Abstract
Proteolysis targeting chimera (PROTAC) technology has received extensive attention due to its “event-driven” mechanism of action. However, existing chimera molecules tend to have higher molecular weight and higher polar surface area and exhibit lower druggable properties. In order to effectively improve the bioavailability, circulation time and other proprietary drug parameters of “chimera molecules”, we combine the characteristics of nanotechnology and chimera technology, and propose the strategy of “Split-and-Mix” proteolysis degradation (SM-PROTAC) technology. However, both peptide- and liposome-based SM-PROTAC showed suboptimal in vivo efficiency. Hence, in this study, we incorporated Polylactic acid (PLA), an FDA-approved biomedical material, as the self-assembled matrix for SM-PROTAC to enhance in vivo effectiveness. PLA-based SM-PROTACs successfully degrade model targets including BRD4, ERα, and CDK4 in cellular assays and display more obvious tumor inhibition in vivo in female mice with a lower target ligand content (~1/10 of small molecule PROTAC controls) and long-term therapeutic potential (1 injection per three days vs 1 injection every day). In summary, PLA-based SM-PROTACs demonstrate promising drug characteristics and present obvious advantages in low-dose and long-term applications, providing additional insights into the pharmaceutical potential of the SM-PROTAC strategy.
Similar content being viewed by others
Introduction
In recent years, PROTAC (Proteolysis Targeting Chimera) technology has sparked a revolutionary new era for small-molecule drug development. PROTAC offers multiple advantages: effectively targeting a wide range of undruggable proteins of interest (POI), exhibiting reversibility, minimizing off-target effects, overcoming drug resistance, prolonging the duration of action, and ultimately reducing the incidence of adverse reactions1,2,3,4,5,6. In particular, inactive or low-active protein modulators can be transformed into effective drug candidates by PROTAC technology7. To date, more than 20 PROTAC molecules are in clinical trials for multiple disease indications8. However, some challenges still exist with traditional PROTAC, such as poor membrane penetration, time-consuming screening, and high metabolic rate in vivo, which hinder the clinical applications of PROTACs9,10,11,12,13.
To address these challenges, researchers have explored nano-delivery as well as prodrug strategies11,14. However, encapsulation-based approaches for intracellular delivery of the entire PROTAC molecule still fail to overcome the pharmacokinetic defects posed by its inherent high metabolic rate and rapid clearance. Therefore, we proposed a “Split-and-Mix” protein degradation strategy (SM-PROTAC) on account of the highly modular structures of PROTAC molecules. Based on self-assembled peptides and liposomes, we confirmed various advantages of SM-PROTAC, including facile screening, high permeability, self-optimized biomolecule spatial recognition, and multifunctional properties15,16,17. Multiple research groups have proposed similar approaches, such as Supra-PROTAC and Nano-PROTAC. Yu et al. employed the self-assembling peptide EIYIYE to demonstrate that this strategy facilitates flexible modulation of ligand ratios and enables sustained therapeutic efficacy in vivo18. Wang et al. utilized the self-assembling motif GNNQQNY to validate its superiority in mitigating the Hook effect inherent in conventional PROTAC molecules19. Kim et al. further leveraged the FF self-assembly module to confirm enhanced delivery efficiency for peptide-based PROTACs20. However, the peptide-based SM-PROTAC has the defects of high effective concentration and potential toxicity, which is difficult to be further applied. While there are issues with the liposome approach, mainly related with the uncontrollable membrane fusion21. Moreover, both the peptide-based and liposome-based SM-PROTAC only exhibited moderate in vivo efficiency22.
Polylactic acid (PLA) is an FDA-approved biocompatible polyester polymer for drug-sustained release and medical implantation23. Normally, PEG with a molecular weight of less than 5000 Da can be phagocytic or excreted through renal filtration membrane, which means that selecting a suitable molecular weight PEG to modify PLA can obtain a biocompatible and biodegradable block PLA-PEG copolymer24,25. The amphiphilic block copolymer can be easily prepared into microspheres, micelles, hydrogels and other nanomaterials in suitable ways, which meets SM-PROTAC’s need for self-assembled carriers (Fig. 1A). Studies have shown that the molecular weight of PEG has little effect on the size of nanomaterials, and can increase the solubility and stability of PLA-PEG and prolong the half-life of the polymer in vivo26. Consequently, in this study, we opted for PLA-PEG as the self-assembly carrier and confirmed the stability of PLA-PEG nanoparticles in vitro and in vivo. In model studies, PLA-based SM-PROTACs successfully degrade BRD4, ERα, and CDK4 at satisfactory concentrations. To delve deeper into the in vivo efficacy of PLA-based SM-PROTAC, we selected ARV-825 as a positive control and modified the drug administration time from once a day to once every 3 days in xenograft mouse models (Fig. 1B). ARV-825 is a small molecule PROTAC that connects OTX-015 to Pomalidomide, which is shown to lead to fast, efficient degradation of BRD427. In our study, PLA-PEG-based SM-PROTAC is of remarkable stability and can effectively inhibit tumor growth with a sustained efficacy over a prolonged duration compared with ARV-825. In general, the PLA-PEG system shows good preparation simplicity, higher action efficiency, and prolonged action time in vivo. Besides, BRD4 stands out as an excellent target for cancer therapy, which underscores the potential of the PLA-PEG system for cancer treatment28,29,30.
A Synthesis and assembly process of PLA-PEG-based SM-PROTAC; B Schematic illustration of PLA-PEG-Based SM-PROTAC inhibiting tumor growth in vivo, with comparative advantages over ARV-825. The mouse image was sourced from the SciDraw database (https://scidraw.io), with original contributions by Ethan Tyler and Lex Kravitz.
Results
Preparation and characterization of PLA-PEG nanoparticles
The PLA-PEG nanoparticles were prepared using a nanoprecipitation strategy (shown in Fig. 2A)31. The particle size is maintained between 100 and 250 nm (Figs. 2B and S1) and shows a negative surface charge (Fig. 2C). The polydispersity index (PDI) value is mainly below 0.2. Moreover, the addition of a stabilizer (Polyvinyl alcohol, PVA) and lyophilizing agent (sucrose) could effectively ensure the stability and consistency of nanoparticles (Fig. S2A)32,33. Therefore, the optimal conditions for constructing PLA-PEG nanoparticles were using 15 mg/ml PLA-PEG, a 1:20 oil-water ratio, 0.25% PVA, and THF solvent, with the addition of 1.5% sucrose. Scanning electron microscopy (SEM) further confirmed the size and the uniform size distribution of the nanoparticles (less than 200 nm, Fig. 2D)34. Besides, we observed PLA-PEG nanoparticles with good cell membrane penetration ability through flow cytometry and confocal laser scanning microscopy (CLSM) (Figs. 2E and S4). The PLA-PEG nanoparticles could remain stable in PBS and FBS for >96 h at room temperature (Fig. S2B). Next, we conducted the fluorescence resonance energy transfer (FRET) assay in cells with CLSM35,36. As shown in Fig. 2F and S6, FRET signals were detected in the PLA-PEG-Cy3_Cy5 group, and the FRET signal could maintain stability in the cell for more than 96 h (Figs. S7, S8, S9). After 96 h, the FRET signal began to weaken gradually, which might be due to the dissociation of PLA-PEG nanoparticles, the decrease in the number of viable cells, or the gradual quenching of dye molecules. The stability of PLA-PEG nanoparticles also serves as a robust foundation for designing drug administration protocols in animal experiments.
A Schematic diagram of nanoparticle preparation method based on PLA-PEG; B PLA-PEG nanoparticle size measured via DLS. The average particle size is 136 nm, and the PDI is 0.154; C Zeta potential of PLA-PEG nanoparticles was measured via DLS. The zeta potential is −0.22 mV; D SEM morphology of PLA-PEG nanoparticles with a scale bar of 200 nm; E Cell membrane penetration results of PLA-PEG nanoparticles taken by CLSM with a scale bar of 10 μm (4 h). PLA-PEG-Cy3 NP represents nanoparticles with Cy3 dye; PLA-PEG-Cy3 refers to the compound before assembly, and such expression generally refers to nanoparticles if not specified in the following. COOH-PEG-Cy3 represents polyethylene glycol coupled with Cy3 dye; PLA-PEG-Cy3_Cy5 NP represents PLA-PEG nanoparticles coupled with both Cy3 and Cy5 dyes (Fig. S5); F CLSM images with scale bar of 50 μm in different fluorescence channels of PLA-PEG-Cy3_Cy5 treated HCT116 cells (24 h).
In addition, we have tried to clarify how the PLA-PEG nanoparticles were internalized or the PLA-PEG nanoparticles could escape from the lysosomes. As illustrated in Fig. S10, no linear correlation was observed between the quantity of PLA-PEG nanoparticles entering cells and the alterations in lysosome levels. Over the first 5 h, there was a minimal increase in the number of lysosomes. However, at 7 h, the lysosome count peaked in tandem with an increase in the number of nanoparticles entering the cells, subsequently initiating a decline (Fig. S10A). The co-localization analysis (Pearson’s coefficient) further suggests that even after nanoparticles enter lysosomes, they possess the ability to efficiently escape from them (Fig. S10B).
Design and validation of SM-PROTAC based on PLA-PEG
PROTAC molecules are highly modular structures that contain a ligand to POI, a ligand to E3 ligase, and a linker to couple the two ligands together. Therefore, in the previous study, we tried to split the modular PROTAC molecule and connect POI and E3 ligands to the self-assembled carrier, and then combine the two functional ligands together in the form of self-assembly (split-and-mix PROTAC, SM-PROTAC)15,16. Based on self-assembled peptides and liposomes, we validate the concept of SM-PROTAC on a variety of targets. Hence, according to the SM-PROTAC strategy, ARV-825 was separated naturally into two functional molecules and covalently coupled to PLA-PEG, respectively (Fig. 3A). To ensure good coupling efficiency, the active ester condensation between PLA-PEG and the functional molecule was adopted (Fig. S3). The two functional molecules were seamlessly combined via co-assembly, yielding the novel SM-PROTAC, PLA-PEG-JQ1_CRBN. Similar to ARV-825, PLA-PEG-JQ1_CRBN can degrade the target protein BRD4 at less than 10 μM within 24 h in both HCT116, HeLa, and MCF-7 cell lines (Figs. 3B, C, D and S11–S14). The degradation of BRD4 started at 12 h and was exhibited in a time-dependent manner (Fig. 3E). We also tested the degradation effects of different ligand proportions. As shown in Fig. 3F, G, H, I, different ligand proportions didn’t cause obvious differences in BRD4 expression. Hence, in subsequent experiments, different SM-PROTACs are all assembled by two functional molecules in a ratio of 1:1. The growth inhibition curves mirrored the effects of BRD4 degradation (Fig. 3J). In addition, PLA-PEG-JQ1_CRBN has also exhibited the capability of degrading target proteins via the ubiquitin-proteasome pathway (Fig. 3K). An obvious increase in the BRD4 content was observed after the use of MG132 from 8 to 24 h (Figs. 3K and S15). The experiment of ligand competition also confirmed this further. As shown in Fig. S16, after the addition of Pomalidomide, the targeting ligand of E3 ligase CRBN, BRD4 was indeed effectively recovered.
A Design strategy of PLA-PEG-based “Split-and-Mix” PROTAC (PLA-PEG-JQ1_CRBN SM-PROTAC), in which the ARV-825 represents the “Split” motif. The ARV-825 is a small molecule PROTAC targeting BRD4, which contains the BRD4-targeting ligand (JQ1 analogue) and CRBN-targeting ligand (thalidomide analogue); B, C, D Concentration gradient west blot of BRD4 degradation by PLA-PEG-JQ1_CRBN respectively in HCT116, HeLa, and MCF-7 cell lines for 24 h; E BRD4 degradation by PLA-PEG-JQ1_CRBN (5 μM) in HCT116 cell line at different time points; F, G, H, I Effects of different proportions of BRD4 and CRBN ligands on the degradation of PLA-PEG nanoparticles. BRD4 Ligand: CRBN Ligand = 2:1(F) and 3:1(G); BRD4 Ligand: CRBN Ligand = 1:2(H) and 1:3(I); J The growth inhibition of PLA-PEG-JQ1_CRBN targeting BRD4 on HCT116, Hela, and MCF-7 cell lines. Error bars represent SEMs of at least three independent measurements; K BRD4 degradation by PLA-PEG-JQ1_CRBN through the ubiquitin-proteasome pathway, where the treating time of the proteasome inhibitor MG132 (10 μM) was 8 h.
The significance of co-assembly for PLA-PEG-based SM-PROTAC
Additional verifications were conducted to further strengthen the significance of the co-assembly of the two functional molecules. Figure 4A depicted a notable difference between the two co-assembly systems and the mixing post-self-assembly. As shown in Fig. 4D, E, the protein BRD4 remained stable even when the drug concentration surpassed 20 μM. While the ligand itself was also insufficient in degrading the target protein (Fig. 4B, C). PLA-based SM-PROTAC showed the best cell growth inhibition ability (Fig. 4F). This observation profoundly underscores the rationality of SM-PROTAC design and the necessity of a self-assembly vector.
A Schematic diagram of co-assembly after mixing and mixing after self-assembly of PLA-PEG coupled with different functional groups (ligand 1 and ligand 2); B, C, D, E Western blot of HCT116 cells treated with different concentrations of compounds for 24 h. PLA-PEG-JQ1 + PLA-PEG-CRBN and PLA-PEG-JQ1 + PLA-PEG-VHL represent mixtures of self-assembled PLA-PEG-JQ1 and PLA-PEG-CRBN or PLA-PEG-VHL nanoparticles; F The growth inhibition rate of different compounds or mixtures on the HCT116 cell line. Error bars represent SEMs of at least three independent measurements.
Validation of PLA-PEG-based SM-PROTAC extension to other targets
To further validate the high efficiency and flexibility of the SM-PROTAC strategy, we switched our ligand from CRBN to VHL (Fig. 5A). As revealed in Fig. 5B, C, D, PLA-PEG-JQ1_VHL can also efficiently degrade BRD4 similarly as PLA-PEG-JQ1_CRBN, at less than 10 μM in cell line HCT116 (Figs. 5B and S11), HeLa (Figs. 5C and S12), and MCF-7 (Figs. 5D and S13). We then applied the PLA system with other protein targets such as CDK4 and ERα (Fig. 5E). Based on our western blot results, the PLA-PEG-based PROTAC also achieved a highly efficient degradation effect on the target proteins CDK4 (~2.5 μM, Fig. 5F, G) and ERα (~5 μM, Fig. 5I, J). The cell growth inhibition curve showed similar trends with the degradation (Fig. 5H, K). The target degradation reliance on the ubiquitin-proteasome system was also demonstrated (Fig. S17). To assess the impact of PLA block length on target protein degradation, we tested varying PLA and PEG ratios using ERα (Fig. S18). Since degradation rates showed no statistically dependence on PLA ratio, we standardized subsequent experiments to the PLA3000-PEG5000 carrier system. These findings suggest PLA polymer could be utilized as a versatile matrix for the SM-PROTAC strategy.
A Schematic diagram of replacing the recruited E3 ligase in the aforementioned BRD4-targeting SM-PROTAC from CRBN ligand to VHL; B, C, D Concentration gradient western blot of BRD4 degradation by PLA-PEG-JQ1_VHL, respectively, in HCT116, HeLa, and MCF-7 cell lines after treating for 24 h. E Design diagram of PLA-PEG extended for the degradation of CDK4 and ERα. Palbociclib and tamoxifen were used for the ligands targeting CDK4 and ERα, respectively. F, G Concentration gradient west blot of CDK4 degradation in HCT116 cell line by PLA-PEG-Pabo_VHL and PLA-PEG-Pabo_CRBN; H The growth inhibition of PLA-PEG-Pabo_VHL and PLA-PEG-Pabo_CRBN on HCT116 cell line; I, J Concentration gradient west blot of ERα degradation in T47D cell line by PLA-PEG-TMXF_VHL and PLA-PEG-TMXF_CRBN; K The growth inhibition of PLA-PEG-TMXF_VHL and PLA-PEG-TNXF_CRBN on T47D cell line. Error bars represent SEMs of at least three independent measurements.
Evaluation of in vivo activity of PLA-PEG-based SM-PROTAC
Encouraged by the high degradation efficiency and the strong stability of the PLA-PEG nanoparticles in vitro, we constructed a cell-derived xenograft (CDX) HCT116 colon tumor model. The tumor-bearing mice were administered with different groups by intraperitoneal injection at a dosage of 10 mg/kg or 20 mg/kg every 3 days for 6 treatments (Fig. 6A). As shown in Fig. 6C (tumor volume), 6D (tumor weight), 6E (inhibition ratio), and 6 F (tumor image), the group of mice treated with PLA-PEG-JQ1_CRBN produced a more distinct tumor growth suppression effect, especially with the high-dose group (20 mg/kg). In contrast, the small molecule inhibitor group (JQ1) and the small molecule PROTAC group (ARV-825) did not show the ability in tumor growth inhibition. This might be attributed to the fact that the drug administration of small-molecule modulators was changed from traditionally once daily to once every 3 days in this study. Indeed, we undertook a daily dosing experiment, where ARV-825 exhibited a measurable degree of tumor suppression (Fig. S19). ARV-825 (and MZ-1) exhibit superior cellular activity compared to SM-PROTAC, as evidenced by their lower DC₅₀ and IC₅₀ values (Fig. S20). This additional evidence underscores the potential drawbacks of ARV-825, including faster metabolism within the body and suboptimal membrane penetration. Besides, WB experiments revealed that the content of the BRD4 protein was the weakest in the tumor tissue in 20 mg/kg PLA-PEG-JQ1_CRBN-treated mice (Fig. 6G). During the treatment, we did not observe obvious changes in the body weight of mice (Fig. 6B).
A The administration plan for Balb/c mice. The CDX model was constructed using the HCT116 cell line, and the drugs were intraperitoneally injected into the mice every 3 days. The treatment groups:①PBS;②JQ1(20 mg/kg)③PLA-PEG-JQ1(20 mg/kg)④PLA-PEG-CRBN (20 mg/kg)⑤ARV-825 (20 mg/kg)⑥PLA-PEG-JQ1_CRBN(10 mg/kg)⑦PLA-PEG-JQ1_CRBN(20 mg/kg); B Body weight changes of mice throughout the treatment. NS refers to no significance; C Average tumor growth curves of each group throughout the treatment; D Weight statistics of tumor tissue at the end point of the experiment; E Calculated tumor growth inhibition rates in different groups of mice. Tumor growth inhibition rates (Tumor weight in PBS group-Tumor weight in each group)/Tumor weight in PBS group*100%; F The images of tumor tissue at the end point of the experiment with scale bar 10 mm; G Western blot of BRD4 in different groups of tumor tissues at the endpoint of the experiment. Data are analyzed by GraphPad Prism 8 (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001).
Furthermore, we focused on some administration methods and test others for a more comprehensively evaluate the advantages of PLA-PEG-based SM-PROTAC. We also incorporated LNP (liposome) into our comparative analysis. As shown in Fig. S22, the results indicated that administration of the tail intravenous injection also produced a positive antitumor effect. Yet, it fell short in effectiveness compared to the intraperitoneal injection (Figs. 6E and S22E). The positive drug, ARV-825, showed better results when administered intravenously compared to intraperitoneal administration. However, it still performs inferior to PLA-PEG-JQ1_CRBN. On the other hand, the antitumor performance of SM-PROTAC when formulated with LNP was relatively modest. Similarly, administering the PLA-PEG system orally also showed limited tumor suppression. Additionally, we attempted to discern the stability disparity between LNP and PLA-PEG utilizing SEM. As depicted in Fig. S24, after incubating LNP and PLA-PEG separately with solutions containing trypsin enzyme and proteases for a specified duration, morphological analysis was conducted. The observations revealed that the PLA-PEG system retained its particle morphology intact even after 72 h, whereas the LNP began to exhibit dispersion, making it challenging to maintain a coherent particle shape. These findings align with the outcomes of prior intracellular FRET experiments (Fig. 2F), reinforcing the stability of PLA-PEG nanoparticles. Moreover, compared to the blank control, none of the major organs (Heart, Liver, Spleen, Lung, and Kidney) displayed notable morphological differences in hematoxylin and eosin (H&E) staining (Figs. S21 and S23).
Toxicity associated with BRD4 degraders arises from multiple mechanisms, including off-target degradation toxicity in normal cells, cross-degradation of BET family proteins, and acquired drug resistance due to high-dose administration. However, for the sake of ease of handling, all groups of drugs in this experimental segment were measured by weight. This means that even though the weight of the drug in the most effective group was 20 mg/kg PLA-PEG-JQ1_CRBN, the actual amount of effective functional molecule was much lower than that of the small molecule. Additionally, the enhanced permeability and retention (EPR) effect of nanoparticles promotes tumor-specific accumulation of SM-PROTAC, thereby minimizing off-target degradation in normal tissues. As observed in Fig. 7 (Figs. S26–S29), compared to the nonspecific distribution and rapid clearance of free dye molecules (Cy5.5 and Cy7), the PLA-PEG nanoparticles exhibited better tumor accumulation and maintained long-term stability. Over time, the nanoparticles were primarily present in metabolic organs (mainly liver/spleen/kidney), yet a strong FRET signal remained at the tumor site. Taken together, our in vivo experiments confirmed that the PLA-PEG-based SM-PROTAC was highly efficient and durable. This further implies that PLA-PEG-based SM-PROTAC holds immense potential for expansive clinical applications in cancer therapy.
A Live Fluorescence imaging of mice. Images were taken utilizing the IVIS live imaging system with excitation and emission wavelengths set to 660 and 790 nm, respectively. B Ex vivo fluorescence imaging of different organs. C, D The residual percent of fluorescence intensity (%) of different parts of mice. Using the fluorescence intensity at 3 h as the initial value. The free dye molecules (Cy5.5 and Cy7) were the positive control.
Discussion
In summary, this study comprehensively demonstrated the advantages of the PLA-PEG system for the development of SM-PROTACs, while concurrently highlighting the inherent simplicity, effectiveness, and programmability of the SM-PROTAC strategy. Against self-assembled peptides and liposomes, the PLA-PEG system not only boasts a simple preparation method that allows for large-scale expansion, but also possesses excellent efficiency in vivo. Notably, even at higher concentrations, experimental groups still exhibited negligible abnormalities attributed to drug injection. Furthermore, the tumor suppression experiments indicated that the PLA-PEG-based SM-PROTAC has a prolonged duration of action in vivo, offering the advantages of lower dosage requirements, higher therapeutic efficacy, and long-lasting effects compared to its corresponding small-molecule PROTAC counterparts. This advantage aligns perfectly with the aspirations for SM-PROTAC development, marking the first comparison between small-molecule PROTACs and their PLA-PEG-based SM-PROTAC. Furthermore, degrader-antibody conjugates technology has achieved advancements due to its superior targeting specificity and reduced toxicity profile, offering valuable insights for SM-PROTAC development37,38,39,40,41,42. SM-PROTAC can leverage its modular assembly capability to efficiently incorporate targeting ligands, including small molecules, peptides, or antibody-based agents, thereby further enhancing degradation selectivity and reducing toxicity and side effects. It is imperative that we actively seek out novel approaches in subsequent research to accumulate more comprehensive metabolic data on PLA-PEG-based SM-PROTACs, thereby advancing their applicability and ensuring consistency and stability across different manufacturing batches.
Methods
Reagents and materials
PLA-PEG-NHS, PLA-PEG-Cy3, PLA-PEG-Cy5, Cy3-NHS or Cy3-NH2, Cy5-NHS or Cy5-NH2 were purchased from Yusi Pharmaceutical Technology Co., Ltd (Chongqing, China). MG132, MLN7243 proteasome inhibitor, JQ1 analogue, Palbocilib analogue, Tamoxifen analogue, Thalidomide analogue, VHL (von Hippel-Lindau) ligand, Polyvinyl alcohol (PVA, 87.5 mol% hydrolyzed), and other HPLC-grade solvents are all commercially available. Dulbecco’s Modified Eagle Medium (DMEM), RPMI Medium 1640, 0.25% Trypsin-EDTA (1×), penicillin-streptomycin (PS), and fetal bovine serum (FBS) were purchased from Thermo Fisher Scientific (Shanghai, China). HCT116, T47D, HeLa, and MCF-7 cell lines were purchased from ATCC. The anti-HA antibody, anti-BRD4 antibody, anti-GAPDH antibody, anti-CDK4 antibody, anti-ERα antibody, anti-tubulin antibody, anti-mouse IgG, and anti-rabbit IgG were purchased from CST (Shanghai, China) or Abcam (Shanghai, China). RIPA lysis buffer, Tris-Base, glycine, SDS, and 4% paraformaldehyde were purchased from Beijing Solarbio Science & Technology Co., Ltd (Beijing, China). Dialysis membranes MD44(3500D) and YA1069(100-500D) were purchased from Beijing Solarbio Science & Technology Co., Ltd (Beijing, China).
PLA-PEG-ligand synthesis and purification
The coupling of PLA-PEG with diverse ligands is depicted in Fig. S3. Specifically, PLA-PEG-NHS was dissolved in DMSO, and subsequently, each ligand bearing amino reaction groups was added in a ratio equivalent to 1.2 times the amount of PLA-PEG-NHS, utilizing DIPEA as the catalyst. Once the reaction progressed for 12 h, the entire reaction system was carefully transferred to a dialysis bag with a molecular weight cutoff of 1000 Da. The dialysis process was then conducted for a duration of 3–4 days, during which it was crucial to ensure that the dialysate (Deionized water) was replaced every 30 min during the initial few hours and then at a reduced frequency over the subsequent hours. After the dialysis process, the liquid contained within the dialysis bag was subjected to freeze-drying to yield the desired PLA-PEG-Ligand product. This final product was subsequently characterized using nuclear magnetic resonance (NMR) spectroscopy for analysis.
UV spectral analysis
In order to quantify different PLA-PEG-Ligand relatively accurately, the functional ligand content was calculated in this paper, except for animal experiments (Measured by the total weight of PLA-PEG-Ligand). That is, before each assembly, the corresponding small molecule ligand is used as the standard sample, and the standard curve is constructed by the UV-visible absorption spectrometer (Nanodrop 2000C, Thermo Fisher Scientific Inc.). The maximum absorption wavelengths of different small molecular ligands in the case of acetonitrile as solvent are: ERα Ligand:335 nm; VHL Ligand: 310 nm; CRBN Ligand: 420 nm; BRD4 Ligand: 334 nm; CDK4 Ligand: 362 nm. These data are also obtained by Nanodrop full-spectrum scanning. Then, at the corresponding maximum absorption wavelength, the absorption value of the corresponding PLA-PEG-Ligand is detected, and the absorption value is brought into the above standard curve to obtain the corresponding functional ligand content.
Characterization of PLA-PEG nanoparticles
PLA-PEG nanoparticles size (PDI) and zeta potential were measured by dynamic light scattering (DLS) via a Nano ZS Zetasizer (Malvern Instruments, UK) at the 0.5 mg/mL concentration. The morphology of PLA-PEG nanoparticles was observed by SEM (ZEISS SUPRA® 5) or TEM (Commercial).
To test the stability of the PLA-PEG nanoparticles in the physiological environment, the PLA-PEG nanoparticles were dispersed in PBS or FBS or trypsin, and protease cocktail and stored at room temperature. After different hours, the PLA-PEG nanoparticles were measured by Nano ZS Zetasizer (Malvern Instruments, UK) or SEM (ZEISS SUPRA® 5).
Cell viability by CCK8 assay
To explore the cytotoxicity of the PLA-PEG nanoparticles in vitro, Different cell lines were seeded into the 96-well cell culture plates for 24 h. Then, the cells were treated with 100 μL of different concentrations of PLA-PEG nanoparticles (or molecule group) for 48 h. The cell viability was then assessed by CCK-8 assay (Cell Counting Kit-8) (Biosharp, BS350B), in brief, 10 μL of CCK8 was added to each well and incubated at 37 °C for 0.5–2.0 h. The absorbance was measured at 450 nm by a microplate reader (BioTek, USA).
Cellular uptake assay
To investigate the cellular uptake ability of the PLA-PEG nanoparticles, HCT116 cells were seeded into the 12-well cell culture plates and incubated with Cy3, Cy5, or Cy3-Cy5 labeled PLA-PEG (or nanoparticles) for 4 h. Then cells were collected and washed with PBS and 0.4% trypan blue, and the intracellular fluorescence intensity was analyzed by flow cytometry (ThermoFisher Attune NxT, USA).
Lysosomal escape capability of NPs in HCT116 cells
To explore the lysosomal escape ability of as-fabricated NPs, HCT116 cells were incubated with PLA-PEG-Cy3_Cy5 NPs and staining by the LysoTracker™ Green DND-26 (1 mM, 0.2 μL) at different time points and DAPI at 37 °C, and using CLSM imaging to localize the NPs’ distribution, as well as their lysosomal escape capability.
Confocal microscopy imaging
To investigate the visualization distribution and cellular uptake ability of the PLA-PEG nanoparticles in cells, HCT116 cells were seeded into coverslips, which were placed in the 24-well cell culture plates. The next day, HCT116 cells were incubated with Cy3, Cy5, or Cy3-Cy5 labeled PLA-PEG (or nanoparticles) for different hours. The details are as follows: the duration of the cell membrane penetration test spans 4 h, whereas the stability test (FRET) encompasses various durations, as detailed in the manuscript. Then, the culture medium was removed and washed with PBS three times, and the cells were fixed with 4% formaldehyde at room temperature for 10 min, then washed with PBS and mounted on slides with a DAPI-containing mounting medium. Finally, samples were imaged using a confocal laser scanning microscope (Zeiss, LSM980, Germany). The FRET channel is characterized by an excitation wavelength of 543 nm, with an emission wavelength spanning the range of 643–756 nm.
Western blot and immunoprecipitation
Cell lines were seeded into the 12-well cell culture plates for 24 h and then incubated with different formulations. First, to obtain the cell lysate protein, the cells were washed with PBS and then lysed by RIPA lysis buffer (Solarbio, R0100), encompassing protease inhibitor cocktail (1:100) (MedChemExpress, HY-K0010) on ice. Then cells were centrifuged at 12,000 g at 4 °C for 15 min. The protein lysate was collected, and the concentrations were tested by the bicinchoninic acid protein assay kit (Elabscience, E-BC-K318-M). Next, the protein samples were denatured at 100 °C and separated by the 10–12% SDS-PAGE gel, then transferred onto a polyvinylidene fluoride (PVDF) membrane by a wet transfer cell (Bio-Rad, USA). After that, the PVDF membrane was blocked with 5% skim milk at room temperature for 1 h and incubated with a specific primary antibody overnight at 4 °C. After being washed, the PVDF membrane was incubated with the secondary antibody at room temperature for 1 h. Finally, the PVDF membrane was stained with ECL Western blotting detection reagents and imaged by BioRad ChemiDoc XRS System. All image data were analyzed with the Image LabTM Software. Primary antibodies included BRD4 (1:1000; Abcam, ab128874), ERα (1:1000; CST, #8644), CDK4 (1:1000; CST, #12790), β-Tubulin (1:1000; CST, #15115) and GAPDH (1:5000; CST, #97166). For immunoprecipitation, cells were seeded into 6-well cell culture plates for 24 h. Then the cells were transfected with pcDNA3.1-HA-ubiquitin for 48 h and treated with the degradation group (with or without 10 μM MG132 or MLN7243 (MedChemExpress, HY-13259) or 50 μM Pomalidomide) for 8 h or 24 h. Subsequently, total cell protein was obtained and determined according to the steps above. Finally, the protein lysate was preincubated with anti-HA rabbit antibody (1 μg) at 4 °C overnight, and 30 μL Protein G Sepharose (Cytiva, Shanghai, China) was added for 2 h at 4 °C. The precipitates were washed with PMSF-containing PBS and analyzed by western blotting.
In vivo xenograft assay
All in vivo experimental procedures were approved by the Peking University Shenzhen Graduate School Animal Care and Use Committee and performed according to the national and international guidelines for the humane treatment of animals (Approval number: AP0023024). Balb/C nude mice (19–20 g, 5–6 weeks old) were purchased from Jiangsu Xishan Biotechnology Co., Ltd. Animal Center (Jiangsu, China). All animal experiments were performed in accordance with the guidelines of the Institutional Animal Care and Use Committee of Shenzhen Glorybay Biotech Co., Ltd. Balb/C nude mice were used to evaluate the antitumor performance of the PLA-PEG nanoparticles in vivo. The 6-week-old female Balb/C nude mice were injected with 100 μL of HCT116 cell suspensions (2 × 106/100 μL, resuspended in PBS). When the tumor volume reached about 50–100 mm3, the mice were randomly divided into 7 groups (n = 5) and were injected with different drugs. The tumor-bearing mice were administered by intraperitoneal injection or intravenous injection. The mice’s tumor volume and body weight were recorded every 3 days, and tumor volume was calculated by equation V (mm3) = (a × b × b) (a and b represent the longest dimension and the shortest dimension of the tumor tissues). At the end of the treatment, the mice’s tumors and major organs (heart, liver, spleen, lung, kidney, and brain) were collected. The weight and the microscopic images of the tumors were collected, and organs were fixed with 4% paraformaldehyde (Biosharp, BL539A) for hematoxylin-eosin (HE) staining. During mouse experiments, when it is necessary to keep the mice unconscious for experimental procedures, the mice will be placed in an anesthesia machine for anesthesia administration, with isoflurane selected as the anesthetic agent.
In vivo fluorescence imaging assay
To facilitate optimal in vivo fluorescence imaging, Cy5.5 and Cy7 fluorescent dyes were selected for this study. These dyes not only exhibit excellent tissue penetration depth but also undergo FRET, enabling stable nanoparticle detection while maintaining structural integrity. After successful tumor seeding of mice, the following procedures were performed: (1) In vivo fluorescence detection. At 3, 24, and 48 h after a single administration, in vivo fluorescence detection (Ex: 660 nm, Em: 710/790 nm) was performed. (2) Fluorescence Detection of isolated organs. At 3, 24, and 48 h after a single administration, organ tissues (heart, liver, spleen, lung, both kidneys, intestine, and tumor) were collected from three animals at each time point for fluorescence detection (Ex: 660 nm, Em: 710/790 nm). (3) Determination of probe fluorescence Intensity in Whole blood. After the in vivo imaging at each time point was completed, 100–200 μL of anticoagulated whole blood was taken from the mouse orbit and placed in an anticoagulated brown 96-well plate for fluorescence photography (Ex: 660 nm, Em: 710/790 nm). During mouse experiments, when it is necessary to keep the mice unconscious for experimental procedures, the mice will be placed in an anesthesia machine for anesthesia administration, with isoflurane selected as the anesthetic agent.
Reporting summary
Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.
Data availability
The authors declare that the data supporting the findings of this study are available within the article, source data, and its Supplementary Information. A reporting summary for this article is available as a Supplementary Information file. Any remaining information can be obtained from the corresponding author upon reasonable request. Source data are provided with this paper.
References
Gu, S. S., Cui, D. R., Chen, X. Y., Xiong, X. F. & Zhao, Y. C. PROTACs: an emerging targeting technique for protein degradation in drug discovery. Bioessays 40, e1700247 (2018).
Békés, M., Langley, D. R. & Crews, C. M. PROTAC targeted protein degraders: the past is prologue. Nat. Rev. Drug Discov. 21, 181–200 (2022).
Lazo, J. S. & Sharlow, E. R. Drugging undruggable molecular cancer targets. Annu Rev. Pharm. Toxicol. 56, 23–40 (2016).
Adams, D. et al. Long-term safety and efficacy of patisiran for hereditary transthyretin-mediated amyloidosis with polyneuropathy: 12-month results of an open-label extension study. Lancet Neurol. 20, 49–59 (2021).
Bedard, P. L., Hyman, D. M., Davids, M. S. & Siu, L. L. Small molecules, big impact: 20 years of targeted therapy in oncology. Lancet 395, 1078–1088 (2020).
Wang, T. et al. Identification and characterization of essential genes in the human genome. Science 350, 1096–1101 (2015).
Zhong, L. et al. Small molecules in targeted cancer therapy: advances, challenges, and future perspectives. Signal Transduct. Tar. 6, 201 (2021).
Chirnomas, D., Hornberger, K. R. & Crews, C. M. Protein degraders enter the clinic - a new approach to cancer therapy. Nat. Rev. Clin. Oncol. 20, 265–278 (2023).
Price, E. et al. Beyond rule of five and PROTACs in modern drug discovery: polarity reducers, chameleonicity, and the evolving physicochemical landscape. J. Med. Chem. 67, 5683–5698 (2024).
Gao, J. et al. Engineered bioorthogonal POLY-PROTAC nanoparticles for tumour-specific protein degradation and precise cancer therapy. Nat. Commun. 13, 4318 (2022).
Wang, W. S. et al. Self-assembled nano-PROTAC enables near-infrared photodynamic proteolysis for cancer therapy. J. Am. Chem. Soc. 145, 16642–16649 (2023).
Chen, Y. et al. Proteolysis-targeting chimera (PROTAC) delivery system: advancing protein degraders towards clinical translation. Chem. Soc. Rev. 51, 5330–5350 (2022).
Liu, Z. et al. An overview of PROTACs: a promising drug discovery paradigm. Mol. Biomed. 3, 46 (2022).
Lu, X. C. et al. Self-assembled PROTACs enable protein degradation to reprogram the tumor microenvironment for synergistically enhanced colorectal cancer immunotherapy. Bioact. Mater. 43, 255–272 (2025).
Song, C. L. et al. Selective protein of interest degradation through the split-and-mix liposome proteolysis targeting chimera approach. J. Am. Chem. Soc. 145, 21860–21870 (2023).
Yang, F. F. et al. Targeted biomolecule regulation platform: a split-and-mix PROTAC approach. J. Am. Chem. Soc. 145, 7879–7887 (2023).
Bemis, T. A., La Clair, J. J. & Burkart, M. D. Unraveling the role of linker design in proteolysis targeting chimeras. J. Med Chem. 64, 8042–8052 (2021).
Chen, N. L. et al. Sulfatase-induced in situ formulation of antineoplastic supra-PROTACs. J. Am. Chem. Soc. 146, 10753–10766 (2024).
Zhang, N. Y. et al. Nano proteolysis targeting chimeras (PROTACs) with anti-hook effect for tumor therapy. Angew. Chem. Int. Ed. 62, e202308049 (2023).
Moon, Y. et al. Self-assembled peptide-derived proteolysis-targeting chimera (PROTAC) nanoparticles for tumor-targeted and durable PD-L1 degradation in cancer immunotherapy. Angew. Chem. Int. Edit. 64, e202414146 (2024).
Shin, W. et al. Visualization of membrane pore in live cells reveals a dynamic-pore theory governing fusion and endocytosis. Cell 173, 934 (2018).
Wang, Y. F. et al. Transportation of AIE-visualized nanoliposomes is dominated by the protein corona. Natl. Sci. Rev. 8, nwab068 (2021).
Kulkarni, R. K., Pani, K. C., Neuman, C. & Leonard, F. Polylactic acid for surgical implants. Arch. Surg. 93, 839–843 (1966).
Dong, Y. & Feng, S. S. Methoxy poly(ethylene glycol)-poly(lactide) (MPEG-PLA) nanoparticles for controlled delivery of anticancer drugs. Biomaterials 25, 2843–2849 (2004).
Mundel, R., Thakur, T. & Chatterjee, M. Emerging uses of PLA-PEG copolymer in cancer drug delivery. 3 Biotech 12, 41 (2022).
Lee, S. Y. et al. Preparation of nano-emulsified paclitaxel using MPEG-PLGA diblock copolymers. Colloid Surf. A 313, 126–130 (2008).
Lu, J. et al. Hijacking the E3 ubiquitin ligase cereblon to efficiently target BRD4. Chem. Biol. 22, 755–763 (2015).
He, Y. H. et al. Brd4 proteolysis-targeting chimera nanoparticles sensitized colorectal cancer chemotherapy. J. Control Release 354, 155–166 (2023).
Asangani, I. A. et al. Therapeutic targeting of BET bromodomain proteins in castration-resistant prostate cancer. Nature 510, 278–282 (2014).
Shi, J. et al. Disrupting the interaction of BRD4 with diacetylated twist suppresses tumorigenesis in basal-like breast cancer. Cancer Cell 25, 210–225 (2014).
Vasir, J. K. & Labhasetwar, V. Biodegradable nanoparticles for cytosolic delivery of therapeutics. Adv. Drug Deliv. Rev. 59, 718–728 (2007).
Saadati, R. & Dadashzadeh, S. Marked effects of combined TPGS and PVA emulsifiers in the fabrication of etoposide-loaded PLGA-PEG nanoparticles: in vitro and in vivo evaluation. Int J. Pharm. 464, 135–144 (2014).
Poller, B., Painter, G. F. & Walker, G. F. Influence of albumin in the microfluidic synthesis of PEG-PLGA nanoparticles. Pharm. Nanotechnol. 7, 460–468 (2019).
Camli, S. T., Buyukserin, F., Balci, O. & Budak, G. G. Size controlled synthesis of sub-100 nm monodisperse poly(methylmethacrylate) nanoparticles using surfactant-free emulsion polymerization. J. Colloid Interface Sci. 344, 528–532 (2010).
Sun, X. R. et al. The blood clearance kinetics and pathway of polymeric micelles in cancer drug delivery. Acs Nano 12, 6179–6192 (2018).
Li, M. et al. Autophagy caught in the act: a supramolecular fret pair based on an ultrastable synthetic host-guest complex visualizes autophagosome-lysosome fusion. Angew. Chem. Int. Ed. 57, 2120–2125 (2018).
Uitdehaag, J. C. et al. A novel kinase degrader antibody conjugate for the treatment of mCRPC. Cancer Res. 85, 5468 (2025).
Hong, K. B. & An, H. Degrader-antibody conjugates: emerging new modality. J. Med. Chem. 66, 140–148 (2023).
Guo, Y. L.; Li, X. X.; Xie, Y.; Wang, Y. X., What influences the activity of degrader-antibody conjugates (DACs). Eur. J. Med. Chem. 268, 116216 (2024).
Dragovich, P. S. Degrader-antibody conjugates. Chem. Soc. Rev. 51, 3886–3897 (2022).
Dong, X. W. et al. Potent in vitro and in vivo efficacy of Hdz-C123A, a GSPT1 degrader-antibody conjugate targeting CD123 in acute myeloid leukemia. Blood 144, 156–156 (2024).
Chen, C. J. et al. Discovery and characterization of DAC-1522, a novel Trop2-targeting degrader-antibody conjugate for precision oncology. Cancer Res. 85, 7005 (2025).
Acknowledgements
This work was funded by the National Key Research and Development Program “Synthetic Biology” Key Special Project of China, 2021YFC2103900, 2021YFA0910803, the Natural Science Foundation of China grants 21933004, 21977010, 22307081, 21977011, 22307084, National Center for Biological Medicine Technology Innovation, NCTIB2022HS01017, the Key-Area Research and Development Program of Guangdong Province 2020B0101350001, 2020B010188001, Natural Science Foundation of Guangdong Province, 2022A1515010996, 2020A1515010766, Shenzhen Science and Technology Program, RCYX20231211090129037, RCJC20200714114433053, JCYJ20200109140406047, JCYJ20220818095808019, JCYJ20230807151059004, JCYJ20210324102809026, Shenzhen Fundamental Research Program GXWD20201231165807007-20200812124825001, Beijing National Laboratory of Molecular Science open grant BNLMS20160112; Shenzhen-Hong Kong Institute of Brain Science-Shenzhen Fundamental Research Institutions grant 2019SHIBS0004, Tian Fu Jin Cheng Laboratory (Advanced Medical Center) Group Racing Project TFJC2023010008; China Postdoctoral Science Foundation under Grant Number 2024M752153.
Author information
Authors and Affiliations
Contributions
M.Z.: Original draft, validation, methodology, investigation, formal analysis, data curation, funding acquisition; H.C.: Validation, methodology, investigation, data curation; M.H., C.S., and Z.J.: Assist in conducting some cell experiments and animal experiments; N.L., Z.Liu., Z.H., and Y.C.: Assist in providing related cells or proteins and other materials; Z.S.: Assist in the morphological characterization of nanoparticles. Y.X: Writing review and editing. Z.Li.: Supervision, funding acquisition, and conceptualization. F.Y.: Validation, supervision, funding acquisition, and conceptualization.
Corresponding authors
Ethics declarations
Competing interests
The authors declare no competing interests.
Peer review
Peer review information
Nature Communications thanks Jinmin Miao, Yan Xiong, and Haibin Zhou for their contribution to the peer review of this work. A peer review file is available.
Additional information
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Source data
Rights and permissions
Open Access This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which permits any non-commercial use, sharing, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if you modified the licensed material. You do not have permission under this licence to share adapted material derived from this article or parts of it. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by-nc-nd/4.0/.
About this article
Cite this article
Zhan, Mm., Chen, H., He, M. et al. Highly efficient and long-acting split-and-mix proteolysis targeting chimera based on self-assembled polylactic acid. Nat Commun 16, 10555 (2025). https://doi.org/10.1038/s41467-025-65590-6
Received:
Accepted:
Published:
Version of record:
DOI: https://doi.org/10.1038/s41467-025-65590-6
