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.
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).
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 [24,25,26,27]. 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.
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.
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)
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).
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.
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)
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.
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.
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 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.