of 1 exerted excellent photocytotoxicity in RGK-1 cells and showed distinct photochemical behavior in the cellular microenvironment compared to 2. The enhanced PDT effect of polymer 1 micelles should be related to its pH-responsive nature and good compatibility with ZnPc.

Conflicts of interest
There are no conicts to declare.