Polymer-engineered PROTAC nanovehicles amplify synergistic effects with temozolomide by BRD4 degradation

Abstract

As the most aggressive primary brain tumor, glioblastoma (GBM) remains therapeutically challenging. Proteolysis-targeting chimeras (PROTACs), capable of degrading target proteins like BRD4, offer a promising strategy for GBM therapy. However, their clinical application is limited by poor solubility, stability, and bioavailability. This study systematically evaluates PLGA, PCL, and poly amino-acid based nanoparticles (NPs) for optimizing ARV-825, a BRD4-degrading PROTAC. This study compares the particle size, polydispersity index (PDI), and encapsulation efficiency of NPs prepared by different methods and carriers, explores the computer-simulated design of cyclic peptide carriers, and reveals the impact of PROTAC's molecular structure and action time on its toxicity. Furthermore, the delivery of ARV-825 using NPs achieves synergistic anti-tumor effects with temozolomide (TMZ) in GBM cells. These findings validate nanovehicles as a strategic solution for PROTAC limitations and provide a blueprint for translating catalytic degradation into clinically viable therapies against GBM.

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1. Introduction

As the most aggressive and lethal primary brain tumor, glioblastoma (GBM) remains a therapeutic enigma despite decades of research.¹ Its hallmark features—including rapid infiltrative growth, molecular heterogeneity, and immunosuppressive microenvironment—render conventional therapies largely ineffective.² There are three critical dimensions of GBM clinical management: therapeutic resistance mechanisms, anatomical barriers to drug delivery, and the urgent unmet need for targeted strategies against core survival pathways. As the cornerstone chemotherapeutic agent for GBM, temozolomide (TMZ) is hampered by dose-limiting systemic toxicity, rapid metabolic clearance, and insufficient penetration across the blood–brain barrier (BBB).^3,4 Even with maximal surgical resection and radiotherapy, tumor recurrence is nearly inevitable, underscoring the urgent need for therapies capable of targeting GBM's core survival mechanisms. Emerging strategies now focus on eliminating oncogenic drivers at the protein level, moving beyond the traditional inhibition of enzymatic activity. This paradigm shift has brought proteolysis-targeting chimeras (PROTACs) to the forefront of GBM therapeutics.^5,6

PROTACs offer a transformative approach by hijacking the ubiquitin-proteasome system to degrade disease-causing proteins.⁷ Unlike small-molecule inhibitors that require continuous target engagement, PROTACs catalytically degrade targets such as bromodomain protein 4 (BRD4), a transcriptional regulator overexpressed in GBM that sustains tumor proliferation and chemoresistance.^8,9 By eradicating rather than inhibiting such proteins, PROTACs overcome the limitations of conventional drugs, including transient efficacy and acquired resistance. However, their clinical translation faces significant hurdles, while poor pharmacokinetics and off-target risks limit therapeutic indices.¹⁰ As highlighted by Craig M. Crews, a pioneer in targeted protein degradation, “delivery systems may resolve these translational bottlenecks by enhancing PROTAC specificity and bioavailability”.^5,7,11,12 The vision aligns with the growing recognition of nanotechnology's role in brain tumor targeting.

NP-based delivery platforms are uniquely suited to address PROTACs’ physicochemical and biological challenges.¹³ Polymeric carriers like poly(lactic-co-glycolic acid) (PLGA) and poly(ε-caprolactone) (PCL) can encapsulate hydrophobic PROTACs, improving their solubility and circulation time while leveraging the enhanced permeability and retention (EPR) effect for tumor-selective accumulation.^14–16 Crucially, controlled release kinetics enable spatiotemporal coordination of PROTAC activity with chemotherapy.¹⁷ For instance, initial burst release of PROTACs could degrade DNA repair proteins like BRD4, sensitizing GBM cells to subsequent TMZ-induced DNA damage,¹⁸ while sustained release maintains therapeutic concentrations below hook effect thresholds. Recent studies further suggest that NP-mediated delivery of PROTACs enhances BBB penetration and synergizes anti-tumor efficacy. Despite these advances, systematic comparisons of nanocarrier materials, formulation methods, and their impact on PROTAC performance remain underexplored—a critical gap this study seeks to address.

Building on Craig M. Crews’ proposition that nanodelivery represents a pivotal direction for advancing PROTAC therapeutics, this study systematically evaluates PLGA and PLA-based NPs to optimize the delivery of ARV-825, a BRD4-degrading PROTAC, for GBM therapy. By correlating polymer properties—including degradation rate and hydrophobicity—with NP characteristics (e.g., particle size, size distribution, drug loading efficiency) and cytotoxicity profiles, we establish design principles for maximizing therapeutic efficacy. Furthermore, we demonstrate that NP-mediated delivery of ARV-825 achieves enhanced synergistic anti-tumor activity with TMZ in GBM cells. This synergy arises from sequential drug action: initial BRD4 degradation by ARV-825 impairs DNA repair mechanisms, thereby amplifying TMZ-induced DNA damage accumulation and driving synergistic anti-tumor effect (Scheme 1).

Scheme 1 PROTAC-based nanovehicles amplify anti-tumor effects with TMZ by degrading BRD4 and inhibiting DNA repair.

2. Materials and methods

2.1 Synthesis and characterization of various polymers

PEG-PCL was synthesized through the polymerisation reaction of ε-caprolactone. PEG-pPhe and PEG-pLys-pPhe were synthesized via amin-initiated NCA ring-opening polymerization. In the chemical synthesis process, the specific procedures of PEG-pPhe are clearly illustrated in the ESI (Fig. S1†). To ensure the accuracy and integrity of the characterization, all synthesized products underwent analysis using ¹H NMR spectra with a frequency of 400 MHz (Oxford Instruments, Abingdon, UK). The chemical composition of Phe-NCA, mPEG_2K-pPhe₈-NH₂ and mPEG_2K-pPhe₁₅-NH₂ intermediate polymers was verified through ¹H NMR spectroscopy (Fig. S2–S4†). The PEG-PLGA and PEG-PCL were purchased (Ruixi Biological Technology, Xi an, China).

2.2 Preparation and characterizations of various NPs

NPs were prepared using nanoprecipitation, thin-film hydration, and dialysis methods. The characterization process involved the measurement of key parameters such as the NPs size distribution, polydispersity index (PDI), and zeta potential, which were determined using a dynamic light scattering (DLS) machine (Zetasizer Nano-ZS, Malvern Panalytical, Malvern, UK). Additionally, to gain a visual understanding of the morphological characteristics of the various NPs, transmission electron microscopy (TEM) was carried out.

2.3 Drug loading efficiency and encapsulation efficiency

To assess the drug loading efficiency (DLE) and encapsulation efficiency (EE) of the NPs, the lyophilized powder of the NPs was first weighed and subsequently dissolved in acetonitrile. The concentration of the ARV-825 was then measured using a high-performance liquid chromatography (HPLC, Agilent Technologies, Santa Clara, CA, USA) method. DLE and EE of NPs were calculated with the following equations.

Total ARV-825 means the total mass of ARV-825 involved in the preparation of NPs.

The HPLC method was carried out using specific parameters. An Agilent C18 column, with a length of 250 mm and an inner diameter of 4.6 mm, was utilized for the separation process. The mobile phase, which is crucial for the elution of analytes, is designated as a flow rate of 1.0 mL min⁻¹ and was composed of a mixture of 30% acetonitrile and 70% KH₂PO₄ buffer solution (pH 3.5). The injection volume of the sample was carefully set at 10 μL. A diode array detector (DAD) was used for the detection with a scan wavelength of 247 nm. The retention time is 5.72 ± 0.03 min.

2.4 Bioinformatics analysis and molecular dynamics simulations

The specific bioinformatics tools and databases used, such as the GEPIA 2 database and the RCSB online platform (https://gepia2.cancer-pku.cn). Additionally, we have specified the statistical methods employed in the analysis. Differential expression was calculated using |log₂ FC| > 1 and FDR-adjusted *p < 0.05 thresholds, with survival curves generated through Kaplan–Meier analysis (median expression cutoff).

The crystal structures of BRD4 and E3 proteins were retrieved from the RCSB online platform (https://www.rcsb.org/). The enCIFer software (version 1.7.3) from the CCDC (Cambridge Crystallographic Data Centre, Cambridge, UK) was used to visualise the cyclic peptide structure and measure specific distances. The interaction between polymers and ARV-825 was simulated using the Materials Studio software.

2.5 Cell viability assay

To evaluate the susceptibility of the GBM cell lines G422 to different drugs, we conducted the cell viability assay. G422 cells were planked in 96-well culture plates at a density of 2 × 10³ cells per well. When reaching 60%–70% confluence, the cells were incubated with different concentrations of drugs or preparations in DMEM for 48 h at 37 °C. After incubation, the medium was removed, and cells were washed with PBS three times. Then, 10 μL per well Cell Counting Kit-8 (CCK-8) solution (10%, Meilunbio, Dalian, China) was added and incubated with cells at 37 °C for 2 h. Then, 96-well plates were shaken by the oscillating table for 10 min. The absorbance of formazan crystals was read at 540 nm using a 6 Multiskan MK3 microplate reader (Thermo Scientific, Waltham, MA, USA). Cells without treatment were considered as the control. The cell viability was calculated based on the normalized absorbance.

2.6. Western blot procedure

Cell samples were lysed in a radioimmunoprecipitation assay (RIPA) lysis buffer supplemented with phenylmethanesulfonyl fluoride (1 mmol L⁻¹). The lysing process involved ultrasound treatment at 40% power within an ice water bath for 10 seconds, which was carried out three times with 10 second intervals in between. After lysis, the resultant supernatant, containing the extracted proteins, was collected through centrifugation at 13 000 rpm for 10 min at 4 °C (ThermoFisher Scientific, Waltham, MA, USA). The protein concentration in the cell samples was determined using a BCA Protein Assay Kit (Beyotime Biotechnology). For subsequent analysis, total protein samples (10 μg per well) underwent separation via SDS-PAGE electrophoresis, initially at a voltage of 80 V for 30 min, followed by a transition to 120 V for a duration of 2 h. In contrast, tissue samples were utilized at a concentration of 20 μg per well. The proteins were then transferred to polyvinylidene fluoride (PVDF) membranes, and then the PVDF membranes were blocked with 5% fat-free milk for 2 h. Primary antibodies were incubated overnight at 4 °C with details of the antibodies used for western blot (WB) provided in Table 1. After the primary incubation, membranes were washed three times, each for 10 min, using TBST buffer. Following these washes, membranes were subjected to incubation with horseradish peroxidase (HRP)-conjugated secondary antibodies (anti-rabbit or anti-mouse antibody) at a dilution of 1 : 1000 for 1.5 h. Additional washing with TBST buffer (pH = 7.4) was performed three times for 10 min each. Protein expression levels were detected employing enhanced chemiluminescence (ECL) autoradiography with ECL plus reagent. A quantitative analysis of the WB images was carried out utilizing ImageJ software, with all gray value statistics documented in the manuscript.

Table 1 Summary of WB antibodies used in this study

Target	Source and catalog number	Dilution ratios
BRD4	Abcam, ab128874	1 : 1000
c-Myc	Abclonal, A19032	1 : 1000
Bcl-2	Abclonal, A0208	1 : 1000
Cleaved-caspase 3	Beyotime, AC033	1 : 1000
GAPDH	Beyotime, AF1186	1 : 1000

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2.7. Cell apoptosis detection

G422 cells were seeded in a 12-well plate with a density of 4 × 10⁴ per well. When reaching 60%–70% confluence, the cells were incubated with different treatments for 48 h. Single cells were obtained from the plate by 0.25% trypsin without EDTA, stained with an apoptosis detection kit (Meilunbio, Dalian, China), incubated for 20 min, and analyzed by flow cytometry (cytoFLEX, Beckman Coulter, Brea, CA, USA).

2.8. γ-H2AX protein immunofluorescence

For immunofluorescence staining of γ-H2AX protein, cells were first seeded in confocal or 12-well plates. Once they reached a specific growth stage, they were treated with drugs as per the experimental design. After treatment, the culture medium was removed, and the cells were gently washed 2–3 times with pre-cooled PBS to eliminate residual medium and impurities. Then, 4% PFA solution was added to fix the cells at room temperature for 10 min. After fixation, the cells were washed 3 times with PBS (5 min each) to stop the fixation reaction. Next, 10% bovine serum albumin was added and left for 1–2 h at room temperature to block non-specific antibody binding sites. The blocking solution was then discarded, and the cells were incubated with the primary antibody against γ-H2AX (Abcam, ab81299), diluted according to the instructions, at 4 °C overnight to allow full binding of the primary antibody to intracellular γ-H2AX. The next day, the cells were washed 3 times with PBS (5 min each). The corresponding fluorescently labeled secondary antibody was added, and the cells were incubated at room temperature for 1.5 h in the dark. After incubation, the cells were rewashed 3 times with PBS (5 min each) to remove unbound secondary antibody. DAPI working solution was added, and the cells were incubated at room temperature for 10 min for nuclear staining, followed by another 3 washes with PBS (5 min each). Finally, the cells were observed under a confocal laser scanning microscope (CLSM, Leica TCS-SP8 STED, Wetzlar, Germany), and images were captured to analyze the fluorescence intensity and distribution of γ-H2AX, providing key data for relevant studies.

2.9. Statistical analysis

Statistical evaluations were conducted using GraphPad Prism v.9.4.1. Comparisons between groups were analyzed through t-tests or one-way ANOVA, with a significance threshold set at P < 0.05. Data are expressed as means ± standard deviation (SD), where *P < 0.05, **P < 0.01, and ***P < 0.001 indicate statistically significant differences.

3. Results and discussion

3.1. Bioinformatics analysis of BRD4 in GBM

In the current field of medical research, bioinformatics analysis has provided powerful support for the in-depth exploration of the pathogenesis of diseases and the search for potential therapeutic targets.^19,20 For GBM, an extremely invasive and challenging malignant brain tumor, the BRD4 gene/protein has attracted significant attention as a key research object.^21,22 There is increasing evidence indicating that BRD4 is associated with transcriptional proliferation and the repair of DNA damage in GBM.²³ We employed bioinformatics methods to predict the feasibility of inhibiting BRD4 in the context of GBM.

Firstly, we analyzed the genes that were highly expressed (log₂ FC > 0) and those that were lowly expressed (log₂ FC < 0) in patients with GBM. As shown in Fig. 1a, BRD4 was identified as a significantly highly expressed gene (log₂ FC > 1). In addition to the differential gene analysis, we also investigated the relationship between BRD4 gene overexpression and overall survival in GBM patients, as shown in Fig. 1b. The overexpressed BRD4 gene was found to be associated with poor overall survival. Additionally, gene analysis of the GSE178471 gene demonstrated that the signaling pathway of BRD4 gene might regulate the DNA damage and repair processes in GBM (Fig. 1c). The analysis predicted from the GEPIA 2 (cancer-pku.cn) database that the BRD4 genes were overexpressed in glioma tissue (Fig. 1d).

Fig. 1 Bioinformatics analysis of BRD4 signaling pathway in GBM. (a) Different gene expression analysis between GBM tissues and normal tissues. (b) Analysis of overall survival associated with BRD4 overexpression in GBM. (c) Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis. (d) Analysis of BRD4 gene expression in GBM tissues and normal tissues. (e) Functional network diagram of BRD4-related genes. (f) GO gene enrichment analysis.

The gene enrichment analysis was further conducted, and the results revealed that the genes highly expressed in GBM might be involved in various biological processes, such as the regulation of transcription elongation by RNA polymerase II, protein localization to chromatin, and the regulation of DNA-templated transcription and elongation (Fig. 1e). Furthermore, the BRD4 gene was mainly related to DNA or RNA-mediated transcription activities, as shown in the gene enrichment analysis results. Notably, in GBM, the highly expressed BRD4 gene was associated with the intrinsic apoptotic signaling pathway in response to DNA damage (Fig. 1f). The convergence of these bioinformatics findings suggested a dual role for BRD4 in both sustaining transcriptional addiction and subverting DNA damage response mechanisms in GBM.

These results collectively demonstrated that the BRD4 signaling pathway is closely related to the occurrence and development of GBM. Therefore, the targeted inhibition of BRD4 signaling may potentiate DNA damage accumulation while disrupting oncogenic transcription, representing a promising therapeutic strategy against this lethal malignancy.

3.2. Utilization of different delivery systems for PROTAC

3.2.1 Impact of preparation methods and polymers on PROTAC-based formulations. In the contemporary pharmaceutical landscape, a variety of drug delivery systems have emerged, with liposomes, micelles, and gels being among the commonly employed ones.²⁴ When it comes to the fabrication of NPs using amphiphilic polymers, techniques such as nanoprecipitation, dialysis, and film hydration are frequently utilized. The amphiphilic polymers that are most prevalently utilized include PEG-PLA, PEG-PCL, PEG-PLGA, and PEG-polypeptide. ARV-825, which has garnered extensive research attention as a BRD4-PROTAC, was chosen as the model drug for encapsulation in our study. The objective was to comprehensively evaluate the impact of diverse methods and carriers on the performance of the BRD4-PROTAC NP drug delivery system. PEG-polypeptide has emerged as a promising candidate for drug delivery applications, owing to its excellent biocompatibility and reliable preparation stability. Our initial investigations focused on discerning the influence of the film hydration method and the dialysis method on the characteristics of formulations self-assembled from PEG_2k-pPhe (Fig. 2a). In the case of PEG_2K-Phe₈, it was observed that the NPs fabricated using the film hydration method (Z-average = 130.0 ± 3.91 nm, PDI = 0.309 ± 0.023) possessed a more favorable particle size distribution, being both smaller and more uniform, in contrast to those prepared via the dialysis method (Z-average = 190.3 ± 1.00 nm, PDI = 0.428 ± 0.036). This property is of particular significance as it can enhance the in vivo circulation kinetics and augment the EPR effect at the tumor site, thereby improving the therapeutic efficacy.^25,26 Furthermore, during the dialysis process, the choice of solvent for the polymeric materials was found to exert a profound impact on the particle size of the resulting NPs. Interestingly, the optimal organic solvents for PEG_2k-pPhe with varying degrees of polymerization differed. This phenomenon can be attributed to the fact that the solubility of PEG_2k-pPhe in organic solvents is highly dependent on its hydrophilicity and hydrophobicity, which in turn is modulated by the degree of polymerization. For PEG_2K-Phe₈, the most favorable NPs were obtained when it was dissolved at a concentration of 2 mg mL⁻¹ in DMF and subjected to the dialysis method, yielding NPs with Z-average = 104.8 ± 1.91 nm. Similarly, for PEG_2K-Phe₁₅, the best results were achieved when it was dissolved at 2 mg mL⁻¹ in DMSO and processed using the dialysis method, with NPs exhibiting Z-average = 107.4 ± 1.45 nm.

Fig. 2 Impact of formulation parameters on NP properties. (a) Particle size distributions under different preparation methods and polymer-solvent combinations (n = 3). (b) Comparative analysis of preparation methods on particle size and encapsulation efficiency (n = 3). (c) Physicochemical characteristics of dialyzed NPs across polymer types (n = 3). (d) HPLC calibration curve for ARV-825 quantification (n = 3). (e) TEM image of PPLGA@ARV. (f) TEM image of PPLA@ARV.

PEG-PLA and PEG-PLGA have found widespread application in the industrial domain, primarily due to their robust preparation stability and enhanced safety profiles.^27–29 These polymers have thus become a standard and versatile choice for drug delivery templates. In light of this, we also undertook a detailed exploration of the factors influencing the self-assembly and performance of NPs derived from PEG-PLA and PEG-PLGA (Fig. 2b). When these polymers were dissolved at a concentration of 5 mg mL⁻¹ in DMSO, it was evident that among the film hydration and dialysis methods, the dialysis method consistently yielded NPs with a smaller particle size, accompanied by higher drug loading and encapsulation efficiencies. Although PEG-PCL demonstrated the highest DLE of 2.04%, a comprehensive comparison revealed that PEG-PLGA exhibited relatively satisfactory drug loading and encapsulation efficiencies across both preparation methods. This finding has significant implications for the rational selection of polymer carriers and preparation methods in the design of optimized drug delivery systems.

It is noteworthy that we discovered that PEG_5K-pLys₁₂-pPhe₂₀ exhibits more favorable properties. Although its particle size was somewhat larger than that of the NPs formed by PEG-PLA and PEG-PLGA, it demonstrated a stronger encapsulation ability for PROTAC, possessing the highest EE and DLE among these materials (Fig. 2c). It should be emphasized that the NPs prepared with the materials used in this study have not achieved the optimal particle size and drug loading. The purpose here was merely to compare the properties among different methods and polymers. If a particular material is selected for drug encapsulation, there is still room for subsequent optimization.

To commence, a standard linear curve was meticulously constructed using HPLC (Fig. 2d). This curve served as a crucial tool for accurately quantifying the drug loading capacity and encapsulation efficiency of the NPs. In order to assess the impact of PEG-PLGA and PEG-PLA on PROTAC drug efficacy, we prepared PLGA@ARV and PLA@ARV using these polymers. TEM images of both showed regular spherical shapes (Fig. 2e and f). DLS and zeta potential of PLGA@ARV and PLA@ARV were measured (Table S1†).

3.2.2 Kinetic simulation of designed cyclic peptides encapsulating PROTAC. Self-designed cyclic peptides present multiple benefits in drug delivery.^30,31 Firstly, they exhibit excellent stability. The cyclic structure restricts protease access to peptide bonds. For example, cyclic peptides containing disulfide bonds can resist degradation, prolonging the drug half-life and ensuring delivery efficiency. Secondly, they possess high affinity and specific binding capabilities. Through precise modulation of the amino acid sequence and cyclization structure, they can target diseased cells. In tumor treatment, for instance, they can recognize receptors on the surface of tumor cells, reducing damage to normal tissues. Thirdly, they are easy to modify and functionalize. Functional groups can be introduced as needed. For example, connecting PEG can improve water solubility, and adding a fluorescent label can monitor drug distribution, flexibly adapting to delivery requirements.^32,33

Given the numerous advantages that self-designed cyclic peptides offer in drug delivery, especially their enhanced specificity, we explored the interaction between designed cyclic peptides and ARV-825 to investigate whether they could be utilized to encapsulate this PROTAC for improved delivery efficacy. Firstly, the crystal structures of BRD4 and E3 proteins were retrieved from the RCSB online platform. The amino acids that are directly connected in space were identified as the key amino acids (Fig. S5a and S5b†). Eventually, based on these key amino acids, the cyclic peptide sequence CYCNCCWWHC (Cys Tyr Cys Asn Cys Cys Trp Trp His Cys) was designed (Fig. S6a and S6b†). The structure of the cyclic decapeptide is shown in Fig. S6b.†

Materials Studio was employed to simulate the interaction between polymers and ARV-825. By utilizing the Mesocite template in Materials Studio, the self-assembling micelles of block polymers and encapsulated drugs were simulated (Fig. S6a†). To enhance the hydrophobic ability and self-assembly capacity, PEG was attached and a linear polymer was designed for comparison (Fig. S6c†). The output results indicate that one ARV-825 molecule penetrates longitudinally into the cyclic peptide. To achieve a visual effect, we simulated an extremely small number of polymer and PROTAC molecules. Although this output has an element of contingency, it provides a feasible approach for designing drug delivery systems. Subsequent optimization of relevant parameters during simulation can be carried out to explore the innovation and feasibility of this method.

3.3 In vitro anti-tumor efficacy of BRD4-PROTAC

The BRD4-MYC pathway plays a crucial role in promoting tumor growth and progression.³⁴ As illustrated in Fig. 3a, BRD4 activates transcription, leading to the expression of c-Myc and subsequent processes that promote tumor proliferation, metabolic reprogramming, DNA repair, and resistance to chemotherapy.^35,36 The pathway also involves the upregulation of Bcl-2, which inhibits apoptosis, further supporting tumor growth. Bioinformatics analysis reveals that in GBM, there is a high degree of correlation among the BRD4 and MYC, BCL2 genes (Fig. S7a and S7b†).

Fig. 3 In vitro cellular pharmacodynamic effects of ARV-825. (a) Schematic diagram of the BRD4-MYC pathway and its impact on tumor growth. (b) WB analysis of the degradation effect of ARV-825 on BRD4. (c) WB analysis of the impact of ARV-825 on downstream proteins. (d) The impact of ARV-825 on the apoptosis of G422 cells. (e) The ratio of early and late apoptosis of G422 cells induced by ARV-825.

ARV-825 is a classic BRD4-PROTAC molecule.³⁷ Firstly, we investigated whether ARV-825 could degrade BRD4 in the GBM cell line G422. The results indicated that it was capable of degrading the expression of BRD4 protein in G422 cells (Fig. 3b), and this effect exhibited a concentration-dependent manner. BRD4 plays a crucial role in genetic regulation and gene transcription, particularly in the BRD4-MYC genetic regulatory pathway where it functions as an upstream dominant factor. Therefore, inhibiting BRD4 can disrupt normal transcriptional functions and induce tumor cell apoptosis. WB experiments demonstrated that ARV-825 could downregulate the expression levels of c-Myc and Bcl-2 proteins and enhance the expression of the apoptotic protein cleaved caspase-3 by degrading the BRD4 protein (Fig. 3c). After confirming that ARV-825 could promote cell apoptosis, we also utilized flow cytometry to specifically monitor the specific impacts of ARV-825 on early and late apoptosis of cells (Fig. 3d and e). The results showed that with the increase in the concentration of ARV-825, both early and late apoptosis of cells gradually increased, and the impact on early apoptosis was somewhat higher than that on late apoptosis. In summary, the above results confirm that ARV-825 can affect the expression of downstream c-Myc and Bcl-2 and promote tumor cell apoptosis by degrading the BRD4 protein.

In addition, it has been clearly established that the BRD4-PROTAC is capable of modulating the cell cycle progression of the G422 tumor cell line.^38,39 We systematically investigated two distinct BRD4-PROTAC variants, specifically ARV-825 and SIAIS171142. Our experimental findings unequivocally demonstrated that both ARV-825 and SIAIS171142 possess the ability to perturb the cell cycle dynamics of tumor cells (Fig. 4a and b), with this effect being directly proportional to the concentration of the administered PROTAC. By virtue of degrading BRD4, both ARV-825 and SIAIS171142 are able to augment the fraction of cells residing in the G1 phase of the cell cycle (Fig. 4c and d). Cells are equipped with an intricate checkpoint machinery. In the event of DNA damage, cells will halt at the G1 phase checkpoint and initiate a cascade of DNA repair processes. If the damage is irreparable, the cells will trigger apoptosis. However, during the repair phase, the number of cells in the G1 phase will increase as they are detained in this stage, awaiting repair or to determine their ultimate fate.⁴⁰ These results are indicative of the beneficial impact of BRD4-PROTAC in terms of DNA damage induction and anti-tumor efficacy.

Fig. 4 Influence of BRD4-PROTAC on the cell cycle. (a) Monitoring the influence of different concentrations of ARV-825 on the G422 cell cycle by flow cytometry. (b) Monitoring the influence of different concentrations of SIAIS171142 on the G422 cell cycle by flow cytometry. (c) Influence of different concentrations of ARV-825 on the G422 cell cycle. (d) Influence of different concentrations of SIAIS171142 on the G422 cell cycle. (e) Cytotoxicity of G422 cells incubated with TMZ for 24 h (n = 3, IC₅₀ = 960.1 μmol L⁻¹). (f, g) Cytotoxicity of G422 cells incubated with lower concentrations of TMZ for 48 h and 72 h (n = 5).

Owing to its unique property of traversing the blood–brain barrier, TMZ occupies a pivotal position in the first-line clinical management of GBM. In an effort to explore the potential synergistic interactions between PROTAC and TMZ, we also undertook an investigation into the cytotoxicity of TMZ against G422 cells. After 24 h incubation period, the IC₅₀ value of TMZ was determined to be 960.1 μmol L⁻¹ (Fig. 4e). When the incubation time of TMZ with G422 cells was extended to 48 or 72 h, at lower concentrations ranging from 0 to 800 μmol L⁻¹, TMZ did not manifest any significant cytotoxic effects on G422 cells (Fig. 4f). Conversely, when TMZ was administered at relatively high concentrations exceeding 1000 μmol L⁻¹, a sharp increase in the cytotoxicity of TMZ towards G422 cells was observed (Fig. 4g). This behavior of TMZ under different concentration and time conditions provided valuable insights for the subsequent exploration of its combination with PROTAC, potentially leading to enhanced therapeutic strategies in the treatment of GBM.

3.4. Comparison of the in vitro anti-tumor efficacy of BRD4-PROTACs with different structures

PROTACs possess a three-segment structure: target-binding ligand, E3 ligase-binding ligand and linker. Due to the variations in the three-segment structure, PROTACs exhibit a diverse range of forms. Even when targeting the same protein, different structures can still be derived from changes in the three-segment configuration.^41,42

Consequently, we investigated the differential effects of three BRD4-PROTACs with distinct structures on tumor cells. The three BRD4-PROTACs, namely ARV-825, SIAIS171142, and GT-03708, have their specific chemical structures as shown in Fig. 5a–c. The IC₅₀ value of ARV-825 after incubation with cells for 48 h was determined to be 1766 nmol L⁻¹ (Fig. 5d). SIAIS171142 exhibited differences in cytotoxicity compared to ARV-825 due to its distinct mode of BRD4 degradation. The detailed investigation of SIAIS171142 is presented in the earlier article.⁴³ SIAIS171142 displayed a hook effect at low concentrations, and after incubation with cells for either 24 or 48 h, the cell survival rate initially decreased and then slightly increased (Fig. 5e). Its IC₅₀ value was approximately 400 nmol L⁻¹, indicating a stronger cytotoxic effect. The IC₅₀ value of GT-03708 after incubation with cells for 48 h was 3621 nmol L⁻¹ (Fig. 5f), demonstrating the weakest cytotoxic effect among the three molecules.

Fig. 5 Comparison of BRD4-PROTACs with different structures. (a) Chemical structural formula of ARV-825. (b) Chemical structural formula of SIAIS171142. (c) Chemical structural formula of GT-03708. (d) Cytotoxicity of G422 cells incubated with ARV-825 for 48 h (n = 3, IC₅₀ = 1766 nmol L⁻¹). (e) Cytotoxicity of G422 cells incubated with SIAIS171142 for 24 h and 48 h (n = 6). (f) Cytotoxicity of G422 cells incubated with GT-03708 for 48 h (n = 3). (g) Degradation of BRD4 in G422 cells incubated with GT-03708 for 48 h. (h) Degradation of BRD4 in G422 cells incubated with SIAIS171142 for different durations.

This is attributed to the relatively poor protein degradation ability of GT-03708 towards BRD4. As illustrated in Fig. 5g, although GT-03708 can degrade BRD4, its degradation capacity was inferior to that of ARV-825. Additionally, we explored the impact of the incubation time of BRD4-PROTACs with G422 cells on the degradation of cellular proteins (Fig. 5h). During the 0–24 h period, prolonging the incubation time enhanced the degradation ability of SIAIS171142 towards BRD4. Specifically, after 24 h of incubation, SIAIS171142 exhibited the strongest degradation effect on BRD4, reducing it to 41% of the control group.

3.5. Synergistic anti-tumor effect of ARV-825 and TMZ in vitro

In the realm of tumor research, exploring the synergistic interactions between different anti-tumor agents holds great promise for enhancing therapeutic efficacy and overcoming drug resistance. ARV-825, a prominent member of the BRD4-PROTAC family, and TMZ, a well-established chemotherapeutic drug, have garnered significant attention due to their potential combined effect against tumors. TMZ causes DNA damage in tumor cells, but tumor cells may develop resistance through mechanisms such as drug efflux and DNA repair processes.⁴⁴ ARV-825, as a PROTAC targeting BRD4, can degrade BRD4 protein, thereby inhibiting DNA repair processes and drug efflux, leading to enhanced chemotherapy effects. The combination of ARV-825 and TMZ not only causes DNA damage but also impairs the tumor cells’ ability to repair this damage, ultimately resulting in more effective tumor cell death (Fig. 6a).

Fig. 6 Synergistic effect of PROTAC and TMZ. (a) Schematic diagram of the synergistic effect of BRD4-PROTAC and TMZ for GBM treatment. (b) Quantitative results of γ-H2AX protein immunofluorescence (n = 3). (c) DNA damage effect of ARV-825 and TMZ on G422 cells (G1: control; G2: 10 μmol L⁻¹ ARV-825; G3: 30 μmol L⁻¹ ARV-825; G4: 2 mmol L⁻¹ TMZ; G5: 5 mmol L⁻¹ TMZ; G6: 10 μmol L⁻¹ ARV-825 + 2 mmol L⁻¹ TMZ). (d) Cytotoxicity of different concentrations of ARV-825 and TMZ on G422 cells. (e) Combination index (CI) of different concentrations of ARV-825 and TMZ.

To begin with, our research focused on ascertaining whether the concurrent application of the two drugs could lead to a synergistic impact on cellular DNA damage. The γ-H2AX protein is widely recognized as a reliable biomarker for DNA damage. In our study, the immunofluorescence staining technique was employed to assess the extent of DNA damage subsequent to various treatment regimens. As evidenced by the staining outcomes and the ensuing quantitative analysis (Fig. 6b and c), it was strikingly observed that the combination of a relatively low concentration of ARV-825 and TMZ (G6: 10 μmol L⁻¹ ARV-825 + 2 mmol L⁻¹ TMZ) induced a more pronounced DNA damage effect compared to either a high concentration of ARV-825 (G3) or a high concentration of TMZ (G5).

Thereafter, we delved deeper into the exploration of the synergistic interaction between different concentrations of ARV-825 and TMZ. By capitalizing on the cytotoxicity data obtained from the diverse concentrations of these two drugs (Fig. 6d and Fig. S8a†), the CI was computed using the CompuSyn software. It is well-established that a CI value less than 0.75 is indicative of a synergistic effect. Our findings conclusively revealed that by precisely modulating the concentration ratio of the two agents, a combination of 50 μmol L⁻¹ ARV-825 and 500 μmol L⁻¹ TMZ manifested a significant synergistic therapeutic effect (Fig. 6e). ARV-825 downregulates BRD4, amplifying TMZ-induced DNA strand breaks.⁴⁵ This discovery holds great promise for the development of more efficacious treatment strategies in the field of pharmacology.

3.6. Anti-tumor effects of PROTAC-based nanovehicles

As shown in Fig. 7a, the process of PROTAC degrading proteins occurs through a unique mechanism and this degradation mode endows PROTAC with high efficiency. PROTAC molecules are designed with three components: a ligand that binds to the target protein, a linker, and another ligand that binds to an E3 ubiquitin ligase. When the PROTAC molecule enters the cell, it simultaneously binds to the target protein and the E3 ligase, bringing them in close proximity. This proximity allows the E3 ligase to attach ubiquitin molecules to the target protein. The ubiquitinated target protein is then recognized and degraded by the proteasome, the cell's protein degradation machinery. Once the target protein is degraded, the PROTAC molecule is released and can then go on to initiate another round of degradation, thus giving it the recyclable characteristic.⁴⁶ However, the application of PROTAC may bring about some drawbacks, such as potential toxicity. Therefore, the exploration of nano-delivery systems aims to optimize the delivery of PROTAC and expand its therapeutic potential.

Fig. 7 The synergistic anti-tumor effect of nano-delivery systems and TMZ. (a) Schematic illustration of BRD4 protein degradation via PROTAC. (b) Cytotoxicity of different concentrations of PPLA@ARV and PPLGA@ARV on G422 cells (n = 4). (c) Cytotoxicity of different concentrations of PPLA@ARV and TMZ on G422 cells (n = 3). (d) CI of different concentrations of PPLA@ARV and TMZ. (e) Cytotoxicity of different concentrations of PPLGA@ARV and TMZ on G422 cells (n = 3). (f) CI of different concentrations of PPLGA@ARV and TMZ.

PLGA and PLA nanoparticles were selected for their gold-standard clinical translatability, high PROTAC payload capacity, and unique degradation-driven endolysosomal escape—a synergy critical for intracellular PROTAC delivery. While novel platforms show conceptual promise, PLGA/PLA provides immediate therapeutic viability for advancing protein degraders toward clinical utility.⁴⁷ To investigate the impact of delivery systems on PROTAC efficacy, two types of NPs were fabricated with PLA and PLGA, and the cytotoxicity of PPLGA@ARV and PPLA@ARV NPs was compared against tumor cells. Both PPLGA@ARV and PPLA@ARV released encapsulated ARV-825 to induce ubiquitin-dependent degradation of the target protein BRD4, thereby exerting cytotoxic effects. Cytotoxicity assays comparing ARV-825, PPLGA@ARV, and PPLA@ARV revealed no significant enhancement in cytotoxicity for the NP formulations compared to free ARV (Fig. 7b). This suggests that simple encapsulation of ARV-825 with PLA or PLGA matrices does not markedly alter its inherent anti-tumor properties.

The cytotoxicity of PPLGA@ARV and PPLA@ARV was further evaluated in combination with TMZ (Fig. 7c, e and Fig. S8b, S8c†) and the CI was calculated to quantify synergistic effects. CI analysis demonstrated synergistic interactions (CI < 0.7) between TMZ and both NP formulations across most concentration ratios (Fig. 7d and f). Notably, PPLGA@ARV exhibited synergistic effects at 24 out of 25 tested concentration ratios, with only one ratio (TMZ = 1500 μmol L⁻¹, PPLGA@ARV = 5 μmol L⁻¹) showing additive effects (CI = 0.95). These results indicate that PLA- and PLGA-based nanoencapsulation enhances the synergistic anti-tumor efficacy of ARV-825 and TMZ.

This enhancement may stem from the inherent advantages of nanocarriers. For instance, PLGA NPs modulate drug release kinetics, enabling sequential delivery: initial ARV-825 released rapidly and degraded BRD4 to impair DNA repair capacity, followed by TMZ-induced O6-methylguanine (O6-MeG) lesions. Under compromised repair mechanisms, these lesions accumulate as lethal double-strand breaks, maximizing synthetic lethality through spatiotemporal synergy. Additionally, NPs may be internalized by tumor-associated macrophages, polarizing them toward a pro-inflammatory M1 phenotype to augment immune checkpoint inhibitor efficacy. However, the precise mechanisms underlying these observations warrant further investigation.

4. Conclusions

This study demonstrates the critical role of nanocarrier design in optimizing PROTAC delivery for GBM therapy. By comparing polymeric NPs (PEG-PLGA, PEG-PLA, PEG-PCL) fabricated via film hydration, dialysis, and nanoprecipitation, we found that preparation methods and polymer hydrophobicity directly influence particle size (104–190 nm), and encapsulation efficiency. Dialysis-synthesized PEG-PLGA NPs achieved optimal ARV-825 loading and stability, enabling sustained BRD4 degradation. Structural variations among PROTACs (ARV-825, GT-03708 vs. SIAIS171142) highlighted the importance of molecular tailoring. Notably, NP formulations maintained PROTAC bioactivity without introducing cytotoxicity, underscoring their translational potential. When combined with TMZ, PLGA-based delivery systems (PPLGA@ARV) exhibited potent synergy (CI < 0.7 at 24/25 ratios), where sequential release of ARV-825 and TMZ disrupted DNA repair and amplified damage accumulation, as evidenced by γ-H2AX immunofluorescence and apoptosis assays. Bioinformatics analysis validated BRD4's dual role in GBM progression—sustaining oncogenic transcription and evading DNA damage responses. These findings align with Craig M. Crews’ vision of nano-delivery as a cornerstone for PROTAC development. Future work should refine BBB-penetrating carriers and integrate immune-modulating agents to maximize clinical efficacy. Our results provide a roadmap for advancing PROTAC delivery and protein degradation therapies against GBM or other intractable tumors.

Author contributions

Yun Guo and Tao Sun contributed to design, concept, and supervise the project; Yun Guo performed the synthesis and characterization and wrote the manuscript; Haoyu You, Yiyang Li, Zheng Zhou, Zonghua Tian and Chen Jiang provided valuable advice for article writing; Tao Sun revised and edited the manuscript. All authors have approved the final article.

Conflicts of interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Data availability

All experimental procedures, characterization data (including DLS, HPLC, and cytotoxicity results), and computational details are provided in the Materials and Methods section, relevant figure captions, and ESI.† Raw datasets generated during this study (e.g., kinetic profiles, simulation input files) are tabulated within the main text or available in the ESI.† No data were deposited in external repositories as all ESI† is included in this article and its supplementary files.

Acknowledgements

The authors acknowledge the support from the National Key R&D Program of China (2023YFC3404103), National Natural Science Foundation of China (82473853, 82361148716), Shanghai Municipal Science and Technology Commission Intergovernmental Cooperation Project (24430710700), Shanghai Natural Science Foundation Project (22ZR1414100), Shanghai Municipal Science and Technology Major Project (Grant 2018SHZDZX01) and ZJLab.

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Footnote

† Electronic supplementary information (ESI) available. See DOI: https://doi.org/10.1039/d5bm00443h

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