The senescence-associated secretory phenotype (SASP), as one of the major metabolic and signaling characteristics of senescent cells, has long been a hot topic in the field of senescence and aging research [1, 2]. SASP can accelerate senescence in an autocrine manner and induce senescence of surrounding cells in a paracrine manner, cumulatively exacerbating the development of age-related diseases such as cancer, cardiovascular diseases, and neurodegenerative disorders [3, 4]. Thus, suppression of SASP has been considered an effective way to treat and/or prevent agerelated diseases [5]. In this context, the development of drugs capable of specifically regulating SASP has become a research frontier in both academic and clinical fields.
Of particular importance are senomorphics, a class of senotherapeutics capable of maintaining the metabolic homeostasis and controlling pathological progression by attenuating SASP secreted by senescent cells [6, 7]. Senomorphics are commonly compounds from natural extraction or chemical synthesis, but most of these compounds lack selectivity for senescent cells and induce adverse effects on normal cells, given the fact that some SASP factors play important roles in many biological processes [7, 8]. Engineering of senotherapeutic prodrugs represents an important alternative strategy to overcome this challenge due to the control of drug activity [9-12]. Previously, researchers have reported a senescence-associated β-galactosidase (SA-β-gal)-activated H₂S donor as an effective senomorphic prodrug [13]. It can be cleaved by the accumulated SA-β-gal in senescent cells, triggering the release of H₂S to alleviate the levels of SASP and reactive oxygen species in senescent cells. Despite the controlled senomorphic activity, SA-β-gal-activated senomorphics still suffer from non-specific activation in some nonsenescent cells due to the limitation of using SA-β-gal to distinguish cellular senescence from other cell states with endogenous expression of β-gal [14]. Therefore, strategic innovations are highly needed to further improve the senomorphic selectivity in the regulation of cellular senescence.
To achieve this goal, we design and report a senescent cell-targeted and activatable senomorphic by combining membrane biomarker-mediated active cell recognition with intracellular biomarker-promoted prodrug activation mechanisms. Specifically, an aptamer targeting L1CAM, a transmembrane protein that plays important roles in various cellular processes and is overexpressed in various senescent cells, was selected as the targeting ligand, and SA-β-gal-activated H₂S donor was used as the prodrug moiety to construct a senescence-targeted activatable senomorphic (Apt-H₂SD). Benefiting from the aptamer moiety, Apt-H₂SD shows active targeting and enhanced cellular uptake in senescent cells over proliferating cells. Under the receptor-mediated endocytosis, Apt-H₂SD could be activated in situ by the accumulated SA-β-gal in the lysosomes of senescent cells, triggering the release of H₂S to regulate the SASP signature. Our results suggest that Apt-H₂SD has the potential to be a useful molecular tool for precise intervention in cellular senescence and the related disorders.

Synthesis of compounds

Compounds 2, and 3 were synthesized according to previously published procedures [9]. To synthesize compound 4, NaH (45 mg, 1.12 mmol) was added to a solution containing compound 3 (200 mg, 0.37 mmol) and p-tolylisothiocyanate (67 mg, 0.45 mmol) in anhydrous tetrahydrofuran (THF) (20 mL) at 0 °C. The mixture was stirred at 0 °C for 30 min and at room temperature for another 4 h. The reaction was quenched with H₂O and extracted with ethyl acetate. The solvent was removed by a rotary evaporator, and the residue was purified by column chromatography to give compound 4 in 48% yield.
For compound 5 (H₂SD), compound 4 (123 mg, 0.18 mmol) was dissolved in a mixture of methanol and dichloromethane (5:3, v/v). Sodium methoxide (65 mg, 1.2 mmol) was then added at 0 °C. The mixture was stirred at 0 °C for 30 min, and the solution was neutralized with Amberlite IRC 50 for 10 min. After removing the solvent with a rotary evaporator, the residue was purified by column chromatography to give compound 5 in 78% yield.
For compound 6 (Apt-H₂SD), azide-modified antiL1CAM aptamer (50 nmol) was dissolved in 50 µL of ddH₂O and mixed with 50 µL of the TEAA buffer (2 M). 150 mL of dimethylformamide (DMF) containing 500 nmol of compound 5 was added to the above mixture. Then 25 µL of Cu-TBTA complex (10 mM in 55% v/v dimethyl sulfoxide) and 25 µL of ascorbic acid (5 mM) were mixed and added. After stirring for 4 h at 37 °C, the mixture was purified by high performance liquid chromatography (HPLC) to obtain Apt-H₂SD. The HPLC procedures and the DNA sequences used in this study are summarized in Table S1 and Table S2, respectively.

H₂S release experiments

The release of H₂S from H₂SD was determined by a methylene blue (MB) colorimetric assay according to the previous method [13]. Briefly, a 5 mM stock solution of H₂SD was added to PBS containing β-gal (0.5 U/mL) and carbonic anhydrase (CA, 25 μg/mL), followed by incubation at 37 °C with shaking. The MB colorimetric assay was performed, and the absorbance at 670 nm was determined to calculate the release of H₂S. A blank solution without the addition of β-gal was also tested as a reference.

Cell culture

BJ cells were purchased from the American Type Culture Collection (ATCC), and cultured in Dulbecco’s modified Eagle’s medium (DMEM) with the addition of 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (PS) in an incubator with a humidified atmosphere of 5% CO₂ at 37 °C.

Senescence induction and characterizations

The hydrogen peroxide (H₂O₂)-induced senescent cell model was established based on previous reports [9, 14, 15]. Briefly, BJ cells were seeded at 6 × 10⁵ cells per well and cultured for 24 h to allow adherence. The cells were then cultured with DMEM containing H₂O₂ (600 µM) for 6 days, after which the medium was replaced with fresh DMEM for subsequent experiments.
To demonstrate the successful induction of senescence, the activity of SA-β-gal in these stimulated cells was first tested using the Senescence β-galactosidase Staining Kit. Briefly, 1×10⁵ proliferating or H₂O₂-induced senescent BJ cells were plated in the 6-well plate for 24 h to allow adherence. The cells were then washed and fixed, followed by SA-β-gal staining according to the manufacture’s procedures. Finally, the cells were imaged using an inverted microscope (Olympus).
Meanwhile, the cell proliferation ability was examined with 5-ethynyl-2′-deoxyuridine (EdU) assay. Briefly, 1×10⁵ BJ cells per well treated with or without H₂O₂ were plated in the 6-well plates. After adherence, the cell proliferation ability was examined according to the manufacturer’s protocols described and imaged using an inverted microscope.

Cytotoxicity experiments

Cell viability was determined using the Cell Counting Kit-8 (CCK-8) assay. Briefly, 4000 cells per well of proliferating/senescent BJ cells were plated in 96-well plates and cultured for 24h, the cells were incubated with freshly prepared DMEM containing different concentrations of Apt-H₂SD (0, 0.125, 0.25, 0.5, 1, and 2 μM) for 24 h, and then cell viability was assessed using the CCK-8 detection assay.

Cell binding analysis

The ability of Apt-H₂SD to target senescent cells was evaluated by flow cytometry and confocal fluorescence imaging. For flow cytometry, proliferating/senescent BJ cells were digested with 0.2% EDTA and washed twice with PBS. Apt-H₂SD and Con-H₂SD labeled with Cy5 (250 nM) were each incubated with 1.5×10⁵ cells in a serumfree DMEM at 4 °C for 45 min. Cells were then centrifuged and washed for flow cytometry (BD FACS Verse).
For confocal fluorescence imaging, proliferating/senescent BJ cells were plated in the confocal dish at 1.0×10⁵ cells per well. After 24 h of culture, the cells were washed twice with PBS and cultured with 200 μL of serum-free DMEM containing 250 nM Cy5-Apt-H₂SD/Con-H₂SD for 45 min at 4 °C. The cells were then washed and stained with Hoechst. Cellular fluorescence was examined by laser scanning confocal microscopy (FV1000 confocal microscope, Olympus).

SASP inhibition analysis

The expression of SASP in senescent BJ cells treated with or without Apt-H₂SD was examined at the mRNA level by reverse transcription quantitative real-time PCR (RTqPCR). After treatment with Apt-H₂SD, all the experimental procedures were referred to the previous report [9, 13]. Primers used in this experiment are summarized in Table S3.

Statistical Analysis

All data were presented as mean ± standard deviation (SD). The difference between two groups was compared by using the Student’s t‐test. The difference among multiple groups was compared by using the one-way analysis of variance (ANOVA) followed by the LSD (Tukey’s) post hoc test. Differences were considered statistically significant if P < 0.05.

The synthesis of Apt-H₂SD is shown in Figure 1. The glycosylated intermediate (compound 3) was first synthesized following previous work [9], providing an ideal platform for conjugation of the H₂S-donating moiety at the benzylic position and the targeting ligand at the terminal alkyne. Subsequent conjugation with p-tolylisothiocyanate and deprotection of the hydroxy groups yielded the cleavable compound 5 (H₂SD). In the presence of SA-β-gal, H₂SD could release carbonyl sulfide (COS), which would be rapidly converted to H₂S by the ubiquitous enzyme carbonic anhydrase (CA) inside the senescent cells to execute the SASP regulation. Finally, anti-L1CAM aptamer was conjugated at the tail via click chemistry to yield the senescence-targeted activatable molecular senomorphic (Apt-H₂SD). All the intermediates were characterized by NMR spectra, and Apt-H₂SD was characterized by MS (Figure S1-S9).

With H₂SD in hand, we proceeded to investigate its enzyme-responsive and H₂S-releasing capabilities. In the presence of β-gal and carbonic anhydrase via methylene blue assay. As shown in Figure 2, H₂SD exhibited rapid release of H₂S upon incubation in the PBS buffer containing β-gal and CA, and the reaction was almost complete within 2 h, with a release efficiency of about 50%. In sharp contrast, no H₂S production was detected in the absence of β-gal within the same time period, suggesting that the presence of CA alone did not induce the cleavage of thiocarbamates. Collectively, these results confirmed the β-gal-activated property of H₂SD.

Having confirmed the controllable activity, we next asked whether Apt-H₂SD could achieve targeted recognition of senescent cells. As a proof-of-concept, oxidative stressinduced cellular senescence was established using BJ cells as the model and H₂O₂ as the stress. SA-β-gal staining results showed that after stimulation with H₂O₂, BJ cells exhibited obvious enlargement in the cell volume, coupled with blue staining (Figure 3A), indicating the accumulation of SA-β-gal. 5-Ethynyl-2′-deoxyuridine (EdU)-based cell proliferation assay further confirmed that these stressed BJ cells lost the proliferative ability, as evidenced by the negative EdU staining (Figure 3B). On the other hand, we have also examined the levels of three senescence-associated markers, including p16, p21, and IL-1β, in these stimulated cells. The results showed a significant elevation in the expression of these markers at the mRNA level (Figure S10). Furthermore, the upregulation of L1CAM in senescent BJ cells was verified by Western blot analysis (Figure 3C). Next, the cell recognition ability of Apt-H₂SD towards senescent cells was evaluated by flow cytometry and confocal fluorescence imaging. As shown in Figure 3D, H₂O₂-induced senescent BJ cells exhibited a significant shift in fluorescence intensity after incubation with Cy5-labeled Apt-H₂SD compared to proliferating BJ cells. Moreover, such fluorescence shift in senescent BJ cells treated with Cy5-labeled Apt-H₂SD was much greater than that in senescent cells treated with Cy5-labeled non-targeting Con-H₂SD, suggesting that Apt-H₂SD could selectively recognize senescent cells over proliferating cells. Consistent with the flow cytometry results, confocal imaging further confirmed the senescent cell targeting ability of Apt-H₂SD. Among the different treatment groups, only senescent cells treated with Cy5-labeled Apt-H₂SD showed bright red fluorescence (Figure 3E). To confirm whether Apt-H₂SD accumulated in lysosomes of senescent cells, lysosomal colocalization analysis was performed. Senescent BJ cells were treated with Cy5-labeled Apt-H₂SD and stained with Lysotracker Green, followed by confocal fluorescence imaging. As we expected, a large overlap between the green and red fluorescence channels was observed (Figure 3F), suggesting the lysosomal accumulation of Apt-H₂SD in senescent cells. Having identified the cell targeting property, we next evaluated the senomorphic activity of Apt-H₂SD in suppressing the secretion of SASP in senescent cells (Figure 4). When BJ cells were stimulated with H₂O₂, the expression of proinflammatory interleukins (IL-6 and IL-1β) and matrix metalloproteinases 3 (MMP3) were upregulated compared to untreated cells. Interestingly, Apt-H₂SD could attenuate the expression of these SASP factors. On the other hand, Apt-H₂SD showed good biocompatibility without causing adverse effects on proliferation and growth in non-senescent cells at the concentrations tested (Figure S11).

Cellular senescence is a complex cellular stress response triggered by endogenous and/or exogenous stimuli [15]. Despite the loss of the proliferative capacity, senescent cells remain metabolically active and retain the potential to secrete various types of SASP (e.g., pro-inflammatory cytokines, growth factors, chemokines, and matrixdegrading enzymes) [16]. These signaling molecules can reprogram the tissue microenvironment and induce inflammation, which in turn promotes second senescence in surrounding cells, cumulatively driving aging and agerelated diseases such as cancer, cardiovascular disease, and neurodegenerative disorders [17, 18]. Thus, SASP has recently emerged as a therapeutic target for age-related diseases, and studies have shown that selective regulation of SASP with senomorphics is an important strategy to delay the aging process and age-related diseases or to improve disease treatment [19]. Traditionally, senomorphics are discovered by high-throughput screening of large libraries composed of small chemicals and natural products. The activity of these agents has been uncontrolled, which can interfere with the signaling pathways in normal cells, resulting in unwanted side effects [8, 20]. Although SA-β-gal-activated senomorphics have been developed to improve the selectivity of senescent cells, precise intervention in cellular senescence is still a major challenge because β-gal is expressed not only in senescent cells but also in certain normal cells and proliferating cancer cells. Therefore, there is an urgent need to develop targeted approaches with enhanced specificity and efficacy for SASP regulation during senescence. Therefore, this work aimed to improve the applicability of senomorphic-based anti-senescence methods, and reported a novel class of senomorphics, designated Apt-H₂SD, with active cell recognition and activatable senomorphic activity, by taking advantage of aptamer-prodrug conjugation strategy. The senomorphic activity of Apt-H₂SD can be switched by SA-β-gal-catalyzed cleavage of the molecular structure, resulting in release of the H₂S donor and subsequent conversion of H₂S by carbonic anhydrase (Figure 2), a ubiquitous enzyme in various mammalian cells. As a proof of concept, BJ cells, a normal human fibroblast cell line, were selected as a model cell to establish the model of cellular senescence, given the fact that fibroblast senescence contributes to the organic aging and the pathology of many important diseases, such as, pulmonary fibrosis, cancer, neurodegeneration, and cardiac diseases [21-28]. The feasibility of Apt-H₂SD to regulate SASP was investigated in oxidative stress-induced senescent BJ cells. Cell binding studies showed that Apt-H₂SD could selectively target and accumulate in senescent BJ cells over proliferating cells through the aptamer-mediated cell recognition (Figure 3). More impressively, treatment with Apt-H₂SD could downregulate the expression of three types of important SASP factors in senescent cells (Figure 4), suggesting its potential for cell-specific SASP regulation. The graphical summary is shown in Figure 5. Nevertheless, more in-depth studies, such as its influence on the secretion of other types of SASP, the expression of SASP at the genetic level, and the duration of action, are still needed to better evaluate the performance of Apt-H₂SD and to elucidate the exact mechanisms underlying the SASP regulation.

Although the research we present here is relatively preliminary, its scientific applications are potentially broad, as cellular senescence not only contributes to aging, but is highly implicated in the initiation and the initiation and progress of many important diseases. Such an aptamerprodrug conjugation strategy may also pave the way for the design and construction of various senomorphics by altering the drug moiety, which is expected to have a profound impact on the treatment and prevention of agerelated diseases.