Animals

BALB/c mice (male, 18–20 g) were purchased from Peking University Health Science Center (Beijing, China). CT26-bearing mice model was established by injecting 1 ×105 CT26 cells in the right underarm of BALB/c mice subcutaneously. All animal experiments were performed under an Animal Ethics Committee of Peking University approved protocol (LA2020345).

Preparation and characterizations of PSRNs and PNRNs

PEG-b-P(EPA-r-PROTAC) was dissolved in dimethyl sulfoxide (DMSO) and added into ddH2O under sonication. After removing DMSO by ultrafiltration, the PSRNs were finished as a yellow suspension. PNRNs was prepared under the same method except that PEG-b-P(EPA-r-PROTAC) was replaced with PEG-b-P(EH-r-PROTAC). For Cy5-labeled formulations, PEG-b-P(EPA)-Cy5 or PEG-b-P(EH)-Cy5 was added additionally during preparation.

The concentration of the preparation was adjusted to 1 mg/mL (calculated based on polymer concentration) for characterization. The particle size and zeta-potential were measured by Zetasizer Nano ZS (Malvern). UV absorption spectrum and fluorescence spectrum were analyzed by UV-Vis (Hitachi) and spectro fluorophotometer (Shimadzu), respectively. For transition pH of PSRNs, count rates at different pH were analyzed using Zetasizer Nano ZS.

TEM analysis

TEM images in different medium were captured by transmission electron microscope (JEM 1400PLUS). 10 µL of formulation at a concentration of 1.0 mg/mL (calculated based on polymer concentration) was dropped onto copper grids treated with de-electrostatic methods. After standing at room temperature for 2 min, surface moisture was removed by clean filter paper gently. Subsequently, 5 µL of deionized water was used for rinsing, followed by the addition of 5 µL of 2% phosphotungstic acid solution (pH 7.0) for negative staining for 3 min. Surface stain was removed by clean filter paper, and the residual stain was dried using a cold air blower for approximately 30 s.

In vitro release of PROTAC

Release of PROTAC in vitro from polymer, PSRNs and PNRNs were evaluated in different medium. Briefly, the formulations were dispersed in the medium (0.5 mL) and incubated at 37 °C with a shaking rate of 100 rpm. At predetermined time points, a sample of the medium (20 µL) was collected, then pre-cooled sodium azide solution (10 µL) and acetonitrile (70 µL) were added immediately. The concentration of the released PROTAC was examined by HPLC (Shimadzu. Mobile phase: A: ACN, 70%, B: H2O with 0.1% (v/v) ammonium hydroxide, 30%; flow rate: 1 mL/min; column temperature: 40 °C; measure wavelength: 359 nm).

Western blot assay

PROTAC-induced protein CDK4/6 degradation in vitro was analyzed by western blot assay. Briefly, CT26, MDA-MB-231, MCF-7 and PANC-1 cells were seeded into 12-well plates at a density of 20 ×104 cells per well. Following an overnight incubation at 37 °C, the culture medium was replaced with fresh medium containing either free PROTAC, PSRNs or PNRNs under the predetermined conditions. The concentration of PROTAC in PSRNs and PNRNs was quantified using UV spectrophotometry at 359 nm. For studying the PROTAC-mediated protein degradation, different concentrations of MG132 were used as the proteasome inhibitors co-incubated with the preparations for 16 h. CA-074Me was used as a cathepsin B inhibitor (20 μM) incubated for 4 h in advance. At the end of experiments, cell lysates were collected. The protein samples were separated by SDS-PAGE and transferred onto a PVDF membrane (Millipore). Then, the membranes were blocked with blocking buffer for 30 min at room temperature, and incubated with primary antibodies (anti-CDK4, 1:2000; anti-CDK6, 1:1000; anti-β-tubulin, 1:10000; anti-Rb phospho S807, 1:1000) at 4 °C overnight. After washing 3 times using tris buffered saline with 1% Tween-20, HRP-conjugated secondary antibodies (HRP-conjugated Goat Anti-Rabbit IgG, 1:10000; HRP-conjugated Goat Anti-Mouse IgG, 1:10000) were added and incubated with the membrane at room temperature for 1 h. Finally, after washing 6 times using tris buffered saline with 1% Tween-20, the membranes were imaged with a gel imager (Tanon). ImageJ was used for quantitative analysis.

Cytotoxicity and cell cycle analysis in vitro

The in vitro cytotoxicity was evaluated using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. CT26, MDA-MB-231, MCF-7, PANC-1, HUVEC and L-929 cells were seeded into 96-well plates at a density of 1500 cells per well. Following an overnight incubation at 37 °C, the culture medium was replaced by fresh medium containing different PROTAC preparations at predetermined concentrations. After 72 h of incubation, MTT was added to each well for a subsequent 4 h incubation. Finally, a microplate reader (Thermo scientific) was used to measure the relative cell viabilities at an absorbance of 490 nm. The IC50 values were calculated by GraphPad.

For cell cycle analysis, CT26 cells were treated with different PROTAC preparations (PROTAC concentration of 1 μM) for 48 h. At the end of experiment, cells were collected and fixed with pre-cold 70% ethanol at 4 °C overnight. After washing with PBS, the cells were stained with propidium iodide at 37 °C for 30 min. Then, cells were measured by flow cytometer (Calibur 2, BD). The cell cycle was analyzed with the FlowJo (v10.6.2).

Cellular uptake and penetration in vitro

The uptake of PSRNs and PNRNs at different pH values were quantified by flow cytometry (Calibur 2, BD). Briefly, CT26 cells were seeded into 12-well plates and incubated with Cy5-labeled preparations for predetermined time points at pH 7.4/6.6, respectively. The intracellular fluorescence intensity was then examined by flow cytometry. PSRNs taken in cells and cleaved intracellular PROTAC were measured by Liquid Chromatography-Mass Spectrometry (LC/MS) (AB SCIEX. Mobile phase: a gradient elution was performed using solvent A (0.1% formic acid in water, v/v) and solvent B (acetonitrile) at a flow rate of 0.4 mL/min). Concentration of PROTAC in cell homogenate after incubating with papain solution for 4 h was regarded as PSRNs internalized.

We further explored the uptake mechanism of PSRNs. Briefly, CT26 cells were plated onto 12-well plates and incubated with different inhibitors (Chlorpromazine and Dynasore, clathrin inhibitor; M-β-CD and Nystatin, caveolin inhibitor; Amiloride, macropinocytosis inhibitor; Cytochalasin D, cytoskeleton inhibitor; Hypertonic sucrose, cell membrane fluidity inhibitor; 4 °C, energy inhibitor) for 1 h. After incubation, medium was replaced with preparations included medium for another 2 h at pH 7.4/6.6, respectively. Pre-incubation with blank medium used as control. The intracellular fluorescence intensity was then examined by flow cytometry.

Tumor spheroids of MCF-7, CT26 and PANC-1 cells (used as high-, medium- and low-level permeability tumor model, respectively) were employed to evaluate penetration of different PROTAC preparations in vitro. Briefly, the 3D tumor spheroids were cultured in 48-well plate onto 2% agarose gel. Then, spheroids were incubated with Cy5-labeled preparations at pH 7.4 and 6.6 for 8 h. Z-stack images of spheroids were captured by confocal laser scanning microscopy (CLSM, Nikon). Fluorescence intensity profiles were analyzed using ImageJ.

Pharmacokinetics and biodistribution in vivo

Health male BALB/c mice were employed to study the pharmacokinetics profiles of PSRNs and PNRNs. The mice were randomly divided into 2 groups (n = 5 per group), and administrated i.v. with Cy5-labeled preparations at a dose of 100 mg/kg, based on polymer concentration. At predetermined time intervals post-administration (0.033, 0.25, 0.5, 1, 3, 6, 12, 24, 48 and 72 h), blood samples were collected and centrifuged at 2000 rpm for plasma. Then an aliquot of plasma (20 µL) was mixed with acidified methanol (200 µL), vortexed for 10 min, then centrifuged again at 12000 rpm for 10 min to precipitate proteins. The supernatant was collected and analyzed using spectro fluorophotometer (λex/em = 640/665 nm). Inject Dose (%) = (FITest/FI100%) × 100%. Fluorescence intensity of indicated time points was recorded as FITest. Fluorescence intensity of 0.033 min was regarded as FI100%. Pharmacokinetic parameters were analyzed by DAS 2.0.

The biodistribution of preparations in vivo was examined using CT26-bearing mice with tumor volumes of 150–200 mm3. Briefly, real-time fluorescence images of mice were collected at predetermined time points (1, 2, 4, 8, 12, 24 and 48 h) after i.v. administration using IVIS imaging system (IVIS Lumina Series III). At 24 and 48 h post-injection, mice were sacrificed. The major organs and tumor tissues were harvested for ex vivo fluorescence imaging. The relative fluorescence intensity derived from the images was used to semi-quantitative analysis of accumulation of preparations in tumors and biodistribution in major organs. To visualize penetration of preparations in tumor, the tumor tissues (24 h) were freezing-sectioned into slices of 10 µm. Followed by fixing with 4% paraformaldehyde and blocking with 5% FBS, slices were cultured with CD31 antibodies (1:600), Alexa Fluor-488 labeled secondary antibody (1:800) and anti-fade mounting medium (Hochest 33342 included). Then captured by automated pathology imaging system (Vectra Polaris, Akoya).

Anti-tumor study in vivoTherapeutic efficacy of PSNRs

CT26 colorectal cancer bearing mice were employed to assess anti-tumor efficacy of PSRNs in vivo. The mice with tumors in the range of 50–100 mm3 were randomly divided into six groups (n = 5 per group): Saline, PEG-b-P(EPA), free PROTAC, PSRNs, PSRNs + α-PD-1 and α-PD-1 alone. Dose: PROTAC of 5 mg/kg, α-PD-1 of 50 μg each. The treatment groups received intravenous injections of either PEG-b-P(EPA), free PROTAC, or PSRNs at a PROTAC dose of 5 mg/kg on days 0, 3, 6, 9 and 12. Additionally, the PSRNs + α-PD-1 and the α-PD-1 alone group received intraperitoneal injections of α-PD-1 on days 1, 4, 7, 10 and 13. Tumor growth and body weight of mice were monitored every 2 days. The tumors, major organs and serums of mice were harvested at the end of the studies. The tumor burden should not exceed 10% of the animal’s body weight. Herein, CDK4/6 protein levels were evaluated by western blot assay with tumor lysate. In addition, tumors and organs were fixed and stained with H&E for investigating anti-tumor efficacy and primary safety evaluation.

The tumor volume was calculated with formula: V = Length × Width × Width/2.

Survival experiment of PSNRs

We employed CT26-bearing mice for survival experiment. Mice with tumor volumes of 50–100 mm3 were randomly divided into six groups (n = 5 per group): Saline, PEG-b-P(EPA), free PROTAC, PSRNs, PSRNs + α-PD-1 and α-PD-1. Dose: PROTAC of 5 mg/kg, α-PD-1 of 50 μg each. Experimental schedule was described above. Tumor growth and body weight of mice were monitored every 2 days.

After treatment, the condition of the mice was monitored continuously. The mice were euthanized when the tumor volume reached 2000 mm3.32,55 The survival time of mice in each group was recorded for survival curve.

Protein degradation efficacy in vitro and in vivoWash-out assay in vitro

To evaluate protein degradation efficiency of PSRNs in different medium, wash-out assay was used. Briefly, CT26 cells were plated onto 12-well plates and then incubated with different PROTAC preparations at pH 7.4 and 6.6 (PROTAC of 1 μM). After incubation for 16 h, PROTAC-containing medium was replaced with regular medium. Cells were cultured continuously. Protein expression level in cell lysate was then analyzed by western blot assay at predetermined time points. ImageJ was used for quantitative analysis.

Protein level in vivo post-single administration

CT26-bearing mice were employed to investigate changes of protein expression at tumor site post-single administration. Briefly, CT26 bearing mice with tumor volume of about 200 mm3 were randomly divided into 2 groups (free PROTAC, PSRNs, n = 12 per group). Mice were sacrificed after 12, 24, 48 and 72 h of injection of different preparations. Tumors were extracted for western blot assay to evaluate changes of CDK4/6 protein expression (tumors from untreated mice used as negative control). ImageJ was used for quantitative analysis.

CDK4/6 protein degradation percentages (%) were calculated as formula below:

$$}\, \% =(}}_-})/}}_\times 100 \%$$

Based on data quantified by ImageJ, the relative protein expression level change curves vs. time were plotted. Relative protein expression level of 100% used as initial protein expression level, and plotted a straight line. A100% represents area under the straight. AUC represents the area under the curve of protein expression changes.

Further, tumor tissues were freezing-sectioned into slices of 10 µm. Followed by fixing with 4% paraformaldehyde and blocking with 5% FBS, slices were cultured with CDK4/6 antibodies, fluorescein labeled secondary antibody and Hochest 33342, in sequence. Then captured by CLSM to visualize CDK4/6 expression level in tumor.

Expression level of PD-L1 in vitro

Briefly, CT26 cells were incubated with different PROTAC preparations for 48 h. At the end of experiment, cells were collected to investigate PD-L1 protein levels by flow cytometer and western blot assay. IFN-γ and palbociclib treated group were used as positive control.

In Vivo immune response analysisFlow cytometry for immune cell in vivo

Spleen and lymph nodes: single cell suspensions were obtained by mechanical digestion.

Tumor tissues: single cell suspensions were obtained by enzyme digestion. Briefly, tumor tissues were firstly cut into pieces, then incubated with enzyme digestion solution (0.5 mg/mL collagenase IV, 0.2 mg/mL DNase and 0.2 mg/mL hyaluronidase in 1640 culture medium containing 5% FBS and 1% penicillin-streptomycin) at 37 °C for 30 min. Finally, single lymphocyte suspensions were separated by lymphocytes isolation kits.

One million cells per condition were stained with fluorochrome conjugated antibodies diluted in PBS for 30 min on ice. For intracellular stain, cell suspensions should be fixed and permeabilizated with 4% paraformaldehyde and permeabilization buffer. Cellometer (Nexcelom) was used to determine the number of cells. Flow cytometry was performed on LSRFortessa (BD Biosciences), and data were analyzed using FlowJo (10.6.2). Flow cytometry gating strategies were performed in Supplementary Fig. 24.

Uptake of PSRNs by immune cells post i.v. administration

To evaluate the uptake of PSRNs by immune cells in CT26 bearing mice. Briefly, CT26-bearing mice with tumor volume of about 150 mm3 were randomly divided into 2 groups (PSRNs and PNRNs, n = 3 per group). After intravenous administration of Cy5-labeled nanoparticles, mice were sacrificed. Tumors were collected and prepared into single lymphocyte suspension. The single-cell suspensions were incubated with appropriate-conjugated antibodies for 30 min on ice. After washing twice with cold PBS, suspensions were fixed. PBS washing was processed prior to flow cytometry analysis. Cy5 signals in Treg cells and CD8+T cells were collected.

Remodeling immunosuppression environment post in situ administration

Inhibitions of CDK4/6 suppress the proliferation of Treg cells. CT26 bearing mice with tumor volume around 150 mm3 were randomly divided into 4 groups (Saline, free PROTAC, PSRNs and palbociclib; Dose: PROTAC of 2 mg/kg; n = 5 per group). Mice were injected intratumorally with saline, free PROTAC, PSRNs and palbociclib (as positive control), respectively, every 2 days. Tumor growth and body weight of mice were monitored every 2 days. After 5 cycles, mice were sacrificed and tumor tissues were collected for immune-environment analysis. Tumor infiltrating lymphocytes (TILs) were prepared into single-cell suspension using mice lymphocytes isolation kit. The single-cell suspensions were incubated with fluorophore-conjugated antibodies: CD45-Pacific blue, CD3-FITC, CD8a-APC, CD4-PE/Cy7, and CD25-PerCP/Cy5.5 for 30 min on ice. After washing with cold PBS 2 times, suspensions were fixed and permeabilizated, and then incubated with Foxp3-PE antibody at 4 °C overnight. PBS washing was processed prior to flow cytometry analysis. Lymph nodes and spleens were also collected, stained and analyzed mentioned above. TGF-β and IFN-γ levels in serum were evaluated by ELISA kit.

In vivo immune response analysis post chem-immunotherapy

Tumors were harvested after chem-immunotherapy. TILs were prepared into single-cell suspension using mice lymphocytes isolation kit. Suspensions were stained by the same method above expect that adding one more fluorophore-conjugated intracellular antibodies (Granzyme B-BV421). PBS washing was processed prior to flow cytometry analysis. Lymph nodes and spleens were also collected, stained and analyzed by method mentioned above. TGF-β and IFN-γ levels in serum and tumor homogenate were evaluated by ELISA kit.

Statistical analysis

The GraphPad Prism 8.0 was used for the statistical analyses. Unpaired t-test and One-way analysis of variance (ANOVA) were used for statistical comparison. Log-rank (Mantel–Cox) test was used for the statistical comparison of the survival study.