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An in situ nanoparticle recombinant strategy for the enhancement of photothermal therapy
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Xinxin Liua, b, c, d, e, Cuixia Zhengb, c, d, Yueyue Kongb, c, d, Hao Wangb, c, d, Lei Wanga, b, c, d, e, *
Chinese Chemical Letters | 2022, 33(1) : 328 - 333
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Chinese Chemical Letters | 2022, 33(1): 328-333
• Communication •
An in situ nanoparticle recombinant strategy for the enhancement of photothermal therapy
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Xinxin Liua, b, c, d, e, Cuixia Zhengb, c, d, Yueyue Kongb, c, d, Hao Wangb, c, d, Lei Wanga, b, c, d, e, *
Affiliations
  • a Luoyang Central Hospital Affiliated to Zhengzhou University, Luoyang 471009, China
  • b School of Pharmaceutical Sciences, Zhengzhou University, Zhengzhou 450001, China
  • c Key Laboratory of Advanced Drug Preparation Technologies, Ministry of Education, School of Pharmaceutical Sciences, Zhengzhou University, Zhengzhou 450001, China
  • d Henan Key Laboratory of Targeting Therapy and Diagnosis for Critical Diseases, Zhengzhou University, Zhengzhou 450001, China
  • e Tumor Immunity and Biomaterials Advanced Medical Center, Zhengzhou University, Luoyang 471009, China
Published: 2022-01-15 doi: 10.1016/j.cclet.2021.07.025
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Photothermal therapy (PTT)-induced immune response has attracted much attention, however, which cannot work at full capacity. In this study, the simvastatin (SV) adjuvant is loaded into gold nanocages (AuNCs) to develop a simple drug delivery system, which can efficiently utilize the tumor-associated antigens (TAAs) for improving immune responses. AuNCs/SV-mediated PTT treatment enhances tumor cells damage and promotes the release of TAAs which are immediately captured by AuNCs/SV to form AuNCs/SV/TAAs recombinant nanoparticle. Impressively, AuNCs/SV/TAAs can accumulate in lymph nodes effectively due to the suitable size of ~55 nm and hyperthermia-induced vasodilative effect. And the co-delivery of antigen and adjuvant is beneficial to stimulating the maturation of dendritic cells for further activating T cells. In a word, the recombinant strategy could make full use of TAAs to produce an individual powerful immunotherapy.

Photothermal therapy  /  Tumor-associated antigens  /  Recombinant nanoparticle  /  T cells activation  /  Individual immunotherapy
Xinxin Liu, Cuixia Zheng, Yueyue Kong, Hao Wang, Lei Wang. An in situ nanoparticle recombinant strategy for the enhancement of photothermal therapy[J]. Chinese Chemical Letters, 2022 , 33 (1) : 328 -333 . DOI: 10.1016/j.cclet.2021.07.025
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Currently, cancer is still one of the diseases with high morbidity and mortality in the world [1–4]. Recently, as an important cancer treatment method, photothermal therapy (PTT) based on nanomaterials has attracted much attention because of its minimally invasive, high efficiency and few adverse reactions [5–9]. PTT could induce dying tumor cells to release tumor-associated antigens (TAAs) and damage-associated molecular model (DAMPs) to stimulate the host immune system [10–12]. However, the immune response induced by hyperthermia is relatively weak and short-lived [13]. At present, how to make better use of TAAs produced in the process of PTT is very important to amplify the immune effect induced by PTT.
Researches have showed that nanoparticles (NPs) could enhance immune response due to the effective delivery of antigen and/or adjuvant [14–17]. For example, large-pore mesoporous organosilica nanospheres could deliver antigens to dendritic cells (DCs) and then activate DCs effectively [18]. Polymer/lipid NPs could also mediate the retention of vaccine cargo in DCs, enhancing DCs activation and the body's immune response [19]. Unfortunately, traditional nanovaccines are always using the pre-extracted antigens which are complicated and costly, limiting their applications [20–22]. Using TAAs produced by PTT to construct recombinant vaccine-like NPs in tumor site can not only solve the above problem, but also improve the therapeutic efficiency of PTT.
As one of the PTT intensifiers, gold nanocages (AuNCs) with hollow mesoporous structure and unique surface property are suitable for small molecule drugs loading and protein capture, which are beneficial for the protection of adjuvant and TAAs [23–27]. Besides, AuNCs can migrate to lymph nodes (LNs) easily due to the size of 20~100 nm and PTT-induced blood vessels dilatation, which are beneficial to activating systemic immune response [28,29].
Herein, AuNCs/SV NPs were constructed by placing simvastatin (SV) adjuvant in the cavity of AuNCs. SV is a mevalonate pathway inhibition-based immunological adjuvant, known to increase antigen presentation via prolonged antigen preservation during endocytosis [30]. With the irradiation of near-infrared (NIR) laser, tumor could be killed by AuNCs/SV directly and released TAAs that would be captured by AuNCs/SV to construct an in situ NPs (AuNCs/SV/TAAs). AuNCs/SV/TAAs of ~55 nm could be advantageous to migrate to the LNs, especially with the help of PTT-induced vasodilation, and ingested by DCs to promote the differentiation of CD4+ T cells and CD8+ T cells (Fig. 1). In general, this work established a possibility that AuNCs-based PTT can be used to amplify PTT-immunotherapy.
The AuNCs/SV were synthesized with the following steps. Briefly, 28.5 nm AgNPs were developed firstly [31]. Then we synthesized AuNCs on the basis of AgNPs with the help of HAuCl4 [32,33]. As shown in Fig. 2a, transmission electron microscope (TEM) image of the AuNCs clearly showed a hollow mesoporous structure. Furthermore, the Au, Ag, N and C elements were distributed in AuNCs (Fig. 2b), suggesting the successful etching on AgNPs for the construction of AuNCs. Then, the size of AuNCs/SV was confirmed to be about 50 nm by dynamic light scattering analysis (DLS, Fig. 2c). The UV absorption spectrum (Fig. 2d) of AuNCs showed a new broad peak between 200 nm and 300 nm, indicating that SV was loaded into AuNCs. Subsequently, the photothermal conversion of AuNCs/SV was detected. When AuNCs solution (50 µg/mL) was irritated with NIR laser (808 nm) for 5 min, its temperature increased from 25.0 ℃ to 55.4 ℃, while the temperature of deionized water has no obvious change (Fig. 2e). As shown in Fig. 2f, when the feed of AuNCs and SV ratio was increased to 1/1, the entrapment efficiency did not increase significantly and the entrapment efficiency of SV reached to ~22%.
Antigen capture and LN-targeting ability of AuNCs/SV are crucial for stimulating effective cancer treatment. We first evaluated the formation of in situ recombinant NPs. The TEM image (Fig. 3a) of AuNCs/SV with laser irradiation showed that there was a halo covered the original AuNCs. And the zeta potential and size (Figs. 3b and c) of AuNCs/SV changed obviously after laser irradiation, hinting that the AuNCs/SV captured TAAs successfully. Gel electrophoresis (Fig. 3d) showed that the proteins captured by AuNCs were different from those of B16-F10 cell lysates. To further confirm the presence of specific antigens on the AuNCs-bound proteins, the mass spectrometry was performed to determine if any of the captured proteins contained antigens expressed by B16-F10 cells. As shown in Fig. 3e, there were many neoantigens and DAMPs being detected which were in favor of the immune effect [34,35]. Then western blotting analysis (Fig. 3f) was adopted on tumor-specific antigens (Tnpo3 and Cpsf3l) that could initiate antitumor immune response. These results proved that the in situ effective recombinant NPs could be successfully constructed.
Afterwards, to confirm that AuNCs could deliver TAAs to LNs, we injected Cy5 labeled-AuNCs intratumourally to study the efficiency of lymphatic drainage. The fluorescence intensity of AuNCs/SV/Cy5 group was obviously stronger than free Cy5 at 12 h post-administration (Figs. 3g and h), showing AuNCs could easily deliver antigens to nearby LNs (axillary LNs), which was instrumental in augmenting systemic immune response.
In addition, we investigated the cellular uptake of isothiocyanate (FITC)-labeled AuNCs in B16-F10 cells by flow cytometry and microscope. As depicted in Fig. 4a, the fluorescence intensity in cells increased in a time-dependent manner. And at 4 h, we found that the presence of AuNCs/SV could enhance the uptake of preparations compared with the FITC group (Fig. 4b). In order to study the effect of 808 nm laser on cell proliferation, MTT (3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide) assay was chosen to evaluate cell viability. AuNCs/SV group showed less cytotoxic to tumor cells (with cell viability higher than 92%) (Fig. 4c). However, after laser irradiation, AuNCs/SV exhibited the significant cytotoxicity, and only approximately 12.1% of the cells were alive at the AuNCs concentration of 60 µg/mL, indicating that the combination of AuNCs/SV and NIR could effectively kill tumor cells. Then the expression level of immunogenic cell death (ICD) makers, including calreticulin (CRT) and high mobility group box 1 (HMGB1), were determined by flow cytometry and enzyme-linked immunosorbent assay respectively [36,37]. Flow cytometry examination showed that AuNCs/SV with laser treatment induced ~3.5-fold higher CRT expression than the control group (Fig. 4d). The extracellular HMGB1 of AuNCs/SV with laser treatment group was ~1.7-fold higher than control group (Fig. 4e). All these results consistently verified that AuNCs-based PTT could effectively induce ICD in vitro. It has reported that DCs support antitumor adaptive immunity by presenting antigen and stimulating T cells [38,39], and then ICD-induced DCs maturation was examined by flow cytometry. The mature DCs frequency of AuNCs/SV+Laser group was higher than other groups (Figs. 4f and g), indicating that tumor-ICD induced by AuNCs/SV with PTT could efficiently promote DCs maturation. Above results suggested that AuNCs/SV were likely to become a promising PTT agent and further trigger downstream immune responses.
Inspired by the highly therapeutic efficiency and the antitumor immune responses in vitro, we next evaluated the synergistic antitumor efficiency of AuNCs/SV with NIR on B16F10-bearing C57 mice. All animal experiments were strictly conducted according to the guidelines stipulated and approved by Henan laboratory animal center. Moreover, when saline, SV, AuNCs, AuNCs+SV or AuNCs/SV were intratumorally injected, the groups containing AuNCs were irradiated with a NIR laser (1.5 W/cm2). Then the tumor volume and body weight were recorded every other day, and the survival time of all experimental groups was still inspected after 5 times of administrations. As shown in Fig. 5a, according to the tumor volume change profiles, both the tumor volumes in saline-treated and SV-treated groups showed a rapidly increase over 14 days, while the AuNCs with laser irradiation exhibited certain tumor growth delay due to PTT-mediated tumor cells damage. However, the AuNCs/SV treated mice with laser irradiation displayed obvious tumor inhibition compare to AuNCs+SV+Laser group and AuNCs+Laser group, which was due to the direct tumor killing effect induced by PTT and the existence of adjuvant SV [40]. After AuNCs/SV with laser treatment, the tumor cells could be lysed into large amount of tumor debris to release TAAs. The in situ tumor vaccine formed by AuNCs/SV and released TAAs could induce strong anti-tumor immune response, and further remove the residual tumor cells, showing prolonged survival time in AuNCs+SV+Laser group (Fig. 5b). The hematoxylin and eosin (H&E) staining of tumors (Fig. S1 in Supporting information) were performed to confirm the therapeutic effects. Furthermore, the weight of all treated mice showed no significant change during the period of treatment (Fig. S2 in Supporting information) and the H&E staining of major organs showed no noticeable organ damage in the group containing AuNCs (Fig. S3 in Supporting information), indicating the excellent biocompatibility and minimal toxicity.
Next, we carefully studied the anti-tumor immune response induced by AuNCs/SV downstream, including mature DCs and activated T cells in tumor and LNs after 3 times of administration. As shown in Fig. S4a (Supporting information), an obviously increase of mature DCs in tumors could be observed in the AuNCs/SV with NIR laser irradiation compared with other groups. This was due to the co-delivery of antigen and adjuvant. CD8+ T cells and CD4+ T cells are the main effector cells of anti-tumor immune response, which can be activated by mature DCs [41,42]. Therefore, we further evaluated the number of infiltrating T cells at tumor site. As expected, compared with any other treatment group, more mature DCs, CD8+ T cells and CD4+ T cells were found in the tumor site after the treatment of AuNCs/SV with NIR laser (Figs. S4b, S4c and S5 in Supporting information), which was owing to the synergistic effect of TAAs and SV.
Especially in PTT induced vasodilation, AuNCs/SV/TAAs of ~55 nm could be advantageous to drained to the LNs and enhance the body's immunity, so some immune cells in LNs were investigated. As shown in Fig. 5c, AuNCs/SV with laser group significantly enhanced the expression of CD40 and CD86 in LNs, which were almost twice higher than control group. These data demonstrated that the construction of in situ recombinant NPs improved DCs activation. After primed by antigens, mature DCs would trigger the following immune response and process antigens into MHC–peptide complex to present to T cells [43,44]. Therefore, as shown in Figs. 5d and e, AuNCs/SV under laser irradiation significantly enhanced CD4+ and CD8+ T-cell proliferation compared to control, SV, AuNCs with laser and the AuNCs+SV with laser groups (Fig S6 in Supporting information), indicating that AuNCs/SV/TAAs NPs promoted the differentiation and proliferation of T lymphocytes in LNs effectively. The results also showed the recombinant NPs were easy to migrate to LNs and activate T cells obviously. Moreover, immunofluorescence assay (Fig. S7 in Supporting information) was conducted to confirm the increase of the percentage of CD8+ T cells, indicating that the co-delivery of TAAs and SV improved the activation of CD8+ T cells in LNs. Altogether, these results demonstrated that the in situ NP recombinant strategy based on AuNCs/SV with 808 nm laser irradiation enhanced PTT effect by improving the immune responses.
In summary, in situ NP recombinant strategy based on AuNCs was developed for enhancing PTT effect. AuNCs/SV with NIR treatment could kill tumor cells directly and promote TAAs release. And the released TAAs could be then captured by AuNCs/SV to construct AuNCs/SV/TAAs which were ingested by DCs, significantly inducing strong antitumor immunity. Besides, AuNCs/SV/TAAs exhibited good LN-targeting ability, which promoted the maturation of DCs and enhanced the cytotoxic T lymphocytes obviously. Moreover, study on pharmacodynamics showed that AuNCs/SV+Laser group could effectively inhibit tumor growth and prolong the survival time of mice. Therefore, we believe that the therapeutic strategy might provide a new way for enhancing PTT-immunotherapy and facilitating the development of autologous anticancer vaccines.
Declaration of competing interest
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The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgments
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This project was financially supported by the National Natural Science Foundation of China (Nos. U1804183, 81901878 and 81874304), China Postdoctoral Science Foundation (No. 2019M662553) and Key Scientific Research Project (Education Department of Henan Province, No. 20HASTIT049).
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Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.cclet.2021.07.025
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Year 2022 volume 33 Issue 1
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Article Info
doi: 10.1016/j.cclet.2021.07.025
  • Receive Date：2021-05-17
  • Online Date：2025-12-12
  • Published：2022-01-15
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  • Received：2021-05-17
  • Revised：2021-07-02
  • Accepted：2021-07-07
Affiliations
    a Luoyang Central Hospital Affiliated to Zhengzhou University, Luoyang 471009, China
    b School of Pharmaceutical Sciences, Zhengzhou University, Zhengzhou 450001, China
    c Key Laboratory of Advanced Drug Preparation Technologies, Ministry of Education, School of Pharmaceutical Sciences, Zhengzhou University, Zhengzhou 450001, China
    d Henan Key Laboratory of Targeting Therapy and Diagnosis for Critical Diseases, Zhengzhou University, Zhengzhou 450001, China
    e Tumor Immunity and Biomaterials Advanced Medical Center, Zhengzhou University, Luoyang 471009, China
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表12种不同金属材料的力学参数
科
Family		 属数
Number of
genus		 种数
Number of
species		 占总种数比例
Percentage of
total species (%)		 属
Genus		 种数
Number of
species		 占总种数比例
Percentage of total
species (%)
鹅膏菌科Amanitaceae 2 11 5.26 鹅膏菌属 Amanita 10 4.78
小菇科 Mycenaceae 2 12 5.74 丝盖伞属 Inocybe 5 2.39
多孔菌科 Polyporaceae 8 14 6.70 蜡蘑属 Laccaria 5 2.39
红菇科 Russulaceae 3 23 11.00 小皮伞属 Marasmius 6 2.87
小菇属 Mycena 11 5.26
光柄菇属 Pluteus 5 2.39
红菇属 Russula 17 8.13
栓菌属 Trametes 5 2.39
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