Squaraine nanoparticles for optoacoustic imaging-guided synergistic cancer phototherapy

: The unique optical properties of squaraine dyes make them promising for cancer phototheranostics, but the reported squaraines for in vivo treatments mainly rely on their photothermal effect, where monotherapy cannot achieve the desired therapeutic effect. Here we generated a type of squaraine capable of killing tumors through both photothermal and photodynamiceffects.We optimized squaraine structure with selenium modulation and formulated it into nanoparticles that showed strong absorption of infrared light, negligible ﬂuorescence, good photothermal conversion (66.6 %), and strong photodynamic effects even after several irradiation cycles. In addition, the nanoparticles could be tracked through their strong optoacoustic signal. In mice, the nanoparticles effectively accumulated in tumors and eliminated them upon irradiation, without causing adverse effects. Our work demonstrates the potential of selenium modulation of squaraine for precise cancer diagnosis and treatment through synergistic photothermal and photodynamic effects.


Introduction
Anti-cancer therapies based on irradiation with nearinfrared (NIR) light are attractive because they are noninvasive and can be tightly controlled in space and time, minimizing off-target adverse effects [1][2][3]. These therapies involve compounds that, after absorption of NIR light, cause local cytotoxic heating (photothermal therapy, PTT) or produce cytotoxic singlet oxygen (photodynamic therapy, PDT) [4][5][6]. In parallel, these compounds undergo thermoelastic expansion, which can be detected optoacoustically to image the diseased tissues for diagnostics and treatment guidance [7][8][9][10][11][12]. Among the most promising compounds for cancer phototheranostics are organic chromophores, which have the merits of easy chemical structure tuning, good biocompatibility, and minimal toxicity [13]. For example, the chromophores indocyanine green (ICG) and methylene blue have been licensed for clinical use, but their low photothermal conversion efficiency and poor photostability hampers their theranostic effectiveness.
A more effective chromophore may be squaraine, which features a resonance-stabilized zwitterionic planar structure with an electron-deficient, four-membered ring at its core [14]. Rigidly planar squaraines strongly absorb in the NIR region, leading to high molar absorption coefficients and bright fluorescence useful for biomedical applications [15,16]. Also, squaraines have been modified in some ways to stabilize them and prevent self-aggregation [13], including chemical modifications of their backbone [17][18][19][20] and encapsulation within rotaxanes [21], micelles [22][23][24], or albumin [25,26]. These used strategies show their improved photothermal performance, but monotherapy is usually not enough to completely eliminate tumors [2]. Endowing photodynamic property with squraines is a relative easy way to achieve combined therapy. Therefore, structural modification of squaraine backbone is further needed to enhance the synergistic photothermal-photodynamic performance.
Heavy atom modulation is used to improve the photodynamic performance of dyes, mainly due to the heavy atom effects causing vibronic spin-orbit coupling of the molecule to increase the probability of intersystem crossing [27,28]. Herein, we propose to introduce heavy atom (selenium) in the squaraine structure to optimize the optical properties for synergistic photothermal-photodynamic treatments of cancers ( Figure 1). We first characterized the physiochemical properties of SQSe and based-nanoparticles (SQSe-NPs) to verify the improved optical and therapeutic performances. Next, we tested the phototoxicity in vitro. We further performed the in vivo OA imaging to evaluate the optimal time point for treatments. Finally, SQSe-NPs were administrated on tumor-bearing mice to evaluate the therapeutic effect. We expect that SQSe-NPs can be used as a high-efficient photo-theranostic agent for precise cancer treatments.

Synthesis and characterization of SQSe
SQSe was synthesized through condensation of 4-methylchalcogenopyrylium salts with squaric acid ( Figure S1), and its chemical structure was verified using MALDI-TOF mass spectrometry, 1 H NMR, and 13 C NMR ( Figures S2-S4). It showed good solubility in organic solvents. Importantly, SQSe exhibited the enhanced and stable NIR absorption, which is attributed to the acceptor engineering (4-methylchalcogenopyrylium salts) on the electron-deficient squaric acid (Figure 2a). The photophysical properties of SQSe were next characterized. The molar absorption coefficient of SQSe in dichloromethane (DCM) was tested with 2.27 × 10 5 L mol −1 cm −1 , which is consistent with the reported squaraine dyes [14]. Its quantum yield (Φ = 0.0028) was only 5.6 % that of indocyanine green, which can be attributed to the selenium and which implies that a greater fraction of excitation energy undergoes nonradiative decay and intersystem crossing. To better understand the effect of the SQSe structure on the optical properties, density functional theory (DFT) calculation was performed for SQSe at the B3LYP/Def2SVP level via the Gaussian 16 program package. The highest occupied molecular orbitals (HOMO) are distributed over the conjugated structure, while the lowest unoccupied molecular orbitals (LUMO) localize mainly to acceptor regions. The narrow bandgap between HOMO and LUMO indicated the charge-transfer characteristics of SQSe with longer wavelength absorption (Figure 2c).

Preparation and characterization of SQSe-NPs
To improve the bioavailability of hydrophobic SQSe, we encapsulated it into amphiphilic PEG 114 -b-PCL 60 to prepare polymeric nanoparticles (SQSe-NPs). We characterized the morphology and size by transmission electron microscopy (TEM) and dynamic light scattering (DLS), which showed around 110 nm of nanosize ( Figure 2d and e). The stability of SQSe-NPs was monitored in phosphate-buffered saline (PBS) and 10 % fetal bovine serum (FBS), which showed no significant changes in nanosize ( Figure 2f). The optical properties of SQSe-NPs were next determined by absorption spectrum and fluorescence spectrum. Figure 2a showed SQSe-NPs absorbed strongly at 700-900 nm without suffering absorption quenching or blue-shifting. Due to the aggregationinduced quenching effect in nanoformulations, SQSe-NPs have negligible fluorescence (Φ = 0.00004) (Figure 2b).

Photothermal and photodynamic effects of SQSe-NPs in vitro
The strong NIR absorption of SQSe-NPs indicated the capacity of NIR light harvesting. To assess the photothermal effect of SQSe-NPs, an 808 nm CW laser was chosen to irradiate various concentrations of SQSe-NPs due to the fact that the absorption of normal tissue and water is lower at this wavelength, which in turn allows the laser to penetrate the tissue to a relatively deeper depth. And the generated heat was monitored by a thermal camera. Figure 3a and b showed the concentration-dependent temperature changes. Under the same laser irradiation, SQSe-NPs (20 μM) can induce huge temperature changes with 34.1 • C while water only has a negligible temperature increase. Such heat generation from SQSe-NPs is sufficient for hyperthermia damage of cancerous cells [3]. Next, we tested the temperature changes of SQSe-NPs with different laser power densities, which showed an obvious laser power intensity dependence (Figure 3c). The photothermal conversion efficiency of SQSe-NPs was calculated as 66.6 %, which was higher than most of the reported squaraines [14,29,30] due to favorable electron transfer ( Figure 3d). Besides, SQSe-NPs showed a stable temperature increase during 4 cycles of laser irradiation, indicating good photostability ( Figure 3e). In addition, the photodynamic effect of SQSe-NPs was also determined by 1,3-Diphenylisobenzofuran (DPBF, a ROS sensor) under the same test tube conditions. The decreased absorption intensity of DPBF means the endowed photodynamic effect of SQSe-NPs, which is attributed to the modulation of selenium ( Figure 3f). The singlet oxygen quantum yield of SQSe-NPs was determined to be ∼0.03 using ICG as a reference [31]. We next evaluated the in vitro phototherapeutic effect on 4T1 breast cancer cells. We used the Cell Counting Kit-8 (CCK8) assay to evaluate the photo-cytotoxicity of SQSe-NPs on these cells after 8 h's incubation. SQSe-NPs showed negligible cytotoxicity even at a concentration of up to 20 μM (Figure 3g). However, upon laser radiation, the cell viability decreased dependent on SQSe-NPs concentration and was nearly 0 % when treated with 20 μM of SQSe-NPs. Next, the pure PDT or PTT effects of SQSe-NPs was investigated by ice water bath to obstruct heat generation or adding glutathione (GSH) to eliminate ROS production. Figure S5 showed that monotherapy had partial inhabitation of cancerous cells growth while synergistic phototherapy had the improved celling killing. To directly visualize the phototherapeutic effect of SQSe-NPs on 4T1 cells, calceinacetoxymethyl (calcein-AM) and propidium iodide were costained with the treated cells to distinguish between live and dead cells. Figure 3i showed the strong photo-cytotoxicity on SQSe-NPs + Laser treated cells while minimizing damage in the control groups. To further explore the photodynamic effect of cancerous cells, 2 ′ ,7 ′ -dichlorodihydrofluorescein diacetate (DCFH-DA) was co-incubated with the treated cells to visualize the ROS generation upon laser irradiation. Figure 3h displayed strong green fluorescence from SQSe-NPs + Laser treated cells but few weak signals from the other 3 control groups.

OA imaging of SQSe-NPs in phantoms and in vivo
To examine the in vitro OA performance of SQSe-NPs, we first tested them with various concentration gradients, which is a good linear fit between the OA signals and concentrations (Figure 4a). Given the promising optoacoustic (OA) properties of SQSe-NPs, 4T1 tumor mouse models were established to explore the possibility of using SQSe-NPs for tumor detection in vivo. Figure 4b and c showed representative 3D OA images of 4T1 tumor-bearing mice at various time points postinjection of SQSe-NPs. It is obvious to see that SQSe-NPs were progressively accumulated in the tumor region due to the enhanced permeability and retention effect. The OA intensity of the tumor reached its maximal accumulation between 8 and 12 h of administration, which can be considered the optimal time point for cancer phototherapy. Subsequently, the OA signals of tumor areas were decreased due to systemic clearance from the tumor vasculature. After 24 h of intravenously injection, the major organs and tumor were isolated and done the OA measurements. Except tumor retention, SQSe-NPs showed higher accumulation in liver and kidney ( Figure S6).

Phototherapeutic effect of SQSe-NPs in vivo
Considering the promising photothermal and photodynamic effects of SQSe-NPs on cell killing, we performed to test of the phototherapy on mice bearing 4T1 tumor xenograft. With regard to this, 4T1 tumor-bearing mice were randomly divided into 4 groups (n = 5 each) with the following treatments respectively: PBS, PBS + laser, SQSe-NPs, SQSe-NPs + laser. The irradiation time was operated at 8 h post-injection based on the in vivo OA imaging results. The temperature changes in tumor areas were monitored by a thermal imaging camera for 10 min. Figure 5a and b shows that the tumor temperature from SQSe-NPs treated mice increased to 49. using SQSe-NPs (Figure 5c and d, S8). Besides, hematoxylin and eosin (H&E) staining was further used to confirm the therapeutic effect on tumors. The tumor slices from treated mice showed cell shrinkage and nucleus fragmentation with severe necrosis due to the phototherapeutic effect, while the other control groups displayed a high number of abnormal cells with typical cancerous features (Figure 5f). In addition, the biosafety of SQSe-NPs was evaluated by body weight monitoring and H&E staining of major organs. The body weight of all the animals showed a normal variation during the observation period, suggesting the absence of any serious side-effects (Figure 5e). H&E staining did not find major signs of toxicity, confirming the promising biocompatibility of SQSe-NPs ( Figure S9). Overall, these results indicate that SQSe-NPs have excellent phototherapeutic performance on tumors with good biosafety.

Conclusions
Here we employed the strategy of selenium modulation to optimize squaraine structure, producing a molecule that strongly absorbs near-infrared wavelengths and efficiently converts excitation energy into local heating and production of reactive oxygen species. A formulation of SQSe into hydrophilic nanoparticles led to efficient tumor killing in a mouse model of cancer, and the nanoparticles could be imaged non-invasively by optoacoustics. Our results establish a new reagent with potential for cancer phototheranostics, and more generally they demonstrate the potential of doping organic dyes with heavy metals for creating strong photothermal and photodynamic effects.

Materials
All chemical agents were purchased from Aladdin Scientific Ltd.

Synthesis of SQSe-NPs
SQSe (1 mg) and PEG 114 -b-PCL 60 (4 mg) were dissolved in DMSO (1 mL), then added dropwise to deionized water (15 mL), which was left stirring for 1 h. The suspension was concentrated on a microcentrifuge filter with a molecular weight cut-off of 100 kDa to obtain the purified SQSe-NPs were obtained for further use.

Characterization
1 H and 13 C NMR spectra were recorded on Bruker 500 Fourier transform spectrometer. Mass spectrometry was determined using a MALDI UltrafleXtreme (Bruker) with a matrix of dihydroxybenzoic acid. The molar absorption coefficient and relative quantum yield of dyes were calculated as previously described, where ICG in ethanol is as the reference solution (0.05) [33]. Nanoparticles were characterized using a transmission electron microscopy (FEI Tecnai F20) and a dynamic light scattering instrument (Malvern Zetasizer). Absorption spectra were recorded on a SHIMADZU UV-3600 Plus UV-Vis-NIR spectrometer. Fluorescent spectra were recorded in a steady state and time-resolved modes using photoluminescence spectrometer (Edinburg FLS1000).

Analysis of photothermal and photodynamic effects of SQSe-NPs
SQSe-NPs in phantoms were irradiated with an 808 nm continuouswave laser (Changchun New Industries Optoelectronics Technology Co., Ltd., 0.6 W/cm 2 ), and the temperature was measured using an infrared thermal camera. The photothermal conversion efficiency (PCE) of SQSe-NPs was calculated as described [34] using the equation = [(hS(T − Tsurr) − QDis]/I(1−10 −A808 ), where A808 refers to the absorbance of SQSe-NPs at 808 nm (0.58) and I refer to the laser power (0.6 W/cm 2 ). The photostability of SQSe-NPs was assessed by several cycles of irradiation to check if the temperature changes can remain the same. The same excitation was performed in the presence of DPBF, a sensor of reactive oxygen species, in order to assess photodynamic performance and determine 1 O 2 quantum yield [31]. In this assay, generation of singlet oxygen leads to a decrease in absorption.

Xenograft tumor models
All animal protocols were approved by the Animal Care and Use Committee of the First Affiliated Hospital of Zhejiang University School of Medicine. 4T1 breast cancer cells were subcutaneously injected into the right legs of female BALB/c mice (6-8 weeks). Approximately 1 × 10 6 cells were injected into each animal. When tumors had grown to a volume of 100 mm 3 , animals were used for further experiments (see below).

OA imaging of SQSe-NPs in vitro and in vivo
The OA imaging studies were operated on a LOIS-3D Pre-Clinical Mouse Imaging System (TomoWave, USA) with 808 nm-fixed pulsed laser and ∼150 μm of spatial resolution. Mice were intravenously injected with SQSe-NPs (0.3 mM, 150 μL), then imaged at various time points (4 h, 8 h, 12 h, 24 h). Animals were also imaged immediately before injection of SQSe-NPs. OA signals were quantitated using 3D Slicer software.

Phototherapy with SQSe-NPs in 4T1 tumor-bearing mice
At 8 h after injection of SQSe-NPs (0.3 mM, 150 μL) or phosphatebuffered saline (PBS) as a negative control, animals were irradiated or not for 10 min at 808 nm (0.6 W/cm 2 ). Five mice were subjected to each treatment. Tumor temperature was recorded using a thermal camera. Tumor volume and body weight of mice were recorded every two days. After 14 days of observation, all mice were sacrificed and the vital organs and tumors were isolated for histology based on staining with hematoxylin and eosin (H&E).

Statistical analysis
Statistical analysis was performed using Origin 2021. Inter-group differences were assessed for significance using One-Way/Two-Way ANOVA with Tukey's HSD test. Results were expressed as mean ±SD, and differences were considered significant if P < 0.05.