POSS Engineering of Multifunctional Nanoplatforms for Chemo-Mild Photothermal Synergistic Therapy

Chemo-mild photothermal synergistic therapy can effectively inhibit tumor growth under mild hyperthermia, minimizing damage to nearby healthy tissues and skin while ensuring therapeutic efficacy. In this paper, we develop a multifunctional study based on polyhedral oligomeric sesquisiloxane (POSS) that exhibits a synergistic therapeutic effect through mild photothermal and chemotherapy treatments (POSS-SQ-DOX). The nanoplatform utilizes SQ-N as a photothermal agent (PTA) for mild photothermal, while doxorubicin (DOX) serves as the chemotherapeutic drug for chemotherapy. By incorporating POSS into the nanoplatform, we successfully prevent the aggregation of SQ-N in aqueous solutions, thus maintaining its excellent photothermal properties both in vitro and in vivo. Furthermore, the introduction of polyethylene glycol (PEG) significantly enhances cell permeability, which contributes to the remarkable therapeutic effect of POSS-SQ-DOX NPs. Our studies on the photothermal properties of POSS-SQ-DOX NPs demonstrate their high photothermal conversion efficiency (62.3%) and stability, confirming their suitability for use in mild photothermal therapy. A combination index value (CI = 0.72) verified the presence of a synergistic effect between these two treatments, indicating that POSS-SQ-DOX NPs exhibited significantly higher cell mortality (74.7%) and tumor inhibition rate (72.7%) compared to single chemotherapy and mild photothermal therapy. This observation highlights the synergistic therapeutic potential of POSS-SQ-DOX NPs. Furthermore, in vitro and in vivo toxicity tests suggest that the absence of cytotoxicity and excellent biocompatibility of POSS-SQ-DOX NPs provide a guarantee for clinical applications. Therefore, utilizing near-infrared light-triggering POSS-SQ-DOX NPs can serve as chemo-mild photothermal PTA, while functionalized POSS-SQ-DOX NPs hold great promise as a novel nanoplatform that may drive significant advancements in the field of chemo-mild photothermal therapy.


Introduction
As a highly promising candidate, photothermal therapy (PTT) has garnered significant attention in recent years.PTT utilizes light and photothermal agents (PTAs).
As one of the main strategies for cancer treatment, chemotherapy plays an important role in clinical cancer treatment.However, the non-specific distribution and resistance of chemotherapeutic drugs significantly limit the clinical applications of chemotherapy.The combination of MPTT with chemotherapy can solve these problems easily.First, MPTT may enable spatiotemporally controlled drug delivery by adjusting the position, power density, and working time of NIR light.Second, its mild temperature can make cancer cells more accessible and vulnerable to chemotherapeutic drugs.Third, the heat generated by PTAs can stimulate intratumoral penetration and the accumulation of drugs due to the increased tumor blood flow and vascular permeability [21].In turn, enhanced chemotherapy efficacy can compensate for insufficient heat damage due to the mild temperature.Therefore, a more excellent anti-tumor effect can be achieved by combining MPTT with chemotherapy.
Squaraine dyes (SQs) are a well-studied class of zwitterionic dyes with stable quinoid structures and high planarity [38].Typically, they consist of an electron-accepting squaric acid and two electron donors in a D-A-D pattern.Due to their advantageous structure, these dyes exhibit exceptional optical properties, including elevated photoconductivity, well-defined absorption in the visible and near-infrared regions, a high molar absorption coefficient, and impressive photothermal conversion efficiency (PCE) [39,40].As a result, SQs find wide applications in various biological fields, such as biosensing [41], bioimaging [42,43], and phototherapy [44,45].However, on account of the strong dipole-dipole and π-π stacking interactions between molecules, SQs tend to aggregate in aqueous solutions, which can negatively impact their photothermal performance and photostability [46].Therefore, there is a challenge when using SQs for biological applications, especially in PTT, as follows: preventing the easy formation of dye aggregates while preserving their photophysical characteristics.
The emergence of polyhedral oligomeric sesquisiloxane (POSS) can effectively address this issue.A typical POSS molecule possesses a three-dimensional cage-like structure consisting of a cubic silica core and eight surrounding organic corner groups.These corner groups can be readily modified into various functional groups, facilitating the convenient preparation of multifunctional polymers with POSS cores [47][48][49].Moreover, due to the nanoscale size of POSS, diverse types of functional POSS-based nanoplatforms can be constructed.Additionally, Wang et al. reported that conjugating porphyrin to POSS resulted in increased photodynamic efficiency and reduced aggregation [50].Consequently, incorporating POSS can mitigate the aggregation caused by π-π stacking interactions between molecules.Furthermore, based on our previous research conducted in our laboratory [51,52], we discovered that introducing PEG could effectively enhance water solubility and achieve amphipathy for POSS-based nanoparticles, thereby significantly improving cell permeability and the biocompatibility of these nanoplatforms while simultaneously enhancing their potential applications in biological imaging [53], drug delivery [54,55], and tissue regeneration [56].
Hence, based on the aforementioned introduction, a multifunctional POSS-based nanoplatform (POSS-SQ-DOX) was developed for mild chemo-photothermal synergistic tumor therapy.Firstly, POSS-PEG was synthesized via click chemistry to enhance the cell permeability and biocompatibility of the nanoplatform.Subsequently, SQ-N and DOX were incorporated into POSS using the same method.In this nanoplatform, SQ-N exhibited excellent photothermal properties and high PCE and served as a PTA in MPTT.Additionally, DOX acted as a chemotherapeutic drug for chemotherapy purposes.As illustrated in Scheme 1, upon in situ injection, POSS-SQ-DOX NPs accumulated within tumor cells where the mild thermal effect induced by near-infrared light at 808 nm worked synergistically with DOX to achieve an enhanced anti-tumor effect.The experimental results demonstrated that tumor growth was significantly inhibited in mice treated with POSS-SQ-DOX NPs compared to those receiving single DOX chemotherapy or MPTT alone.Furthermore, this treatment approach proved safe while ensuring highly effective anti-tumor outcomes.Overall, this study explored the synergistic anti-tumor effect of DOX chemotherapy and photothermal therapy working under mild temperatures, highlighting the great potential for utilizing the chemo-mild photothermal synergistic therapy system based on DOX and SQ-modified POSS-based nanoparticles for tumor treatments, bringing a novel strategy to combat tumors.
Hence, based on the aforementioned introduction, a multifunctional POSS-based noplatform (POSS-SQ-DOX) was developed for mild chemo-photothermal synergisti mor therapy.Firstly, POSS-PEG was synthesized via click chemistry to enhance the permeability and biocompatibility of the nanoplatform.Subsequently, SQ-N and D were incorporated into POSS using the same method.In this nanoplatform, SQ-N ex ited excellent photothermal properties and high PCE and served as a PTA in MPTT.ditionally, DOX acted as a chemotherapeutic drug for chemotherapy purposes.As i trated in Scheme 1, upon in situ injection, POSS-SQ-DOX NPs accumulated within tu cells where the mild thermal effect induced by near-infrared light at 808 nm worked ergistically with DOX to achieve an enhanced anti-tumor effect.The experimental res demonstrated that tumor growth was significantly inhibited in mice treated with PO SQ-DOX NPs compared to those receiving single DOX chemotherapy or MPTT al Furthermore, this treatment approach proved safe while ensuring highly effective a tumor outcomes.Overall, this study explored the synergistic anti-tumor effect of D chemotherapy and photothermal therapy working under mild temperatures, highligh the great potential for utilizing the chemo-mild photothermal synergistic therapy sys based on DOX and SQ-modified POSS-based nanoparticles for tumor treatments, bring a novel strategy to combat tumors.Scheme 1. Schematic illustration of POSS-SQ-DOX NPs' preparation and the chemo-mild ph thermal therapeutic mechanism in vivo.

Preparation and Characterization of POSS-SQ-DOX NPs
PEG, SQ-N, and DOX were all chemically attached to POSS nanoparticles thro click chemistry, resulting in the formation of POSS-SQ-DOX NPs (Schemes S1-S3).structures of these compounds were confirmed using 1 H NMR and FITR (Figures S1-The structure of POSS-SQ-DOX NPs is shown in Figure 1a.Additionally, transmis electron microscope (TEM) and dynamic laser light scattering (DLS) techniques were ployed to verify the nanostructures of POSS-SQ-DOX NPs.TEM analysis revealed tha size of POSS-SQ-DOX NPs was approximately 25 nm, while DLS measurements sho a size of 24 nm, which are both larger than that of pure POSS-SH [57] (Figure 1b,c).thermore, the particle size stability of POSS-SQ-DOX NPs was monitored over a two-w period (Figure 1d), demonstrating minimal changes in nanoparticle size and indica excellent stability for at least two weeks.Additionally, UV-Vis absorption spectra and orescence spectra were used to investigate the optical properties of POSS-SQ-DOX N Scheme 1. Schematic illustration of POSS-SQ-DOX NPs' preparation and the chemo-mild photothermal therapeutic mechanism in vivo.

Preparation and Characterization of POSS-SQ-DOX NPs
PEG, SQ-N, and DOX were all chemically attached to POSS nanoparticles through click chemistry, resulting in the formation of POSS-SQ-DOX NPs (Schemes S1-S3).The structures of these compounds were confirmed using 1 H NMR and FITR (Figures S1-S4).The structure of POSS-SQ-DOX NPs is shown in Figure 1a.Additionally, transmission electron microscope (TEM) and dynamic laser light scattering (DLS) techniques were employed to verify the nanostructures of POSS-SQ-DOX NPs.TEM analysis revealed that the size of POSS-SQ-DOX NPs was approximately 25 nm, while DLS measurements showed a size of 24 nm, which are both larger than that of pure POSS-SH [57] (Figure 1b,c).Furthermore, the particle size stability of POSS-SQ-DOX NPs was monitored over a two-week period (Figure 1d), demonstrating minimal changes in nanoparticle size and indicating excellent stability for at least two weeks.Additionally, UV-Vis absorption spectra and fluorescence spectra were used to investigate the optical properties of POSS-SQ-DOX NPs.As depicted in Figure 1e, distinct UV absorption peaks near 818 nm and a fluorescence emission peak at 949 nm were observed for POSS-SQ-DOX NPs.The presence of near-infrared absorption characteristics provided a foundation for utilizing POSS-SQ-DOX NPs as PTAs in mild photothermal therapy.Notably, compared to SQ-N alone, its incorporation into POSS nanoparticles significantly enhanced both absorption and emission intensities for SQ-N molecules within the hybrid system due to the increased intermolecular distances between SQ-N units and weakened π-π stacking interactions (Figure S5).Furthermore, no significant changes in either the position or intensity of the absorption peak were observed during storage at room temperature for two weeks, indicating that POSS-SQ-DOX NPs displayed optical stability under suitable conditions (Figure 1f).Moreover, contact angle measurements confirmed the enhanced hydrophilicity of POSS-SQ-DOX NPs.In comparison to POSS-SH, DOX, and SQ-N, POSS-SQ-DOX NPs exhibited superior hydrophilicity due to the PEG modification (Figure S6).In summary, we successfully synthesized NIR-absorbing and highly stable POSS-SQ-DOX NPs, making them a suitable and robust system for the following research.
As depicted in Figure 1e, distinct UV absorption peaks near 818 nm and a fluoresc emission peak at 949 nm were observed for POSS-SQ-DOX NPs.The presence of infrared absorption characteristics provided a foundation for utilizing POSS-SQ-NPs as PTAs in mild photothermal therapy.Notably, compared to SQ-N alone, its i poration into POSS nanoparticles significantly enhanced both absorption and emi intensities for SQ-N molecules within the hybrid system due to the increased interm ular distances between SQ-N units and weakened π-π stacking interactions (Figure Furthermore, no significant changes in either the position or intensity of the absorp peak were observed during storage at room temperature for two weeks, indicating POSS-SQ-DOX NPs displayed optical stability under suitable conditions (Figure Moreover, contact angle measurements confirmed the enhanced hydrophilicity of P SQ-DOX NPs.In comparison to POSS-SH, DOX, and SQ-N, POSS-SQ-DOX NPs exhi superior hydrophilicity due to the PEG modification (Figure S6).In summary, we suc fully synthesized NIR-absorbing and highly stable POSS-SQ-DOX NPs, making th suitable and robust system for the following research.

Photothermal Performance of POSS-SQ-DOX NPs
In order to evaluate the photothermal performance of POSS-SQ-DOX NPs, an 80 laser was employed for irradiation, and the resulting temperature changes were recor As shown in Figure 2a, it was observed that the temperature increments of POSS-SQ-NPs exhibited a positive correlation with their concentrations under the irradiation 808 nm laser (0.5 W/cm 2 ).Specifically, when the concentration of POSS-SQ-DOX reached 200 µg/mL, the temperature increased from 29.3 °C to 81.1 °C within 210 s (

Photothermal Performance of POSS-SQ-DOX NPs
In order to evaluate the photothermal performance of POSS-SQ-DOX NPs, an 808 nm laser was employed for irradiation, and the resulting temperature changes were recorded.As shown in Figure 2a, it was observed that the temperature increments of POSS-SQ-DOX NPs exhibited a positive correlation with their concentrations under the irradiation of an 808 nm laser (0.5 W/cm 2 ).Specifically, when the concentration of POSS-SQ-DOX NPs reached 200 µg/mL, the temperature increased from 29.3 • C to 81.1 • C within 210 s (∆T = 51.8• C).In addition, at a concentration of 50 µg/mL, the temperature elevation demonstrated an upward trend as the laser power densities increased (Figure 2b).These results collectively indicate that POSS-SQ-DOX NPs possess efficient light-to-thermal energy conversion capabilities and enable precise control over heat generation.Additionally, a high PCE value (η) of up to 62.3% was calculated for POSS-SQ-DOX NPs [58,59] (Figure 2c,d).Moreover, thermal images provided a more intuitive visualization of the temperature variations associated with POSS-SQ-DOX NPs (Figure 2e).In addition, significant chemosensitization effects occur when the tumor site temperature reaches 39.0-43.0• C because, when the temperature is increased from 37.0 to 43.0 • C, the permeability of the cell membrane is significantly enhanced, which promotes the uptake of nano-drugs by cells [5].Thus, a laser power of 0.5 W/cm 2 and a concentration of 50 µg/mL were selected for the following experiments.Furthermore, photothermal conversion stability referred to their ability to withstand multiple excitation cycles without irreversible loss, which was an important parameter for characterizing organic dye molecules [60].The stability of photothermal conversion was evaluated through heating-cooling cycle experiments.As shown in Figure 2f, the POSS-SQ-DOX NPs solution was irradiated with an 808 nm laser for 210 s and subsequently allowed to cool naturally to room temperature while recording the temperatures.It was evident that the temperature of POSS-SQ-DOX NPs exhibited negligible changes during five heating-cooling cycles, suggesting excellent stability and reproducibility of POSS-SQ-DOX NPs.Hence, all these experiments indicate that POSS-SQ-DOX NPs could serve as a highly efficient PTA in PTT due to their high PCE and good photochemical stability.

Evaluation Synergies Effect In Vitro
The cytotoxicity of different formulations (free DOX, free SQ-N, and different ratios of DOX and SQ-N) in HeLa cells was evaluated using Cell Counting Kit-8 (CCK-8) assays.Both single DOX and SQ-N +L exhibited cytotoxic effects on HeLa cells.However, the efficacy was suboptimal with an IC 50 value of 19.94 µg/mL for DOX alone and 17.45 µg/mL for SQ-N +L (Figure 3a, Table 1).To determine the optimal ratio of DOX and SQ-N in nanoparticles, we assessed the impact of varying DOX contents on cell viability at a constant total concentration (Figure 3b).Furthermore, combination index (CI) values were calculated to confirm the synergistic effect between different ratios and CI < 1 indicated synergism.Our results demonstrate clear synergistic effects within the range of DOX:SQ-N +L = 1:3-3:1, with the strongest synergistic effect observed at a dose ratio of DOX:SQ-N +L was 2:1 (n/n), resulting in an IC 50 value as low as 9.32 µg/mL for DOX and 4.33 µg/mL for SQ-N when prepared as POSS-SQ-DOX NPs.In summary, by utilizing a ration of DOX:SQ-N +L = 2:1 during preparation, not only could we ensure a synergistic therapeutic effect but also reduce chemotherapy drugs dosage while alleviating potential side effects.

Evaluation Synergies Effect In Vitro
The cytotoxicity of different formulations (free DOX, free SQ-N, and different ra of DOX and SQ-N) in HeLa cells was evaluated using Cell Counting Kit-8 (CCK-8) assa Both single DOX and SQ-N +L exhibited cytotoxic effects on HeLa cells.However, efficacy was suboptimal with an IC50 value of 19.94 µg/mL for DOX alone and 17.45 µg/ for SQ-N +L (Figure 3a, Table 1).To determine the optimal ratio of DOX and SQ-N nanoparticles, we assessed the impact of varying DOX contents on cell viability at a c stant total concentration (Figure 3b).Furthermore, combination index (CI) values w calculated to confirm the synergistic effect between different ratios and CI < 1 indica synergism.Our results demonstrate clear synergistic effects within the range of DOX:S N +L = 1:3-3:1, with the strongest synergistic effect observed at a dose ratio of DOX:SQ +L was 2:1 (n/n), resulting in an IC50 value as low as 9.32 µg/mL for DOX and 4.33 µg/ for SQ-N when prepared as POSS-SQ-DOX NPs.In summary, by utilizing a ration DOX:SQ-N +L = 2:1 during preparation, not only could we ensure a synergistic therapeu effect but also reduce chemotherapy drugs dosage while alleviating potential side effe

In Vitro Tumor Therapy
It is essential to assess the biocompatibility of POSS-SQ-DOX NPs prior to conducting further in vitro and in vivo anti-tumor therapy experiments.Moreover, all experiments were performed under 808 nm laser irradiation with a power density of 0.5 W/cm 2 .The toxicity of POSS-SQ-DOX NPs was evaluated using the CCK-8 method.Figure 4a shows that even at a concentration of 200 µg/mL, the cell viability remained above 90% for POSS-SQ NPs, indicating their safety for both normal L929 cells and tumor HeLa cells.However, when incubated with POSS-SQ-DOX NPs or a free DOX treatment for 24 h, the viability of HeLa cells was only slightly above 40%, suggesting that the toxicity of POSS-SQ-DOX NPs primarily stemmed from the presence of the DOX drug (Figure 4b).Furthermore, as depicted in Figure 4c,d, it could be visually confirmed that chemo-mild photothermal synergistic therapy exhibited better efficacy compared to single chemotherapy or PTT alone.Additionally, the IC 50 value for DOX was determined to be 10.94 µg/mL for POSS-SQ-DOX NPs, which is lower than the 19.94 µg/mL of free DOX.These results collectively indicate that chemo-mild photothermal synergistic therapy exerts a stronger inhibitory effect on HeLa cell proliferation with reduced amounts of chemotherapy drugs compared to single chemotherapy or PTT.This further supports the notion that chemo-mild photothermal synergistic therapy could achieve enhanced anti-tumor therapeutic effects.
Additionally, live-dead cell-staining experiments were carried out to further confirm the synergistic anti-tumor effect of chemo-mild photothermal synergistic therapy (Figure 4e).In these experiments, living cells were stained with Calcein AM (green), and dead cells were stained with PI (red).Compared to the DOX group, the POSS-SQ-DOX NPs group exhibited enhanced cytotoxicity against HeLa cells due to the superior cell permeability of the nanoparticles.Furthermore, the PI-positive rate was calculated for each group.As shown in Figure 4f, the PI-positive rate of the POSS-SQ-DOX +L group was 1.9-fold higher than that of the POSS-SQ +L group and 1.7-fold higher than that of the POSS-SQ-DOX group, confirming that POSS-SQ-DOX NPs achieved effective cellular ablation under 808 nm laser irradiation.These findings collectively demonstrate how POSS-SQ-DOX NPs could effectively exert a synergistic effect in chemo-mild photothermal therapy by more efficiently inhibiting tumor cell proliferation and inducing apoptosis.

Anti-Tumor Therapy In Vivo
To assess the feasibility of chemo-mild photothermal therapy in tumors, we established a HeLa tumor-bearing mouse model and utilized the following five formulations for our studies: PBS, DOX, POSS-SQ +L, POSS-SQ-DOX, and POSS-SQ-DOX +L.In subsequent experiments, the concentrations of DOX, POSS-SQ NPs, and POSS-SQ-DOX NPs were 12 µg/mL, 18 µg/mL, and 50 µg/mL, respectively.All experiments were carried out under irradiation from an 808 nm laser (0.5 W/cm 2 ).Notably, significant thermal signals were observed in tumor areas following the injection of POSS-SQ NPs and POSS-SQ-DOX NPs, while there was no clear single detection with PBS or DOX alone (Figure 5a).Real-time temperature changes at the tumor site were also recorded to further validate these thermal imaging results (Figure 5b).Tumor volume change data were documented, as shown in Figure 5c-g.While single chemotherapy (DOX group) and mild photothermal therapy (POSS-SQ +L group) exhibited certain inhibitory effects on tumor growth, they still resulted in an increased tumor volume with only moderate therapeutic efficacy.However, the inhibitory effect on tumors was slightly better in the POSS-SQ-DOX group compared to the DOX group due to the enhanced cell permeability provided by the use of POSS-SQ-DOX NPs.Moreover, the smallest tumor volume was observed in the group of POSS-SQ-DOX +L, indicating that this combination had a more pronounced efficacy in suppressing tumor growth.Furthermore, in sharp contrast with other experimental groups, both the tumor weight and the tumor inhibition ratio (TIR) were meticulously recorded and calculated (Figure 5h,i).The TIR value of the POSS-SQ-DOX +L group was found to be 72.7%, which exhibited a remarkable increase of 2.35 times compared to DOX alone, which is 1.46 times higher than POSS-SQ +L and 1.53 times higher than POSS-SQ-DOX.Additionally, the changes in body weight of tumor-bearing mice in each group were recorded, and it was found that the weight change trend of different groups of tumor-bearing mice was similar.Furthermore, on day 12, the body weight of the tumor-bearing mice was similar between the experimental and control groups, which demonstrated the excellent biosafety profile of POSS-SQ-DOX NPs (Figure 5j).Based on these comprehensive findings, it can be inferred that chemo-mild photothermal therapy had superior tumor-suppressive effects when compared to single chemotherapy or photothermal therapy alone without any associated toxicity.
Int. J. Mol.Sci.2024, 25, x FOR PEER REVIEW 7 of HeLa cells was only slightly above 40%, suggesting that the toxicity of POSS-SQ-D NPs primarily stemmed from the presence of the DOX drug (Figure 4b).Furthermor depicted in Figure 4c,d, it could be visually confirmed that chemo-mild photothermal ergistic therapy exhibited better efficacy compared to single chemotherapy or PTT al Additionally, the IC50 value for DOX was determined to be 10.94 µg/mL for POSS DOX NPs, which is lower than the 19.94 µg/mL of free DOX.These results collecti indicate that chemo-mild photothermal synergistic therapy exerts a stronger inhibi effect on HeLa cell proliferation with reduced amounts of chemotherapy drugs comp to single chemotherapy or PTT.This further supports the notion that chemo-mild ph thermal synergistic therapy could achieve enhanced anti-tumor therapeutic effects.Additionally, live-dead cell-staining experiments were carried out to further con the synergistic anti-tumor effect of chemo-mild photothermal synergistic therapy (Fi 4e).In these experiments, living cells were stained with Calcein AM (green), and d cells were stained with PI (red).Compared to the DOX group, the POSS-SQ-DOX group exhibited enhanced cytotoxicity against HeLa cells due to the superior cell per ability of the nanoparticles.Furthermore, the PI-positive rate was calculated for group.As shown in Figure 4f, the PI-positive rate of the POSS-SQ-DOX +L group was   The therapeutic effects of all groups were further evaluated through H&E, Ki-67, and TUNEL staining (Figure 6a-c).According to the results of H&E staining, tumor cells in the PBS were in good condition.However, the DOX, POSS-SQ +L, and POSS-SQ-DOX groups showed varying degrees of tumor cell lysis, indicating that a single drug or mild photothermal therapy had inhibitory effects on the tumor.In contrast, extensive damage and destruction with significant cell lysis were observed in the POSS-SQ-DOX +L group.Moreover, compared to other groups, treatment with POSS-SQ-DOX NPs with laser irradiation resulted in a significant reduction in Ki67-positive cells and an increase in TUNEL-positive cells within tumors.This may be because the cell membrane integrity was severely disrupted under the synergistic treatment of chemotherapy and MPTT, which led to cell membrane lysis and nuclear erosion and ultimately resulted in the apoptosis of tumor cells (Figure S7).These findings confirm that chemo-mild photothermal synergistic therapy is superior to chemotherapy or photothermal alone.Consistently with previous studies, these results highlight the excellent anti-tumor effects of POSS-SQ-DOX NPs by inhibiting cell proliferation and promoting cell apoptosis.Overall, these results demonstrate that this system has an excellent synergistic effect and achieves an excellent anti-tumor therapeutic outcome.

In Vivo Safety Evaluation
To further evaluate the in vivo biosafety of POSS-SQ-DOX NPs, histological analyse and hemanalysis were conducted.HeLa tumor-bearing mice were injected with POSS-SQ DOX NPs at the tumor site and subsequently exposed to 808 nm laser irradiation at a powe density of 0.5 W/cm −2 for 5 min.After 12 days post-irradiation, the major organs, includin the hearts, livers, spleens, lungs, and kidneys, were dissected from the mice and stained with H&E (Figure 7a).The results revealed no discernible organ damage in the POSS-SQ DOX +L-treated mice compared to those treated with PBS alone, indicating its excellent bi ocompatibility and minor side effects for anti-tumor therapy.To further evaluate the poten tial toxicity of POSS-SQ-DOX NPs in vivo, two groups of mice were sacrificed for a blood routine assay after 12 days of treatment.As shown in Figure 7b-g, there was no significan difference in the parameters of blood routines between the PBS group and the POSS-SQ DOX +L group, and all data were within the normal range.Normal white blood cell count and lymphocyte counts indicated that there was no infection or inflammation, and the nor mal red blood cell counts and platelet counts indicated that there was no severe tissue dam age.Altogether, the POSS-SQ-DOX NPs displayed excellent biocompatibility and biosafet and could be applied in chemo-mild PTT against cancers.

In Vivo Safety Evaluation
To further evaluate the in vivo biosafety of POSS-SQ-DOX NPs, histological analyses and hemanalysis were conducted.HeLa tumor-bearing mice were injected with POSS-SQ-DOX NPs at the tumor site and subsequently exposed to 808 nm laser irradiation at a power density of 0.5 W/cm −2 for 5 min.After 12 days post-irradiation, the major organs, including the hearts, livers, spleens, lungs, and kidneys, were dissected from the mice and stained with H&E (Figure 7a).The results revealed no discernible organ damage in the POSS-SQ-DOX +L-treated mice compared to those treated with PBS alone, indicating its excellent biocompatibility and minor side effects for anti-tumor therapy.To further evaluate the potential toxicity of POSS-SQ-DOX NPs in vivo, two groups of mice were sacrificed for a blood routine assay after 12 days of treatment.As shown in Figure 7b-g, there was no significant difference in the parameters of blood routines between the PBS group and the POSS-SQ-DOX +L group, and all data were within the normal range.Normal white blood cell counts and lymphocyte counts indicated that there was no infection or inflammation, and the normal red blood cell counts and platelet counts indicated that there was no severe tissue damage.Altogether, the POSS-SQ-DOX NPs displayed excellent biocompatibility and biosafety and could be applied in chemo-mild PTT against cancers.

Characterization of POSS-SQ-DOX NPs
1 H NMR spectra were recorded with a Bruker AVANCE III-HD 600 MHz NMR spectrometer (Bruker, Billericca, MA, USA).Fourier transform infrared (FTIR) spectra were obtained using a Thermo Nicolet 8700 spectrometer (Thermo Scientific, Waltham, MA, USA).The morphologies of POSS-SQ-DOX NPs were observed using transmission electron microscopy (TEM, Thermo Scientific, Waltham, MA, USA) with an accelerating voltage of 120 kV.The size distribution of POSS-SQ-DOX NPs was assessed via dynamic light scattering (DLS, Brookhaven Instruments Co., New York, NY, USA).Ultraviolet-visible (UV-vis) absorption spectra were measured with a Lambda 950 UV-vis spectrometer (PerkinElmer, Waltham, MA, USA).

Stability of POSS-SQ-DOX NPs
To evaluate the nanoparticles' stability, POSS-SQ-DOX NPs were placed at 37 • C in PBS, and the sizes were detected at different times using DLS.

Optical Performance of POSS-SQ-DOX NPs
The optical performance of POSS-SQ-DOX NPs was evaluated using UV-Vis and fluorescence spectra.The UV scanning spectra were in the range of 600-1000 nm of the POSS-SQ-DOX NP solution (200 µg/mL), and the excitation wavelength was 850 nm of fluorescence spectra.Furthermore, the optical stability of POSS-SQ-DOX NPs was verified by measuring the solution after the dilution process, with storage for 7 days and storage for 14 days at 37 • C.

Cytotoxicity and Combination Index Evaluation
CCK-8 assays were carried out to evaluate the in vitro cytotoxicity and killing effect of SQ-N, DOX, POSS-SQ NPs, and POSS-SQ-DOX NPs on the L929 cell line and HeLa cell line (accession number: SCSP-504).After seeding cells in a 96-well plate (1 × 10 4 cells/well) for 18 h in a humidified 5% CO 2 atmosphere at 37 • C, the culture medium was replaced by a serum-free medium containing a series different concentrations of SQ-N, DOX, POSS-SQ NPs and POSS-SQ-DOX NPs.After incubation for another 8 h, half of the parallel wells were exposed to 808 nm laser irradiation (0.5 W/cm 2 ) for 5 min, while the other half was kept in the dark.After a further 16 h incubation, 2-(2-methoxy-4-nitrophenyl)-3-(4nitrophenyl)-5-(2, 4-benzene disulfonic acid) 2h tetrazolium monosodium salt (WST-8) was added into the wells and mixture were incubated for 4 h.After this, 100 µL of dimethyl sulfoxide (DMSO) was added into each well to dissolve the formazan crystal.Eventually, the absorbance was measured at a wavelength of 450 nm using a microplate reader.The cell viability (%) was calculated using the following Equation (1): The concept of the combination index (CI) was introduced to evaluate the synergistic, additive, and antagonistic effects of two different drugs at different dosages according to the Chou-Talalay method, and the formula for CI is shown in Equation ( 2

Live/Dead Cell Double-Staining Assay
The in vitro anti-tumor efficacy of DOX, POSS-SQ NPs, and POSS-SQ-DOX NPs was evaluated using Calcein-AM/PI (propidium iodide) staining and a CKX 53 fluorescence microscope (Olympus, Tokyo, Japan).First, HeLa cells were incubated with PBS, DOX (12 µg/mL in DMEM, 1 mL), POSS-SQ NPs (18 µg/mL in DMEM, 1 mL), and POSS-SQ-DOX NPs (50 µg/mL in DMEM, 1 mL) in confocal dishes for 4 h.Then, the cells were kept in the dark or irradiated with an 808 nm laser (0.5 W/cm −2 ) for 2 min.And then after 6 h of continuous incubation, the cells were co-stained with calcein AM/propidium iodide to mark viable cells and dead cells, respectively.Cells were imaged with the CKX53 fluorescence microscope via the green/red fluorescence channel.

In Vivo Synergistic Effects
Animal experiments were carried out following strict compliance with the provisions of the "Guidelines for the Use and Management of Experimental Animals" (IACUC).In order to evaluate the synergistic anti-tumor effect of chemotherapy and photothermal therapy, we selected mice with a tumor volume of about 120 mm 3 and randomly divided them into five groups (n = 3).The experimental groups were "PBS", "DOX", "POSS-SQ +L", "POSS-SQ-DOX", and "POSS-SQ-DOX +L".In total, 200 µL of the PBS, DOX (12 µg/mL), POSS-SQ NPs (18 µg/mL), and POSS-SQ-DOX NPs (50 µg/mL) solution were injected into mice via intratumoral injection and the groups of "POSS-SQ +L", and "POSS-SQ-DOX +L" were treated with 808 nm laser irradiation for 2 min immediately (tumor site, 0.5 W/cm 2 ), and then synchronously imaged every 10 s with a T540 infrared thermal camera (FLIR, Wilsonville, Oregon, USA).After that, all groups were treated with the same operation again every three days, and the tumor volume and body weight of the tumor-bearing mice in each group were measured and recorded for a total of 12 consecutive days.All animal studies were approved by the e Institutional Animal Care and Use Committee guidelines of Shanghai Jiao Tong University.

Histological Studies
All the mice in the three groups were euthanized on the 12th day after therapy.The tumors and main normal organs (heart, liver, spleen, lungs, and kidney) were resected, sliced, and stained.Cell states in the tumor sites and other main normal organs were analyzed using hematoxylin-eosin (H&E) staining; the tissues were immersed in 4% formalin solution, embedded in paraffin after dehydration, and sliced at a 4 µm thickness.For cell proliferation detection, serial tissue sections with a thickness of 4 µm were cut and then stained with an antibody against Ki67 (Servicebio, Wuhan, China) for 1 h.For cell apoptosis detection, the tumor sections were stained with terminal transferase-mediated dUTP nick end labeling (TUNEL) per the manufacturer's instructions (Promega, Madison, Wisconsin, USA).All slides were digitized with a high-resolution whole-slide scanner (Pannoramic DESK, Kongerizoge Street, Budapest, Hungary).

Blood Routine Assay
Healthy BALB/c nude mice were randomly assigned to two groups, labeled as "PBS" and "POSS-SQ-DOX +L" (n = 3 per group).After 12 days, the blood was sampled and detected for the blood routine examination.

Ethical Statement
The animal study protocol was approved by the Ethics Committee for Animal Experiments Affairs in Shanghai Renji Hospital, China (ra-2019-091).

Statistical Analysis
All statistical analyses were performed using GraphPad Prism 9.0 (GraphPad software, San Diego, California, USA).Statistical significance was determined using one-way analysis of variance (ANOVA) for multiple groups.All data are presented as the mean ± SD. p < 0.05 was considered statistically significant.

Conclusions
In summary, POSS-based multifunctional nanoparticles (POSS-SQ-DOX NPs) were rationally designed and successfully applied for chemo-mild photothermal synergistic anti-tumor therapy.POSS-SQ-DOX NPs were strong in stability, good in biocompatibility, and had a high photothermal conversion efficiency, which allowed POSS-SQ-DOX NPs to be used as a PTA and drug transport platform in chemo-mild photothermal therapy.Meanwhile, the calculation of the combination index (0.72, <1.0) indicated the existence of a synergistic anti-tumor effect.Furthermore, due to the better cell permeability and the presence of synergistic therapy, POSS-SQ-DOX NPs could enter into tumor cells more easily, which was conducive to the nanoparticles for more effective mild photothermal therapy.Additionally, chemotherapy also solved the problem of the poor therapeutic effect caused by low temperatures during mild photothermal therapy.Therefore, both in vitro and in vivo experiments showed the effect of chemo-mild photothermal synergistic therapy better than single chemotherapy and photothermal therapy.In conclusion, the developed POSS-SQ-DOX NPs, as a comprehensive nanotherapeutic agent, have great potential to induce chemo-mild photothermal synergistic therapy, improve anti-tumor efficacy and reduce side effects, which is expected to be further applied in clinical tumor treatments.

Figure 1 .
Figure 1.(a) The structure of POSS-SQ-DOX NPs; (b) TEM image of POSS-SQ-DOX NPs; (c data of POSS-SQ-DOX NPs; (d) The results of POSS-SQ-DOX NP size monitoring within two w (e) UV-vis absorption and fluorescence spectra of POSS-SQ-DOX NPs (200 µg/mL) in an aqu solution; (f) Results of UV-vis absorption spectra of POSS-SQ-DOX NPs within two weeks.The bar is 100 µm.

Figure 1 .
Figure 1.(a) The structure of POSS-SQ-DOX NPs; (b) TEM image of POSS-SQ-DOX NPs; (c) DLS data of POSS-SQ-DOX NPs; (d) The results of POSS-SQ-DOX NP size monitoring within two weeks; (e) UV-vis absorption and fluorescence spectra of POSS-SQ-DOX NPs (200 µg/mL) in an aqueous solution; (f) Results of UV-vis absorption spectra of POSS-SQ-DOX NPs within two weeks.The scale bar is 100 µm.
temperatures.It was evident that the temperature of POSS-SQ-DOX NPs exhibited ne gible changes during five heating-cooling cycles, suggesting excellent stability and rep ducibility of POSS-SQ-DOX NPs.Hence, all these experiments indicate that POSS-DOX NPs could serve as a highly efficient PTA in PTT due to their high PCE and go photochemical stability.

Figure 3 .Figure 3 .
Figure 3. Cell viability of HeLa cells incubated with (a) free DOX and SQ-N +L at different conc trations and (b) different ratios of free DOX and SQ-N +L.

Figure 4 .
Figure 4. (a) Cell viability of L929 cells and HeLa cells incubated with POSS-SQ NPs at diff concentrations; (b) Cell viability of HeLa cells incubated with free DOX and POSS-SQ-DOX N different concentrations; cell viability of HeLa cells incubated (c) with or without DOX and (d) or without 808 nm laser irradiation at different concentrations; (e) Confocal fluorescence imagi HeLa cells co-stained with calcein AM (green color, staining live cells) and propidium iodide color, staining dead cells) after incubation with PBS, DOX (12 µg/mL), POSS-SQ +L (18 µg/ POSS-SQ-DOX and POSS-SQ-DOX +L (50 µg/mL).The scale bar is 100 µm; (f) Quantitative ana of dead HeLa cells following different treatments.(Data are presented as the mean ± SD, n = 3 p < 0.0001).

Figure 4 .
Figure 4. (a) Cell viability of L929 cells and HeLa cells incubated with POSS-SQ NPs at different concentrations; (b) Cell viability of HeLa cells incubated with free DOX and POSS-SQ-DOX NPs at different concentrations; cell viability of HeLa cells incubated (c) with or without DOX and (d) with or without 808 nm laser irradiation at different concentrations; (e) Confocal fluorescence imaging of HeLa cells co-stained with calcein AM (green color, staining live cells) and propidium iodide (red color, staining dead cells) after incubation with PBS, DOX (12 µg/mL), POSS-SQ +L (18 µg/mL), POSS-SQ-DOX and POSS-SQ-DOX +L (50 µg/mL).The scale bar is 100 µm; (f) Quantitative analysis of dead HeLa cells following different treatments.(Data are presented as the mean ± SD, n = 3, **** p < 0.0001).

Figure 5 .
Figure 5. (a) IR thermal images of tumor-bearing mice upon 808 nm laser irradiation (0.5 W/cm 2 ) for different durations; (b) The changes in temperatures in tumor sites in every treatment group; (c-g) Tumor growth curves and the representative tumor pictures of mice in every group; (h) The tumor weight and (i) tumor inhibition rate of mice in different treatment groups; (j) Body weight curve of tumor-bearing mice in every group.(Data are presented as the mean ± SD, n = 3, ** p < 0.01, *** p < 0.001, and **** p < 0.0001).

Figure 5 .
Figure 5. (a) IR thermal images of tumor-bearing mice upon 808 nm laser irradiation (0.5 W/cm 2 ) for different durations; (b) The changes in temperatures in tumor sites in every treatment group; (c-g) Tumor growth curves and the representative tumor pictures of mice in every group; (h) The tumor weight and (i) tumor inhibition rate of mice in different treatment groups; (j) Body weight curve of tumor-bearing mice in every group.(Data are presented as the mean ± SD, n = 3, ** p < 0.01, *** p < 0.001, and **** p < 0.0001).

Table 1 .
5050and CI values of DOX and SQ-N +L at different dose ratios to HeLa cells under 808 nm NIR laser irradiation.