Enzyme-sequential responsive core-satellite nanomedicine enables activatable near-infrared-II photoacoustic imaging-guided chemotherapy cascade-enhanced radiotherapy

Abstract

The standard treatment for various types of cancers typically involves the combination of concurrent localized radiotherapy and systemic chemotherapy. However, no treatment options have been reported that utilize chemotherapy cascade-enhanced radiotherapy. In this study, we report a core-satellite nanomedicine designed to enhance radiotherapeutic effects through a cascade mechanism by triggering the release of a potent chemotherapeutic agent in response to trypsin. We synthesized a functional enzyme-sequential responsive nanomedicine, DOX@Gel-DEVD-AuNR, which consists of gelatin nanoparticles loaded with the chemotherapeutic drug doxorubicin (DOX). These nanoparticles are covalently linked to gold nanorods (AuNR) via a caspase-3 specific DEVD peptide substrate. Upon trypsin activation, the DOX@Gel-DEVD-AuNR formulation releases DOX, thereby enhancing chemotherapy efficacy against tumors. Simultaneously, it activates caspase-3, inducing the aggregation of AuNRs, which in turn activates a near-infrared-II photoacoustic signal. This signal is crucial for determining the optimal timing for X-ray irradiation. The resulting large-size AuNRs aggregates promote their accumulation within tumors by preventing the migration and backflow of AuNRs, thereby improving radiotherapeutic effects. Consequently, when combined with image-guided X-ray irradiation, DOX@Gel-DEVD-AuNR induces significant cytotoxicity in cancer cells and effectively inhibits tumor growth. Our study underscores the potential application of enzyme catalysis-mediated chemistry in activating nanomedicine for activatable image-guided chemotherapy cascade-enhanced radiotherapy.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12951-025-03531-7.

Introduction

Cancer remains a highly lethal disease worldwide, presenting a significant and persistent health challenge. Despite substantial investments in human resources, materials, and financial capital to develop innovative therapeutic approaches such as chemotherapy [1], radiotherapy [2, 3], immunotherapy [4], photodynamic therapy [5], starvation therapy [6], photothermal therapy [7], gas therapy [8], and magnetic hyperthermia [9]; current clinical monotherapy options have only achieved limited success. This limitation arises primarily from the complex nature, diverse characteristics, and heterogeneity of tumors, which hinder the effectiveness of single-treatment strategies. Consequently, in recent years, the focus of cancer research has shifted from monotherapy to combination therapy, emphasizing the synergistic interactions between two or more treatments to improve therapeutic outcomes [10, 11]. These synergistic interactions have given rise to multimodal synergistic therapy, which leverages cooperative enhancement effects to achieve remarkable superadditive outcomes (commonly expressed as “1 + 1 > 2”). Such outcomes surpass the efficacy of any monotherapy or the theoretical additive effects of combining them, offering a promising avenue for advancing cancer treatment [12].

However, the limited correlation between treatment modalities often compromises the effectiveness of combination therapies in amplifying therapeutic outcomes. Consequently, the current situation necessitates the development of more efficient and effective strategies to tackle these challenges and enhance cancer treatment in a secure and dependable manner. One promising approach is the utilization of cascade reactions, which were initially developed in engineering for cascade synthesis [13, 14]. These reactions involve a sequential chain of events, where one reaction triggers the subsequent reaction, and are facilitated by catalysts to achieve high efficiency, excellent atomic economy, and minimal waste [15]. This environmentally-friendly methodology has garnered significant attention in biomedicine, particularly in cancer treatment, owing to advancements in materials chemistry and nanomedicine [16]. Referred to as cascade therapy, this approach integrates two or more treatment modalities, where the output of one treatment acts as a substrate or catalyst for the subsequent treatment [17, 18]. By harnessing cascade reactions, cascade therapy provides a precise and efficient technique for tumor ablation, offering a promising solution to the persistent challenges in cancer therapy [19].

Radiotherapy and chemotherapy remain the most widely utilized cancer treatment modalities in clinical practice. In order to enhance the therapeutic efficacy of these treatments, Lin et al.. developed a nanodrug called Hf-TP-SN, based on the principle of cascade therapy [20]. This nanodrug was constructed by conjugating 7-ethyl-10-hydroxycamptothecin (SN38), an active metabolite of irinotecan, to Hf-TP-OH metal–organic frameworks (nMOFs) through a 3,5-dimethoxylbenzyl carbonate linkage. This specific linkage was designed to be cleaved by hydroxyl radicals (·OH), enabling controlled drug release. Under X-ray irradiation, the electron-dense Hf₁₂ secondary building units (SBUs) within the nMOFs acted as radiosensitizers, significantly enhancing ·OH production. The generated ·OH triggered the cleavage of the 3,5-dimethoxylbenzyl carbonate linkage through hydroxylation, followed by 1,4-elimination process which results in a fivefold increase in SN38 release compared to its molecular counterpart. This mechanism achieved radiotherapy cascade-enhanced chemotherapy and demonstrated the potential of Hf-TP-SN for improving treatment outcomes.

Building upon this concept, Lin et al.. have further advanced their design by developing Hf-DBP-QP-SN, a nanodrug that incorporates a quaterphenyl dicarboxylate ligand (QP-SN) conjugated with SN38 through the same ROS-responsive 3,5-dimethoxylbenzyl carbonate linkage [21]. Similar to its predecessor, Hf-DBP-QP-SN utilized the electron-dense Hf₁₂ SBUs to generate ·OH under X-ray irradiation. These radicals initiated a cascade reaction involving ortho hydroxylation of the 3,5-dimethoxylbenzyl carbonate and subsequent 1,4-elimination to achieve tumor-specific release of SN38. This design further exemplified the efficacy of radiotherapy cascade-enhanced chemotherapy and highlighted the potential of such nanodrugs for precise and efficient cancer treatment.

Radiotherapy cascade-enhanced chemotherapy represents a novel approach to optimize the efficacy of cancer treatment. However, to our knowledge, there has been no utilization of chemotherapy cascade-enhanced radiotherapy in cancer therapy. Furthermore, establishing reliable methods for accurately determining the optimal initiation point for X-ray irradiation remains a pivotal challenge. In this study, we developed an enzyme-sequential responsive core-satellite nanomedicine system, namely DOX@Gel-DEVD-AuNR, comprising gelatin nanoparticles (NPs) loaded with the chemotherapeutic drug doxorubicin (DOX), and decorated with gold nanorods (AuNR). The “core” component (DOX@Gel) and the “satellite” component (AuNR) were covalently linked through a caspase-3 specific Asp-Glu-Val-Asp-Cys(S-S-NH₂)-(alkynyl)-cyanobenzothiazole (CBT) peptide substrate (DEVD). After intravenous administration, DOX@Gel-DEVD-AuNR underwent degradation in response to overexpression of trypsin within the tumor microenvironment, leading to the release of DOX from the DOX@Gel-DEVD-AuNR system. The released DOX induced apoptosis in tumor cells through chemotherapy by activating caspase-3, which cleaved the amide bond between DEVD and Cys (i.e., DEVD↓P) in DOX@Gel-DEVD-AuNR to release Cys(S-S-NH₂)-(AuNR)-CBT. Upon reduction of its disulfide bond, Cys(S-S-NH₂)-(AuNR)-CBT underwent a click condensation reaction with CBT-Cys, resulting in a cyclized dimeric product that self-assembles into large AuNR aggregates. These aggregates enhanced NPs’ accumulation and retention within tumor by preventing migration and backflow [22]. The plasmonic coupling effect between neighboring AuNRs within the aggregates activated near-infrared-II (NIR-II, 1000–1700 nm) photoacoustic (PA) signals. X-ray irradiation was applied when the intensity of the NIR-II PA signal reached its peak, generating abundant reactive oxygen species (ROS) to induce apoptosis and thereby achieving an enzyme-sequential activatable image-guided chemotherapy cascade-enhanced radiotherapy (Fig. 1).

Fig. 1.

Schematic illustration of DOX@Gel-DEVD-AuNR nanomedicine-mediated enzyme-sequential activatable NIR-II photoacoustic imaging-guided chemotherapy cascade-enhanced radiotherapy

Results and discussion

Fabrication and characterization of DOX@Gel-DEVD-AuNR

The core-satellite DOX@Gel-DEVD-AuNR nanomedicine was fabricated through the encapsulation of doxorubicin (DOX) within gelatin nanoparticles (Gel NPs), followed by surface modification using caspase-3-cleavable Asp-Glu-Val-Asp-Cys(S-S-NH₂)-(alkynyl)-cyanobenzothiazole (CBT) peptide substrate (DEVD) functionalized gold nanorods (AuNRs). As shown in Fig. 2A, The Gel NPs were initially synthesized using a modified two-step desolvation technique [23]. Subsequently, DOX was incorporated into the Gel NPs through sonication to obtain the DOX@Gel NPs, which served as the “core” of nanomedicine. To construct the “satellite” structure of nanomedicine, a DEVD peptide and a caspase-3 inactivated DDVD peptide, both containing an alkynyl group and an amino group, were first synthesized using the solid-phase synthesis method, as illustrated in Scheme S1-S11. The peptides and their intermediate products (1–13) were confirmed through the utilization of ¹H NMR spectroscopy and mass spectrometry techniques, as depicted in Figure S1-S15. Following that, AuNRs with the length of 20 nm and width of 4 nm were prepared (Figure S16). The AuNRs were functionalized with hydrophilic azido and thiolated polyethylene glycol (N₃-PEG-SH, MW = 400 Da) via a covalent Au-S bond (AuNR-PEG-N₃). The grafted PEG brushes provided enough azido groups for conjugating the DEVD peptide, which contains an alkynyl group, to achieve NH₂-DEVD-AuNR through the Cu(I)-catalyzed “click” reaction (Scheme S12), thus serving as a “satellite” of nanomedicine. Finally, core–satellite DOX@Gel-DEVD-AuNR were obtained by grafting AuNR-DEVD-NH₂ onto the surface of DOX@Gel NPs via a carboxyl-amine coupling strategy. The control nanomedicine (DOX@Gel-DDVD-AuNR) was prepared using the same procedure as DOX@Gel-DEVD-AuNR, with the exception of substituting AuNR-DEVD-NH₂ with AuNR-DDVD-NH₂. The transmission electron microscopy (TEM) imaging revealed that.

Fig. 2.

Characterization of DOX@Gel-DEVD-AuNR nanomedicine. (A) Schematic illustration of synthesis procedure of DOX@Gel-DEVD-AuNR nanomedicine. Transmission electron microscopy (TEM) of (B) Gel NPs, (C) DOX@Gel, (D) DOX@Gel-DEVD-AuNR, Inset: Scanning electron microscope (SEM) image. (E) HAADF-STEM image and corresponding elemental mapping demonstrate the distribution of C, N, and Au. (F) The particle size analysis of G 1: Gel NPs, G 2: DOX@Gel, and G 3: DOX@Gel-DEVD-AuNR. (G) UV-vis-NIR spectra of AuNR and DOX@Gel-DEVD-AuNR

both Gel NPs (Fig. 2B) and DOX@Gel NPs (Fig. 2C) exhibited a spherical morphology, while DOX@Gel-DEVD-AuNR (Fig. 2D) displayed a core-satellite structure. The morphology of the DOX@Gel-DEVD-AuNR was confirmed by high-angle annular dark-field scanning transmission electron microscopy (HAADF-STEM) and elemental mapping analysis, as depicted in Fig. 2E. This analysis revealed the distribution of carbon (C), nitrogen (N), and gold (Au). The particle size of Gel NPs, approximately 200 nm, remained unchanged before and after DOX loading. However, after modification with AuNR, the particle size of DOX@Gel NPs increased to approximately 236 nm (Fig. 2F). Notably, aside from retaining the inherent properties of AuNR, the absorption intensity below 600 nm increased due to the addition of DOX (Fig. 2G).

Evaluation of the sequential responsive property of DOX@Gel-DEVD-AuNR in vitro

We tested the changes in the morphological, spectroscopic, and PA properties of core-satellite DOX@Gel-DEVD-AuNR nanomedicine upon trypsin and active recombinant mouse caspase-3 addition to assess the capability of DOX@Gel-DEVD-AuNR nanomedicine for monitoring and quantifying enzyme-activated drug release and NIR-II PA signal “turn on”. Initially, we investigated the trypsin’s ability to degrade Gel NPs responsively. The introduction of trypsin led to efficient structural degradation and size change of Gel NPs within 60 min (Figure S17). Even after a 20-minute exposure to trypsin, a significant portion of Gel NPs remained degraded as confirmed by TEM observation in Fig. 3A-C. This provided both theoretical and experimental support for exploring the release behavior of DOX from DOX@Gel-DEVD-AuNR nanomedicine in response to trypsin (Fig. 3D). Following that, the morphological changes of DOX@Gel-DEVD-AuNR nanomedicine and the release of DOX triggered by trypsin were investigated. The addition of trypsin efficiently degraded DOX@Gel-DEVD-AuNR, resulting in the release of DOX, as confirmed by TEM observation in Fig. 3E, F. The released DOX from the DOX@Gel-DEVD-AuNR nanomedicine was collected after incubation with trypsin for different durations and quantified using standard absorption curve (Figure S18). The cumulative release of DOX reached approximately 50% within the initial 20 min of incubation with trypsin, followed by an additional release of 43.8% during the subsequent period (Fig. 3G).

Fig. 3.

Enzyme-sequentially responsive evaluation of DOX@Gel-DEVD-AuNR nanomedicine. TEM images of Gel NPs before ((A) and after the addition of trypsin for 20 min (B) and 60 min (C). (D) Enzyme-activated release of DOX and aggregation of AuNRs in DOX@Gel-DEVD-AuNR nanomedicine. TEM images of DOX@Gel-DEVD-AuNR nanomedicine before (E) and after (F) the addition of trypsin. (G) Time-dependent profile of cumulative release of DOX from DOX@Gel-DEVD-AuNR nanomedicine in the absence (−) and presence (+) of trypsin (n = 3). (H) TEM images of Gel-DEVD-AuNR after (F) the addition of GSH and caspase-3. (I) UV-vis-NIR spectra of DOX@Gel-DEVD-AuNR in the absence and presence of trypsin, GSH and caspase-3. (J) PA intensity of trypsin treated DOX@Gel-DDVD-AuNR and DOX@Gel-DEVD-AuNR at 1250 nm in the presence of GSH (5 mM) and varying levels of caspase-3 activity

The investigation into the aggregation of AuNRs mediated by active caspase-3 began with the preparation of the trypsin-treated DOX@Gel-DEVD-AuNR dispersion. This dispersion was centrifuged, and the precipitate obtained was re-dispersed in a caspase buffer (referred to as Gel-DEVD-AuNR). To facilitate efficient disulfide reduction, the sample was treated with 5 mM glutathione (GSH) for 2 h at 37 °C. Following this, an active recombinant mouse caspase-3 (10 U) was introduced to initiate the enzymatic reaction at 37 °C. Active caspase-3 cleaved the DEVD-Cys(S-S-NH₂)-(AuNR)-CBT from DOX@Gel-DEVD-AuNR, thereby exposing a Cys residue on Cys(S-S-NH₂)-(AuNR)-CBT. This exposed Cys residue rapidly condensed with the CBT motif on another Cys(S-S-NH₂)-(AuNR)-CBT, leading to cross-linking [24–27]. Consequently, aggregates of AuNRs were formed, as depicted in Fig. 3H. This aggregation induced a red-shift in the absorption wavelength, which was attributed to plasmonic coupling effects among neighboring AuNRs, as shown in Fig. 3I. The treatment of DOX@Gel-DDVD-AuNR, which was used as a control due to its unresponsiveness to caspase-3, was carried out in the same manner as that of DOX@Gel-DEVD-AuNR, no noticeable red-shift in the absorption wavelength (Figure S19) and aggregation of AuNRs were observed (Figure S20), thereby further emphasizing the pivotal role played by the caspase-3-cleavable DEVD peptide substrate in this process. In addition, the NIR-II PA signal responsive “turn on” capability of caspase-3 was investigated in vitro. Various caspase-3 contents ranging from 0 to 10 U were incubated with Gel-DEVD-AuNR in a caspase assay buffer for 5 h. The NIR-II PA intensities exhibited a gradual increase at 1250 nm as the enzymatic activity was enhanced. In comparison to the intrinsic signal at 1250 nm of DOX@Gel-DEVD-AuNR, the NIR-II PA signal showed a remarkable enhancement of 7.77-fold when DOX@Gel-DEVD-AuNR was incubated with 10 U caspase-3. Conversely, there was minimal change observed in the NIR-II PA signal of DOX@Gel-DDVD-AuNR before and after incubation (Fig. 3J). The collective results confirmed the capability of the core-satellite DOX@Gel-DEVD-AuNR system to achieve enzyme-triggered release of DOX and activate NIR-II PA signal “turn on”.

Enzyme-sequentially responsive nanomedicine enables the aggregation of AuNRs and activates NIR-II PA signal

Activation of caspase-3 serves as the trigger for AuNRs aggregation and initiation of NIR-II PA signals, thus elucidating the source behind caspase-3 activation is a pivotal aspect addressed in this study. Previous investigations have demonstrated that Gel NPs can be degraded by trypsin, facilitating DOX release. Given the abundance of trypsin in cancer cells, we investigated whether DOX@Gel-DEVD-AuNR specifically activates caspase-3 in cancer cells. To achieve this objective, DOX@Gel-DEVD-AuNR was labeled with Cy5.5 and subsequently incubated with CT26 cells for different durations. The resulting fluorescence signal of the CT26 cells was then detected using a confocal laser scanning microscope (CLSM). As shown in Fig. 4A, the intensity of the red fluorescence signal exhibited a positive correlation with the incubation time, demonstrating a significant enhancement of 3.69-fold after 9 h compared to 3 h (Figure S21), indicating superior uptake at longer durations.

Fig. 4.

DOX@Gel-DEVD-AuNR nanomedicine mediated caspase-3 activation in vitro. (A) Representative confocal fluorescence images of CT26 cell incubated with cy5.5-labeled DOX@Gel-DEVD-AuNR (2.5 mg/mL, 20 µL) at different time points and (B) corresponding western blot bands and (C) cleaved caspase-3/ß-actin (n = 3). (D) Bio-TEM images of CT26 cell after treated with DOX@Gel-DEVD-AuNR for 24 h. (E) PA intensity at 1250 nm of CT26 cell after treated with DOX@Gel-DEVD-AuNR or DOX@Gel-DDVD-AuNR for 24 h (n = 3)

DOX release occurs after the internalization of DOX@Gel-DEVD-AuNR and is triggered by trypsin overexpression in tumor cells, leading to caspase-3 activation through chemotherapy. This process induces aggregation of AuNRs at the cellular level, facilitating the turn-on of NIR-II PA signals. To test this hypothesis, western blotting assay was employed to characterize the relative expression levels of cleaved caspase-3 (activated form of caspase-3). Importantly, an overall increase in the expression levels of cleaved caspase-3 (Fig. 4B, C) and an increased NIR-II PA signal (Figure S22A) were observed with prolonged incubation time. This suggested a positive correlation between caspase-3 activation and the quantity of DOX. Moreover, the released DOX can sufficiently activate caspase-3 to turn on the NIR-II photoacoustic signal (Figure S22B). The bio-TEM images revealed conspicuous aggregates of AuNRs in cells incubated with DOX@Gel-DEVD-AuNR (Fig. 4D), whereas no such aggregates were observed in cells incubated with DOX@Gel-DDVD-AuNR (Figure S23). The NIR-II PA signal intensity of cells incubated with DOX@Gel-DEVD-AuNR was found to be 5.23 times greater than that of cells incubated with DOX@Gel-DDVD-AuNR (Fig. 4E). This significant difference indicated that the aggregation of AuNRs at the cellular level, as well as the “turn on” of NIR-II PA signals, is co-regulated by trypsin and cleaved caspase-3. The activation of caspase-3 was notably facilitated by trypsin-induced DOX release, emphasizing the cascade interplay between these molecular components in modulating the observed signal intensity.

Cellular-level evaluation of chemotherapy cascade-enhanced radiotherapy

To verify the chemotherapy cascade-enhanced radiotherapy capacity of DOX@Gel-DEVD-AuNR nanomedicine, an ROS probe called 2’,7’-dichlorofluorescein diacetate (DCFH-DA) was utilized to visualize intracellular ROS levels. Upon reaction with ROS, DCFH-DA generates a green fluorescent signal. The CT26 cells treated with DOX@Gel-DEVD-AuNR and exposed to 6 Gy X-ray irradiation (DOX@Gel-DEVD-AuNR + X-ray group) exhibited significantly higher levels of cellular ROS compared to the CT26 cells treated with X-ray irradiation alone, DOX@Gel-DEVD-AuNR alone, and the DOX@Gel-DDVD-AuNR + X-ray group by 2.99-fold, 3.21-fold, and 1.46-fold respectively (Fig. 5A, Figure S24).

Fig. 5.

Cellular-level evaluation of chemotherapy cascade-enhanced radiotherapy. (A) Confocal fluorescence microscope images of CT26 cells stained with DCFH-DA and DAPI following various treatments. (C) Confocal fluorescence microscope images of CT26 cells stained with γ-H2AX and DAPI following different treatments. (C) The flow cytometry analyses of CT26 cells after different treatments. (G 1: PBS, G 2: X-ray, G 3: DOX@Gel-DEVD-AuNR (2.5 mg/mL, 20 µL), G 4: DOX@Gel-DDVD-AuNR (2.5 mg/mL, 20 µL) + X-ray, G 5: DOX@Gel-DEVD-AuNR (2.5 mg/mL, 20 µL) + X-ray)

The generation of ROS was amplified through chemotherapy-induced cascade radiosensitization, resulting in increased DNA damage. To assess the extent of DNA double-strand breaks, immunofluorescence staining of γ-H2AX was employed, with the intensity of intracellular red fluorescence serving as an indicator. Notably, the intracellular red fluorescence intensity exhibited a consistent trend with the green fluorescence signal intensity detected by DCFH-DA (Fig. 5B, Figure S25), suggesting that the release of DOX from DOX@Gel-DEVD-AuNR nanomedicine, triggered by trypsin activity in tumor cells, enhances DNA double-strand damage via cascade enhanced radiotherapy. To further investigate the cascade effects of chemotherapy and radiotherapy in vitro, flow cytometry assay was performed. As anticipated, the DOX@Gel-DEVD-AuNR + X-ray group demonstrated the highest apoptotic rate (69.9%) among all experimental groups, approximately 1.5 times greater than that of the.

DOX@Gel-DDVD-AuNR + X-ray group. The significant increase in apoptosis can be attributed to the release of DOX triggered by trypsin, leading to the activation of caspase-3. Caspase-3 cleaved the DEVD sequence, promoting the assembly of AuNRs into large aggregates. This aggregation enhanced the retention and enrichment of radiosensitizers within tumor cells, thereby amplifying the therapeutic effect. To substantiate this mechanism, inductively coupled plasma-mass spectrometry (ICP-MS) was employed to quantify the accumulation efficiency of gold within cancer cells, based on gold content. After treating CT26 cells with either DOX@Gel-DEVD-AuNR or DOX@Gel-DDVD-AuNR for an equivalent duration. The results revealed a 2.46-fold increase in gold content in cancer cells treated with DOX@Gel-DEVD-AuNR compared to those treated with DOX@Gel-DDVD-AuNR (Figure S26). The present finding validated that the release of DOX triggered by trypsin induces caspase-3 activation, thereby facilitating the formation of gold aggregates and consequently enhancing gold accumulation in cancer cells, leading to improved radiosensitization.

Evaluation of activatable NIR-II p.a. image-guided chemotherapy cascade-enhanced radiotherapy in vivo

Image-guided tumor therapy combines diagnostic and therapeutic capabilities into a single entity, thus facilitating the advancement of precision medicine [28, 29]. Based on the promising results obtained from nanomedicine dispersions and cellular-level studies, we further investigated the in vivo NIR-II PA image-guided chemotherapy cascade-enhanced radiotherapy potential of the DOX@Gel-DEVD-AuNR nanomedicine. NIR-II PA imaging was performed at various time intervals following tail vein injection of DOX@Gel-DEVD-AuNR to determine the optimal time point for therapeutic intervention. The PA signal intensity at 1250 nm within the tumor region exhibited a gradual increase over time, reaching its peak at 21 h post-injection (Fig. 6A, B). Consequently, this time point was selected for subsequent X-ray irradiation. For comparison, the in vivo NIR-II imaging capability of DOX@Gel-DDVD-AuNR was also assessed. Unlike DOX@Gel-DEVD-AuNR, the PA intensity at 1250 nm in the tumor region remained relatively unchanged at all time points post-injection of DOX@Gel-DDVD-AuNR (Figure S27). These results provided further evidence that the activation of the NIR-II PA signal was attributed to the cleaved caspase-3 mediated cleavage of the DEVD sequence, activated by the release of DOX from the intratumoral nanomedicine degraded by trypsin. This process subsequently induced the aggregation of AuNRs, thereby turning on the NIR-II PA signal due to the plasmonic coupling effect between neighboring AuNRs.

Fig. 6.

NIR-II PA Image-Guided Anticancer therapeutic effect evaluation in vivo. (A) Representative US, photoacoustic (PA) imaging at a wavelength of 1250 nm, and merged PA and US images obtained after intravenous administration of DOX@Gel-DEVD-AuNR. (B) Corresponding post-injection PA intensity at 1250 nm was assessed at different time points. (C)  Representative photographs, (D) relative tumor volume, (E) survival rate, (F) body weight curves, and H&E staining of tumor sections with different groups (n = 3). (G 1: PBS, G 2: X-ray, G 3: DOX@Gel-DEVD-AuNR, G 4: DOX@Gel-DDVD-AuNR + X-ray, G 5: DOX@Gel-DEVD-AuNR + X-ray)

The in vivo antitumor efficacy of DOX@Gel-DEVD-AuNR nanomedicine was assessed using a tumor-bearing mouse model. Based on the findings from in vitro experiments and in vivo imaging, DOX@Gel-DEVD-AuNR was administered intravenously to the mice, followed by X-ray irradiation 21 h post-injection. Control groups included PBS, X-ray alone, DOX@Gel-DEVD-AuNR alone, and DOX@Gel-DDVD-AuNR + X-ray. Tumor volume and body weight were monitored and recorded every three days over the course of 18 days. At the end of the treatment period, mice treated with X-ray alone (relative tumor volume ~ 10.69) or DOX@Gel-DEVD-AuNR alone (relative tumor volume ~ 9.08) exhibited only mild therapeutic effects compared to the control group (relative tumor volume ~ 13.84). In contrast, the combination of DOX@Gel-DEVD-AuNR and X-ray irradiation demonstrated significantly enhanced antitumor efficacy. Notably, the DOX@Gel-DEVD-AuNR + X-ray group achieved substantial tumor growth inhibition, with a relative tumor volume of approximately 3.53. This outcome was superior to that of the DOX@Gel-DDVD-AuNR + X-ray group, which exhibited a relative tumor volume of 5.60 (Fig. 6C, D). The enhanced therapeutic efficacy of DOX@Gel-DEVD-AuNR + X-ray can be attributed to the increased accumulation of AuNRs at the tumor site, which was facilitated by chemotherapy induced activation of caspase-3. This had been confirmed through ICP-MS analysis, showing an uptake value of 11.59 [Au]% ID/g at 21 h after injection for DOX@Gel-DEVD-AuNR and 5.27 [Au]% ID/g for DOX@Gel-DDVD-AuNR (Figure S28). However, DOX@Gel-DEVD-AuNR and DOX@Gel-DDVD-AuNR were metabolized rapidly in other major organs. Only a small amount of them was detected after 10 days (Figure S29 and Figure S30). This phenomenon can be ascribed to the small size of DOX@Gel-DEVD-AuNR and DOX@Gel-DDVD-AuNR, which were unable to self-assemble into larger aggregates in normal tissues.

This increased accumulation of AuNRs, functioning as radiosensitizers, amplified the radiosensitization effect. Consequently, the tumor inhibition rate for the DOX@Gel-DEVD-AuNR + X-ray group (75%) was approximately 1.25 times, 2.21 times, and 3.26 times higher than that of the DOX@Gel-DDVD-AuNR + X-ray group (60%), the DOX@Gel-DEVD-AuNR alone group (34%), and the X-ray alone group (23%), respectively. The results demonstrated that compared to the DOX@Gel-DDVD-AuNR + X-ray group, the DOX@Gel-DEVD-AuNR + X-ray group exhibited a more potent inhibitory effect on tumor growth.

The combination of DOX@Gel-DEVD-AuNR and X-ray irradiation mediated chemotherapy cascade-enhanced radiotherapy significantly prolonged the lifespan of the treated animals (Fig. 6E). Furthermore, throughout the experiment, none of the mice exhibited a significant reduction in body weight, indicating that the nanomedicine used in the study has negligible side effects (Fig. 6F). To evaluate the therapeutic efficacy and safety of this approach, the major organs and tumors from each mouse were harvested and subjected to histopathological analysis using hematoxylin and eosin (H&E) staining. The results demonstrated significant apoptotic and necrotic tumor tissue in the group treated with DOX@Gel-DEVD-AuNR and X-ray irradiation (Fig. 6G). This group exhibited more pronounced damage to the tumor tissue compared to other treatment groups, highlighting the efficacy of this chemotherapy cascade-enhanced radiotherapy platform in treating tumor tissue.

Conclusions

In conclusion, we developed a DOX@Gel-DEVD-AuNR nanomedicine with a core–satellite architecture that enables trypsin-activated drug release in vivo. This mechanism activated caspase-3, which subsequently triggered the aggregation of AuNR, thereby achieving synergistically enhanced chemotherapy and radiotherapy in a cascade manner. Studies conducted in test tubes, in vitro, and in vivo have demonstrated that trypsin stimulation facilitated the release of DOX from DOX@Gel-DEVD-AuNR. The released DOX then activated caspase-3, inducing the aggregation of AuNR, which in turn initiated the NIR-II PA signal and enhanced the sensitivity of radiotherapy. In subcutaneous CT26 colon tumor models, the tumor inhibition efficiency of DOX@Gel-DEVD-AuNR combined with X-ray irradiation was 1.25-fold higher than that of DOX@Gel-DDVD-AuNR with X-ray irradiation, due to the chemotherapy cascade-enhanced radiotherapy. This work underscored the potential of DOX@Gel-DEVD-AuNR nanomedicine in activatable image-guided chemotherapy cascade-enhanced radiotherapy through enzyme-sequentially triggered release of therapeutic agents.

Experimental section

Preparation of DOX-Loaded Gelatin Nanoparticles (DOX@Gel NPs): Gelatin nanoparticles (Gel NPs) were synthesized using a modified two-step desolvation method. Initially, 625 mg of gelatin type A was dissolved in 12.5 mL of deionized water at 40 °C. To this solution, 12.5 mL of acetone was added dropwise at a rate of 6 mL/min while stirring continuously at 600 rpm for 10 min. Following this, the supernatant containing the low molecular weight gelatin fraction was removed by discarding approximately half of the solution. The pH of the remaining solution was then adjusted to approximately 2.5 using 1 M HCl. Subsequently, 20.75 mL of acetone was added slowly dropwise at a rate of 1 mL/min under continuous stirring at 1000 rpm. To cross-link the gelatin nanoparticles, a mixture of 1 mL acetone and 30 µL of 50% glutaraldehyde solution was added dropwise at a rate of 0.05 mL/min while maintaining the solution at 40 °C under constant stirring at 1000 rpm. After incubating for 16 h, excess acetone and glutaraldehyde were removed by rotary evaporation until the volume was reduced to approximately 5 mL. The resulting solution was purified through dialysis against water to eliminate any residual acetone and glutaraldehyde, and then diluted to a final volume of 50 mL with PBS buffer. For the preparation of DOX-loaded gelatin NPs (DOX@Gel), 2.5 mL of Gel NPs solution, 1 mL of DOX solution (concentration: 5 mg/mL), and 1.375 mL of ddH₂O were sonicated together for 30 min. This was followed by centrifugation to remove free DOX molecules, and the precipitate was dissolved in PBS buffer (pH 7.4).

Preparation of DOX@Gel-DEVD-AuNR

To activate the carboxyl groups on the surface of gelatin nanoparticles, 1 mL of DOX@Gel (10 mg/mL) in PBS was mixed with 2 mg of EDC and 2 mg of NHS under continuous stirring for 30 min at a pH of approximately 6. Following this activation step, the solution’s pH was adjusted to approximately 7.2, and it was then added dropwise to the AuNR-DEVD-NH₂ solution. This mixture was stirred continuously for 2 h at a constant temperature. Subsequently, 0.2 mL of EDC stock solution (20 mg/mL) and 0.2 mL of NHS stock solution (20 mg/mL) were introduced into the DOX@Gel-DEVD-AuNR solution at a pH of approximately 6 and maintained for 30 min. The pH of the mixture was then adjusted to approximately 7.2, after which 0.05 mL of water containing 20 mg of methyl-PEG-amine (PEG-NH₂, 5 kDa) was added to the DOX@Gel-DEVD-AuNR mixture. The reaction was allowed to proceed at room temperature for 2 h. Finally, the resulting DOX@Gel-DEVD-AuNR complexes were purified through three rounds of ultracentrifugation. DOX@Gel-DDVD-AuNR was prepared in the same way as the DOX@Gel-DEVD-AuNR, except for replacing AuNR-DEVD-NH₂ with AuNR-DDVD-NH₂.

Statistical analysis

The data were presented as mean ± standard deviation (SD). A t-test or One-Way ANOVA was used for statistical comparison of the data. Statistical significance was indicated by * P < 0.05, ** P < 0.01, ***P < 0.001, ****P < 0.0001. ns: no significance.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Author contributions

WX, XY, MW, ZJ contributed equally. QF, JS, conceived and designed experiments. QF, revised the manuscript. WX, QF, drafted the initial version and conducted imaging experiments. XY, conducts experiments on material synthesis and cancer treatment. MW, provided assistance for all experiments. ZJ, HZ, GM, LZ, participated in the experimental process, analyzed results. X.W., and Q.F. acquired the funds. All the authors reviewed the manuscript.

Data availability

No datasets were generated or analysed during the current study.

Declarations

Competing interests

The authors declare no competing interests.