Thermal Stimuli-Responsive Nanoplatform Based on Phase Change Material for Synergistic Enhancement of p53 Protein Expression in HCC

doi:10.21203/rs.3.rs-3209111/v1

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Thermal Stimuli-Responsive Nanoplatform Based on Phase Change Material for Synergistic Enhancement of p53 Protein Expression in HCC

https://doi.org/10.21203/rs.3.rs-3209111/v1

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TP53 is one of the most important tumor suppressor genes and is known as the “genetic guardian”. Targeting the MDM2-p53 loop is an attractive tumor treatment strategy. Herein, an organic PCM with a melting point of 43.5℃ was prepared by using biocompatible fatty acids and alcohols. PCM was used to co-encapsulate a photosensitizer (IR780) and a novel MDM2-p53 inhibitor (APG-115). The final prepared multifunctional temperature responsive release nanoplatform (PIA NPs) can not only effectively deliver APG-115 and IR780 but also act as a “gatekeeper” to protect the delivered drug, avoid hydration of IR780, and prevent drug leakage. Under 808 nm laser irradiation, the protected IR780 can melt the PCM by generating the photothermal effect, thereby achieving drug release regulated by exogenous stimuli (light). Furthermore, the oxidative stress signal (ROS) generated by the PDT effect of IR780 can promote the expression of p53 protein, collaborating with APG-115 to improve the therapeutic efficiency of HCC. PIA NPs have shown superior therapeutic effects in vivo and vitro. This PDT/PTT/MDM2-p53 inhibitor combined therapy strategy provides an unprecedented approach to the treatment of HCC via synergistically enhancing p53 expression.

Hepatocellular carcinoma

phototherapy

phase change material

p53

APG-115

As is well known, the TP53 tumor suppressor gene is the most common inactivated gene in cancer. The p53 protein (also known as p53) encoded by TP53 is a vital transcription factor that inhibits tumor growth. Almost all malignant tumor cells exhibit abnormalities in p53, including p53 mutations and the loss of p53 tumor suppressor function caused by overexpression of MDM2[1–3]. The p53 can be activated by multiple signal stimulation, such as DNA damage, which in turn determines cell survival or death[1–3]. High levels of p53 can cause damage to the growth of normal cells, therefore, intracellular p53 levels are strictly regulated by proteases[4, 5]. Under normal and non-stress conditions, p53 protein can maintain normal levels by binding to its inhibitors (MDM2 protein, etc.). However, in tumor cells, p53 loses its tumor suppressor function due to mutations in the TP53 gene or abnormalities in the tumor suppressor function of the p53 protein. TP53 mutations occur in approximately 50% of malignant tumors, and their prevalence ranges from less than 5% (cervical cancer) to as high as 80–90% (small cell lung cancer, ovarian cancer)[6, 7]. The p53 may be deactivated due to the overexpression of MDM2, leading to the loss of its tumor suppressor function[8]. The interaction between MDM2 and p53 forms a feedback loop, and in this feedback loop, MDM2 can block the transcription of downstream target genes mediated by p53 by combining with each other. On the other hand, MDM2 can promote the ubiquitination degradation of p53 by affecting the activity of E3 ubiquitin ligase. Moreover, MDM2 can also promote SUMOylation and nuclear output of p53 by interacting with the SUMO E3 ligase PIASy[4, 9]. In recent years, a series of MDM2-p53 inhibitors (such as RG7112, Nutlin-3a, etc.) have been designed for this negative feedback loop. These MDM2-p53 inhibitors can competitively bind to the pocket structure in the hydrophobic cracks of MDM2 (occupied by the amino acid side chains of leucine 26, tryptophan 23, and phenylalanine 19 in p53), leading to upregulated expression of the p53 protein, and thus exerting a powerful antitumor effect[10]. APG-115 is a novel type of MDM2-p53 inhibitor that has the advantages of slow isomerization, high MDM2 affinity (Ki less than 1 nM), and excellent oral pharmacokinetic characteristics[11]. APG-115 can enhance radiosensitivity to gastric adenocarcinoma in vitro and vivo[12]. The activation of p53 mediated by APG-115 can also enhance antitumor immunity in the TME and is independent of the TP53 state of the tumor itself[13, 14]. In addition, APG-115 has shown superior efficacy in the treatment of acute myeloid leukemia[15, 16]. Although these targeted drugs are effective for certain patient populations, they can cause some systemic toxic reactions and easily develop drug resistance. The off-target effect and emerging drug resistance are significant challenges in the field of tumor targeted therapy[17, 18]. In addition, MDM2-p53 inhibitors can activate p53 in tumor cells expressing wild-type p53 but possess no such effect on tumor cells with p53 mutations or deletions[19]. Therefore, exploring joint or collaborative therapeutic strategies is of great significance for improving the therapeutic efficiency of tumors.

Members of the p53 family play an important role in PDT-activated cell death, which is beneficial for guiding combined or synergistic therapeutical strategies for tumors. The selective activation of p53 family proteins via the PDT effect is expected to become a novel approach to enhance the efficiency of tumor treatment. The light damage caused by PDT leads to that mitochondria, endoplasmic reticulum and lysosomes producing excessive ROS, thus effectively activating p53, upregulating the expression of p53 protein, and promoting p53-related apoptosis[20]. There is currently controversy regarding the relationship between p53 expression and PDT. Many studies have shown that wild-type p53 is a related messenger of cell death signals in PDT. Compared with tumor cell lines expressing wild-type p53, glioma cell lines expressing mutant p53 show poorer therapeutic effects on PDT[21]. Fisher et al. found that wild-type p53 cells have a more pronounced therapeutic effect on fluorescent protein-mediated PDT[22]. In another study, it was also pointed out that colon cancer cells expressing wild-type p53 have higher sensitivity to PDT mediated by photosensitizers. However, some studies also suggest that the expression of p53 is not related to PDT[23]. Therefore, further exploration is needed to clarify the interrelationships between PDT and p53 expression.

Herein, an organic PCM with a melting point of 43.5℃ was prepared by adjusting different proportions of organic fatty acids (oleic acid) and fatty alcohols (hexadecanol). PCM was used to encapsulate APG-115 and the photosensitizer IR780, and the resulting nanoparticles (PIA NPs) were synthesized after modification with l-α-lecithin and DSPE-PEG2000. In this system, the introduction of PCM can effectively avoid the hydration of IR780, and deliver drugs efficiently. PIA NPs can achieve temperature responsive drug release effect under external stimuli (808 nm light illumination), synergistically enhancing the expression of wild-type p53 protein and efficiently treating HCC (cell proliferation inhibition, apoptosis induction, invasion and migration inhibition) via an exciting strategy based on phototherapy and APG-115 (Scheme 1).

oleic acid (OA) and hexadecanol (HEX) were selected as the synthetic materials for organic PCM, and the melting points of different OA/HEX ratios (1:2, 1:3, 1:4) were measured using differential scanning calorimetry (DSC). As shown in Table 1 and Fig S1, the melting point and solidification point of PCM gradually increase with the increase of HEX content. To reduce the damage of photothermal effects on normal tissues and take into account the influence of normal body temperature on the carrier state, we selected PCM (melting point: 43.5℃) with OA/HEX (1:2) as the nanocarrier for subsequent research (Fig S1c).

Table 1

The melting points of PCM prepared with different OA/HEX ratios using DSC.
OA/HEX	Melting point (℃)
1: 2	43.5
1: 3	46.1
1: 4	48.6

The mixture of PCM, IR780, and APG-115 was ultrasonicated at 60℃, followed by rapid cooling in an ice bath, allowing hydrophobic PCM to quickly solidify into nanoparticles during the cooling process, encapsulating IR780 and APG-115. Subsequently, l-α-lecithin and DSPE-PEG2000 were used to hydrophilically modify nanoparticles. Thus, PIA NPs encapsulated with IR780 and APG-115 were prepared. As shown in Figs. 1a and 1b, TEM images of PIA NPs present monodisperse, round, or oval particles, which proved the successful preparation of nanoparticles. PIA NPs appeared green in PBS solution (IR780 was successfully encapsulated), and the DLS was approximately 194.75 ± 14.92 nm (Fig. 1c). This size is conducive to targeting tumor tissue through the EPR effect after PIA NPs enter the body. As shown in Fig. 1d, the zeta potential (-47.07 ± 2.78 mV) of nanoparticles after hydrophilic modification of l-α-lecithin and DSPE-PEG2000 is lower than that of unmodified nanoparticles (IR780@PCM, IR780/APG-115@PCM), demonstrating the good stability of PIA NPs. As shown in Fig. 1e, the absorption spectrum shows that PIA NPs have obvious absorption peaks at 262 nm and 800 nm, corresponding to the characteristic absorption peaks of APG-115 and IR780, respectively, indicating that APG-115 and IR780 are effectively encapsulated in PIA NPs. It is worth noting that the characteristic absorption peak of IR780 shifts from 780 nm red to 800 nm, which may be due to drug aggregation, intramolecular interactions, and intermolecular π-π interactions. This phenomenon makes the NIR absorption peak of PIA NPs closer to the excitation wavelength of the laser (808 nm), which is more favorable for subsequent PTT and PDT. PIA NPs also exhibit strong fluorescence intensity when excited at 780 nm, which further indicates that IR780 is effectively encapsulated in nanoparticles and that PIA NPs can be employed for NIR fluorescence imaging (Fig. 1f). Subsequently, the encapsulation efficiency and loading rate were calculated using the absorption spectra of PIA NPs and the standard curves of IR780 and APG-115 (Figs. S2, S3). The encapsulation efficiency of IR780 in PIA NPs was approximately 79.04 ± 1.75%, and the loading rate was approximately 3.18 ± 0.06%. The encapsulation efficiency of APG-115 was 81.12 ± 1.55%, and the loading rate was 9.30 ± 0.21%. Organic PCM has achieved effective encapsulation of APG-115 and IR780.

IR780 is a lipophilic heptamethyl cyanine dye with stronger fluorescence intensity than the traditional photosensitizer ICG approved for clinical application[24, 25]. However, IR780 has many drawbacks such as poor water solubility, rapid clearance in vivo, and susceptibility to photooxidation bleaching in aqueous solutions, severely limiting its clinical application. As shown in Fig. 2a, after exposure to light illumination, IR780 rapidly undergoes oxidative bleaching in an aqueous solution, with the color changing from green to yellow and the absorption peak significantly decreasing (almost approaching 0), indicating its poor photostability. The change in the characteristic absorption peaks of PIA NPs aqueous solution was much smaller than that of the IR780 group, and no significant color change was observed (Fig. 2b). This may be attributed to the fact that IR780 encapsulated by organic PCM has a lipid shell on its surface, and the shell effectively isolates contact with O₂ and water, avoiding oxidative bleaching of IR780. In addition, PIA NPs (120 µg/mL) show remarkable photothermal performance after five heating cycles under 808 nm laser irradiation, further demonstrating the excellent photothermal stability of PIA NPs (Fig. S5). The photothermal properties of the prepared nanoparticles are crucial for realizing temperature-sensitive drug release under light stimulation. Due to the strong absorption peak at 800 nm, PIA NPs have great application prospects in NIR PTT. As shown in Fig. 2c, with an 808 nm laser illuminating PIA NPs and IR780 aqueous solution with equal IR780 concentration for 12 minutes (600 mW/cm²), the temperature of PIA NPs can rise to 48.7℃, while IR780 aqueous solution can only rise to approximately 25℃. This is probably because the fact that IR780 is prone to oxidative bleaching in aqueous solutions, which seriously affects its photothermal performance. Subsequently, different concentrations of PIA NPs were irradiated with an 808 nm laser, and the results show that the photothermal effect of the nanoparticles is significant concentration-dependent, and even though lower concentrations (15 µg/mL) of PIA NPs also possess good photothermal performance (Fig. 2d). When the temperature is higher than the melting point of PCM, the photothermal effect generated by PIA NPs can melt PCM, thereby releasing the encapsulated drugs. As shown in Fig. 2e, after 10 minutes of illumination, the detected APG-115 concentration gradually increased, with a cumulative release rate of 21.93%. However, the APG-115 concentration remained basically unchanged without light illumination. When the light is turned on again, the cumulative release rate of APG-115 continues to increase. After three cycles of reciprocating, the concentration of APG-115 showed a stepwise increase, with a cumulative release rate of up to 65.63%. Meanwhile, the cumulative drug release rate of PIA NPs without illumination is only 1.87%. This indicates that PIA NPs effectively achieve temperature-responsive release of drug photostimulation, avoid early drug leakage, enhance drug targeting ability, and improve treatment effectiveness. The production ability of ROS is the key to the therapeutic effect of PDT[26]. To evaluate the ROS production ability of PIA NPs, SOSG was used as a fluorescent probe for ¹O₂, and a mixed solution of PIA NPs and SOSG was irradiated with an 808 nm laser (100 mW/cm²). The generation of ¹O₂ in PIA NPs aqueous solution was detected by observing the fluorescence intensity change of SOSG at 531 nm. As shown in Fig. 2f, the fluorescence intensity gradually increases with time, proving that PIA NPs have a certain ability to produce ¹O₂ and can be employed for PDT. The absorbance of DPBF mixed with PIA NPs gradually decreases with 808 nm laser irradiation, which further confirms the excellent ¹O₂ generation capacity of PIA NPs (Fig. S4).

Nanoparticles should be evaluated for uptake by tumor cells before in vitro experiments. Due to the high fluorescence performance of IR780 in the near-infrared region, the fluorescence signal of IR780 can be used to evaluate the cellular uptake ability of PIA NPs. PIA NPs were coincubated with HepG2 and Huh7 cells for 6 hours, followed by 633 nm laser stimulation of tumor cells. As shown in Fig. 3a, a large amount of red fluorescence signals were observed in the cytoplasm of tumor cells under a confocal microscope, indicating that PIA NPs can be effectively internalized and absorbed by HepG2 and Huh7 cells. Subsequently, we evaluated the biological toxicity of the carrier using the HepG2 cell line expressing wild-type p53 and the Huh7 cell line expressing mutant p53. IR780@PCM NPs are served as the main carrier to evaluate cytotoxicity because the drug release of PIA NPs can be obviously affected without the photothermal effect of IR780. As shown in Fig. S6, the MTT results indicate that IR780@PCM NPs exhibit excellent biosafety for HepG2 and Huh7 cells. Even if the concentration of IR780@PCM NPs was greater than 200 µg/mL, both cell lines still maintained high cell survival rates (greater than 80%). Then, HepG2 cells expressing wild-type p53 were utilized to evaluate the therapeutic effect of PIA NPs. As shown in Fig. 3b, the cell survival rate of PIA NPs without laser irradiation is higher than that of APG-115. The IC50 values of the APG-115 and PIA NPs without laser irradiation were 22.01 µg/mL and 53.75 µg/mL, respectively (APG-115 concentration), which may be attributed to the effective encapsulation of APG-115 by PCM affecting the release of APG-115 from PIA NPs. Notably, PIA NPs also show certain dark toxicity. When the APG-115 concentration in PIA NPs was 80 µg/mL, the cell survival rate of HepG2 cells was 41.03%. With increasing APG-115 concentration, PIA NPs will also play a role in killing tumor cells, which may be because the main components of the carrier (OA, HEX, DSPE-PEG2000, and lecithin) can be destroyed by intracellular oxidative enzymes after PIA NPs are endocytosed by tumor cells, resulting in the release of APG-115, thus exerting the cytotoxicity of the drug. More importantly, PIA NPs showed strong phototoxicity to HepG2 cells. When the concentration of IR780 was 5 µg/mL (APG-115 concentration: 14.6 µg/mL), the cell survival rate was only 32.28%. Furthermore, the IC50 value of the PIA NPs irradiation group (IR780: 1.98 µg/mL, APG-115: 5.79 µg/mL) was lower than that of the IR780 nanoparticles irradiation group (IR780: 4.67 µg/mL), which may be because the photothermal effect of IR780 caused PIA NPs to release APG-115, and phototherapy cooperated with APG-115 to enhance the killing effect on HepG2 cells (Fig. 3c). Additionally, APG-115 increases the expression of wild-type p53 protein, which may increase sensitivity to PDT[27]. The effect of PIA NPs on HepG2 cell apoptosis was further validated using flow cytometry apoptosis experiments. As shown in Figs. 3d and 3e, the survival rate of HepG2 cells in the blank control group was as high as 91.8%, with only a small portion of cells experiencing apoptosis (early apoptosis: 2.29%, late apoptosis: 5.50%). After 24 hours of treatment with APG-115, the apoptosis rate increased to 17.25%. When IR780 nanoparticles were treated with light (600 mW/cm², 3 min), the apoptosis rate reached 29.2%. After incubating HepG2 cells with PIA NPs and exposure to light, 43.5% of cells underwent apoptosis (early apoptosis: 14.8%, late apoptosis: 28.7%). This further proves that PIA NPs can efficiently kill HepG2 cells through phototherapy combined with APG-115.

To more intuitively verify the killing effect of PIA NPs on HepG2 cells, we further evaluated the therapeutic effect of PIA NPs using live cell staining experiments. As shown in Fig. 4a, the PIA NPs with light illumination can observe the most visible red fluorescence signal and its ratio of red fluorescence signal to green signal is greater than that of other groups. This result indicates that PIA NPs can effectively kill HepG2 cells under 808 nm laser irradiation. It should be noted that due to the large amount of light killing HepG2 cells, the ability of dead tumor cells to adhere to the wall is weakened. In the subsequent PBS flushing process, a large number of necrotic or apoptotic cells were shed, which will affect the experimental results. Therefore, the staining probe was added to cells after laser irradiation in this study, and the drug incubation time of HepG2 cells was controlled at 12 hours. In addition, due to the limited aperture range of the laser, the laser probe is approximately 1.5 cm away from the bottom of the six-hole plate during the laser illumination, and the aperture cannot cover the entire small hole of the six-hole plate, causing the red fluorescence signal in the illumination area to be greater than that in the surrounding area, resulting in an uneven distribution of cell death. However, the staining experiment of living and dead cells can still prove that PIA NPs with laser illumination can rapidly lead to HepG2 necrosis or apoptosis, and the therapeutic effect is superior to other groups. This phenomenon of rapidly killing tumor cells may be related to the large amount of ROS generated during the PDT process attacking the cell membranes and lysosomes of tumor cells[28]. Subsequently, an EdU staining experiment was used to verify the effect of PIA NPs on the proliferation ability of HepG2 cells. As shown in Figs. 4b and S7, the proportion of EdU-positive cells in the PIA NPs irradiation group (8.11%) was far less than that in other groups, including APG-115 (37.62%) and IR780 nanoparticles with light irradiation (18.95%), which further proves the APG-115 combined phototherapy strategy can effectively inhibit HepG2 cell proliferation. Subsequently, the Transwell experiment was used to further investigate the effect of PIA NPs on the migration and invasion ability of HepG2 cells. As shown in Figs. 4c and 4d, the PIA NPs light group and IR780 light group significantly inhibited the migration and invasion of HepG2 cells, and the inhibitory effect of the PIA NPs light group was stronger than that of the IR780 light group.

PIA NPs exhibit excellent killing effects on HepG2 cells, mainly relying on the PDT effect of IR780 and the killing effect of APG-115 on cells. Firstly, we used DCFH-DA to detect ROS generation in different treatment groups. As shown in Figs. 5a and 5b, the green fluorescence intensity of the IR780 light irradiation group and PIA NPs light irradiation group was significantly higher than that of the other groups, indicating that the photosensitizer IR780 in nanoparticles can produce a large amount of ROS under light conditions, thereby causing necrosis and inducing cell apoptosis. Subsequently, to verify the effect of PDT on p53 protein expression, HepG2 cells with different concentrations of IR780 were incubated using 808 nm laser irradiation (100 mW/cm²). After illumination was completed, the expression of the p53 protein was measured after further incubation for 12 hours. The results showed that with the increase of IR780 concentration, the expression of p53 protein gradually increased, indicating that PDT can enhance the expression of p53 protein (Fig. 5c). This may be because after the photosensitizer is ingested by tumor cells, it will damage the cell membrane of mitochondrial proteins, endoplasmic reticulum, and organelles under light conditions, and then the accumulation of ROS activates the cascade signal of p53 and promotes the expression of downstream apoptosis genes[23]. As shown in Fig. 5d, the expression levels of p53 and Bax proteins in the PIA NPs illumination group were significantly higher than those in the other groups, while the expression of Bcl-2 protein, which inhibits apoptosis, was significantly reduced. These results proved that PDT combined with APG-115 can significantly increase the expression of p53 protein and promote cell apoptosis. PDT can induce three main pathways of cell death: apoptosis, necrosis, and autophagy-related cell death. The mode of death mainly depends on the location of photosensitizers in the cell, the dosage of light, the concentration of photosensitizers, and so on[29]. When the laser power is too high or the concentration of photosensitizers is too high, it can cause a large amount of cell necrosis, which will cause certain difficulties in protein extraction and thus affect the expression of p53. Therefore, it is necessary to regulate the laser power and the concentration of photosensitizers. In the early stage of mitochondrial damage, ROS produced by light-damaged mitochondria, endoplasmic reticulum, and lysosomes activate p53 and promote p53-related apoptosis. According to reports, PDT-induced cell apoptosis is caused by the inactivation of the antiapoptotic Bcl-2 protein, as well as the activation and translocation of the proapoptotic Bax protein to the mitochondrial membrane[30]. ROS is not only an upstream signal that triggers p53 activation but also a downstream factor that mediates cell apoptosis. However, some studies suggest that there is no correlation between PDT and p53 expression[23]. The possible reason is that these studies used high doses of light, so the death mode of tumor cells after PDT is necrosis caused by organelle and cell membrane damage rather than the activation of cell pathways. It should be noted that whether it is the activation of cellular pathways (increased expression of p53 protein) induced by low-dose illumination, induction of cell apoptosis, or the direct killing of tumor cells by high-dose light, the outcome will lead to tumor cell necrosis or apoptosis, playing a role in killing the tumor. Considering that high-power light can cause damage to normal tissues around tumors, it is of great significance for us to choose low-power light therapy combined with MDM2-p53 inhibitors for treatment.

Multimode imaging has certain significance for the diagnosis and treatment of HCC. As shown in Fig. 6a, 8 hours after injection of PIA NPs through the tail vein, the photoacoustic signal reached its maximum value and then began to show a downward trend. This indicates that PIA NPs have certain photoacoustic imaging performance and can be effectively enriched in tumor tissue. Subsequently, using an 808 nm laser to irradiate the tumor site enriched with PIA NPs, it was found that the temperature at the tumor site in the PIA NPs group significantly increased (over 15℃) within 2 minutes compared to the saline injection group, which was sufficient to melt the PCM and release the encapsulated drug (Figs. 6b, 6c). This further proves that PIA NPs can effectively enrich the tumor site and produce good photothermal imaging results. The real-time fluorescence imaging results of tumor-bearing nude mice showed that the fluorescence intensity of the tumor tissue gradually increased after injection of PIA NPs, reaching its maximum quantity at 8 hours, and then gradually decreased, further confirming the superior passive targeting performance of PIA NPs (Figs. 6d, 6e). Multimode imaging can be applied to the early diagnosis and intraoperative navigation of HCC.

The biosafety of nanomaterials is crucial for their clinical conversion. We evaluated the biological safety of PIA NPs by observing the weight changes of nude mice during the 18-day treatment period. As shown in Fig. 7a, it can be seen that the body weight of nude mice maintained a small growth trend within 18 days, and the body weight of nude mice in each group was normal, with no deaths of nude mice, proving that PIA NPs have a certain degree of biological safety. By observing the changes in tumor volume of nude mice in different groups (control group, IR780 + light, APG-115, PIA NPs, and PIA NPs + light), the in vivo therapeutic effect of different treatment groups on HepG2 tumor-bearing nude mice was evaluated. The results showed that the PIA NPs group had the best in vivo treatment effect, further demonstrating the excellent synergistic therapeutic effect of PDT, PTT, and MDM2-p53 inhibitors (APG-115) (Figs. 7b, 7e-i). Furthermore, the APG-115 group can also significantly inhibit tumor volume in mice, demonstrating its potential as a novel and efficient MDM2-p53 inhibitor in the treatment of wild-type p53-bearing nude mice[12, 14]. The tumor volume of nude mice in the dark light group of PIA NPs was also significantly reduced, possibly due to the enrichment of PIA NPs in the tumor site of nude mice and their uptake by cells. Over a longer enrichment time, DSPE-PEG2000 and PCM shells were digested and degraded by various intracellular enzymes, releasing a portion of APG-115 and IR780. The strong inhibitory effect of APG-115 on wild-type p53 expression in tumors and the combined dark toxicity of IR780 with concentration accumulation significantly inhibited tumor growth. After treatment, the weight of the tumor in vitro further confirmed the excellent in vivo therapeutic effect of PIA NPs (Figs. 7c, 7d). After two treatments, one nude mouse from each group was selected, and the tumor tissue was removed for fixation, sectioning, and staining. As shown in Fig. 7j, the H&E staining results of tumor tissues in each group indicate that the nude mice in the PIA NPs light group showed extensive tumor tissue necrosis or apoptosis, showing the most severe damage compared to the other groups. The Ki67 staining results showed that the brown area was the smallest in the PIA NPs light group, indicating that the PIA NPs light group can effectively inhibit tumor proliferation in nude mice (Fig. 7k). In vitro experiments, we demonstrated that both APG-115 and IR780 light can promote the expression of p53 protein. As shown in Fig. 7l, the p53 immunohistochemistry experiment in HepG2 tumor-bearing nude mice further demonstrated that the APG-115 group and IR780 light group can promote the expression of p53 protein. When these two methods are combined, the PIA NPs light group exhibits the most significant p53 protein expression ability. After 18 days of treatment, the hearts, livers, spleens, lungs, and kidneys of each group of nude mice were removed, and H&E tissue sections were stained to analyze the biological toxicity of PIA NPs to normal tissues from a morphological perspective. As shown in Fig. S8, the H&E staining analysis results showed that there were no significant pathological changes in the important organs of each group, indicating that PIA NPs do not have significant cytotoxic effects on important organs within a certain concentration range and that PIA NPs have excellent biocompatibility.

This study successfully prepared an organic PCM with a melting point of 43.5℃ using biocompatible fatty acids (OA) and fatty alcohols (HEX) and used it to deliver the photosensitizer IR780 and a novel MDM2-p53 inhibitor (APG-115). After the hydrophilic modification of DSPE-PEG2000 and l-α-lecithin, the finally prepared temperature-responsive drug-releasing nanoparticles (PIA NPs) showed appropriate size and better PDT and PTT properties. PCM can play the role of a “gatekeeper”, protecting the delivered medication from premature release without laser irradiation, and it can protect the photosensitizer IR780 from hydration. Under 808 nm laser irradiation, the excellent photothermal performance of IR780 causes PCM to melt, thereby releasing the delivered drug. At the same time, IR780 protected by PCM can generate ROS through PDT, which together with APG-115 promotes the expression of wild-type p53 protein, thereby improving the killing efficiency of tumors. In vitro experiments, under light conditions, PIA NPs can effectively inhibit the proliferation, invasion, and migration of HepG2 cells, and promoted their apoptosis or necrosis. In vitro experiments, PIA NPs not only effectively inhibited the growth of tumor-bearing nude mice but also served as diagnostic reagents for multimodal imaging to guide the treatment of HCC. This integrated multimodal diagnostic and therapeutic strategy of temperature-responsive release of PDT/PTT in collaboration with APG-115 to enhance p53 expression provides a new approach to the treatment of HCC.

4.1 Preparation of PCM with different melting points: Dissolve 1-hexadecanol (HEX) and oleic acid (OA) in anhydrous ethanol in different mass ratios (HEX: OA = 2.0, 3.0, 4.0), mix well under ultrasonic vibration for 10 minutes, vacuum dry, remove ethanol, and cool to obtain a uniform solid PCM. Place in a 4℃ refrigerator for refrigeration.

4.2 Modification of PCM: First, 30 mg PCM, 20 mg 1-α-Lecithin and 6 mg DSPE-mPEG2000 were codispersed in 5 mL of deionized water and sonicated in a 60℃ water bath for 10 minutes. Subsequently, the mixture was quickly immersed in ice water to allow the PCM to solidify rapidly. After freeze-drying, the modified PCM was obtained.

4.3 Preparation and Modification of PIA NPs: First, l-α-Lecithin (40 mg) and DSPE-mPEG2000 (12 mg) were dissolved in 5 mL of deionized water and sonicated for 20 minutes. Two milligrams of IR780 iodide, 8 mg of APG-115, and 60 mg of PCM were dissolved in 2 mL of ethanol and added to the above solution. Ultrasound was performed in a 60°C water bath for 10 minutes. The mixed solution was quickly poured into ice water after ultrasound, allowing the PCM to solidify quickly. Subsequently, the samples were centrifuged 3 times using a low-temperature high-speed centrifuge (4℃, 11000 rpm) for 20 minutes each time. After PBS redissolves and centrifugation of the precipitate, it is filtered through a 220 nm microporous filter membrane to obtain the final PIA NPs

4.4 Drug release experiment of PIA NPs: Place 5 mL of 2 mg/mL PIA NPs PBS suspension in a dialysis bag (molecular weight: 3000 Da). Subsequently, the dialysis bag was placed in 50 mL of PBS solution, stirred, and shaken well at 100 r/min at 37°C. PIA NPs were illuminated with an 808 nm laser (600 mW/cm²) every 10 minutes, 2.0 mL of liquid was removed from the dialysis bag, the UV visible light absorption value at 262 nm was measured, and the amount of drug released into the medium was calculated through the drug standard curve.

4.5 EdU cell proliferation detection: HepG2 cells were inoculated into a six-well plate (approximately 4 × 10⁵ cells were treated with drugs (APG-115 [9.85 µg/mL], IR780 [3.37 µg/mL], PIA NPs [IR780: 3.37 µg/mL, APG-115: 9.85 µg/mL]) after 12 hours of drug incubation. The light group was irradiated with an 808 nm laser (600 mW/cm²) for 3 minutes. EdU working solution (2X) was mixed with an equal volume of cell culture solution (the final concentration of EdU was 10 µM) and added to each group of cells in a six-well plate. After incubation at 37℃ for 2 hours, EdU-labeled cells were completed. The culture medium was removed, the cells were rinsed with PBS three times, and fixed with paraformaldehyde for 20 minutes. Subsequently, the fixed solution was removed, washed with PBS three times (5 minutes each time), and incubated with the permeable solution for 10 minutes. After removing the transparent solution, 400 µL of click reaction solution was added to each well, gently shaken to evenly cover the sample, and incubated in the dark for 30 minutes. The click reaction solution was removed, the cells were washed with PBS 3 times, and photos were taken under a fluorescence microscope.

4.6 Live photoacoustic imaging: Anesthetize mice with isoflurane. Inject 100 through the tail vein µ L PIA NPs (APG-115: 5.00 mg/kg, IR780: 1.71 mg/kg) were injected, and photoacoustic images were obtained using a photoacoustic imager at 0 h, 4 h, 8 h, and 12 h.

4.7 Establishment of animal subcutaneous tumor model and in vivo treatment: Subcutaneous injection of HepG2 cells (100 µL, 4 × 10⁷/mL) into the right back of nude mice, diluted with matrix gel and serum-free medium at a ratio of 1:1. When the tumor volume reached approximately 75 mm³, the nude mice were divided into five groups (saline injection control group, APG-115 group, IR780 nanoparticle light group, PIA NPs nonlight group, and PIA NPs light group). Different drugs (APG-115 [5.00 mg/kg], IR780 [1.71 mg/kg], PIA NPs [APG-115: 5.00 mg/kg, IR780: 1.71 mg/kg]) were injected into the tail vein of nude mice. After 8 hours, the tumor site of the nude mice was irradiated with an 808 nm laser (600 mW/cm²) for 10 minutes, and treatment was performed every 3 days. The tumor volume and body weight of the nude mice were recorded. Tumor volume: long × wide × high × 0.52. After two treatments, each group randomly selected a portion of nude mice to obtain in vitro tumor tissue, which was stained with H&E, Ki67, and p53 to analyze histological changes. After 18 days of treatment, the nude mice were euthanized, their main organs were obtained, and H&E staining was performed. Histological changes were observed under a microscope.

4.8 Statistical methods: The experimental data were analyzed and plotted using software GraphPad Prism 8 and Origin 2021 software. According to the results, the mean ± standard deviation (mean ± SD) was selected for data description, and t-test (two groups) was used for parameter testing. P < 0.05 indicates a significant difference, * P < 0.05, ** P < 0.01, *** P < 0.001.

Supporting Information

Supporting Information is available free of charge on the ACS Publications website at DOI:

The melting points of different OA/HEX ratios. The freezing points of different OA/HEX ratios. The standard curve of IR780 and APG-115. Absorption spectra of DPBF in the presence of PIA NPs. Photothermal cycle curve of PIA NPs. Cell viability of Hep3B cells after treatment with Ce6, sorafenib, and MCS NPs without light irradiation. Cell viability of HepG2 cells and Huh7 cells after incubation with IR780@PCM NPs without light irradiation. Quantitative analysis of the percentage of EdU-positive cells in each group. After two treatments, the main organ slices of different groups were stained with H&E.

Author contributions

Wenjing Xu: investigation, visualization, designed the experiments, writing original draft; Ting Zhang: data curation and formal analysis; Wenning Zhang: Westernblot experiment; Weilu Jia: methodology; Weilu Jia: formal analysis; Shiwei Liu: methodology, revising, Hao peng: methodology, Haidong Zhang: investigation; Rui Yang: resources; Yewei Zhang: conceptualization, funding acquisition, project administration. All authors reviewed the manuscript.

Funding

The work was funded by the National Natural Science Foundation of China (62141109, 62227803), the Social Development Project of Jiangsu Province (BE2022812), and the Preface Leading Technical Basic Research Special Project of Jiangsu Province (BK20212012).

Availability of data and materials

The datasets used and analyzed during the current study are available from the corresponding author on reasonable request.

Ethics approval and consent to participate

This project was under the approval of the institutional review board at the Ethics Committee of Southeast University.

Competing interests

The authors declare no competing interests.

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Scheme 1 is available in Supplementary Files section.

No competing interests reported.

Scheme1.png
Scheme 1. The anti-HCC mechanism diagram of the nano-drug delivery system based on PCM codelivery of IR780 and APG-115 synergistically enhancing p53 protein expression.
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Thermal Stimuli-Responsive Nanoplatform Based on Phase Change Material for Synergistic Enhancement of p53 Protein Expression in HCC

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