Photosensitizer Conjugate for the

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Acetylated Hyaluronic Acid/Photosensitizer Conjugate for the Preparation of Nanogels with Controllable Phototoxicity: Synthesis, Characterization, Autophotoquenching Properties, and in Witro Phototoxicity against HeLa Cells Fangyuan Li, Byoung-chan Bae, and Kun Na* Department of Biotechnology, The Catholic University of Korea, 43-1 Yeokkok2-dong, Wonmi-gu, Bucheon-si, Gyeonggi-do 420-743, Republic of Korea. Received March 6, 2010; Revised Manuscript Received June 1, 2010

A proposal is herein examined for a novel yet simple design of a polymeric nanogel, with tumor targeting properties and a controllable phototoxicity, utilizing a low molecular weight-hyaluronic acid (HALM)/photosensitizer conjugate. HALM was acetylated prior to being dissolved in DMSO (Ac-HALM) and then was conjugated with different amounts of pheophorbide a (Pba), resulting in the formulation of self-organizing nanogels in aqueous solutions (Ac-HA-Pba 1, 2, and 3). The nanogels observed were below 200 nm in size, with a monodispersed size distribution. The nanogels displayed auto photoquenching qualities in PBS, while their fluorescent intensity strongly correlated with the amount of Pba in the organic solvent (DMSO or DMF). The critical self-quenching concentration (CQC) of the conjugates was found to have decreased as the content of Pba rose. Although Pba was conjugated with HA, the nanogel’s photoactivity, in terms of fluorescent properties, singlet oxygen generation, and photocytotoxicity, was approximately maintained. Confocal imaging and FACS analysis showed that Ac-HA-Pba nanogels were rapidly internalized into HeLa cells via an HA-induced endocytosis mechanism, a process which could be blocked with the application of an excess of HA polymer. The results of the study indicate that HAbased nanogels can potentially be applied in photodynamic therapy (PDT).

INTRODUCTION Photodynamic therapy (PDT), a promising therapeutic modality for cancer treatment, involves the administration of photosensitizers (PSs) coupled with specific wavelengths of light irradiation, having the effect of significantly increasing production of cytotoxic singlet oxygen, as well as other reactive oxygen species (ROS), causing malignant cell death (1, 2). However, problems with water-insolubility and nonspecific distribution after intravenous injection of most PSs limit the range of application in spite of their great efficacy and compel researchers to overcome these hurdles in order to make fuller use of the technique. To date, a variety of polymer vehicles have been extensively investigated for use with hydrophobic PSs with the objective of achieving not only stable aqueous dispersion but also sitespecific delivery. In previous studies, it was shown that the attachment of polyethylene glycol to a conjugate between poly L-lysine and chlorine6 was able to reduce the aggregation of PS and increase tumor targeting (3). In addition, two water-soluble polymer conjugates of Zn-protoporphyrin, ZnPP-poly(styrenemaleicacid) and ZnPP-poly(ethylene glycol), were synthesized to make ZnPP water-soluble and investigated for use as an aqueous solution (4). Lee et al. also reported enhanced tumor targeting activity in self-assembled chitosan-based nanoparticles encapsulating water-insoluble protophorphyrin IX (5). The passive targeting of these polymeric nanoparticles is referred to as the enhanced permeability and retention (EPR) effect, with theory based on the high permeability of blood vessels at tumor sites. However, this targeting modality may release a considerable quantity of drugs before any substantial uptake by malignant cells. To further enhance selective accumulation of PSs into * Corresponding author. Tel: (82) (2) 2164-4832. Fax: (82) (2) 21644865. E-mail: [email protected].

target cells, surface modification, using monoclonal antibodies or specific tumor-seeking molecules, has been attached to PS loaded nanoparticles (6-8). Although antibody targeting is regarded as a promising strategy, there have been reports that antibody targeting does not increase tumor localization (9, 10) and does not address the difficulty in further conjugation without a subsequent decrease in binding capabilities. Hyaluronic acid (HA), the only nonsulfated glycosaminoglycan composed of repeating disaccharide units of N-acetyl-Dglucosamine and D-glucuronic acid (11), highlights itself as a potential drug carrier due to its unique properties: to render an appropriate size alongside aqueous stability for passive targeting tumor tissues using the EPR effect; to control active targeting to tumor cells through the selective interaction between HA and its receptors; and to release drugs in targeted cells with enhanced therapeutic efficacy. Naturally occurring HA is widely present in the extracellular matrix (ECM), connective tissues, and bodily fluids and plays a vital role in many pathophysiological processes including cell adhesion, proliferation, and migration. It is also known that HA specific receptors such as CD44 (12-14) and RHAMM (15) overexpress on the surface of various tumor cells, enhancing the binding and internalization of HA. As a result, HA has found success in an extraordinarily broad range of biomedical applications such as molecular imaging (16, 17), drug delivery (18, 19), and tissue engineering (20). Unfortunately, the strong hydrophility of HA renders itself unpopular for conjugation with hydrophobic drugs, as a result of chemical reactions in polar organic solvents. Some physiochemical methods have been developed to solve this problem (18, 19). However, most synthesis approaches using carboxyl groups of HA probably affect the interaction between HA/CD44 and specific receptor-mediated endocytosis of cancer cells (21, 22). Pheophorbide a (Pba), chosen as an attractive secondgeneration PS, absorbs at longer wavelengths and rapidly exudes

10.1021/bc100116v  2010 American Chemical Society Published on Web 06/29/2010

Acetylated HA Nanogels for Photodynamic Therapy Scheme 1. Schematic Diagram of the PDT Strategy of Ac-HA-Pbaa

a Ac-HA-Pba nanogels in aqueous solution intelligently quenched both the dye’s fluorescence and singlet oxygen generation as a result of the hydrophobic (π-π) interaction between Pba’s themselves, known as the fluorescence resonance energy transfer effect (FRET). However, the dequenching behavior of nanogels destroyed cancer cells under illumination after internalization due to the cleavage of the HA backbone caused by various intracellular enzymes in cellular compartments.

a very high singlet oxygen yield (23). It has been reported that Pba exhibits strong antitumor effects on human lung cancers cells (24), hepatoma carcinoma cells (25), and uterine sarcoma cells (26). The resulting Ac-HA-Pba nanogel, in an aqueous solution, quenched both the dye’s fluorescence and singlet oxygen generation through a hydrophobic (π-π) interaction between Pbas themselves, such as the fluorescence resonance energy transfer (FRET) effect. However, the dequenching behavior of the nanogel destroyed cancer cells under illumination after internalization as a result of cleavage of the HA backbone, caused by various intracellular enzymes in cellular compartments (27) and reactive oxygen species (28, 29) (Scheme 1). In this study, we synthesized Ac-HA-Pba utilizing a simple two-step reaction method: (1) the acetylation of HA (Ac-HA) capable of being dissolved in both distilled water (DW) and anhydrous dimethyl solfoxide (DMSO) and (2) the conjugation of Ac-HA and Pba through a conventional carbodiimide reaction, in which hydroxyl groups of HA were arranged to be substituted. Physicochemical properties, such as particle size and zeta potential, were then examined. We also detected the photoquenching properties of the Ac-HA-Pba nanogel, targetspecific activities, as well as in Vitro phototoxicity with HeLa cells.

EXPERIMENTAL PROCEDURES Materials. Oligo hyaluronic acid (HALM, MW 5800 Da) was kindly provided by the Bioland Company (Cheonan, Korea). Pba was obtained from Frontier Scientific Inc. (Salt Lake City, UT). Acetic anhydride (AA), pyridine, 1, 3-dicyclohexyl carbodiimide (DCC), 4-dimethylaminopyridine (DMAP), formamide, N,N-dimethylformamide (N,N-DMF), and anhydrous dimethyl sulfoxide (DMSO) were purchased from the SigmaAldrich Co. (St. Louis, MO, USA). The dialysis membrane was sourced from Spectrum Laboratories Inc. (Rancho Dominguez, CA). Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), antibiotics (penicillin/streptomycin), and Dulbecco’s phosphate buffer saline (DPBS) were obtained from GibcoBRL (Invitrogen Corp., Carlsbad, CA). The Live/Dead assay kit containing two fluorescent probes, ethidium ho-

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modimer-1 (EthD-1), and CalceinAM were purchased from the Molecular Probe Inc. (Eugene, OR, USA). All other chemicals and solvents were of analytical grade. Preparation of Ac-HA-Pba Conjugates. In order to prepare the amphiphilic HALM polymer, acetyl moieties were chemically introduced to HA. HALM (0.5 g) was dissolved in 10 mL of formamide via vigorous stirring. After adding pyridine (55 µL) and AA (50 µL), the mixture was stirred for a further 12 h. The reactant mixture was placed in a molecular porous dialysis membrane (molecular weight cutoff size 3.5 kDa) against distilled water for three days. The white HALM-acetate (Ac-HALM) was obtained by lyophilization. The conjugation of Pba into Ac-HALM was conducted via a conventional carbodiimide reaction: three different concentrations of Pba (10 mg, 5 mg, and 2.5 mg) were separately added to 10 mL of anhydrous DMSO containing 50 mg of Ac-HALM. The mixtures were vigorously stirred for 48 h followed by the addition of predetermined amounts of DCC and DMAP. Using distilled water, the reactants were then filtered and dialyzed for three days with a dialysis membrane (MWCO 10 kDa). The dialysis was repeated 3 times to remove unreacted Pba. The final green products were obtained by lyophilization. All of the above reactions were carried out at room temperature. The synthesized Ac-HA-Pba conjugations were analyzed through 1H NMR spectroscopy. Characterization of the Ac-HA-Pba Nanogel. Selforganized nanogels were prepared by the dispersion of the conjugates into aqueous solution. After filtration with a 0.45 µm syringe filter, the particle size and zeta potential of each sample (1 mg/mL) were determined utilizing a dynamic light scattering instrument (Zetasizer 3000, Malvern Instruments Ltd., UK). The particle production yield was shown to be approximately 75%. As a means of observing the morphology of the nanogel, a droplet of nanogel suspension in distilled water (DW) was deposited on carbon stickers and the water evaporated in a vacuum oven. The dried sample was visualized with a Ptcoating by a field emission scanning electron microscope (FESEM; S-4700, Hitachi, Japan). Average size was also assessed by measuring the diameter of particles in the SEM images. Measurement of the Critical Self-Quenching Concentration. The critical self-quenching concentration (CQC) of Ac-HA-Pba nanogels was measured using a fluorescence probe technique. Pyrene dissolved in acetone was diluted in distilled water to give a concentration of 12 × 10-7 M. Acetone was removed by distillation under vacuum at 60° for 1 h. The acetone-free pyrene solution and each nanogel solution of different concentrations were mixed and incubated for 6 h without light. The final pyrene in each sample solution was 6 × 10-7 M. The excitation spectra of the pyrene were measured at a fixed emission wavelength of 390 nm, and the excitation wavelength was set at 339 nm for the emission spectra of pyrene which were recorded on a RF spectrofluorometer (RF-5301PC, Shimadzu, Japan). The slit width was 10 nm for both excitation and emission. Optical Spectroscopy. The absorption spectra were acquired using a UV/visible spectrophotometer (UV-2450, Shimadzu, Japan) in a quartz cuvette of 1 cm path length to compare Ac-HA-Pba conjugates with free Pba in DMSO. To estimate the self-quenching effect, Ac-HA-Pba was dissolved in DMSO or PBS, and Pba concentrations were adjusted to 20, 10, 5, 2, 1, 0.5, 0.2, and 0.1 µg/mL. The fluorescence spectra were taken at room temperature in 1 cm × 1 cm optical quartz cells using a Xenon short arc lamp (XBO 150, Ushino, Japan) in a spectrofluorephotometer. All samples were then plated onto 96-well plates to take direct fluorescence images utilizing KODAK Image Station.

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To measure the fluorescence quantum yield (FOY), six Ac-HA-Pba suspensions in DMSO with different Pba concentrations of 10, 5, 2, 1, 0.5, and 0.2 µg/mL were prepared to plot a graft of integrated fluorescence intensity against absorbance. Absorbance at the excitation wavelength was recorded from the UV/vis absorbance spectrum and marked as X data, whereas integrated fluorescence intensity, obtained from the fully corrected fluorescence spectrum, was tabled as Y data. The FQY of Ac-HA-Pba conjugates was calculated using free Pba with a known FQY value, according to the following equation: 2 ) ΦX)ΦST(GradX/GradST)(ηX2/ηST

where the subscripts ST and X denote standard and test, respectively, ΦX is the FQY, Grad is the gradient from the plot of integrated fluorescence intensity against absorbance, and η is the refractive index of the solvent. Singlet Oxygen Generation. The generation of singlet oxygen (1O2) was observed chemically by the detection of 9,10dimethylanthracene (DMA) utilizing fluorescent spectroscopy as an independent method. It is known that DMA reacts irreversibly with 1O2 in many organic solvents and water, which causes a decrease in the intensity of the DMA absorption band at 360 nm (30-32). Twenty micromolars of DMA was mixed with each solution (N,N-DMF, or DW) of Ac-HA-Pba. The solutions were irradiated with a 670 nm laser source (Institute of Electronics) at 10mW/cm2. Singlet oxygen-induced reduction of DMA fluorescence intensity (Ex 360 nm and Em 380-550 nm) was recorded at 0, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, and 120 s using an RF-spectrofluorometer. Singlet oxygen quantum yields (Φ∆) were then calculated according to the gradient from the curve of DMA fluorescent intensity (or DMA concentration) against the time of light exposure, utilizing the following equation: Φ∆U)Φ∆ST(SU/SST) where U and St denote unknown and standard, respectively, and S represents the slope. Free Pba was used as the standard. Ac-HA-Pba Nanogels in the Cell Culture System. Cell Culture. HeLa cells were cultured in suspension in DMEM medium supplemented with 10% FBS and 1% antibiotics at 37° in a balanced air humidified incubator with an atmosphere of 5% CO2. Cells were maintained in an exponential growth phase through periodic dilutions with fresh medium. Confocal Microscopy. To observe cellular uptake, HeLa cells were seeded at a density of 1.0 × 105 cells per well on 18 × 18 mm sterile cover glasses inserted into 12-well plates. The medium was replaced with serum-free DMEM containing Ac-HA-Pba or free Pba (Pba concn 15 µg/mL). After 6 h, the cells were rinsed with DPBS, fixed with 4% paraformaldehyde solution for 10 min, and stained with 4, 6-diamidino2-phenylindole (DAPI 1 µL, 3.63 mM) for 2 min. Cover glasses were then placed on the slide glasses. Internalization of Pba into live cells was monitored by fluorescence using a confocal laser scanning microscope (Zeiss, LSM 510 Meta, Germany). Fluorescence images were analyzed using LSM image browser software (Zeiss, Germany). Fluorescence ActiVated Cell Sorting (FACS). To confirm the competitive inhibition of cellular uptake, HeLa cells were exposed to HA polymer (3 mg/mL) and Ac-HA-Pba (Pba concn 0.3 µg/mL) simultaneously and compared with those exposed only to Ac-HA-Pba. The cells were incubated for 1 h, washed, harvested, and resuspended with DPBS. Flow cytometry was performed using a FACScan flow cytometer (Beckman, San Jose, CA, USA). Ten thousand cells (gated events) were counted for each sample, and free Pba fluorescence was detected with logarithmic settings (FL4; Em ) 670 nm).

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Cells were counted as positive if their fluorescence (FL4) was higher than that of cells from an untreated cell suspension. Each experiment was analyzed statistically using the CXP Analysis Program. Near-Infrared (NIR) Fluorescent Images. HeLa cells were grown in a 96-well culture plate at 1 × 104 cells per well in a 200 µL culture medium. Ac-HA-Pba with two different concentrations were treated to HeLa cells or serum-free DMEM at predetermined incubation times of 0 min, 20 min, 1 h, 6 h, and 12 h, and the plates were observed through NIR fluorescence emissions on the KODAK Image Station. MTT Assay. HeLa cells at 1 × 104 HeLa cells per well were seeded in 200 µL of cell culture medium in 96-well plates. After cell attachment, the medium were replaced with a serum-free culture. The cells were treated with Ac-HA-Pba or equivalently free Pba and incubated for 6 h. The medium were replaced again with serum-free DMEM, followed by light illumination. The cells were divided into three groups under distinctly different conditions: (1) no irradiation; (2) irradiation for 1 min with a 1 mW/cm2 laser; (3) irradiation for 5 min with a 1 W/cm2 laser. Cell viability was determined following incubation for 24 h using 3-[4,5-dimethylthiazol-2-yl]-3,5-diphenyltetrazolium bromide dye (MTT dye, 2 mg/mL) uptake at 570 nm on an ELISA reader. Dark toxicity of Ac-HA-Pba and free Pba was also assessed against HeLa cells incubated for 72 h in a completely lightproof plate. LiVe/Dead Viability/Cytotoxicity Assay. A two-color fluorescence cell viability assay was used to measure two recognized parameters of cell viability-intracellular esterase activity and plasma membrane integrity. It is known that nonfluorescent CalceinAM is converted into a green-fluorescent calcein after acetoxymethyl ester hydrolysis by intracellular esterases in live cells and that EthD-1 marks the nuclei by staining dead cells red once the integrity of the cell membrane is compromised (33). HeLa cells on glass coverslips were removed from the culture medium, rinsed carefully with Hanks1× balanced salt solution, and placed in the microscope chamber at 37 °C. The cells were then covered with Hanks1× balanced salt solution (2 mL) containing 10 mM pH buffer HEPES (4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid), 2 µM CalceinAM, 4 µM EthD-1, and Ac-HA-Pba (Pba concn 0.15 µg/mL). The cells were exposed at a small location for 10 min to a 670 nm laser with a power intensity of 1 mW/cm2. Finally, confocal microscopic images were taken with an oil immersion 60× objective lens in two channels (488-nm excitation of the calcein converted from CalceinAM, 568-nm excitation of the EthD-1), allowing for the concurrent comparison of cells exposed to light alongside those without exposure.

RESULTS AND DISCUSSION Synthesis of Ac-HA-Pba Conjugates. HA, a linear polysaccharide widely used in drug delivery systems, was selected for the production of PS-conjugated nanogels, from various block copolymers, due to its biocompatibility, biodegradability, and tumor-targeting receptors. However, the possibilities for chemosynthesis of HA, with hydrophobic drugs or other polymers in a single organic phase, are severely limited due to the poor solubility of HA in most organic solvents. To increase the hydrophobicity of HA, partially acetylated derivatives were prepared in formamide through the reaction between the hydroxyl groups of HA and acetic anhydride (AA), while adding pyridine as a catalyzer (Figure 1). Previously, we have described acetylated modification with other polysaccharides, such as pullulan, to increase hydrophobicity (34-36). By changing the feeding amount of AA for the reaction, control of the degree of AA modification in polymer-acetate could be achieved (37). In the present study, acetylation was confined to

Acetylated HA Nanogels for Photodynamic Therapy


Figure 1. Synthetic scheme of AC-HA-Pba conjugates. Table 1. Ac-HA-Pba Conjugates with Three Different Pba Contents Were Synthesized, Showing the Characteristics of Self-Organized Ac-HA-Pba Nanogels in an Aqueous Solution

Ac-HA-Pba1 Ac-HA-Pba2 Ac-HA-Pba3

Pba contenta

average size (nm)b

potential (mV)c

CQC (×10-6 M)d

0.31 0.17 0.08

125.1 ( 1.1 139.1 ( 0.5 150.4 ( 1.5

-33.8 ( 0.8 -27.1 ( 1.6 -21.2 ( 1.0

0.26 0.83 2.10

a

The degree of substitution of Pba molecules per 1 unit (2 glucose rings) of HA. b Mean diameter (intensity average) measured by dynamic light scattering. c Potential detected by zeta potential. d Critical self-quenching concentration determined using pyrene.

a low extent because the conversion of HA to HA-acetate (AcHA) was required merely to make HA soluble in DMSO for the second conjugation of HA and Pba. Typically, the proton signal of the acetamido methyl group in disaccharide units of HA polymer was observed at δ ) 1.9 ppm (-CH3) in the 1H NMR spectrum. Because of the introduction of acetyl groups, the signals from AC-HA slightly increased when compared with those of native HA. Thus, by a comparison of the intensities of peaks from δ ) 1.9 ppm from HA and Ac-HA in D2O, the degree of acetylation was determined as 0.4 acetyl groups per unit (2 glucose rings) of HA. The synthesis of Ac-HA with Pba was successfully carried out using carbodiimide coupling chemistry as schematically shown in Figure 1. The chemical structures of Ac-HA-Pba conjugates were confirmed by 1H NMR. The characteristic peaks of Pba such as dCH- from 4 pyrrole subunits were identified, while the methyl resonance of the acetamido moiety of HA was 2 ppm (Figure S1, Supporting Information). The degrees of substitution of Pba molecules per unit (2 glucose rings) of HA are in the range of 0.31 to 0.08 (Table 1). Many researchers have reported that most synthesis protocol designs of HA nanoparticles are based on there carboxyl groups, which may impair the binding of HA to its receptors when tumor targeting (22). Also, Petra et al. reported that after the hydroxyl groups were modified, the degradation rate of HA derivates is almost the same for the original HA (38). From this paper, we considered that the modification of hydroxyl groups in HA is better than that of the carboxyl groups to maintain the nature of HA. It should be noted that the simple two-step reaction scheme utilized in the current study was based on the hydroxyl group of HA. Size Distribution and Morphology. Ac-HA-Pba conjugates showed self-organizing behavior in aqueous solutions due to the strong hydrophobicity of grafted Pba, the driving force

Figure 2. Size distribution histogram measured by dynamic light scattering (A) and morphology observed by a field-emission scanning electron microscope (B) of Ac-HA-Pba1 nanogels.

of aggregation. It is likely that Pba self-organized to form a hydrophobic inner core surrounded by a hydrophilic outer shell of HA. The mean particle size of Ac-HA-Pba1 nanogels was 125 nm with a monodispersed size distribution (Figure 2A). The samples showed a decreasing particle size with increasing Pba content (Table 1). The size of the tumor vascular pore openings has been reported to be significantly larger than those observed along vessels in normal tissues (39). The average size of tumor vascular pores is generally 400-600 nm (40). Therefore, the greater pore size leads to prolonged retention of Ac-HA-Pba nanogels in tumors. The negative charge, determined by zeta potential measurement, confirmed the presence of HA at the surface of the nanogels (Table 1). This negative surface charge may also help the enhancement of retention times within the body. The FE-SEM images of Ac-HA-Pba1 nanogels revealed that they were homogeneously distributed in spherical shapes with sizes of 40-80 nm (Figure 2B). The smaller size of Ac-HA-Pba1 nanogels when observed through FE-SEM, rather than DLS data, indicates that Ac-HA-Pba1 nanogels have huge water retention properties. Critical Self-Quenching Concentration (CQC). Pyrene, as a fluorescence probe, was used to investigate the CQCs of Ac-HA-Pba nanogels (41). The excitation and emission spectra of pyrene were recorded by an RF spectrofluorometer (Figure 3). In general, changes in pyrene intensity and (0,0) band shift as a function of material concentration were employed to measure critical micelle concentrations (CMC) and critical aggregation concentrations (CAC). Polymers with high concentrations spontaneously formed nanoparticles in an aqueous solution, and pyrene could partition into the polycore of the nanoparticles due to its hydrohopicity, resulting in an increase in pyrene intensity. At the dilution of the polymer suspension, excitation and emission were more weakly exhibited, in line with a reduction of naonoparticle formations. When the


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Figure 3. Critical self-quenching concentration (CQC) of Ac-HA-Pba measured by pyrene: emission spectra of pyrene in Ac-HA-Pba1 suspension (A) and the plot of intensity ratio of I1/I3 as a function of the logarithm of concentration (B).

concentration was adjust below the CAC, excitation and emission dropped to their lowest levels, both in suspension and in DW. Interestingly, in the case of Ac-HA-Pba suspensions, the excitation and emission spectra of pyrene were the reverse of the general result (Figure 3). The emission intensity of pyrene gradually decreased with an increase in Ac-HA-Pba1 concentration, while in DW, these were shown to be at their highest levels (the red line in Figure 3A). It is assumed that Pba located in the hydrophobic core interfered with the fluorescent intensity of pyrene due to the strong quenching effect of the hydrophobic interaction between Pba and pyrene, known as the fluorescence resonance energy transfer effect (FRET). Thus, we designated a new term, the critical self-quenching concentration (CQC) (Figure 3B). Table 1 shows that the CQC values of Ac-HA-Pba nanogels decreased alongside an increased Pba content. Photoactivities of Ac-HA-Pba1 Nanogels. As regards photophysical and photochemical properties, the typical absorption spectra of Ac-HA-Pba conjugates in DMSO were overlapped by that of free Pba with the same Pba concentration of 10 µg/mL (Figure S2, Supporting Information). Also, the fluorescent intensity of Ac-HA-Pba dissociated in DMSO clearly declined alongside a decrease in dye concentration (Figure 4A), whereas in DW it was unclear (Figure 4B), as a likely result of the fact that Ac-HA-Pba begins to form nanogels in DW, while the close geometry between conjugated Pba in the core of nanogels creates the self-quenching effect (42). Combining the absorbance and the emission intensity in DMSO, the fluorescence quantum yield of Ac-HA-Pba conjugates was found to be almost the same when compared with that of standard Pba (Figure S3, Supporting Information). This demonstrates that the conjugation of Ac-HA and Pba does not affect any fluorescent properties of free Pba.

Figure 4. Fluorescence spectra, near-infrared fluorescence images, and optical images of Ac-HA-Pba1 in DMSO (A) and PBS (B).

Figure 5. Changes in DMA fluorescent intensity according to time in the presence of Ac-HA-Pba1 conjugates in N,N-DMF (b) or DW (2), as well as the same concentration of free Pba in N,N-DMF (O). All irradiations were performed using 670 nm light at a fluorescencerate of 10 mW/cm2.

DMA was used in the experiment as a detector to evaluate singlet oxygen generation (Figure S4, Supporting Information, and Figure 5). Ac-HA-Pba completely dissolved in DMF resulting in a sharp decline in DMA fluorescent intensity, indicating the rapid generation of singlet oxygen upon laser irradiation. The experiment mixed DMA with free Pba dispersed in N,N-DMF was used as the standard. Singlet oxygen quantum yields (Φ∆) of Ac-HA-Pba1, 2, and 3 were calculated as 0.552, 0.550, and 0.546, respectively, by a comparison of the slope for Ac-HA-Pba with the corresponding slope for free Pba (Φ ) 0.56). However, singlet oxygen generation can significantly change in a different medium. As self-assembly of Ac-HA-Pba in an aqueous solution leads to fluorescent



Figure 6. Confocal microscopy images of fixed HeLa cells incubated with Ac-HA-Pba (A-D) or free Pba (a-d) for 6 h. (A,a) Merged image of panels B/C or b/c; (B,b) DIC image; (C,c) DAPI image; (D,d) Pba image. The scale length of each picture is 20 µm.

Figure 7. Flow cytometry results of nontreated control cells (A), Ac-HA-Pba treated cells (B), and HA polymer and Ac-HA-Pba cotreated cells (C).

quenching, the Ac-HA-Pba nanogels could not be activated into the triplet state by mutual energy transfer, resulting in the concealment of the ability to generate ROS (43). The absence of differences in absorption/fluorescence/singlet oxygen generation for Ac-HA-Pba and free Pba denotes that conjugation with HA maintains the PDT effect of Pba. Though the three samples show similar properties of fluorescence and efficiency of singlet oxygen generation, Ac-HA-Pba1 was chosen for further in Vitro studies due to it being of the smallest size and the lowest CQC. A small size and lower CQC allow for easier tumor cellular targeting and reduced side effects from photosensitizers. Cell Uptake of Ac-HA-Pba Nanogels. The cellular uptake behavior of Ac-HA-Pba was monitored by confocal microscopy following incubation for 6 h (Figure 6). The internalization and passive diffusion of Ac-HA-Pba nanogels and Pba, into HeLa cells, were very rapid and effective. The red fluorescence of free Pba induced the formulation of ambiguous clusters, while

Figure 8. In Vitro fluorescent image of Ac-HA-Pba induced by timedependent cellular uptake and degradation of HA: Ac-HA-Pba was exposed to HeLa cells (a, c) or culture medium (b, d) for a predetermined incubation time. Pba concns are 1.0 µg/mL (a, b) or 0.5 µg/mL (c, d), respectively.

Ac-HA-Pba appeared as perinuclear punctuate spots in the cytoplasm, suggesting that Ac-HA-Pba was internalized via endocytosis. To confirm whether the uptake of Ac-HA-Pba nanogels is involved with HA-receptors, competition inhibition studies were performed with an HA polymer with strong binding properties to specific HA-receptors on the surface of tumor cells, potentially reducing the uptake of Ac-HA-Pba nanogels into HeLa cells (44, 45). As shown in Figure 7, HeLa cells incubated with Ac-HA-Pba for 1 h showed a high fluorescence of 99.38%. However, with competition as a result of excess HA polymer (non acetylated HA), the peak of the fluorescence shifted, and the value dropped to 14.90%. The results suggest a significantly greater cellular uptake of Ac-HA-Pba (Figure 7B) than that of Ac-HA-Pba added with HA polymer (Figure 7C), as a consequence of HA receptor-mediated endocytosis. Thus, HAbased nanogels not only selectively target cancer cells but also improve the internalization of photosensitizers within targeted cancer cells. The Ac-HA-Pba nanogel was designed for internalization within cancer cells via HA receptor-mediator endocytosis. The


Figure 9. Viability of HeLa cells treated with Ac-HA-Pba (0, O, ∆) and Pba (9, b, 2) after photoirradiation for 0 min (0, 9), 1 min (O, b), or 5 min (∆, 2) using 670 nm light at a fluorescence rate of 1 mW/cm2 (A). Dark toxicity of Ac-HA-Pba (black bar) and free Pba (gray bar) (B).

internalized nanogel goes to the lysozome, which has a lot of enzymes passing through the endosome for polysaccharide cleavage, and at last, photoactivity appears in the lysozome as a result of the removal of the FRET effect (Scheme 1). In an attempt to prove this theory, nanogels were coincubated with HeLa cells, and the fluorescence image as a function of time was observed with a KODAK image station (Figure 8). As incubation time increased, the fluorescence became more pronounced due to an increase in Pba concentrations. The results

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of confocal images and FACS indicate that the cellular uptake of Ac-HA-Pba nanogels correlated well with the theory. However, the nanogels incubated in a cell culture medium without cells showed no fluorescence. Form the above results, we conclude that Ac-HA-based nanogels are internalized by HA receptor-mediated endocytosis and then disintegrated by enzymes in the intracellular microenvironment, leading to the release of fluorescence exhibiting Pba. Phototoxicity of Ac-HA-Pba against Cancer Cell Lines. The efficacy of Ac-HA-Pba nanogels as PDT agents depends on the production of singlet oxygen (46). As the singlet oxygen quantum yield is similar to that of free Pba, tumor cells treated with Ac-HA-Pba may not show a significant difference from those treated with Pba. As shown in Figure 9A, the group with no light and no drug treatment was set as 100%, and the other data were normalized for comparison. The nanogel samples of different Pba concentrations (0.0025, 0.0250, 0.2500, and 2.5000 µg/mL) showed a cytotoxicity similar to that of free Pba in each group with laser treatment. An increase in Pba concentrations or the prolongation of illumination time brought about higher cell death. However, samples treated only with Ac-HA-Pba nanogels or laser excitation did not cause cytotoxicity against HeLa cells. The dark toxicity was also evaluated by the MTT assay. With a long incubation time of 72 h, Ac-HA-Pba nanogels went inside HeLa cells, causing intracellular HA degradation and releasing Pba. Thus, Ac-HA-Pba nanogels displayed a slightly lower dark toxicity than that of free Pba in the range of the concentrations used (Figure 9B). To further assess the phototoxicity of Ac-HA-Pba naonogels, their tumor cell killing activity was also determined by the live and dead assay. Stained live and dead cells treated with CalceinAM and EthD-1 were visualized as green and red light emissions, respectively, through fluorescence microscopy (Figure 10). No toxicity was found in the control cells treated with light only. Unlike the control, HeLa cells treated by Ac-HA-Pba showed small, bright, red dots where also treated by a laser (Figure 9A). It was clearly evident that the combination of treatment with Ac-HA-Pba nanogels and exposure to laser light resulted in selected cell death.

CONCLUSIONS A HA/photosensitizer conjugate system has seldom been reported as a potential photosensitizer carrier for photodynamic

Figure 10. Detection of photodamage by fluorescence microscopy with fluorescent probes (double-staining with CalceinAM and EthD-1: the Ac-HA-Pba group (A-D) and the control group without drug treatment (a-d) were irradiated with a 1 mW/cm2 laser for 10 min at a small dot, in which the red dead cells might appear. (A,a) Compound pictures of c/d or C/D; (B,b) optical images; (C,c) control living cells without irradiation; green fluorescence of CalceinAM; (D,d) dead cells after PDT treatment; red fluorescence of EthD-1.


therapy. In this study, we introduced a simple method for the solubilization of HALM in an organic solvent. The method was utilized to conjugate Pba to HALM (Ac-HA-Pba conjugate) and self-organized nanogels, with the diameter below 150 nm, was prepared by simple dispersion in an aqueous solution. The results displayed autoquenching properties on photoactivity. As the nanogel was incubated with cells, the autoquenching behavior disappeared due to the degradation of the HA backbone by attacks from enzymes in the presence of intracellular compartments such as the endosome and lysosome. The nanogels also show HA-induced tumor homing properties, resulting in a rapid internalization rate. Moreover, the cytotoxicity of the nanogel is similar to that of free Pba with light, while the nanogel reveals very low cytotoxicity against cancer cells without light. Therefore, Ac-HA-Pba nanogels may be instrumental in the design of new photodynamic therapies utilizing a minimum of unfavorable phototoxicity.

ACKNOWLEDGMENT This work was financially supported by Fundamental R&D Program for Core Technology of Materials, Ministry of Knowledge Economy, Republic of Korea. Supporting Information Available: Additional data associated with this article. Figure S1 showing 1H NMR spectra, Figure S2 showing absorption spectra, Figure S3 showing fluorescence quantum yields, and Figure S4 showing singlet oxygen generation. This material is available free of charge via the Internet at http://pubs.acs.org.

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