Near-infrared photothermal liposomal nanoantagonists for amplified cancer photodynamic therapy
Haitao Sun,a,1 Meixia Feng,b,1 Siyu Chen,c,1 Ruizhi Wang,a Yu Luo,d Bo Yin,e,* Jingchao Li,f,* Xiaolin Wanga,*
Introduction
Photodynamic therapy (PDT) has demonstrated to be a promising strategy for cancer treatment, while its therapeutic efficacy is often compromised due to excessive concentrations of glutathione (GSH) as a reactive oxygen species (ROS) scavenger in cancer cells. Herein, we report the development of near-infrared (NIR) photothermal liposomal nanoantagonists (PLNA) for amplified PDT through reducing intracellular GSH biosynthesis. Such PLNA were constructed via encapsulating a photosensitizer, indocyanine green (ICG) and a GSH synthesis antagonist, L-buthionine sulfoximine (BSO) into a thermal responsive liposome. Under NIR laser irradiation at 808 nm, PLNA generates mild heat via ICG-mediated photothermal conversion effect, which leads to the destruction of thermal responsive liposomes for a controlled release of BSO in tumor microenvironment, ultimately reducing GSH levels. This amplifies intracellular oxidative stresses and thus synergizes with PDT to afford an enhanced therapeutic efficacy. Both in vitro and in vivo data verify that PLNA-mediated phototherapy has an at least 2-fold higher efficacy in killing cancer cells and inhibiting tumor growth compared to sole PDT. This study thus demonstrates a NIR photothermal drug delivery nanosystem for amplified photomedicine. a high concentration of 10 mM in tumor microenvironment, greatly compromises antitumor effect of PDT.7 To overcome this limitation, Photodynamic therapy (PDT) that utilizes photosensitizers to produce cytotoxic reactive oxygen species (ROS) under photoirradiation to induce cell death and tissue damage has emerged as a promising strategy for cancer therapy.1, 2 Via integrating ROS-responsive component, PDT can also be used for photoactivation of prodrugs, allowing synergistic antitumor treatment.3 Nevertheless, the curative effect of PDT is often restricted because of the existence of antioxidant systems in tumor microenvironment.4, 5 Cancer cells highly express different types of antioxidant enzymes (e.g. glutathione peroxidase, superoxide dismutase, and catalase) and ROS scavenger (e.g. glutathione (GSH)), which can not only sustain the intracellular balance of redox state, but also inadvertently weaken the effect of external ROS.6 As the dominating antioxidant to defend against oxidative stress, GSH with some strategies that can reduce the antioxidant levels have been adopted for improved PDT efficacy.8-10 For example, manganese dioxide nanosheets that can reduce intracellular GSH levels have been used to improve the PDT efficacy of photosensitizers.11 In addition, β-phenylethyl isothiocyanate, a natural GSH scavenger has been combined with photosensitizer-loaded nanoparticles for amplified cancer PDT.12 However, systemic administration of GSH scavengers will disturb intracellular balance of redox states in normal cells, thus leading to side effects.
Nanomaterials-based drug delivery systems have shown a great promise for cancer therapy in preclinical animal models.13-15 In particular, stimuli-responsive nanocarriers that can achieve on- demand release of drugs are receiving tremendous attention because they not only improve the therapeutic efficacy, but also reduce drug-induced toxicity.16-18 A larger number of nanocarriers that are responsive to endogenous biomarkers, such as ROS,19 acid enzymes,26 and hypoxia existed in tumor microenvironment have been extensively developed.27-29 However, because these tumor-associated biomarkers also exist in most healthy tissues, the endogenous stimuli-responsive nanocarriers still have the issue of off-target release. In contrast, external stimuli, such as light,30, 31 magnetic fields,32, 33 and ultrasound waves hold fine control performance for both pharmacokinetics and releases of drugs.34, 35 Among them, light has the advantages of noninvasiveness, easy accessibility, excellent controllability, and good flexibility in both spatial and temporal manners, and thus have been widely used for controlled drug release.17, 18 In this regard, near-infrared (NIR) light is more adequate because of its lower tissue absorption, less photon scattering, deeper tissue penetration and less phototoxicity compared to ultraviolet (UV) and visible light.36-38 For example, photothermal conversion effect of gold nanoparticles mediated by NIR light can increase the local temperature, which triggers dehybridization of DNA helices on the nanoparticle surface, allowing the release of anticancer drug bound to consecutive cytosine- guanine base pairs.39 Alternatively, NIR light can be used to trigger the switch of azobenzene molecules between trans and cis isomer through NIR-to-UV-visible transduction, achieving photo-controlled release of doxorubicin from mesoporous silica nanoparticles.40 However, the use of photo-responsive nanocarriers for on-demand release of GSH scavengers/inhibitors for enhanced PDT has not been reported.
In this study, we report the synthesis of NIR photothermal liposomal nanoantagonists (PLNA) with on-demand release of antagonists for enhanced cancer PDT. Such PLNA comprise indocyanine green (ICG) and L-buthionine sulfoximine (BSO) encapsulated in a thermosensitive liposome. ICG, as a photosensitizer approved by the US Food and Drug Administration (FDA) for clinical imaging and diagnosis, have been widely used for both photothermal therapy (PTT) and PDT.41, 42 BSO is a well-known GSH biosynthesis antagonist that inhibits the γ-glutamylcysteine synthetase (γ-GCS), a major enzyme for GSH synthesis, and thus effectively reduces intracellular GSH levels and disrupts the redox homeostasis.43, 44 Thermosensitive liposomes consisted of 1,2- distearoyl-sn-glycero-3-phosphoethanolamine-N- [methoxy(polyethylene glycol)-2000] (DSPE-PEG2000) and 1,2- dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) are used as biocompatible nanocarriers for on-demand cargo release because of the transition temperature of DPPC at 41-42 °C.45, 46
The synthesis of PLNA and their working mechanism for enhanced PDT are showed in Figure 1. Under laser irradiation, ICG within PLNA mediates photothermal conversion effect to increase the local temperature to induce the phase transition of thermosensitive liposomes from gel phase to fluid phase, eventually leading to on- demand release of BSO. In addition, ICG exerts PDT to generate ROS under NIR laser irradiation. The released BSO decreases the GSH levels in tumor microenvironment, which can amplify the generation of ROS for ICG-mediated PDT, leading to enhanced therapeutic efficacy. In view of the NIR fluorescence property of ICG, PLNA also enable convenient in vivo tracking and imaging of injected nanocarriers. This study thus provides a feasible and innovative strategy to specifically enhance the PDT efficiency via precisely amplifying oxidative stresses in tumor microenvironment.
Results and discussion
Synthesis and characterization of PLNA
To achieve photo-controlled release of BSO for enhanced PDT, PLNA was synthesized via a hydration-sonication method.46 First, DPPC (MW = 734.0) and DSPE-PEG2000 (MW = 2805.5) were co-dissolved in chloroform at a weight ratio of 25:5, and the mixture was then evaporated using a rotary evaporator to form a thin film. Next, the obtained film was hydrated with phosphate buffer solution (PBS) solution containing 5 mg BSO and 5 mg ICG at 65 °C for 2 h. The obtained mixture solution was sonicated and extruded through a polycarbonate membrane, followed by ultrafiltration three times to remove free ICG and BSO. ICG loaded nanocarriersVieww iAtrhtioclue tOntlhinee addition of BSO (termed as LI) were also preDpOaIr:e1d0.1a0c3c9o/rDd0inTgB0t1o43th7Ke similar steps and used as the control counterpart.
The synthesized PLNA and LI was characterized using different technologies. The transmission electron microscopy (TEM) was performed to investigate morphological features of PLNA and LI. As showed in the TEM images, both nanoparticles exhibited uniform size distribution with spherical shapes (Figure 2a and Figure S1). The hydrodynamic size of PLNA was measured to be 26.3 ± 0.6 nm by dynamic light scattering (DLS), which was slightly larger than that of LI (22.3 ± 0.8 nm) (Figure 2b). The polydispersity indexes (PDI) of PLNA and LI were calculated to be 0.22 and 0.25, respectively, suggesting the good monodispersity for both nanoparticles. Both PLNA and LI were found to have an excellent colloidal stability because their hydrodynamic sizes remained negligibly changed after being dispersed in PBS for 15 days (Figure S2). Zeta potentials of these nanoparticles were measured to be similar (-16.5 mV for LI and -17.0 mV for PLNA) (Figure 2c).
As shown in the absorption spectra of LI and PLNA, the characteristic absorption peaks of both nanoparticles were almost identical to ICG, indicating the loading of BSO displayed a negligent influence on the optical property of ICG (Figure 2d). The ability of generating singlet oxygen (1O2) for nanoparticles exposed upon the NIR laser (808 nm) was evaluated by spectrofluorometer using an 1O2 indicator, singlet oxygen sensor green (SOSG).47 As depicted in Figure 2e, the fluorescence intensity of SOSG for PLNA, LI and free ICG contained solution was elevated by 3.6-, 3.3-, and 2.6-fold after 10 min of NIR laser irradiation, respectively, which indicated their effective generation of 1O2. Considering that an increase in the temperature of PLNA under NIR laser irradiation can induce the phase transition of thermosensitive liposomes, the NIR photo- controlled release of BSO was verified using high-performance liquid chromatography (HPLC). The PBS solution containing PLNA was exposed with 808 nm laser at a power density of 0.6 W cm-2 for 10 min and the released BSO was then analyzed. An elution peak of BSO at 8.2 min was observed in PLNA solution after laser irradiation (Figure 2f), suggesting the photo-controlled release of BSO. In contrast, no obvious elution peak was observed in PLAN solution without laser irradiation.
In vitro enhanced PDT efficacy and intracellular inhibition of GSH synthesis
The cellular uptake of PLNA and LI by murine 4T1 cancer cells was first investigated using flow cytometry assay. After incubation after 24 h, the cell uptake assay showed the fluorescence intensity of PLNA- and LI-cultured cancer cells was almost identical, about 97.5 and 93.9-fold higher compared to that of the cells without nanoparticle treatment, respectively, (Figure 3a and Figure S3), suggesting the excellent internalization for both nanoparticles. The therapeutic efficacy of PLNA was then studied and compared with that of LI. Note that PLNA alone caused nontoxicity to 4T1 cells even at the ICG content up to 20 μg mL-1 for 24 and 48 h of incubation (Figure 3b), which suggested PLNA did not afford therapeutic effect without laser irradiation. To identify the therapeutic effect, 4T1 cancer cells treated with nanoparticles were irradiated twice by 808 nm NIR laser with a power density of 0.6 W cm-2. The first irradiation was used to increase local temperature and thus allowed on-demand release of BSO, and the second one was performed to exert PDT effect. During the laser irradiation, the temperature was controlled to below 43 oC, which not only triggered the release of BSO, but also minimized the damage caused by PTT effect. The cell viability in 4T1 cancer cells after treatments with PLNA and LI with laser illumination was found to show a significant reduction, indicating PDT effect (Figure 3c). The cell viability in PLNA + Laser group was 28.8%, which was 2.2-fold lower than that in LI + Laser group (62.7%). Apoptosis rate of cells was further verified using flow cytometry, showing the apoptosis rate of cells treated with PLNA + Laser was the highest, in accordance with the results of cell viability assay (Figure S4). These results demonstrated the superior therapeutic effect of PLNA over LI. The underlying mechanism of enhanced PDT for PLNA was proposed (Figure 3d). The released BSO inhibited γ-GCS-mediated GSH biosynthesis and thus decreased the intracellular GSH levels, ultimately increasing the generation of ROS during PDT. To verify the mechanism, intracellular GSH and ROS levels after different treatments were evaluated. The intracellular GSH levels were assessed using reduced glutathione assay kit. The results showed that the intracellular GSH levels in LI and PLNA group after NIR laser irradiation group decreased by 86.9% and 60.5% compared to the control group, respectively (Figure 3e). Such a much lower GSH level in PLNA + Laser group relative to that in LI + Laser group should be due to the inhibitory action of released BSO. In contrast, negligible changes in GSH levels was found in 4T1 cancer cells after treatments with LI and PLNA without laser irradiation. Then, the intracellular oxidative stress levels were studied using flow cytometry and confocal laser scanning microscopy with 2’,7’- dichlorodihydrofluorescein diacetate (DCFH-DA) as a ROS fluorescent indicator. The intracellular ROS level of 4T1 cells treated with PLNA plus laser irradiation was the highest, which was at least 1.5-fold higher relative to other treatment groups (Figure 3f and Figure S5). As shown in the fluorescence images, 4T1 cell treated with PLNA plus laser irradiation showed a much stronger intracellular green fluorescence signal of ROS than those treated with LI plus laser irradiation (Figure 3g). These results verified that PLNA with laser irradiation obviously reduced intracellular GSH levels and further increased ROS levels for an enhanced PDT effect.
In vivo tumor accumulation and biodistribution
To confirm the optimized time points for cancer therapy, in vivo NIR fluorescence imaging was performed to detect the accumulation of nanomaterials in tumor tissues of 4T1 tumor-bearing living mice. Mice were intravenously injected with PBS solution of LI or PLNA, and NIR fluorescence images were acquired using IVIS system at different post-injection time points (0, 12, 24, 48 h). As depicted in Figure 4a, the fluorescence signals in tumor sites of living mice after injection of both nanoparticles gradually raised and reached the peaks at 24 h post-injection time-point. Similarly, the fluorescence intensity of tumor sites for LI- and PLNA-injected mice (24 h) was 7.2- and 8.2- fold higher relative to that before injection (0 h), respectively (Figure 4b). These results indicated the excellent enhanced permeability and retention (EPR) effect of the constructed nanomaterial for tumor accumulation.
The biodistribution of nanoparticles in 4T1 tumorV-ibeweaArritnicgle Omnilicnee was also evaluated. Ex vivo NIR fluorescencDeOimI: 1a0g.i1n0g39s/hDo0wTBe0d14t3h7aKt obvious fluorescence signals could be observed in the tumor, liver and kidney of mice at 48 h post-injection of LI and PLNA (Figure 4c). Quantification of fluorescence intensity indicated that both LI and PLNA after intravenous injection had the maximum accumulation in hepatic tissue, followed by kidney and tumor (Figure 4d).
In vivo enhanced PDT
The in vivo therapeutic efficacy of PLNA and LI was evaluated in 4T1 tumor-bearing mouse models. The mice were randomly divided into five groups: (i) saline-injected group (control), (ii) LI group, (iii) PLNA group, (iv) LI + Laser group, and (v) PLNA + Laser group. The mice in each group were intravenously injected with saline, PBS solution of LI or PLNA at day 0. The achieve photothermal controlled BSO release, the tumors of living mice were exposed under 808 nm laser illumination at 18 h post-injection (Figure 5a). During NIR laser irradiation, the temperatures of tumor sites gradually increased to allow on-demand release of BSO, while the highest temperatures were controlled below 43 °C to minimize the photothermal ablation (Figure S6). At 24 h post-injection of LI and PLNA, the tumors were exposed with 808 nm laser again for 10 min for PDT. The nanoparticle injection and NIR laser irradiation were repeated on day 3.
To confirm in vivo pharmacological actions of released BSO, the oxidative stress and GSH levels in tumor tissues after different treatments were evaluated. Generations of 1O2 in tumor sites were tested by detecting the fluorescence signals of SOSG. It was observed that the tumor slices in PLNA+ Laser group displayed a much stronger green fluorescence signal than those in LI + Laser groups, while negligible green fluorescence signals were observed in the tumor slices for control, LI and PLNA groups (Figure 5b). This indicated the obviously higher 1O2 generation after PLNA-mediated PDT relative to LI-mediated PDT. The levels of GSH in tumor tissues after different treatments were then investigated using reduced glutathione assay kit. Without 808 nm laser irradiation, the GSH levels in LI and PLNA group were similar to that in control group, suggesting these nanoparticles did not have obvious influence on GSH levels (Figure 5c). With 808 nm laser irradiation, the GSH levels in LI + Laser and PLNA + Laser group reduced by 2.2- and 6.6-fold compared to control group, respectively. The decrease of GSH levels in LI + Laser group should be attributed to the GSH scavenging by ROS generated from PDT effect. Note that the GSH level in PLNA + Laser group was 2.9- fold lower than that in LI + Laser group, suggesting the action of PLNA in inhibiting GSH synthesis after laser irradiation.
Encouraged by the effective amplified oxidative stress caused by PLNA-mediated GSH inhibition, the in vivo therapeutic efficacy of nanoparticles was then evaluated by monitoring tumor growth and measuring tumor weights. Sole LI and PLNA injection without laser irradiation showed negligible influence on the tumor growth compared to control group (Figure 5d), while the tumor growth in these groups with laser irradiation was significantly inhibited. More importantly, the tumor growth in PLNA + Laser group was much slower than that in LI + Laser group. After treatments for 16 days, the tumor weights in PLNA + Laser group was 2.0-, 3.8-, 4.1-, and 4.3-fold lower than those in LI + Laser, PLNA, LI, and control groups, respectively (Figure 5e). Hematoxylin and eosin (H&E) staining and immunohistochemical TdT-mediated dUTP-biotin nick end labeling (TUNEL) staining further indicated much severer necrosis and/or apoptosis in PLNA + Laser group relative to that in LI + Laser group, while no obvious damage was observed in the other groups (Figure 5f). These results verified the superior therapeutic efficacy of PLNA over LI.
In vivo biosafety evaluation of PLNA-mediated cancer therapy
To verify the in vivo biosafety of PLNA-mediated antitumor treatment, blood biochemistry test, histological analysis, and body weight monitoring were respectively conducted. The serum levels of important liver and kidney function indicators including alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), urea and creatinine (Crea) in PLNA + Laser group were almost the same to those in control group (Figure 6a). The levels of vital blood biochemistry parameters including red blood cells (RBC), white blood cells (WBC), hematocrit (HCT), hemoglobin (HGB), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), hemoglobin concentration (MCHC), platelet (PLT), red cell distribution width (RDW-SD), and red cell volume distribution width (RDW-CV) in mice after PLNA-mediated cancer therapy were almost the identical to those in the control healthy mice. The major organs containing heart, liver, spleen, lung and kidney from mice after treatment were extracted for histological analysis. H&E staining images showed that the histological morphologies of these organs in the PLNA + Laser group were normal, similar to those in saline control group (Figure 6b). In addition, the body weights of mice in PLNA + Laser group did not have obvious changes over 16 days compared to control group (Figure S7). These results confirmed the good biosafety of PLNA- mediated cancer therapy.
Conclusions
In this study, we have developed a photothermal liposomal nanoantagonist (PLNA) that can achieve NIR photo-controlled release of antagonist (BSO) for enhanced cancer PDT. Due to the encapsulation of photosensitizer (ICG) in nanocarriers, PLNA showed good photodynamic property. Such PLNA with a small size (~26.4 nm) could effectively accumulated into tumors of living mice. Under NIR laser irradiation, PLNA initiated a temperature elevation for on- demand release of BSO through photothermal conversion effect to selectively reduce GSH levels in the tumor microenvironment, which synergistically amplified the production of cytotoxic 1O2 during PDT process. As such, PLNA-mediated PDT afforded a much higher therapeutic efficacy in inhibiting growth of tumors relative to traditional PDT, without inducing obvious side effects to living mice. Therefore, this study provided a new strategy to precisely enhance the therapeutic effect of PDT via regulating the intratumoral oxidative stresses. In view of their feasible controllability and good biocompatibility, PLNA may be a promising nanomedicine for precise cancer therapy in the future.
Experimental Section
Materials
DPPC, DSPE-PEG2000 and ICG were purchased frVoiemw ArStihclaenOgnhlinaei Aicheng biological technology Co., LTD (ShangDhOaIi:, 1C0h.1in03a9)./DCh0TloBr0o1f4o3r7mK and BSO were purchased from Shanghai Lingfeng chemical reagent Co. LTD (Shanghai, China) and hanghai Ruicheng Bio-Tech Co., LTD (Shanghai, China), respectively. Fetal bovine serum (FBS), Dulbecco’s Modified Eagle’s Medium (DMEM), and penicillin were brought from Gibco (New York, USA). DCFH-DA was obtained from MedChemExpress (Princeton, USA) and cell counting kit-8 (CCK-8) was brought from 7Sea Pharmatech, Co., LTD (Shanghai, China). Reduced glutathione (GSH) assay kit was purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China) and Annexin V/propidium iodide (PI) apoptosis assay kit was purchased from MultiSciences (LIANKE) Biotech, Co., LTD (Hangzhou, China).
Synthesis of LI and PLNA
In order to develop liposomes containing ICG (LI) and ICG/BSO (PLNA), respectively, DPPC and DSPE-PEG2000 with a mass ratio of 25:5 were dissolved in 10 mL chloroform and then evaporated via a rotary evaporator to form a thin film. Then, ICG (5mg) and ICG/BSO (5mg/5mg) dissolved in 10 mL of ultrapure water at 65 °C was added into the formed thin film, followed by magnetic stir at 65 °C for 2 h. The obtained aqueous solutions were sonicated for 60 min and extruded through a polycarbonate membrane (200 nm). The LI and PLNA were obtained after ultrafiltration by a centrifugal filter device (MWCO = 3500 Da) three times to remove free ICG and BSO. Finally, the purified LI and PLNA were stored at 4 °C for further use.
Characterization
TEM was conducted on a JEMe2010 electron microscope with an operating voltage of 200 kV. The UV-vis-NIR absorption spectra were recorded using a UV-3600 Shimadzu spectrometer. Dynamic light scattering (DLS) and zeta potential measurement were conducted on a Zetasizer Nanoseries (Nano ZS90, Malvern). The temperature monitoring and thermal imaging were conducted by a thermo- camera (FLIR A325SC camera). HPLC was used to evaluate the BSO release. Fluorescence images were recorded using confocal laser scanning microscopy images (Olympus Company, Japan). In vivo NIR fluorescence imaging was performed using an IVIS imaging system (IVIS-CT machine, PerkinElmer).
In vitro 1O2 generation
To measure the generation of 1O2 for LI and PLNA under laser irradiation, 1 µL SOSG agent (500 µM) was added into 1 mL PBS solution of free ICG, LI and PLNA (ICG = 20 μg mL−1). The mixture was then irradiated by an 808nm laser (0.6 W cm−2) for different time. The spectrofluorometer were used to measure the fluorescence intensity (F) of each sample at 520 nm. The initial fluorescence intensity (F0) of each sample at 520 nm before laser irradiation were also calculated. Generation of 1O2 was represented as the relative fluorescence intensity (F/F0).
Cell culture
4T1 cancer cells were cultured in RPMI 1640 cell culture medium containing 10% FBS, 100 U mL−1 penicillin and 100 μg mL−1 streptomycin at 37 °C and 5% CO2.
Intracellular ROS Detection
4T1 cancer cells seeded in confocal cell culture dishes were treated with LI or PLNA (ICG = 20 µg mL−1) for 24 h. DCFH-DA probe (0.01 mM) was then added into the treated cell culture medium. The 4T1 cells were cultured with the probe for 30 min. After that, the cell culture medium was washed with PBS for three times and the cells were subsequently irradiated with an 808 nm laser at a power density of 0.6 W cm−2 for 10 min, followed by culture the cells for 6h. The second irradiation (808 nm, 0.6 W cm−2) was then performed for 10 min. During the entire course of the irradiation, the laser irradiation was controlled to maintain the temperature below 43 °C to allow BSO release and minimize the hyperthermia effect. Immediately after second laser irradiation, the cells were washed with PBS for three times. Then fluorescence images of cells were captured using CLSM and their fluorescence intensity was measured using flow cytometry assay.
Cellular uptake assay
The cellular uptake of LI and PLNA were quantified by flow cytometry assay. 4T1 cells were seeded in 12-wells culture plates and cultured for 24 h and then the cells were incubated with PBS, LI and PLNA (ICG = 20 µg mL−1) for another 24 h. After that, the cells were subsequently digested, collected, and analyzed using flow cytometry.
Detection of intracellular GSH levels
The intracellular GSH levels were measured using the reduced glutathione (GSH) assay kit. In Brief, 4T1 cells were incubated with PBS, LI and PLNA (ICG = 20 μg mL-1) for 24 h. Then the cells were exposed under 808 nm laser irradiation at a power density of 0.6 W cm-2 for 10 min. During laser irradiation, the temperature of cells was controlled below 43 °C to minimize the hyperthermia effect. The cells were cultured for another 6 h. After that, the cells were lysed by repeated cycle of freezing and thawing for three times and subsequently centrifuged to collect the supernatants for measuring the intracellular GSH according to the standard protocol.
In vitro cytotoxicity assay
4T1 cancer cells were seeded into 96-well cell culture plates and cultured for 24 h. The cells were then incubated with PLNA (ICG = 20 μg mL-1) for 24 and 48 h, respectively. CCK-8 assay was used to evaluate the cell viability of cells after treatments.
In vitro therapeutic efficacy
4T1 cancer cells seeded in 96-well cell culture plates were treated with PBS, LI or PLNA (ICG = 20 µg mL−1) for 24 h. Then the treated cells were irradiated with an 808 nm laser at a power density of 0.6 W cm−2 for 10 min, followed by culture the cells for 6 h. Then the cells were exposed under 808 nm laser irradiation (0.6 W cm−2) again for 10 min. During the laser irradiation, the temperature was controlled below 43 °C to minimize the hyperthermia effect. TVhieew cAertilclsle Ownelirnee cultured for another 12 h and then the cell vDiaObIi:li1t0y.1w0a39s/aDs0sTeBs0se14d3b7Ky CCK-8 assay. For apoptosis assay, 4T1 cells seeded in 24-well plates were treated as above described, and were subsequently digested, collected, and stained with calcein-AM/PI. The apoptosis percentages of the cells were then analyzed using flow cytometry.
Tumor model
All animal experiments were approved with the Administrative Committee of Laboratory Animals of Institute Pasteur of Shanghai. Female Balb/c mice (4 weeks old) were purchased from Shanghai SLAC Laboratory Animal Co., Ltd (Shanghai, China). To establish the tumor models, 4T1 cancer cells (106 cells) in 100 μL PBS were subcutaneously injected into the left flank of the mice. The 4T1 tumor-bearing mice were used for further experiments when the tumor volume grew to about 100 mm3.
In vivo NIR fluorescence imaging of tumor
4T1 tumor-bearing mice were randomly divided into two groups (n = 3) and the mice in each group were intravenously injected with 200 μL PBS solution of LI and PLNA (ICG = 2 mg kg-1), respectively. Then the mice were anesthetized using 2% isoflurane in oxygen, and NIR fluorescence images were acquired using an IVIS system (excitation: 710 nm; emission: 790 nm) before (0 h) and at various post-injection time points (12 h, 24 h, 48 h). The corresponding fluorescence intensities of tumor sites were quantitatively evaluated by Living Image software.
Ex vivo biodistribution
After 48 h post-injection of LI or PLNA, 4T1 tumor-bearing mice were euthanized. Tumor and major organs (heart, liver, spleen, lung, and kidney) of mice were extracted for ex vivo fluorescence imaging using the IVIS imaging system (excitation: 710 nm; emission: 790 nm). The fluorescence intensities of the tumors and major organs were quantitatively measured using the Living Image software.
In vivo 1O2 generation evaluation
4T1 tumor-bearing mice were randomly divided into five groups (n = 3) and were intravenously injected with 200 μL saline, PBS solution of LI and PLNA (ICG = 2 mg kg-1), respectively. At 18 h post-injection, the tumors of living mice were exposed under 808 nm laser irradiation (0.6 W cm-2) for 10 min. At 24 h post-injection, 30 µL SOSG solution (50 µM) was intraperitoneal injected into mice in each group. At 24 h post-injection, the tumors were irradiated by 808 nm laser (0.6 W cm-2) again for 10 min. Laser irradiation was always controlled to maintain the tumor temperature below 43 °C. After laser irradiation, the mice were euthanized to extract tumors. The collected tumors were fixed in 4% paraformaldehyde and then cryosectioned into slides at a thickness of 10 µm. The tumor slides in different groups were stained with DAPI and then observed using CLSM.
In vivo measurement of GSH levels
4T1 tumor-bearing mice were randomly divided into five groups (n = 3) and them in each group were intravenously injected with 200 μL saline, PBS solution of LI and PLNA (ICG = 2 mg kg-1), respectively. At 18 and 24 h post-injection, the tumors of living mice were exposed under 808 nm laser irradiation (0.6 W cm-2) for 10 min, respectively. The mice were then euthanized and tumors were subsequently extracted and frozen with liquid nitrogen. The GSH levels in tumor tissues were measured using reduced glutathione (GSH) assay kit according to the manufacture protocol.
In vivo therapeutic efficacy evaluation
4T1 tumor-bearing mice were randomly divided into five groups (n = 7). The mice in each group were intravenously injected with 200 μL saline, PBS solution of LI or PLNA (ICG = 2 mg kg-1) at day 0. The tumors of living mice were exposed under 808 nm laser irradiation (0.6 W cm-2) for 10 min at 18 h post-injection. At 24 h post-injection of LI and PLNA, the tumors were irradiated by 808 nm laser (0.6 W cm-2) again for 10 min. The injection of nanoparticles and laser irradiation were repeated on day 3 as above described. During NIR laser irradiation, the temperatures of tumor sites were controlled below 43 °C. After treatments, the tumor sizes and body weights of mice were measured every 2 days. The tumor volume was calculated as “volume = length × width2/2”. Relative tumor volume was shown as V/V0 (V0 was the initial tumor volume). In addition, the tumors of mice in each group were collected for for H&E and TUNEL staining.
In vivo biosafety evaluation
Biosafety of PLNA-mediated PDT was evaluated using blood routine testing and H&E staining. The mice in the control and PLNA-mediated treatment group (n = 5) were sacrificed after 30 days of treatment. Blood samples and major organs (heart, liver, spleen, lung, kidney) of mice were extracted for blood routine testing and H&E staining.
Statistical analysis
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