Near-Infrared Light-Activated Photochemical Internalization of

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Near-Infrared Light-Activated Photochemical Internalization of Reduction-Responsive Polyprodrug Vesicles for Synergistic Photodynamic and Chemotherapy kangning Zhu, Guhuan Liu, Jinming Hu, and Shiyong Liu Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.7b00693 • Publication Date (Web): 05 Jul 2017 Downloaded from http://pubs.acs.org on July 7, 2017

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Near-Infrared Light-Activated Photochemical Internalization of Reduction-Responsive Polyprodrug Vesicles for Synergistic Photodynamic and Chemotherapy Kangning Zhu, Guhuan Liu, Jinming Hu*, and Shiyong Liu*.

CAS Key Laboratory of Soft Matter Chemistry, Hefei National Laboratory for Physical Sciences at the Microscale, iChem (Collaborative Innovation Center of Chemistry for Energy Materials), Department of Polymer Science and Engineering, University of Science and Technology of China, Hefei, Anhui 230026, China.

KEYWORDS. Near-infrared light, upconversion nanoparticles, polyprodrug vesicles, photodynamic therapy, chemotherapy.

ABSTRACT. The use of intracellular reductive microenvironment to control the release of therapeutic payloads has emerged as a popular approach to design and fabricate intelligent nanocarriers. However, these reduction-responsive nanocarriers are generally trapped within endolysosomes after internalization and are subjected to unwanted disintegration, remarkably compromising the therapeutic performance.

Herein,

amphiphilic polyprodrugs of

poly(N,N-dimethylacrylamide-co-EoS)-b-PCPTM were synthesized via sequential reversible addition-fragmentation chain transfer (RAFT) polymerization, where EoS and CPTM are Eosin Y- and camptothecin (CPT)-based monomers, respectively. An oil-in-water (O/W) 1

ACS Paragon Plus Environment

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emulsion method was applied to self-assemble the amphiphilic polyprodrugs into hybrid vesicles in the presence of hydrophobic oleic acid (OA)-stabilized upconversion nanoparticles (UCNPs, NaYF4:Yb/Er), rendering it possible to activate the EoS photosensitizer under a near-infrared (NIR) laser irradiation with the generation of singlet oxygen (1O2) through the energy transfer between UCNPs and EoS moieties. Notably, the in situ generated singlet oxygen (1O2) can not only exert its photodynamic therapy (PDT) effect but also disrupt the membranes of endolysosomes and thus facilitate the endosomal escape of internalized nanocarriers (i.e., photochemical internalization (PCI)). Cell experiments revealed that the hybrid vesicles could be facilely taken up by endocytosis. Although the internalized hybrid vesicles were initially trapped within endolysosomes, a remarkable endosomal escape into the cytoplasm was observed under 980 nm laser irradiation as a result of the PCI effect of 1O2. The escaped hybrid vesicles subsequently underwent GSH-triggered CPT release in the cytosol, thereby activating the chemotherapy process. The integration of PDT module into the design of reduction-responsive nanocarriers provides a feasible approach to enhance the therapeutic performance.

INTRODUCTION Chemotherapy has been clinically used for the treatment of cancers.1,

2

However,

traditional chemotherapy suffers from severe adverse effects due to the inherent physicochemical properties of anticancer drugs such as poor water-solubility, short blood circulation time, and non-specific targeting to pathological sites.3 To circumvent these drawbacks, a variety of drug delivery nanocarriers have been developed to alter the pharmacokinetics of therapeutic drugs and elevate the therapeutic efficacy.4-9 Of these, the 2


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use of pathological microenvironments to spatiotemporally control the release of therapeutic payloads from nanocarriers has emerged as a promising strategy to treat cancers due to significantly decreased systemic toxicities.10-14 For example, mildly acidic pH,15-20 redox potential,21-25 overexpressed enzymes,26-31 and hypoxia32-35 have been extensively applied to fabricate drug delivery nanocarriers capable of releasing payloads under a specific stimulus. Among all the specific pathological triggering events, the reductive microenvironment in the cytoplasm has received broad interest since a remarkably distinct glutathione (GSH) concentration has been observed in the cytosol (1-10 mM) and the extracellular fluids (20-40 µM).21, 36, 37 More favorably, this difference is further amplified within cancer cells, making it possible to selectively exert therapeutic effects in cancer cells by virtue of high GSH concentrations within cancer cells.38, 39 As such, numerous GSH-responsive nanocarriers have been developed by taking advantage of the GSH-mediated thiol exchange reactions of disulfide bonds. Despite tremendous achievements, it should be mentioned that these GSH-responsive nanocarriers generally enter into cells by endocytosis and cannot efficiently escape from endolysosomes. Unfortunately, these nanocarriers together with the encapsulated therapeutic payloads experience disintegration and eventually end up within endolysosomes prior to entering into the cytoplasm, resulting in eventually the failure of therapy. Therefore, to elevate the therapeutic efficacy of reduction-sensitive nanocarriers, it is of pivotal importance to facilitate the entrance of reduction-responsive nanocarriers into the cytoplasm rather than trapping within endolysosomes, where cytosolic abundant GSH could then take effect, thereby triggering the release of encapsulated payloads on demand. Indeed, to avoid being retained within endolysosomes, several approaches have been employed including the 3


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incorporation of specific targeting motifs,40 the introduction of endosome-escapable moieties,41, 42 and the modulation of the morphologies of nanocarriers43-45 and so on. On the other hand, photodynamic therapy (PDT) has been used as a complementary option to classic chemotherapy.46, 47 In this regard, photosensitizers are incorporated into drug delivery nanocarriers and cytotoxic reactive oxygen species (ROS) such as singlet oxygen (1O2) could be generated under photo-irradiation, leading to the death of cancer cells. Moreover, the PDT efficacy could be further optimized if a longer irradiation wavelength (e.g., near-infrared (NIR) light) is applied, exhibiting higher tissue penetration and lower toxicity and avoiding the activation of endogenous chromophores. In comparison with the difficulties in developing novel fluorophores with long emission wavelengths, the advent of upconversion nanoparticles (UCNPs) provides an alternative strategy to resolve this concern, which are characterized by the fact that a long excitation wavelength gives rise to a low emission wavelength.48, 49 Hence, the excitation of UCNPs using an NIR light renders it possible to activate a photosensitizer through an energy transfer mechanism, provided that the emission of UCNPs could well match with the absorption of the photosensitizer, resulting in the generation of 1O2 under NIR irradiation without recourse to new NIR fluorophores. Notably, besides well-known PDT effect, 1O2 can also efficiently disrupt the membranes of endolysosomes, facilitating the release of internalized nanocarriers from endolysosomes and this unique phenomenon was coined as photochemical internalization (PCI).50-52 Therefore, we envisioned that if reduction-responsive nanocarriers were integrated with specific photosensitizers, after endocytosis, the internalized nanocarriers could efficiently produce 1O2 under photo illumination and thus exert PDT effect. The generated 1O2 could destabilize the 4


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membranes of endolysosomes and in turn accelerate the endosomal escape of internalized nanocarriers into the cytoplasm, in which abundant GSH further mediated the disintegration of the reduction-responsive nanocarriers and then triggered the release of encapsulated therapeutic payloads, thereby treating cancers in a spatiotemporally controlled manner through the combination of PDT and chemotherapy. To verify our assumption, herein, a photosensitizer, Eosin Y (EoS), was incorporated into the shells of GSH-responsive vesicles, which were assembled from polyprodrug amphiphiles originating from camptothecin (CPT)-based monomers (CPTM) through an oil-in-water (O/W) approach under pulse sonication. Moreover, hydrophobic oleic acid (OA)-stabilized UCNPs were also introduced during the self-assembly process to enable the activation of EoS photosensitizer using an NIR laser. The hybrid vesicles were taken up readily by endocytosis. Under an NIR laser irradiation, the in situ generated 1O2 from the hybrid vesicles can simultaneously exert PDT and PCI effects, killing cancer cells and promoting the endosomal escape of internalized hybrid vesicles. Moreover, the following GSH-triggered release of intact CPT drug within the cytoplasm can further eradicate cancer cells in a synergistic manner (Scheme 1).

5


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Scheme 1. Schematic illustration for the fabrication of UCNP-loaded hybrid vesicles from P(DMA-co-EoS)-b-PCPTM diblock copolymers via an O/W emulsion and a subsequent solvent evaporation procedure. Under 980 nm laser irradiation, energy transfer from UCNPs to EoS moieties occurs within the hybrid vesicles, resulting in the activation of EoS sensitizers with the production of singlet oxygen (1O2). The generated 1O2 simultaneously exert PDT and PCI effects, promoting the endosomal escape of hybrid vesicles into the cytosol, where the GSH-triggered release of CPT from the vesicle bilayers is then actuated, thereby synergistically killing cancer cells via the combination of PDT and chemotherapy. EXPERIMENTAL SECTION

Materials. N,N-dimethylacrylamide (DMA) was purchased from Aldrich and distilled over CaH2 prior to use. Eosin Y, 4-vinylbenzyl chloride, 2′,7′-dicholrofluorescin diacetate (DCF),

3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium

bromide

(MTT),

N,N-dimethyl-4-nitrosoaniline (RNO), and imidazole were purchased from Aldrich and used as received. Fluorescein diacetate (FDA) and propidium iodide (PI) were purchased from 6


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Molecular Probes® and used as received. Fetal bovine serum (FBS), penicillin, streptomycin, and Dulbecco’s modified Eagle medium (DMEM) were purchased from GIBCO and used as received. Camptothecin (CPT), 1,4-dioxane, DMSO and all other reagents were purchased from Sinopharm Chemical Reagent Co. Ltd. and used as received unless otherwise noted. Water was deionized with a Milli-Q SP reagent water system (Millipore) to a specific resistivity

of

18.4

MΩ

cm.

4-Cyanopentanoicacid

dithiobenzoate

(CPADB),53

reduction-responsive CPT-based monomer (CPTM),43 Eosin Y-based Monomer (EoS),54 and UCNPs (NaYF4:Yb(18%)/Er(2%))55 with an average diameter of 10 nm (Figure S1) were synthesized according to literature procedures.

Scheme 2. Synthetic routes employed for the preparation of P(DMA-co-EoS)-b-PCPTM diblock copolymers (BP1-BP4) via sequential RAFT polymerizations.

Sample Preparation. 7


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Synthesis of P(DMA-co-EoS) via RAFT polymerization (Scheme 2). Typically, CPADB (188 mg, 0.67 mmo1), DMA (2 g, 20.2 mmol), EoS (307 mg, 0.402 mmol), and AIBN (11 mg, 0.067 mmol) were dissolved in DMSO (4 mL) with a magnetic stirring bar. The tube was carefully degassed by three freeze-pump-thaw cycles and then sealed under vacuum. After stirring for 8 h at 70 oC, the reaction tube was quenched into liquid nitrogen, exposed to air, and precipitated into an excess of diethyl ether. The above dissolution-precipitation cycle was repeated three times. The final product was dried in a vacuum oven overnight at room temperature, yielding a red solid (1.2 g, yield: 60%). The molecular weight and molecular weight distribution of P(DMA-co-EoS) were determined by GPC using DMF as the eluent, revealing an Mn of 2 kDa and Mw/Mn of 1.18. The Degree of polymerization (DP) of P(DMA-co-EoS) was determined to be ~ 20 by

1

H NMR

spectroscopy (Figure S2a) and the molar content of EoS moieties was determined to be 2% by UV-Vis using a standard calibration curve (Figure S3). Thus, the as-synthesized polymer was denoted as P(DMA0.98-co-EoS0.02)20. PDMA20 macroRAFT agent without EoS labeling was synthesized under otherwise identical conditions without the addition of EoS monomer.

Synthesis of P(DMA-co-EoS)-b-PCPTM Diblock Copolymers (Scheme 2). Using the preparation

of

P(DMA0.98-co-EoS0.02)20-b-PCPTM20

(BP1)

as

an

example,

P(DMA0.98-co-EoS0.02)20 macroRAFT agent (9 mg, 0.0046 mmol), CPTM (84 mg, 0.14 mmol), and AIBN (0.18 mg, 0.00115 mmol) were dissolved in a 400 µL mixed solvent of DMSO/1,4-dioxane (1/1, v/v) with a magnetic stirring bar. The tube was carefully degassed by three freeze-pump-thaw cycles and then sealed under vacuum. After stirring at 70 oC for 24 h, the reaction tube was quenched into liquid nitrogen, exposed to air and precipitated into 8


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an excess of diethyl ether. The above dissolution-precipitation cycle was repeated three times. The final product was dried in a vacuum oven overnight at room temperature to afford a red solid (50 mg, yield: 56%). The molecular weights and molecular weight distributions of P(DMA-co-EoS)20-b-PCPTMn were determined by GPC using DMF as the eluent, revealing an Mn of 14.7 kDa and Mw/Mn of 1.19 (Table 1). The DP of PCPTM block was determined to be 20 by 1H NMR analysis in CDCl3 (Figure S2b). Thus, the synthesized diblock copolymer was denoted as P(DMA0.98-co-EoS0.02)20-b-PCPTM20 (BP1). According to similar procedures, P(DMA0.98-co-EoS0.02)20-b-PCPTM25

(BP2),

PDMA20-b-PCPTM25

(BP3),

and

P(DMA0.98-co-EoS0.02)20-b-PCPTM38 (BP4) were also synthesized. The structural parameters of synthesized diblock copolymers (BP1-BP4) are summarized in Table 1.

Fabrication of UCNP-Loaded Hybrid Assemblies. UCNP-loaded hybrid polymeric assemblies were fabricated from amphiphilic P(DMA-co-EoS)-b-PCPTM diblock copolymers in the presence of UCNPs via an O/W emulsion and chloroform (CHCl3) was employed as the

oil

phase,

followed

by

a

solvent

evaporation

procedure.

Briefly,

P(DMA-co-EoS)-b-PCPTM copolymers (2 mg) were dissolved in 100 µL of CHCl3 and 140 µL of UCNPs in CHCl3 (~0.2 mg UCNPs) was added. The mixture was then injected into 8 mL of deionized water and was sonicated in a pulse-pause manner for varying durations. After removal of the residual CHCl3 under vacuum, the unloaded UNCPs were removed by filtrating the as-assembled hybrid vesicles through a cellulose filter membrane (pore size: 450 nm). UCNP-loaded hybrid assemblies were achieved and stored at 4 oC for further use.

In Vitro CPT Release Profiles of UCNP-Loaded Hybrid Vesicles. Typically, 4 mL of UCNP-loaded hybrid polymeric vesicles were equally divided into four batches. Each batch 9


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contained 1 mL of hybrid vesicle dispersions and was placed in a dialysis tube (molecular weight cutoff: 3,500 Da). Then, the dialysis tubes were immersed in 10 mL of PBS media at pH 7.4 with varying GSH concentrations (0, 2 µM, 5 mM, and 10 mM) under gentle stirring at 37 oC. Periodically, aliquots of the incubation media were taken and replaced with the same volume of fresh PBS buffer solutions. HPLC was employed to assay the CPT contents in the media.

In Vitro Determination of Singlet Oxygen (1O2). UCNP-loaded hybrid vesicle dispersions were mixed with 50 mM of N,N-dimethyl-4-nitrosoaniline (RNO) and 100 mM of imidazole in PBS medium (20 mM, pH 7.4), and then irradiated with a 980 nm laser (1.5 W/cm2) for varying durations. The generation of singlet oxygen (1O2) by these hybrid vesicles resulted in the bleaching of RNO with characteristic absorption decrease at 440 nm, which could thus be employed to probe the 1O2 generation.56, 57

Cellular Internalization of UCNP-Loaded Hybrid Polymeric Vesicles. HepG2 cells were plated onto glass-bottom Petri dishes at a density of 80,000 cells per dish, then cultured in DMEM supplemented with 10% fetal bovine serum (FBS), penicillin (100 units/mL), and streptomycin (100 µg/mL) for 48 h at 37 ºC in CO2-air (5:95). UCNP-loaded hybrid BP2 vesicles were added and the equivalent CPT concentration was finally adjusted to 10 µg/mL. The cells were incubated for 1 h, 3 h, 8 h, and 12 h, respectively. After rinsing with PBS buffer, the endolysosomes were stained with LysoTracker red, fluorescence images were taken using a confocal laser scanning microscopy. CPT and LysoTraker red were excited by 405 nm and 594 nm lasers and the emission channels were set to be 420-470 nm and 605-720 nm, respectively. 10


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Determination of Singlet Oxygen (1O2) in HepG2 Cell. HepG2 cells were plated onto glass-bottom Petri dishes at a density of 80,000 cells per dish, then cultured in DMEM supplemented with 10% fetal bovine serum (FBS), penicillin (100 units/mL), and streptomycin (100 µg/mL) for 48 h at 37 ºC in CO2-air (5:95). UCNP-loaded hybrid vesicles were added and the equivalent CPT concentration was finally adjusted to 10 µg/mL. After the cells were incubated for 8 h, 2′,7′-dicholrofluorescin diacetate (DCF) was added. After 30 min, cells were rinsed with PBS buffer, followed by irradiation with a 980 nm laser. Fluorescence images were taken using a confocal laser scanning microscopy. DCF was excited by 488 nm laser and the emission channel was set to be 500-550 nm. The 1O2 generation of UCNP-loaded hybrid vesicles was roughly evaluated by the fluorescein intensity changes using an Imaging-Pro Plus software. The cells incubated with UCNP-loaded hybrid vesicles without 980 nm laser irradiation were used as a negative control.

Intracellular Trafficking of UCNP-Loaded Hybrid Polymeric Vesicles. HepG2 cells were plated onto glass-bottom Petri dishes at a density of 80,000 cells per dish, then cultured in DMEM supplemented with 10% fetal bovine serum (FBS), penicillin (100 units/mL), and streptomycin (100 µg/mL) for 28 h at 37 ºC in CO2-air (5:95). UCNP-loaded hybrid BP2 vesicles were added and the equivalent CPT concentration was finally adjusted to 10 µg/mL. The cells were incubated for 8 h, irradiated with 980 nm laser (1.5 W/cm2) for 15 min, and then incubated for additional 0 h, 1 h, 5 h, and 9 h, respectively. Afterward, cell endolysosomes were stained with LysoTracker Red. After rinsing with PBS buffer, fluorescence images were taken using a confocal laser scanning microscopy. The blue 11


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channel (CPT) and the red channel (LysoTracker red) were excited by 405 nm and 594 nm lasers and the emission channels were set to be 420-470 nm and 605-720 nm, respectively. The cells incubated with UCNP-loaded hybrid vesicles without 980 nm laser irradiation were utilized as a control.

In Vitro Cytotoxicity Assay of UCNP-Loaded Hybrid Vesicles. HepG2 cells were first cultured in DMEM supplemented with 10% FBS, penicillin (100 units/mL), and streptomycin (100 mg/mL) at 37 oC in a CO2-air (5:95) incubator for 48 h. For the cytotoxicity assay, HepG2 cells were seeded in a 96-well plate at an initial density of ca.5,000 cells/well in 100 µL of complete DMEM medium. After incubation for 24 h, DMEM was replaced with fresh medium and the cells were treated with UCNP-loaded hybrid vesicle dispersions at varying concentrations. After incubation for another 8 h, the treated cells were rinsed with PBS buffer and 180 µL of fresh culture medium was added. After with or without 980 nm laser irradiation (1.5 W/cm2) for 15 min, the cells were incubated in a humidified environment with 5% CO2 at 37 oC for another 24 h. After that, PBS solution of MTT reagent (20 µL, 5.0 g/L) was added to each well and the cells were further incubated at 37 oC for 4 h. The medium in each well was then removed and replaced with 150 µL of DMSO. The plate was gently agitated for 15 min before the absorbance at 570 nm was recorded by a microplate reader (Thermo Fisher). Each experiment was conducted in quadruple and the data are shown as the mean plus a standard deviation (± SD).

Live/Dead Assay of HepG2 Cells. A live/dead assay was performed for the analysis of cell viability after cellular uptake of UCNP-loaded hybrid vesicles without or with 980 nm laser irradiation. UCNP-loaded hybrid vesicles ([CPT] = 10 µg/mL) were added to HepG2 12


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cells and incubated for 8 h. The cells were rinsed with PBS and 180 µL of fresh medium was added, which was or was not irradiated with a 980 nm laser (1.5 W/cm2) for 15 min. After irradiation, the cells were incubated for another 24 h and were stained with fluorescein diacetate (FDA) and propidium iodide (PI) for 30 min to visualize the populations of live and dead cells. After rinsing with PBS buffer, the cells were imaged by confocal microscopy using 488 nm and 543 nm lasers as the excitation wavelengths and the emission detection channels were set to 500–530 nm and 560-700 nm, respectively. Characterization. Nuclear magnetic resonance (NMR) spectra were recorded on a Bruker AV300 NMR spectrometer (resonance frequency of 300 MHz for 1H and 75 MHz for 13

C) operated in the Fourier transform mode. Molecular weights and molecular weight

distributions were determined by gel permeation chromatography (GPC) equipped with Waters 1515 pump and Waters 2414 differential refractive index detector (set at 45 oC). It used a series of two linear Styragel columns (HR2 and HR4) at an oven temperature of 45 oC. The eluent was DMF at a flow rate of 1.0 mL/min. A series of low dispersity polystyrene standards were employed for calibration. UV-Vis absorbance was carried out on a TU-1910 double beam UV-Vis spectrophotometer (Puxi General Instrumental Company, China). Fluorescence experiments were performed on an F-4600 (Hitachi) fluorospectrometer. The slit widths for both excitation and emission monochromators were set to 5 nm. Dynamic laser light scattering (DLS) measurements were conducted on a Zetasizer Nano ZS (Malvern). Scattered light was collected at a fixed angle of 173o for a duration of 5 min. Hydrodynamic diameters, , were averaged over three consecutive measurements. Thermogravimetric analysis (TGA) was performed in air using a Perkin Elmer Diamond TG/DTA instrument at a 13


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heating rate of 10 oC/min. Transmission electron microscopy (TEM) and scanning electron microscopy (SEM) measurements were conducted on a JEOL 2010 high-resolution transmission electron microscope and a JEOL JSM-6700 ﬁeld emission scanning electron microscope, respectively. The samples for TEM observations were prepared by dropping 20 µL of aqueous dispersions of hybrid assemblies onto copper grids successively coated with thin films of Formvar and carbon. The samples for SEM observations were prepared according to similar procedures used for TEM. Confocal laser scanning microscopy (CLSM) images were acquired on a Leica TCS SP5 microscope. Table 1. Structural Parameters of Polyprodrug Amphiphiles Used in This Study.

Mn,NMR

Mn,GPC

(KDa) a

(KDa) b


14.2

14.7

1.19

50.1

BP2


17.2

18.3

1.21

51.6

BP3

PDMA20-b-PCPTM25

16.9

18.4

1.21

51.6

BP4


24.9

26.4

1.23

53.7

Entry

Samplesa

BP1

a

1

b

Mw/Mnb

DLC/ %c

c

Determined by H NMR spectroscopy; Calculated from GPC using DMF as the eluent; Drug loading content (DLC) determined by UV-Vis spectroscopy against a standard calibration curve.

RESULTS AND DISCUSSION Fabrication of UCNP-Loaded Hybrid Nanostructures. Amphiphilic diblock copolymers composed of polyprodrug blocks (e.g., PCPTM) were synthesized via sequential RAFT polymerizations. Specifically, the copolymerization of DMA and EoS monomers yielded EoS-labeled P(DMA-co-EoS) precursor, which was further employed as the macroRAFT

agent

to

polymerize

CPTM

monomer

with

the

generation

of

P(DMA-co-EoS)-b-PCPTM diblock copolymers (Scheme 2). The degrees of polymerization 14


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(DPs) of the PCPTM blocks were calculated from 1H NMR spectroscopy and the typical 1H NMR spectrum of the BP1 diblock copolymer is shown in Figure S2. The molar content of EoS in the P(DMA-co-EoS) was quantitatively determined to be 2% by UV-Vis spectroscopy against a standard calibration curve (Figure S3). The structural parameters of the as-synthesized block copolymers (BP1-BP4) are summarized in Table 1. Note that the drug loading contents (DLCs) of all four polyprodrug amphiphiles (BP1-BP4) were higher than 50 wt%, which was quite difficult to be achieved by conventional physical encapsulation procedures and would be beneficial for drug delivery applications.11, 58

Figure 1. (a) Intensity average hydrodynamic diameter distribution, f(Dh), and (b) TEM image of UCNP-loaded hollow hybrid BP2 nanoparticles through pulse sonication for 2 min (pulse on 10 s and pulse off 10 s). (c,d) SEM images of the UCNP-loaded hybrid BP2 nanoparticles.

15


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Previously, we found that polyprodrug amphiphiles of poly(ethylene oxide)-b-PCPTM (PEO-b-PCPTM) could self-assemble into four types of nanostructures including spherical micelles, smooth disks, staggered lamellae, and large compound vesicles (LCM) via a co-solvent protocol, exhibiting distinct intracellular trafficking pathways.43 Herein, using 1,4-dioxane as a co-solvent, the co-self-assembly of diblock copolymers (BP2 or BP4) and oleic acid (OA)-stabilized UCNPs failed to obtain uniform nanostructures (Figure S4). To resolve this problem, we assembled the as-prepared polyprodrug amphiphiles with OA-stabilized UCNPs through an oil-in-water (O/W) emulsion approach under pulse sonication. Specifically, OA-stabilized UCNPs and polyprodrug amphiphiles (BP1-BP4) were dispersed in CHCl3 and the oil phases were injected into an aqueous solution, followed by pulse sonication for varying durations and CHCl3 removal under vacuum. Interestingly, we found that the sonication time plays a crucial role in the final morphologies and sizes of the assembled hybrid nanostructures. For example, when pulse sonication was applied for a total duration of 2 min (pulse on 10 s and pulse off 10 s), hybrid hollow nanostructures were obtained with an intensity average hydrodynamic diameter, , of ~ 720 nm, as confirmed by DLS measurements, TEM, and SEM observations (Figure 1). By contrast, if the sonication time was extended to 5 min under otherwise identical conditions, hybrid vesicles with a of 136 nm were obtained (Figure 2). Note that vesicles (also referred to as polymersomes) have been widely applied for nanocarriers and nanoreactors.59-61 In addition to sonication time, the block lengths of hydrophobic PCPTM exhibited a significant influence on the morphologies of the final nanostructures as well. For instance, the use of BP1 and BP4 diblock copolymers with shorter and longer PCPTM blocks than that of BP2 failed to obtain 16


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well-defined hybrid vesicles (Figure S5). Further, free UCNPs were observed for the BP1 and BP4 hybrid assemblies (Figure S5), in sharp contrast to the hybrid BP2 vesicles without evident free UCNPs (Figure 2). The loading content of UCNPs within hybrid BP2 vesicles was calculated to be ~11.5 wt% by thermogravimetric analysis (TGA, Figure S6), close to the original feed ratio of UCNPs to BP2 copolymer (wt/wt = 1/10) and the approximate invisibility of free UCNPs in TEM observation (Figure 2). Therefore, hybrid BP2 vesicles achieved by pulse sonication for 5 min were then employed for the following studies considering the appropriate size and uniform morphology.

Figure 2. (a) Hydrodynamic diameter distribution of UCNP-loaded hybrid BP2 vesicles fabricated through pulse sonication for 5 min (pulse on 10 s and pulse off 10 s). (b,c) TEM and (d) SEM images of the UCNP-loaded hybrid BP2 vesicles.

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Energy Transfer from UCNPs to EoS Photosensitizer. The loading of UCNPs within the hybrid BP2 vesicles may render it possible to activate the EoS photosensitizer by using an NIR light source with superior tissue penetration. A prerequisite was the efficient energy transfer between UCNPs and EoS moieties. To determine whether an efficient energy transfer could take place between UCNPs and EoS moieties, the upconversion emission of UCNPs under a 980 nm laser irradiation and the absorbance of EoS residues were recorded (Figure 3a). Upon exciting the UCNPs, characteristic emission peaks with two green bands located at 524 nm and 543 nm and one red band at 655 nm were observed (Figure 3a). Notably, the green bands overlapped quite well with the absorption spectrum of EoS moieties, implying a potential FRET pair of UCNPs and EoS moieties.

a)

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100 80

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Figure 3. (a) Normalized upconversion emission spectrum of UCNPs (red line) in n-hexane under 980 nm laser irradiation and absorbance spectrum of BP2 in DMSO (black line). (b) Upconversion luminescence of the aqueous dispersions of UCNP-loaded hybrid BP2 vesicles 18


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and UCNP-loaded hybrid BP3 assemblies under 980 nm laser irradiation. (c) Evolution of UV-Vis spectra of the aqueous dispersions (0.2 g/L) of UCNP-loaded hybrid BP2 vesicles with RNO (50 mM) and imidazole (100 mM) under 980 nm laser irradiation. (d) Time dependence of 1O2 generation by BP2 vesicles with and without loading of UCNPs under 980 nm laser irradiation. The generation of 1O2 was determined by the bleaching of RNO at 440 nm. To confirm the presence of energy transfer between UCNPs and EoS moieties, the emission spectra of UCNP-loaded hybrid vesicles were recorded (Figure 3b). Notably, although the red band centered at 655 nm remained unchanged, in the absence of EoS receptors, the emission peak of UCNP-loaded BP3 vesicles at 549 nm could be clearly observed, whereas it significantly dropped for hybrid BP2 vesicles, indicating that energy transfer indeed occurred between UCNPs and EoS moieties under 980 nm laser irradiation (Figure 3b). If we arbitrarily defined the energy transfer efficiency (E%) as the following equation,62, 63

% =

− × 100%

where I0 and I are the fluorescence intensities at 549 nm of hybrid BP3 vesicles (without EoS labeling) and hybrid BP2 vesicles (with EoS labeling) under 980 nm irradiation, respectively. The energy transfer efficiency (E%) was calculated to be ~86%, indicative of a quite efficient energy transfer between UCNPs and EoS moieties, enabling activation of EoS photosensitizer and thus the generation of cytotoxic 1O2 under 980 nm irradiation.

In Vitro 1O2 Generation. To determine whether 1O2 was in situ produced under 980 nm irradiation, an established RNO/imidazole assay was applied to probe the generation of 1O2 in aqueous solution. In this protocol, 1O2 could be captured by imidazole with the formation of a transannular peroxide intermediate, which then reacted with RNO and induced the bleaching 19


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of RNO. Thus, the amount of 1O2 produced by UCNP-loaded hybrid vesicles could be determined by the decreased absorbance at 440 nm.56, 57, 64 Upon irradiation of the hybrid BP2 vesicles in the presence of RNO and imidazole with a 980 nm laser (1.5 W/cm2), the absorbance of RNO at 440 nm gradually decreased, whereas BP2 assemblies without UCNPs loading exhibited negligible absorbance changes (Figures 3c,d and S7). This result demonstrated that EoS photosensitizer cannot be directly activated by NIR irradiation and the 1

O2 generation under 980 nm laser irradiation can be solely attributed to the energy transfer

between UCNPs and EoS moieties that in turn activated the EoS residues.

Figure 4. Incubation time dependence of (a) intensity average hydrodynamic diameters, , and (b) normalized scattered light intensities of aqueous dispersions (20 mM PBS buffer, pH 7.4) of UCNP-loaded hybrid BP2 vesicles (0.2 g/L) in the presence of varying amounts of GSH. (c) TEM image of the hybrid BP2 vesicles after incubation with 10 mM GSH for 24 h. (d) In vitro release profiles of CPT from UCNP-loaded hybrid BP2 vesicles in 20


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the presence of varying GSH concentrations.

In Vitro GSH-Triggered CPT Release. Hybrid BP2 vesicles consisting of hydrophobic PCPTM polyprodrug blocks, which could be triggered to release intact anticancer drug CPT under reductive microenvironment.24,

65

Upon incubation with different concentrations of

GSH, although the exhibited negligible changes within 30 h incubation time in the absence and presence of GSH, the corresponding scattering intensities underwent steady decrease and the higher GSH concentrations led to more prominent drops in the scattering intensities (Figure 4a,b). TEM image of hybrid BP2 vesicles subjected to incubation with 10 mM GSH for 24 h suggested the formation of irregular aggregates, which could be ascribed to the GSH-mediated thiol exchange reactions rather than the complete disintegration of hydrophobic units (Figure 4a). The in vitro CPT release profiles of hybrid BP2 vesicles was then studied by co-incubation the hybrid BP2 vesicles with varying GSH concentrations, suggesting that 14% CPT was released within 60 h in the absence of GSH, presumably due to the spontaneous hydrolysis of PCPTM blocks. In the presence of 2 µM GSH, approximately 20% CPT was released; however, the released CPT contents were increased to 41% and further to 89% in the presence of 5 mM and 10 mM GSH (corresponding to the cytoplasmic GSH concentration), respectively.

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Figure 5. (a) Characterization of in situ generation of reactive oxygen species in HepG2 cells after incubation with UCNP-loaded hybrid BP2 vesicles in the presence and absence of 980 nm laser irradiation (1.5 W/cm2) for 15 min. The green channel was excited at 488 nm and collected between 500 nm and 550 nm. (b) Relative fluorescence intensities of DCF within HepG2 cells with or without 980 nm laser irradiation (1.5 W/cm2, 15 min) were quantified from CLSM observations. Error bars represent mean ± SD, n = 4.

Cellular Internalization of UCNP-Loaded Hybrid Vesicles. As mentioned previously, reduction-responsive nanocarriers primarily dump their payloads within the cytoplasm and therefore endosomal escape of the internalized nanocarriers was of crucial importance to augment the therapeutic efficacy. Given hybrid BP2 vesicles bearing EoS moieties that can produce 1O2 under NIR light irradiation, we surmised that the generated 1O2 can not only kill cancer cells due to PDT effect but also facilitate the endosomal escape of internalized hybrid vesicles via a PCI process. First, we co-incubated HepG2 cells with hybrid BP2 vesicles for varying times (1-12 h) and monitored the fluorescence changes in the blue channel ascribed to the emission of CPT moieties of hybrid BP2 vesicles (Figure S8). Upon extending the incubation time, the fluorescence intensities of the blue channel gradually increased and 22


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intense blue emission was observed after 8 h incubation, suggesting that hybrid BP2 vesicles were successfully taken up by HepG2 cells. However, the internalized hybrid BP2 vesicles were mainly colocalized with LysoTracker red, a commercially available staining reagent for acidic organelles (e.g., endolysosomes), manifesting that the internalized hybrid BP2 vesicles were retained within endolysosomes, which may lead to the disintegration of hybrid vesicles and compromise the therapeutic performance. Considering that 8 h incubation time resulted in sufficient cellular internalization of hybrid BP2 vesicles and a longer incubation time may incur unwanted side effects (e.g., vesicle disassembly), 8 h incubation time was then chosen for the following assessments. Next, we assessed the capability of 1O2 generation of internalized hybrid BP2 vesicles within HepG2 cells. Using commercially available DCF as a probe, the generation of 1O2 could be discerned by the activated green emission. Interestingly, under 980 nm laser irradiation for 15 min, intense green emission was observed within HepG2 cells, whereas no detectable green emission was observed without 980 nm laser irradiation (Figure 5). Quantitative analysis revealed that the emission intensity of green channel under 980 nm laser irradiation was 5.4-fold higher than that without irradiation (Figure 5b), clearly suggesting the formation of 1O2.

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Figure 6. (a, c) Representative CLSM images recorded for HepG2 cells after incubation with UCNP-loaded hybrid BP2 vesicles ([CPT] = 10 µg/mL) for 8 h, followed by rinsing with PBS buffer three times, (a) without or (c) with 980 nm laser irradiation (1.5 W/cm2) for 15 min, and additional incubation for 0 h, 1 h, 5 h, and 9 h, respectively. Late endosomes/lysosomes were stained with LysoTracker red (red channel). (b, d) colocalization fluorescence ratios of blue channel (CPT) and the red channel (LysoTracker red) were quantified from CLSM results.

Intracellular Trafficking of UCNP-Loaded Hybrid Vesicles. Given that 1O2 could be generated within cancer cells under 980 nm laser irradiation, we hypothesized that the in situ produced 1O2 may facilitate the endosomal escape of internalized hybrid BP2 vesicles, 24


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thereby enabling the intracellular transport to the cytoplasm and thus activating the release of CPT drug. After incubation the hybrid BP2 vesicles with HepG2 cells for 8 h, the cells were subjected to further incubation for varying times (0-9 h). The blue channel of hybrid BP2 vesicles and the red channel of LysoTracker red overlapped quite well and the colocalization ratios remained higher than 80% even after an additional 9 h incubation (Figure 6a,b), revealing that the internalized hybrid BP2 vesicles themselves were not eligible for efficient endosomal escape. In striking contrast, after exposure to 980 nm laser irradiation for 15 min, the colocalization ratio between the blue channel and red channel dramatically dropped to less than 33%, indicating a successful endosomal escape from endolysosomes (Figure 6c,d). Therefore, the generated 1O2 under 980 nm laser irradiation could successfully promote the endosomal escape of hybrid BP2 vesicles, probably via a PCI mechanism. Note that this result would be favorable for the following GSH-triggered release of CPT drug within the cytosol.

In Vitro Cytotoxicity of UCNP-Loaded Hybrid Vesicles. The inhibiting proliferation against cancer cells of UCNP-loaded hybrid BP2 vesicles was first analyzed by a Live/Dead assay and the untreated HepG2 cells were employed as a control (Figure 7a). In the absence of UCNP-loaded hybrid BP2 vesicles, exposure the cancer cells to 980 nm laser irradiation for 15 min did not induce significant cell death, suggesting that a short period of NIR laser irradiation was cytocompatible. However, previous studies have demonstrated that even a low dosage of UV light may be detrimental to cell growth.66,

67

Therefore, it was quite

advantageous to utilize an NIR laser irradiation to activate the EoS photosensitizer, exhibiting better tissue penetration and lower cytotoxicity. Incubation HepG2 cells with UCNP-loaded 25


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hybrid BP2 vesicles led to appreciable cell death, whereas a large population of cancer cells were still survived, which could be a result of poor endosomal escape and the internalized hybrid BP2 vesicles cannot efficiently release therapeutic CPT drugs within endolysosomes (Figure 7c). By contrast, after irradiating the HepG2 cells with a 980 nm laser for 15 min, the pronouncedly enhanced cytotoxicity was evidenced by the overwhelming red emission of propidium iodide that can only penetrate the cell membranes of dead cells (Figure 7d). We tentatively ascribed this remarkable result to the PCI-facilitated endosomal escape of internalized hybrid BP2 vesicles that in turn reinforce the anticancer performance.

Figure 7. Apoptosis of HepG2 cells induced by UCNP-loaded hybrid BP2 vesicles was detected by the Live/Dead assay. Representative CLSM images of HepG2 cells after incubation for 24 h in the (a,b) absence and (c,d) presence of UCNP-loaded hybrid BP2 vesicles (a,c) without and (b,d) with 980 nm laser irradiation for 15 min (1.5 W/cm2). Cells were first incubated with hybrid BP2 vesicles for 8 h, followed by washing with PBS buffer three times, irradiation with a 980 nm laser (1.5 W/cm2) for 15 min, and incubation for additional 24 h. (e) In vitro cytotoxicity against HepG2 cells of UCNP-loaded hybrid BP2 vesicles under different conditions.

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Next，the cytotoxicity of UCNP-loaded hybrid BP2 vesicles was quantitatively assayed by an MTT method (Figure 7e). Although a decreased cell viability was observed for HepG2 cells after incubation with the hybrid BP2 vesicles without 980 nm laser irradiation, nearly 50% cells were still alive in the presence of an identical CPT concentration of 10 µg/mL. Moreover, hybrid BP3 vesicles without Eos photosensitizer exhibited similar cytotoxicity without 980 nm light irradiation (Figure S9). This result was primarily ascribed to the chemotherapeutic effect of the hybrid vesicles comprising PCPTM prodrug blocks, which were taken up into the cells and intracellular GSH-triggered release of CPT was then actuated, thereby killing cancer cells in an on-demand manner. Interestingly, after incubation with hybrid BP2 vesicles, the cytotoxicity was remarkably decreased to ~26% after 980 nm laser irradiation for 15 min and a further decrease to 5% was observed if the irradiation period was extended to 30 min at an identical CPT concentration. Significantly, these results concurred quite well with the Live/Dead assay (Figure 7a-d) and the increased cytotoxicity likely stemmed from the synergistic contributions of the PDT effect of generated 1O2 as well as the PCI-promoted endosomal escape of hybrid BP2 vesicles that elevated the chemotherapeutic performance. CONCLUSIONS In summary, hybrid vesicles comprising hydrophilic PDMA coronas covalently labeling with EoS photosensitizer and hydrophobic PCPTM polyprodrug bilayers have been successfully fabricated via the co-self-assembly of amphiphilic polyprodrug copolymers and UCNPs. Upon illumination with an NIR laser, an energy transfer from excited UCNPs to EoS moieties then occurred by taking advantage of their close proximity, resulting in the 27


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generation of cytotoxic 1O2. Remarkably, the generated 1O2 can simultaneously exert PDT and PCI effects, killing cancer cells and facilitating the endosomal escape of internalized hybrid vesicles into the cytoplasm. The abundant GSH within the cytosol then triggered the release of CPT drug via the cleavage of disulfide bonds, further eliminating cancer cells through a chemotherapy process. The combination of PDT and chemotherapy represents an applicable method to improve therapeutic efficacy.

ASSOCIATED CONTENT

Supporting Information. Additional TEM images,

1

H NMR spectroscopy, UV-Vis

absorbance, TGA measurement, confocal laser scanning microscopy images, and cytotoxicity data. This materials is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Authors E-mail: [email protected] (J.H.) E-mail: [email protected] (S.L.)

ACKNOWLEDGMENTS The financial support from National Natural Science Foundation of China (NNSFC) Project (51690150, 51690154, 21674103, and 51673179), International S&T Cooperation Program of China (ISTCP) of MOST (2016YFE0129700), and the Fundamental Research Funds for the Central Universities (WK2060200023) is gratefully acknowledged.

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For Table of Contents Use Only

Near-Infrared Light-Activated Photochemical Internalization of Reduction-Responsive Polyprodrug Vesicles for Synergistic Photodynamic and Chemotherapy

Kangning Zhu, Guhuan Liu, Jinming Hu*, and Shiyong Liu*

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Near-Infrared Light-Activated Photochemical Internalization of

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