Black TiO 2 -based nanoparticles as Toll-like receptor stimulators delivery system for enhanced photothermal-immunotherapy of pancreatic cancer

doi:10.21203/rs.3.rs-3742375/v1

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Research Article

Black TiO 2 -based nanoparticles as Toll-like receptor stimulators delivery system for enhanced photothermal-immunotherapy of pancreatic cancer

https://doi.org/10.21203/rs.3.rs-3742375/v1

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Background

The tumor-specific immune responses, essential for removing residual lesions and preventing tumor metastases, can be stimulated by tumor-associated antigens (TAAs) released following photothermal therapy (PTT). However, due to the immunosuppressed microenvironment of pancreatic ductal adenocarcinoma (PDAC), the TAAs released by PTT are difficult to induce an effective immune response. In this work, we prepared the mesoporous silica (mSiO₂) coated black titanium dioxide (bTiO₂) photothermal nanoparticles (NPs) for enhanced photothermal-immunotherapy toward PDAC, in which resiquimod (R848) was loaded and DOTA-Gd was conjugated. The NPs are specified as bTiO₂@mSiO₂@Gd/R848 and abbreviated to NPs/R848. R848 as a kind of Toll-like receptor 7/8 agonist can induce a strong immune response against tumors. Furthermore, DOTA-Gd serves as a magnetic resonance imaging (MRI) contrast agent to improve the T₁-weighted MRI performance of the NPs.

Results

In vitro results of this study show that NPs/R848 could thermally ablate tumor cells and efficiently trigger dendritic cells (DCs) maturation. The results of in vivo investigations demonstrate that the combined use of photothermal-immunotherapy exhibits a significant inhibitory effect on tumor growth. Besides, it promoted maturation of DCs and enhanced infiltration of CD8+, CD4 + T cells to improve the tumor microenvironment.

Conclusions

Our study anticipate that by encouraging the maturation of DCs, this strategy will enhance the tumor microenvironment and enable the successful photothermal-immunotherapy of PDAC.

Pancreatic ductal adenocarcinoma

Nanoparticles

Photothermo-immunotherapy

Toll-like receptor 7/8 agonist

Pancreatic ductal adenocarcinoma (PDAC) is currently ranked as the seventh most prevalent cause of cancer-related mortality globally. Unfortunately, the current five-year survival rate for this particular disease remains below 10%, and its incidence is steadily increasing year by year (Siegel et al. 2022; Sung et al. 2021; Rawla et al. 2019). At present, the primary therapeutic approach for patients diagnosed with PDAC involves surgical excision of the affected region, followed by adjuvant radiotherapy and chemotherapy. However, this method is frequently considered unsatisfactory in its treatment outcomes (Neoptolemos et al. 2018; Vincent et al. 2011).

The utilization of nanoparticles (NPs) with a significant photothermal conversion efficiency in the field of photothermal therapy (PTT), to eliminate malignant cells and induce immunogenic cell death (ICD), has garnered considerable interest in the past few decades (Balakrishnan et al. 2020; Wang et al. 2019; Duan et al. 2019). However, because of restrictions on heat shock protein and light penetration depth, a single PTT may not be sufficient to completely ablate a tumor, which could lead to metastasis or recurrence(Huang et al. 2021; Han and Choi. 2021). Additionally, the immunosuppressed microenvironment of PDAC makes it difficult for released TAAs from PTT to induce an effective immune response (Liu et al. 2022; Nguyen et al. 2012). Immunotherapy is regarded as an additional crucial cancer treatment method because it stimulates the immune system to combat malignancies. Current immunotherapeutic approaches for cancer encompass a variety of methods such as specific immunomodulators, immune checkpoint blockade (ICB), cancer vaccines, and therapy with chimeric antigen receptor T cells (Zhang and Zhang 2020a). However, PDAC has distinct biological features that make it difficult to treat with a single immunotherapy, including a lack of blood supply, an immunosuppressed microenvironment, and high pressure for dense fibrous matrix within tumors (Hou et al. 2022; Gao et al. 2019). The results of our earlier investigations of the treatment of PDAC have demonstrated that PTT can disrupt the fibrous matrix of PDAC models, thereby enhancing the permeability of blood vessels and facilitating improved penetration of therapeutic drugs (Xu et al. 2022). It is challenging to induce an effective immune response with the TAAs generated by PTT in the immunosuppressed microenvironment of PDAC. DCs, as the major antigen-presenting cells (APCs), can induce the formation of specific cytotoxic T lymphocytes (CTLs). Due to their immaturity and low vitality, DCs in the PDAC microenvironment are unable to deliver tumor antigens to effector T cells and initiate an immune response against the tumor (Muller et al. 2022). Immune adjuvants, as non-specific immune enhancers, can function through the following mechanisms: (1) promoting antigen recognition. (2) activating DCs and macrophages. (3) inducing cytokine production (Lee and Suresh 2022). R848 is a Toll-like receptor 7/8 agonist that is recognized by ligands in antigen-presenting intracellular bodies. Its function is to stimulate intracellular signaling pathways that facilitate the maturation of DCs, resulting in the secretion of chemokines and pro-inflammatory cytokines. Effective humoral and cellular immune responses are also induced by its activation of effector T cells. Recent evidence suggests that it modulates the immune responses of both the host and tumor, leading to increased survival and reduced cachexia in murine models of PDAC (Michaelis et al. 2019; Kim et al. 2019). Significant treatment outcomes for malignancies have been observed when photodynamic therapy, immunotherapy, or chemotherapy were combined with PTT (Xu et al. 2022; Li et al. 2019; Zhou et al. 2018). Hence, the synergistic effect of PTT and the immune adjuvant R848 could increase the rate of antitumor response and impede tumor growth even further.

As a result of advancements in nanotechnology, a variety of NPs with therapeutic visualization capabilities have been designed and implemented in positron emission tomography, computed tomography, ultrasonic imaging, and magnetic resonance imaging (MRI), among others (Zhang et al. 2020b; Kim et al. 2017; Salvanou et al. 2022). NPs with visual capabilities can observe the absorption, distribution, metabolism, and excretion processes of NPs within the human body in real-time. Furthermore, these NPs can even facilitate the observation of changes in metastasis and tumor growth, enabling the evaluation of the efficacy of tumor treatment (Wang et al. 2022; Zhang et al. 2022). The integration of diagnosis and treatment under the precision medicine model has been further advanced with the advent of visual NPs.

Here, as illustrated in Scheme 1, bTiO₂ NPs with good photothermal characteristics were employed as the core; they were then loaded with an agonist (R848) and conjugated with an MRI contrast agent (DOTA-Gd) after being coated with mSiO₂ to provide drug loading sites. The NPs exhibited thermally ablate tumor cells and stimulated the release of TAAs via PTT. Toll-like receptor 7/8 agonist R848 enhanced the immunosuppressive microenvironment of the tumor, promoted the maturation of DCs to enhance the efficacy of TAAs presentation, and stimulated T cell activation to induce an antitumor immune response. Meanwhile, DOTA-Gd has facilitated the acquisition of T₁-weighted MRI capabilities by the NPs, enabling the visualization of photothermo-immunotherapy for PDAC.

The synthesis of bTiO₂@mSiO₂@Gd/R848 NPs and mechanism of bTiO₂@mSiO₂@Gd/R848 for enhanced photothermo-immunotherapy of Pancreatic Cancer.

2.1 Agents Reagents: TiO₂ NPs, tetraethyl orthosilicate (TEOS), sodium borohydride (NaBH₄), hexadecyltrimethylammonium bromide (CTAB), cyclohexane (C₆H₁₂), concentrated hydrochloric acid (HCl), sodium hydroxide (NaOH), ethanol (C₂H₅OH), 3-(4,5-di-methylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), Rhodamine B (C₂₈H₃₁C_lN₂O₃), and dimethyl sulfoxide (DMSO) were acquired from Aladdin Industrial Inc. (Shanghai, China). The mono-N-hydroxysuccinimide ester (DOTA-NHS Ester) was supplied by Yare Bio Co., Ltd (Shanghai, China). Macklin Biochemical Co., Ltd (Shanghai, China) provided the 3-aminopropyltriethoxysilane (APTES) and Gadolinium acetate hydrate (C₆H₁₁GdO₇). Fetal bovine serum (FBS), DMEM medium, RPMI-1064 medium, and Trypsin-EDTA 0.25% were obtained from Gibco (Grand Island, USA). The phosphate buffer saline (PBS) and penicillin-streptomycin solutions were acquired from GE Healthcare HyClone (LA, USA). Solarbio Biotech Co., Ltd. (Beijing, China) supplied both formaldehyde solution and Hoechst 33258 (C₂₅H₂₄N₆O·3HCl). Beyotime Biotech Co., Ltd (Shanghai, China) provided the calcein-AM/PI double stain kit. Resiquimod (R848) was obtained from InvivoGen (San Diego, CA, USA). Binding buffer was purchased from Immunostep (Salamanca, Spain).

2.2 Synthesis of mSiO₂ coated bTiO₂ based nanoparticles

2.2.1 Synthesis of bTiO₂ nanoparticles

The TiO₂ was reduced to bTiO₂ employing NaBH₄ as the reducing agent. Initially, 1 g of TiO₂ powder was mixed with NaBH₄ at a 1:1 mass ratio for 30 min before being placed in a tube furnace heated with 10°C min^− 1. Under an argon atmosphere, the temperature was raised to 350°C, and the reaction lasted for 3 h. The produced bTiO₂ NPs were washed with ultrapure water, centrifuged thrice to eliminate NaBH₄, and then dried for further use.

2.2.2 Synthesis of bTiO₂@mSiO₂ nanoparticles

Ultrasound was employed to disperse 100 mg of bTiO₂ NPs in 20 mL of water. 0.3 mL of 0.1 mM NaOH solution and 500 mg of CTAB were added to the bTiO₂ solution, which was then heated to 60℃ with magnetic stirring. 20 mL of cyclohexane containing TEOS (20 V/V%) was added after 2 h of magnetic stirring, and the mixture was then continuously stirred at 60℃ for 24 h. CTAB was removed from mSiO₂-coated bTiO₂ by heating it to 80°C in acetone following centrifugation and alternating washings with ethanol and water. Centrifugation was then employed to collect the as-prepared bTiO₂@mSiO₂ NPs.

2.2.3 Synthesis of DOTA-Gd modified bTiO₂@mSiO₂

The surface of bTiO₂@mSiO₂ was amino-functionalized by APTES. In particular, 20 mg bTiO₂@mSiO₂ NPs were dispersed in 20 mL of ethanol, followed by the addition of 2 mL of APTES and heating to 80℃ while stirring for 12 h. After centrifugation, the prepared bTiO₂@mSiO₂-NH₂ NPs were collected and subjected to modification using gadolinium acetate hydrate and DOTA-NHS-Ester. The PH value of the gadolinium acetate hexahydrate solution was adjusted to 6.5 by adding NaOH after an excessive quantity of DOTA-NHS-ester was introduced. Following a 24-hour stirring period, the dispersion of bTiO₂@mSiO₂-NH₂ was introduced into the reaction system. The pH value was subsequently adjusted to a range of 7.5 to 8.5, and the mixture was stirred for an additional 24 h at ambient temperature. The DOTA-Gd modified bTiO₂@mSiO₂@Gd NPs were obtained after centrifugation.

2.2.4 Synthesis of bTiO₂@mSiO₂@Gd/R848 nanoparticles

R848 was loaded by physical adsorption. Briefly, bTiO₂@mSiO₂@Gd and R848 were mixed in sterile water. After stirring for 24 h, the bTiO₂@mSiO₂@Gd/R848 NPs were obtained by centrifugation.

2.3 Characterization

Transmission electron microscopy (TEM, FEI Tecnai F20) was utilized to examine the micro-morphologies of NPs. The zeta potential and hydrated particle size distribution of the NPs were determined using a nano-size zeta potential analyzer (Nano ZS, Malvern Instruments Ltd, England). The UV-visible absorption spectra of the NPs were examined using a UV-visible spectrophotometer (T10CS, Persee General Equipment Co., Ltd, China). The Fourier transform infrared spectrometer (FTIR, Thermo Nicolet 6700, US) was employed to acquire the infrared (IR) spectra of the NPs. The elemental composition was examined using an inductively coupled plasma optical emission spectrometry (ICP-OES) system developed by Spectro Analytical Instruments GmbH of Germany. MR scanners were utilized to examine the relaxation and MRI characteristics of NPs. Furthermore, to assess the photothermal performance of bTiO₂@mSiO₂@Gd/R848 NPs, cuvettes were filled with 1 mL of bTiO₂@mSiO₂@Gd/R848 dispersions with varying concentrations of Ti (0-200 µg mL^− 1) and exposed to 808 nm near infrared (NIR) radiation for 600 s at 1.5 W cm^− 2. To determine the power-dependent photothermal performance in the NIR, 1 mL of bTiO₂@mSiO₂@Gd/R848 dispersions containing 150 µg mL^− 1 Ti were exposed to an 808 nm NIR for 600 s at different power densities (varying from 0.5 to 2.0 W cm^− 2). To assess photothermal stability, 6 on/off repeating cycles of 808 nm NIR (1.5 W cm^− 2) were applied to 1 mL of bTiO₂@mSiO₂@Gd/R848 every 300 s. The imaging acquired through the use of the IR thermal imaging equipment was compiled for subsequent comparisons, and the temperature values continually collected were analyzed and represented as time-temperature curves.

2.4 Cytotoxicity of the nanoparticles

To examine the cytotoxicity of bTiO₂@mSiO₂@Gd/R848 and bTiO₂@mSiO₂@Gd NPs, Panc02 cells were cultured for 24 h in 96-well plates after seeding. After removing the old culture medium, various amounts of NPs (ranging from 50 to 300 µg mL^− 1) were introduced to each well and left to incubate for 24 h in the incubator. Following that, MTT reagent (20 µL; 5 mg mL^− 1 in PBS) was introduced to each well and left for another 4 h. After removing the mediums, DMSO was employed to dissolve the formazan crystals. Cell viabilities were calculated by detecting the absorbance of formazan solutions using the microplate absorbance reader.

2.5 Characterization of cellular uptake

The uptake of bTiO₂@mSiO₂@Gd/R848 NPs by Panc02 cells was observed by fluorescence confocal microscopy. Rhodamine B, a fluorescent dye, was utilized to label the bTiO₂@mSiO₂@Gd/R848 NPs. Before being treated with DMEM and bTiO₂@mSiO₂@Gd NPs for 4, 8, and 12 h, respectively, Panc02 cells were cultivated in a 35 mm petri dish and incubated for 24 h. Before being fixed in 4% paraformaldehyde, the cells were washed twice in PBS. After that, the cells were washed with PBS and then treated with Hoechst 33342 (2 µg mL^− 1) to stain the nucleus.

2.6 In vitro PTT evaluation

Panc02 cells were cultured for 12 h in 96-well plates with DMEM, bTiO₂@mSiO₂@Gd, or bTiO₂@mSiO₂@Gd/R848 NPs at 150 µg mL^− 1 Ti concentrations. The media were subsequently substituted with fresh DMEM. Each well was exposed to an 808 nm NIR laser for 5 or 10 min at 0.5, 1.0, or 1.5 W cm^− 2 power densities, respectively. Cell viability was then evaluated using MTT. In addition, the CAM/PI staining approach was utilized to directly evaluate the cell state following therapy. Panc02 cells were specifically exposed to 808 nm NIR (1.5 W cm^− 2) radiation for 0, 5, or 10 min after being cultured for 12 h in DMEM or bTiO₂@mSiO₂@Gd/R848 NPs with Ti (150 µg mL^− 1). The cells were then examined using fluorescence microscopy after being stained with CAM/PI.

2.7 PTT synergistic immunotherapy promotes DCs maturation in vitro

The transwell method was implemented as a means to examine the impact of NPs on the in vitro maturation of DCs. The upper section included Panc02 cells after various treatments, while the lower section contained DCs. The various treatments were as follows: Following incubation with DMEM (1), bTiO₂@mSiO₂@Gd NPs (2), and bTiO₂@mSiO₂@Gd/R848 NPs (3), the Panc02 cells were irradiated with or without 808 nm NIR (1.5 W cm^− 2), respectively. Each cell was cultured for an additional 32 h. After collecting and staining DCs in the lower chamber with CD11c, CD86, and CD80, flow cytometry was employed to examine the expression of co-stimulatory molecules CD86 and CD80 on DCs in various treatment groups.

2.8 In vivo toxicity evaluation of nanoparticles

Female C57BL/6 mice (6 − 8 weeks) were randomly divided into three groups to assess the in vivo toxicity of the NPs. The mice were administered intravenous injections of PBS, bTiO₂@mSiO₂@Gd, or bTiO₂@mSiO₂@Gd/R848, and their weights were then measured during a feeding period of 20 days. After 20 days, all mice were sacrificed. Routine blood tests were performed on the mice in each group, and their major organs, such as the lung, heart, liver, and spleen, were fixed in a 10% formalin solution and histologically examined using hematoxylin and eosin (H&E) staining.

2.9 In vivo MR evaluation

The mice were injected with 1 × 10⁶ Panc02 cells orthotopically to establish the PDAC models. Both PBS and NPs/R848 were administered intratumorally to the mice. The MR images of the tumors were captured by a 3.0T MRI system (MAGNETOM Prisma, Siemens).

2.10 In vivo PTT synergistic immunotherapy

Six groups of female C57BL/6 mice (6–8 weeks) were randomly assigned. To generate the PDAC models, 1 × 10⁶ Panc02 cells were injected orthotopically into the mice. The tumors were then given time to grow until they reached 250 mm³ before experiments. To assess the synergistic immunotherapy of PTT in vivo, the following treatments were administered to the tumor-injected animals: (1) In the NPs/R848 or NPs/R848 + Laser groups, bTiO₂@mSiO₂@Gd/R848 was administered, followed by 808 nm NIR laser irradiation or not. (2) The NPs or NPs + Laser groups were given bTiO₂@mSiO₂@Gd before being exposed to an 808 nm NIR laser or not. (3) The PBS or PBS + Laser group received PBS before being irradiated with an 808 nm NIR laser or not. The body weights and tumor volumes of various groups were assessed on alternate days over 14 days. Furthermore, the main tumors retrieved from the mice on the third day of treatment were embedded in paraffin wax after being preserved in neutral buffered formalin (10%). Subsequently, the embedded tissue samples underwent sectioning and were subjected to blocking using a 2.5% Normal Goat Serum Blocking Solution. Following this, the samples were stained with H&E, CD80, CD86, CD4, and CD8.

Data were presented as mean ± standard deviation and compared using the student’s t-test. P < 0.05 was set as the significance level.

4.1 Characterization of as-prepared nanoparticles

Scheme 1 depicts the synthesis of bTiO₂@mSiO₂@Gd/R848 NPs. To improve drug loading efficiency, core-shell nanocomposites were formed by coating bTiO₂ NPs with mSiO₂. In addition, Gadolinium (Gd) was subsequently used to integrate the MRI function with the nanoparticles. The DOTA-NHS-Ester reagent was attributed to conjugate amino groups through the NHS-ester and chelate Gd³⁺ ion by DOTA (Wang et al. 2018). Finally, R848 was loaded on NPs by physical adsorption. TEM analysis revealed the morphological characteristics of bTiO₂@mSiO₂@Gd and bTiO₂@mSiO₂@Gd/R848 NPs (Fig. 1a-b), respectively. According to the TEM analysis, the average size of the bTiO₂@mSiO₂@Gd/R848 NPs was around 149.8 nm. According to Fig. 1c, which displays the FTIR spectra of several samples, bTiO₂@mSiO₂@Gd exhibited peak shifts at C = O (1576 cm^− 1) and C-N (1483 cm^− 1), suggesting that an amide reaction took place between bTiO₂@mSiO₂-NH₂ and DOTA-Gd, leading to the successful connection of DOTA-Gd. The FTIR spectrum of bTiO₂@mSiO₂@Gd/R848 changed significantly in comparison to bTiO₂@mSiO₂@Gd, this change was ascribed to the loading of R848. The mean hydrated sizes of bTiO₂@mSiO₂, bTiO₂@mSiO₂-NH₂, bTiO₂@mSiO₂@Gd, and bTiO₂@mSiO₂@Gd/R848 were approximately 191.3 nm, 240.7 nm, 288.9 nm, 290.5 nm, respectively (Fig. 1d). The results indicate that particle sizes gradually increase with the modification of related components. In addition, the zeta potentials of R848, bTiO₂@mSiO₂, bTiO₂@mSiO₂-NH₂, bTiO₂@mSiO₂@Gd and bTiO₂@mSiO₂@Gd/R848 were about − 16 mV, -26.2 mV, 19.4 mV, 31.7 mV, -8.5 mV, respectively (Fig. 1e). The surface charge of bTiO₂@mSiO₂@Gd exhibited a reduction from 31.7 to -8.5 mV after the introduction of agonist R848 as determined by the DLS. R848 is successfully loaded onto bTiO₂@mSiO₂@Gd, as evidenced by the partial neutralization of its positive charge by the negative charge of R848. The bTiO₂@mSiO₂@Gd/R848 with negative surface charge prolongs blood circulation and efficiently accumulates into tumor tissues through enhanced permeability and retention effect, making it potentially useful for in vivo studies (Zhang et al. 2022). As shown in the UV-visible spectra of various samples, there was no substantial change in the absorption peak of bTiO₂@mSiO₂@Gd/R848 (Fig. 1f).

4.2 MR and photothermal performance properties

The MR characteristics of NPs were assessed utilizing a 0.5 T MR scanner system. Magnevist, a commercial contrast agent, was utilized as the control. Figure 2a and 2b show that at the same dose of Gd, the r₁ values for bTiO₂@mSiO₂@Gd/R848 and Magnevist were 6.623 and 4.099 mM^− 1 s^− 1, respectively. The corresponding values for r₂ were 7.772 mM^− 1 s^− 1 and 4.284 mM^− 1 s^− 1, respectively. The r₂/r₁ ratio of bTiO₂@mSiO₂@Gd/R848 is around 1.17, suggesting that the NPs exhibit potential as MRI contrast agents due to their superior T₁-weighted contrast efficacy (r₂/r₁ < 3) (Guo et al. 2017). Figure 2c displays the T₁-weighted MR imaging of bTiO₂@mSiO₂@Gd/R848.

Previous studies have demonstrated that bTiO₂ NPs exhibit a significant photothermal capability (Wang et al. 2018). The PTT performance of the bTiO₂@mSiO₂@Gd/R848 NPs that were synthesized by modifying bTiO₂ NPs with silicon oxide was assessed in this investigation. After being exposed to 808 nm NIR light for 10 min at various power densities, the temperatures of 1 mL bTiO₂@mSiO₂@Gd/R848 dispersions (150 µg mL^− 1) rapidly increase, as illustrated in Fig. 3a. As the 808 nm NIR power density increased, there was a corresponding acceleration in the rise of dispersion temperature. Following a 10-min exposure to 1.5 W cm^− 2 of 808 nm NIR radiation, the temperature of 1 mL bTiO₂@mSiO₂@Gd/R848 dispersions (150 µg mL^− 1) increased to around 50℃. Within minutes of exposure, cancer cells can experience fast ablation at this temperature (Qi et al. 2019). Subsequently, the dispersions were exposed to 1.5 W cm^− 2 of 808 nm NIR radiation at various concentrations, varying from 0 to 200 µg mL^− 1. As depicted in Fig. 3b, an increase in the concentration of Ti in dispersions leads to a more rapid rise in the temperature of the dispersions. The temperature curves in Fig. 3a and 3b are consistent with the real-time photothermal images shown in Fig. 3c I and II. The photothermal stability of NPs is also very important. As shown in Fig. 3d, bTiO₂@mSiO₂@Gd/R848 were exposed to on/off cycles of 808 nm NIR (1.5 W cm^− 2) every 5 min for 1 h. Throughout the irradiation cycle, the temperature curve for heating and cooling remained constant, indicating that bTiO₂@mSiO₂@Gd/R848 NPs are stable when exposed to NIR radiation.

4.3 Cell uptake of nanoparticles

The uptake and internalization of NPs by cancer cells are crucial for cancer therapy. The cell nuclei stained with Rhodamine B-labeled NPs in red and Hoechst 33342 in blue were observed and recorded using fluorescence confocal microscopy. The increase in co-incubation time resulted in a corresponding increase in cellular absorption of NPs, as illustrated in Fig. 4. The findings suggest that Panc02 cells can internalize bTiO₂@mSiO₂@Gd/R848 NPs.

4.4 Cytotoxicity assay

Although earlier research has established the reduced toxicity of bTiO₂ NPs in both in vitro and in vivo settings (Xu et al. 2022), it is important to note that the inclusion of CTAB as a template during nanocomposite synthesis and the loading of R848 further necessitates an evaluation of the cytotoxicity of bTiO₂@mSiO₂@Gd/R848 NPs. The cytotoxic effects of bTiO₂@mSiO₂@Gd and bTiO₂@mSiO₂@Gd/R848 on Panc02 cells are illustrated in Fig. 5a. After incubation for 24 h, more than 80% of the cells were viable for NPs containing Ti concentrations between 50 and 300 µg mL^− 1, indicating that the NPs have minimal cytotoxicity and can be applied in biotherapy.

4.5 PTT performance of pancreatic cancer in vitro

The systematic evaluation of the PTT efficacy of the NPs in vitro was conducted using the MTT assay, as illustrated in Fig. 5b. Before being exposed to 808 nm NIR radiation under various settings, Panc02 cells were maintained in DMEM, bTiO₂@mSiO₂@Gd (NPs), and bTiO₂@mSiO₂@Gd/R848 (NPs/R848) for 12 h. Following a 10-min exposure with 808 nm NIR at 1.5 W cm^− 2 a power density, the cell survival rates were 80.1%±2.9% for the Laser group, 40.9%±4.0% for the NPs group, and 39.7%±1.4% for the NPs/R848 group. Tumor cells were effectively photothermally destroyed by both bTiO₂@mSiO₂@Gd and bTiO₂@mSiO₂@Gd/R848 NPs, with the substance R848 having no discernible effect on this outcome, according to the results.

Moreover, the live/dead cell staining assay was employed to investigate the PTT performance of NPs on Panc02 cells. As depicted in Fig. 5c, live cells were labeled with green fluorescence (Calcein-AM), while dead cells were indicated with red fluorescence (PI). After being exposed to a 1.5 W cm^− 2 NIR laser, most of the panc02 cells in DMEM survived, however many Panc02 cells in bTiO₂@mSiO₂@Gd/R848 died. An increase in irradiation duration resulted in a greater destruction of Panc02 cells. The results obtained from the live/dead cell staining are in agreement with the outcomes of the MTT test. The aforementioned findings suggest that bTiO₂@mSiO₂@Gd/R848 NPs exhibit a strong photothermal killing effect on Panc02 cells.

4.6 PTT synergistic immunotherapy promoted DCs maturation in vitro

DCs have an essential role as a link between the adaptive and innate immune responses, making them one of the most important APCs. Both naive cytotoxic T cells (CD8+) and helper T cells (CD4+) can be activated by mature DCs. To examine the in vitro impact of bTiO₂@mSiO₂@Gd/R848 NPs on the promotion of DCs maturation, the transwell system is implemented. The results presented in Fig. 6 indicate that the maturation of DCs was significantly increased from 17.3–48.5% in the Panc02 cells co-cultured with NPs/R848 without irradiation (NPs/R848 (-) group), in comparison to the NPs (-) group where R848 was absent. In addition, at the same dose of R848 as the NPs/R848 group, NPs/R848 combined with irradiation (NPs/R848 (+) group) increased DCs maturation by 53.4%. Moreover, the maturation of DCs was observed to increase from 0.59–17.3% in the NPs (-) group without irradiation, in comparison to the control (-) group. This finding suggests that bTiO₂@mSiO₂@Gd NPs, in the absence of the R848 drug, also serve as an immune adjuvant and have the ability to stimulate DCs maturation.

4.7 In vivo toxicity evaluation of nanoparticles

The body weights, blood routine indicators, and organ histology of mice were evaluated to determine the toxicity of the treatments. Following injection, mice in each group were weighed within 20 days (Fig. 7a). Figure 7b and 7c show the blood routine indexes and histology of the organs after injection for 20 days. There were no appreciable changes in blood routine indicators or significant losses in body weights when compared to the PBS-injected control group. Moreover, the bTiO₂@mSiO₂@Gd and bTiO₂@mSiO₂@Gd/R848 injected groups exhibited no discernible tissue injury or other lesions, including necrosis, inflammation, or pulmonary fibrosis. These findings demonstrate that the treatments are safe and well-tolerated in PDAC models.

4.8 Photothermal imaging, MRI, and PTT synergistic immunotherapy in vivo

Photothermal imaging of bTiO₂@mSiO₂@Gd/R848 and PBS-injected tumor-bearing mice exposed to 808 nm NIR is presented in Fig. 8a. Compared to a control group of mice that were injected with PBS and exposed to 808 nm NIR radiation, the group that received injections of NPs/R848 showed a significantly rapid increase in tumor site temperature after the injections. Figure 8b illustrates the temperature profile at the sites of Tumors when exposed to NIR irradiation. Exposure to temperatures above 50°C has been reported to cause the ablation of cancer cells within a few minutes (Qi et al. 2019). Previous research indicated that exposing cells to 50°C effectively killed tumor cells in the NPs/R848 group following NIR irradiation. Figure 8c depicts MRI images of mice from different groups. The intratumoral T₁-weighted MRI of the NPs/R848 group showed a strong signal, confirming that NPs/R848 has T₁-weighted MRI performance, in comparison to the PBS group.

Tumor-injected mice were divided into six groups and treated for 14 days to evaluate PTT synergistic immunotherapy in vivo. Changes in tumor volume after treatment are illustrated in Fig. 9a. The tumors exhibited a significant increase in growth rate upon treatment with either PBS or PBS combined with NIR irradiation (PBS + Laser). In comparison to the control group (PBS), tumor growth was slightly suppressed in mice treated with bTiO₂@mSiO₂@Gd (NPs) or bTiO₂@mSiO₂@Gd/R848 (NPs/R848). However, mice injected with bTiO₂@mSiO₂@Gd or bTiO₂@mSiO₂@Gd/R848 and exposed to NIR irradiation (NPs + Laser) or (NPs/R848 + Laser) displayed tumor growth inhibition. Notably, the NPs/R848 + Laser group demonstrated significantly stronger antitumor efficacy because of the combined impact of immunotherapy and PTT. To assess the toxicity of the treatments, the body weight of the mice was measured after 12 days; no statistically significant variations were observed between groups (Fig. 9b), suggesting that the treatments are tolerable and safe for use in PDAC models. Figure 9c depicts tumor images with varying degrees of antitumor activity across different treatment groups. Figure 9d further illustrates the variation in cell damage between these groups.

4.9 Immune Response in Vivo

In vitro experiments indicate that the Panc02 cells residue post NIR-induced PTT with NPs/R848 significantly accelerates the maturation of DCs in comparison to the other groups. Based on these findings, it appears that NPs/R848 can be used as an adjuvant in photothermal-immunotherapy to stimulate the immune response against cell residues containing TAAs. In further in vivo experiments, the levels of DCs maturation were investigated through immunofluorescence staining. In comparison to the other groups, the NPs/R848 + Laser group demonstrated greater DCs maturation (Fig. 10a). This outcome is in line with the flow analysis, which showed that in vitro, the NPs/R848 + Laser group caused higher DCs maturation than the other groups. This indicates that more DCs are drawn to the tumor site as APCs to engulf the TAAs and DAMPs released by apoptotic tumor cells and mature with the aid of released R848. Having the ability to eliminate cancer cells, CD8 + and CD4 + T cells function as the ultimate effector T cells for tumor immunity. The tumor microenvironment causes immunologic tolerance, which in turn causes CTLs to become dysfunctional and exhausted during carcinogenesis. Hence, further immunofluorescence staining was carried out to determine if NPs/R848 altered tumor CTL activation for photothermo-immunotherapy. Figure 10b shows that the NPs/R848 + Laser group had considerably increased levels of CD4 + and CD8 + T cell expression compared to the other groups. This suggests that the combinatorial therapy had a positive effect on CD8 + and CD4 + expression, as well as their associated immune responses, leading to a remarkable improvement in antitumor efficiency. All of these results indicate that this combinatorial therapy effectively promotes DCs maturity and increases the number of CD8 + and CD4 + T cells, stimulates an antitumor immune response, and improves the immunosuppressed microenvironment of PDAC.

In conclusion, the present investigation involves the development of bTiO₂@mSiO₂@Gd/R848 as a PTT synergistic immunotherapeutic agent for PDAC and capable of inducing direct harm to cancer cells via PTT, promoting DCs maturation, and thereby stimulating T cell-associated immune responses. As evidenced by in vitro tests, this synergistic therapy successfully stimulates DCs maturation and destroys tumor cells upon NIR-induced PTT. In vivo results further showed that synergistic therapy activates T cell-related immune responses to inhibit tumor growth. Furthermore, the incorporation of DOTA-Gd into the NPs enables the use of T₁-weighted MRI to monitor the progress of tumor treatment. This work emphasizes the tremendous potential of combining immunotherapy and bTiO₂-based PTT as an excellent synergistic therapeutic method to treat PDAC while expanding the use of bTiO₂ in photothermal-immunotherapy.

Ethics approval and consent to participate

For animal experiments, animal care and handling procedures were in agreement with the guidelines of the Regional Ethics Committee for Animal Experiments at Ningbo University.

Consent for publication

Not applicable.

Availability of data and materials

All data generated or analysed during this study are included in this published article.

Competing interests

The authors declare that they have no competing interests.

Funding

This study was supported by the National Natural Science Foundation of China (No.82172004), Ningbo Public welfare science and technology project (No.2021S181) and Project of Ningbo Leading Medical & Health Discipline (No.2022-B15-A).

Author contributions

L.X., R.W. and J.N. contributed equally to this work. L.X. prepared nanoparticles and wrote the manuscript. R.W., J.N. and L.J. did a part of animal experiments. K.X., Y.Z., L.H. and C.C. conducted a statistical analysis of the study. L.W. and L.Z. analyzed the results of MRI. W.Z. analyzed the Figures of tissue staining. J.W. provided financial support, supervised the study, and reviewed the manuscript. All authors readand approved the final manuscript.

Acknowledgments

The authors thank Ningbo Cixi Institute of Biomedical Engineering for the help of synthesis and characterization of nanoparticles.

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You are reading this latest preprint version

Black TiO 2 -based nanoparticles as Toll-like receptor stimulators delivery system for enhanced photothermal-immunotherapy of pancreatic cancer

Status:

Version 1

Abstract

Background

Results

Conclusions

Figures

1. Introduction

2. Materials and methods

2.2 Synthesis of mSiO2 coated bTiO2 based nanoparticles

2.2.1 Synthesis of bTiO2 nanoparticles

2.2.2 Synthesis of bTiO2@mSiO2 nanoparticles

2.2.3 Synthesis of DOTA-Gd modified bTiO2@mSiO2

2.2.4 Synthesis of bTiO2@mSiO2@Gd/R848 nanoparticles

2.3 Characterization

2.4 Cytotoxicity of the nanoparticles

2.5 Characterization of cellular uptake

2.6 In vitro PTT evaluation

2.7 PTT synergistic immunotherapy promotes DCs maturation in vitro

2.8 In vivo toxicity evaluation of nanoparticles

2.9 In vivo MR evaluation

2.10 In vivo PTT synergistic immunotherapy

3. Statistical analysis

4. Results and discussion

4.1 Characterization of as-prepared nanoparticles

4.2 MR and photothermal performance properties

4.3 Cell uptake of nanoparticles

4.4 Cytotoxicity assay

4.5 PTT performance of pancreatic cancer in vitro

4.6 PTT synergistic immunotherapy promoted DCs maturation in vitro

4.7 In vivo toxicity evaluation of nanoparticles

4.8 Photothermal imaging, MRI, and PTT synergistic immunotherapy in vivo

4.9 Immune Response in Vivo

5. Conclusions

Declarations

References

Additional Declarations

Supplementary Files

Status:

Version 1

2.2 Synthesis of mSiO₂ coated bTiO₂ based nanoparticles

2.2.1 Synthesis of bTiO₂ nanoparticles

2.2.2 Synthesis of bTiO₂@mSiO₂ nanoparticles

2.2.3 Synthesis of DOTA-Gd modified bTiO₂@mSiO₂

2.2.4 Synthesis of bTiO₂@mSiO₂@Gd/R848 nanoparticles