Self-assembled Aldehyde Dehydrogenase-Activatable Nano-prodrug for Cancer Stem Cell-Enriched Tumor Detection and Treatment

doi:10.21203/rs.3.rs-4319425/v1

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Self-assembled Aldehyde Dehydrogenase-Activatable Nano-prodrug for Cancer Stem Cell-Enriched Tumor Detection and Treatment

https://doi.org/10.21203/rs.3.rs-4319425/v1

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published 31 Oct, 2024

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Cancer stem cells (CSCs), characterized by high tumorigenicity and drug-resistance, are often responsible for tumor progression and metastasis. Aldehyde dehydrogenases (ALDH), often overexpressed in CSC-enriched tumors, present a potential target for specific anti-CSC treatment. In this study, we report a self-assembled nano-prodrug (PS-CHO@ATRA-SS-ATRA) composed of ALDH-activatable photosensitizer (PS-CHO) and disulfide-linked all-trans retinoic acid (ATRA-SS-ATRA) for diagnose and targeted treatment of CSC-enriched tumors. The ATRA-SS-ATRA can load with PS-CHO and self-assemble into a stable nanoparticle PS-CHO@ATRA-SS-ATRA, which can be disassembled into ATRA and PS-CHO in CSC by high level of GSH. As for released PS-CHO, overexpressed ALDH catalyzes the oxidation of aldehydes (-CHO) to carboxyl (-COOH) under CSC-enriched microenvironment, activating the generation of reactive oxygen species (ROS) and fluorescence emission. This ROS generation leads to direct killing of CSCs and is accompanied by a noticeable fluorescence enhancement for real-time monitoring of the CSC-enriched microenvironment. Moreover, the released ATRA, as a differentiation agent, reduce the CSCs stemness and improve the CSC-enriched microenvironment, offering a synergistic effect for enhanced anti-CSC treatment of PS-CHO in inhibition of in vivo tumor growth and metastasis.

Physical sciences/Materials science/Biomaterials/Biomedical materials

Physical sciences/Chemistry/Materials chemistry/Biomaterials/Biomedical materials

Tumor heterogeneity is an ongoing and formidable obstacle in cancer treatment due to a variety of cells in solid tumor bearing distinct phenotypic profiles with varying sensitivity to traditional therapies^1–3. Cancer stem cells (CSCs) have been considered as a driving source of tumor heterogeneity, possessing a natural characteristic of stemness that induces self-renewal, multi-differentiation potential, and drug resistance^4–6. Clinical research has highlighted that CSCs, characterized by their stemness, are different from normal cancer cells and yet similar to normal cells^{7, 8}. This similarity lies in their quiescent and silent state, rendering them less susceptible to conventional anti-cancer treatments^{9, 10}. Therefore, CSC-enriched tumor is insensitive to traditional chemotherapy or radiotherapy, which resulted in limited therapeutic effect^11–13. However, these residual CSCs within lesion locations are often referred to as a "timebomb" due to their potential to undergo differentiation into mature cancer cells with elevated stemness, and this process can result in tumor metastasis and recurrence^14–16. Thus, it is of practical importance to explore a specific and highly effective strategy for direct targeting and elimination of CSC-enriched tumors.

Through studying the unique stemness characteristics of CSC and their associated biomarkers, the overexpression of aldehyde dehydrogenase (ALDH) is found to be closely associated with poor survival rates in CSC-enriched tumors, including leukemia, pancreas, breast, and liver cancers^17–21. All these tumors often exhibit a high level of ALDH, which is considered as an important and specific biomarker in CSC-enriched tumor^{22, 23}. In organisms, ALDH is an enzyme responsible for oxidizing xenobiotic aldehydes (-CHO) into the corresponding carboxylates (-COOH)^24–26. This unique enzymatic activity may provide a promising potential for designing ALDH-responsive prodrug with selective toxicity activation for the specific and safe treatment of CSC-enriched tumors.

Photodynamic therapy (PDT) has emerged as a promising non-invasive and highly efficient cancer treatment approach, utilizing reactive oxygen species (ROS) generated from photosensitizers (PSs) to induce cancer cell death^{27, 28}. In comparison to traditional PSs, activatable PSs (aPSs) can be considered as a PDT prodrug, which remain inactive and non-toxic in their "OFF" state but transition to an "ON" state in response to specific stimuli present in the tumor microenvironment^29–31. This activation triggers ROS generation, leading to cell death in targeted tumor cells³², Moreover, aPSs function as sensitive fluorescent probes, providing real-time information about the drug activation and tumor microenvironment^{33, 34}. Therefore, the development of ALDH-responsive aPSs, capable of ROS generation and fluorescence monitoring in the CSC-enriched microenvironment, holds significant potential for the precise and high-efficiency treatment of CSC-enriched tumors.

Recent research has demonstrated that PDT can directly eliminate CSC-enriched tumors through ROS-induced cell apoptosis^{35, 36}. However, prolonged, or excessive use of apoptosis-related drugs, including PSs, can contribute to the enrichment of CSC stemness, leading to poor drug sensitivity and high drug resistance^{4, 37–39}. To address this challenge, differentiation therapy has been explored as an effective synergetic treatment to reduce CSC stemness and improve the CSC tumor environment, offering an indirect approach to decreasing CSC^40–42. Among various differentiation agents, all-trans retinoic acid (ATRA), an FDA-approved differentiation agent, has shown significant efficacy in reducing CSC stemness in CSC-enriched tumors^{43, 44}. However, ARTA has insignificant cytotoxicity of CSC and thus it is not effective for in vivo tumor inhibition in CSC-enrich tumor. Thus, the combination of ALDH-activatable PS and ATRA may integrate their advantage to overcome drug resistance of PDT and achieve the specific and high-efficiency therapeutic effect in CSC-enriched tumors.

In this work, we present a self-assembled nano-prodrug composed of an ALDH-responsive aPSs (PS-CHO) and differentiation agent ATRA for precise and highly effective diagnose and PDT of CSC-enriched tumor. As shown in Fig. 1, PS-CHO contains an ALDH-responsive aldehyde group (-CHO) resulting in poor PDT effect and weak fluorescent signal. ATRA, conjugated with disulfide bond (-SS-) to form ATRA-SS-ATRA, demonstrates good self-assembly behavior^45–47, allowing it to load the hydrophobic PS-CHO and create a nan-prodrug (PS-CHO@ATRA-SS-ATRA). Under high ALDH expression, PS-CHO is transformed into PS-COOH, activating PS-CHO and inducing cell death specifically through ROS generation. Moreover, the activation of PS-CHO also results in the restoration of its fluorescent signal, enabling real-time monitoring of the CSC-enriched microenvironment. In PS-CHO@ATRA-SS-ATRA, ATRA-SS-ATRA not only acts as a good carrier for PS-CHO but also triggers by a high level of GSH to release ATRA. The released ATRA can reduce the stemness of CSC and improve the CSC-enriched microenvironment, offering a synergistic effect for enhanced anti-CSC treatment of PS-CHO. For targeted CSC-enriched tumor, PS-CHO@ATRA-SS-ATRA is decorated with hyaluronic acid (DSPE-PEG2000-HA), which targets CD44 receptors highly expressed on CSC⁴⁸. The systematic administration of PS-CHO@HA-ATRA-SS-ATRA has demonstrated good tumor-specific accumulation in different CSC-enriched tumor mice models, resulting in significant inhibition of tumor growth and minimized tumor metastasis in normal tissues, particularly in the lung and liver. The current study presents the first proof-of-concept example for CSC-activated PDT prodrug with the adjuvant differentiation therapy, offering a precise and safe approach for treating various CSC-enriched cancer.

Synthesis and characterization of PS-CHO and ATRA-SS-ATRA

The synthetic route of PS-CHO and ATRA-SS-ATRA is depicted in Fig. 2a-i. In general, compound 1 was synthesized according to the previous literature⁴⁹, and then the -COOH on compound 1 was reduced to -CH₂OH (compound 2) in 78.1% yield. Then, compound 2 was modified with -CHO by Duff reaction to offer compound 3 in 91.2% yield. Reaction between compound 3 and malononitrile derivatives by aldol reaction yielded compound 4, which was oxidized to give PS-CHO (compound 5) in 24.4% yield. In addition, the small molecular self-assembly prodrug ATRA-SS-ATRA (compound 6) was obtained from ARTA upon reaction with 2,2'-disulfide diethanol by using 4-dimethylaminopyridine (DMAP)/triphosgene in 82.0% yield (Fig. 2a-ii). Meanwhile, we also obtained PS-COOH according to the previous literature⁴⁹. All these compounds were confirmed by ¹H nuclear magnetic resonance (¹H NMR) and electro spray ionization-mass spectroscopy (ESI-MS) (Supplementary Figs. 1–10) to reveal their right structure and high purity.

ALDH-responsive behavior and ROS generation of PS-CHO in physiological conditions

ALDH is an enzyme responsible for the oxidation of aldehydes to carboxylic acids in vivo. Therefore, the ALDH-responsiveness of PS-CHO was first assessed by the qualitative analysis of ESI-MS. As shown in Supplementary Fig. 11, a new ionic peak emerged at 817.13 [M + H⁺] after incubation with ALDH, which was assigned to PS-COOH. Moreover, the qualitative analysis of ALDH-responsiveness was also investigated by high-performance liquid chromatography (HPLC). As shown in Fig. 2b, the retention time of PS-CHO was emerged at 5.06 min. After incubation with ALDH, the intensity of PS-CHO was decreased, while a new retention time was observed at 2.78 min for PS-COOH. In addition, quantitative analysis of PS-CHO in high level of ALDH was also performed by HPLC. In Supplementary Fig. 12, the cumulative percentage of PS-COOH reached 50.7% in PBS solution (10% DMSO, due to poor solubility of PS-CHO in PBS solution) after 4 h incubation, and this relatively moderate transformation rate may be caused by poor water solubility of PS-CHO. All these results suggested that ALDH could effectively oxidize PS-CHO to PS-COOH in physiological conditions.

We next investigated the fluorescence properties of PS-CHO with or without ALDH. As shown in Fig. 2c, PS-CHO showed a relatively weak fluorescent signal at 778 nm when excited at 550 nm. After incubation with ALDH, an obvious fluorescence enhancement was observed at 767 nm, which gradually increased with time and reached a plateau in 4 h (Fig. 2d and Supplementary Fig. 13a). The higher ALDH concentration also triggered higher fluorescence intensity at 767 nm (Supplementary Figs. 13b and 14), which belonged to PS-COOH. In addition, the specificity of ALDH to PS-CHO was also evaluated in vitro. Only ALDH could induce significant fluorescence enhancement of PH-CHO, and the signal is stable over pH 4 to 10 (Supplementary Fig. 15a and 15b). Inspired by the good ALDH-responsive behavior of PS-CHO in physiological conditions, we next assessed the ROS generation under high ALDH microenvironments using a ROS probe (dichlorodihydrofluorescein, DCFH). In Fig. 2e, DCFH solution incubated with free PS-CHO displayed no obvious fluorescence enhancement at 525 nm under light irradiation, indicating its poor ROS generation capacity. However, under the same condition, after incubating PS-CHO with ALDH, a significant fluorescence enhancement was detected (Figs. 2f and Supplementary Fig. 16), indicating the ROS production capability after the reaction.

As PS-CHO is a hydrophobic molecule with poor water solubility, PS-CHO-based nano-formulation by ATRA-SS-ATRA was used to improve the biocompatibility. The small molecular self-assembly behavior of ATRA-SS-ATRA was first assessed by dynamic light scattering (DLS) and transmission electron microscopy (TEM). As shown in Supplementary Fig. 17, the nanoparticles of ATRA-SS-ATRA displayed spherical morphology with an obvious good size distribution (average size: 143 nm) and a low polydispersity index (PDI, 0.115). The DLS revealed that the nanoparticles have slightly negative surface charge (ζ potential: -0.178 mV, Supplementary Fig. 18). Based on this finding, the nano-formulation of PS-CHO (nano-prodrug, PS-CHO@ATRA-SS-ATRA) was prepared by nanoprecipitation with ATRA-SS-ATRA (Fig. 2g). DLS and TEM results indicate that PS-CHO@ATRA-SS-ATRA has an average size of 166.4 nm (PDI = 0.129) with an obvious Tyndall effect (Fig. 2h and 2i) and display spherical morphology with the positive ζ potential (8.78 mV) (Supplementary Fig. 18). The stability was also investigated by DLS, and the size distribution and PDI remained stable after 30 days of storage (Fig. 19a and 19b). In addition, due to the presence of -SS- in ATRA-SS-ATRA, PS-CHO@ATRA-SS-ATRA disassembled after incubation with 10 mM GSH (Supplementary Fig. 20). This GSH-responsive disassembly behavior could also induce PS-CHO release from nanoparticles, and the PS-CHO release behavior was investigated by HPLC. With increasing GSH concentrations, PS-CHO@ATRA-SS-ATRA progressively released PS-CHO and reached a maximum cumulative release of 83.1% after 4 h incubation (Fig. 2j). Meanwhile, a similar drug release rate of ATRA (77.8%) was also detected in Supplementary Fig. 21. The above results indicated that high level of GSH could trigger disassembly behavior, further releasing both PS-CHO and ATRA under physiological conditions.

Intracellular ALDH-responsive behavior and ROS generation of PS-CHO@ATRA-SS-ATRA

We first investigated the ALDH-responsive behavior of small molecular PS-CHO and nano-prodrug PS-CHO@ATRA-SS-ATRA by fluorescence imaging in CSC-enriched 4T1 cells. As shown in Supplementary Fig. 22, no obvious fluorescent signal was detected in small molecular PS-CHO dissolved in 1% DMSO for cell experiments due to its poor solubility, while there was a strong fluorescent signal in the nano-prodrug PS-CHO@ATRA-SS-ATRA. Recently, solid evidence proved that hypoxia microenvironment is a typical characteristic of CSC-enriched microenvironment, which could induce the higher expression of ALDH and upregulate the CSC-enriched microenvironment. Based on these findings, we evaluated the ALDH-responsive behavior in 4T1 cells under hypoxic condition. As shown in Fig. 3a and Supplementary Fig. 23, the fluorescence intensity of PS-CHO@ATRA-SS-ATRA was stronger under hypoxic condition due to the elevated level of ALDH, and this intensity was gradually increased in a time- and dose-dependent manner, providing a real-time reporting of CSC-enriched microenvironment.

As a control, there was no obvious fluorescent signal in 3T3 cells (normal cells) under hypoxic conditions, indicating the low side-effect of PS-CHO@ATRA-SS-ATRA in normal cells (Fig. 3b). Based on the good ALDH-responsive activation and high-efficiency drug release behavior of PS-CHO@ATRA-SS-ATRA in CSC-enriched 4T1 cells, we next investigated its ROS generation by using a ROS probe (DCFH-DA) as the indicator. In 4T1 cells, weak green fluorescence was detected in ATRA-SS-ATRA, free PS-CHO and PS-CHO@ATRA-SS-ATRA without light irradiation, and PS-CHO with light irradiation also displayed a poor green fluorescence due to the poor cell uptake. In contrast, the nano-prodrug, PS-CHO@ATRA-SS-ATRA, exhibited much stronger fluorescence in 4T1 cells after light irradiation, suggesting that the high level of ALDH could transform PS-CHO into PS-COOH and thus trigger the ROS generation (Fig. 3c). In normal cells, only faint fluorescence was detected in all formulations, suggesting that our nano-prodrug was not able to produce ROS and therefore exhibited good biosafety (Supplementary Fig. 24).

Cytotoxicity of PS-CHO@ATRA-SS-ATRA

Inspired by good ROS activation of PS-CHO@ATRA-SS-ATRA on CSC-enriched cancer cells, we next performed CCK8 assays in different types of cells to evaluate whether the ALDH-responsive behavior of PS-CHO could result in potential cytotoxicity. In both CSC-enriched cancer cells (4T1) and no-CSC normal cells (3T3), free ATRA-SS-ATRA, PS-CHO and PS-CHO@ATRA-SS-ATRA displayed very low cytotoxicity without light irradiation, and PS-CHO with light irradiation also showed relatively low cytotoxicity due to the poor cell uptake (Fig. 3d and 3e). In contrast, PS-CHO@ATRA-SS-ATRA with light irradiation exhibited high cytotoxicity in 4T1 cells but poor in 3T3 cells, which was due to its good selectivity, as well as good cell uptake by ALDH-responsive activation and the improvement of water solubility. In addition, flow cytometry analysis and LIVE/DEAD cell death assay indicated that free ATRA-SS-ATRA, PS-CHO, and PS-CHO@ATRA-SS-ATRA merely induced any cell death, and relatively low cell death was observed in free PS-CHO with light irradiation. Under the same condition, PS-CHO@ATRA-SS-ATRA treatment showed the highest level of cell death (Fig. 3f and Supplementary Fig. 25), suggesting that PS-CHO@ATRA-SS-ATRA could induce high cytotoxicity of CSC-enriched cancer cells due to ALDH-responsive activation.

In vitro elimination and differentiation of CSC on CSC-related tumor spheres

The cell cytotoxicity suggested that PS-CHO@ATRA-SS-ATRA showed good therapeutic efficacy on CSC-enriched cancer cells. To further confirm the good CSC elimination of PS-CHO@ ATRA-SS-ATRA, we successfully constructed the CSC-enriched 4T1 tumor sphere model, which could mimic the in vivo CSC-enriched microenvironment. In 4T1 tumor sphere model, the CSC elimination and CSC-enriched microenvironment were mainly investigated by four key indicators, including tumorsphere formation, hypoxia conditions (HIF-1α), ALDH-positive cell proportions (ALDH⁺), and stemness-related gene (Sox2, Nanog and Oct4). To assess the synergic effect of ATRA, the nano-prodrug without ATRA (PS-CHO@DSPE-PEG2000, Supplementary Fig. 26) was also prepared by loading PS-CHO with DSPE-PEG2000 and severe as a negative control. As shown in Supplementary Fig. 22, the cell uptake of PS-CHO@DSPE-PEG2000 was determined by fluorescence imaging and flow cytometry, and the qualitative and quantitative analysis revealed that the fluorescence intensity of PS-CHO@DSPE-PEG2000 was similar to that of PS-CHO@ATRA-SS-ATRA, suggesting similar cell uptake of PS-CHO@DSPE-PEG2000 and PS-CHO@ATRA-SS-ATRA by 4T1 cells.

As tumorspheres have been used to determine the percentage of CSCs present in a tumor population, and thus it also used for evaluating the ability of such cells to generate stemness, and the numbers of tumorspheres can be used to visualize the effectiveness of anti-CSC treatment^50–52. In Fig. 4a and 4b, a large amount and volume of tumorspheres was observed in control group and free PS-CHO, suggesting insignificant CSC elimination. In addition, all ATRA-contained formulations exhibited smaller and lower dispersive tumorspheres, because ATRA could provide an effective differentiation-inducing effect to reduce the stemness of CSC, further improving CSC-enriching environment. As control, PS-CHO@DSPE-PEG2000 with light irradiation also reduced the number and volume of tumor spheres and further inhibited tumorsphere formulation, indicating that PS-CHO@DSPE-PEG2000 could be effectively triggered by ALDH and transformed into activated PSs (PS-COOH) for eliminating CSC. It was also observed that PS-CHO@ATRA-SS-ATRA with light irradiation displayed stronger inhibition of tumorsphere formulation than that of PS-CHO@DSPE-PEG2000, which was contributed to the improvement of CSC-enriched microenvironment from ATRA (Fig. 4a). The elevated stemness of CSCs could sharply reduce the oxygen concentration in the CSC-enriched microenvironment, which upregulated the expression of hypoxia-inducible factors (HIFs)^{53, 54}. Therefore, HIFs can serve as an important indicator for the CSC stemness and numbers of CSC formulation. Based on this finding, CSC elimination was also investigated by HIF-1α expression of 4T1 tumorspheres. The HIF-1α was labeled green dye and cell nuclei were stained with blue dye (Hoechst). As shown in Fig. 4c, a strong green, fluorescent signal was observed for the control group and free PS-CHO without light irradiation, and there was a much weaker green fluorescent signal in the formulation contained ATRA or PS-CHO with light irradiation, especially in PS-CHO@ATRA-SS-ATRA. All these results suggested that both ATRA and PS-CHO could effectively eliminate the CSCs and further induce the downregulation of HIF-1α, achieving the improvement of therapeutic effects on CSC-enriched microenvironments.

ALDH is known as a CSC biomarker, and the higher proportions of ALDH positive (ALDH⁺) cancer cells are usually associated with the stronger CSC-enriched microenvironment⁵⁵. After treatment with our nano-prodrug, the CSC elimination was monitored by the proportions of ALDH⁺ in 4T1 tumorspheres. As shown in Fig. 4d, PBS (negative control) exhibited high proportions of ALDH⁺ (26.7%) in 4T1 tumorspheres, and similar results were also detected in free PS-CHO without light irradiation (25.19%). In ATRA-contained formulation, the proportions of ALDH⁺ were decreased to 16.33% and 15.98% due to the differentiation-inducing effect of ATRA. As control, PS-CHO@DSPE-PEG2000 with light irradiation showed lower proportions of ALDH⁺ (8.39%) than that of ATRA-contained formulation, which was due to good elimination of CSC by ALDH-activated PDT treatment. Among these formulations, PS-CHO@ATRA-SS-ATRA displayed the lowest proportions of ALDH⁺ (2.86%) that was close to ALDH inhibitor (DEAB, 0.73%, positive control). Finally, the stemness-related genes (Sox2, Nanog and Oct4) were also evaluated by 4T1 tumorsphere model. As shown in Fig. 4e-4g, all ATRA-contained formulation exhibited an obvious decline of Sox2, Nanog and Oct4 gene expression, indicating good improvement of CSC-enriched microenvironment. As expected, PS-CHO@ATRA-SS-ATRA exhibited highest down-regulation of Sox2, Nanog and Oct4 genes in all formulations, further confirming that the ALDH-activated nano-prodrug could effectively eliminate the CSC and reduce the CSC-enriched microenvironment.

Preparation and in vitro drug release behaviors of nano-prodrug PS-CHO@ATRA-SS-ATRA and CSC-targeting nano-prodrug PS-CHO@HA-ATRA-SS-ATRA

To further enhance drug accumulation of the CSC-enriched tumor and maintain high stability in systemic circulation, we decorated a layer of hyaluronic acid (HA) with DSPE-PEG2000 (DSPE-PEG2000-HA) on the surface of PS-CHO@ATRA-SS-ATRA to render active CSC targeting capability. After coating, DLS suggested that the hydration diameter of PS-CHO@HA-ATRA-SS-ATRA slightly increased from 166.4 nm to 188.6 nm with a low PDI (0.129), which was also confirmed by TEM (Fig. 5a and 5b). The ζ potential was decreased from 8.78 mV to -17.7 mV (Supplementary Fig. 18). Moreover, the nano-formulation with HA decoration also showed a similar release profile as the uncoated one (Supplementary Fig. 27) and exhibited a slightly higher cytotoxicity against 4T1 cells (Supplementary Fig. 28). To further prove the HA decoration of PS-CHO@ATRA-SS-ATRA could effectively improve the therapeutics effects by active-targeting capacity, as shown in Fig. 5c, a short-term drug administration was investigated by CCK8 assays. Both 4T1 and 3T3 cells were first treated with PS-CHO@ATRA-SS-ATRA or PS-CHO@HA-ATRA-SS-ATRA for 4 h, and then the medium was replaced with fresh medium for another 48 h. As compared with PS-CHO@ATRA-SS-ATRA, PS-CHO@HA-ATRA-SS-ATRA could induce higher cytotoxicity in 4T1 cells, while very low cytotoxicity was observed in 3T3 cells when PS-CHO@HA-ATRA-SS-ATRA concentration was high, owing to the good CSC targeting in CSC-enriched cancer cells.

In vivo ALDH-responsiveness of PS-CHO@HA-ATRA-SS-ATRA

We first evaluated the hemocompatibility of our nano-prodrug, as shown in Supplementary Fig. 29. Small molecular PS-CHO displayed relatively poor hemocompatibility that the hemolysis rate is over 20% due to poor water solubility, whereas the nano-formulations that contained DSPE-PEG2000-HA showed the lowest hemolysis rate (less than 2%), indicating that the poor biocompatibility of small molecular PS-CHO. Subsequently, the in vivo drug release was detected by IVIS imaging system, and the ALDH-activated PS-COOH could serve as fluorescent dye for monitoring drug distribution. In Fig. 5d, an obvious fluorescent signal was detected on tumor site after 1 h intravenous injection of PS-CHO@HA-ATRA-SS-ATRA, which increased with time and remained strong after 12 h injection. In main organs, we only observed some fluorescent signals at liver site after the first 3 h injection, whereas there was no obvious signal in other sites at the period of 12 h injection (Fig. 5d). As a positive control, PS-COOH was also transformed into the HA-modified nanoparticle (PS-COOH@HA-ATRA-SS-ATRA, Supplementary Fig. 30) that displayed “always on” fluorescent signal. In Fig. 5d, we found that a stronger fluorescent signal was first detected in the liver, and then gradually accumulated in the abdomen site. Note that the fluorescent signal in liver and abdomen site gradually increased to the highest 6 h after the systemic administration and then maintained stable. At tumor site, an obvious fluorescent signal was visible at 4 h and became strong at 6 h, but this fluorescent signal was much lower than that of PS-CHO@HA-ATRA-SS-ATRA. This result indicated that PS-CHO@HA-ATRA-SS-ATRA mainly triggered PS-CHO in tumor site that might display good biosafety. Moreover, the mice were sacrificed to obtain the main organs for ex vivo imaging after 12 h injection, as shown in Fig. 5e and 5f, PS-CHO@HA-ATRA-SS-ATRA exhibited the strongest fluorescent signal on tissue, whereas PS-COOH@HA-ATRA-SS-ATRA was mainly distributed in metabolic organ (such as liver and kidney), which may cause potential phototoxicity and other side effects. These encouraging results illustrated the advantage of ALDH-responsive capability of PS-CHO@HA-ATRA-SS-ATRA, which was mainly activated on the CSC-enriched tumor and displayed good therapeutic effect by high selectivity and good biosafety.

In vivo anti-tumor efficiency on a 4T1 subcutaneous model

Inspired by good tumor accumulation and effective ALDH-responsiveness of our nano-prodrug, we further investigated their effects in tumor treatment (Fig. 6a). Compared with Saline (G1, Fig. 6b), systemic administration of ATRA-SS-ATRA (G3) and PS-CHO@ATRA-SS-ATRA without light irradiation (G4) exhibited negligible inhibition of tumor growth after 21-days treatment (Fig. 6d and 6e). Small molecular PS-CHO with light irradiation (G2) also exhibited poor tumor growth inhibition (Fig. 6c), whereas the nano-formulation of PS-CHO without ATRA, PS-CHO@HA-DSPE-PEG2000 that PS-CHO modified with DSPE-PEG2000-HA (G5, Supplementary Fig. 31), could obviously inhibit the tumor growth (Fig. 6f). On the other hand, PS-CHO@ATRA-SS-ATRA with light irradiation (G6) exhibited some better tumor growth inhibition than that of G5, which was contributed to the synergistic therapeutic effect of ATRA (Fig. 6g). As expected, the CSC-targeted nano-prodrug PS-CHO@HA-ATRA-SS-ATRA (G7) resulted in almost complete response of tumor growth (Fig. 6h), and most tumors in mice were reduced greatly and even faded away. The summarized analysis in Fig. 6i also revealed the best tumor inhibition of G7. On 21 days, all mice were sacrificed and dissected to collect tumor tissue and main organs, and the photograph and weight of dissected tumor tissues also revealed that G7 displayed the best tumor growth inhibition in all groups (Fig. 6j and 6k). Moreover, similar results were also proved by H&E staining of ex vivo tumor tissues, and there was an obvious histological damage in the tumor tissue after the treatment of G7 (Fig. 6l). As shown in Fig. 6m and Supplementary Fig. 32, no obvious loss in body weight and no obvious histological damage in the main organs were observed after G7 treatment, indicating the safe profile of this CSC-targeted nano-prodrug. As a result, mice with G7 treatment exhibited the highest tumor growth inhibition and better biosafety during a period of 21-day treatment.

In vivo anti-tumor efficiency and anti-metastasis on a TNBC-orthotopic mice model

Clinic research revealed the aggressive nature of TNBC, characterized by higher rates of distant metastasis and recurrence, is influenced by the residual CSC in TNBC tumor ^[56]. This small subpopulation of CSC within TNBC plays a critical role in initiating and driving the metastatic process, often affecting sites like the lungs, liver, and bones ^[57]. Therefore, we further investigated the anti-tumor growth and anti-metastatic effect of PS-CHO@HA-ATRA-SS-ATRA on TNBC-orthotopic mice model (Fig. 7a). After 30 days treatment, there was no obvious difference of tumor growth in Saline (G1, control group), PS-CHO with light irradiation (G2), ATRA-SS-ATRA (G3), and PS-CHO@ATRA-SS-ATRA without light irradiation (G4) (Fig. 7b and Supplementary Fig. 33a-d). Especially in G2, small molecular PS-CHO with light irradiation displayed poor tumor inhibition due to its poor biocompatible, while the nano-formulations of PS-CHO with light irradiation (G5: PS-CHO@HA-DSPE-PEG2000; G6: PS-CHO@ATRA-SS-ATRA, and G7: PS-CHO@HA-ATRA-SS-ATRA) can effectively suppress the tumor growth after 30-days treatment (Fig. 7b and Supplementary Fig. 33e-f). As compared with G5 and G6 groups, much stronger inhibition was observed in tumor growth after CSC-targeted nano-prodrug G7 treatment (Fig. 7b and Supplementary Fig. 33g). On day 30, all mice were sacrificed and dissected to obtain the tumor tissues and main organ, and tumor weight and photograph of different groups revealed the similar results of in vivo tumor inhibition (Fig. 7c and 7d). In addition, the anti-tumor efficacy was also confirmed by H&E staining, as shown in Supplementary Fig. 34, nano-prodrug G5 (without ARTA) and G6 (without HA targeting) groups exhibited obvious histological damage in tumor tissues, indicating good inhibition by the effective ALDH-responsive PDT treatment. As control, nano-prodrug G7 group (contained HA and ATRA) showed the deepest histological damage in all groups due to effective synergic effect of ATRA and good CSC-targeting of HA.

In clinics, the distant metastasis from TNBC is often diagnosed at lung and liver, which was a leading cause of death in TNBC treatment. Therefore, we investigated the inhibition of distant metastases on the TNBC-orthotopic mice model by different immunohistochemical staining. In lung tissue, the Bouin’s fluid was first stained to mark the lung metastatic nodules, and fewer metastatic nodules were observed for all nano-formulation groups, while other groups displayed more obvious lung nodules. In comparison with G1-G4, the nano-formulation G5 was able to effectively inhibit the distant metastases on the surface of lung tissue. Both G6 and G7 exhibited the poorest metastatic nodules, especially for targeted nano-prodrug that the number of lung nodules was negligible with less than 5 lung metastases per mouse, suggesting a strong anti-metastasis effect of G7 (Fig. 7e). Furthermore, only a few nodules were detected after PS-CHO@HA-ATRA-SS-ATRA treatment (Fig. 7f), which was confirmed by H&E staining, suggesting the effective inhibition of lung metastasis. In the liver tissue, the H&E staining was performed to investigate its distant metastatic nodule, as shown in Fig. 7g, obvious tumor metastatic nodule was observed in G1-G4 groups, while there was no obvious metastatic nodule in G7, and this result was similar with that of lung metastases.

To confirm the good anti-CSC effect of our nano-prodrug, we analyzed the ALDH-expression of tumor tissue by immunofluorescence staining. As shown in Fig. 7h, G1, G2, G3 and G4 showed relatively strong green, fluorescent signal in tumor tissues. As a control, G5 also displayed relatively poor fluorescent signal in tumor tissue, which was due to PS-CHO@HA -DSPE-PEG2000 could effectively accumulate into CSC and then activate PS-CHO to kill the CSC. As expected, the tumor tissues treated with nano-formulation G6 and G7 (simultaneously contained ATRA and PS-CHO) hardly observed green fluorescence, indicating fewer ALDH-overexpressed CSC in tumor microenvironment. In addition, the expression of stemness-related genes (Sox2, Nanog and Oct4) in tumor tissues was also investigated after different groups treatment. As shown in Supplementary Fig. 35, G7 was able to effectively down-regulate the expression of stemness-related gene and decrease the generation of CSC. Collectively, these results suggested that reduced CSC in tumor tissue were able to effectively inhibit distant metastasis in vivo. During the treatment period of 30 days, although G2 (free PS-CHO) caused slow growth in body weight, there were no obvious changes in other groups, including G7 (Supplementary Fig. 36). In addition, there was no obvious histological damage in the main organs after the treatment of G7 (Supplementary Fig. 37). After G7 treatment, 80% of mice survived for 60 days, indicating the long-term anti-metastatic effect. The above results proved that our nano-prodrug exhibited good biosafety and could prolong the survival rate of mice with distant metastatic foci (Supplementary Fig. 38).

We have explored a simple and convergent self-assembled nano-prodrug that contained an ALDH-activatable PS and differentiation agents ATRA for CSC-associated tumor treatment. The ALDH-activatable PS (PS-CHO) exhibited good ALDH-responsiveness with triggered ROS activation and fluorescence generation in vitro. To improve the biocompatibility and in vivo therapeutic effect, ATRA was linked with -SS- bond to form a GSH-response ATRA-SS-ATRA, which exhibited good small molecular self-assembly behavior, serving as a good nano-carrier to load PS-CHO. The PS-CHO@ATRA-SS-ATRA not only effectively enhanced the pharmacokinetics of PS-CHO due to its good water dispersibility but also reduced the CSC stemness and improved the CSC-enriched tumor microenvironment by the synergistic effect of ATRA. In CSC-enriched tumor cells, PS-CHO@ATRA-SS-ATRA dissembled into PS-CHO and ATRA by high level of GSH, and PS-CHO was transformed into PS-COOH under high expression of ALDH that generated the fluorescence for reporting the real-time information about the CSC microenvironment. Moreover, PS-COOH exhibited good ROS generation that can directly eliminate CSC and indirectly improve CSC-enriched microenvironment. More importantly, such distinct anti-CSC mechanism of two drugs satisfies the demand for promoting a maximized synergistic effect in CSC-enriched mice model, exhibiting the good inhibition of tumor growth and metastasis. These findings provide a promising perspective for exploring simple, convergent, and effective strategies to improving the therapeutic effect in a wide range of CSC-enriched tumors.

Preparation of PS-CHO@ATRA-SS-ATRA

The nanoparticles PS-CHO@ATRA-SS-ATRA were prepared by nanoprecipitation, and small molecular PS-CHO (5 mg) and ATRA-SS-ATRA (5 mg) were added into dimethyl sulfoxide (DMSO, 1 mL) and then slowly injected to 19.5 mL of ddH₂O under vigorous sonication (~ 10 min). This crude product of PS-CHO@ATRA-SS-ATRA was loaded with dialysis tube (MWCO = 1000 Da) and then dialyzed for 12 h in ddH₂O. As for PS-CHO@HA-ATRA-SS-ATRA, PS-CHO (2 mg), ATRA-SS-ATRA (2 mg) and DSPE-PEG2000-HA (1 mg) were added into DMSO (0.5 mL) and then slowly added to 9.5 mL of ddH₂O under vigorous sonication (~ 10 min) and then dialyzed for 12 h in ddH₂O (MWCO = 5000 Da).

The nanoparticles PS-CHO@DSPE-PEG2000 and PS-CHO@HA-DSPE-PEG2000 were also prepared by nanoprecipitation. The DMSO solution of PS-CHO (5 mg, 1 mL) was slowly added into 19 mL ddH₂O containing 1 mg of DSPE-PEG2000-HA or DSPE-PEG2000, and this solution was mixed under sufficient stirring. This crude product was subjected to dialysis (MWCO = 2000 Da) against ddH₂O to remove the DMSO and the residual drug

ALDH-responsive behavior of PS-CHO in PBS solutions

The ALDH-responsive behavior of PS-CHO was first investigated by HPLC, which was performed by C18 column under 25 ^oC condition. The mobile phase was methanol and water at the ratio of 9:1. The PS-CHO was first incubated with 50 µg/mL of ALDH for 1 h in PBS solution (contained 10% DMSO), and then it was analyzed by HPLC. In addition, the ALDH-responsive behavior of PS-CHO was also detected by fluorescence imaging, PS-CHO, PS-COOH, and PS-CHO incubated with ALDH (50 µg/mL) were dissolved in PBS solution (contained 10% DMSO) under 37°C, and the fluorescence spectra were measured by fluorescence spectrometry. For different concentrations (0–100 µg/mL) or times (0–240 min), PS-CHO (20 µg/mL) was incubated with different concentrations (0–100 µg/mL) for 2 h or with 50 µg/mL of ALDH for different times. Finally, the ALDH-responsive behavior of PS-CHO was also detected by HR-MS, The PS-CHO (20 µg/mL) was first incubated with 50 µg/mL of ALDH in PBS solution (contained 10% DMSO), and after 1 h incubation, the mixture was analyzed by HR-MS.

ROS generation of PS-CHO in PBS solutions

Firstly, the investigation of ALDH-responsive behavior of PS-CHO was carried out by high-performance liquid chromatography (HPLC) analysis. HPLC was conducted using a C18 column at a constant temperature of 25°C. The mobile phase comprised methanol and water in a 9:1 ratio, with a flow rate set at 1 mL/min. Firstly, PS-CHO was incubated with ALDH at a concentration of 50 µg/mL in a PBS solution containing 10% DMSO. Following a 1-hour incubation, the mixture was subjected to HPLC analysis. Furthermore, the ALDH-responsive behavior of PS-CHO was assessed through fluorescence imaging. Solutions of PS-CHO, PS-COOH, and PS-CHO incubated with 50 µg/mL of ALDH were prepared in PBS containing 10% DMSO at 37°C. The fluorescence spectra of these solutions were then recorded using fluorescence spectrometry. In addition, varying concentrations (0-100 µg/mL) of PS-CHO were incubated with ALDH for 2 h, and PS-CHO (20 µg/mL) was incubated with 50 µg/mL of ALDH for different durations (0–240 min). The respective mixtures were subsequently analyzed. Then, the ALDH-responsive behavior of PS-CHO was further characterized using high-resolution mass spectrometry (HR-MS). PS-CHO (20 µg/mL) was incubated with 50 µg/mL of ALDH in a PBS solution containing 10% DMSO. After 1 h, the mixture was analyzed again by HR-MS.

Drug release behavior of PS-CHO@ATRA-SS-ATRA

The nanoparticle PS-CHO@ATRA-SS-ATRA (20 µg/mL) was incubated with different level of GSH (0–10 mM), which then extracted and detected by HPLC using C18 column under 25 ^oC condition, and the mobile phase was methanol and water at the ratio of 9:1. In detail, 2 mL of each sample was added into a 500 Da dialysis bag and dialyzed against phosphate-buffered saline (PBS) at 37 ^oC in a shaker. At predetermined time intervals (0, 1, 2, 5, 10, 20, 30, 60, and 120 mins), 1 mL solution in each sample was taken out and 1 mL of fresh PBS was restored its solutions. The concentration of PS-CHO was measured by HPLC.

Intracellular ALDH-responsive behavior of PS-CHO@ATRA-SS-ATRA in normal and cancer cells

The intracellular ALDH-responsive behavior of PS-CHO@ATRA-SS-ATRA was investigated by cell imaging in different cells (normal cells: 3T3, cancer cells: 4T1). All these cells were added into 8-well confocal plate under normal or hypoxic conditions, allowing for suitable growth density (60%). Then, PS-CHO@ATRA-SS-ATRA was added into plates by the different concentration (0, 5, 10, and 20 µg/mL), and then incubated for another 4 h. After PBS washing, the intracellular fluorescence intensity was recorded by using CLSM. In addition, PS-CHO@ATRA-SS-ATRA (10 µg/mL) was incubated with 4T1 cells for different times (0–4 h), which recorded the fluorescence intensity by CLSM. For ALDH-responsive behavior of PS-CHO@DSPE-PEG2000, the protocol was the same with the ALDH-responsive behavior of PS-CHO@ATRA-SS-ATRA. The fluorescence intensity was recorded by CLSM after incubation with 4 h.

Intracellular ROS detection of PS-CHO@ATRA-SS-ATRA

The ROS generation of different formulations was investigated by ROS indicator (DCFH-DA). Different cells (normal cells: 3T3, cancer cells: 4T1) were seeded in 8-well plates, allowing for suitable growth density (70%). After the cell adhesion, different formulations (ATRA-SS-ATRA, PS-CHO, PS-CHO + L, PS-CHO@ATRA-SS-ATRA, and PS-CHO@ATRA-SS-ATRA + L, 20 µg/mL) were added and incubated for 24 h. Then, all treated cells were washed by PBS for 3 times and then irradiated with or without laser. All these cells were stained by DCFH-DA for 30 mins and observed in a fluorescent microscope. All laser condition was performed at 40 mW/cm² (5 min).

In vitro cytotoxicity of PS-CHO@ATRA-SS-ATRA

Different cells (normal cells: 3T3, cancer cells: 4T1) were added into 96-well plates (100 µL, 5000 cells for each well). After the cell adhesion, a gradient concentration of different drugs (PBS, ATRA-SS-ATRA, PS-CHO, PS-CHO + L, PS-CHO@ATRA-SS-ATRA, and PS-CHO@ATRA-SS-ATRA + L, 20 µg/mL) were added and incubated for 48 h, and all these cells was irradiated with or without white light (40 mW/cm²) for 5 min. Then, all treated cells were washed by PBS and added10 µL of CCK-8 solution (diluted in 200 µL PBS). After 4 h incubation at 37 ^oC, the absorption values at 450 nm were measured by a UV-vis instrument. The cell viability was determined by comparison with the PBS groups. As for short-term drug administration, different cells were firstly incubated with different concentrations (5 and 20 µg/mL) of PS-CHO@ATRA-SS-ATRA + L and PS-CHO@HA-ATRA-SS-ATRA + L for 4 h and then replaced with fresh medium and continue to incubate for another 48 h. Finally, the CCK8 assay was performed according to the standard protocol. All laser condition was performed at 40 mW/cm² (4 min).

Cell death of PS-CHO@ATRA-SS-ATRA by Live/dead Staining

4T1 cells were added in 24-well plates (500 µL, 5 × 10⁴ cells for each well). After the cell adhesion, different drug formulations (PBS, ATRA-SS-ATRA, PS-CHO, PS-CHO + L, PS-CHO@ATRA-SS-ATRA, and PS-CHO@ATRA-SS-ATRA + L, 20 µg/mL) were added and incubated for 4 h. Then, all treated cells were irradiated with white light irradiation (40 mW/cm²) for 5 min. After another 24 h incubation, the cells were washed by PBS and stained by Calcein-AM/PI for 30 mins. Then the cells were washed by PBS and observed in a fluorescent microscope.

Cell apoptosis of PS-CHO@ATRA-SS-ATRA by Flow Cytometry Assay

4T1 cells were added in 6-well plates (2 mL, 2 × 10⁵ cells for each well). After the cell adhesion, different drug formulations (PBS, ATRA-SS-ATRA, PS-CHO, PS-CHO + L, PS-CHO@ATRA-SS-ATRA, and PS-CHO@ATRA-SS-ATRA + L, 20 µg/mL) were added and incubated for 4 h, and then all treated cells were irradiated with or without white light irradiation (40 mW/cm²) for 4 min. After 24 h incubation, the adherent and suspended cells were all collected and performed according to the protocol. After being stained by Annexin-V-FITC/PI in dark for 30 mins, the cells were performed by flow cytometry and measure the apoptosis rates.

In vitro tumorspheres formation assay

4T1 cells were added in ultra-low adhesion 24-well plates (500 µL, 1 × 10⁴ cells for each well) and incubated under hypoxic conditions. Then different drug formulations (PBS, PS-CHO + L, ATRA-SS-ATRA, PS-CHO@ATRA-SS-ATRA, PS-CHO@DSPE-PEG2000 + L, and PS-CHO@ATRA-SS-ATRA + L) at the concentration of 20 µg/mL were added and incubated for 5 days, the cells were observed and the numbers of tumorspheres were recorded by using microscope.

ALDH-positive rate of tumorsphere cells

4T1 cells were added in ultra-low adhesion 24-well plates (500 µL, 1 × 10⁴ cells for each well) and incubated under hypoxic conditions. Then different drugs (PBS, PS-CHO + L, ATRA-SS-ATRA, PS-CHO@ATRA-SS-ATRA, PS-CHO@DSPE-PEG2000 + L, and PS-CHO@ATRA-SS-ATRA + L) at the concentration of 20 µg/mL were added and incubated for 3 days, all treated cells were collected and washed by PBS, and then stained with ALDEFLUOR kit for flow cytometry. The untreated cells were added with N, N-diethylaminobenzaldehyde (DEAB) to determine the ALDH-positive cells in other groups.

Stemness-related genes expression of tumorsphere cells

4T1 cells were added in ultra-low adhesion 24-well plates (500 µL, 1 × 10⁴ cells for each well) and incubated under hypoxic conditions. Then different drugs (PBS, PS-CHO + L, ATRA-SS-ATRA, PS-CHO@ATRA-SS-ATRA, PS-CHO@DSPE-PEG2000 + L, and PS-CHO@ATRA-SS-ATRA + L) at the concentration of 20 µg/mL were added and incubated for 3 days, RNAs in the cells were extracted by an RNA extraction kit. The stemness-related gene (Sox2, Nanog and Oct4) were quantified by qPCR analysis.

HIF-1α expression by immunofluorescence

4T1 cells were added in ultra-low adhesion 24-well plates (500 µL, 1 × 10⁴ cells for each well) under hypoxic conditions and allowed for the tumorsphere formation. Then the tumorspheres were collected and transferred to 48-well plates (1 tumorsphere per well) and exposed to different drugs (PBS, PS-CHO + L, ATRA-SS-ATRA, PS-CHO@ATRA-SS-ATRA, PS-CHO@DSPE-PEG2000 + L, and PS-CHO@ATRA-SS-ATRA + L) at the concentration of 20 µg/mL. After 3 days incubation, all treated cells were fixed with 4% paraformaldehyde and incubated with 0.5% Triton-X-100, and then washed with PBS for 3 times. Then, the tumorsphere cells were blocked by goat serum, and the incubated with the primary antibody of HIF-1α at 4 ^oC overnight. After that these treated cells were incubated with Alexa Fluor 488 conjugated secondary antibodies for 1 h and the nucleus was stained by DAPI. The HIF-1α expression was determined and observed by a fluorescent microscope.

In vivo imaging and drug release behavior of PS-CHO@ATRA-SS-ATRA

The CSC-enriched subcutaneous tumor model was established by the injection of 4T1 cells, which were subcutaneously injected into the right groins of female BALB/c mice (5 × 10⁵ 4T1 cells; 6 weeks old). After the tumor volume reached about 500 mm³, different formulations (PS-CHO@HA-ATRA-SS-ATRA and PS-COOH@HA-ATRA-SS-ATRA) were performed by intravenous injection into the 4T1 subcutaneous tumor mice (n = 3). At several time intervals (0, 1, 2, 3, 4, 6, 8, and 12 h), the mice were subjected to in vivo imaging by IVIS system. After 12 h, the mice were sacrificed, and the tumors and organs (hearts, livers, spleens, lungs, and kidneys) were collected for ex vivo imaging. The fluorescent signals were monitored through a 740 nm band-pass filter and excited by a 540 nm LED.

In vivo anti-tumor efficacy in a 4T1 subcutaneous model

The CSC-enriched subcutaneous tumor model was established by the injection of 4T1 cells, which were subcutaneously injected into the right groins of female BALB/c mice (5 × 10⁵ 4T1 cells; 6 weeks old). After the tumors reached about 100 mm³, the mice were randomly distributed into 7 groups (n = 5) (G1-G7: Saline, PS-CHO + L, ATRA-SS-ATRA, PS-CHO@ATRA-SS-ATRA, PS-CHO@HA-DSPE-PEG2000 + L, PS-CHO@ATRA-SS-ATRA + L, and PS-CHO@HA-ATRA-SS-ATRA + L, 5 mg/kg), and then all groups were intravenously injected for three times on day 0, 3, and 6. After 12 h injection, the mice of G2, G5, G6, and G7 were irradiated by white light (60 mW/cm²; 5 min). At the period of 21-days, the tumor volume was recorded for each mouse every 2 days and then calculated according to (longest width) × (longest length)²/2. The tumor volumes and body weights were recorded every 3 days. On day 21, all mice were sacrificed and dissected to obtain the tumor tissue and main organs. The tumors were weighted and made into slices for H&E and ALDH immunofluorescence staining. The organs were made into slices for H&E staining. Another batch of mice were received the same treatment for survival analysis until day 60.

In vivo anti-tumor efficacy and anti-metastasis in 4T1-orthotopic mice model

4T1 cells (5 × 10⁶ cells/mL) were orthotopically injected into the right second mammary pad of female nude mice. After injection, all mice were randomly divided into 7 groups and intravenously injected G1-G7 groups (Saline, PS-CHO + L, ATRA-SS-ATRA, PS-CHO@ATRA-SS-ATRA, PS-CHO@HA-DSPE-PEG2000 + L, PS-CHO@ATRA-SS-ATRA + L, and PS-CHO@HA-ATRA-SS-ATRA + L, 5 mg/kg) for three times on day 0, 3, and 6. After 12 h injection, the mice of G2, G5, G6, and G7 were irradiated by white light (60 mW/cm²; 5 min). At the period of 30-days, the tumor volume was recorded for each mouse every 3 days, which were calculated according to (longest width) × (longest length)²/2. At 30 days, and all mice were sacrificed and dissected to obtain the tumor tissue and main organs. In addition, the body weight of all groups was also recorded at the treatments of 30-days. For tumor inhibition and intratumoral ALDH expression, the tumor tissues of all groups were stained by H&E staining of stemness-related genes (Sox2, Nanog and Oct4). For the distant metastases, the lung and liver tissues after treatments were stained by H&E staining and Bouin’s fluid. Another batch of mice were received the same treatment for survival analysis until day 60.

Reporting summary

Further information on research design is available in the Nature Research Reporting Summary linked to this article.

Data availability

The data generated in this study are available within the article, Supplementary Information, and Source Data. Source data are provided with this paper.

Acknowledgments

We acknowledge the National University of Singapore (grant no. A-0009163-01-00), Liu Bin - Tan Chin Tuan Centennial Professorship (grant no. E-467-00-0012-02), and National Natural Science Foundation of China (grant no. 82073779, 32000992) for funding support.

Author contributions

B.L. and Y.P. supervised the project. W.B.L. and J.W.T. conceived and designed the experiments. F.Z., Z.Y.L. and C.Z.W. conducted animal experiments. M.W., D.D.W., J.H.Z., W.T.S., and Y.F. T took part in the discussion and gave important suggestions. W.B.L. J.W.T., B.L., and Y.P. co-wrote the paper.

Competing interests

The authors declare no competing interests.

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Self-assembled Aldehyde Dehydrogenase-Activatable Nano-prodrug for Cancer Stem Cell-Enriched Tumor Detection and Treatment

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Abstract

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Introduction

Results

Synthesis and characterization of PS-CHO and ATRA-SS-ATRA

Cytotoxicity of PS-CHO@ATRA-SS-ATRA

Discussion

Methods

Preparation of PS-CHO@ATRA-SS-ATRA

ALDH-responsive behavior of PS-CHO in PBS solutions

ROS generation of PS-CHO in PBS solutions

Drug release behavior of PS-CHO@ATRA-SS-ATRA

Intracellular ALDH-responsive behavior of PS-CHO@ATRA-SS-ATRA in normal and cancer cells

Intracellular ROS detection of PS-CHO@ATRA-SS-ATRA

Cell death of PS-CHO@ATRA-SS-ATRA by Live/dead Staining

Cell apoptosis of PS-CHO@ATRA-SS-ATRA by Flow Cytometry Assay

ALDH-positive rate of tumorsphere cells

Stemness-related genes expression of tumorsphere cells

HIF-1α expression by immunofluorescence

Declarations

References

Additional Declarations

Supplementary Files

Status:

Journal Publication

Version 1